NORE1A induction by membrane-bound CD40L (mCD40L) contributes to CD40L-induced cell death and G1 growth arrest in p21-mediated mechanism

Membrane-bound CD40L (mCD40L) but not soluble CD40L (sCD40L) has been implicated in direct cell death induction and apoptosis in CD40-expressing carcinomas. In this study, we show that mCD40L but not sCD40L induces NORE1A/Rassf5 expression in an NFκB-dependant mechanism. NORE1A expression appeared to contribute to mCD40L-induced cell death and enhance cell transition from G1 to S phase of the cell cycle in a p21-dependent mechanism. The upregulation of p21 protein was attributed to NORE1A expression, since NORE1A inhibition resulted in p21 downregulation. p21 upregulation was concomitant with lower p53 expression in the cytoplasmic fraction with no detectable increase at the nuclear p53 level. Moreover, mCD40L-induced cell death mediated by NORE1A expression appeared to be independent of mCD40L-induced cell death mediated by sustained JNK activation since NORE1A inhibition did not affect JNK phosphorylation and vice versa. The presented data allow better understanding of the mechanism by which mCD40L induces cell death which could be exploited in the clinical development of CD40-targeted anti-cancer therapies.We, and others, have demonstrated that the outcome of CD40 receptor activation by its ligand CD40L/CD154 in carcinomas predominantly relies on the way the ligand is presented to the receptor at the cell surface, where membrane-bound CD40L (mCD40L) but not soluble CD40L (sCD40L) induces cell growth arrest and apoptosis. mCD40L-induced cell death occurs through a mechanism that involves the downregulation of pro-survival signals, mainly the PI3K/AKT pathway and the sustained activation of the pro-apoptotic JNK pathway, leading to caspase activation and subsequently apoptosis.^{1, 2} However, the role of CD40L in cell cycle regulation and growth arrest are still largely elusive; cell cycle regulation is a complex process that ensures correct cell division and is controlled by multiple mechanisms, governed by key regulatory proteins, including the cyclin-dependent kinases (CDKs) and their regulatory inhibitors. CDKs are a family of serine/threonine protein kinases that are activated at specific points of the cell cycle and are regulated by several different mechanisms.³CDK inhibitors function through inhibition of the G1 CDK cyclin complexes.⁴ Furthermore, the CDK inhibitor p21 has the ability to also inhibit DNA synthesis by binding to and inhibiting proliferating cell nuclear antigen.^{5, 6, 7} p21 protein expression is tightly regulated by the p53 tumour suppressor gene.⁸ The expression of p21 is also reported to be regulated by the recently identified Ras-associated factor 5 (NORE1A/RASSF5),⁹ a protein that is 41.8 kD and a member of the RASSF family of tumour suppressors.¹⁰ NORE1A expression is frequently downregulated by promoter methylation in human tumours.^{11, 12} Structurally, NORE1A contains a Ras-binding domain and was originally identified by a yeast two-hybrid screen.¹³ NORE1A can directly bind the Ras oncoprotein in a GTP-dependent manner.¹⁴ Exogenous expression of NORE1A has been reported to promote apoptosis by Ras-dependant and -independent mechanisms.¹² Furthermore, NORE1A protein expression results in a decrease in the number of cells in S phase of the cell cycle,¹⁵ indicating that NORE1A might function as a tumour suppressor; here, we show for the first time that mCD40L but not sCD40L induces the expression of NORE1A in an NFκB-dependant manner. NORE1A expression was found to contribute to mCD40L-induced cell death since inhibition of mCD40L-induced NORE1A expression resulted in reduced cell death by mCD40L. The role of CD40-induced NORE1A expression in cell cycle regulation was also examined and found to be mediated by p21 upregulation.