FAM83D promotes cell proliferation and motility by downregulating tumor suppressor gene FBXW7

Abstract

Amplification of chromosome 20q is frequently found in various types of human cancers, including breast cancer. The list of candidate oncogenes in 20q has expanded over the past decade. Here, we investigate whether FAM83D (family with sequence similarity 83, member D) on chromosome 20q plays any role in breast cancer development. The expression level of FAM83D is significantly elevated in breast cancer cell lines and primary human breast cancers. High expression levels of FAM83D are significantly associated with poor clinical outcome and distant metastasis in breast cancer patients. We show that ectopic expression of FAM83D in human mammary epithelial cells promotes cell proliferation, migration and invasion along with epithelial-mesenchymal transition (EMT). Ablation of FAM83D in breast cancer cells induces apoptosis and consequently inhibits cell proliferation and colony formation. Mechanistic studies reveal that overexpression of FAM83D downregulates FBXW7 expression levels through a physical interaction, which results in elevated protein levels of oncogenic substrates downstream to FBXW7, such as mTOR, whose inhibition by rapamycin can suppress FAM83D-induced cell migration and invasion. The results demonstrate that FAM83D has prognostic value for breast cancer patients and is a novel oncogene in breast cancer development that at least in part acts through mTOR hyper-activation by inhibiting FBXW7.

Figure 1. The expression of FAM83D is elevated in human breast cancers

(A) Genomic amplification on chromosome 20q was refined by integrative analysis of public copy number datasets for breast cancers, indicating that FAM83D is located at a peak of a sub-amplicon. (B) Expression profile of FAM83D in breast cancer cell lines. FAM83D mRNA levels relative to normal breast epithelial cell line 184A1 were determined by qRT-PCR. Gene expression was normalized to GAPDH. Data are presented as means ± Standard deviation. (C) Protein level of FAM83D in cultured breast cancer cell lines. (D) FAM83D mRNA expression levels are significantly elevated in breast tumors in comparison to normal breast tissues, using three public expression datasets. FAM83D expression is measured as log₂ (probe intensities). The p-values were obtained from Mann-Whitney U or Kruskal-Wallis tests.

Figure 2. Elevated expression of FAM83D is associated with poor disease-free survival of breast cancer patients in four different cohorts (A) GSE1456, (B) GSE3494, (C) GSE6532, and (D) GSE20685

FAM83D expression is measured as log₂ (probe intensities). The p-values were obtained from a long-rank test.

Figure 3. Ectopic expression of FAM83D enhances cell proliferation, migration and invasion

(A) MCF10A cells with FLAG-tagged FAM83D (MCF10A-FAM83D) were established with stable expression of FAM83D and confirmed by Western blot using an anti-Flag antibody. (B) Proliferation of MCF10A-FAM83D is significantly accelerated compared to normal MCF10A control cells measured by MTT assay. (C) MCF10A-FAM83D (FAM83D) closed the scratched area significantly faster than MCF10A control cells (control). The representative photographs are shown in left panel. Quantification of the data is presented as relative migration area in each cell line (right panel). (D) Migration and invasion ability of MCF10A-FAM83D cells (FAM83D) is significantly enhanced compared to MCF10A control cells (control) using the transwell assay. The representative photographs are shown in the left panel and the average number of migrated cells/field in the right panel. In b-d, data are presented as means ± standard deviation from three independent experiments, each performed in triplicate. * indicates p < 0.05 and ** indicates p < 0.01, which were obtained by a t-test. (E) High FAM83D mRNA levels reduce distant metastasis-free survival for breast cancer patients in two independent datasets (GSE6532 and GSE20685). The p-values in the graphs were obtained from a long-rank test.

Figure 4. Overexpression of FAM83D in MCF10A cells leads to an epithelial-mesenchymal transition

(A) Representative micrographs of MCF10A-FAM83D and MCF10A control cells under bright field display significant differences in morphology. (B) Western analysis of E-cadherin and vimentin indicates EMT in MCF10A-FAM83D compared to MCF10A control cells. Loading control is β-actin. (C) Immunofluorescent staining of E-cadherin (red, upper panels) and vimentin (green, lower panels) demonstrates EMT in MCF10A-FAM83D compared to MCF10A control cells. DNA is stained by DAPI (blue).

Figure 5. Biological consequences of FAM83D knockdown in BT549 cells

(A) FAM83D expression was analyzed by Western blotting using anti-FAM83D antibody in both stable shRNA and transient siRNA knockdown in BT549 cells. Loading was controlled by β-actin levels. (B) BT549 cells with stable FM83D knockdown (shFAM83D) show a significant reduction in proliferation in comparison to control cells (shCtrl). BT549-shFAM83D and control cells were seeded in six-well dish in triplicates and cell numbers were counted by hemocytometer at indicated time points. (C) Knockdown of FAM83D induces cell apoptosis in BT549 cells. BT549 cells were transfected with siFAM83D or control siRNA (siCtrl) in a 96-well plate in triplicates for the indicated times. Apoptosis was determined by a Caspase3/7 assay. (D) Knockdown of FAM83D reduces colony formation in BT549 cells. (E) Knockdown of FAM83D significantly decreases cell invasiveness. Representative photographs from the Matrigel-coated transwell invasion assay of BT549 shCtrl control cells and shFAM83D cells. Data are presented as means ± standard deviation from three independent experiments, each performed in triplicate. * indicates p<0.05 and ** indicates p< 0.01, which were obtained from t-test.

Figure 6. FAM83D downregulates FBXW7 via physical interaction

(A) FLAG-FAM83D is specifically co-immunoprecipitated with HA-FBXW7. (B) Ectopic FBXW7 (HA-FBXW7) binds to endogenous FAM83D in vivo. (C) FAM83D protein levels are not regulated by FBXW7. (D) Overexpression of FAM83D in MCF10A represses FBXW7 expression and leads to elevated expression of known FBXW7 targets, including c-Myc, c-Jun and mTOR. (E) Ectopic overexpression of FAM83D (FLAG-FAM83D) reduces FLAG-FBXW7 protein levels in a dose-dependent manner. Amounts of transfected plasmids are indicated above the blot. (F) Proteasome inhibition by MG132 suppresses reduction of FLAG-FBXW7 by FAM83D ectopic overexpression. In co-immunoprecipitated experiments, 5% of extracts used were shown as loading controls (WCL). In Western-blotting experiments, β-actin serves as the loading control. In a and b, “-“ indicates empty vector.

Figure 7. mTOR inhibition by rapamycin alleviates the enhanced migration and invasion caused by ectopic overexpression of FAM83D

(A) Inhibition of mTOR by rapamycin significantly suppresses cell motility induced by FAM83D ectopic overexpression in scratch-healing assay. The representative photographs are shown in left panel. Quantification of the data was presented as relative migration area in each cell line (right panel). (B and C) Enhanced cell migration and invasion caused by FAM83D ectopic overexpression were rescued by treatment of Rapamycin. Uncoated (B) or Matrigel-coated (C) transwell assays on MCF10A control cells and MCF10A-FAM83D cells treated with DMSO control and rapamycin. In a-c, data are presented as means ± Standard deviation from three independent experiments each performed in triplicates. ** indicates p<0.01, which were obtained from t-test. (D) Schematic illustration of the proposed oncogenic function of FAM83D in breast cancer development through inhibition of the FBXW7 tumor suppressor by physical interaction.