Abstract A54: Regulating MUC16 expression to decrease ovarian tumor proliferation and increase efficacy of therapeutic antibodies.

The membrane-spanning mucin MUC16 promotes proliferation and metastasis of and protects ovarian cancer cells from innate immune responses and antibody-mediated cytotoxicity. We are hypothesizing that studying the regulation of MUC16 as well as developing techniques to prevent production of this mucin by ovarian tumor cells will be important in curtailing the growth of these tumors as well as increasing the efficacy of antibody-based therapeutics. We first investigated the downregulation of MUC16 by siRNA. Four different siRNA constructs were used to downregulate expression of this mucin. When the VK8 antibody was used in flow cytometry experiments to monitor knockdown, a complete loss of the mucin was observed between 48-72 h after transfection of the siRNA. MUC16 was recovery was observed by 96 h post-transfection. While the results with VK8 showed conclusive loss of the mucin, when flow cytometry was conducted with OC125 as the detecting antibody, the maximum inhibition was between 40-60%. The disparity in the data could not be attributed to the differences in the antigen affinities or the concentrations of the two detecting antibodies. The data with siRNA is in stark contrast with cell lines transfected with intracytoplasmic scFv of a MUC16-capturing antibody. In this case complete loss of MUC16 is observed irrespective of the detecting antibody used in the flow cytometry experiments. Even though the knockdown of MUC16 was not complete when analysis was conducted with OC125 antibody, the resulting cells showed a significant decrease in cell proliferation. A trend towards decreased immunoprotection of the cancer cells in the MUC16 siRNA knockdown cells was also observed when the cancer cells were subjected to lysis by natural killer cells isolated from healthy volunteers. To maximize downregulation of MUC16 and to develop a strategy that may potentially be used to decrease expression of this mucin in vivo, we developed a triblock copolymer poly(ethylene glycol)-b-poly{N-[N-(2-aminoethyl)-2-aminoethyl] aspartamide}-b-poly(ε-caprolactone) (PEG-b-PAsp(DET)-b-PCL) capable of self-assembling into micelles that were loaded with MUC16 siRNA. In vitro experiments showed efficient knockdown of the mucin that was dependent on concentration of siRNA used. Even when the siRNA was delivered via the PEG-PAsp-PCL nanoparticles and VK8 antibody showed complete knockdown, only approximately 60-70% knockdown was observed using OC125. Using a different approach, we also investigated if the expression of MUC16 by ovarian, breast and pancreatic cancer cells can be regulated by small molecule inhibitors of DNA methylation and histone deacetylation. The DNA methyltransferase inhibitor, 5-aza-2'deoxycytidine (5-azadC) was found to increase by 2-3-fold the expression of MUC16 in all of the approximately fifteen cell lines tested in our studies. Treatment of most cell lines with the histone deacetylase inhibitor (HDAC), trichostatin also resulted in increase in MUC16 expression, although the increase was not as robust and consistent between the cell lines tested. However, MassARRAY and CHIP analysis of the MUC16 5'-flanking region showed no epigenetic modifications of the promoter of this mucin. Overall our results indicate that while siRNA can be used to inhibit MUC16 expression the data should be interpreted with caution as differences in the activities of the antibodies used in the assays have different specificities for this mucin. These conclusions also suggest the presence of different isoforms and splice variants of the mucins that are likely to be detected with distinct affinities and specificities by the MUC16 antibodies. Our experiments with 5-azadC and trichostatin indicate MUC16 expression is not regulated via direct epigenetic regulation of its gene. Instead, transcription factors and signaling events required to control MUC16 expression are likely regulated via epigenetic mechanisms.

Citation Format: Mildred Felder, Arvinder Kapur, Ryan Serbin, San Zheng, May Xiong, Norishige Yamada, Manish Patankar. Regulating MUC16 expression to decrease ovarian tumor proliferation and increase efficacy of therapeutic antibodies. [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 A54.