A Natural Compound Screen for SPOP Downregulated Breast Cancer

Abstract

Since breast cancer is the second leading cause of cancer deaths among American women there is a compelling need to uncover novel and superior treatment regimens for women suffering from this disease. Interestingly, several studies have indicated that up to 70% of breast cancer patients have downregulation of the E3 ubiquitin ligase Speckle Type POZ Protein (SPOP) thus indicating that SPOP is a critical tumor suppressor gene in breast cancer. Our lab has previously shown that SPOP targets GLI3 for ubiquitination to promote SHH signaling in prostate cancer patients, a finding that we hypothesized to be true for breast cancer as well since SHH signaling is often found to be hyper-activated in advanced breast cancer. We further hypothesized that SPOP downregulated breast cancer cells therefore will likely have a differential response to targeted therapy and that SHH targeted drugs could prove to be beneficial for SPOP downregulated breast cancer patients. First of all, to study the effect of downregulated SPOP, a lentivirus carrying an shRNA against SPOP was generated and infected into MCF-7 cells. Proliferation assays were utilized to confirm that downregulated SPOP plays a role in the increased proliferation of breast cancer cells. Next, quantitative PCR was performed to determine the effect of downregulated SPOP on the expression of GLI3-dependent SHH target genes. Finally, a natural compound library was utilized to find a novel therapeutic strategy for SPOP downregulated breast cancer. Our findings confirmed that SPOP knockdown increases proliferation of MCF-7 cells in vitro. Furthermore, quantitative PCR confirmed that GLI3 target genes are upregulated in MCF-7 cells when SPOP expression is low. Finally, our natural compound library screen identified novel therapeutic compounds that specifically target SPOP downregulated MCF-7 cells in a manner that involves disruption of GLI3-dependent SHH signaling. These findings therefore provide critical insight into novel treatment strategies for patients suffering from SPOP downregulated breast cancer.