Abstract A38: Identification through functional genomics screening of factors whose downregulation enhances the side population in ovarian cancer.

Abstract

Abstract Cancer stem cells (CSC) play a role in chemoresistance and cancer relapse. CSCs are enriched for a side population (SP) of cells as detected by efflux of hoechst dye. In this research, we performed a functional genomics screen to identify genes whose downregulation generates a SP of serous ovarian cancer cells. We transfected a library of 81,000 shRNA lentiviral plasmids (Cellecta) targeting 15,000 genes into two serous ovarian cancer cell lines, CH1 and SKOV3, which harbor minimal SP fractions (less than 0.1%). Following transfection, we sorted cells to obtain the SP fraction, expanded these from independently plated single SP cells and extracted DNA. We amplified the transduced shRNAs by PCR and reconstructed shRNA-lentiviral plasmids, followed by transfection into CH1 or SKOV3 to determine reproducibility. Furthermore, we transfected different sequences of shRNAs targeting the identified genes and confirmed the consequent increase in the SP fraction. As a result, suppression of GeneA, GeneB and GeneC markedly increased the SP in CH1 cells (control; 0.08% vs GeneA; 1.1%, GeneB; 1.0%, GeneC; 2.0%). Suppression of GeneD, GeneE and GeneF markedly increased the SP of SKOV3 cells (control; 0.07% vs GeneD; 1.4%, GeneE; 1.1%, GeneF; 1.0%). Moreover, we overexpressed five genes (GeneA, GeneC, GeneD, GeneE and GeneF) in two serous ovarian cancer cell lines, A2780 and IGROV1, which contain more abundant SP fractions and found a marked decrease of the SP fraction (IGROV1: control, 6.8% vs 0.65%, 2.1%, 4.2%, 0.34%, 0.35%, p<0.001, respectively, A2780: control, 3.48% vs 0.01%, 0.15%, 0.22%, 0.08%, 0.21%, p<0.001, respectively). We next analyzed phenotypic characteristics of CH1 cells in which GeneA, GeneB or GeneC is suppressed. In clonogenicity assays of single cells, we found that the SP had higher clonogenicity than the main population (MP) and control cells (control shRNA; 1%, sh-GeneA-SP vs. MP; 10.7% vs. 5.5%, sh-GeneB-SP vs MP; 8.5% vs. 2.2%, sh-GeneC-SP 3.0% vs. 1.0%, p<0.05, respectively). The CH1 SP had higher tumorigenicity than the MP following subcutaneous injection of 1,000 cells into NOD/SCID mice (control shRNA, 1/4; sh-GeneA-SP vs. MP, 2/4 vs. 1/4; sh-GeneB-SP vs MP, 4/4 vs. 1/4; sh-GeneC-SP vs MP, 4/4 vs. 0/4, respectively). Suppression of these genes significantly elevated cisplatin IC50 values of CH1 cells as compared to the control-control, 40.9nM; sh-GeneA, 110nM; sh-GeneB, 102.3nM; sh-GeneC, 120.3nM; p<0.05, respectively. In addition, suppression of GeneB contributed to paclitaxel resistance while suppression of GeneC enhanced resistance to gemcitabine and paclitaxel. (p<0.05). Also we investigated characteristics of SKOV3 cells in which GeneD, GeneE or GeneF was suppressed. In clonogenicity assays of single cells, we found that SP cells had higher clonogenicity than MP and control cells (p<0.05, respectively). Suppression of these three genes significantly increased the IC50 values of SKOV3 for liposomal doxorubicin, gemcitabine and paclitaxel (p<0.05, respectively). Suppression of GeneD significantly increased cisplatin IC50 values compared to control cells (p<0.05). In conclusion, we identified six genes, whose down-regulation contributed to an increase in the SP of serous ovarian cancer, through functional genomics screening. Moreover, suppression of these genes was associated with CSC phenotypes, including clonogenicity, chemoresistance and in vivo tumorigenicity. These results are important with regard to revealing the molecular mechanisms of tumor recurrence. (Gene names are masked owing to potential conflict with a patent.) Citation Format: Noriomi Matsumura, Koji Yamanoi, Susan K. Murphy, Junzo Hamanishi, Kaoru Abiko, Ken Yamaguchi, Tsukasa Baba, Masafumi Koshiyama, Ikuo Konishi. Identification through functional genomics screening of factors whose downregulation enhances the side population in ovarian cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr A38.