TMEM88通过DVL激活Wnt通路促进非小细胞肺癌恶性表型的机制

TMEM88 is a newly identified protein which is expressed on the membrane of Xenopus cells. It inhibits the Wnt signaling pathway through interaction with DVL-1. We found that TMEM88 is expressed in the cytoplasm of lung cancer cells, and its overexpression is significantly correlated with poor prognosis and lymph node metastasis of patients with lung cancer. Lung cancer cell line with high activity of Wnt signaling pathway also showed overexpression of TEME88 in the cytoplasm. We hypothesize that the cytoplasmic TMEM88 is a potential activator of the Wnt pathway, and it may promote the phosphorylation of DVLs in the form of TMEM88/DVLs/CK1 (CK2 or PAR-1) complex and upregulate the activity of Wnt signaling pathway. We intend to adopt various blockers and activators to bi-directionally regulate the expression of TMEM88, DVLs, and CK1, etc. We will use co-immunoprecipitation, electrophoretic mobility shift assay, luciferase assay and quantitative real-time PCR to clarify, both in vivo and in vitro, the interaction patterns of the cytoplasmic TMEM88 with various DVL subtypes, CK1 (or CK2 or PAR-1) and elucidate their influence to the canonical/non-canonical Wnt signaling pathway. Our goal is to reveal the molecular mechanism that how cytoplasmic TMEM88 promotes the proliferation, invasion and metastasis of non-small-cell lung cancer (NSCLC), and further provide the experimental basis for the discovery of new markers of NSCLC and the development of targeted medicine.

TMEM88是一个新近被发现的存在于爪蟾细胞膜上的蛋白，通过DVL-1抑制Wnt信号通路。而我们发现TMEM88存在于肺癌等细胞浆中，且其高表达与肺癌淋巴结转移等不良预后明显相关，在Wnt通路高活性的肺癌细胞浆中TMEM88高表达，故提出胞浆中TMEM88是Wnt通路潜在的激活子，可能以TMEM88/DVLs/CK1（CK2或PAR-1）复合物形式促进DVLs磷酸化而正向调控Wnt信号通路。我们拟双向调控TMEM88、DVLs、CK1等的表达，使用各种阻断剂和激活剂，用免疫共沉淀、电泳迁移率实验、荧光素酶报告基因及实时定量PCR等方法，在体内外阐明细胞浆中的TMEM88与DVL各亚型及CK1(或CK2或PAR-1)的相互作用模式和对经典/非经典Wnt信号通路的影响，揭示胞浆中TMEM88促进非小细胞肺癌增殖、侵袭转移的分子机制，为寻找新的非小细胞肺癌标记物和开发靶向治疗药物提供实验基础。

TMEM88是一个潜在的跨膜蛋白，在胚胎发育和干细胞分化方面发挥着重要作用，然而其在人类组织尤其是恶性肿瘤组织中的表达和亚细胞定位情况尚不清楚，其所发挥的生物学作用也不得而知。我们通过对肺癌，乳腺癌，结肠癌，胃癌，肝癌等恶性上皮性肿瘤进行免疫组织化学染色和Western blot实验发现，TMEM88肺癌等组织中存在着明显的胞浆过表达,仅有极少数膜表达，而且其胞浆高表达与肺癌的低分化，高TNM分期，淋巴结转移及不良预后明显正相关。TMEM88仅在极少的肺癌组织中呈现胞膜表达。我们选择了TMEM88细胞膜表达的LK2及TMEM88细胞浆表达的A549，H1299等细胞，通过MTT,transwell，裸鼠成瘤等功能实验证实了TMEM88在膜表达细胞中抑制肺癌细胞的侵袭和增殖，而在浆表达细胞中促进肺癌细胞的侵袭，但对增殖没有影响。我们通过co-IP和激光共聚焦实验证实TMEM88可以和Wnt通路核心蛋白DVL分别在LK2的细胞膜和H1299的细胞浆处存在共定位。TMEM88在膜表达细胞中，可以通过与DVL结合抑制经典Wnt信号通路而抑制肺癌细胞的侵袭和增殖；而在浆表达细胞中，TMEM88对Wnt信号无明显影响，却能通过与DVL结合，促进P38和GSK3β的磷酸化，而稳定Snail蛋白，进而下调Occludin和Zo-1促进肺癌细胞的侵袭。综上，我们提出不同定位的TMEM88发挥着不同的生物学功能，为肺癌的防治提供了新的理论基础和潜在的治疗靶点。主要论文发表在Cancer research上，受此课题资助共发表SCI论文16篇，其中JCR 1区1篇，IF:8.556；JCR 2区3篇，累计影响因子达54.975