Ovarian cancer ranks fifth in cancer deaths among women and accounts for the most deaths of any cancer involving the female reproductive system. In 2019, there were an estimated 22,000 new cases and 14,000 from ovarian cancer. Two oncogenes, MYC and kRAS, whose amplification is associated with more aggressive disease, have been put forth as potential targets in ovarian cancer to mediate cell death or enhance chemotherapeutic efficacy. The CRISPR CAS9 system allows for gene editing and thus the knocking out of specifically selected genes. Here, we investigate the effects of knocking, MYC, kRAS, or both, in ovarian cell lines with amplified kRAS (OV56, SKOV-3) in terms of their morphology and chemotherapeutic efficacy. kRAS and MYC targeted knockdowns in OV56 and SKOV-3 were carried out via CRISPR-CAS9 transfection. Knockout of target oncogenes was verified via PCR and gel electrophoresis and protein expression was monitored by western blot. Once knockouts were verified, culture conditions were changed to form cell aggregates in suspension by changing culture medium and vessels to grow "spheroids" that more closely mimic in vivo ovarian tumors and stem cells. Various stably transfected OV56 cell lines were seen to have a morphology change. Possible morphology changes while grown into spheroids, is being monitored. Changes in cell cytotoxicity of pre and post knockouts of both cells line when treated with chemotherapeutics are being compared. Successful transfection of OV56 and SKOV-3 cells with MYC and kRAS combination knockouts will allow for future investigations.
Download Full Text (586 KB)
Zuniga, Jorge and Ferrotta, Annalisa, "Therapeutic Effects of Knocking out Validated Oncogene Targets kRAS and MYC in Ovarian Cencer Cell Lines" (2020). Research Days Posters Spring 2020. 100.