Abstract 4380: Splicing factor SRSF3 knockdown-induced breast cancer cell growth suppression and apoptosis is partially reversed by Z-VAD-FMK and its expression correlates malignancy of breast cancer.

Abstract

Abstract Cancer-specific alternative splicing as well as aberrant splicing factor expression is seen in tumors, compared to normal tissues. Splicing factor, arginine/serine-rich 3 (SRSF3; SRp20) belongs to a family of highly conserved serine-arginine-rich proteins with multiple functions in RNA processing such as spliceosome assembly and alternative splicing. We have reported that knockdown (KD) of SRSF3 inhibited breast cancer cell growth and nearly complete KD of SRSF3 induced apoptosis in breast cancer cells via activating intrinsic apoptotic pathway (T-T Ho et al., Proceedings of the 2010 CTRC-AACR San Antonio Breast Cancer Symposium). To further determine whether SRSF3 KD-induced cell growth suppression and apoptosis is caspase-dependent, we treated triple negative breast cancer MDA-MB-231/LUCsh and MDA-MB-231/SRSF3sh2 cell lines [MDA-MB-231 cell lines carrying doxycycline (Doxy)-inducible luciferase shRNA and SRSF3shRNA2, respectively] with Doxy and a general caspase inhibitor Z-VAD-FMK and then evaluated cell survival using MTT assay. While we did not observe any effect of Z-VAD-FMK on the growth of MDA-MB-231/LUCsh cells, Z-VAD-FMK partially abolished SRSF3 KD-induced cell growth inhibition by 24.1% and 42.3%, respectively, following 10 μM or 50 μM Z-VAD-FMK treatment in MDA-MB-231/SRSF3sh2 cells compared to controls. In addition, co-treatment of Doxy and 10 μM or 50 μM of Z-VAD-FMK in MDA-MB-231/SRSF3sh2 cells resulted in 55.3% and 75.3%, respectively, reduction in apoptosis compared to Doxy treatment alone, while no effect of treatment was observed on control MDA-MB-231/LUCsh cells, as assessed by Hoechst 33342 dye staining. We are presently assessing the downstream targets of SRSF3 and investigating the mechanistic basis for the apoptotic effect of KD of SRSF3. Of note, we found that the expression of SRSF3 was upregulated in immortalized human mammary epithelial cells (HMECs), compared to isogenic finite HMECs, and that the level of SRSF3 correlated with the transformation state of cells. To evaluate the clinical significance of this splicing factor, we noticed a significant increase (p=0.001) of SRSF3 expression in breast tumor tissues compared to normal breast tissue, as assessed by immunohistochemical staining for SRSF3 of tissue microarrays. Indeed, we noted a correlation between SRSF3 staining and higher tumor grade (p=0.016), suggesting that expression of SRSF3 is associated with malignancy of human breast tumors. In summary, our results indicate that SRSF3 plays a major role in the survival of some breast cancer cell lines and is overexpressed in some breast cancers. We conclude that this splicing factor, which appears to behave as an oncogene, may represent a novel therapeutic target for the treatment of some breast cancers. Citation Format: Tsui-Ting Ho, Szilard Asztalos, Neena Majumdar, Ahmet D. Arslan, Martha R. Stampfer, Elizabeth L. Wiley, Seema A. Khan, Nilanjana Banerji, William McDonald, Xiaolong He, Debra A. Tonetti, William T. Beck. Splicing factor SRSF3 knockdown-induced breast cancer cell growth suppression and apoptosis is partially reversed by Z-VAD-FMK and its expression correlates malignancy of breast cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4380. doi:10.1158/1538-7445.AM2013-4380