MAGEA2调控p53和ERα信号通路诱导他莫昔芬耐药性乳腺癌细胞上皮细胞间质转化和转移的作用机制研究

Although survival of estrogen receptor positive (ER+) breast cancer patients has been improved by the onset of tamoxifen treatment, eventual resistance to this therapy is a common and devastating result for over 30% of patients. Unfortunately, there are presently no effective cures or predictor tools for tamoxifen resistant (TR) breast cancer. Providing new approaches to identify tamoxifen resistant predictors whist improving tamoxifen clinical effectiveness is critical if we are to improve outcome. Our recent studies demonstrated that expression of MAGEA2 is up-regulated in tamoxifen resistant tumor cells. Expression of MAGEA2 in tumor cell lines grown in vitro or as xenografts led to continued proliferation in the presence of tamoxifen. Importantly, our preliminary data showed that tamoxifen stimulates MAGEA2 expressing breast cancer cells to undergo epithelial-mesenchymal transition (EMT) and metastasis both in vitro and in vivo. In a series of ER+, tamoxifen-treated breast cancer, we showed a highly significant association between MAGEA expression and reduced overall patient survival, confirming the clinical significance of our work. At the molecular level, we demonstrated that MAGEA2 protein forms complexes with p53 and ERα, resulting in repression of the p53 activity but increased ERα-phosphorylation and transcriptional activity. However, it is still unclear how MAGEA2 regulates p53 and ERα activities. Our preliminary data showed that inhibition of AMPK activity in tamoxifen resistant breast cancer cells significantly up-regulates the activity of p53 deacetylase SIRT1, whereas AMPK activator metformin treatment inhibits MAGEA2 induced tamoxifen resistance. Therefore, we hypothesized that MAGEA2 inhibits AMPK to up-regulate SIRT1 mediated p53 repression. Our preliminary result also showed that MAGEA2 up-regulates ERα signaling pathway related kinases after tamoxifen treatment, including EGFR, Src, FAK. It could be the potential mechanism of MAGEA2 mediated ERα regulation. In this project, we will employ a wide range of molecular biology techniques including co-immunoprecipitation and inducible shRNA knock-down, together with our recently built metastatic breast cancer animal model to study the effect of MAGEA2 on p53 and ERα activities, and examine its potential role in breast cancer EMT and metastasis. Overall, our result will discover new therapeutic targets for metastatic tamoxifen resistant breast cancer treatment.

我们体内外研究发现MAGEA2的过度表达引起乳腺癌细胞他莫昔芬耐药,进一步实验显示MAGEA2能抑制他莫昔芬诱导p53调节的生长停滞,同时激活ERα介导细胞增殖,并且预实验显示其参与调控耐药癌细胞EMT和转移。但MAGEA2调控p53和ERα的机制尚未报道。预实验显示耐药癌细胞的AMPK活性下调导致p53脱乙酰酶SITR1被激活,并且AMPK活化剂二甲双胍逆转MAGEA2诱导他莫昔芬耐药。我们提出MAGEA2通过抑制AMPK上调SIRT1活性从而抑制p53的新机制。预实验也表明MAGEA2在他莫昔芬刺激下上调ERα信号通路相关激酶Src,FAK等的活性,我们推测其为MAGEA2有待发现的ERα功能调节方式。本课题拟采用免疫共沉淀,shRNA和我们建立的转移模型等揭示MAGEA2调节p53和ERα活性的分子机理,阐述其调控耐药癌细胞EMT和转移的功能,为耐药性治疗提供新的分子靶点。

雌激素受体(Estrogen Receptor,ER)阳性乳腺癌患者常在治疗过程中对内分泌治疗药物他莫昔芬产生耐药性。我们前期发现MAGEA2参与雌激素受体阳性乳腺癌患者他莫昔芬耐药和癌症进展的调控,但背后机制不明。在本项目的支持下,我们利用蛋白组学和代谢组学联合分析技术以及一系列生物研究实验,发现了MAGEA2下游相关转录因子EHF通过上调鞘脂代谢酶神经酰胺激酶(Ceramide kinase,CERK)的表达,进而通过催化神经酰胺磷酸化(Ceramide-1-phosphate,C1P)来抑制他莫昔芬诱导的神经酰胺(Ceramide,Cer)的累积，进而阻止了其对ER参与调控的PI3K-AKT信号轴介导的细胞增殖通路的抑制,并同时下调p53-caspase-3介导的细胞凋亡的激活,最终导致ER阳性的乳腺癌细胞对他莫昔芬耐药。临床数据分析也发现EHF或CERK的高表达与ER阳性乳腺癌病人预后较差和癌症进展加快正相关,且它们之间的表达也存在正相关的联系。原位乳腺癌动物实验也表明,利用Cripsr-cas9基因工程技术敲除MAGEA2下游分子CERK的表达可有效逆转乳腺肿瘤对他莫昔芬的耐药性,并有效抑制耐药细胞的生长,上皮间质转化(EMT)和转移。总的来说,我们的研究首次揭示了MAGEA2参与调控的EHF-CERK信号轴,通过驱动鞘脂代谢重编程来调节乳腺癌他莫西芳耐药的机制,并发现了有效逆转肿瘤耐药和生长进展的治疗靶点,具有一定的临床转化意义。重要的是,我们的发现首次阐明了鞘脂代谢在癌症生长和进展中起关键作用,提示我们血清中鞘脂代谢产物的水平可能是一种良好的肿瘤生物标志物,值得进一步推行相关临床研究。