Abstract 2947: Knockdown of splicing factor SRp20 induces apoptosis in ovarian tumor cells

Abstract

Abstract SRp20 is a member of the serine/arginine-rich (SR) protein family with multiple functions in RNA processing such as alternative splicing and polyadnylation. Our previous studies found that SRp20, together with another splicing factor, polypyrimidine tract-binding protein (PTB), is up-regulated in human ovarian tumors compared to normal ovarian surface epithelia (He, X et al, Clin Cancer Res, 10:4652). In order to determine whether overexpressed SRp20 is required for ovarian tumor cell growth, we studied the effects of SRp20 knockdown in several ovarian cancer cell lines. The knockdown of SRp20 expression was achieved by lentivirus-delivered, doxycycline (Dox)-induced siRNAs. We identified two effective siRNAs against SRp20, SRp20si1 and SRp20si2, which can suppress SRp20 expression by approximately 50% and 90%, respectively. In ovarian cancer cell lines A2780, SKOV3 and IGROV1, knockdown of SRp20 by either SRp20 siRNA caused substantial inhibition of cell growth and colony formation in soft agar. The degree of inhibition correlated with the extent of SRp20 suppression. Massive down-regulation of SRp20 expression by SRp20si2 led to almost complete loss of A2780's capability to form colonies in soft agar. Moreover, Hoechst 33342 staining revealed that the percentage of apoptotic cells was remarkably increased when SRp20 was knocked down ∼90% by SRp20si2. In contrast, moderate suppression (∼50%) of SRp20 by SRp20si1 did not trigger apoptosis. These results suggest that overexpression of SRp20 is required for ovarian cancer cell growth and survival. To determine which apoptotic pathway is activated by SRp20 knockdown, we examined the cleaved fragments of several caspases by immunoblotting and found that caspases 9, 3 and 7 but not caspases 2 and 8were cleaved in cells with SRp20 knocked down by SRp20si2. These results imply that the intrinsic apoptotic pathway is activated by massive SRp20 knockdown. In support of this, we also observed a substantial down-regulation of Bcl-2, a major anti-apoptotic regulator of the intrinsic apoptotic pathway, in the above cells. How SRp20 regulates Bcl-2 expression is unclear at present and currently under investigation. (Supported in part by CA40570 and CA138762 from NCI (to WTB), in part by OCRF (to XH), and in part by UIC). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2947.