候选抑癌基因p140Cap的失活机制及其对结肠癌侵袭转移的研究

Colorectal cancer (CRC), the forth most commonly diagnosed cancer in the world, is from uncontrolled cell growth in the colon, rectum, or appendix. Invasion and metastasis present are the major factors for its prognosis. It is perfectly believed that metastasis prevention is the only way to improve survival rate with advanced cancer. DNA methylation is an important regulator of gene transcription and a large body of evidence has demonstrated that aberrant DNA methylation is associated with unscheduled gene silencing. Hypermethylation is one of the major epigenetic modifications that repress transcription via promoter region of tumor suppressor genes. .Integrins are receptors that mediate attachment between a cell and the tissues surrounding it, which may be other cells or the extracellular matrix (ECM). They also play a role in cell signaling and thereby regulate cellular shape, motility, and the cell cycle. p140Cap (p130Cas associated protein), a novel adaptor protein in integrin family, was recently reported as a potential negative regulator of tumor growth. .Our preliminary data already showed that p140Cap was dramatically reduced in cancer tissues compared with matched normal tissues (Fig 1); demethylation and acetylation restored p140Cap expression in cancer cells (Fig 2); p140Cap overexpression induced anoikis and cell cycle arrest (Fig 3); cell population with low p140Cap expression gained an enhanced adhesion capability (Fig 4). In this proposal, combined bisul?te restriction analysis (COBRA), bisul?te genomic sequencing (BGS), methylation- specific PCR analysis (MSP) and chromatin immunoprecipitation (ChIP) will be used to clarify the mechanisms of p140Cap silencing. In order to observe the role of p140Cap in cell proliferation, anoikis, invasion, migration and drug resistance, we will perform constructs transfection, siRNA transfection, dual-luciferase reporter assay and flow cytometry assay. The correlation among p140Cap silencing, clinical features and pathological features in CRC patients respectively will be assessed at the same time. Furthermore, xenograft model in nude mice will be established to confirm the role of p140Cap during CRC progression in vivo..Based on the previous studies and our preliminary data, this study would elucidate for the first time that p140Cap acts as a tumor suppressor in CRC and plays an important role in CRC cell proliferation, anoikis, invasion and migration. Moreover, we propose to understand the mechanisms of p140Cap silencing during CRC progression by epigenetics analyses. With good design and strong experimental basis, this study would be well done and provide novel opinions and methods for diagnosis, prognosis and treatment of CRC.

转移是导致90％以上肿瘤病人死亡的原因，但转移的分子机制尚无深入认识，故无法对其进行有效干预。DNA高甲基化可导致抑癌基因表达静默，在肿瘤的发生中起到重要作用。p140Cap为一个p130Cas相关蛋白，研究表明其可抑制乳腺癌的生长和转移。本课题组预实验发现，p140Cap在肠癌中低表达，去甲基化和乙酰化可诱导其重新表达；且p140Cap过表达可诱导肿瘤细胞失巢凋亡和阻断细胞周期，是一个潜在的抑癌基因。本研究拟通过MSP、BGS、COBRA和ChIP等方法评估p140Cap失活表达机制；质粒转染、siRNA干扰和流式细胞术等技术探讨p140Cap对结肠癌细胞增殖、失巢凋亡、侵袭和转移的影响；结合临床资料评估p140Cap失活表达与肿瘤患者临床病理特征的关系；采用活体动物构建移植瘤模型，体内进一步验证p140Cap的上述作用与机制。预计完成本课题可为肠癌的诊断、预后及治疗提供新思路和方法。