FAM83A confers EGFR-TKI resistance in breast cancer cells and in mice
Sun-Young Lee, … , Ren Xu, Mina J. Bissell
Sun-Young Lee, … , Ren Xu, Mina J. Bissell
Published August 13, 2012
Citation Information: J Clin Invest. 2012;122(9):3211-3220. https://doi.org/10.1172/JCI60498.
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Research Article

FAM83A confers EGFR-TKI resistance in breast cancer cells and in mice

Abstract

Breast cancers commonly become resistant to EGFR–tyrosine kinase inhibitors (EGFR-TKIs); however, the mechanisms of this resistance remain largely unknown. We hypothesized that resistance may originate, at least in part, from molecular alterations that activate signaling downstream of EGFR. Using a screen to measure reversion of malignant cells into phenotypically nonmalignant cells in 3D gels, we identified FAM83A as a candidate cancer-associated gene capable of conferring resistance to EGFR-TKIs. FAM83A overexpression in cancer cells increased proliferation and invasion and imparted EGFR-TKI resistance both in cultured cells and in animals. Tumor cells that survived EGFR-TKI treatment in vivo had upregulated FAM83A levels. Additionally, FAM83A overexpression dramatically increased the number and size of transformed foci in cultured cells and anchorage-independent growth in soft agar. Conversely, FAM83A depletion in cancer cells caused reversion of the malignant phenotype, delayed tumor growth in mice, and rendered cells more sensitive to EGFR-TKI. Analyses of published clinical data revealed a correlation between high FAM83A expression and breast cancer patients’ poor prognosis. We found that FAM83A interacted with and caused phosphorylation of c-RAF and PI3K p85, upstream of MAPK and downstream of EGFR. These data provide an additional mechanism by which tumor cells can become EGFR-TKI resistant.

Authors

Sun-Young Lee, Roland Meier, Saori Furuta, Marc E. Lenburg, Paraic A. Kenny, Ren Xu, Mina J. Bissell

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Figure 2

FAM83A levels positively determine cell growth and tumorigenic potential.

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FAM83A levels positively determine cell growth and tumorigenic potential...
(A) Generation of a T4-2 cell line stably depleted of FAM83A by shRNA (83Ash). (B) FAM83A shRNA expression reverted T4-2 cells to an acinar-like phenotype in 3D lrECM cultures. α6 integrin (red) was used to indicate basal polarity; blue, DAPI. (C) Colony size. Luciferase shRNA was used as control. (D) Number of T4-2 cells expressing either FAM83A shRNA or FAM83A-overexpressing construct versus control (vector) invaded through lrECM-coated Transwell filters after 48 hours (n = 4). (E) Generation of an MDA-MB468 cell line stably depleted of FAM83A by shRNA. (F) Control versus FAM83A-depleted MDA-MB468 cells grown in 3D lrECM cultures for 5 days. Cells were stained with ethidium bromide to indicate apoptotic cells (blue, Hoechst 33258). (G) Number of viable cells after growth in 3D lrECM for 5 days (n = 4). (H) Number of cells invaded through lrECM-coated Transwell filters after 24 hours (n = 3). (I) Growth of control versus FAM83A-depleted MDA-MB468 cells, measured by MTT assay for 9 days (n = 12). P < 0.05, 2-tailed ANOVA. (J) Clonogenic potential of control versus FAM83A-depleted MDA-MB468 cells measured by colony formation assay for 10 days. Shown are representative images of colonies formed. (K) Number of colonies formed (n = 6). (L) Tumorigenic foci formed by 3T3 fibroblasts overexpressing FAM83A through loss of contact inhibition of growth. Representative images of 3T3 cells transduced by control and FAM83A-overexpressing lentivirus and cultured for 2 weeks. (M) Quantification of 3T3 foci shown in L (n = 4). (N) Representative images of T4-2 cells in duplicates in 2 rows transduced by empty vector, FAM83A-overexpressing, or FAM83A shRNA lentivirus and cultured on soft agar for 6 weeks. (O) Number of colonies formed on soft agar (n = 6). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, Student’s t test. Scale bars: 50 μm.