Clofazimine treatment modulates key non-coding RNAs associated with tumor progression and drug resistance in lethal Prostate Cancer

Prostate cancer (PCa) is the most commonly diagnosed cancer and the second-leading cause of cancer death among men in the United States, representing 24.3% of all new cancer cases in the US. Metastatic castration-resistant prostate cancer (mCRPC) is a clinically advanced form of PCa that is associated with increased aggressiveness, cancer stemness, morbidity, and the risk of developing resistance to taxanes, currently the first-line chemotherapeutic agents for mCRPC.Development of new target-directed drugs to treat mCRPC poses significant challenges, given the recognition that monospecific inhibitors have limited efficacy. Hence, there is an urgent medical need to develop new strategies that block major oncogenic signaling pathways driving the most lethal forms of PCa. Clofazimine (CLF) is a potential immunomodulator drug that is FDA-approved for the treatment of leprosy. Recently, using a phenotype-based high-throughput drug screening, we demonstrated the in vitro (cell lines), in vivo (mouse xenograft models), and ex vivo (patient-derived primary tumor cells) efficacy of CLF in drug-resistant forms of chronic myeloid leukemia and multiple myeloma. In this study, we demonstrate that CLF is effective as a single agent and in combination with docetaxel (DTX) in a panel of PCa cell lines representing the diversity of CRPC patients. We also found that CLF reduces aldehyde dehydrogenase activity, which is a marker for cancer stem-like cells (CSCs), a subtype of cancer cells with self-renewal and differentiation capacities (acquisition of mesenchymal phenotype or epithelial to mesenchymal transdifferentiation/EMT) that significantly contribute to tumor aggressiveness and the development of drug resistance. Further, using a microfluidic assay, we showed the impact of CLF on cancer cell migration and metastatic potential. Drug-induced changes were investigated using bulk tumor and single-cell RNA sequencing followed by functional analysis of top gene/pathway signatures, where CLF treatment was found to modulate cellular pathways associated with apoptosis, ER stress, oxidative phosphorylation, and mitochondrial dysfunction. Most importantly, CLF modulates the expression of several non-coding RNAs, including MALAT1 and NEAT1, that are linked to tumor cell proliferation, cell migration, and drug resistance. In silico validation of the non-coding RNA signature was performed using multiple patient datasets. Our results support the preclinical development of CLF against lethal PCa and provide novel insights into its mechanism of action.