CD40 ligand induces RIP1-dependent,necroptosis-like cell death in low-grade serous but not serous borderline ovarian tumor cells

Ovarian high-grade serous carcinomas (HGSCs) and invasive low-grade serous carcinomas (LGSCs) are considered to be distinct entities. In particular, LGSCs are thought to arise from non-invasive serous borderline ovarian tumors (SBOTs) and show poor responsiveness to conventional chemotherapy. The pro-apoptotic effects of CD40 ligand (CD40L) have been demonstrated in HGSC, though the underlying mechanisms are not fully understood. Conversely, the therapeutic potential of the CD40L-CD40 system has yet to be evaluated in LGSC. We now show that CD40 protein is focally expressed on tumor cells in two of five primary LGSCs compared with no expression in eight primary SBOTs. Treatment with CD40L or agonistic CD40 antibody decreased the viability of LGSC-derived MPSC1 and VOA1312 cells, but not SBOT3.1 cells. Small interfering RNA (siRNA) targeting CD40 was used to show that it is required for these reductions in cell viability. CD40L treatment increased cleaved caspase-3 levels in MPSC1 cells though, surprisingly, neither pan-caspase inhibitor nor caspase-3 siRNA reversed or even attenuated CD40L-induced cell death. In addition, CD40-induced cell death was not affected by knockdown of the mitochondrial proteins apoptosis-inducing factor (AIF) and endonuclease G (EndoG). Interestingly, CD40L-induced cell death was blocked by necrostatin-1, an inhibitor of receptor-interacting protein 1 (RIP1), and attenuated by inhibitors of RIP3 (GSK''872) or MLKL (mixed lineage kinase domain-like; necrosulfonamide). Our results indicate that the upregulation of CD40 may be relatively common in LGSC and that CD40 activation induces RIP1-dependent, necroptosis-like cell death in LGSC cells.Epithelial ovarian cancer accounts for approximately 90% of all ovarian malignancies and is the leading cause of gynecological cancer death in developed countries.^{1, 2} Recently, differences in molecular alterations and clinicopathological features have established a dualistic model dividing ovarian serous carcinomas into high-grade serous carcinoma (HGSC) and low-grade serous carcinoma (LGSC) subtypes. HGSCs are more common and are thought to develop directly from the ovarian surface epithelium or from serous tubal intra-epithelial carcinomas in the fallopian tube. In contrast, LGSCs are rare and are generally considered to develop from benign serous cystadenomas through serous borderline ovarian tumors (SBOT). SBOTs are slow-growing, non-invasive epithelial neoplasms that have a better prognosis compared with other types of ovarian cancer.^{3, 4, 5} Our previous studies have shown that the inhibition of p53 or treatment of epidermal growth factor or transforming growth factor-β1 increases SBOT cell invasion by inducing epithelial–mesenchymal transition, which suggests a possible mechanism that mediates the progression from SBOT to LGSC.^{6, 7, 8, 9} However, many of SBOTs recur as LGSCs that display poor responsiveness to conventional chemotherapy and for which survival rates are <50%.^{1, 3, 10} Thus, the development of novel, targeted therapeutic strategies is likely required to significantly improve patient survival.CD40, a transmembrane glycoprotein belonging to the tumor necrosis factor receptor superfamily, is expressed by a wide range of cell types including immune, endothelial and epithelial cells. Engagement of CD40 with its ligand, CD40L, has been shown to have important roles in a variety of physiological and pathological processes, especially in immunity.^{11, 12} In addition, CD40 expression has been demonstrated in several types of cancer, including colon, lung, cervical, bladder and prostate cancer.¹³ However, reported functions of CD40 in tumor cells vary, with both pro-apoptotic and anti-proliferative effects observed depending on the cellular context.^{14, 15, 16} Alternatively, some studies have shown that CD40 activation may promote the neoplastic transformation and growth of normal cells.^{17, 18, 19} Expression of CD40 has been demonstrated in ovarian cancer cell lines and tumor samples, but not in normal ovarian tissue, suggesting that CD40 may have an important role in ovarian tumors.^{20, 21, 22, 23, 24} Indeed, CD40L-CD40 signaling has been shown to induce growth-inhibitory effects in HGSC cells,^{20, 21, 23, 24, 25} however, the therapeutic potential of CD40 in LGSC and SBOT has not been evaluated.In the present study, we report for the first time elevated CD40 expression in a significant proportion of LGSCs compared with SBOTs. Moreover, CD40 expression is elevated in LGSC-derived MPSC1 and VOA1312 cells compared with SBOT3.1 cells, and CD40 activation induces cell death via CD40 only in LGSC-derived cells. Neither pan-caspase inhibitor nor caspase-3 small interfering RNA (siRNA) has any effect on CD40L-induced MPSC1 cell death. Moreover, CD40L-induced cell death was unaffected by individual or combined knockdown of the mitochondrial proteins apoptosis-inducing factor (AIF) and endonuclease G (EndoG). Interestingly, our results suggest that receptor-interacting protein 1 (RIP1), RIP3 and MLKL are involved in CD40-induced MPSC1 cell death. These results demonstrate that CD40 induces RIP1-dependent, necroptosis-like cell death in LGSC cells.