Bidirectional modulation of endogenous EpCAM expression to unravel its function in ovarian cancer

Abstract

Background: The epithelial cell adhesion molecule (EpCAM) is overexpressed on most carcinomas. Dependent on the tumour type, its overexpression is either associated with improved or worse patient survival. For ovarian cancer, however, the role of EpCAM remains unclear.

Methods: Cell survival of ovarian cancer cell lines was studied after induction or repression of endogenous EpCAM expression using siRNA/cDNA or artificial transcription factors (ATF) consisting of engineered zinc-fingers fused to either a transcriptional activator or repressor domain.

Results: Two ATFs were selected as the most potent down- and upregulator, showing at least a two-fold alteration of EpCAM protein expression compared with control. Downregulation of EpCAM expression resulted in growth inhibition in breast cancer, but showed no effect on cell growth in ovarian cancer. Induction or further upregulation of EpCAM expression decreased ovarian cancer cell survival.

Conclusion: The bidirectional ATF-based approach is uniquely suited to study cell-type-specific biological effects of EpCAM expression. Using this approach, the oncogenic function of EpCAM in breast cancer was confirmed. Despite its value as a diagnostic marker and for immunotherapy, EpCAM does not seem to represent a therapeutic target for gene expression silencing in ovarian cancer.

Figure 1

Downregulation of EpCAM expression by ATFs inhibits cell growth in breast cancer cells. SKBR3 cells were transduced to express the indicated ATFs. At days 4 and 21 after transduction, the percentage of GFP-positive (A) and the relative EpCAM protein expression (B; pMX set at 100) was measured by flow cytometry. (C) Clonogenic survival assay: 4 days after transduction cells were equally replated in 6-well plates and allowed to grow colonies for 16 days when surviving colonies were stained. MFI, mean fluorescence intensity.

Figure 2

Phenotypical effect of up- and downregulation of EpCAM expression in ovarian cancer. SKOV3 cells were transduced to express the indicated ATFs in 6- and 96-well plates. (A) At day 4 after transduction, cells were harvested and analysed for EpCAM mRNA (left; pMX set at 1) and protein (right; pMX set at 100) expression (mean±s.e.m., n⩾5; t-test **P<0.01, ***P<0.001). (B) At day 4 after transduction, cells were fixed, stained (inset) and 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay was performed at days 4, 5 and 6 after transduction (left) or cells were replated at equal cell numbers and MTT assay was performed for the four following days (right). GAPDH, glyceraldehyde 3-phosphate dehydrogenase; MFI, mean fluorescence intensity; OD, optical density.

Figure 3

SiRNA-based silencing of EpCAM expression in ovarian cancer has no effect on cell growth. SKOV3 cells were transfected with irr-siRNA or EpCAM-siRNA. At days 1 to 3 after transfection, EpCAM mRNA, protein expression (A) and 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay (B) were analysed (SD set at 100=delivery agent). MFI, mean fluorescence intensity; OD, optical density.

Figure 4

Increased EpCAM expression levels correlate with cell growth inhibition. SKOV3 cells were transduced with pMX and ZF_BVP64. At day 4 after transduction, cells were equally replated. Next day, siRNA transfection was performed, and subsequently at 3 days, EpCAM protein (A) and 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay (B) were analysed. (C) SKOV3 cells were transduced with a constant amount of ZF_BVP64 virus supernatant mixed with pMX and/or ZF_ASKD. At day 4 after transduction, EpCAM protein expression was measured (inset) and transduced cells were replated at equal cell numbers, and MTT assay was performed for the four following days.

Figure 5

Induction of EpCAM expression in EpCAM-negative ovarian cancer cell line. A2780 cells were transduced to express the indicated ATFs in 6- and 96-wellsplates. (A) At day 4, transduced cells were fixed and stained, or 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT assay was performed at days 4, 5 and 6 after transduction. (B) Transduced cells were harvested at day 4 after transduction, replated at equal cell numbers in 96-well plates and MTT assay was performed for the four following days. OD, optical density.