Abstract A13: Mechanism-based chemoprevention of triple-negative breast cancer by blockade at multiple cell cycle regulatory points

Abstract

Abstract Effective chemoprevention strategies currently only target the development of ER-positive breast cancer, but do not prevent triple negative breast cancer (TNBC) which represents approximately 20% of all breast cancers. TNBC mostly affects young women and the standard of care therapy (highly cytotoxic chemotherapy) is too toxic to use as a prevention strategy. Therefore, there is a critical need to identify effective and tolerable drugs for the prevention of triple negative breast cancer. Naturally occurring and synthetic retinoids, which are vitamin A analogues, have been successfully used for the prevention and treatment of cancers. However, naturally occurring retinoids are of limited use because of their toxicity. Thus, drugs that specifically target RXR nuclear receptors (“rexinoids”) and activate RXR without activating RAR nuclear receptors are being studied as potential cancer preventive drugs. One of the most promising rexinoids is the compound, LG100268. We have demonstrated that LG100268 is highly effective in preventing ER-negative/HER2-positive mammary tumors in MMTV-ErbB2 mice, but less preventive in models of TNBC (p53-null mice and C3(1)-SV40 Tag mice). We have also previously demonstrated that rexinoids suppress growth of breast cells by inhibiting cyclin D-dependent pathways. For this study, we hypothesized that more effective TNBC cancer prevention could be achieved by combining kinase inhibitors and LG100268 to enhance the cancer preventive effect of the rexinoid. To identify such kinases we carried out a ‘whole kinome’ high-throughput siRNA screen in combination with LG100268 in immortalized, human mammary epithelial cells overexpressing telomerase (HME-hTert). The siRNA library targets each of the 636 known human kinases and was arrayed in pools of three individual siRNAs per kinase. The screen was performed in quadruplicate, with a non-targeting siRNA pool as negative control. After 4 days of growth cells were stained with DAPI and cell number was determined by automated microscopy and image analysis (the Z'-score of the image-based assays exceeded 0.5, suggesting that our assay had a robust signal-to-noise ratio). A total of 41 kinases whose inhibition by siRNA and LG100268 resulted in > 2SD of the value for growth inhibition with siRNA and vehicle alone were considered “hits” and carried forward in validation studies. Transfections with siRNAs to candidate genes were repeated to confirm gene silencing using quantitative real-time PCR (qRT-PCR), with ≥70% knockdown considered acceptable. Results showed highly effective knock-down of candidates and confirmed enhanced growth suppression. In particular knockdown of CDK1, 2 and 5 enhanced the chemopreventive effect of the rexinoid LG100268. CDK1 and 2 regulate cell cycle through Cyclin B- and Cyclin E-dependent pathways. CDK inhibitors (either CDKs siRNA or the CDK inhibitor Dinaciclib) in combination with LG100268 additively suppressed mammary epithelial growth in vitro and hyperplastic nodule formation of the C3(1)-SV40 Tag TNBC mouse model in vivo. These results suggest that inhibition of multiple cell cycle regulatory points, such as those controlled by distinct cyclin dependent kinases, is a promising strategy to prevent triple negative breast cancer. These results enhance our understanding of TNBC tumorigenesis and also provide the foundation to develop combination therapy for prevention of TNBC development. These studies were supported by an NCI R01 and NCI R25T grant (RO1CA078480, PB; R25CA057730, BL) Citation Format: Beate C. Litzenburger, Ivan P. Uray, Jamal Hill, Khandan Keyomarsi, Powel H. Brown. Mechanism-based chemoprevention of triple-negative breast cancer by blockade at multiple cell cycle regulatory points. [abstract]. In: Proceedings of the Twelfth Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2013 Oct 27-30; National Harbor, MD. Philadelphia (PA): AACR; Can Prev Res 2013;6(11 Suppl): Abstract nr A13.