Cell proliferation and migration are cellular processes essential to normal cell function; errors in these cellular processes impact the ability of a cell to maintain cellular homeostasis. The current study examines the role of Asef, a GEF for Rac1, in cell proliferation and migration in the presence or absence of EGF in a triple negative breast cancer epithelial cell line. Overexpression of Asef reduced stress fiber formation and increased cell migration and proliferation in MDA-MB-231 epithelial cells. These data indicate a role for Asef in the EGFR/Rac1 signaling cascade involved in regulating cellular behavior.
","acknowledgements":"The authors thank the SMC School of Science for their support of this project and the Cell Biology Education Consortium (CBEC) for feedback and comments on the manuscript.
","authors":[{"affiliations":["Saint Mary's College of California"],"departments":["Biology"],"credit":["investigation","methodology","writing_originalDraft"],"email":"na21@stmarys-ca.edu","firstName":"Natalie","lastName":"Alaniz","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":"0009-0004-5090-7951"},{"affiliations":["Saint Mary's College of California"],"departments":["Biology"],"credit":["conceptualization","formalAnalysis","methodology","project","supervision","writing_reviewEditing","investigation"],"email":"knk8@stmarys-ca.edu","firstName":"Khameeka N.","lastName":"Kitt","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":false,"WBId":null,"orcid":"0009-0003-2381-877X"}],"awards":[],"conflictsOfInterest":"The authors declare that there are no conflicts of interest present.
","dataTable":null,"extendedData":[],"funding":"The authors thank the SMC School of Science Summer Endowment fund. Undergraduate student research participation and publication support were funded by the Cell Biology Education Consortium (CBEC): Path to Publication (Award ID #2316122) through the National Science Foundation (NSF).
","image":{"url":"https://portal.micropublication.org/uploads/72bfc566efea79327b9d8ca28f7d6633.png"},"imageCaption":"Human MDA-MB-231 breast cancer epithelial cells were transfected with Asef-HA (green) and allowed to express for 24 hours before treatment with 10 ng/ml of EGF for 24 hours. (A) In the absence of EGF, Asef and Rac1 (red) localize to the cytoplasm and membrane. In the presence of EGF for 24 hours, Rac1 colocalizes with Asef at the cell membrane (arrows). Scale bar: 40 µm. (B) Asef localizes to the cytoplasm and membrane ruffles (arrows) in cells with and without EGF treatment. In the absence of EGF, EGFR (red) localizes to the cell membrane and cytoplasm in Asef-HA expressing cells (arrowhead). Cells treated with EGF for 24 hours caused a minor shift in EGFR localization from the membrane to vesicle-like structures inside the cell (arrowhead). Scale bar: 40 µm. (C) Asef localizes to the cytoplasm and membrane ruffles (arrows) in cells with and without EGF treatment. Cortical Actin (red) appears unchanged in Asef-HA expressing cells (arrowhead). Cells expressing Asef-HA show reduced stress fibers extending across the cell (asterisks) compared to untransfected cells (arrows) in the presence or absence of EGF for 24 hours. Scale bar: 40 µm. (D) MDA-MB-231 cell monolayers overexpressing HA-only or Asef-HA were scratched and imaged over 24 hours in the presence or absence of EGF. The width of the wound was measured using ImageJ and plotted to examine the wound width over time. Asef-HA expressing cells regardless of EGF treatment migrated faster than control cells, however, Asef-HA expressing cells without EGF treatment showed a significant increase in migration at 12 and 24 hour time points compared to HA only expressing cells. (**P < 0.005; n=3 measurements of each group). (E) MDA-MB-231 cell monolayers overexpressing HA-only or Asef-HA were serum- starved and treated with 10 ng/ml of EGF for 24 hours. Following EGF-treatment, MTT reagent was added, cells were solubilized with DMSO, and absorbance was read at 570 nm. Asef-HA expressing cells in the absence of EGF treatment showed a significant increase in cell proliferation compared to untransfected cells (*P < 0.05; n=3 measurements of each group). (F) Model of the Rho GTPase signaling pathway demonstrates how Asef can be recruited downstream of EGFR signaling to regulate Rac1 activation to stimulate cell proliferation and migration.
","imageTitle":"Asef-HA overexpression in MDA-MB-231 breast epithelial cancer cells causes changes in protein localization, cell migration, and cell proliferation.
","methods":"Cell Culture
Triple negative ductal breast carcinoma epithelial cell line MDA-MB-231 (ATCC) were grown in Dulbecco's Modified Eagle Serum (DMEM) high glucose media (Thermo Fisher Scientific) with 10% fetal bovine serum and 1% penicillin-streptomycin-glutamine (Thermo Fisher Scientific). Cells were incubated under the following conditions: 5% CO2 and 37˚C.
Transfection
MDA-MB-321 cells were transfected with pcDNA 3.1-HA Asef (human) full length plasmid (provided by Tetsu Akiyama lab, University of Tokyo, (Kawasaki et al., 2000)) using Lipofectamine 2000 diluted in Opti-MEM (Thermo Fisher Scientific) following product recommendations. As a control for proliferation and scratch assays, cells were transfected with pcDNA 3.1-HA plasmid alone. Transfected cells were incubated for 24 hours in antibiotic-free media before being replated in various well formats for various assays.
Immunofluorescence
Transfected MDA-MB-231 cells (with or without Asef-HA) were placed on rat tail collagen-coated coverslips in serum-free media and treated with or without 10 ng/mL of EGF for 24 hours. Cells were fixed using 4% paraformaldehyde 48 hours post-transfection. To localize HA, Rac1, and EGFR, cells were blocked and labeled with primary antibodies for HA (Roche), Rac1 (BD Biosciences) and EGFR (Thermo Fisher Scientific) followed by secondary antibodies conjugated to Alexa Fluor 488 and 594 (Thermo Fisher Scientific). Actin filaments and nuclei were labeled using Phalloidin 594 and Hoechst 33342 DNA stains, respectively (Thermo Fisher Scientific). Cells were imaged using the Leica STELLARIS Inverted Spectral Live Cell Confocal Microscope.
Scratch Assay (Wound Healing)
Transfected MDA-MB-231 cells (with or without Asef-HA) were placed on rat tail collagen-coated 12 well plates and allowed to form a monolayer in serum free media. Monolayers were scratched 24 hours later using a sterile 200 µL pipette tip, washed using phosphate-buffered saline (PBS) to remove debris, and incubated in serum-free media treated with or without 10 ng/mL of EGF for 24 hours. Images were captured at 0, 4, 8, 12 and 24 hours. The area between both sides of the scratch were quantified after each image time stamp to determine the wound closure distance using ImageJ software. Distance of wound closure for each group at each time point was measured in µm using ImageJ (pixels per micron was determined using scale bar) and plotted to display the width of the wound closure over time.
Proliferation (MTT) Assay
Transfected MDA-MB-231 cells (with or without Asef-HA) were placed in a 96-well tissue culture-treated plate in serum free media. Cells were treated with or without 10 ng/mL of EGF for 24 hours before adding 10 µL of a 5 mg/mL stock of MTT reagent per well and incubated for 3 hours. After three hours, the MTT reagent was removed and 100µL DMSO was added to each well to solubilize cells, and the optical density was measured at 570 nm (Tecan Plate Reader). Percent viability was calculated for each well and normalized against the control (HA-only).
Statistical Analysis
For the scratch assay, a multiple unpaired t-test analysis was performed to determine the significance at each time point. Three biological replicates (independent wells) were analyzed for each group. For the proliferation (MTT) assay, an unpaired t-test was used to determine significance between the treated groups compared to the control. Three biological replicates (independent wells) were analyzed for each group. All data was graphed using GraphPad Prism.
","reagents":"Plasmid | Description | Source |
pcDNA 3.1-HA- Asef | Full length Asef (human) | Tetsu Akiyama lab, University of Tokyo |
Cell Line | Description | Source |
MDA-MB-231 | Human Epithelial Breast Tissue isolated from Mammary Gland; Adenocarcinoma | ATCC Catalog #: HTB-26 |
Antibodies | Dilution | Source |
Phalloidin 594 | 1:400 | Thermo Fisher Scientific Catalog #: A12381 |
Alexa Goat anti Rabbit 488 | 1:800 | Thermo Fisher Scientific Catalog #: A-11008 |
Alexa Goat anti Mouse 594 | 1:800 | Thermo Fisher Scientific Catalog #: A-11005 |
HA (Rabbit) | 1:500 | Abcam Catalog #: ab9110 |
Rac1 (Mouse) | 1:200 | BD Biosciences Catalog #: 610651 |
EGFR (Mouse) | 1:200 | Thermo Fisher Scientific Catalog #: MA513269 |
Hoechst 33342 DNA stain | 1 µg/ml final | Thermo Fisher Scientific Catalog #: H3570 |
Breast cancer is not a single disease, but rather a group of diverse diseases with different subtypes. Breast cancers are organized into subtypes distinguished by the absence or presence of certain proteins on the cancer cells (Fragomeni et al., 2018). Triple negative breast cancers (TNBCs) are a subtype of breast cancer characterized by the absence of three receptors: estrogen receptor (ER), progesterone receptors (PR), and human epidermal growth factor 2 (HER2) (Yin et al., 2020). The TNBC subtype is both invasive and aggressive and occurs more frequently in younger women and African American women (Hines et al., 2011). Understanding the specific subtype of breast cancer is crucial for determining the most effective treatment options. The ductal carcinoma TNBC epithelial cell line, MDA-MD-231, is commonly used to study invasive breast cancer and metastasis due to its highly invasive and migratory nature (Amaro et al., 2016).
Cell adhesion, proliferation and migration are three critical cellular processes required for tissues to maintain homeostasis. When one or all of these cellular processes become unregulated, for example in cancer, this can lead to uncontrolled cell growth and metastasis (Hanahan & Weinberg, 2011). Various molecules have been identified that are involved in maintaining the balance of these cellular processes under normal conditions. Rho GTPases are a family of proteins that act as molecular switches that can be activated by guanine nucleotide exchange factors (GEFs) (Lawson & Ridley, 2017). The Rho GTPase Rac1 has been shown to regulate the actin cytoskeleton and cell proliferation by working downstream of the receptor tyrosine kinase, epidermal growth factor receptor (EGFR) and its ligand, epidermal growth factor (EGF), to regulate various kinase pathways (Dise et al., 2008; Itoh et al., 2008). Dimerization of EGFR leads to the phosphorylation of different proteins along these pathways involved in regulating gene expression and activation of proteins involved in promoting cell migration, adhesion, and proliferation (Wee & Wang, 2017). Adenomatous Polyposis Coli (APC)-stimulated guanine nucleotide-exchange factor, Asef, is a GEF that activates Rac1 to promote cell migration by reducing cell-cell adhesions and remodeling the actin cytoskeleton (Kawasaki et al., 2000, 2010). In colorectal cancer, APC can activate Asef, leading to enhanced migration and invasion (Mitin et al., 2007).
The MDA-MB-231 cell line serves as a valuable model for studying breast cancer proliferation and invasion due to the presence or absence of molecules involved in maintaining normal cell function. MDA-MB-231 cells express Rac1 and are responsive to EGF stimulation (Davidson et al., 1987; Veber et al., 1994). However, it is currently unknown how EGFR activation impacts the regulatory molecules, Asef and Rac1, along with cell migration and proliferation in MDA-MB-231 cells. The current study aims to examine the role of Asef in cell proliferation and migration in the presence of EGF in MDA-MB-231 cells. We hypothesize that due to the regulatory nature of Asef on Rac1, overexpression of Asef in the presence of EGFR signaling pathway will lead to changes in the protein localization and an increase in proliferation and cell migration due to activation of downstream signaling pathways.
Asef-HA (Kawasaki et al., 2000) was overexpressed in MDA-MB-231 cells using a lipid-based transfection approach and protein localization of Rac1, EGFR, and actin was observed in untreated and EGF-treated cells. Using immunofluorescence, Asef-HA overexpressing cells show Asef both in the cytosol and at the cell membrane co-localizing with endogenous EGFR and Rac1 protein in untreated cells and cells treated with EGF for 24 hours (Figure 1A-B). The increased protein localization for Asef and Rac1 at the membrane suggests a possible interaction of the proteins to control cell migration (Figure 1A). Kawasaki, et al. previously showed that Asef promotes the activation of the Rac1 protein, exchanging GDP for GTP, in endothelial cells (Kawasaki et al., 2010). Additional studies are required to determine if there is a direct interaction between the overexpressed Asef-HA protein and Rac1-GTP at the membrane.
EGFR protein appears to internalize into small vesicular structures after EGF stimulation (Figure 1B), suggesting activation of the receptor and downstream cellular pathways (Pennock & Wang, 2003). Due to the shift of EGFR from the membrane to internal vesicular structures in the presence of EGF and previous studies which have examined the impact of EGFR internalization on actin remodeling and cell migration (Pinilla-Macua et al., 2025), actin organization in Asef-HA expressing cells was examined. Actin organization provides a critical scaffold to cellular organization and structure and is represented by two different and interconnected cytoskeletal structures: cortical and stress fibers (Vallenius, 2013). Cortical actin forms a meshwork for actin filaments beneath the cell membrane and is involved in controlling cell shape by locally modifying cortical tension (Chalut & Paluch, 2016). Stress fibers are organized as bundles of actin filaments and play a role in cell migration, adhesion, and mechanosensing, particularly in the context of cancer. These structures are involved in cell migration and linked to cell stiffness, which can either promote or inhibit migration depending on the specific context and cell type (Fischer et al., 2021; Tavares et al., 2017). To determine the impact of Asef on actin filament organization, MDA-MB-231 cells were transfected with Asef-HA. Stress fibers appear to be reduced in Asef-HA expressing cells (asterisks), with or without EGF, as compared to cortical actin, which remains unchanged in both untreated and EGF-treated cells (Figure 1C). The loss of stress fiber formation in Asef-HA cells in comparison to untransfected cells suggests the cells have lost their stability and may be more susceptible to movement.
The lack of stress fiber formation in Asef-HA expressing cells suggests that the cells may be more migratory in nature. To determine if overexpressed Asef leads to an increase in cell migration, a wound healing assay was performed in MDA-MB-231 cells overexpressing Asef-HA. Data indicate that cells overexpressing Asef-HA with or without EGF for 24 hours migrate faster compared to HA only expressing cells (Figure 1D). Quantitative analysis shows a significant increase in proliferation at 12 and 24 hours between HA-only cells and Asef-HA without EGF stimulation in comparison to cells under EGF stimulation (Figure 1D). These data support the hypothesis that overexpression of Asef-HA in MDA-MB-231 cells activates pathways that lead to increased cell migration, by possibly impacting actin organization and turnover at the leading edges of cells (Tian et al., 2015). This is further validated by the reorganization and distribution of key molecules involved in migration, namely EGFR, Rac1, and actin filament organization (Figure 1A-C) and that Rac1 serves as a relay protein between EGFR and cell migration (Dise et al., 2008). The lack of significance between HA-only cells and Asef-HA with EGF treatment suggests that the EGF concentration was not sufficient to induce a response. Previous studies have shown that EGF at a 10 fold higher concentration appears to have more of an impact on MDA-MB-231 cell migration (Kozlova et al., 2016) than lower concentrations. Future studies will determine if higher EGF concentrations lead to significant changes in cell proliferation and migration over basal conditions in cells overexpressing Asef.
Internalization of EGFR via EGF binding, as observed in Figure 1B, may lead to activation of cyclins and/or other cell division regulators to promote an increase in cell proliferation. Furthermore, the EGFR pathway is frequently implicated in cancer development and progression, as it can drive uncontrolled cell growth and division (X. Song et al., 2020; Z. Song et al., 2016). To determine if the activation of EGFR leads to possible increase in MDA-MB-231 cell proliferation due to downstream pathways becoming activated, a MTT assay was performed on control and Asef-HA transfected cells. Data show a significant increase in proliferation in Asef-HA without EGF-stimulation compared to HA-only cells and there was no significant change in Asef-HA with 10 ng/ml EGF for 24 hours (Figure 1E). Similar to the wound healing assay, the lack of significance between control cells and Asef-HA with EGF treatment suggests that the EGF concentration may not be sufficient. These data support the hypothesis that overexpression of Asef-HA in MDA-MB-231 cells may activate pathways that lead to cell proliferation (Lo & Hung, 2006; Pennock & Wang, 2003). Additional studies are required to determine which pathways are responsible for the increased cell proliferation and the impact of EGFR signaling on cell proliferation.
Our findings show that Asef overexpression impacts actin filament remodeling and increases cell migration and proliferation in a metastatic breast cancer cell line. Mutated versions of APC and Asef have been implicated in colon cancer cell migration and invasion (Yang et al., 2021), and we extend the role of Asef into a highly invasive and aggressive epithelial breast cancer subtype. Asef is primarily known for its role in APC-Wnt signaling (Kawasaki et al., 2000); however, Asef and Rac1 were shown to be involved in the EGFR pathway and may be a possible molecular target to consider in understanding the aggressiveness of triple negative breast cancer (Itoh et al., 2008). Future work aims to explore the connection of Asef to signaling proteins involved in controlling cell proliferation and how Asef regulates Rac1 and actin organization in the presence of EGF to control cell motility.
","references":[{"reference":"Amaro A, Angelini G, Mirisola V, Esposito AI, Reverberi D, Matis S, et al., Pfeffer. 2016. A highly invasive subpopulation of MDA-MB-231 breast cancer cells shows accelerated growth, differential chemoresistance, features of apocrine tumors and reduced tumorigenicityin vivo. Oncotarget 7: 68803-68820.
","pubmedId":"","doi":"10.18632/oncotarget.11931 "},{"reference":"Chalut KJ, Paluch EK. 2016. The Actin Cortex: A Bridge between Cell Shape and Function. Developmental Cell 38: 571-573.
","pubmedId":"","doi":"10.1016/j.devcel.2016.09.011 "},{"reference":"Davidson NE, Gelmann EP, Lippman ME, Dickson RB. 1987. Epidermal Growth Factor Receptor Gene Expression in Estrogen Receptor-Positive and Negative Human Breast Cancer Cell Lines. Molecular Endocrinology 1: 216-223.
","pubmedId":"","doi":"10.1210/mend-1-3-216 "},{"reference":"Dise RS, Frey MR, Whitehead RH, Polk DB. 2008. Epidermal growth factor stimulates Rac activation through Src and phosphatidylinositol 3-kinase to promote colonic epithelial cell migration. American Journal of Physiology-Gastrointestinal and Liver Physiology 294: G276-G285.
","pubmedId":"","doi":"10.1152/ajpgi.00340.2007 "},{"reference":"Fischer RS, Sun X, Baird MA, Hourwitz MJ, Seo BR, Pasapera AM, et al., Waterman. 2021. Contractility, focal adhesion orientation, and stress fiber orientation drive cancer cell polarity and migration along wavy ECM substrates. Proceedings of the National Academy of Sciences 118: 10.1073/pnas.2021135118.
","pubmedId":"","doi":"10.1073/pnas.2021135118 "},{"reference":"Fragomeni SM, Sciallis A, Jeruss JS. 2018. Molecular Subtypes and Local-Regional Control of Breast Cancer. Surgical Oncology Clinics of North America 27: 95-120.
","pubmedId":"","doi":"10.1016/j.soc.2017.08.005 "},{"reference":"Hanahan D, Weinberg RA. 2011. Hallmarks of Cancer: The Next Generation. Cell 144: 646-674.
","pubmedId":"","doi":"10.1016/j.cell.2011.02.013 "},{"reference":"Hines LM, Risendal B, Byers T, Mengshol S, Lowery J, Singh M. 2011. Ethnic Disparities in Breast Tumor Phenotypic Subtypes in Hispanic and Non-Hispanic White Women. Journal of Women's Health 20: 1543-1550.
","pubmedId":"","doi":"10.1089/jwh.2010.2558"},{"reference":"Itoh RE, Kiyokawa E, Aoki K, Nishioka T, Akiyama T, Matsuda M. 2008. Phosphorylation and activation of the Rac1 and Cdc42 GEF Asef in A431 cells stimulated by EGF. Journal of Cell Science 121: 2635-2642.
","pubmedId":"","doi":"10.1242/jcs.028647 "},{"reference":"Kawasaki Y, Jigami T, Furukawa S, Sagara M, Echizen K, Shibata Y, Sato R, Akiyama T. 2010. The Adenomatous Polyposis Coli-associated Guanine Nucleotide Exchange Factor Asef Is Involved in Angiogenesis. Journal of Biological Chemistry 285: 1199-1207.
","pubmedId":"","doi":"10.1074/jbc.M109.040691 "},{"reference":"Kawasaki Y, Senda T, Ishidate T, Koyama R, Morishita T, Iwayama Y, Higuchi O, Akiyama T. 2000. Asef, a Link Between the Tumor Suppressor APC and G-Protein Signaling. Science 289: 1194-1197.
","pubmedId":"","doi":"10.1126/science.289.5482.1194"},{"reference":"Kozlova N, Samoylenko A, Drobot L, Kietzmann T. 2015. Urokinase is a negative modulator of Egf‐dependent proliferation and motility in the two breast cancer cell lines MCF‐7 and MDA‐MB‐231. Molecular Carcinogenesis 55: 170-181.
","pubmedId":"","doi":"10.1002/mc.22267 "},{"reference":"Lawson CD, Ridley AJ. 2017. Rho GTPase signaling complexes in cell migration and invasion. Journal of Cell Biology 217: 447-457.
","pubmedId":"","doi":"10.1083/jcb.201612069 "},{"reference":"Lo HW, Hung MC. 2006. Nuclear EGFR signalling network in cancers: linking EGFR pathway to cell cycle progression, nitric oxide pathway and patient survival. British Journal of Cancer 94: 184-188.
","pubmedId":"","doi":"10.1038/sj.bjc.6602941"},{"reference":"Mitin N, Betts L, Yohe ME, Der CJ, Sondek J, Rossman KL. 2007. Release of autoinhibition of ASEF by APC leads to CDC42 activation and tumor suppression. Nature Structural & Molecular Biology 14: 814-823.
","pubmedId":"","doi":"10.1038/nsmb1290 "},{"reference":"Pennock S, Wang Z. 2003. Stimulation of Cell Proliferation by Endosomal Epidermal Growth Factor Receptor As Revealed through Two Distinct Phases of Signaling. Molecular and Cellular Biology 23: 5803-5815.
","pubmedId":"","doi":"10.1128/MCB.23.16.5803-5815.2003"},{"reference":"Pinilla-Macua I, Surve S, Sorkin A. 2025. Cell migration signaling through the EGFR-VAV2-Rac1 pathway is sustained in endosomes. Journal of Cell Science 138: 10.1242/jcs.263541.
","pubmedId":"","doi":"10.1242/jcs.263541 "},{"reference":"Song X, Liu Z, Yu Z. 2020. <p>EGFR Promotes the Development of Triple Negative Breast Cancer Through JAK/STAT3 Signaling</p>. Cancer Management and Research Volume 12: 703-717.
","pubmedId":"","doi":"10.2147/CMAR.S225376 "},{"reference":"Song Z, Fusco J, Zimmerman R, Fischbach S, Chen C, Ricks DM, et al., Gittes. 2016. Epidermal Growth Factor Receptor Signaling Regulates β Cell Proliferation in Adult Mice. Journal of Biological Chemistry 291: 22630-22637.
","pubmedId":"","doi":"10.1074/jbc.M116.747840 "},{"reference":"Tavares S, Vieira AF, Taubenberger AV, Araújo M, Martins NP, Brás-Pereira C, et al., Janody. 2017. Actin stress fiber organization promotes cell stiffening and proliferation of pre-invasive breast cancer cells. Nature Communications 8: 10.1038/ncomms15237.
","pubmedId":"","doi":"10.1038/ncomms15237"},{"reference":"Tian X, Tian Y, Gawlak G, Meng F, Kawasaki Y, Akiyama T, Birukova AA. 2015. Asef controls vascular endothelial permeability and barrier recovery in the lung. Molecular Biology of the Cell 26: 636-650.
","pubmedId":"","doi":"10.1091/mbc.E14-02-0725 "},{"reference":"Vallenius T. 2013. Actin stress fibre subtypes in mesenchymal-migrating cells. Open Biology 3: 130001.
","pubmedId":"","doi":"10.1098/rsob.130001 "},{"reference":"Veber N, Prévost G, Planchon P, Starzec A. 1994. Evidence for a growth effect of epidermal growth factor on MDA-MB-231 breast cancer cells. European Journal of Cancer 30: 1352-1359.
","pubmedId":"","doi":"10.1016/0959-8049(94)90186-4 "},{"reference":"Wee P, Wang Z. 2017. Epidermal Growth Factor Receptor Cell Proliferation Signaling Pathways. Cancers 9: 52.
","pubmedId":"","doi":"10.3390/cancers9050052"},{"reference":"Yang X, Zhong J, Zhang Q, Feng L, Zheng Z, Zhang J, Lu S. 2021. Advances and Insights of APC-Asef Inhibitors for Metastatic Colorectal Cancer Therapy. Frontiers in Molecular Biosciences 8: 10.3389/fmolb.2021.662579.
","pubmedId":"","doi":"10.3389/fmolb.2021.662579 "},{"reference":"Yin L, Duan JJ, Bian XW, Yu Sc. 2020. Triple-negative breast cancer molecular subtyping and treatment progress. Breast Cancer Research 22: 10.1186/s13058-020-01296-5.
","pubmedId":"","doi":"10.1186/s13058-020-01296-5 "}],"title":"Examining the Role of Asef and Epidermal Growth Factor on Cell Migration and Proliferation in Triple Negative Breast Cancer Epithelial Cells
","reviews":[{"reviewer":{"displayName":"Lori Hensley"},"openAcknowledgement":false,"status":{"submitted":true}}],"curatorReviews":[]},{"id":"2f00739b-b5c6-4d42-9653-595014b4038c","decision":"accept","abstract":"Cell proliferation and migration are cellular processes essential to normal cell function; errors in these cellular processes impact the ability of a cell to maintain cellular homeostasis. The current study examines the role of Asef, a GEF for Rac1, in cell proliferation and migration in the presence or absence of EGF in a triple negative breast cancer epithelial cell line. Overexpression of Asef reduced stress fiber formation and increased cell migration and proliferation in MDA-MB-231 epithelial cells. These data indicate a role for Asef in the EGFR/Rac1 signaling cascade involved in regulating cellular behavior.
","acknowledgements":"The authors thank the SMC School of Science for their support of this project and the Cell Biology Education Consortium (CBEC) for feedback and comments on the manuscript.
","authors":[{"affiliations":["Saint Mary's College of California"],"departments":["Biology"],"credit":["investigation","methodology","writing_originalDraft"],"email":"na21@stmarys-ca.edu","firstName":"Natalie","lastName":"Alaniz","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":"0009-0004-5090-7951"},{"affiliations":["Saint Mary's College of California"],"departments":["Biology"],"credit":["conceptualization","formalAnalysis","methodology","project","supervision","writing_reviewEditing","investigation"],"email":"knk8@stmarys-ca.edu","firstName":"Khameeka N.","lastName":"Kitt","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":false,"WBId":null,"orcid":"0009-0003-2381-877X"}],"awards":[],"conflictsOfInterest":"The authors declare that there are no conflicts of interest present.
","dataTable":{"url":null},"extendedData":[],"funding":"The authors thank the SMC School of Science Summer Endowment fund. Undergraduate student research participation and publication support were funded by the Cell Biology Education Consortium (CBEC): Path to Publication (Award ID #2316122) through the National Science Foundation (NSF).
","image":{"url":"https://portal.micropublication.org/uploads/6beb4dbe909a226e9706715cb55882f5.png"},"imageCaption":"Human MDA-MB-231 breast cancer epithelial cells were transfected with Asef-HA (green) and allowed to express for 24 hours before treatment with 10 ng/ml of EGF for 24 hours. (A) In the absence of EGF, Asef and Rac1 (red) localize to the cytoplasm and membrane. In the presence of EGF for 24 hours, Rac1 colocalizes with Asef at the cell membrane (arrows). Scale bar: 40 µm. (B) Asef localizes to the cytoplasm and membrane ruffles (arrows) in cells with and without EGF treatment. In the absence of EGF, EGFR (red) localizes to the cell membrane and cytoplasm in Asef-HA expressing cells (arrowhead). Cells treated with EGF for 24 hours caused a minor shift in EGFR localization from the membrane to vesicle-like structures inside the cell (arrowhead). Scale bar: 40 µm. (C) Asef localizes to the cytoplasm and membrane ruffles (arrows) in cells with and without EGF treatment. Cortical Actin (red) appears unchanged in Asef-HA expressing cells (arrowhead). Cells expressing Asef-HA show reduced stress fibers extending across the cell (asterisks) compared to untransfected cells (arrows) in the presence or absence of EGF for 24 hours. Scale bar: 40 µm. (D) MDA-MB-231 cell monolayers overexpressing HA-only or Asef-HA were scratched and imaged over 24 hours in the presence or absence of EGF. The width of the wound was measured using ImageJ and plotted to examine the wound width over time. Asef-HA expressing cells regardless of EGF treatment migrated faster than control cells, however, Asef-HA expressing cells without EGF treatment showed a significant increase in migration at 12 and 24 hour time points compared to HA only expressing cells. (**P < 0.005; n=3 measurements of each group). (E) MDA-MB-231 cell monolayers overexpressing HA-only or Asef-HA were serum- starved and treated with 10 ng/ml of EGF for 24 hours. Following EGF-treatment, MTT reagent was added, cells were solubilized with DMSO, and absorbance was read at 570 nm. Asef-HA expressing cells in the absence of EGF treatment showed a significant increase in cell proliferation compared to untransfected cells (*P < 0.05; n=3 measurements of each group). (F) Model of the Rho GTPase signaling pathway demonstrates how Asef can be recruited downstream of EGFR signaling to regulate Rac1 activation to stimulate cell proliferation and migration.
","imageTitle":"Asef-HA overexpression in MDA-MB-231 breast epithelial cancer cells causes changes in protein localization, cell migration, and cell proliferation.
","methods":"Cell Culture
Triple negative ductal breast carcinoma epithelial cell line MDA-MB-231 (ATCC) were grown in Dulbecco's Modified Eagle Serum (DMEM) high glucose media (Thermo Fisher Scientific) with 10% fetal bovine serum and 1% penicillin-streptomycin-glutamine (Thermo Fisher Scientific). Cells were incubated under the following conditions: 5% CO2 and 37˚C.
Transfection
MDA-MB-321 cells were transfected with pcDNA 3.1-HA Asef (human) full length plasmid (provided by Tetsu Akiyama lab, University of Tokyo, (Kawasaki et al., 2000)) using Lipofectamine 2000 diluted in Opti-MEM (Thermo Fisher Scientific) following product recommendations. As a control for proliferation and scratch assays, cells were transfected with pcDNA 3.1-HA plasmid alone. Transfected cells were incubated for 24 hours in antibiotic-free media before being replated in various well formats for various assays.
Immunofluorescence
Transfected MDA-MB-231 cells (with or without Asef-HA) were placed on rat tail collagen-coated coverslips in serum-free media and treated with or without 10 ng/mL of EGF for 24 hours. Cells were fixed using 4% paraformaldehyde 48 hours post-transfection. To localize HA, Rac1, and EGFR, cells were blocked and labeled with primary antibodies for HA (Roche), Rac1 (BD Biosciences) and EGFR (Thermo Fisher Scientific) followed by secondary antibodies conjugated to Alexa Fluor 488 and 594 (Thermo Fisher Scientific). Actin filaments and nuclei were labeled using Phalloidin 594 and Hoechst 33342 DNA stains, respectively (Thermo Fisher Scientific). Cells were imaged using the Leica STELLARIS Inverted Spectral Live Cell Confocal Microscope.
Scratch Assay (Wound Healing)
Transfected MDA-MB-231 cells (with or without Asef-HA) were placed on rat tail collagen-coated 12 well plates and allowed to form a monolayer in serum free media. Monolayers were scratched 24 hours later using a sterile 200 µL pipette tip, washed using phosphate-buffered saline (PBS) to remove debris, and incubated in serum-free media treated with or without 10 ng/mL of EGF for 24 hours. Images were captured at 0, 4, 8, 12 and 24 hours. The area between both sides of the scratch were quantified after each image time stamp to determine the wound closure distance using ImageJ software. Distance of wound closure for each group at each time point was measured in µm using ImageJ (pixels per micron was determined using scale bar) and plotted to display the width of the wound closure over time.
Proliferation (MTT) Assay
Transfected MDA-MB-231 cells (with or without Asef-HA) were placed in a 96-well tissue culture-treated plate in serum free media. Cells were treated with or without 10 ng/mL of EGF for 24 hours before adding 10 µL of a 5 mg/mL stock of MTT reagent per well and incubated for 3 hours. After three hours, the MTT reagent was removed and 100µL DMSO was added to each well to solubilize cells, and the optical density was measured at 570 nm (Tecan Plate Reader). Percent viability was calculated for each well and normalized against the control (HA-only).
Statistical Analysis
For the scratch assay, a multiple unpaired t-test analysis was performed to determine the significance at each time point. Three biological replicates (independent wells) were analyzed for each group. For the proliferation (MTT) assay, an unpaired t-test was used to determine significance between the treated groups compared to the control. Three biological replicates (independent wells) were analyzed for each group. All data was graphed using GraphPad Prism.
","reagents":"Plasmid | Description | Source |
pcDNA 3.1-HA- Asef | Full length Asef (human) | Tetsu Akiyama lab, University of Tokyo |
Cell Line | Description | Source |
MDA-MB-231 | Human Epithelial Breast Tissue isolated from Mammary Gland; Adenocarcinoma | ATCC Catalog #: HTB-26 |
Antibodies | Dilution | Source |
Phalloidin 594 | 1:400 | Thermo Fisher Scientific Catalog #: A12381 |
Alexa Goat anti Rabbit 488 | 1:800 | Thermo Fisher Scientific Catalog #: A-11008 |
Alexa Goat anti Mouse 594 | 1:800 | Thermo Fisher Scientific Catalog #: A-11005 |
HA (Rabbit) | 1:500 | Abcam Catalog #: ab9110 |
Rac1 (Mouse) | 1:200 | BD Biosciences Catalog #: 610651 |
EGFR (Mouse) | 1:200 | Thermo Fisher Scientific Catalog #: MA513269 |
Hoechst 33342 DNA stain | 1 µg/ml final | Thermo Fisher Scientific Catalog #: H3570 |
Breast cancer is not a single disease, but rather a group of diverse diseases with different subtypes. Breast cancers are organized into subtypes distinguished by the absence or presence of certain proteins on the cancer cells (Fragomeni et al., 2018). Triple negative breast cancers (TNBCs) are a subtype of breast cancer characterized by the absence of three receptors: estrogen receptor (ER), progesterone receptors (PR), and human epidermal growth factor 2 (HER2) (Yin et al., 2020). The TNBC subtype is both invasive and aggressive and occurs more frequently in younger women and African American women (Hines et al., 2011). Understanding the specific subtype of breast cancer is crucial for determining the most effective treatment options. The ductal carcinoma TNBC epithelial cell line, MDA-MD-231, is commonly used to study invasive breast cancer and metastasis due to its highly invasive and migratory nature (Amaro et al., 2016).
Cell adhesion, proliferation and migration are three critical cellular processes required for tissues to maintain homeostasis. When one or all of these cellular processes become unregulated, for example in cancer, this can lead to uncontrolled cell growth and metastasis (Hanahan & Weinberg, 2011). Various molecules have been identified that are involved in maintaining the balance of these cellular processes under normal conditions. Rho GTPases are a family of proteins that act as molecular switches that can be activated by guanine nucleotide exchange factors (GEFs) (Lawson & Ridley, 2017). The Rho GTPase Rac1 has been shown to regulate the actin cytoskeleton and cell proliferation by working downstream of the receptor tyrosine kinase, epidermal growth factor receptor (EGFR) and its ligand, epidermal growth factor (EGF), to regulate various kinase pathways (Dise et al., 2008; Itoh et al., 2008). Dimerization of EGFR leads to the phosphorylation of different proteins along these pathways involved in regulating gene expression and activation of proteins involved in promoting cell migration, adhesion, and proliferation (Wee & Wang, 2017). Adenomatous Polyposis Coli (APC)-stimulated guanine nucleotide-exchange factor, Asef, is a GEF that activates Rac1 to promote cell migration by reducing cell-cell adhesions and remodeling the actin cytoskeleton (Kawasaki et al., 2000, 2010). In colorectal cancer, APC can activate Asef, leading to enhanced migration and invasion (Mitin et al., 2007).
The MDA-MB-231 cell line serves as a valuable model for studying breast cancer proliferation and invasion due to the presence or absence of molecules involved in maintaining normal cell function. MDA-MB-231 cells express Rac1 and are responsive to EGF stimulation (Davidson et al., 1987; Veber et al., 1994). However, it is currently unknown how EGFR activation impacts the regulatory molecules, Asef and Rac1, along with cell migration and proliferation in MDA-MB-231 cells. The current study aims to examine the role of Asef in cell proliferation and migration in the presence of EGF in MDA-MB-231 cells. We hypothesize that due to the regulatory nature of Asef on Rac1, overexpression of Asef in the presence of EGFR signaling pathway will lead to changes in the protein localization and an increase in proliferation and cell migration due to activation of downstream signaling pathways.
Asef-HA (Kawasaki et al., 2000) was overexpressed in MDA-MB-231 cells using a lipid-based transfection approach and protein localization of Rac1, EGFR, and actin was observed in untreated and EGF-treated cells. Using immunofluorescence, Asef-HA overexpressing cells show Asef both in the cytosol and at the cell membrane co-localizing with endogenous EGFR and Rac1 protein in untreated cells and cells treated with EGF for 24 hours (Figure 1A-B). The increased protein localization for Asef and Rac1 at the membrane suggests a possible interaction of the proteins to control cell migration (Figure 1A). Kawasaki, et al. previously showed that Asef promotes the activation of the Rac1 protein, exchanging GDP for GTP, in endothelial cells (Kawasaki et al., 2010). Additional studies are required to determine if there is a direct interaction between the overexpressed Asef-HA protein and Rac1-GTP at the membrane.
EGFR protein appears to internalize into small vesicular structures after EGF stimulation (Figure 1B), suggesting activation of the receptor and downstream cellular pathways (Pennock & Wang, 2003). Due to the shift of EGFR from the membrane to internal vesicular structures in the presence of EGF and previous studies which have examined the impact of EGFR internalization on actin remodeling and cell migration (Pinilla-Macua et al., 2025), actin organization in Asef-HA expressing cells was examined. Actin organization provides a critical scaffold to cellular organization and structure and is represented by two different and interconnected cytoskeletal structures: cortical and stress fibers (Vallenius, 2013). Cortical actin forms a meshwork for actin filaments beneath the cell membrane and is involved in controlling cell shape by locally modifying cortical tension (Chalut & Paluch, 2016). Stress fibers are organized as bundles of actin filaments and play a role in cell migration, adhesion, and mechanosensing, particularly in the context of cancer. These structures are involved in cell migration and linked to cell stiffness, which can either promote or inhibit migration depending on the specific context and cell type (Fischer et al., 2021; Tavares et al., 2017). To determine the impact of Asef on actin filament organization, MDA-MB-231 cells were transfected with Asef-HA. Stress fibers appear to be reduced in Asef-HA expressing cells (asterisks), with or without EGF, as compared to cortical actin, which remains unchanged in both untreated and EGF-treated cells (Figure 1C). The loss of stress fiber formation in Asef-HA cells in comparison to untransfected cells suggests the cells have lost their stability and may be more susceptible to movement.
The lack of stress fiber formation in Asef-HA expressing cells suggests that the cells may be more migratory in nature. To determine if overexpressed Asef leads to an increase in cell migration, a wound healing assay was performed in MDA-MB-231 cells overexpressing Asef-HA. Data indicate that cells overexpressing Asef-HA with or without EGF for 24 hours migrate faster compared to HA only expressing cells (Figure 1D). Quantitative analysis shows a significant increase in proliferation at 12 and 24 hours between HA-only cells and Asef-HA without EGF stimulation in comparison to cells under EGF stimulation (Figure 1D). These data support the hypothesis that overexpression of Asef-HA in MDA-MB-231 cells activates pathways that lead to increased cell migration, by possibly impacting actin organization and turnover at the leading edges of cells (Tian et al., 2015). This is further validated by the reorganization and distribution of key molecules involved in migration, namely EGFR, Rac1, and actin filament organization (Figure 1A-C) and that Rac1 serves as a relay protein between EGFR and cell migration (Dise et al., 2008). The lack of significance between HA-only cells and Asef-HA with EGF treatment suggests that the EGF concentration was not sufficient to induce a response. Previous studies have shown that EGF at a 10 fold higher concentration appears to have more of an impact on MDA-MB-231 cell migration (Kozlova et al., 2016) than lower concentrations. Future studies will determine if higher EGF concentrations lead to significant changes in cell proliferation and migration over basal conditions in cells overexpressing Asef.
Internalization of EGFR via EGF binding, as observed in Figure 1B, may lead to activation of cyclins and/or other cell division regulators to promote an increase in cell proliferation. Furthermore, the EGFR pathway is frequently implicated in cancer development and progression, as it can drive uncontrolled cell growth and division (X. Song et al., 2020; Z. Song et al., 2016). To determine if the activation of EGFR leads to possible increase in MDA-MB-231 cell proliferation due to downstream pathways becoming activated, a MTT assay was performed on control and Asef-HA transfected cells. Data show a significant increase in proliferation in Asef-HA without EGF-stimulation compared to HA-only cells and there was no significant change in Asef-HA with 10 ng/ml EGF for 24 hours (Figure 1E). Similar to the wound healing assay, the lack of significance between control cells and Asef-HA with EGF treatment suggests that the EGF concentration may not be sufficient. These data support the hypothesis that overexpression of Asef-HA in MDA-MB-231 cells may activate pathways that lead to cell proliferation (Lo & Hung, 2006; Pennock & Wang, 2003). Additional studies are required to determine which pathways are responsible for the increased cell proliferation and the impact of EGFR signaling on cell proliferation.
Our findings show that Asef overexpression impacts actin filament remodeling and increases cell migration and proliferation in a metastatic breast cancer cell line. Mutated versions of APC and Asef have been implicated in colon cancer cell migration and invasion (Yang et al., 2021), and we extend the role of Asef into a highly invasive and aggressive epithelial breast cancer subtype. Asef is primarily known for its role in APC-Wnt signaling (Kawasaki et al., 2000); however, Asef and Rac1 were shown to be involved in the EGFR pathway and may be a possible molecular target to consider in understanding the aggressiveness of triple negative breast cancer (Itoh et al., 2008). Future work aims to explore the connection of Asef to signaling proteins involved in controlling cell proliferation and how Asef regulates Rac1 and actin organization in the presence of EGF to control cell motility.
","references":[{"reference":"Amaro A, Angelini G, Mirisola V, Esposito AI, Reverberi D, Matis S, et al., Pfeffer. 2016. A highly invasive subpopulation of MDA-MB-231 breast cancer cells shows accelerated growth, differential chemoresistance, features of apocrine tumors and reduced tumorigenicityin vivo. Oncotarget 7: 68803-68820.
","pubmedId":"","doi":"10.18632/oncotarget.11931 "},{"reference":"Chalut KJ, Paluch EK. 2016. The Actin Cortex: A Bridge between Cell Shape and Function. Developmental Cell 38: 571-573.
","pubmedId":"","doi":"10.1016/j.devcel.2016.09.011 "},{"reference":"Davidson NE, Gelmann EP, Lippman ME, Dickson RB. 1987. Epidermal Growth Factor Receptor Gene Expression in Estrogen Receptor-Positive and Negative Human Breast Cancer Cell Lines. Molecular Endocrinology 1: 216-223.
","pubmedId":"","doi":"10.1210/mend-1-3-216 "},{"reference":"Dise RS, Frey MR, Whitehead RH, Polk DB. 2008. Epidermal growth factor stimulates Rac activation through Src and phosphatidylinositol 3-kinase to promote colonic epithelial cell migration. American Journal of Physiology-Gastrointestinal and Liver Physiology 294: G276-G285.
","pubmedId":"","doi":"10.1152/ajpgi.00340.2007 "},{"reference":"Fischer RS, Sun X, Baird MA, Hourwitz MJ, Seo BR, Pasapera AM, et al., Waterman. 2021. Contractility, focal adhesion orientation, and stress fiber orientation drive cancer cell polarity and migration along wavy ECM substrates. Proceedings of the National Academy of Sciences 118: 10.1073/pnas.2021135118.
","pubmedId":"","doi":"10.1073/pnas.2021135118 "},{"reference":"Fragomeni SM, Sciallis A, Jeruss JS. 2018. Molecular Subtypes and Local-Regional Control of Breast Cancer. Surgical Oncology Clinics of North America 27: 95-120.
","pubmedId":"","doi":"10.1016/j.soc.2017.08.005 "},{"reference":"Hanahan D, Weinberg RA. 2011. Hallmarks of Cancer: The Next Generation. Cell 144: 646-674.
","pubmedId":"","doi":"10.1016/j.cell.2011.02.013 "},{"reference":"Hines LM, Risendal B, Byers T, Mengshol S, Lowery J, Singh M. 2011. Ethnic Disparities in Breast Tumor Phenotypic Subtypes in Hispanic and Non-Hispanic White Women. Journal of Women's Health 20: 1543-1550.
","pubmedId":"","doi":"10.1089/jwh.2010.2558"},{"reference":"Itoh RE, Kiyokawa E, Aoki K, Nishioka T, Akiyama T, Matsuda M. 2008. Phosphorylation and activation of the Rac1 and Cdc42 GEF Asef in A431 cells stimulated by EGF. Journal of Cell Science 121: 2635-2642.
","pubmedId":"","doi":"10.1242/jcs.028647 "},{"reference":"Kawasaki Y, Jigami T, Furukawa S, Sagara M, Echizen K, Shibata Y, Sato R, Akiyama T. 2010. The Adenomatous Polyposis Coli-associated Guanine Nucleotide Exchange Factor Asef Is Involved in Angiogenesis. Journal of Biological Chemistry 285: 1199-1207.
","pubmedId":"","doi":"10.1074/jbc.M109.040691 "},{"reference":"Kawasaki Y, Senda T, Ishidate T, Koyama R, Morishita T, Iwayama Y, Higuchi O, Akiyama T. 2000. Asef, a Link Between the Tumor Suppressor APC and G-Protein Signaling. Science 289: 1194-1197.
","pubmedId":"","doi":"10.1126/science.289.5482.1194"},{"reference":"Kozlova N, Samoylenko A, Drobot L, Kietzmann T. 2015. Urokinase is a negative modulator of Egf‐dependent proliferation and motility in the two breast cancer cell lines MCF‐7 and MDA‐MB‐231. Molecular Carcinogenesis 55: 170-181.
","pubmedId":"","doi":"10.1002/mc.22267 "},{"reference":"Lawson CD, Ridley AJ. 2017. Rho GTPase signaling complexes in cell migration and invasion. Journal of Cell Biology 217: 447-457.
","pubmedId":"","doi":"10.1083/jcb.201612069 "},{"reference":"Lo HW, Hung MC. 2006. Nuclear EGFR signalling network in cancers: linking EGFR pathway to cell cycle progression, nitric oxide pathway and patient survival. British Journal of Cancer 94: 184-188.
","pubmedId":"","doi":"10.1038/sj.bjc.6602941"},{"reference":"Mitin N, Betts L, Yohe ME, Der CJ, Sondek J, Rossman KL. 2007. Release of autoinhibition of ASEF by APC leads to CDC42 activation and tumor suppression. Nature Structural & Molecular Biology 14: 814-823.
","pubmedId":"","doi":"10.1038/nsmb1290 "},{"reference":"Pennock S, Wang Z. 2003. Stimulation of Cell Proliferation by Endosomal Epidermal Growth Factor Receptor As Revealed through Two Distinct Phases of Signaling. Molecular and Cellular Biology 23: 5803-5815.
","pubmedId":"","doi":"10.1128/MCB.23.16.5803-5815.2003"},{"reference":"Pinilla-Macua I, Surve S, Sorkin A. 2025. Cell migration signaling through the EGFR-VAV2-Rac1 pathway is sustained in endosomes. Journal of Cell Science 138: 10.1242/jcs.263541.
","pubmedId":"","doi":"10.1242/jcs.263541 "},{"reference":"Song X, Liu Z, Yu Z. 2020. <p>EGFR Promotes the Development of Triple Negative Breast Cancer Through JAK/STAT3 Signaling</p>. Cancer Management and Research Volume 12: 703-717.
","pubmedId":"","doi":"10.2147/CMAR.S225376 "},{"reference":"Song Z, Fusco J, Zimmerman R, Fischbach S, Chen C, Ricks DM, et al., Gittes. 2016. Epidermal Growth Factor Receptor Signaling Regulates β Cell Proliferation in Adult Mice. Journal of Biological Chemistry 291: 22630-22637.
","pubmedId":"","doi":"10.1074/jbc.M116.747840 "},{"reference":"Tavares S, Vieira AF, Taubenberger AV, Araújo M, Martins NP, Brás-Pereira C, et al., Janody. 2017. Actin stress fiber organization promotes cell stiffening and proliferation of pre-invasive breast cancer cells. Nature Communications 8: 10.1038/ncomms15237.
","pubmedId":"","doi":"10.1038/ncomms15237"},{"reference":"Tian X, Tian Y, Gawlak G, Meng F, Kawasaki Y, Akiyama T, Birukova AA. 2015. Asef controls vascular endothelial permeability and barrier recovery in the lung. Molecular Biology of the Cell 26: 636-650.
","pubmedId":"","doi":"10.1091/mbc.E14-02-0725 "},{"reference":"Vallenius T. 2013. Actin stress fibre subtypes in mesenchymal-migrating cells. Open Biology 3: 130001.
","pubmedId":"","doi":"10.1098/rsob.130001 "},{"reference":"Veber N, Prévost G, Planchon P, Starzec A. 1994. Evidence for a growth effect of epidermal growth factor on MDA-MB-231 breast cancer cells. European Journal of Cancer 30: 1352-1359.
","pubmedId":"","doi":"10.1016/0959-8049(94)90186-4 "},{"reference":"Wee P, Wang Z. 2017. Epidermal Growth Factor Receptor Cell Proliferation Signaling Pathways. Cancers 9: 52.
","pubmedId":"","doi":"10.3390/cancers9050052"},{"reference":"Yang X, Zhong J, Zhang Q, Feng L, Zheng Z, Zhang J, Lu S. 2021. Advances and Insights of APC-Asef Inhibitors for Metastatic Colorectal Cancer Therapy. Frontiers in Molecular Biosciences 8: 10.3389/fmolb.2021.662579.
","pubmedId":"","doi":"10.3389/fmolb.2021.662579 "},{"reference":"Yin L, Duan JJ, Bian XW, Yu Sc. 2020. Triple-negative breast cancer molecular subtyping and treatment progress. Breast Cancer Research 22: 10.1186/s13058-020-01296-5.
","pubmedId":"","doi":"10.1186/s13058-020-01296-5 "}],"title":"Examining the Role of Asef and Epidermal Growth Factor on Cell Migration and Proliferation in Triple Negative Breast Cancer Epithelial Cells
","reviews":[],"curatorReviews":[]},{"id":"219550e3-b20e-415e-9e94-8f54a6d48979","decision":"revise","abstract":"Cell proliferation and migration are cellular processes essential to normal cell function; errors in these cellular processes impact the ability of a cell to maintain cellular homeostasis. The current study examines the role of Asef, a GEF for Rac1, in cell proliferation and migration in the presence or absence of EGF in a triple negative breast cancer epithelial cell line. Overexpression of Asef reduced stress fiber formation and increased cell migration and proliferation in MDA-MB-231 epithelial cells. These data indicate a role for Asef in the EGFR/Rac1 signaling cascade involved in regulating cellular behavior.
","acknowledgements":"The authors thank the SMC School of Science for their support of this project and the Cell Biology Education Consortium (CBEC) for feedback and comments on the manuscript.
","authors":[{"affiliations":["Saint Mary's College of California, Moraga, CA, United States"],"departments":["Biology Department"],"credit":["investigation","methodology","writing_originalDraft"],"email":"na21@stmarys-ca.edu","firstName":"Natalie","lastName":"Alaniz","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":"0009-0004-5090-7951"},{"affiliations":["Saint Mary's College of California, Moraga, CA, United States"],"departments":["Biology Department"],"credit":["conceptualization","formalAnalysis","methodology","project","supervision","writing_reviewEditing","investigation"],"email":"knk8@stmarys-ca.edu","firstName":"Khameeka N.","lastName":"Kitt","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":false,"WBId":null,"orcid":"0009-0003-2381-877X"}],"awards":[],"conflictsOfInterest":"The authors declare that there are no conflicts of interest present.
","dataTable":{"url":null},"extendedData":[],"funding":"The authors thank the SMC School of Science Summer Endowment fund. Undergraduate student research participation and publication support were funded by the Cell Biology Education Consortium (CBEC): Path to Publication (Award ID #2316122) through the National Science Foundation (NSF).
","image":{"url":"https://portal.micropublication.org/uploads/6beb4dbe909a226e9706715cb55882f5.png"},"imageCaption":"Human MDA-MB-231 breast cancer epithelial cells were transfected with Asef-HA (green) and allowed to express for 24 hours before treatment with 10 ng/ml of EGF for 24 hours. (A) In the absence of EGF, Asef and Rac1 (red) localize to the cytoplasm and membrane. In the presence of EGF for 24 hours, Rac1 colocalizes with Asef at the cell membrane (arrows). Scale bar: 40 µm. (B) Asef localizes to the cytoplasm and membrane ruffles (arrows) in cells with and without EGF treatment. In the absence of EGF, EGFR (red) localizes to the cell membrane and cytoplasm in Asef-HA expressing cells (arrowhead). Cells treated with EGF for 24 hours caused a minor shift in EGFR localization from the membrane to vesicle-like structures inside the cell (arrowhead). Scale bar: 40 µm. (C) Asef localizes to the cytoplasm and membrane ruffles (arrows) in cells with and without EGF treatment. Cortical Actin (red) appears unchanged in Asef-HA expressing cells (arrowhead). Cells expressing Asef-HA show reduced stress fibers extending across the cell (asterisks) compared to untransfected cells (arrows) in the presence or absence of EGF for 24 hours. Scale bar: 40 µm. (D) MDA-MB-231 cell monolayers overexpressing HA-only or Asef-HA were scratched and imaged over 24 hours in the presence or absence of EGF. The width of the wound was measured using ImageJ and plotted to examine wound width over time. Asef-HA expressing cells, regardless of EGF treatment, migrated faster than control cells; however, Asef-HA expressing cells without EGF treatment showed a significant increase in migration at 12 and 24 hour time points compared to HA only expressing cells. (**P < 0.005; n=3 measurements of each group). (E) MDA-MB-231 cell monolayers overexpressing HA-only or Asef-HA were serum-starved and treated with 10 ng/ml of EGF for 24 hours. Following EGF-treatment, MTT reagent was added, cells were solubilized with DMSO, and absorbance was read at 570 nm. Asef-HA expressing cells in the absence of EGF treatment showed a significant increase in cell proliferation compared to untransfected cells (*P < 0.05; n=3 measurements of each group). (F) Model of the Rho GTPase signaling pathway demonstrating how Asef could be recruited downstream of EGFR signaling to regulate Rac1 activation and stimulate cell proliferation and migration.
","imageTitle":"Asef-HA overexpression in MDA-MB-231 breast epithelial cancer cells causes changes in protein localization, cell migration, and cell proliferation.
","methods":"Cell Culture
Triple negative ductal breast carcinoma epithelial cell line MDA-MB-231 (ATCC) were grown in Dulbecco's Modified Eagle Serum (DMEM) high glucose media (Thermo Fisher Scientific) with 10% fetal bovine serum and 1% penicillin-streptomycin-glutamine (Thermo Fisher Scientific). Cells were incubated under the following conditions: 5% CO2 and 37˚C.
Transfection
MDA-MB-321 cells were transfected with pcDNA 3.1-HA Asef (human) full length plasmid (provided by Tetsu Akiyama lab, University of Tokyo, (Kawasaki et al., 2000)) using Lipofectamine 2000 diluted in Opti-MEM (Thermo Fisher Scientific) following product recommendations. As a control for proliferation and scratch assays, cells were transfected with pcDNA 3.1-HA plasmid alone. Transfected cells were incubated for 24 hours in antibiotic-free media before being replated in various well formats for various assays.
Immunofluorescence
Transfected MDA-MB-231 cells (with or without Asef-HA) were placed on rat tail collagen-coated coverslips in serum-free media (DMEM with 1% penicillin-streptomycin-glutamine) and treated with or without 10 ng/mL of EGF for 24 hours. Cells were fixed using 4% paraformaldehyde 48 hours post-transfection. To localize HA, Rac1, and EGFR, cells were blocked and labeled with primary antibodies for HA (Roche), Rac1 (BD Biosciences) and EGFR (Thermo Fisher Scientific) followed by secondary antibodies conjugated to Alexa Fluor 488 and 594 (Thermo Fisher Scientific). Actin filaments and nuclei were labeled using Phalloidin 594 and Hoechst 33342 DNA stains, respectively (Thermo Fisher Scientific). Cells were imaged using the Leica STELLARIS Inverted Spectral Live Cell Confocal Microscope.
Scratch Assay (Wound Healing)
Transfected MDA-MB-231 cells (with or without Asef-HA) were placed on rat tail collagen-coated 12 well plates and allowed to form a monolayer in serum free media. Monolayers were scratched 24 hours later using a sterile 200 µL pipette tip, washed using phosphate-buffered saline (PBS) to remove debris, and incubated in serum-free media treated with or without 10 ng/mL of EGF for 24 hours. Images were captured at 0, 4, 8, 12 and 24 hours. The area between both sides of the scratch were quantified after each image time stamp to determine the wound closure distance using ImageJ software. Distance of wound closure for each group at each time point was measured in µm using ImageJ (pixels per micron was determined using scale bar) and plotted to display the width of the wound closure over time.
Proliferation (MTT) Assay
Transfected MDA-MB-231 cells (with or without Asef-HA) were placed in a 96-well tissue culture-treated plate in serum free media. Cells were treated with or without 10 ng/mL of EGF for 24 hours before adding 10 µL of a 5 mg/mL stock of MTT reagent per well and incubated for three hours. After three hours, the MTT reagent was removed and 100µL DMSO was added to each well to solubilize cells, and the optical density was measured at 570 nm (Tecan Plate Reader). Percent viability was calculated for each well and normalized against the control (HA-only).
Statistical Analysis
For the scratch assay, a multiple unpaired t-test analysis was performed to determine the significance at each time point. Three biological replicates (independent wells) were analyzed for each group. For the proliferation (MTT) assay, an unpaired t-test was used to determine significance between the treated groups compared to the control. Three biological replicates (independent wells) were analyzed for each group. All data was graphed using GraphPad Prism.
","reagents":"Plasmid | Description | Source |
pcDNA 3.1-HA- Asef | Full length Asef (human) | Tetsu Akiyama lab, University of Tokyo |
Cell Line | Description | Source |
MDA-MB-231 | Human Epithelial Breast Tissue isolated from Mammary Gland; Adenocarcinoma | ATCC Catalog #: HTB-26 |
Antibodies | Dilution | Source |
Phalloidin 594 | 1:400 | Thermo Fisher Scientific Catalog #: A12381 |
Alexa Goat anti Rabbit 488 | 1:800 | Thermo Fisher Scientific Catalog #: A-11008 |
Alexa Goat anti Mouse 594 | 1:800 | Thermo Fisher Scientific Catalog #: A-11005 |
HA (Rabbit) | 1:500 | Abcam Catalog #: ab9110 |
Rac1 (Mouse) | 1:200 | BD Biosciences Catalog #: 610651 |
EGFR (Mouse) | 1:200 | Thermo Fisher Scientific Catalog #: MA513269 |
Hoechst 33342 DNA stain | 1 µg/ml final | Thermo Fisher Scientific Catalog #: H3570 |
Breast cancer is not a single disease, but rather a group of diverse diseases with different subtypes. Breast cancers are organized into subtypes distinguished by the absence or presence of certain proteins on the cancer cells (Fragomeni et al., 2018). Triple negative breast cancers (TNBCs) are a subtype of breast cancer characterized by the absence of three receptors: estrogen receptor (ER), progesterone receptors (PR), and human epidermal growth factor 2 (HER2) (Yin et al., 2020). The TNBC subtype is both invasive and aggressive and occurs more frequently in younger women and African American women (Hines et al., 2011). Understanding the specific subtype of breast cancer is crucial for determining the most effective treatment options. The ductal carcinoma TNBC epithelial cell line, MDA-MD-231, is commonly used to study invasive breast cancer and metastasis due to its highly invasive and migratory nature (Amaro et al., 2016).
Cell adhesion, proliferation and migration are three critical cellular processes required for tissues to maintain homeostasis. When one or all of these cellular processes become unregulated, for example in cancer, this can lead to uncontrolled cell growth and metastasis (Hanahan & Weinberg, 2011). Various molecules have been identified that are involved in maintaining the balance of these cellular processes under normal conditions. Rho GTPases are a family of proteins that act as molecular switches that can be activated by guanine nucleotide exchange factors (GEFs) (Lawson & Ridley, 2017). The Rho GTPase Rac1 has been shown to regulate the actin cytoskeleton and cell proliferation by working downstream of the receptor tyrosine kinase, epidermal growth factor receptor (EGFR) and its ligand, epidermal growth factor (EGF), to regulate various kinase pathways (Dise et al., 2008; Itoh et al., 2008). Dimerization of EGFR leads to the phosphorylation of different proteins along these pathways involved in regulating gene expression and activation of proteins involved in promoting cell migration, adhesion, and proliferation (Wee & Wang, 2017). Adenomatous Polyposis Coli (APC)-stimulated guanine nucleotide-exchange factor, Asef, is a GEF that activates Rac1 to promote cell migration by reducing cell-cell adhesions and remodeling the actin cytoskeleton (Kawasaki et al., 2000, 2010). In colorectal cancer, APC can activate Asef, leading to enhanced migration and invasion (Mitin et al., 2007).
The MDA-MB-231 cell line serves as a valuable model for studying breast cancer proliferation and invasion due to the presence or absence of molecules involved in maintaining normal cell function. MDA-MB-231 cells express Rac1 and are responsive to EGF stimulation (Davidson et al., 1987; Veber et al., 1994). However, it is currently unknown how EGFR activation impacts the regulatory molecules, Asef and Rac1, along with cell migration and proliferation in MDA-MB-231 cells. The current study aims to examine the role of Asef in cell proliferation and migration in the presence of EGF in MDA-MB-231 cells. We hypothesize that due to the regulatory nature of Asef on Rac1, overexpression of Asef in the presence of EGFR signaling pathway will lead to changes in the protein localization and an increase in proliferation and cell migration due to activation of downstream signaling pathways.
Asef-HA (Kawasaki et al., 2000) was overexpressed in MDA-MB-231 cells using a lipid-based transfection approach and protein localization of Rac1, EGFR, and actin was observed in untreated and EGF-treated cells. Using immunofluorescence, Asef-HA overexpressing cells show Asef both in the cytosol and at the cell membrane co-localizing with endogenous EGFR and Rac1 protein in untreated cells and cells treated with EGF for 24 hours (Figure 1A-B). The increased protein localization for Asef and Rac1 at the membrane suggests a possible interaction of the proteins to control cell migration (Figure 1A). Kawasaki, et al. previously showed that Asef promotes the activation of the Rac1 protein, exchanging GDP for GTP, in endothelial cells (Kawasaki et al., 2010). Additional studies are required to determine if there is a direct interaction between the overexpressed Asef-HA protein and Rac1-GTP at the membrane.
EGFR protein appears to internalize into small vesicular structures after EGF stimulation (Figure 1B), suggesting activation of the receptor and downstream cellular pathways (Pennock & Wang, 2003). Due to the shift of EGFR from the membrane to internal vesicular structures in the presence of EGF and previous studies which have examined the impact of EGFR internalization on actin remodeling and cell migration (Pinilla-Macua et al., 2025), actin organization in Asef-HA expressing cells was examined. Actin organization provides a critical scaffold to cellular organization and structure and is represented by two different and interconnected cytoskeletal structures: cortical and stress fibers (Vallenius, 2013). Cortical actin forms a meshwork for actin filaments beneath the cell membrane and is involved in controlling cell shape by locally modifying cortical tension (Chalut & Paluch, 2016). Stress fibers are organized as bundles of actin filaments and play a role in cell migration, adhesion, and mechanosensing, particularly in the context of cancer. These structures are involved in cell migration and linked to cell stiffness, which can either promote or inhibit migration depending on the specific context and cell type (Fischer et al., 2021; Tavares et al., 2017). To determine the impact of Asef on actin filament organization, MDA-MB-231 cells were transfected with Asef-HA. Stress fibers appear to be reduced in Asef-HA expressing cells (asterisks), with or without EGF, as compared to cortical actin, which remains unchanged in both untreated and EGF-treated cells (Figure 1C). The loss of stress fiber formation in Asef-HA cells in comparison to untransfected cells suggests the cells have lost their stability and may be more susceptible to movement.
The lack of stress fiber formation in Asef-HA expressing cells suggests that the cells may be more migratory in nature. To determine if overexpressed Asef leads to an increase in cell migration, a wound healing assay was performed in MDA-MB-231 cells overexpressing Asef-HA. Data indicate that cells overexpressing Asef-HA with or without EGF for 24 hours migrate faster compared to HA only expressing cells (Figure 1D). Quantitative analysis shows a significant increase in proliferation at 12 and 24 hours between HA-only cells and Asef-HA without EGF stimulation in comparison to cells under EGF stimulation (Figure 1D). These data support the hypothesis that overexpression of Asef-HA in MDA-MB-231 cells activates pathways that lead to increased cell migration, by possibly impacting actin organization and turnover at the leading edges of cells (Tian et al., 2015). This is further validated by the reorganization and distribution of key molecules involved in migration, namely EGFR, Rac1, and actin filament organization (Figure 1A-C) and that Rac1 serves as a relay protein between EGFR and cell migration (Dise et al., 2008). The lack of significance between HA-only cells and Asef-HA with EGF treatment suggests that the EGF concentration was not sufficient to induce a response. Previous studies have shown that EGF at a 10 fold higher concentration appears to have more of an impact on MDA-MB-231 cell migration (Kozlova et al., 2016) than lower concentrations. Future studies will determine if higher EGF concentrations lead to significant changes in cell proliferation and migration over basal conditions in cells overexpressing Asef.
Internalization of EGFR via EGF binding, as observed in Figure 1B, may lead to activation of cyclins and/or other cell division regulators to promote an increase in cell proliferation. Furthermore, the EGFR pathway is frequently implicated in cancer development and progression, as it can drive uncontrolled cell growth and division (X. Song et al., 2020; Z. Song et al., 2016). To determine if the activation of EGFR leads to possible increase in MDA-MB-231 cell proliferation due to downstream pathways becoming activated, a MTT assay was performed on control and Asef-HA transfected cells. Data show a significant increase in proliferation in Asef-HA without EGF-stimulation compared to HA-only cells and there was no significant change in Asef-HA with 10 ng/ml EGF for 24 hours (Figure 1E). Similar to the wound healing assay, the lack of significance between control cells and Asef-HA with EGF treatment suggests that the EGF concentration may not be sufficient. These data support the hypothesis that overexpression of Asef-HA in MDA-MB-231 cells may activate pathways that lead to cell proliferation (Lo & Hung, 2006; Pennock & Wang, 2003). Additional studies are required to determine which pathways are responsible for the increased cell proliferation and the impact of EGFR signaling on cell proliferation.
Our findings show that Asef overexpression impacts actin filament remodeling and increases cell migration and proliferation in a metastatic breast cancer cell line. Mutated versions of APC and Asef have been implicated in colon cancer cell migration and invasion (Yang et al., 2021), and we extend the role of Asef into a highly invasive and aggressive epithelial breast cancer subtype. Asef is primarily known for its role in APC-Wnt signaling (Kawasaki et al., 2000); however, Asef and Rac1 were shown to be involved in the EGFR pathway and may be a possible molecular target to consider in understanding the aggressiveness of triple negative breast cancer (Itoh et al., 2008). Future work aims to explore the connection of Asef to signaling proteins involved in controlling cell proliferation and how Asef regulates Rac1 and actin organization in the presence of EGF to control cell motility.
","references":[{"reference":"Amaro A, Angelini G, Mirisola V, Esposito AI, Reverberi D, Matis S, et al., Pfeffer. 2016. A highly invasive subpopulation of MDA-MB-231 breast cancer cells shows accelerated growth, differential chemoresistance, features of apocrine tumors and reduced tumorigenicityin vivo. Oncotarget 7: 68803-68820.
","pubmedId":"","doi":"10.18632/oncotarget.11931 "},{"reference":"Chalut KJ, Paluch EK. 2016. The Actin Cortex: A Bridge between Cell Shape and Function. Developmental Cell 38: 571-573.
","pubmedId":"","doi":"10.1016/j.devcel.2016.09.011 "},{"reference":"Davidson NE, Gelmann EP, Lippman ME, Dickson RB. 1987. Epidermal Growth Factor Receptor Gene Expression in Estrogen Receptor-Positive and Negative Human Breast Cancer Cell Lines. Molecular Endocrinology 1: 216-223.
","pubmedId":"","doi":"10.1210/mend-1-3-216 "},{"reference":"Dise RS, Frey MR, Whitehead RH, Polk DB. 2008. Epidermal growth factor stimulates Rac activation through Src and phosphatidylinositol 3-kinase to promote colonic epithelial cell migration. American Journal of Physiology-Gastrointestinal and Liver Physiology 294: G276-G285.
","pubmedId":"","doi":"10.1152/ajpgi.00340.2007 "},{"reference":"Fischer RS, Sun X, Baird MA, Hourwitz MJ, Seo BR, Pasapera AM, et al., Waterman. 2021. Contractility, focal adhesion orientation, and stress fiber orientation drive cancer cell polarity and migration along wavy ECM substrates. Proceedings of the National Academy of Sciences 118: 10.1073/pnas.2021135118.
","pubmedId":"","doi":"10.1073/pnas.2021135118 "},{"reference":"Fragomeni SM, Sciallis A, Jeruss JS. 2018. Molecular Subtypes and Local-Regional Control of Breast Cancer. Surgical Oncology Clinics of North America 27: 95-120.
","pubmedId":"","doi":"10.1016/j.soc.2017.08.005 "},{"reference":"Hanahan D, Weinberg RA. 2011. Hallmarks of Cancer: The Next Generation. Cell 144: 646-674.
","pubmedId":"","doi":"10.1016/j.cell.2011.02.013 "},{"reference":"Hines LM, Risendal B, Byers T, Mengshol S, Lowery J, Singh M. 2011. Ethnic Disparities in Breast Tumor Phenotypic Subtypes in Hispanic and Non-Hispanic White Women. Journal of Women's Health 20: 1543-1550.
","pubmedId":"","doi":"10.1089/jwh.2010.2558"},{"reference":"Itoh RE, Kiyokawa E, Aoki K, Nishioka T, Akiyama T, Matsuda M. 2008. Phosphorylation and activation of the Rac1 and Cdc42 GEF Asef in A431 cells stimulated by EGF. Journal of Cell Science 121: 2635-2642.
","pubmedId":"","doi":"10.1242/jcs.028647 "},{"reference":"Kawasaki Y, Jigami T, Furukawa S, Sagara M, Echizen K, Shibata Y, Sato R, Akiyama T. 2010. The Adenomatous Polyposis Coli-associated Guanine Nucleotide Exchange Factor Asef Is Involved in Angiogenesis. Journal of Biological Chemistry 285: 1199-1207.
","pubmedId":"","doi":"10.1074/jbc.M109.040691 "},{"reference":"Kawasaki Y, Senda T, Ishidate T, Koyama R, Morishita T, Iwayama Y, Higuchi O, Akiyama T. 2000. Asef, a Link Between the Tumor Suppressor APC and G-Protein Signaling. Science 289: 1194-1197.
","pubmedId":"","doi":"10.1126/science.289.5482.1194"},{"reference":"Kozlova N, Samoylenko A, Drobot L, Kietzmann T. 2015. Urokinase is a negative modulator of Egf‐dependent proliferation and motility in the two breast cancer cell lines MCF‐7 and MDA‐MB‐231. Molecular Carcinogenesis 55: 170-181.
","pubmedId":"","doi":"10.1002/mc.22267 "},{"reference":"Lawson CD, Ridley AJ. 2017. Rho GTPase signaling complexes in cell migration and invasion. Journal of Cell Biology 217: 447-457.
","pubmedId":"","doi":"10.1083/jcb.201612069 "},{"reference":"Lo HW, Hung MC. 2006. Nuclear EGFR signalling network in cancers: linking EGFR pathway to cell cycle progression, nitric oxide pathway and patient survival. British Journal of Cancer 94: 184-188.
","pubmedId":"","doi":"10.1038/sj.bjc.6602941"},{"reference":"Mitin N, Betts L, Yohe ME, Der CJ, Sondek J, Rossman KL. 2007. Release of autoinhibition of ASEF by APC leads to CDC42 activation and tumor suppression. Nature Structural & Molecular Biology 14: 814-823.
","pubmedId":"","doi":"10.1038/nsmb1290 "},{"reference":"Pennock S, Wang Z. 2003. Stimulation of Cell Proliferation by Endosomal Epidermal Growth Factor Receptor As Revealed through Two Distinct Phases of Signaling. Molecular and Cellular Biology 23: 5803-5815.
","pubmedId":"","doi":"10.1128/MCB.23.16.5803-5815.2003"},{"reference":"Pinilla-Macua I, Surve S, Sorkin A. 2025. Cell migration signaling through the EGFR-VAV2-Rac1 pathway is sustained in endosomes. Journal of Cell Science 138: 10.1242/jcs.263541.
","pubmedId":"","doi":"10.1242/jcs.263541 "},{"reference":"Song X, Liu Z, Yu Z. 2020. <p>EGFR Promotes the Development of Triple Negative Breast Cancer Through JAK/STAT3 Signaling</p>. Cancer Management and Research Volume 12: 703-717.
","pubmedId":"","doi":"10.2147/CMAR.S225376 "},{"reference":"Song Z, Fusco J, Zimmerman R, Fischbach S, Chen C, Ricks DM, et al., Gittes. 2016. Epidermal Growth Factor Receptor Signaling Regulates β Cell Proliferation in Adult Mice. Journal of Biological Chemistry 291: 22630-22637.
","pubmedId":"","doi":"10.1074/jbc.M116.747840 "},{"reference":"Tavares S, Vieira AF, Taubenberger AV, Araújo M, Martins NP, Brás-Pereira C, et al., Janody. 2017. Actin stress fiber organization promotes cell stiffening and proliferation of pre-invasive breast cancer cells. Nature Communications 8: 10.1038/ncomms15237.
","pubmedId":"","doi":"10.1038/ncomms15237"},{"reference":"Tian X, Tian Y, Gawlak G, Meng F, Kawasaki Y, Akiyama T, Birukova AA. 2015. Asef controls vascular endothelial permeability and barrier recovery in the lung. Molecular Biology of the Cell 26: 636-650.
","pubmedId":"","doi":"10.1091/mbc.E14-02-0725 "},{"reference":"Vallenius T. 2013. Actin stress fibre subtypes in mesenchymal-migrating cells. Open Biology 3: 130001.
","pubmedId":"","doi":"10.1098/rsob.130001 "},{"reference":"Veber N, Prévost G, Planchon P, Starzec A. 1994. Evidence for a growth effect of epidermal growth factor on MDA-MB-231 breast cancer cells. European Journal of Cancer 30: 1352-1359.
","pubmedId":"","doi":"10.1016/0959-8049(94)90186-4 "},{"reference":"Wee P, Wang Z. 2017. Epidermal Growth Factor Receptor Cell Proliferation Signaling Pathways. Cancers 9: 52.
","pubmedId":"","doi":"10.3390/cancers9050052"},{"reference":"Yang X, Zhong J, Zhang Q, Feng L, Zheng Z, Zhang J, Lu S. 2021. Advances and Insights of APC-Asef Inhibitors for Metastatic Colorectal Cancer Therapy. Frontiers in Molecular Biosciences 8: 10.3389/fmolb.2021.662579.
","pubmedId":"","doi":"10.3389/fmolb.2021.662579 "},{"reference":"Yin L, Duan JJ, Bian XW, Yu Sc. 2020. Triple-negative breast cancer molecular subtyping and treatment progress. Breast Cancer Research 22: 10.1186/s13058-020-01296-5.
","pubmedId":"","doi":"10.1186/s13058-020-01296-5 "}],"title":"Examining the Role of Asef and Epidermal Growth Factor on Cell Migration and Proliferation in Triple Negative Breast Cancer Epithelial Cells
","reviews":[],"curatorReviews":[]},{"id":"a516151e-233e-4fa3-ab1e-5fa9072ff45c","decision":"edit","abstract":"Cell proliferation and migration are cellular processes essential to normal cell function; errors in these cellular processes impact the ability of a cell to maintain cellular homeostasis. The current study examines the role of Asef, a GEF for Rac1, in cell proliferation and migration in the presence or absence of EGF in a triple negative breast cancer epithelial cell line. Overexpression of Asef reduced stress fiber formation and increased cell migration and proliferation in MDA-MB-231 epithelial cells. These data indicate a role for Asef in the EGFR/Rac1 signaling cascade involved in regulating cellular behavior.
","acknowledgements":"The authors thank the SMC School of Science for their support of this project and the Cell Biology Education Consortium (CBEC) for feedback and comments on the manuscript.
","authors":[{"affiliations":["Saint Mary's College of California, Moraga, CA, United States"],"departments":["Biology Department"],"credit":["investigation","methodology","writing_originalDraft"],"email":"na21@stmarys-ca.edu","firstName":"Natalie","lastName":"Alaniz","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":"0009-0004-5090-7951"},{"affiliations":["Saint Mary's College of California, Moraga, CA, United States"],"departments":["Biology Department"],"credit":["conceptualization","formalAnalysis","methodology","project","supervision","writing_reviewEditing","investigation"],"email":"knk8@stmarys-ca.edu","firstName":"Khameeka N.","lastName":"Kitt","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":false,"WBId":null,"orcid":"0009-0003-2381-877X"}],"awards":[],"conflictsOfInterest":"The authors declare that there are no conflicts of interest present.
","dataTable":{"url":null},"extendedData":[],"funding":"The authors thank the SMC School of Science Summer Endowment fund. Undergraduate student research participation and publication support were funded by the Cell Biology Education Consortium (CBEC): Path to Publication (Award ID #2316122) through the National Science Foundation (NSF).
","image":{"url":"https://portal.micropublication.org/uploads/a7e30098fc0811eef71cf03e5f47606a.png"},"imageCaption":"Human MDA-MB-231 breast cancer epithelial cells were transfected with Asef-HA (green) and allowed to express for 24 hours before treatment with 10 ng/ml of EGF for 24 hours. (A) In the absence of EGF, Asef and Rac1 (red) localize to the cytoplasm and membrane. In the presence of EGF for 24 hours, Rac1 colocalizes with Asef at the cell membrane (arrows). Scale bar: 40 µm. (B) Asef localizes to the cytoplasm and membrane ruffles (arrows) in cells with and without EGF treatment. In the absence of EGF, EGFR (red) localizes to the cell membrane and cytoplasm in Asef-HA expressing cells (arrowhead). Cells treated with EGF for 24 hours caused a minor shift in EGFR localization from the membrane to vesicle-like structures inside the cell (arrowhead). Scale bar: 40 µm. (C) Asef localizes to the cytoplasm and membrane ruffles (arrows) in cells with and without EGF treatment. Cortical Actin (red) appears unchanged in Asef-HA expressing cells (arrowhead). Cells expressing Asef-HA show reduced stress fibers extending across the cell (asterisks) compared to untransfected cells (arrows) in the presence or absence of EGF for 24 hours. Scale bar: 40 µm. (D) MDA-MB-231 cell monolayers overexpressing HA-only or Asef-HA were scratched and imaged over 24 hours in the presence or absence of EGF. The width of the wound was measured using ImageJ and plotted to examine wound width over time. Asef-HA expressing cells, regardless of EGF treatment, migrated faster than control cells; however, Asef-HA expressing cells without EGF treatment showed a significant increase in migration at 12 and 24 hour time points compared to HA only expressing cells. (**P < 0.005; n=3 measurements of each group). (E) MDA-MB-231 cell monolayers overexpressing HA-only or Asef-HA were serum-starved and treated with 10 ng/ml of EGF for 24 hours. Following EGF-treatment, MTT reagent was added, cells were solubilized with DMSO, and absorbance was read at 570 nm. Asef-HA expressing cells in the absence of EGF treatment showed a significant increase in cell proliferation compared to untransfected cells (*P < 0.05; n=3 measurements of each group). (F) Model of the Rho GTPase signaling pathway demonstrating how Asef could be recruited downstream of EGFR signaling to regulate Rac1 activation and stimulate cell proliferation and migration.
","imageTitle":"Asef-HA overexpression in MDA-MB-231 breast epithelial cancer cells causes changes in protein localization, cell migration, and cell proliferation.
","methods":"Cell Culture
Triple negative ductal breast carcinoma epithelial cell line MDA-MB-231 (ATCC) were grown in Dulbecco's Modified Eagle Serum (DMEM) high glucose media (Thermo Fisher Scientific) with 10% fetal bovine serum and 1% penicillin-streptomycin-glutamine (Thermo Fisher Scientific). Cells were incubated under the following conditions: 5% CO2 and 37˚C.
Transfection
MDA-MB-321 cells were transfected with pcDNA 3.1-HA Asef (human) full length plasmid (provided by Tetsu Akiyama lab, University of Tokyo, (Kawasaki et al., 2000)) using Lipofectamine 2000 diluted in Opti-MEM (Thermo Fisher Scientific) following product recommendations. As a control for proliferation and scratch assays, cells were transfected with pcDNA 3.1-HA plasmid alone. Transfected cells were incubated for 24 hours in antibiotic-free media before being replated in various well formats for various assays.
Immunofluorescence
Transfected MDA-MB-231 cells (with or without Asef-HA) were placed on rat tail collagen-coated coverslips in serum-free media (DMEM with 1% penicillin-streptomycin-glutamine) and treated with or without 10 ng/mL of EGF for 24 hours. Cells were fixed using 4% paraformaldehyde 48 hours post-transfection. To localize HA, Rac1, and EGFR, cells were blocked and labeled with primary antibodies for HA (Roche), Rac1 (BD Biosciences) and EGFR (Thermo Fisher Scientific) followed by secondary antibodies conjugated to Alexa Fluor 488 and 594 (Thermo Fisher Scientific). Actin filaments and nuclei were labeled using Phalloidin 594 and Hoechst 33342 DNA stains, respectively (Thermo Fisher Scientific). Cells were imaged using the Leica STELLARIS Inverted Spectral Live Cell Confocal Microscope.
Scratch Assay (Wound Healing)
Transfected MDA-MB-231 cells (with or without Asef-HA) were placed on rat tail collagen-coated 12 well plates and allowed to form a monolayer in serum free media. Monolayers were scratched 24 hours later using a sterile 200 µL pipette tip, washed using phosphate-buffered saline (PBS) to remove debris, and incubated in serum-free media treated with or without 10 ng/mL of EGF for 24 hours. Images were captured at 0, 4, 8, 12 and 24 hours. The area between both sides of the scratch were quantified after each image time stamp to determine the wound closure distance using ImageJ software. Distance of wound closure for each group at each time point was measured in µm using ImageJ (pixels per micron was determined using scale bar) and plotted to display the width of the wound closure over time.
Proliferation (MTT) Assay
Transfected MDA-MB-231 cells (with or without Asef-HA) were placed in a 96-well tissue culture-treated plate in serum free media. Cells were treated with or without 10 ng/mL of EGF for 24 hours before adding 10 µL of a 5 mg/mL stock of MTT reagent per well and incubated for three hours. After three hours, the MTT reagent was removed and 100µL DMSO was added to each well to solubilize cells, and the optical density was measured at 570 nm (Tecan Plate Reader). Percent viability was calculated for each well and normalized against the control (HA-only).
Statistical Analysis
For the scratch assay, a multiple unpaired t-test analysis was performed to determine the significance at each time point. Three biological replicates (independent wells) were analyzed for each group. For the proliferation (MTT) assay, an unpaired t-test was used to determine significance between the treated groups compared to the control. Three biological replicates (independent wells) were analyzed for each group. All data was graphed using GraphPad Prism.
","reagents":"Plasmid | Description | Source |
pcDNA 3.1-HA- Asef | Full length Asef (human) | Tetsu Akiyama lab, University of Tokyo |
Cell Line | Description | Source |
MDA-MB-231 | Human Epithelial Breast Tissue isolated from Mammary Gland; Adenocarcinoma | ATCC Catalog #: HTB-26 |
Antibodies | Dilution | Source |
Phalloidin 594 | 1:400 | Thermo Fisher Scientific Catalog #: A12381 |
Alexa Goat anti Rabbit 488 | 1:800 | Thermo Fisher Scientific Catalog #: A-11008 |
Alexa Goat anti Mouse 594 | 1:800 | Thermo Fisher Scientific Catalog #: A-11005 |
HA (Rabbit) | 1:500 | Abcam Catalog #: ab9110 |
Rac1 (Mouse) | 1:200 | BD Biosciences Catalog #: 610651 |
EGFR (Mouse) | 1:200 | Thermo Fisher Scientific Catalog #: MA513269 |
Hoechst 33342 DNA stain | 1 µg/ml final | Thermo Fisher Scientific Catalog #: H3570 |
Breast cancer is not a single disease, but rather a group of diverse diseases with different subtypes. Breast cancers are organized into subtypes distinguished by the absence or presence of certain proteins on the cancer cells (Fragomeni et al., 2018). Triple negative breast cancers (TNBCs) are a subtype of breast cancer characterized by the absence of three receptors: estrogen receptor (ER), progesterone receptors (PR), and human epidermal growth factor 2 (HER2) (Yin et al., 2020). The TNBC subtype is both invasive and aggressive and occurs more frequently in younger women and African American women (Hines et al., 2011). Understanding the specific subtype of breast cancer is crucial for determining the most effective treatment options. The ductal carcinoma TNBC epithelial cell line, MDA-MD-231, is commonly used to study invasive breast cancer and metastasis due to its highly invasive and migratory nature (Amaro et al., 2016).
Cell adhesion, proliferation and migration are three critical cellular processes required for tissues to maintain homeostasis. When one or all of these cellular processes become unregulated, for example in cancer, this can lead to uncontrolled cell growth and metastasis (Hanahan & Weinberg, 2011). Various molecules have been identified that are involved in maintaining the balance of these cellular processes under normal conditions. Rho GTPases are a family of proteins that act as molecular switches that can be activated by guanine nucleotide exchange factors (GEFs) (Lawson & Ridley, 2017). The Rho GTPase Rac1 has been shown to regulate the actin cytoskeleton and cell proliferation by working downstream of the receptor tyrosine kinase, epidermal growth factor receptor (EGFR) and its ligand, epidermal growth factor (EGF), to regulate various kinase pathways (Dise et al., 2008; Itoh et al., 2008). Dimerization of EGFR leads to the phosphorylation of different proteins along these pathways involved in regulating gene expression and activation of proteins involved in promoting cell migration, adhesion, and proliferation (Wee & Wang, 2017). Adenomatous Polyposis Coli (APC)-stimulated guanine nucleotide-exchange factor, Asef, is a GEF that activates Rac1 to promote cell migration by reducing cell-cell adhesions and remodeling the actin cytoskeleton (Kawasaki et al., 2000, 2010). In colorectal cancer, APC can activate Asef, leading to enhanced migration and invasion (Mitin et al., 2007).
The MDA-MB-231 cell line serves as a valuable model for studying breast cancer proliferation and invasion due to the presence or absence of molecules involved in maintaining normal cell function. MDA-MB-231 cells express Rac1 and are responsive to EGF stimulation (Davidson et al., 1987; Veber et al., 1994). However, it is currently unknown how EGFR activation impacts the regulatory molecules, Asef and Rac1, along with cell migration and proliferation in MDA-MB-231 cells. The current study aims to examine the role of Asef in cell proliferation and migration in the presence of EGF in MDA-MB-231 cells. We hypothesize that due to the regulatory nature of Asef on Rac1, overexpression of Asef in the presence of EGFR signaling pathway will lead to changes in the protein localization and an increase in proliferation and cell migration due to activation of downstream signaling pathways.
Asef-HA (Kawasaki et al., 2000) was overexpressed in MDA-MB-231 cells using a lipid-based transfection approach and protein localization of Rac1, EGFR, and actin was observed in untreated and EGF-treated cells. Using immunofluorescence, Asef-HA overexpressing cells show Asef both in the cytosol and at the cell membrane co-localizing with endogenous EGFR and Rac1 protein in untreated cells and cells treated with EGF for 24 hours (Figure 1A-B). The increased protein localization for Asef and Rac1 at the membrane suggests a possible interaction of the proteins to control cell migration (Figure 1A). Kawasaki, et al. previously showed that Asef promotes the activation of the Rac1 protein, exchanging GDP for GTP, in endothelial cells (Kawasaki et al., 2010). Additional studies are required to determine if there is a direct interaction between the overexpressed Asef-HA protein and Rac1-GTP at the membrane.
EGFR protein appears to internalize into small vesicular structures after EGF stimulation (Figure 1B), suggesting activation of the receptor and downstream cellular pathways (Pennock & Wang, 2003). Due to the shift of EGFR from the membrane to internal vesicular structures in the presence of EGF and previous studies which have examined the impact of EGFR internalization on actin remodeling and cell migration (Pinilla-Macua et al., 2025), actin organization in Asef-HA expressing cells was examined. Actin organization provides a critical scaffold to cellular organization and structure and is represented by two different and interconnected cytoskeletal structures: cortical and stress fibers (Vallenius, 2013). Cortical actin forms a meshwork for actin filaments beneath the cell membrane and is involved in controlling cell shape by locally modifying cortical tension (Chalut & Paluch, 2016). Stress fibers are organized as bundles of actin filaments and play a role in cell migration, adhesion, and mechanosensing, particularly in the context of cancer. These structures are involved in cell migration and linked to cell stiffness, which can either promote or inhibit migration depending on the specific context and cell type (Fischer et al., 2021; Tavares et al., 2017). To determine the impact of Asef on actin filament organization, MDA-MB-231 cells were transfected with Asef-HA. Stress fibers appear to be reduced in Asef-HA expressing cells (asterisks), with or without EGF, as compared to cortical actin, which remains unchanged in both untreated and EGF-treated cells (Figure 1C). The loss of stress fiber formation in Asef-HA cells in comparison to untransfected cells suggests the cells have lost their stability and may be more susceptible to movement.
The lack of stress fiber formation in Asef-HA expressing cells suggests that the cells may be more migratory in nature. To determine if overexpressed Asef leads to an increase in cell migration, a wound healing assay was performed in MDA-MB-231 cells overexpressing Asef-HA. Data indicate that cells overexpressing Asef-HA with or without EGF for 24 hours migrate faster compared to HA only expressing cells (Figure 1D). Quantitative analysis shows a significant increase in proliferation at 12 and 24 hours between HA-only cells and Asef-HA without EGF stimulation in comparison to cells under EGF stimulation (Figure 1D). These data support the hypothesis that overexpression of Asef-HA in MDA-MB-231 cells activates pathways that lead to increased cell migration, by possibly impacting actin organization and turnover at the leading edges of cells (Tian et al., 2015). This is further validated by the reorganization and distribution of key molecules involved in migration, namely EGFR, Rac1, and actin filament organization (Figure 1A-C) and that Rac1 serves as a relay protein between EGFR and cell migration (Dise et al., 2008). The lack of significance between HA-only cells and Asef-HA with EGF treatment suggests that the EGF concentration was not sufficient to induce a response. Previous studies have shown that EGF at a 10 fold higher concentration appears to have more of an impact on MDA-MB-231 cell migration (Kozlova et al., 2016) than lower concentrations. Future studies will determine if higher EGF concentrations lead to significant changes in cell proliferation and migration over basal conditions in cells overexpressing Asef.
Internalization of EGFR via EGF binding, as observed in Figure 1B, may lead to activation of cyclins and/or other cell division regulators to promote an increase in cell proliferation. Furthermore, the EGFR pathway is frequently implicated in cancer development and progression, as it can drive uncontrolled cell growth and division (X. Song et al., 2020; Z. Song et al., 2016). To determine if the activation of EGFR leads to possible increase in MDA-MB-231 cell proliferation due to downstream pathways becoming activated, a MTT assay was performed on control and Asef-HA transfected cells. Data show a significant increase in proliferation in Asef-HA without EGF-stimulation compared to HA-only cells and there was no significant change in Asef-HA with 10 ng/ml EGF for 24 hours (Figure 1E). Similar to the wound healing assay, the lack of significance between control cells and Asef-HA with EGF treatment suggests that the EGF concentration may not be sufficient. These data support the hypothesis that overexpression of Asef-HA in MDA-MB-231 cells may activate pathways that lead to cell proliferation (Lo & Hung, 2006; Pennock & Wang, 2003). Additional studies are required to determine which pathways are responsible for the increased cell proliferation and the impact of EGFR signaling on cell proliferation.
Our findings show that Asef overexpression impacts actin filament remodeling and increases cell migration and proliferation in a metastatic breast cancer cell line. Mutated versions of APC and Asef have been implicated in colon cancer cell migration and invasion (Yang et al., 2021), and we extend the role of Asef into a highly invasive and aggressive epithelial breast cancer subtype. Asef is primarily known for its role in APC-Wnt signaling (Kawasaki et al., 2000); however, Asef and Rac1 were shown to be involved in the EGFR pathway and may be a possible molecular target to consider in understanding the aggressiveness of triple negative breast cancer (Itoh et al., 2008). Future work aims to explore the connection of Asef to signaling proteins involved in controlling cell proliferation and how Asef regulates Rac1 and actin organization in the presence of EGF to control cell motility.
","references":[{"reference":"Amaro A, Angelini G, Mirisola V, Esposito AI, Reverberi D, Matis S, et al., Pfeffer. 2016. A highly invasive subpopulation of MDA-MB-231 breast cancer cells shows accelerated growth, differential chemoresistance, features of apocrine tumors and reduced tumorigenicityin vivo. Oncotarget 7: 68803-68820.
","pubmedId":"","doi":"10.18632/oncotarget.11931 "},{"reference":"Chalut KJ, Paluch EK. 2016. The Actin Cortex: A Bridge between Cell Shape and Function. Developmental Cell 38: 571-573.
","pubmedId":"","doi":"10.1016/j.devcel.2016.09.011 "},{"reference":"Davidson NE, Gelmann EP, Lippman ME, Dickson RB. 1987. Epidermal Growth Factor Receptor Gene Expression in Estrogen Receptor-Positive and Negative Human Breast Cancer Cell Lines. Molecular Endocrinology 1: 216-223.
","pubmedId":"","doi":"10.1210/mend-1-3-216 "},{"reference":"Dise RS, Frey MR, Whitehead RH, Polk DB. 2008. Epidermal growth factor stimulates Rac activation through Src and phosphatidylinositol 3-kinase to promote colonic epithelial cell migration. American Journal of Physiology-Gastrointestinal and Liver Physiology 294: G276-G285.
","pubmedId":"","doi":"10.1152/ajpgi.00340.2007 "},{"reference":"Fischer RS, Sun X, Baird MA, Hourwitz MJ, Seo BR, Pasapera AM, et al., Waterman. 2021. Contractility, focal adhesion orientation, and stress fiber orientation drive cancer cell polarity and migration along wavy ECM substrates. Proceedings of the National Academy of Sciences 118: 10.1073/pnas.2021135118.
","pubmedId":"","doi":"10.1073/pnas.2021135118 "},{"reference":"Fragomeni SM, Sciallis A, Jeruss JS. 2018. Molecular Subtypes and Local-Regional Control of Breast Cancer. Surgical Oncology Clinics of North America 27: 95-120.
","pubmedId":"","doi":"10.1016/j.soc.2017.08.005 "},{"reference":"Hanahan D, Weinberg RA. 2011. Hallmarks of Cancer: The Next Generation. Cell 144: 646-674.
","pubmedId":"","doi":"10.1016/j.cell.2011.02.013 "},{"reference":"Hines LM, Risendal B, Byers T, Mengshol S, Lowery J, Singh M. 2011. Ethnic Disparities in Breast Tumor Phenotypic Subtypes in Hispanic and Non-Hispanic White Women. Journal of Women's Health 20: 1543-1550.
","pubmedId":"","doi":"10.1089/jwh.2010.2558"},{"reference":"Itoh RE, Kiyokawa E, Aoki K, Nishioka T, Akiyama T, Matsuda M. 2008. Phosphorylation and activation of the Rac1 and Cdc42 GEF Asef in A431 cells stimulated by EGF. Journal of Cell Science 121: 2635-2642.
","pubmedId":"","doi":"10.1242/jcs.028647 "},{"reference":"Kawasaki Y, Jigami T, Furukawa S, Sagara M, Echizen K, Shibata Y, Sato R, Akiyama T. 2010. The Adenomatous Polyposis Coli-associated Guanine Nucleotide Exchange Factor Asef Is Involved in Angiogenesis. Journal of Biological Chemistry 285: 1199-1207.
","pubmedId":"","doi":"10.1074/jbc.M109.040691 "},{"reference":"Kawasaki Y, Senda T, Ishidate T, Koyama R, Morishita T, Iwayama Y, Higuchi O, Akiyama T. 2000. Asef, a Link Between the Tumor Suppressor APC and G-Protein Signaling. Science 289: 1194-1197.
","pubmedId":"","doi":"10.1126/science.289.5482.1194"},{"reference":"Kozlova N, Samoylenko A, Drobot L, Kietzmann T. 2015. Urokinase is a negative modulator of Egf‐dependent proliferation and motility in the two breast cancer cell lines MCF‐7 and MDA‐MB‐231. Molecular Carcinogenesis 55: 170-181.
","pubmedId":"","doi":"10.1002/mc.22267 "},{"reference":"Lawson CD, Ridley AJ. 2017. Rho GTPase signaling complexes in cell migration and invasion. Journal of Cell Biology 217: 447-457.
","pubmedId":"","doi":"10.1083/jcb.201612069 "},{"reference":"Lo HW, Hung MC. 2006. Nuclear EGFR signalling network in cancers: linking EGFR pathway to cell cycle progression, nitric oxide pathway and patient survival. British Journal of Cancer 94: 184-188.
","pubmedId":"","doi":"10.1038/sj.bjc.6602941"},{"reference":"Mitin N, Betts L, Yohe ME, Der CJ, Sondek J, Rossman KL. 2007. Release of autoinhibition of ASEF by APC leads to CDC42 activation and tumor suppression. Nature Structural & Molecular Biology 14: 814-823.
","pubmedId":"","doi":"10.1038/nsmb1290 "},{"reference":"Pennock S, Wang Z. 2003. Stimulation of Cell Proliferation by Endosomal Epidermal Growth Factor Receptor As Revealed through Two Distinct Phases of Signaling. Molecular and Cellular Biology 23: 5803-5815.
","pubmedId":"","doi":"10.1128/MCB.23.16.5803-5815.2003"},{"reference":"Pinilla-Macua I, Surve S, Sorkin A. 2025. Cell migration signaling through the EGFR-VAV2-Rac1 pathway is sustained in endosomes. Journal of Cell Science 138: 10.1242/jcs.263541.
","pubmedId":"","doi":"10.1242/jcs.263541 "},{"reference":"Song X, Liu Z, Yu Z. 2020. <p>EGFR Promotes the Development of Triple Negative Breast Cancer Through JAK/STAT3 Signaling</p>. Cancer Management and Research Volume 12: 703-717.
","pubmedId":"","doi":"10.2147/CMAR.S225376 "},{"reference":"Song Z, Fusco J, Zimmerman R, Fischbach S, Chen C, Ricks DM, et al., Gittes. 2016. Epidermal Growth Factor Receptor Signaling Regulates β Cell Proliferation in Adult Mice. Journal of Biological Chemistry 291: 22630-22637.
","pubmedId":"","doi":"10.1074/jbc.M116.747840 "},{"reference":"Tavares S, Vieira AF, Taubenberger AV, Araújo M, Martins NP, Brás-Pereira C, et al., Janody. 2017. Actin stress fiber organization promotes cell stiffening and proliferation of pre-invasive breast cancer cells. Nature Communications 8: 10.1038/ncomms15237.
","pubmedId":"","doi":"10.1038/ncomms15237"},{"reference":"Tian X, Tian Y, Gawlak G, Meng F, Kawasaki Y, Akiyama T, Birukova AA. 2015. Asef controls vascular endothelial permeability and barrier recovery in the lung. Molecular Biology of the Cell 26: 636-650.
","pubmedId":"","doi":"10.1091/mbc.E14-02-0725 "},{"reference":"Vallenius T. 2013. Actin stress fibre subtypes in mesenchymal-migrating cells. Open Biology 3: 130001.
","pubmedId":"","doi":"10.1098/rsob.130001 "},{"reference":"Veber N, Prévost G, Planchon P, Starzec A. 1994. Evidence for a growth effect of epidermal growth factor on MDA-MB-231 breast cancer cells. European Journal of Cancer 30: 1352-1359.
","pubmedId":"","doi":"10.1016/0959-8049(94)90186-4 "},{"reference":"Wee P, Wang Z. 2017. Epidermal Growth Factor Receptor Cell Proliferation Signaling Pathways. Cancers 9: 52.
","pubmedId":"","doi":"10.3390/cancers9050052"},{"reference":"Yang X, Zhong J, Zhang Q, Feng L, Zheng Z, Zhang J, Lu S. 2021. Advances and Insights of APC-Asef Inhibitors for Metastatic Colorectal Cancer Therapy. Frontiers in Molecular Biosciences 8: 10.3389/fmolb.2021.662579.
","pubmedId":"","doi":"10.3389/fmolb.2021.662579 "},{"reference":"Yin L, Duan JJ, Bian XW, Yu Sc. 2020. Triple-negative breast cancer molecular subtyping and treatment progress. Breast Cancer Research 22: 10.1186/s13058-020-01296-5.
","pubmedId":"","doi":"10.1186/s13058-020-01296-5 "}],"title":"Examining the Role of Asef and Epidermal Growth Factor on Cell Migration and Proliferation in Triple Negative Breast Cancer Epithelial Cells
","reviews":[],"curatorReviews":[]},{"id":"30c2b267-ed90-4048-8d7b-ff30bee4782e","decision":"publish","abstract":"Cell proliferation and migration are cellular processes essential to normal cell function; errors in these cellular processes impact the ability of a cell to maintain cellular homeostasis. The current study examines the role of Asef, a GEF for Rac1, in cell proliferation and migration in the presence or absence of EGF in a triple negative breast cancer epithelial cell line. Overexpression of Asef reduced stress fiber formation and increased cell migration and proliferation in MDA-MB-231 epithelial cells. These data indicate a role for Asef in the EGFR/Rac1 signaling cascade involved in regulating cellular behavior.
","acknowledgements":"The authors thank the SMC School of Science for their support of this project and the Cell Biology Education Consortium (CBEC) for feedback and comments on the manuscript.
","authors":[{"affiliations":["Saint Mary's College of California, Moraga, CA, United States"],"departments":["Biology Department"],"credit":["investigation","methodology","writing_originalDraft"],"email":"na21@stmarys-ca.edu","firstName":"Natalie","lastName":"Alaniz","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":"0009-0004-5090-7951"},{"affiliations":["Saint Mary's College of California, Moraga, CA, United States"],"departments":["Biology Department"],"credit":["conceptualization","formalAnalysis","methodology","project","supervision","writing_reviewEditing","investigation"],"email":"knk8@stmarys-ca.edu","firstName":"Khameeka N.","lastName":"Kitt","submittingAuthor":true,"correspondingAuthor":true,"equalContribution":false,"WBId":null,"orcid":"0009-0003-2381-877X"}],"awards":[],"conflictsOfInterest":"The authors declare that there are no conflicts of interest present.
","dataTable":{"url":null},"extendedData":[],"funding":"The authors thank the SMC School of Science Summer Endowment fund. Undergraduate student research participation and publication support were funded by the Cell Biology Education Consortium (CBEC): Path to Publication (Award ID #2316122) through the National Science Foundation (NSF).
","image":{"url":"https://portal.micropublication.org/uploads/a7e30098fc0811eef71cf03e5f47606a.png"},"imageCaption":"Human MDA-MB-231 breast cancer epithelial cells were transfected with Asef-HA (green) and allowed to express for 24 hours before treatment with 10 ng/ml of EGF for 24 hours. (A) In the absence of EGF, Asef and Rac1 (red) localize to the cytoplasm and membrane. In the presence of EGF for 24 hours, Rac1 colocalizes with Asef at the cell membrane (arrows). Scale bar: 40 µm. (B) Asef localizes to the cytoplasm and membrane ruffles (arrows) in cells with and without EGF treatment. In the absence of EGF, EGFR (red) localizes to the cell membrane and cytoplasm in Asef-HA expressing cells (arrowhead). Cells treated with EGF for 24 hours caused a minor shift in EGFR localization from the membrane to vesicle-like structures inside the cell (arrowhead). Scale bar: 40 µm. (C) Asef localizes to the cytoplasm and membrane ruffles (arrows) in cells with and without EGF treatment. Cortical Actin (red) appears unchanged in Asef-HA expressing cells (arrowhead). Cells expressing Asef-HA show reduced stress fibers extending across the cell (asterisks) compared to untransfected cells (arrows) in the presence or absence of EGF for 24 hours. Scale bar: 40 µm. (D) MDA-MB-231 cell monolayers overexpressing HA-only or Asef-HA were scratched and imaged over 24 hours in the presence or absence of EGF. The width of the wound was measured using ImageJ and plotted to examine wound width over time. Asef-HA expressing cells, regardless of EGF treatment, migrated faster than control cells; however, Asef-HA expressing cells without EGF treatment showed a significant increase in migration at 12 and 24 hour time points compared to HA only expressing cells. (**P < 0.005; n=3 measurements of each group). (E) MDA-MB-231 cell monolayers overexpressing HA-only or Asef-HA were serum-starved and treated with 10 ng/ml of EGF for 24 hours. Following EGF-treatment, MTT reagent was added, cells were solubilized with DMSO, and absorbance was read at 570 nm. Asef-HA expressing cells in the absence of EGF treatment showed a significant increase in cell proliferation compared to untransfected cells (*P < 0.05; n=3 measurements of each group). (F) Model of the Rho GTPase signaling pathway demonstrating how Asef could be recruited downstream of EGFR signaling to regulate Rac1 activation and stimulate cell proliferation and migration.
","imageTitle":"Asef-HA overexpression in MDA-MB-231 breast epithelial cancer cells causes changes in protein localization, cell migration, and cell proliferation
","methods":"Cell Culture
Triple negative ductal breast carcinoma epithelial cell line MDA-MB-231 (ATCC) were grown in Dulbecco's Modified Eagle Serum (DMEM) high glucose media (Thermo Fisher Scientific) with 10% fetal bovine serum and 1% penicillin-streptomycin-glutamine (Thermo Fisher Scientific). Cells were incubated under the following conditions: 5% CO2 and 37˚C.
Transfection
MDA-MB-321 cells were transfected with pcDNA 3.1-HA Asef (human) full length plasmid (provided by Tetsu Akiyama lab, University of Tokyo, (Kawasaki et al., 2000)) using Lipofectamine 2000 diluted in Opti-MEM (Thermo Fisher Scientific) following product recommendations. As a control for proliferation and scratch assays, cells were transfected with pcDNA 3.1-HA plasmid alone. Transfected cells were incubated for 24 hours in antibiotic-free media before being replated in various well formats for various assays.
Immunofluorescence
Transfected MDA-MB-231 cells (with or without Asef-HA) were placed on rat tail collagen-coated coverslips in serum-free media (DMEM with 1% penicillin-streptomycin-glutamine) and treated with or without 10 ng/mL of EGF for 24 hours. Cells were fixed using 4% paraformaldehyde 48 hours post-transfection. To localize HA, Rac1, and EGFR, cells were blocked and labeled with primary antibodies for HA (Roche), Rac1 (BD Biosciences) and EGFR (Thermo Fisher Scientific) followed by secondary antibodies conjugated to Alexa Fluor 488 and 594 (Thermo Fisher Scientific). Actin filaments and nuclei were labeled using Phalloidin 594 and Hoechst 33342 DNA stains, respectively (Thermo Fisher Scientific). Cells were imaged using the Leica STELLARIS Inverted Spectral Live Cell Confocal Microscope.
Scratch Assay (Wound Healing)
Transfected MDA-MB-231 cells (with or without Asef-HA) were placed on rat tail collagen-coated 12 well plates and allowed to form a monolayer in serum free media. Monolayers were scratched 24 hours later using a sterile 200 µL pipette tip, washed using phosphate-buffered saline (PBS) to remove debris, and incubated in serum-free media treated with or without 10 ng/mL of EGF for 24 hours. Images were captured at 0, 4, 8, 12 and 24 hours. The area between both sides of the scratch were quantified after each image time stamp to determine the wound closure distance using ImageJ software. Distance of wound closure for each group at each time point was measured in µm using ImageJ (pixels per micron was determined using scale bar) and plotted to display the width of the wound closure over time.
Proliferation (MTT) Assay
Transfected MDA-MB-231 cells (with or without Asef-HA) were placed in a 96-well tissue culture-treated plate in serum free media. Cells were treated with or without 10 ng/mL of EGF for 24 hours before adding 10 µL of a 5 mg/mL stock of MTT reagent per well and incubated for three hours. After three hours, the MTT reagent was removed and 100µL DMSO was added to each well to solubilize cells, and the optical density was measured at 570 nm (Tecan Plate Reader). Percent viability was calculated for each well and normalized against the control (HA-only).
Statistical Analysis
For the scratch assay, a multiple unpaired t-test analysis was performed to determine the significance at each time point. Three biological replicates (independent wells) were analyzed for each group. For the proliferation (MTT) assay, an unpaired t-test was used to determine significance between the treated groups compared to the control. Three biological replicates (independent wells) were analyzed for each group. All data was graphed using GraphPad Prism.
","reagents":"Plasmid | Description | Source |
pcDNA 3.1-HA- Asef | Full length Asef (human) | Tetsu Akiyama lab, University of Tokyo |
Cell Line | Description | Source |
MDA-MB-231 | Human Epithelial Breast Tissue isolated from Mammary Gland; Adenocarcinoma | ATCC Catalog #: HTB-26 |
Antibodies | Dilution | Source |
Phalloidin 594 | 1:400 | Thermo Fisher Scientific Catalog #: A12381 |
Alexa Goat anti Rabbit 488 | 1:800 | Thermo Fisher Scientific Catalog #: A-11008 |
Alexa Goat anti Mouse 594 | 1:800 | Thermo Fisher Scientific Catalog #: A-11005 |
HA (Rabbit) | 1:500 | Abcam Catalog #: ab9110 |
Rac1 (Mouse) | 1:200 | BD Biosciences Catalog #: 610651 |
EGFR (Mouse) | 1:200 | Thermo Fisher Scientific Catalog #: MA513269 |
Hoechst 33342 DNA stain | 1 µg/ml final | Thermo Fisher Scientific Catalog #: H3570 |
Breast cancer is not a single disease, but rather a group of diverse diseases with different subtypes. Breast cancers are organized into subtypes distinguished by the absence or presence of certain proteins on the cancer cells (Fragomeni et al., 2018). Triple negative breast cancers (TNBCs) are a subtype of breast cancer characterized by the absence of three receptors: estrogen receptor (ER), progesterone receptors (PR), and human epidermal growth factor 2 (HER2) (Yin et al., 2020). The TNBC subtype is both invasive and aggressive and occurs more frequently in younger women and African American women (Hines et al., 2011). Understanding the specific subtype of breast cancer is crucial for determining the most effective treatment options. The ductal carcinoma TNBC epithelial cell line, MDA-MD-231, is commonly used to study invasive breast cancer and metastasis due to its highly invasive and migratory nature (Amaro et al., 2016).
Cell adhesion, proliferation and migration are three critical cellular processes required for tissues to maintain homeostasis. When one or all of these cellular processes become unregulated, for example in cancer, this can lead to uncontrolled cell growth and metastasis (Hanahan & Weinberg, 2011). Various molecules have been identified that are involved in maintaining the balance of these cellular processes under normal conditions. Rho GTPases are a family of proteins that act as molecular switches that can be activated by guanine nucleotide exchange factors (GEFs) (Lawson & Ridley, 2017). The Rho GTPase Rac1 has been shown to regulate the actin cytoskeleton and cell proliferation by working downstream of the receptor tyrosine kinase, epidermal growth factor receptor (EGFR) and its ligand, epidermal growth factor (EGF), to regulate various kinase pathways (Dise et al., 2008; Itoh et al., 2008). Dimerization of EGFR leads to the phosphorylation of different proteins along these pathways involved in regulating gene expression and activation of proteins involved in promoting cell migration, adhesion, and proliferation (Wee & Wang, 2017). Adenomatous Polyposis Coli (APC)-stimulated guanine nucleotide-exchange factor, Asef, is a GEF that activates Rac1 to promote cell migration by reducing cell-cell adhesions and remodeling the actin cytoskeleton (Kawasaki et al., 2000, 2010). In colorectal cancer, APC can activate Asef, leading to enhanced migration and invasion (Mitin et al., 2007).
The MDA-MB-231 cell line serves as a valuable model for studying breast cancer proliferation and invasion due to the presence or absence of molecules involved in maintaining normal cell function. MDA-MB-231 cells express Rac1 and are responsive to EGF stimulation (Davidson et al., 1987; Veber et al., 1994). However, it is currently unknown how EGFR activation impacts the regulatory molecules, Asef and Rac1, along with cell migration and proliferation in MDA-MB-231 cells. The current study aims to examine the role of Asef in cell proliferation and migration in the presence of EGF in MDA-MB-231 cells. We hypothesize that due to the regulatory nature of Asef on Rac1, overexpression of Asef in the presence of EGFR signaling pathway will lead to changes in the protein localization and an increase in proliferation and cell migration due to activation of downstream signaling pathways.
Asef-HA (Kawasaki et al., 2000) was overexpressed in MDA-MB-231 cells using a lipid-based transfection approach and protein localization of Rac1, EGFR, and actin was observed in untreated and EGF-treated cells. Using immunofluorescence, Asef-HA overexpressing cells show Asef both in the cytosol and at the cell membrane co-localizing with endogenous EGFR and Rac1 protein in untreated cells and cells treated with EGF for 24 hours (Figure 1A-B). The increased protein localization for Asef and Rac1 at the membrane suggests a possible interaction of the proteins to control cell migration (Figure 1A). Kawasaki, et al. previously showed that Asef promotes the activation of the Rac1 protein, exchanging GDP for GTP, in endothelial cells (Kawasaki et al., 2010). Additional studies are required to determine if there is a direct interaction between the overexpressed Asef-HA protein and Rac1-GTP at the membrane.
EGFR protein appears to internalize into small vesicular structures after EGF stimulation (Figure 1B), suggesting activation of the receptor and downstream cellular pathways (Pennock & Wang, 2003). Due to the shift of EGFR from the membrane to internal vesicular structures in the presence of EGF and previous studies which have examined the impact of EGFR internalization on actin remodeling and cell migration (Pinilla-Macua et al., 2025), actin organization in Asef-HA expressing cells was examined. Actin organization provides a critical scaffold to cellular organization and structure and is represented by two different and interconnected cytoskeletal structures: cortical and stress fibers (Vallenius, 2013). Cortical actin forms a meshwork for actin filaments beneath the cell membrane and is involved in controlling cell shape by locally modifying cortical tension (Chalut & Paluch, 2016). Stress fibers are organized as bundles of actin filaments and play a role in cell migration, adhesion, and mechanosensing, particularly in the context of cancer. These structures are involved in cell migration and linked to cell stiffness, which can either promote or inhibit migration depending on the specific context and cell type (Fischer et al., 2021; Tavares et al., 2017). To determine the impact of Asef on actin filament organization, MDA-MB-231 cells were transfected with Asef-HA. Stress fibers appear to be reduced in Asef-HA expressing cells (asterisks), with or without EGF, as compared to cortical actin, which remains unchanged in both untreated and EGF-treated cells (Figure 1C). The loss of stress fiber formation in Asef-HA cells in comparison to untransfected cells suggests the cells have lost their stability and may be more susceptible to movement.
The lack of stress fiber formation in Asef-HA expressing cells suggests that the cells may be more migratory in nature. To determine if overexpressed Asef leads to an increase in cell migration, a wound healing assay was performed in MDA-MB-231 cells overexpressing Asef-HA. Data indicate that cells overexpressing Asef-HA with or without EGF for 24 hours migrate faster compared to HA only expressing cells (Figure 1D). Quantitative analysis shows a significant increase in proliferation at 12 and 24 hours between HA-only cells and Asef-HA without EGF stimulation in comparison to cells under EGF stimulation (Figure 1D). These data support the hypothesis that overexpression of Asef-HA in MDA-MB-231 cells activates pathways that lead to increased cell migration, by possibly impacting actin organization and turnover at the leading edges of cells (Tian et al., 2015). This is further validated by the reorganization and distribution of key molecules involved in migration, namely EGFR, Rac1, and actin filament organization (Figure 1A-C) and that Rac1 serves as a relay protein between EGFR and cell migration (Dise et al., 2008). The lack of significance between HA-only cells and Asef-HA with EGF treatment suggests that the EGF concentration was not sufficient to induce a response. Previous studies have shown that EGF at a 10 fold higher concentration appears to have more of an impact on MDA-MB-231 cell migration (Kozlova et al., 2016) than lower concentrations. Future studies will determine if higher EGF concentrations lead to significant changes in cell proliferation and migration over basal conditions in cells overexpressing Asef.
Internalization of EGFR via EGF binding, as observed in Figure 1B, may lead to activation of cyclins and/or other cell division regulators to promote an increase in cell proliferation. Furthermore, the EGFR pathway is frequently implicated in cancer development and progression, as it can drive uncontrolled cell growth and division (X. Song et al., 2020; Z. Song et al., 2016). To determine if the activation of EGFR leads to possible increase in MDA-MB-231 cell proliferation due to downstream pathways becoming activated, a MTT assay was performed on control and Asef-HA transfected cells. Data show a significant increase in proliferation in Asef-HA without EGF-stimulation compared to HA-only cells and there was no significant change in Asef-HA with 10 ng/ml EGF for 24 hours (Figure 1E). Similar to the wound healing assay, the lack of significance between control cells and Asef-HA with EGF treatment suggests that the EGF concentration may not be sufficient. These data support the hypothesis that overexpression of Asef-HA in MDA-MB-231 cells may activate pathways that lead to cell proliferation (Lo & Hung, 2006; Pennock & Wang, 2003). Additional studies are required to determine which pathways are responsible for the increased cell proliferation and the impact of EGFR signaling on cell proliferation.
Our findings show that Asef overexpression impacts actin filament remodeling and increases cell migration and proliferation in a metastatic breast cancer cell line. Mutated versions of APC and Asef have been implicated in colon cancer cell migration and invasion (Yang et al., 2021), and we extend the role of Asef into a highly invasive and aggressive epithelial breast cancer subtype. Asef is primarily known for its role in APC-Wnt signaling (Kawasaki et al., 2000); however, Asef and Rac1 were shown to be involved in the EGFR pathway and may be a possible molecular target to consider in understanding the aggressiveness of triple negative breast cancer (Itoh et al., 2008). Future work aims to explore the connection of Asef to signaling proteins involved in controlling cell proliferation and how Asef regulates Rac1 and actin organization in the presence of EGF to control cell motility.
","references":[{"reference":"Amaro A, Angelini G, Mirisola V, Esposito AI, Reverberi D, Matis S, et al., Pfeffer. 2016. A highly invasive subpopulation of MDA-MB-231 breast cancer cells shows accelerated growth, differential chemoresistance, features of apocrine tumors and reduced tumorigenicityin vivo. Oncotarget 7: 68803-68820.
","pubmedId":"","doi":"10.18632/oncotarget.11931 "},{"reference":"Chalut KJ, Paluch EK. 2016. The Actin Cortex: A Bridge between Cell Shape and Function. Developmental Cell 38: 571-573.
","pubmedId":"","doi":"10.1016/j.devcel.2016.09.011 "},{"reference":"Davidson NE, Gelmann EP, Lippman ME, Dickson RB. 1987. Epidermal Growth Factor Receptor Gene Expression in Estrogen Receptor-Positive and Negative Human Breast Cancer Cell Lines. Molecular Endocrinology 1: 216-223.
","pubmedId":"","doi":"10.1210/mend-1-3-216 "},{"reference":"Dise RS, Frey MR, Whitehead RH, Polk DB. 2008. Epidermal growth factor stimulates Rac activation through Src and phosphatidylinositol 3-kinase to promote colonic epithelial cell migration. American Journal of Physiology-Gastrointestinal and Liver Physiology 294: G276-G285.
","pubmedId":"","doi":"10.1152/ajpgi.00340.2007 "},{"reference":"Fischer RS, Sun X, Baird MA, Hourwitz MJ, Seo BR, Pasapera AM, et al., Waterman. 2021. Contractility, focal adhesion orientation, and stress fiber orientation drive cancer cell polarity and migration along wavy ECM substrates. Proceedings of the National Academy of Sciences 118: 10.1073/pnas.2021135118.
","pubmedId":"","doi":"10.1073/pnas.2021135118 "},{"reference":"Fragomeni SM, Sciallis A, Jeruss JS. 2018. Molecular Subtypes and Local-Regional Control of Breast Cancer. Surgical Oncology Clinics of North America 27: 95-120.
","pubmedId":"","doi":"10.1016/j.soc.2017.08.005 "},{"reference":"Hanahan D, Weinberg RA. 2011. Hallmarks of Cancer: The Next Generation. Cell 144: 646-674.
","pubmedId":"","doi":"10.1016/j.cell.2011.02.013 "},{"reference":"Hines LM, Risendal B, Byers T, Mengshol S, Lowery J, Singh M. 2011. Ethnic Disparities in Breast Tumor Phenotypic Subtypes in Hispanic and Non-Hispanic White Women. Journal of Women's Health 20: 1543-1550.
","pubmedId":"","doi":"10.1089/jwh.2010.2558"},{"reference":"Itoh RE, Kiyokawa E, Aoki K, Nishioka T, Akiyama T, Matsuda M. 2008. Phosphorylation and activation of the Rac1 and Cdc42 GEF Asef in A431 cells stimulated by EGF. Journal of Cell Science 121: 2635-2642.
","pubmedId":"","doi":"10.1242/jcs.028647 "},{"reference":"Kawasaki Y, Jigami T, Furukawa S, Sagara M, Echizen K, Shibata Y, Sato R, Akiyama T. 2010. The Adenomatous Polyposis Coli-associated Guanine Nucleotide Exchange Factor Asef Is Involved in Angiogenesis. Journal of Biological Chemistry 285: 1199-1207.
","pubmedId":"","doi":"10.1074/jbc.M109.040691 "},{"reference":"Kawasaki Y, Senda T, Ishidate T, Koyama R, Morishita T, Iwayama Y, Higuchi O, Akiyama T. 2000. Asef, a Link Between the Tumor Suppressor APC and G-Protein Signaling. Science 289: 1194-1197.
","pubmedId":"","doi":"10.1126/science.289.5482.1194"},{"reference":"Kozlova N, Samoylenko A, Drobot L, Kietzmann T. 2015. Urokinase is a negative modulator of Egf‐dependent proliferation and motility in the two breast cancer cell lines MCF‐7 and MDA‐MB‐231. Molecular Carcinogenesis 55: 170-181.
","pubmedId":"","doi":"10.1002/mc.22267 "},{"reference":"Lawson CD, Ridley AJ. 2017. Rho GTPase signaling complexes in cell migration and invasion. Journal of Cell Biology 217: 447-457.
","pubmedId":"","doi":"10.1083/jcb.201612069 "},{"reference":"Lo HW, Hung MC. 2006. Nuclear EGFR signalling network in cancers: linking EGFR pathway to cell cycle progression, nitric oxide pathway and patient survival. British Journal of Cancer 94: 184-188.
","pubmedId":"","doi":"10.1038/sj.bjc.6602941"},{"reference":"Mitin N, Betts L, Yohe ME, Der CJ, Sondek J, Rossman KL. 2007. Release of autoinhibition of ASEF by APC leads to CDC42 activation and tumor suppression. Nature Structural & Molecular Biology 14: 814-823.
","pubmedId":"","doi":"10.1038/nsmb1290 "},{"reference":"Pennock S, Wang Z. 2003. Stimulation of Cell Proliferation by Endosomal Epidermal Growth Factor Receptor As Revealed through Two Distinct Phases of Signaling. Molecular and Cellular Biology 23: 5803-5815.
","pubmedId":"","doi":"10.1128/MCB.23.16.5803-5815.2003"},{"reference":"Pinilla-Macua I, Surve S, Sorkin A. 2025. Cell migration signaling through the EGFR-VAV2-Rac1 pathway is sustained in endosomes. Journal of Cell Science 138: 10.1242/jcs.263541.
","pubmedId":"","doi":"10.1242/jcs.263541 "},{"reference":"Song X, Liu Z, Yu Z. 2020. <p>EGFR Promotes the Development of Triple Negative Breast Cancer Through JAK/STAT3 Signaling</p>. Cancer Management and Research Volume 12: 703-717.
","pubmedId":"","doi":"10.2147/CMAR.S225376 "},{"reference":"Song Z, Fusco J, Zimmerman R, Fischbach S, Chen C, Ricks DM, et al., Gittes. 2016. Epidermal Growth Factor Receptor Signaling Regulates β Cell Proliferation in Adult Mice. Journal of Biological Chemistry 291: 22630-22637.
","pubmedId":"","doi":"10.1074/jbc.M116.747840 "},{"reference":"Tavares S, Vieira AF, Taubenberger AV, Araújo M, Martins NP, Brás-Pereira C, et al., Janody. 2017. Actin stress fiber organization promotes cell stiffening and proliferation of pre-invasive breast cancer cells. Nature Communications 8: 10.1038/ncomms15237.
","pubmedId":"","doi":"10.1038/ncomms15237"},{"reference":"Tian X, Tian Y, Gawlak G, Meng F, Kawasaki Y, Akiyama T, Birukova AA. 2015. Asef controls vascular endothelial permeability and barrier recovery in the lung. Molecular Biology of the Cell 26: 636-650.
","pubmedId":"","doi":"10.1091/mbc.E14-02-0725 "},{"reference":"Vallenius T. 2013. Actin stress fibre subtypes in mesenchymal-migrating cells. Open Biology 3: 130001.
","pubmedId":"","doi":"10.1098/rsob.130001 "},{"reference":"Veber N, Prévost G, Planchon P, Starzec A. 1994. Evidence for a growth effect of epidermal growth factor on MDA-MB-231 breast cancer cells. European Journal of Cancer 30: 1352-1359.
","pubmedId":"","doi":"10.1016/0959-8049(94)90186-4 "},{"reference":"Wee P, Wang Z. 2017. Epidermal Growth Factor Receptor Cell Proliferation Signaling Pathways. Cancers 9: 52.
","pubmedId":"","doi":"10.3390/cancers9050052"},{"reference":"Yang X, Zhong J, Zhang Q, Feng L, Zheng Z, Zhang J, Lu S. 2021. Advances and Insights of APC-Asef Inhibitors for Metastatic Colorectal Cancer Therapy. Frontiers in Molecular Biosciences 8: 10.3389/fmolb.2021.662579.
","pubmedId":"","doi":"10.3389/fmolb.2021.662579 "},{"reference":"Yin L, Duan JJ, Bian XW, Yu Sc. 2020. Triple-negative breast cancer molecular subtyping and treatment progress. Breast Cancer Research 22: 10.1186/s13058-020-01296-5.
","pubmedId":"","doi":"10.1186/s13058-020-01296-5 "}],"title":"Examining the Role of Asef and Epidermal Growth Factor on Cell Migration and Proliferation in Triple Negative Breast Cancer Epithelial Cells
","reviews":[],"curatorReviews":[]}]}},"species":{"species":[{"value":"acer saccharum","label":"Acer saccharum","imageSrc":"","imageAlt":"","mod":"TreeGenes","modLink":"https://treegenesdb.org","linkVariable":""},{"value":"achillea millefolium","label":"Achillea millefolium","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"acinetobacter baylyi","label":"Acinetobacter baylyi","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"actinobacteria bacterium","label":"Actinobacteria bacterium","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"adelges tsugae","label":"Adelges tsugae","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"adenocaulon chilense","label":"Adenocaulon chilense","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"aedes japonicus","label":"Aedes japonicus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"aegorhinus vitulus","label":"Aegorhinus vitulus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"alaimidae","label":"Alaimidae","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"allobates femoralis","label":"Allobates femoralis","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"alnus glutinosa","label":"Alnus glutinosa","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"alosa aestivalis","label":"Alosa aestivalis","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"alosa pseudoharengus","label":"Alosa pseudoharengus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"alternaria alternata","label":"Alternaria alternata","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"amynthas agrestis","label":"Amynthas Agrestis","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"ancylostoma caninum","label":"Ancylostoma caninum","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"ancylostoma ceylanicum","label":"Ancylostoma ceylanicum","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"anemone multifida","label":"Anemone multifida","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"anguilla rostrata","label":"Anguilla rostrata","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"anisakis simplex","label":"Anisakis simplex","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"anomala albopilosa","label":"Anomala albopilosa","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"anthomyiidae sp","label":"Anthomyiidae sp","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"anthomyiidae sp","label":"Anthomyiidae sp","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"arabidopsis","label":"Arabidopsis","imageSrc":"arabidopsis.png","imageAlt":"Arabidopsis graphic by Zoe Zorn CC BY 4.0","mod":"TAIR","modLink":"https://arabidopsis.org","linkVariable":""},{"value":"architeuthis dux","label":"Architeuthis dux","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"arion vulgaris","label":"Arion vulgaris","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"armeria","label":"Armeria","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"artemia","label":"Artemia","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"arthrobacter sp.","label":"Arthrobacter sp.","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"ascaridia","label":"Ascaridia","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"ascaridia galli","label":"Ascaridia galli","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"asparagopsis taxiformis","label":"Asparagopsis taxiformis","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"astatotilapia burtoni","label":"Astatotilapia burtoni","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"avena sativa","label":"Avena sativa","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"aves","label":"Aves","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"bacillus","label":"Bacillus (firmicutes)","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"bacillus cereus","label":"Bacillus cereus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"bacillus mycoides","label":"Bacillus mycoides","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"bacillus subtilis","label":"Bacillus subtilis","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"bacillus thuringiensis","label":"Bacillus thuringiensis","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"bacillus toyonensis","label":"Bacillus toyonensis","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"bacillus wiedmannii","label":"Bacillus wiedmannii","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"bacteria","label":"Bacteria","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"bacteriophage","label":"Bacteriophage","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"bactrocera","label":"Bactrocera sp.","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"batrachospermum gelatinosum","label":"Batrachospermum gelatinosum","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"betula lenta","label":"Betula lenta","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"betula nigra","label":"Betula nigra","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"bombus dahlbohmii","label":"Bombus dahlbohmii","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"bombus terrestris","label":"Bombus terrestris","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"bombyx mori","label":"Bombyx mori","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"bos taurus","label":"Bos Taurus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"brachygobius doriae","label":"Brachygobius doriae","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"brassica oleracea","label":"Brassica oleracea","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"brassica rapa","label":"Brassica rapa","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"brugia malayi","label":"Brugia malayi","imageSrc":"","imageAlt":"","mod":"WormBase","modLink":"www.wormbase.org","linkVariable":""},{"value":"burkholderia thailandensis","label":"Burkholderia thailandensis","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"buttiauxella","label":"Buttiauxella","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"caenorhabditis brenneri","label":"Caenorhabditis brenneri","imageSrc":"","imageAlt":"","mod":"WormBase","modLink":"www.wormbase.org","linkVariable":""},{"value":"caenorhabditis briggsae","label":"Caenorhabditis briggsae","imageSrc":"","imageAlt":"","mod":"WormBase","modLink":"www.wormbase.org","linkVariable":""},{"value":"c. elegans","label":"Caenorhabditis elegans","imageSrc":"c-elegans.jpg","imageAlt":"C. elegans graphic by Zoe Zorn CC BY 4.0","mod":"WormBase","modLink":"https://wormbase.org","linkVariable":""},{"value":"caenorhabditis inopinata","label":"Caenorhabditis inopinata","imageSrc":"","imageAlt":"","mod":"WormBase","modLink":"www.wormbase.org","linkVariable":""},{"value":"caenorhabditis japonica","label":"Caenorhabditis japonica","imageSrc":"","imageAlt":"","mod":"WormBase","modLink":"www.wormbase.org","linkVariable":""},{"value":"caenorhabditis nigoni","label":"Caenorhabditis nigoni","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"caenorhabditis remanei","label":"Caenorhabditis remanei","imageSrc":"","imageAlt":"","mod":"WormBase","modLink":"www.wormbase.org","linkVariable":""},{"value":"caenorhabditis tropicalis","label":"Caenorhabditis tropicalis","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"calidifontibacillus","label":"Calidifontibacillus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"calidifontibacillus erzuremensis","label":"Calidifontibacillus erzuremensis","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"calliphora sp","label":"Calliphora sp","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"caltha sagittata","label":"Caltha sagittata","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"cambarus latimanus","label":"Cambarus latimanus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"candida albicans","label":"Candida albicans","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"canis familiaris","label":"Canis familiaris","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"cannabis sativa","label":"Cannabis sativa","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"caretta caretta","label":"Caretta caretta","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"cassiopea xamachana","label":"Cassiopea xamachana","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"caulobacter vibrioides","label":"Caulobacter vibrioides","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"cephalopods","label":"Cephalopoda","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"cerastium arvense","label":"Cerastium arvense","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"ceriodaphnia","label":"Ceriodaphnia","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"ceroglossus suturalis","label":"Ceroglossus suturalis","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"chaetoceros","label":"Chaetoceros","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"chamaecrista fasciculata","label":"Chamaecrista fasciculata","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"chilicola chalcidiformis","label":"Chilicola chalcidiformis","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"chitinimonas","label":"Chitinimonas","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"chlamydomonas reinhardtii","label":"Chlamydomonas reinhardtii","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"chromobacterium","label":"Chromobacterium","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"chrysemys picta","label":"Chrysemys picta","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"chrysoperla rufilabris","label":"Chrysoperla rufilabris","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"citrus","label":"Citrus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"clavibacter sp.","label":"Clavibacter sp.","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"colinus virginianus","label":"Colinus virginianus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"crassostrea virginica","label":"Crassostrea virginica","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"crithidia fasciculata","label":"Crithidia fasciculata","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"cutibacterium acnes","label":"Cutibacterium acnes","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"cyanobacteria","label":"Cyanobacteria","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"daphnia","label":"Daphnia","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"daphnia pulex","label":"Daphnia pulex","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"diabrotica virgifera","label":"Diabrotica virgifera","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"diabrotica virgifera virgifera virus 1","label":"Diabrotica virgifera virgifera virus 1","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"d. discoideum","label":"Dictyostelium discoideum","imageSrc":"dicty.png","imageAlt":"D. discoideum","mod":"dictyBase","modLink":"http://dictybase.org","linkVariable":""},{"value":"diptera","label":"Diptera","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"dotocryptus bellicosus","label":"Dotocryptus bellicosus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"drechmeria coniospora","label":"Drechmeria coniospora","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"drosophila","label":"Drosophila","imageSrc":"drosophila.png","imageAlt":"Drosophila graphic by Zoe Zorn CC BY 4.0","mod":"FlyBase","modLink":"https://flybase.org/doi/","linkVariable":"doi"},{"value":"dryopteris campyloptera","label":"Dryopteris campyloptera","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"dryopteris expansa","label":"Dryopteris expansa","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"dryopteris intermedia","label":"Dryopteris intermedia","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"dugesia dorotocephala","label":"Dugesia dorotocephala","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"elasmobranchii","label":"Elasmobranchii","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"embryophyta","label":"Embryophyta","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"enoploteuthis chunii","label":"Enoploteuthis chunii","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"enterobacter aerogenes","label":"Enterobacter aerogenes","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"enterococcus raffinosus","label":"Enterococcus raffinosus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"epichloë coenophiala","label":"Epichloë coenophiala","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"equus caballus","label":"Equus caballus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"erigeron sp","label":"Erigeron sp","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"eristalis","label":"Eristalis","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"eruca vesicaria","label":"Eruca vesicaria","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"erwinia carotovora","label":"Erwinia carotovora","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"erythronium americanum","label":"Erythronium americanum","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"escherichia coli","label":"Escherichia coli","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"eukaryota","label":"Eukaryotes","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"felis catus","label":"Felis catus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"francisella novicida","label":"Francisella novicida","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"francisella tularensis","label":"Francisella tularensis","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"fraxinus americana","label":"Fraxinus americana","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"fucus distichus","label":"Fucus distichus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"fungi","label":"Fungi","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"gasteropelecus sp.","label":"Gasteropelecus sp.","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"geranium sp","label":"Geranium sp","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"girardia","label":"Girardia","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"glaucomys volans","label":"Glaucomys volans","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"glycine max","label":"Glycine max","imageSrc":"","imageAlt":"","mod":"Soybase","modLink":"https://soybase.org","linkVariable":""},{"value":"glyptemys insculpta","label":"Glyptemys insculpta","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"gossypium hirsutum","label":"Gossypium hirsutum","imageSrc":"","imageAlt":"","mod":"CottonGen","modLink":"https://www.cottongen.org/","linkVariable":""},{"value":"gromphadorhina portentosa","label":"Gromphadorhina portentosa","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"gryllodes sigillatus","label":"Gryllodes sigillatus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"haliotis rufescens","label":"Haliotis rufescens","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"hepacivirus hominis","label":"Hepatitis C Virus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"herpes simplex virus type 1","label":"Herpes simplex virus type 1","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"human","label":"Human","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"human coronavirus oc43","label":"Human coronavirus OC43","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"hydra vulgaris","label":"Hydra vulgaris","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"hydropsyche sp","label":"Hydropsyche sp","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"hymenoptera","label":"Hymenoptera","imageSrc":"","imageAlt":"","mod":"Hymenoptera Genome Database","modLink":"https://hymenoptera.elsiklab.missouri.edu/","linkVariable":""},{"value":"hypochaeris radicata","label":"Hypochaeris radicata","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"hypodynerus vespiformis","label":"Hypodynerus vespiformis","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"iflaviridae","label":"Iflaviridae","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"iflavuris","label":"Iflavirus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"ipomoea hederacea","label":"Ipomoea hederacea","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"ischnomera","label":"Ischnomera","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"ischnomera ruficollis","label":"Ischnomera ruficollis","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"julidochromis marlieri","label":"Julidochromis marlieri","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"juniperus virginiana","label":"Juniperus virginiana","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"kluyveromyces marxianus","label":"Kluyveromyces marxianus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"l. casei","label":"L. casei","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"lacticaseibacillus casei","label":"Lacticaseibacillus casei","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"larentiinae sp","label":"Larentiinae sp","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"laurus nobilis","label":"Laurus nobilis","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"lepidoptera","label":"Lepidoptera","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"leucanthemum vulgare","label":"Leucanthemum vulgare","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"linepithema humile","label":"Linepithema humile","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"liometopum occidentale","label":"Liometopum occidentale","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"lolium arundinaceum","label":"Lolium arundinaceum","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"lumbriculus variegatus","label":"Lumbriculus variegatus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"lumbricus terrestris","label":"Lumbricus terrestris","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"lupinus polyphyllus","label":"Lupinus polyphyllus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"lycorma delicatula","label":"Lycorma delicatula","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"lynx rufus","label":"Lynx rufus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"magnaporthe oryzae","label":"Magnaporthe oryzae","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"mammalia","label":"Mammalia","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"manihot esculenta","label":"Manihot esculenta","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"medicago lupulina","label":"Medicago lupulina","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"meloidogyne","label":"Meloidogyne","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"mimus polyglottos","label":"Mimus polyglottos","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"bryophyta","label":"Mosses","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"mouse","label":"Mouse","imageSrc":"","imageAlt":"","mod":"MGI","modLink":"https://informatics.jax.org","linkVariable":""},{"value":"m. minutoides","label":"Mus minutoides","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"mycobacterium smegmatis","label":"Mycobacterium smegmatis","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"nakaseomyces glabratus","label":"Nakaseomyces glabratus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"nauphoeta cinerea","label":"Nauphoeta cinerea","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"neurospora","label":"Neurospora","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"n. benthamiana","label":"Nicotiana benthamiana","imageSrc":"","imageAlt":"","mod":"Solgenomics Network","modLink":"https://solgenomics.net/organism/Nicotiana_benthamiana/genome","linkVariable":""},{"value":"nicotiana tabacum","label":"Nicotiana tabacum","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"noctuidae","label":"Noctuidae","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"noctuidae sp","label":"Noctuidae sp","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"nothobranchius furzeri","label":"Nothobranchius furzeri","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"onchocerca volvulus","label":"Onchocerca volvulus","imageSrc":"","imageAlt":"","mod":"WormBase","modLink":"www.wormbase.org","linkVariable":""},{"value":"orconectes virilis","label":"Orconectes virilis","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"ormia ochracea","label":"Ormia ochracea","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"o. sativa","label":"Oryza sativa","imageSrc":"","imageAlt":"","mod":"Gramene","modLink":"https://www.gramene.org/","linkVariable":""},{"value":"other","label":"Other","imageSrc":"","imageAlt":"","mod":null,"modLink":null,"linkVariable":null},{"value":"oxalis enneaphylla","label":"Oxalis enneaphylla","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"paenarthrobacter nicotinovorans","label":"Paenarthrobacter nicotinovorans","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"paenarthrobacter nicotinovorans","label":"Paenarthrobacter nicotinovorans","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"pantoea","label":"Pantoea","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"pantoea agglomerans","label":"Pantoea agglomerans","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"papaver sp","label":"Papaver sp","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"paramecium bursaria","label":"Paramecium bursaria","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"partitiviridae","label":"Partitiviridae","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"pelodiscus sinensis","label":"Pelodiscus sinensis","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"perezia recurvata","label":"Perezia recurvata","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"petromyzon marinus","label":"Petromyzon marinus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"photinus pyralis","label":"Photinus pyralis","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"photinus pyralis associated partiti-like virus","label":"Photinus pyralis associated partiti-like virus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"photinus pyralis iflavirus 1","label":"Photinus pyralis iflavirus 1","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"physcomitrium patens","label":"Physcomitrium patens","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"pinus strobus","label":"Pinus strobus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"pinus taeda","label":"Pinus taeda","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"platycheirus","label":"Platycheirus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"plectus sambesii","label":"Plectus sambesii","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"pogonomyrmex occidentalis","label":"Pogonomyrmex occidentalis","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"poncirus trifoliata","label":"Poncirus trifoliata","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"populus deltoides","label":"Populus deltoides","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"potato virus y","label":"Potato virus Y","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"primula magellanica","label":"Primula magellanica","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"pristionchus pacificus","label":"Pristionchus pacificus","imageSrc":"","imageAlt":"","mod":"WormBase","modLink":"www.wormbase.org","linkVariable":""},{"value":"prunus persica","label":"Prunus persica","imageSrc":"","imageAlt":"","mod":"Genome Database for Rosaceae","modLink":"https://www.rosaceae.org/","linkVariable":""},{"value":"psalmopoeus iriminia","label":"Psalmopoeus iriminia","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"pseudanabaena sp.","label":"Pseudanabaena sp.","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"pseudomonas","label":"Pseudomonas","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"pseudomonas aeruginosa","label":"Pseudomonas aeruginosa","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"pseudomonas glycinae","label":"Pseudomonas glycinae","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"pseudomonas putida","label":"Pseudomonas putida","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"pseudomonas syringae","label":"Pseudomonas syringae","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"pterophyllum scalare","label":"Pterophyllum scalare","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"python regius","label":"Python regius","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"quercus macrocarpa","label":"Quercus macrocarpa","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"ralstonia solanacearum","label":"Ralstonia solanacearum","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"ranitomeya imitator","label":"Ranitomeya imitator","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"ranunculus peduncularis","label":"Ranunculus peduncularis","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"rat","label":"Rat","imageSrc":"","imageAlt":"","mod":"RGD","modLink":"https://rgd.mcw.edu","linkVariable":""},{"value":"rheinheimera","label":"Rheinheimera","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"ribes rubrum","label":"Ribes rubrum","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"sars-cov-2","label":"SARS-CoV-2","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"s. cerevisiae","label":"Saccharomyces cerevisiae","imageSrc":"yeast.png","imageAlt":"Yeast graphic by Zoe Zorn CC BY 4.0","mod":"SGD","modLink":"https://yeastgenome.org","linkVariable":""},{"value":"saccharomyces paradoxus","label":"Saccharomyces paradoxus ","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"s. uvarum","label":"Saccharomyces uvarum","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"schistosoma","label":"Schistosoma","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"schizosaccharomyces japonicus","label":"Schizosaccharomyces japonicus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"s. pombe","label":"Schizosaccharomyces pombe","imageSrc":"pombe.png","imageAlt":"Pombe graphic by Zoe Zorn © Caltech","mod":"PomBase","modLink":"https://www.pombase.org/reference/PMID:","linkVariable":"pmId"},{"value":"schmidtea mediterranea","label":"Schmidtea mediterranea","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"senecio sp","label":"Senecio sp","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"simocephalus","label":"Simocephalus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"siraitia grosvenorii","label":"Siraitia grosvenorii","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"solanum lycopersicum","label":"Solanum lycopersicum","imageSrc":"","imageAlt":"","mod":"Solgenomics Network","modLink":"https://solgenomics.net/organism/1/view/","linkVariable":""},{"value":"sorghum","label":"Sorghum","imageSrc":"","imageAlt":"","mod":"SorghumBase","modLink":"https://www.sorghumbase.org","linkVariable":""},{"value":"spiroplasma eriocheiris","label":"Spiroplasma eriocheiris","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"staphylococcus aureus","label":"Staphylococcus aureus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"staphylococcus epidermidis","label":"Staphylococcus epidermidis","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"steinernema carpocapsae","label":"Steinernema carpocapsae","imageSrc":"","imageAlt":"","mod":"WormBase","modLink":"https://wormbase.org","linkVariable":""},{"value":"steinernema hermaphroditum","label":"Steinernema hermaphroditum","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"stenotrophomonas geniculata","label":"Stenotrophomonas geniculata","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"streptococcus gordonii ","label":"Streptococcus gordonii ","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"streptococcus mutans","label":"Streptococcus mutans","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":" streptococcus pneumoniae","label":"Streptococcus pneumoniae","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"s. purpuratus","label":"Strongylocentrotus purpuratus","imageSrc":"","imageAlt":"","mod":"Echinobase","modLink":"https://www.echinobase.org","linkVariable":""},{"value":"strongyloides ratti","label":"Strongyloides ratti","imageSrc":"","imageAlt":"","mod":"WormBase","modLink":"www.wormbase.org","linkVariable":""},{"value":"sulfolobus","label":"Sulfolobus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"symphoricarpos albus","label":"Symphoricarpos albus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"syncirsodes","label":"Syncirsodes","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"synechococcus elongatus","label":"Synechococcus elongatus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"syrphidae","label":"Syrphidae","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"tarantobelus jeffdanielsi","label":"Tarantobelus jeffdanielsi","imageSrc":"","imageAlt":"","mod":"WormBase","modLink":"www.wormbase.org","linkVariable":""},{"value":"taraxacum officinale","label":"Taraxacum officinale","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"tatochila theodice","label":"Tatochila theodice","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"tetrahymena","label":"Tetrahymena","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"tetramorium immigrans","label":"Tetramorium immigrans","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"tomato brown rugose fruit virus","label":"ToBRFV","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"trachemys scripta","label":"Trachemys scripta","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"tribolium castaneum","label":"Tribolium castaneum","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"trichoptera","label":"Trichoptera","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"trichuris muris","label":"Trichuris muris","imageSrc":"","imageAlt":"","mod":"WormBase","modLink":"www.wormbase.org","linkVariable":""},{"value":"trifolium repens","label":"Trifolium repens","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"trypoxylus dichotomus","label":"Trypoxylus dichotomus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"tsuga canadensis","label":"Tsuga canadensis","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"ulva expansa","label":"Ulva expansa","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"universal","label":"Universal","imageSrc":"","imageAlt":"","mod":null,"modLink":null,"linkVariable":null},{"value":"vargula hilgendorfii","label":"Vargula hilgendorfii","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"vespula vulgaris","label":"Vespula vulgaris","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"virus","label":"Virus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"watasenia scintillans","label":"Watasenia scintillans","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"wolbachia pipientis","label":"Wolbachia pipientis","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"xenopus","label":"Xenopus","imageSrc":"xenopus.png","imageAlt":"Xenopus graphic by Zoe Zorn CC BY 4.0","mod":"XenBase","modLink":"https://xenbase.org","linkVariable":""},{"value":"xenorhabdus griffiniae","label":"Xenorhabdus griffiniae","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"yramea cytheris","label":"Yramea cytheris","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"zaprionus indianus","label":"Zaprionus indianus","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"zea mays","label":"Zea mays","imageSrc":"","imageAlt":"","mod":"MaizeGDB","modLink":"https://www.maizegdb.org","linkVariable":""},{"value":"zebrafish","label":"Zebrafish","imageSrc":"zebrafish.png","imageAlt":"Zebrafish graphic by Zoe Zorn CC BY 4.0","mod":"ZFIN","modLink":"https://zfin.org","linkVariable":""}]}},"pageContext":{"id":"4400f90e-3087-40e8-8a7c-2d22ce471dfc","citedBy":[],"parsedCsv":{"csvHeader":[],"csvData":[]}}}, "staticQueryHashes": ["2114697108"]}