The role of the AR-CCRK axis in doxorubicin chemoresistance in breast cancer

Abstract

Breast cancer (BC) is the top female cancer worldwide and in Hong Kong. Chemotherapy remains one of the most important pillars of treatment in breast cancer. However, chemoresistance development in BC cells renders chemotherapy useless, which greatly limits therapeutic options, especially in triple-negative breast cancer (TNBC). The androgen receptor (AR) has shown to play a role in carcinogenesis in multiple BC subtypes. Androgens, under estrogen deprivation, has been shown to contribute to BC carcinogenesis. It has been shown to interact with cell cycle-related kinase (CCRK) in other cancer models, including hepatocellular carcinoma (HCC) and colorectal cancer (CRC), to cause chemoresistance. CCRK is a recently discovered cyclin-dependent kinase (CDK), which has been shown to be activated by AR. However, its role in BC has not been delineated previously, and its downstream molecular pathway has not been illustrated in the context of BC. pSTAT3 is a regulator of cell regulation and proliferation. Its role in carcinoma progression, proliferation, metastasis and chemoresistance has been previously demonstrated. ß-catenin is a downstream molecule of the Wnt pathway, and its role in multiple cancers has been demonstrated. Both proteins have been previously identified in other cancer models to interact with both AR and CCRK to confer chemoresistance. Hence, we hypothesized that pSTAT3 and ß-catenin are downstream proteins of AR and CCRK which drive the development of chemoresistance in BC cells. We also hypothesized that AR and CCRK would play a role in causing chemoresistance in BC. Through in vitro studies, we showed that decreased levels of CCRK were associated with chemoresistance and delineated the downstream molecular pathway through which CCRK acted in BC cells, namely through pSTAT3 and ß-catenin. Reduced levels of CCRK were achieved via knockdown (KD) in TNBC cell lines, and subsequently confirmed by Western blot and polymerase chain reaction (qRT-PCR). Reduced resistance to doxorubicin was demonstrated in the KD cell lines compared with the control cell lines, supportive of the role of CCRK in chemoresistance. We further demonstrated, by Western blot and qRT-PCR, that reduced CCRK expression was associated with decreased expression of both pSTAT3 and ßcatenin, suggesting both proteins are downstream signalling molecules of CCRK. Through in vivo studies, we demonstrated that high expression of AR and CCRK are independent prognostic factors of BC, associated with relapse, disease progression, metastasis and poor overall survival (OS) and disease specific survival (DSS). This corresponded with results of our in vitro study. In summary, the results of this study demonstrate that CCRK play a role in chemoresistance via activation of pSTAT3 and ß-catenin. High co-expression of AR and CCRK in BC were found to be independent prognostic factors in predicting relapse, metastasis and disease progression of the BC patients. They also showed statistically significant association with poorer overall survival, disease-specific survival and disease-free survival.