内质网应激调控HPV相关口咽鳞癌放射敏感性的机制研究

We previously reported that HPV infection commonly occur in oroopharynx and HPV infected OSCC is very sensitive to radiotherapy, and endoplasmic reticulum stress (ERS) enhances radiosensitivity of oropharyngeal squamous cell carcinomas (OSCC) through the modulation of apoptosis-related factors Bax and HIF-1α, and then inhibition of radiation-induced apoptosis. Given the fact that HPV infection activates endoplasmic reticulum stress (ERS) amd regulates expression of Bcl-2 family members, as well as that ERS level is significantly associated with HPV infection and HIF-1α expression. We hypothesized that ERS may be involved in increased sensitivity to radiation treatment of OSCC, via the modulation of Bcl-2 family members and HIF-1α protein level. To achieve this, we plan to investigate the radiation-induced ERS and radiosensitivity in the HPV infected (HPV+) OSCC compared with its parental tumor without HPV infection (HPV-). First, we will construct stable HPV (+) oropharyngeal cancer cell lines by infecting HPV- OSCC cell lines (FaDu and Detroit562) with the HPV16 oncogene E6 and E7 from Pa317 LXSN16E6/E7. Tumor-bearing mice model with HPV (+) OSCC will then be established. We plan to evaluate the difference in radiosensitivity between the HPV+ OSCC cells and HPV- cells through clonogenic assay and MTT methods. Meanwhile, we will use tunicamycin, the inducer of ERS alone or combined with X-ray radiation to treat the HPV (+) and HPV (-) caner cell lines. siRNAs against signal molecules involved in ERS will be transfected into the cancer cells to see if the radiosensitity effect is reversed after siRNA treatment. Finally we weill implant HPV (-) and HPV (+) OSCC cells into naked mice to confirm the above in-vitro results through quantitation of the ERS level and tumor growth before and after radiation treatment.

课题组前期工作提示HPV感染是介导人口咽鳞癌(OSCC)放射敏感性的内在因素，并证实内质网应激（ERS）通过调节Bax及HIF-1α蛋白表达抑制放射诱导的OSCC细胞凋亡进而调控其放射敏感性，此外结合既往研究结论HPV感染可激活ERS进而调节Bcl-2家族蛋白调控细胞凋亡，且ERS及HPV与HIF-1α表达具有相关性，提出假设：ERS通过调节Bcl-2家族蛋白及活化生存相关蛋白HIF-1α参与调控HPV相关OSCC的放射敏感性，而其对放射诱导ERS的特异性反应是HPV(+)/(-) OSCC放射敏感性差异的内在机制。课题组将建立稳定转染的HPV(+)OSCC细胞系及动物模型，通过克隆实验及肿瘤生长曲线等方法明确HPV(+)/(-)OSCC的放射敏感性差异。应用ERS激动剂以及转染各信号分子反义siRNA、免疫印迹等方法，明确ERS介导HPV相关OSCC细胞放射敏感性主要作用途径及机制。

内质网应激（Endoplasmic Reticulum Stress, ERS）在恶性肿瘤发生、发展以及对放化疗敏感性的调控中扮演着重要角色。课题组首先从临床研究中明确HPV感染与口咽鳞癌的放射敏感性相关，且ERS感应蛋白IRE1α与口咽癌HPV感染存在相关性，此外ERS相关信号通路PERK和IRE1α过表达与口咽癌不良预后相关。 .我们进一步从细胞学研究探求ERS相关信号通路IRE1α-GRP78与HPV相关口咽鳞癌放疗敏感性的相关性。我们转染HPV16E6 plasmid构建HPV(+)口咽癌细胞FaDuS和Detroit562S，克隆形成实验示FaDuS和Detroit562S放疗敏感性明显高于母代细胞FaDu和Detroit562。放疗以时间依赖的方式诱导口咽癌细胞IRE1α-GRP78活化，且IRE1α延迟活化可能是HPV(-)口咽癌放射抗拒的内在原因，沉默IRE1α抑制射线诱导GRP78增加HPV(-)口咽癌放射敏感性。结果示IRE1α活化调控HPV相关口咽鳞癌放射敏感性。.针对HPV(-)口咽癌细胞的放射抗拒性，因此我们的研究重点侧重于寻求提高HPV(-)口咽癌放射敏感性的有效作用靶点。我们构建放射抗拒HPV(-)口咽癌细胞系，结果示PERK-eIF2α在放射抗拒细胞FaDuR和Detroit562R中过表达。克隆形成实验结果示沉默PERK-eIF2α增加口咽癌细胞放射敏感性，且抑制射线诱导NF-κB磷酸化及DNA双链断裂修复，而应用NF-κB激活剂后抵消上述作用。研究结果显示ERS相关通路IRE1α-GRP78和PERK-eIF2α介导生存蛋白调控DNA双链断裂修复及自吞噬诱导口咽癌细胞放射抗拒。.最后我们从临床应用的角度选取有效治疗措施抑制ERS，我们将着眼点定位于近年来头颈部肿瘤治疗热点EGFR靶向治疗。沉默EGFR诱导口咽癌细胞放射抗拒，此外抑制射线诱导的口咽癌细胞ERS信号通路PERK-eIF2α-GRP94及IRE1α-GRP78活化。进一步沉默EGFR和EGFR靶向抑制剂西妥昔单抗抑制射线诱导DNA双链断裂修复及自吞噬，而应用eIF2α磷酸化活化剂Salubrinal及ERS激活剂Tunicamycin抵消了这一作用。本研究结果示抑制EGFR表达增加口咽癌放射敏感性，具体机制与抑制内质网应激通路PERK-eIF2α-GRP94和IRE1α