新基因LASS2/TMSG1与液泡型ATP酶C亚基ATP6V0C作用抑制前列腺癌侵袭和转移的分子机制

LASS2/TMSG1 gene is a novel tumor metastasis suppressor gene which was firstly cloned by our laboratory from non-metastatic and metastatic cancer cell variants of human prostate carcinoma using mRNA differential display in 1999. LASS2/TMSG1 could regulate vacuolar ATPase activity and intracellular pH through the intimate interaction of its Homeodomain and the C subunit of vacuolar ATPase（ATP6V0C）,while the precise molecular mechanism of the interaction between LASS2/TMSG1 and ATP6V0C in prostate carcinoma is still unclear. Thus, our study is as follows. ①The effect of LASS2/TMSG1 or the truncated variants or the mutants on the expression and the activity of LASS2/TMSG1 or ATP6V0C, and the behavior of cancer cells will be studied to explore the functional domain of LASS2/TMSG1.②GST pull-down experiments will be performed with GST-fused LASS2/TMSG1 and in vitro transcribed/translated myc-tagged full-length ATP6V0C or deletions of ATP6V0C to find the site requiring for ATP6V0C binding to LASS2/TMSG1.Next, we will examin whether the effect of LASS2/TMSG1 on prostate cancer is ATP6V0C-dependent and analyze the cellular signaling transduction pathways that potentially influenced by LASS2/TMSG1 using PCR Array.③The influence of ATP6V0C overexpression or depletion on the expression and function of LASS2/TMSG1 will be studied. Furthermore,whether phosphorylation of LASS2/TMSG1 is required for its interaction with ATP6V0C in vitra and vivo will be investigated. ④Whether LASS2/TMSG1 and ATP6V0C will become potential biomarkers for prostate cancer aggressiveness will be explored using blood and cancer samples from nude mice and patients.

LASS2/TMSG1基因是我室于1999年应用mRNA差异显示技术从人前列腺癌细胞系中首先克隆出的一种新的肿瘤转移抑制基因。研究内容：①研究LASS2及其缺失体和突变体对LASS2和ATP6V0C表达量及功能的影响，以及对前列腺癌细胞生物学功能的影响，以探寻LASS2的功能位点；②采用GST pull-down实验研究GST-LASS2和Myc标记的ATP6V0C或其截短体之间的作用，以明确ATP6V0C蛋白与LASS2的结合位点；研究LASS2抑制前列腺癌侵袭和转移的作用是否依赖于ATP6V0C；采用PCR array法寻找LASS2新的作用通路；③研究ATP6V0C过表达或基因沉默对LASS2表达量及其功能的影响，并探寻LASS2磷酸化是否是其与ATP6V0C相互作用所必需的；④利用裸鼠和前列腺癌患者的标本，探寻LASS2和ATP6V0C是否可以成为判断前列腺癌预后的新的标志物。

LASS2/TMSG1基因是我室于1999年从人前列腺癌细胞系PC-3M中克隆出的一种新的肿瘤转移抑制基因，它能够与液泡型ATP酶（V-ATPase）的c亚基ATP6V0C直接结合而抑制前列腺癌的侵袭和转移。在本课题中，我们成功构建了表达LASS2/TMSG1全长、7个缺失体（T1-T7）及其4个突变体（M1-M4）的pcDNA3真核表达载体，并稳定转染到高转移潜能前列腺癌PC-3M-1E8细胞系中。结果发现：①LASS2/TMSG1在体内可以和ATP6V0C、STK38和SCYL2发生相互作用，而且LASS2/TMSG1通过和ATP6V0C、STK38和SCYL2相互作用促进β-catenin降解；②LASS2/TMSG1促进β-catenin降解是通过泛素化-蛋白酶体途径，LASS2/TMSG1磷酸化是β-catenin降解所必需的，并且LASS2 /TMSG1第248位丝氨酸是LASS2/TMSG1关键的磷酸化位点；③LASS2 /TMSG1第248位丝氨酸去磷酸化后（突变为丙氨酸，S248A）能促进前列腺癌细胞增殖 、迁移、侵袭和转移，其分子机制是通过负调控Wnt/β-catenin信号通路促进前列腺癌细胞增殖；同时使ATP6V0C表达量增加，进而促进前列腺癌的侵袭和转移。④LASS2/TMSG1是判断前列腺癌预后的重要的分子标记物，磷酸化是LASS2/TMSG1重要的翻译后修饰，而且LASS2/TMSG1蛋白第248位丝氨酸是抑制前列腺癌侵袭和转移的重要功能位点。