Studies on the Mechanisms of a9-Nicotinic Acetylcholine Receptor Confers Tamoxifen-Induced Drug Resistance in Human Breast Cancer Cells

Background: Tamoxifen (TAM) has been the mainstay of endocrine therapy for estrogen receptor-positive (ER+) breast cancer. However, approximately 40% of breast cancer patients eventually acquire TAM resistance. Thus, it is necessary to develop effective methods for enhancing the efficacy of TAM in breast cancer treatment. Preliminary results: Long-term smoking affects of efficacy of breast cancer therapy. Our preliminary results showed that α9-nicotinic acetylcholine receptor (nAchR) mRNA levels were dramatically increased in surgically dissected (Figs. 1-3, > 80-fold, n = 276) and laser capture microdissected (Figure 4, >80-fold, n = 20) cell clusters isolated from breast tumor tissues compared to normal cells. We found that α9-nAchR was up-regulated by ERα and NFκB under nicotine stimulation in human breast cancer cells (Figs. 5-6). Human neuroendocrine neuron-specific enolase (ENO) can be used as a marker in in vitro screens to identify novel anti-cancer agents. Our results demonstrated that ENO-1 expression was transcriptionally up-regulated by ERα and NFκB which are induced specifically upon lower doses of TAM (50 nM) treatment (Figures 7-8). Our in vivo and in vitro studies demonstrated that changing ENO-1 mRNA expression by either an siRNA or a Tet-Off technique significantly modulates the therapeutic effects of TAM (Figs. 7-12). These results suggest that excess expression of ENO-1 exerts a negative regulatory effect on the transcription of some pro-apoptotic factor (such as c-Myc) that confers TAM-induced drug resistance (Fig. 13). Our results demonstrate that inhibition of ENO-1 may be a novel approach for overcoming TAM resistance in breast cancer. A three-year proposal is required and direct evidence of ENO-1-mediated breast cancer carcinogenesis will be investigated. Year-1 proposal: Evaluation of ENO-1 and 　9-nAchR mRNA expression levels and their relationship to TAM-induced drug resistance in human breast tumor tissue. In year-1, we first want to investigate the expression levels of α9-nAchR and ENO-1 in breast tumor tissue. Surgical and Laser capture microscopic (LCM) dissected human breast cancer cells will be isolated for detection of the expression levels of ENO-1 and α9-nAchR mRNAs (Figs. 2 and 3). Real-time PCR analysis and promoter luciferase activity assays established in our study could be used as research tools to investigate whether the ENO-1 expression is regulated by sex hormones (such as estrogen, E2) or tobacco smoking (such as nicotine). In addition, siRNA knock-down and adenoviral Tet-off overexpression breast cancer cells lines have been established in our lab (Figs. 10-12) and adapted as a research model for evaluating the synergistic effects of α9-nAchR and ENO-1-mediated TAM-induced drug resistance on breast cancer therapy. Year-2 proposal: Studies on the α9-nAchR-mediated signaling pathways that affect TAM-induced drug-resistance in human breast cancer cells. In year-2, TAM-resistant cell lines (rTAM cells) will be established to study cell survival signals related to α9-nAchR. Nicotine or hormone (E2)-induced transcription factors (such as ERα and NFκB) that regulate ENO-1-mediated genes involved in TAM-induced drug-resistance will be further investigated. It has been demonstrated that ENO-1 can bind to the P2 element in the c-Myc promoter and compete with the TATA-box binding protein (TBP) to suppress transcription of c-Myc. Such results suggest that excess expression of ENO-1 triggered by nicotine or hormones (E2) exerts a negative regulatory effect on c-Myc transcription, which then confers TAM-induced drug resistance. This hypothesis was supported by our in vivo preliminary results demonstrating that changing ENO-1 expression, either by an siRNA or the Tet-Off technique, significantly modulates the sensitivity of human breast cancer cells to TAM therapy (Figs. 10-12). The pro-apoptotic effects of c-Myc expression on TAM-induced drug resistance will be investigated in the ENO-1 knockdown (siRNA) or overexpression (Tet-Off) human breast cancer cells. Year-3 proposal: In vivo study on the α9-nAchR-induced modulatory effects of ENO-1 expression on clinical therapeutic effects of TAM. In year-3, we will explore the in vivo mechanisms of TAM-induced drug resistance by α9-nAchR-induced ENO-1 expression. The ENO-1 knock-down and overexpression cell lines, which were established in year-1, will be transplanted into SCID mice (preliminary data shown as figures 7-9). The TAM-resistant cell line (rTAM) will also be implanted into SCID mice as a positive control. The tumor-xenografted mice will receive nicotine in their drinking water in combination with TAM treatment and the tumor growth rate will be evaluated in relation to ENO-1 expression status. The c-Myc-associated pro-apoptotic signals will further be investigated. Nicotine or hormone (E2)-induced transcription factors that regulate ENO-1 expression, which were identified in the first-year (ex. ERα), will further be evaluated in the TAM-treated tumors. In year-3, we will test novel strategies for inhibiting ENO-1 expression to determine if they promote human breast cancer cell sensitization to TAM treatment (Figs. 7H and 8E). This will helpful for the development of clinical therapeutics like cancer chemopreventive agents. All these ideas we have proposed above require further experimentation and the preliminary results we have acquired will be adapted for additional experimental designs in the following 3-year proposal.