小GTP酶Rab23通过Rac1调控乳腺癌细胞迁移和侵袭的作用及机制

Breast cancer is the most common cancer in women in the United States and in other industrialized countries, and the second leading cause of death for women with cancer. Despite improved techniques for breast cancer screening, as many as 7% of women present with metastatic disease at the time of diagnosis. While most women are diagnosed at an early stage, 20-80% of these patients, depending on the initial stage and the treatment strategy followed, will develop distant metastasis within 5 years of their initial diagnosis. Metastasis from primary tumors to other tissues accounts for more than 90% of breast cancer related mortalities. Accordingly, prevention of breast cancer metastasis at the earliest stage is important for the management and prediction of breast cancer progression. It has been reported that Rab23, a member of the Rab family of small GTPase, was found overexpression at both the RNA and protein level and strongly association with the invasion of gastric cancer and squamous cell carcinoma. However, at least at present, our comprehension of the role of Rab23 in the cell invasion remains only fragmentary. Supported by NSFC grant (No 30672368 ), we found high expression of Rab23 in breast cancer firstly, and then, we noted that overexpression Rab23 can significantly increase cell migration and invasion for Bcap-37 breast cancer cells in the Wound Heal assy and Transwell assay, however, knockdown of Rab23 by siRNA significantly reduced cell migration and invasion. We also observed that the expression of several important molecules,such as Rac1, MMP2 and MMP9 could change with Rab23 level, suggesting that Rac1 could be somehow regulated by Rab23. However, the molecular mechanism by which Rab23 enhance migration and invasion of breast cancer cell still remains unclear. Therefore, the aim of this study is to comprehensively clarify the mechanism underlying Rab23 promoting cell migration and invasion of s breast cancer. With stable cell lines of different Rab23 activity, we are to analyze the correlation of expression of Rab23 and Rac1 in breast cancer, to explore whether Rac1 play a central role in the regulation of breast cancer cell migration and invasion enhanced by Rab23, to identify the role of integrin β1 in the Rab23-induced cell migration and invasion, to find Rac1's downstream effectors, which play an important role in mediating cell invasion promoted by Rab23. The anticipated results will clarify the molecular mechanism underlying Rab23 enhanced the cell migration and invasion of breast cancer cell through regulating Rac1, help understanding the new functions of Rab23, and possiblly provide a new strategy and new target for the treatment of breast cancer.

乳腺癌患者死亡原因90%系转移所致。肿瘤细胞侵袭是发生转移的早期关键因素，是防治乳腺癌转移的重点。文献表明，小GTP酶Rab23与胃癌、鳞癌侵袭密切相关，但机制尚不清楚。我们前期在国科金（Rab23调控Hedgehog信号通路及其在乳腺癌中作用的研究）的研究中发现，Rab23在乳腺癌中高表达，划痕及Transwell实验明确Rab23可显著促进乳腺癌细胞的迁移与侵袭，初步的机制探讨发现Rab23可调控侵袭关键分子Rac1的表达。本课题拟在此基础上，深入研究Rab23促进乳腺癌迁移与侵袭的机制：明确Rac1在Rab23促进乳腺癌侵袭中的关键作用；分析Rab23对整合素β1的调控；揭示Rab23调控Rac1的作用机制；明确Rac1受Rab23调控后促侵袭的效应分子。预期结果将明确Rab23调控乳腺癌细胞侵袭的分子机制，有助于深入认识Rab23的功能，并为防治乳腺癌转移提供新的策略和治疗靶点。

乳腺癌在女性所患肿瘤中占第二位，而且乳腺癌总体5年转移率高达80%。肿瘤转移是乳腺癌患者死亡的最主要原因，而乳腺癌细胞的侵袭是发生转移的早期关键事件，是防治乳腺癌转移的重点。我们在国家自然科学基金项目（Rab23调控Hedgehog信号通路及其在乳腺癌中作用的研究，No 30672368）资助下，研究发现过表达Rab23可以显著促进乳腺癌细胞Bcap-37的迁移和侵袭，但是，Rab23是通过哪些下游分子、如何调控乳腺癌细胞侵袭的机制尚不清楚。.本研究的目标在于拟通过检测Rab23在乳腺癌组织和细胞中的表达，在体外培养乳腺癌细胞实验中明确Rab23促进乳腺癌侵袭的作用，并观察Rac1在Rab23促进乳腺癌侵袭中的关键作用及其效应分子，分析Rab23调控Rac1的机制，分析Rab23对其他重要的侵袭相关分子的影响及作用，为全面阐明Rab23促进乳腺癌侵袭作用的分子机制奠定基础。已按计划达到预期目标。.1、检测了Rab23对乳腺癌细胞侵袭和迁移的影响。通过检测正常乳腺组织、乳腺癌及不同乳腺癌细胞系中Rab23的表达，检测乳腺癌细胞系中Rab23不同表达水平对乳腺癌细胞侵袭和迁移的影响，明确了Rab23能够明显促进乳腺癌细胞的侵袭与迁移。.2、明确了Rac1在介导Rab23促进乳腺癌侵袭中的关键作用。研究发现不同方法抑制或降低Rac1活性后均可显著下调Rab23对乳腺癌细胞侵袭及迁移的促进作用，表明Rac1是Rab23促进乳腺癌侵袭与迁移的关键分子。.3、阐明了Rab23调控Rac1的作用方式及二者之间的相互作用机制。虽然激光共聚焦检测证实Rab23与Rac1存在共定位，但免疫共沉淀发现二者之间无直接相互作用，而与Integrin β1和Tiam1有直接相互作用，且抑制Integrin β1可明显减少Rab23对Rac1的调控，表明Rab23对Rac1的调控是通过Integrin β1和Tiam1完成的。.4、探讨Rab23表达对紫外线照射诱导乳腺癌Bcap-37细胞自噬的影响。过表达Rab23明显降低紫外线照射诱导乳腺癌Bcap-37细胞的自噬水平，而降低Rab23的表达增强紫外线照射诱导乳腺癌Bcap-37细胞的自噬水平。