ERAA对舌鳞癌细胞顺铂化疗敏感性的影响

Endoplasmic reticulum stress (ERS) is an important form of cell response to environment stress, such as nutrient deprivation, hypoxia, chemotherapy and radiotherapy. Under low stress, ERS stimulates the unfolded protein response (UPR) to protect the cells, however, a severe or prolonged stress induces cell apoptosis. The ability of certain chemotherapies to cause cell death in cancer cell lines that display resistance to apoptosis may be dependent on autophagy. ER-activated autophagy (ERAA) pathway serves the function of mitigating ER stress and suppressing cell death, inhibition of ERAA may be explored for sensitizing resistant tumor cells to cytotoxic agents. But autophagy in the ERS of tongue squamous cell carcinoma(TSCC) are poorly investigated. Therefore, the aim of this study is to investigate the molecular mechanisms and function of ERAA in TSCC induced by cisplatin. .In our previous studies, we found (i): autophagy gene Beclin1 was associated with the resistance of TSCC to cisplatin; (ii): cisplatin induced ERS and autophagy of TSCC; (iii): Co-administration of cisplatin with autophagy antagonist 3-MA caused synergistic cell Killing; (iiii): During ERS of TSCC, Akt phosphorylation was depressed. As we know, Akt dephosphorylation depresses mTORC1 phosphorylation at Ser2448 and its activation. In the recent studies, some scientists found ERS induced the expression of HIF-1α by repressing mTORC1 phosphorylation, further, induced the expression of BNIP3 and Noxa, both pro-apoptosis members of BH3-only subfamily; BNIP3 and Noxa displaced the pro-survival Bcl-2 family member, Mcl-1, Bcl-2 or Bcl-xL from Beclin1, and then free Beclin1 allowed for autophagy initiation. So we presume that mTORC1/HIF-1α/BH3-only(BNIP3/Noxa) pathway induced the resistance of TSCC to cisplatin by regulating Beclin1..In the present study, two human TSCC cell lines (Tca8113 which is sensitive to cisplatin and CAL-27 resistant to cisplatin) will be induced ERS by cisplatin and then siRNA HIF-1α, siRNA Beclin1 or 3-MA will be combined with cisplatin respectively in vitro, the effects of these meatures on the autophagy and apoptosis associated genes (mTORC1, HIF-1α, BNIP3, Noxa, LC3-I/II, p62, Grp78 and caspase-3,4,9) will be detected by real-time PCR and western blot, and the apoptosis will be detected by flow cytometer, at the same time, the ERS and autophagy ultrastructures will be observed by transmission electron microscopy. At last, animal model using CAL-27 cell line will be established, the proliferation repression of combination siRNA Beclin1 and cisplatin on the CAL-27 xenograft will be detected and the molecular mechanisms will be discussed. We will turn out mTORC1/HIF-1α/BH3-only(BNIP3/Noxa) pathway is responsible for TSCC resistance to cisplatin and Co-administration of cisplatin with autophagy antagonist caused synergistic cell killing by weakening autophagic cell protection, which will provide a new strategy for the comprehensive treatment of TSCC.

内质网应激（ERS）可诱导癌细胞凋亡，ERS激活自噬参入调控癌细胞凋亡，但自噬在顺铂诱导舌鳞癌细胞ERS过程中的作用尚不清楚。我们前期研究发现自噬基因Beclin1与舌鳞癌对顺铂耐药有关，最近研究发现ERS通过抑制mTORC1磷酸化诱导HIF-1α过表达，后者诱导BH3-only亚家族的促凋亡蛋白BNIP3和Noxa的表达，后二者和Beclin1竞争与Bcl-2家族抗凋亡蛋白结合，释放出游离的Beclin1活化自噬发挥促生存作用，但该通路是否与舌鳞癌对顺铂耐药有关尚不明确；本课题拟用对顺铂敏感和耐药的两个舌鳞癌细胞系为研究对象，顺铂诱导ERS，利用siRNA(HIF-1a、Beclin1)和3-MA进行体外干预并检测干预措施对舌鳞癌细胞自噬和凋亡的影响；进一步探索顺铂+自噬阻断剂对舌鳞癌移植瘤的生长抑制作用及机制，证实ERS激活自噬影响舌鳞癌对顺铂化疗敏感性，为舌鳞癌的综合治疗提供新思路。

头颈鳞状细胞癌（head and neck squamous cell carcinoma，HNSCC）经典治疗手段是手术和放疗，然而近30年来，其5年生存率一直维持在50%左右。临床采用以顺铂为基础的化疗方案作为晚期HNSCC的辅助治疗手段，但部分患者对其不敏感或治疗后耐药，5年生存率提高有限（2%~8%），所以有必要探索其不敏感或耐药的机制。HNSCC为实体瘤，随着肿瘤生长，癌细胞经常面临代谢产物的蓄积和营养要素的缺乏以及放化疗的毒性从而启动应激反应；内质网是细胞内蛋白质合成、折叠、修饰和运输以及Ca2+存储的主要场所，所以内质网应激（endoplasmic reticulum stress，ERS）是细胞对外界环境变化做出反应的重要组成，既往研究发现ERS启动非折叠蛋白反应（unfolded protein response，UPR），使细胞渡过应激期，当导致ERS的刺激因素强烈而持久不能解除时，启动内源性凋亡通路。最近研究发现，ERS还激活自噬（ER-activated autophagy，ERAA）缓解应激，ERAA通过自噬体-溶酶体途径降解细胞内过剩蛋白质和受损细胞器从而促进细胞生存。Beclin1基因是第一个发现的人自噬基因，位于17q21，生理条件下在人正常组织高表达；在人肿瘤组织和肿瘤细胞系低表达；进一步研究表明Beclin1是一个重要的自噬基因，其表达缺失在早期促进肿瘤形成；肿瘤形成后Beclin1促进了肿瘤恶性进展和治疗抵抗。本课题在前期研究的基础上，采用顺铂诱导舌鳞癌细胞Tca8113和CAL-27发生ERS，利用siRNA (HIF-1α、Beclin1)和自噬阻断剂3-MA进行体外干预并检测干预措施对舌鳞癌细胞自噬和凋亡的影响；在细胞水平探索ERAA的作用及其机制，证实mTORC1/HIF-1α/BH3-only通路调控Beclin1自噬复合物是舌鳞癌耐受顺铂的重要机制，在此基础上我们还发现，abl与TFEB相关作用，介导溶酶体参入调控舌鳞癌细胞对顺铂细胞的敏感性和耐药。上述研究成果为HNSCC尤其是对顺铂耐药的HNSCC的综合治疗提供新的思路。