FTO在蒽环类药物诱导的乳腺癌免疫性细胞死亡中的作用及机制

Anthracycline was the first-line chemotherapy agent to breast cancer. But drug resistance limited their further development and applications. Accumulating preclinical and clinical evidence indicates that the success of anthracyclines depend (at least in part) on their ability to stimulate anticancer immune responses. Our previous study found that several mouse cancer cells could be divided into immune responsive (IR) and immune non-responsive (IN) tumor based on their response for anthracyclines treatment. Our further study showed that RIG-I/MVAS/type I interferon signaling on IR cancer cell mediated immunogenic cell death and then stimulated anticancer immune responses, but IN could not activate this pathway and ICD . Moreover, after anthracycline treatment, the expression of FTO (one type of RNA demethylase) was down-regulated which was related with production of type I interferon by IR tumor cell, but the mechanism was not clear. Accordingly, we propose a scientific hypothesis that the production of type I interferon from anthracyclines treated IR cancer cells and subsequent induction of ICD was due to increase of export of MAVS mRNA from the nucleus caused by down-regulate of FTO mediated mRNA methylation (m6A). In this study, RNA FISH, reporter gene, and antitumor vaccination experiments will be used to verify type I interferon autocrine from IR cancer cells through FTO-mediated mRNA methylation. Furthermore, we will evaluate the value of this pathway as a prognostic indicator for anthracycline-based adjuvant chemotherapy and we will find a way to improve the efficacy of IN cancer. The result of this study will provide a scientific basis for elucidating the mechanism of anthracycline resistant, optimizing postoperative adjuvant chemotherapy and improving the sensitivity of chemotherapy, which is of great scientific and clinical significance.

蒽环类药物是乳腺癌一线化疗药物，但部分患者对其不敏感。我们前期实验发现，化疗后，敏感肿瘤通过RIG-I/MVAS/I型干扰素通路诱导免疫性细胞死亡，启动抗肿瘤免疫反应；而不敏感肿瘤无法激活此通路。我们进一步的数据表明，蒽环类药物作用敏感肿瘤后，RNA去甲基化酶(FTO)下调，且与肿瘤细胞自分泌I型干扰素有关，但具体机制不清。据此，我们提出科学假设：蒽环类药物下调敏感肿瘤的FTO，导致mRNA的甲基化（m6A）增多，促进MAVS的mRNA从核内输出，产生I型干扰素，继而诱导免疫性细胞死亡。本研究拟采用RNA FISH、报告基因、抗肿瘤疫苗动物模型等方法，获得蒽环类药物通过FTO调控mRNA甲基化来介导肿瘤细胞自分泌I型干扰素的直接证据；找到诱导不敏感肿瘤免疫性细胞死亡的方法；评估FTO作为乳腺癌化疗疗效指标的价值。本项目将为阐明乳腺癌化疗耐药机制提供新的思路，具有重要的科学和临床意义。

肿瘤化疗仍然是肿瘤联合治疗的核心。化疗导致的死亡肿瘤细胞释放抗原激活免疫系统，使机体产生抗肿瘤免疫反应，造成 “免疫性细胞死亡”。蒽环类化疗药物通过RIG-I/MAVS通路促进肿瘤细胞自分泌I型干扰素是ICD启动的关键机制。m6A是一种可逆性的RNA甲基化,可以促进mRNA从细胞核输出。FTO具有去甲基酶活性。本项目研究中研究者以DOX诱导抗肿瘤免疫反应的敏感和不敏感肿瘤细胞为模型，应用体外稳定沉默或过表达细胞株，以及抗肿瘤疫苗，原位移植瘤等动物模型，发现DOX下调FTO，介导了ICD；进一步用MeRIP-qPCR和m6A sequencing等技术发现DOX可以使MAVS的m6A甲基化增加，促进其mRNA从核内转移至胞浆中，与胞浆中的dsRNA结合从而引起肿瘤细胞自分泌I型干扰素，诱导ICD；最后分析公共数据库中乳腺癌相关数据并收集乳腺癌新辅助化疗前后的标本，经过免疫组化检测，发现FTO的表达水平与I型干扰素下游分子及免疫细胞浸润相关。本项目研究成果为提高化疗药物疗效提供新的思路和方法。