The Role Of Androgen Receptor On Proliferation In Triple-negative Breast Cancer

Background:Cancer has become one of the most important serious diseases in China, which affects human being’s health. The morbidity and mortality rate of cancer are increasing in recent years, and cancer has become an important public health issue in China. According to newly cancer data in China, colorectal, lung and bronchus, breast, stomach and esophagus cancer are the most common cancer among female cancers, accounting for nearly 60% of all cases. Breast cancer has the highest morbidity rate among all types of cancer. Many studies have focus on the mechanism of the development of tumor and the comprehensive therapy of breast cancer.Studies have showed that the development of tumor involves multiple steps. The newly study has showed that tumors involved in three broad phases:the breakthrough phase, the expansion phase and the expansion phase. Every phase is driven by multiple genes. The development of breast cancer is associated with hormone receptors. According to the expression of hormone receptor, there are four subtypes of breast cancer:Luminal A, Luminal B, human epithelial receptor-2 (HER-2) and basal-like.80% triple-negative breast cancer (TNBC) belongs to basal-like breast cancer.TNBC makes up about 10 to 20% of invasive breast cancers, which has lowest 5-year survival rate and worst overall survival compared with other breast cancer subtypes. As it lacks estrogen receptor (ER) and progesterone receptor (PR) expression as well as HER-2 amplification, treatments targeting ER or HER-2 are not effective. Up to now, there is no optimal standard of care for the management of TNBC. Although TNBC is defined by its lack of the hormone receptors traditionally associated with breast cancer,it expresses other hormone receptors like the insulin-like growth factor (IGFR) and androgen receptor (AR). AR,also belongs to nuclear hormone receptor family, interacts with molecules in the nuclear, which is widely expressed in up to one-third of TNBC. It has been demonstrated that AR promotes TNBC cells proliferation and AR inhibition can decrease cells viability both in vivo and vitro.In order to verify the safety and effectiveness, a phase II trial in patients with ER-/PR-/AR+ metastatic breast cancer has shown a clinical benefit rate of 19% for bicalutamide. Another phase II study assessing the safety of enzalutamide in AR-positive TNBC or advanced breast cancer is ongoing (NCT01889238). Moreover, a recent case report showed that a 55-year old woman with AR-positive metastatic TNBC received clinical response after 4 months treatment of oral bicalutamide 150 mg every day.As a ligand-dependent transcription factor, AR affects cancer cell growth, as well as regulates multiple genes expression. To better investigate the role of androgen-activated AR in TNBC, we assessed cell cycle related genes upon AR agonist and antagonist, including p53, p73, p21 and Cyclin D1. p53 is classical tumor suppressor, and low level of p53 present in proliferation cancer cells. We found that p53 is mutated in multiple breast cancer cell lines. As the member of p53 family. unlike p53, p73 is rarely mutant or lost in cancers. Cyclin-dependent kinase p21 is tumor suppressor, which promotes cell cycle arrest, blocks cell division, and down-regulation of p21 often occurs in breast cancer. p73 and p21 can promote anti-proliferation activity and induce apoptosis by p53-independent mechanisms.Aims:AR, a steroid hormone receptor, has recently emerged as prognostic and treatment-predictive marker in breast cancer. Previous studies have shown that AR is widely expressed in up to one-third of TNBC. However, the role of AR in TNBC is still not fully understood, especially in TNBC cells. In our study, we designed to investigate the effect of AR in breast cancer growth and the link between AR and cell cycle related genes in TNBC using in vitro and in vivo models. Further, we also wish to examine the efficacy of AR inhibition bicalutamide as a targeted therapy in AR-positive TNBC.Methods:1. To investigate the effects of AR agonist 5-a-dihydrotestosterone (DHT) and antagonist bicalutamide on cell viability, we assessed the effects of DHT and nonsteroidal antagonist bicalutamide on TNBC cells (MDA-MB-231, Hs578T). The effects of AR on cell viability and apoptosis were determined by MTT assay, cell counting and flow cytometry analysis2. The protein expression of AR p53, p73, p21 and Cyclin D1 were analyzed by western blotting after treatment of DHT and bicalutamide.3. The bindings of AR to p73 and p21 promoter were detected by ChIP assay.4. MDA-MB-231 cells were transplanted into nude mice and the tumor growth curves were determined and expression of AR, p73 and p21 were detected by Immunohistochemistry (IHC) staining after treatment of DHT or bicalutamide.Results:1. MTT assay showed that AR agonist DHT induces TNBC breast cancer cells proliferation and inhibits apoptosis in vitro. On the contrary,AR antagonist, bicalutamide, causes apoptosis and exerts inhibitory effects on the growth of breast cancer.2. The effect of AR on cellular proliferation was also assessed by cell counting assay, which was consistent with the MTT assays. The cell number of MDA-MB-231 and Hs578T was increasing after administration of DHT, and the bicalutamide inhibits the proliferation of MDA-MB-231 and Hs578T cells.3. DHT dependent activation of AR involves regulation in the cell cycle related genes, including p73, p21 and Cyclin D1. DHT-activated AR induced the expression of p53, while impairing AR signaling with bicalutamide led to a decrease p53 protein level. The expression of p73 and p21 in protein levels were increased after bicalutamide treatments. Conversely, DHT-activated AR decreased both p73 and p21 protein levels in both cell lines.4. Further investigations indicate the modulation of AR on p73 and p21 mediated by direct binding of AR to their promoters, and DHT could make these binding more effectively.5. DHT dramatically promoted the growth of MDA-MB-231 tumor in vivo while bicalutamide treatment resulted in the inhibition of tumor growth. DHT promotes the TNBC tumor growth through decreasing p73 and p21 levels, while bicalutamide elevated the p73 and p21 expression, resulting in reduction of tumor volume.Conclusion:The study demonstrates the tumorigenesis role of AR and the inhibitory effect of bicalutamide in AR-positive TNBC both in vitro and in vivo, suggesting that AR inhibition could be a potential therapeutic approach for AR-positive TNBC patients.