Abstract B19: Functional genomics approach links anchorage-independence with tumor dormancy in ovarian cancer

Abstract

Abstract Although a number of genes are aberrantly regulated in ovarian cancer, little is known about which genes are functionally important. The aim of this study was to identify new genes relevant to anchorage-independent growth of ovarian cancer cells through a genome-wide functional screening approach. We performed lentiviral transduction of a library of 81,000 shRNAs (Cellecta, Inc.) targeting 15,000 genes into an immortalized human ovarian surface epithelium cell line and 7 ovarian cancer cell lines (OVCA420, OVCA433, OVCA429, CAOV3, SKOV8, Tyk-nu and DOV13) which do not generate anchorage-independent colonies in soft agar. Among these, only OVCA420 generated colonies larger than 100 micrometers after shRNA library transduction. We extracted DNA from these colonies, amplified the shRNA constructs with PCR, and inserted the amplicons back into the original lentiviral vector, thus generating a secondary library of 68 shRNAs. Subsequent analysis, performed to examine the reproducible formation of anchorage-independent colonies using the secondary library, identified 5 shRNAs targeting ABHD2, CYB5R3, MR1, ELAC2 and CCL22. Interestingly, population doubling time was increased following transduction of these shRNAs (44.8, 50.1, 43.9, 28.0 and 33.2 hours, respectively) compared to the original or control shRNA-transducted OVCA420 cells (22.4 and 23.7 hours, respectively). Furthermore, side populations detected by efflux of Hoechst 33342 dye were drastically increased (50.5, 46.6, 55.5, 76.5 and 41.1%, respectively) compared to the original or control shRNA-transducted OVCA420 cells (7.3 and 10.4%, respectively). Although these data are not conclusive, they support that a phenotypic change occurs as ovarian cancer cells leave the primary tumor and are suspended in ascites. We found that repression of any one of these particular genes was sufficient to avoid anoikis. This change was accompanied by suppression of cell proliferation and increased efflux of Hoechst 33342 dye, which indicates activation of pumping function, suggesting a change to chemoresistant dormant cells. In summary, our functional genomics approach suggests a molecular mechanism underlying the process of anchorage independence, tumor dormancy and chemoresistance in ovarian cancer. Citation Format: Noriomi Matsumura, Koji Yamanoi, Yasuaki Amano, Junzo Hamanishi, Tsukasa Baba, Susan K. Murphy, Ikuo Konishi. Functional genomics approach links anchorage-independence with tumor dormancy in ovarian cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr B19.