转移抑制基因nm23-H1通过抑制Src激酶活性增加肺癌放疗敏感性的作用研究

Cancer radiotherapy sensitivity is closely related to the effect of radiotherapy. Studies showed that epithelial-mesenchymal transition (EMT) contributes greatly to the radiotherapy resistance in cancer cells. Oncogenic Src kinase activity increases aberrantly in cancer and is constitutive active in metastasis cancer. Inhibition of Src kinase activity could reduce the metastasis by inhibiting EMT process and increase the radiotherapy sensitivity in cancer. We previously found that tumor metastasis suppressor gene nm23-H1 could suppress EMT during early stage in lung cancer, loss of nm23-H1 expression leads to the radiotherapy resistance in lung cancer cells; nm23-H1 could repress Src kinase activity by downregulation its Tyr416 phosphorylation; Src kinase inhibitor PP2 could suppress the radiotherapy resistance induced by the loss expression of nm23-H1. These data indicated that nm23-H1 could inhibit Src kinase activity by downregulation its Tyr416 phosphorylation, thereby increase radio-sensitivity by reducing EMT levels. In this study, we will further confirm the role of nm23-H1 in radiotherapy sensitivity through immunohistochemistry, in vivo and in vitro experiments; we will elucidate the molecular mechanisms of Src kinase activity regulation by nm23-H1 through in vitro kinase activity assay, Co-IP, western blot and other experiments. This work will help to clarify the role and mechanisms of nm23-H1 in radiotherapy sensitivity predication in lung cancer and provide important evidence and target for clinical treatment especially radiotherapy.

肿瘤放疗敏感性与放疗疗效密切相关，上皮细胞间质转化（EMT）可以降低肿瘤放疗敏感性。癌基因Src激酶活性在转移瘤中持续活化，抑制Src激酶活性可以抑制EMT，增加放疗敏感性。本课题组前期工作发现肿瘤转移抑制基因nm23-H1可以抑制肺癌细胞EMT，其表达缺失会增加肺癌细胞的放疗抵抗；nm23-H1抑制SrcTyr416位磷酸化水平进而抑制其活性；Src抑制剂PP2可以抑制nm23-H1表达缺失引起的肺癌细胞放疗抵抗，提示nm23-H1可以通过抑制Src激酶活性增加肺癌细胞的放疗敏感性。本项目将通过体内和体外实验，并结合临床组织标本进一步验证nm23-H1在肺癌放疗敏感性中的作用；通过体外激酶活性分析、免疫共沉淀、免疫印迹等方法对nm23-H1调控Src激酶活性的分子机制进行探讨。该研究将为nm23-H1在肺癌放疗敏感性预测中的作用提供重要依据，为肺癌个体化放疗和放射增敏提供新的思路和靶点。

肺癌是当今世界死亡率最高的恶性肿瘤。上皮细胞间质转化（EMT）可以降低肿瘤放疗敏感性，增加放疗抵抗。肿瘤治疗抵抗包括放化疗抵抗、复发和转移的主要原因是癌干细胞（CSCs）或肿瘤起始细胞的存在。lncRNA在肿瘤中的研究主要集中在其表达对细胞周期、增殖和转移中的作用，而在CSCs或癌症干性调控中的作用和分子机制尚不明确。ITGB1是一种新发现的lncRNA，参与胆囊癌和乳腺癌的迁移和侵袭，但在肺癌中的表达和功能尚不明确。本研究中，我们通过慢病毒感染靶向ITGB1表达（shRNA-ITGB1），利用Real time PCR、Western blot、Transwell invasion、Wound healing、细胞克隆形成、肿瘤干细胞微球富集分析及小鼠体内成瘤等方法，以非小细胞肺癌为研究对象，阐述了：（1）ITGB1促进肺癌细胞干细胞微球成球能力和相关干性基因Sox2, Nanog, Oct-4, c-Myc和CD133的表达；（2）ITGB1降表达可以抑制肺癌细胞克隆形成，抑制细胞侵袭和迁移潜能，通过上调E-cadherin和下调Vimentin及Fibronectin抑制EMT过程；（3）ITGB1通过转录因子Snail促进肺癌细胞EMT过程；（4）转录因子Snail介导了ITGB1在肺癌细胞干性调控和迁移及侵袭能力中的作用。本研究提示linc-ITGB1可能是肺癌转移中潜在的促进因子，可以增强肺癌干性和EMT。癌症干性或肿瘤干性是临床上肿瘤转移、复发和治疗抵抗（包括放疗、化疗和生物治疗）的重要原因，本研究为肺癌干性调控的分子机制和肿瘤放疗抵抗（放疗敏感性降低）提供了新的靶点和思路。