ABSTRACT
BACKGROUND: MUC16 (CA125) is a large transmembrane mucin protein (> 200 kDa) aberrantly expressed in approximately 80% of human epithelial ovarian cancers (EOC). MUC16 expression in EOC cells is associated with increased tumorigenesis and inhibiton of genotoxic drug-induced apoptosis. However, the mechanism by which MUC16 mediates these effects is unknown. In the present study, we investigated the mechanisms by which MUC16 attenuates TRAIL-induced apoptosis. METHODS: MUC16 expression was down-regulated by stably expressing an anti-MUC16 single-chain antibody (scFv) targeted to the endoplasmic reticulum (ER), which prevents cell surface localization of MUC16 in OVCAR3 cells. We also generated a MUC16 C-terminal domain (MUC16CTD) construct that was stably expressed in MUC16 negative SKOV3 cells. RESULTS: We show that MUC16 attenuates apoptosis, activation of caspase-8 and mitochondria activation in EOC cells in response to TRAIL. MUC16 decreases TRAIL receptor R2 (DR5) expression and inhibits pro-caspase-8 activation at the death-inducing signaling complex (DISC). MUC16CTD expression is sufficient to attenuate the TRAIL signaling cascade. MUC16 knockdown decreases caspase-8 inhibitor cFLIP mRNA levels, increases cFLIP degradation, and consequently increases TRAIL-induced apoptosis. Down-regulation of cFLIP following treatment of MUC16-expressing OVCAR3 cells with cFLIP siRNA also increases TRAIL-induced apoptosis. CONCLUSIONS: These findings indicate that MUC16 protects EOC cells against TRAIL-induced apoptosis through multiple mechanisms including the blockade of TRAIL R2 expression and the regulation of cFLIP expression at both the transcriptional and the protein level.
Subject(s)
CA-125 Antigen/genetics , CASP8 and FADD-Like Apoptosis Regulating Protein/biosynthesis , Membrane Proteins/genetics , Neoplasms, Glandular and Epithelial/genetics , Ovarian Neoplasms/genetics , Receptors, TNF-Related Apoptosis-Inducing Ligand/biosynthesis , TNF-Related Apoptosis-Inducing Ligand/biosynthesis , Apoptosis/drug effects , Apoptosis/genetics , CA-125 Antigen/metabolism , Carcinogenesis/genetics , Carcinoma, Ovarian Epithelial , Caspase 8/metabolism , Cell Line, Tumor , Drug Resistance, Neoplasm , Gene Expression Regulation, Neoplastic , Humans , Membrane Proteins/metabolism , Neoplasms, Glandular and Epithelial/drug therapy , Neoplasms, Glandular and Epithelial/pathology , Ovarian Neoplasms/drug therapy , Ovarian Neoplasms/pathology , Proteolysis , RNA, Small Interfering , Receptors, TNF-Related Apoptosis-Inducing Ligand/metabolism , TNF-Related Apoptosis-Inducing Ligand/metabolism , Transcription, GeneticABSTRACT
Most human genes produce multiple mRNA isoforms through alternative splicing. However, the biological relevance of most splice variants remains unclear. In this study, we evaluated the functional impact of alternative splicing in cancer cells. We modulated the splicing pattern of 41 cancer-associated splicing events and scored the effects on cell growth, viability and apoptosis, identifying three isoforms essential for cell survival. Specifically, changing the splicing pattern of the spleen tyrosine kinase gene (SYK) impaired cell-cycle progression and anchorage-independent growth. Notably, exposure of cancer cells to epithelial growth factor modulated the SYK splicing pattern to promote the pro-survival isoform that is associated with cancer tissues in vivo. The data suggest that splicing of selected genes is specifically modified during tumor development to allow the expression of isoforms that promote cancer cell survival.
Subject(s)
Alternative Splicing , Cell Survival , Gene Expression Regulation , Intracellular Signaling Peptides and Proteins/genetics , Mitosis , Protein-Tyrosine Kinases/biosynthesis , Protein-Tyrosine Kinases/genetics , Apoptosis , Cell Line, Tumor , Epidermal Growth Factor/metabolism , Humans , Syk KinaseABSTRACT
OBJECTIVES: MUC16 (CA125) protein is a high molecular weight mucin overexpressed in the majority of epithelial ovarian cancers (EOC) but not in the epithelium of normal ovaries suggesting that it might play a role in EOC pathogenesis. Here, we explored the phenotypic consequences of MUC16 knockdown and expression of its C-terminal domain with the aim of establishing a role for MUC16 in tumorigenesis. METHODS: MUC16 was down-regulated by stably expressing an anti-MUC16 endoplasmic reticulum-targeted single-chain antibody which prevented MUC16 cell surface localization in NIH:OVCAR3 cells. In addition, we generated epitope tagged, N-terminal region-deleted MUC16 constructs with (MUC16TMU) and without (MUC16CTD) cytoplasmic tail deletions and stably expressed them in SKOV3 cells. RESULTS: Although MUC16 knockdown did not affect the cell growth rate, knockdown cells reached a stationary growth phase after 4 days whereas control cells continued to grow for up to 7 days. Colony formation assays in soft agar demonstrated that MUC16 knockdown cells had >8-fold reduction in their ability to form colonies. Importantly, MUC16 knockdown completely prevents the formation of subcutaneous tumors in nude mice. Conversely, we show that ectopic expression of the MUC16CTD enhances SKOV3 tumor cell growth, colony formation in soft agar and enhances tumor growth and metastases in SCID mice. In addition, MUC16CTD expression increases cell motility, invasiveness, and metastatic property. Deletion of the cytoplasmic tail from the MUC16CTD completely abolished its ability to enhance tumor cell growth, cell motility and invasiveness. Furthermore, the increased invasive properties of MUC16CTD-expressing cells correlated with decreased expression of E-cadherin and increased expression of N-cadherin and vimentin. CONCLUSION: These findings provide the first evidence for a critical role of MUC16 in tumor cell growth, tumorigenesis and metastases.
Subject(s)
CA-125 Antigen/biosynthesis , Membrane Proteins/biosynthesis , Animals , CA-125 Antigen/genetics , Cadherins/biosynthesis , Cadherins/metabolism , Carcinoma, Ovarian Epithelial , Cell Growth Processes/physiology , Cell Line, Tumor , Cell Movement/physiology , Down-Regulation , Female , Gene Knockdown Techniques , Humans , Membrane Proteins/genetics , Mice , Mice, Nude , Mice, SCID , Neoplasm Metastasis , Neoplasms, Glandular and Epithelial/genetics , Neoplasms, Glandular and Epithelial/metabolism , Neoplasms, Glandular and Epithelial/pathology , Neoplastic Stem Cells/metabolism , Neoplastic Stem Cells/pathology , Ovarian Neoplasms/genetics , Ovarian Neoplasms/metabolism , Ovarian Neoplasms/pathology , Transfection , Transplantation, Heterologous , Vimentin/biosynthesisABSTRACT
OBJECTIVE: Little is known about the biological functions of CA125/MUC16 tumor antigen. Here, we examined the role of CA125/MUC16 in regulating the sensitivity of epithelial ovarian carcinoma (EOC) cells to different drugs. METHODS: An endoplasmic reticulum targeted single-chain antibody (scFv) was used to down-regulate cell surface expression of CA125/MUC16 in NIH:OVCAR3 cells and the C-terminal domain (CTD) of MUC16 was ectopically expressed in CA125-negative SKOV3 cells. Sensitivity to genotoxic agents and to inhibitors of microtubule depolymerization was examined in NIH:OVCAR3 and SKOV3 cell sublines. Cell viability was determined by XTT assay, apoptosis by propidium iodide staining and caspase activation by Western blot and fluorogenic assay. RESULTS: Down-regulation of cell surface MUC16 decreases cisplatin IC(50) by 5-fold in NIH:OVCAR3 cells but does not affect paclitaxel IC(50). We found that the sensitivity to other genotoxic agents such as cyclophosphamide, doxorubicine and etoposide was also increased by down-regulation of MUC16. Caspase-9 and caspase-3 activation also significantly augmented in cisplatin-treated NIH:OVCAR3 cells expressing the anti-MUC16 scFv. Ectopic expression of MUC16 CTD has the opposite effect. Cisplatin sensitivity and caspases activation are decreased by the ectopic expression of MUC16 CTD in SKOV3 cells. CONCLUSIONS: CA125/MUC16 selectively modulates the sensitivity of EOC cells to genotoxic agents. The MUC16 CTD appears to be sufficient to promote cisplatin resistance.