他汀类药物下调自噬底物蛋白P62表达治疗肺腺癌溶骨性骨转移的机制研究

Five-year relative survival of lung cancer bone metastasis was less than 5%, and it was lack of effective treatment medicine. Recently it was showed statins had wide antitumor activity.Also statins had bisphosphonate-like manner by preventing mevalonate production, it probably becomes a new kind of medicine for bone metastasis treatment. We previously revealed statins could inhibit tumor migration and invasion of lung adenocarcinoma and induce autophagy leading to metabolic stress, where P62 protein may be one of key functional protein. P62 protein is a substrate of autophagy, and autophagy could degrade p62 protein. But whether statins could induce autophagy by downregulating P62 protein was not clear. Meawhile, latest research showed P62 protein could activate nuclear factor-κb(NF-κb), increasing bone-resorption of osteoclast,causing osteolytic metastasis finally.So downregulation of P62 protein could inhibit bone metastasis, but whether statins depend on P62 protein regulation was not clear. In this study firstly expression of P62 mRNA and protein after statins incubation on lung adenocarcinoma cell SPC-A-1 and A549 were assessed by RT-PCR and westernblot assay.Secondly we upregulate P62 protein expression by plasmid transfection or downregulate P62 protein expression by short hairpin RNA interferrence(shRNA) of lung adenocarcinoma cell SPC-A-1 and A549, and detect tumor migration ang invasion by Transwell migration assay and Matrigel invasive assay after statins incubation. Meanwhile we analyse P62, mTOR1, LC3Ⅱ/Ⅰptotein expression,activation of NF-κb by Electrophoretic mobility shift assay(EMSA) to explore possible signal channel of statins function. Finally we inject shRNA lung adenocarcinoma cell SPC-A-1 to ventriculus sinister of rat osteolytic metastasis model, then evaluate antitumor effect of statins by bioluminescence imaging and X-ray/CT imaging; and discuss the molecular mechanism by flow cytometry analysis, counting osteoclast number with TRAP staining and immunohistochemical staining, to offer new theory evidence for clinical treatment of lung cancer bone metastasis.

肺癌骨转移患者生存率低，有效治疗药物少。近年来发现他汀类药物有广泛抗癌作用，且与双膦酸盐作用机制相似，有望成为新的骨转移癌治疗药物。前期研究提示他汀类药物能抑制肺癌细胞迁移及侵袭，并诱导细胞自噬，其中自噬底物蛋白P62是关键调节蛋白之一,但他汀类药物是否通过下调P62蛋白表达抑制肺癌细胞转移不明确。本研究采用Westernblot检测肺癌细胞经他汀类药物处理后，P62、mTOR1及NF-κb蛋白水平变化，明确他汀类药物是否依赖P62蛋白调节及相关信号传导通路；然后质粒转染上调、shRNA干扰下调P62蛋白表达后，采用划痕实验、基底膜侵袭实验测他汀类药物抑制肺癌细胞迁移及侵袭的变化；最后于溶骨性转移小鼠模型心内注射shRNA P62肺癌细胞，运用活体成像及CT成像，评价下调P62蛋白表达对他汀类药物抗癌作用的影响，并对骨转移组织行流式检测、免疫组化及TRAP染色，分析其抑制肺癌骨转移的机制

30%-40%的肺癌晚期患者出现骨转移，以腺癌多见，5年生存率低于5%，临床有效治疗药物少。近年来发现他汀类药物有望成为新的骨转移癌治疗药物，而P62蛋白与肿瘤及骨代谢关系密切，我们预测他汀类药物有可能通过调控P62蛋白发挥抑制骨转移的作用。首先，我们采用PCR及Western检测8对肺腺癌骨转移活体组织，发现肿瘤组织中P62、Beclin1 、LC3 mRNA及蛋白水平均较正常骨组织升高；对66例肺腺癌骨转移术后术后石蜡标本进行免疫组化检测，发现P62蛋白高表达与多发骨转移、病理性骨折相关。一般认为，作为自噬底物蛋白，P62与LC3的表达呈负相关，但本研究中，Western与免疫组化检测两者并无明显关联，这提示P62蛋白还可能通过自噬以外的途径调控肿瘤骨转移。其次，体外研究采用人肺腺癌A549和SPC-A-1细胞，通过RNA干扰下调P62蛋白表达，不能降低肿瘤细胞增殖，但是采用划痕实验、基底膜侵袭实验，下调P62蛋白表达能够显著下调肿瘤细胞侵袭及迁移能力；同时，体内研究也表明下调P62蛋白表达能够抑制裸鼠种植瘤生长，这说明P62蛋白与肺腺癌转移关系密切。然后，我们判定他汀药物是否通过调控P62蛋白发挥抑癌作用：1、采用不同浓度的他汀药物处理肿瘤细胞，Western检测发现氟伐他汀、阿托伐他汀能够下调P62蛋白表达，同时下游Nf-κb激活受到抑制；2、我们同时检测了自噬的变化：选用阿托伐他汀处理后，western检测结果发现LC3II/I比例升高，电镜结果显示A549细胞的自噬小体数量增加，同时免疫荧光显示也能增加GFP-LC3阳性细胞的比例，说明药物处理后肿瘤细胞发生自噬，但P62蛋白表达并不完全与LC3相反，说明P62蛋白变化并不完全依靠自噬调节。利用BafiIomycin-A1抑制自噬，结果发现阿托伐他汀仍然能够下调P62表达。总之，他汀类药物能够下调P62蛋白的表达，进而抑制肺腺癌骨转移，相关机制可能包括2种：1、依赖于自噬的调节。他汀类药物诱发肺腺癌细胞自噬，造成底物蛋白P62降解下调；2、不依赖于自噬的调节。他汀类药物直接下调P62蛋白表达，下游Nf-κb激活受到抑制，降低肿瘤细胞侵袭及迁移能力，从而抑制肿瘤转移。