跨膜MARCH9泛素连接酶促进结肠癌转移及其分子机制

MARCH9 E3 ligase is the membrane-associated enzyme that induces membrane protein ubiquitination and promotes its lysosomal degradation in the cell. However, its role in tumorigenesis remains unknown thus far. We have recently found that, by using the Cancer Genome Atlas (TCGA), MARCH9 E3 ligase is overexpressed in colon cancer tissue. Further, we found that the enzyme was highly expressed in metastatic colon cancer cell lines. Knockdown of MARCH9 significantly decreased metastatic colon cancer cell migration and invasion in vitro, whereas ectopic expression of the protein in colon cancer cells with low metastatic potential surprisingly promoted their transwell migration. These results suggest that MARCH9 may serve as the promoter of metastatic cell behavior in colon cancer. In our study, we will explore this hypothesis by focusing on the work as follows: .1).To evaluate the effect of MARCH9 expression on colon cancer metastasis using cell line and CAM model;.2).To identify and characterize MARCH9 substrate(s) that contribute(s) to colon metastasis;.3).To test the in vivo function of MARCH9 expression on colon cancer liver metastasis by using orthotropic graft colon cancer mouse model;.4).To evaluate whether MARCH9 expression is correlated with colon cancer metastasis in clinical settings..Hopefully, our work may define the role of MARCH9 E3 ligase in colon cancer metastasis and contribute to develop a new strategy to inhibit this malignancy.

MARCH9泛素连接酶介导膜蛋白泛素化并促进其在溶酶体内降解，但该蛋白在肿瘤中的作用迄今未明。我们前期通过TCGA数据库发现MARCH9蛋白在结肠癌组织过度表达；随后实验证实MARCH9蛋白表达下调能够抑制高转移结肠癌细胞体外迁移及侵袭，而其过度表达则促进低转移肿瘤细胞的迁移。我们由此推测MARCH9蛋白具有促进结肠癌细胞远端转移的作用。为了证实这一假说，本项目拟从体外评价MARCH9蛋白差异表达对结肠癌细胞转移的影响；筛选和鉴定肿瘤转移相关的MARCH9蛋白底物，并评价其在MARCH9蛋白介导结肠癌转移中的作用，最终揭示MARCH9蛋白通过调节其下游底物促进肿瘤转移的分子机制；动物模型研究MARCH9蛋白对结肠癌细胞肝转移的影响及机制；最后结合临床标本评价MARCH9蛋白表达与结肠癌转移的相关性。本项目完成将发现结肠癌转移新机制及干预新靶点，为今后抗结肠癌转移治疗及疾病预后提供科学依据。

肿瘤转移是恶性肿瘤发展过程中最凶险的阶段。因此，控制肿瘤转移是提高临床疗效的关键性措施。但迄今为止，临床上仍缺乏特异、有效的抗肿瘤转移药物。鉴定肿瘤转移新机制及治疗靶点对于开发治疗新药及制定有效的治疗策略具有重要的科学意义及临床价值。基于前期研究基础，本项目设想膜相关泛素连接酶MARCH9在肠癌组织异常过表达能够促进胞内特定蛋白泛素化而导致下游肿瘤相关信号通路过度激活，最终促进肠癌远端转移。为了证实上述研究假说，本研究联合基因过表达技术、RNA干扰技术、CRISPR基因编辑技术及不同生化手段，从临床前模型层面探索MARCH9泛素连接酶表达对肠癌细胞转移的影响及调控分子机制。研究结果发现MARCH9蛋白在肠癌等等肿瘤组织中异常高表达，且其表达水平与患者预后密切相关；MARCH9泛素连接酶能够促进肿瘤胞内PTPRE磷酸酶泛素化及活性，后者通过激活SRC及相关信号通路，最终促进肠癌细胞远端转移。在此基础上，本项目成功开发了靶向“MARCH9-PTPRE-SRC”信号轴的抑制性多肽，其被证实能够阻断MARCH9泛素连接酶与PTPRE磷酸酶的相互作用，从而抑制下游促癌蛋白SRC的活性。鉴于促癌蛋白SRC的小分子抑制剂达沙替尼（Dasatinib）已获准临床应用，本项目设计的PTPRE磷酸酶抑制性多肽具有良好的临床转化前景。总体上，本项目成功鉴定一种以“MARCH9-PTPRE-SRC”信号轴为基础的肠癌转移新机制及干预新靶点，并成功开发了靶向上述相关信号通路的抑制性多肽，达到预期目标。