EFEMP1 promotes ovarian cancer cell growth, invasion and metastasis via activated the AKT pathway

Abstract

EFEMP1, a kind of extracellular matrix (ECM) protein, has been suggested to correlate with the development of different types of carcinoma. However, its functions in ovarian cancer remain unclear. In our study, we performed cDNA microarray analysis and identified EFEMP1 dramatically elevated in the highly invasive subclone, compared with the low invasive subclone. Lentivirus transfection experiments were constructed afterwards. The results demonstrated that knockdown of EFEMP1 significantly inhibited ovarian cancer cell proliferation and induced cell cycle arrest at the G1/G0 phase. We also found that decreased the activity of phospho-AKT could suppress cell invasion and metastasis. Meanwhile, the increased phospho-AKT activity induced by the overexpression of EFEMP1 had significantly enhanced the abilities of ovarian cancer cells to invade and migrate. In addition, the vivo nude mice model confirmed that EFEMP1 was tightly correlated with the development of tumor. The results of RT2 Profiler EMT PCR array further indicated that decreased EFEMP1 suppressed epithelial-to-mesenchymal transition (EMT). Collectively, by activating AKT signaling pathway, EFEMP1 contributed to ovarian cancer invasion and metastasis as a positive regulator. Overall, EFEMP1 had showed the potential use in the development of new therapeutic strategies for ovarian cancer.

Keywords: EFEMP1; EMT; invasion; metastasis; ovarian cancer.

Figure 1. EFEMP1 expression in the highly invasive subclone and the low invasive subclone

(A) Scatter plot showing fold-change values versus P-values for differential gene expression between the highly invasive subclones and the low invasive subclones. The vertical lines correspond to 1.5-fold up and down, respectively, and the horizontal line represents a p-value of 0.05. The red point in the plot represents the differentially expressed genes with statistical significance. (B, C) EFEMP1 expression as measured by Western blot (B) and q-RT-PCR (C). (D) The fluorescence images of EFEMP1-GFP (Magnification × 200). *P < 0.05 versus control.

Figure 2. Identification of the efficiencies of EFEMP1 overexpression and knockdown after transfection (S1 and S21: non-infected control, S1-NC and S21-NC: infected with control-shRNA, S1-124: infected with EFEMP1-shRNA 124, S1-125: infected with EFEMP1-shRNA 125, S1-126: infected with EFEMP1-shRNA 126, and S21-exEFEMP1: infected with pLVX- EFEMP1-Puro vector)

(A) EFEMP1 protein expression in lentivirus-infected cells as measured by Western blot. (B) EFEMP1 mRNA expression in lentivirus-infected cells as measured by q-RT-PCR. (C) PCR products as measured by agarose gel electrophoresis. (D) EFEMP1 protein expression in lentivirus-infected cells as measured by ICC staining (Magnification × 200). *P < 0.05 versus control.

Figure 3. Effects of EFEMP1 on cell growth and cell colony formation

(A) Knockdown of EFEMP1 inhibited cell proliferation of the highly invasive subclone. (B) Overexpression of EFEMP1 promoted cell proliferation of the low invasive subclone. (C) The colony images of lentivirus-infected cells as examined by soft agar colony formation assay. (D) EFEMP1 knockdown decreased the colony forming capacity of highly invasive subclone, while overexpression of EFEMP1 increased the colony forming capacity of low invasive subclone. *P < 0.05 versus control.

Figure 4. Effects of EFEMP1 on cell cycle

(A) Cell cycle analysis as measured by flow cytometry in lentivirus-infected cells. (B) EFEMP1 knockdown suppressed cell by blocking their progression from the G1/G0 phase to S phase, while EFEMP1 overexpression promoted cell proliferation by increasing the S phase cells. (C) Cell cycle-related proteins Cyclin A, Cyclin B1, Cyclin D1 and Cyclin E in lentivirus-infected cells as measured by Western blot. (D) The average band intensities of Cyclin A, Cyclin B1, Cyclin D1 and Cyclin E normalized to GAPDH. *P < 0.05 versus control.

Figure 5. Effects of EFEMP1 on cell migration and invasion

(A) Cell migration images of lentivirus-infected cells as measured by Boyden chambers without Matrigel. (Magnification × 200). (B) Cell invasion images of lentivirus-infected cells by Boyden chambers coated with Matrigel. (Magnification × 200). (C) EFEMP1 knockdown suppressed cell migrating abilities, while EFEMP1 overexpression promoted cell migrating abilities. (D) EFEMP1 knockdown suppressed cell invading abilities, while EFEMP1 overexpression promoted cell invading abilities. *P < 0.05 versus control.

Figure 6. Effects of EFEMP1 on tumor growth

(A) Tumor growths of lentivirus-infected cells observed continuously for 8 weeks. (B) Photograph of xenografts dissected from nude mice after subcutaneous inoculation. *P < 0.05 versus control.

Figure 7. Effects of EFEMP1 on metastasis in nude mice

(A) Immunohistochemistry were performed on subcutaneous transplantation tumor of lentivirus-infected cells. (B) H&E staining were performed on lung metastasis after inoculation through tail vein. (Magnification × 200 and × 100).

Figure 8. Effects of EFEMP1 on AKT pathway

(A) EFEMP1 high expressed cells were serum-starved and treated with DMSO or the indicated inhibitors, SB202190, PD98059, and LY294002, for 24 h. The migration and invasion assay were performed by Boyden chambers without or with Matrigel. (B) After treating with LY294002, unphosphorylated and phosphorylated forms of AKT were detected by immunoblot analysis. (C) The average band intensities were normalized to β-actin. (D) After RNA interference, immunoblot analysis were performed to detect the effect of decreased EFEMP1 on the activities of AKT. (E) After overexpression transfection, immunoblot analysis were performed to detect the effect of increased EFEMP1 on the activities of AKT. (F) The average band intensities were normalized to β-actin. *P < 0.05 versus control.

Figure 9. Effects of EFEMP1 on EMT genes correlated to tumor progression

(A) The microarray image of 84 genes related to tumor progression by RT² Profiler™ PCR Array. (B) After RNA interference, EMT marker, including E-cadherin, N-cadherin and vimentin as measured by Western blot. (C) The top 9 genes (> 5 folds) of the microarray as measure by real-time qPCR in the EFEMP1 shRNA infected group. (D) After overexpression transfection, EMT marker, including E-cadherin, N-cadherin and vimentin as measured by Western blot. (E) The top 9 genes (> 5 folds) of the microarray as measure by real-time qPCR in the S21-exEFEMP1 infected group. (F) The average band intensities were normalized to GAPDH. *P < 0.05 versus control.