PKCe及MTDH/AEG1的相互作用与乳腺癌转移及化疗耐药的关系

PKCe Thr/Ser kinase is the focus of tumor research because it acts as intracelular receptor of natural carcinogen of TPA/PMA. Although its overexpression has been found closely correlated with the initiation, metastasis and chemoresistance of breast cancer, the mechanisms of overexpression have not been reported to our best knowledge. Our preliminary experiments showed that PKCe overexpression is not only the result of high level of mRNA, but also the decreased degradation of the protein. To further study the mechanisms of PKCe overexpression in breast cancer metastasis and chemoresistance, co-immunoprecipetaion and protein mass spectrometry analysis were applied to identified the key point interaction signaling molecules of PKCe. MTDH/AEG1 oncoprotein was found to be one of the molecules interacting with PKCe in breast epithelial cells. MTDH/AEG1 overexpression has been reported in many malignant tumors,including breast cancer. Its overexpresion also highly related with matastasis and chemoresistance of tumors. Both PKCe and MTDH/AEG1 are involving in Ras and PI3K signaling pathway and play as pivotal regulators. In this project, a series plasmid mutants of both PKCe and MTDH/AEG1 will be constructed and co-immunoprecipetation will be applied to identified the anmino acid sequences that responsible for the interaction of PKCe and MTDH/AEG1. Combining with the functional analysis to clarify the molecular mechanisms of PKCe and MTDH/AEG1 interaction in breast cancer metastasis and chemoresistance.

PKCe因其作为自然致癌物佛波酯的细胞内受体而备受关注，近年发现其过量表达与乳腺癌的发生、转移及化疗耐药密切相关，但其过量表达的机制仍未见报导。我们前期的工作表明PKCe在乳腺癌细胞中过量表达不仅与其mRNA的增高有关，而且与其蛋白质的降解减缓密不可分。为了进一步研究PKCe参与乳腺癌转移及化疗耐药的机制，我们采用免疫共沉淀并结合蛋白质质谱分析，发现MTDH/AEG1癌基因与PKCe在乳腺上皮细胞内以复合体形式存在。MTDH/AEG1的过量表达与多种恶性肿瘤的发生、转移及化疗药物耐药有关。由于PKCe与MTDH/AEG1均为Ras、PI3K等信号通路的关键调控分子，本课题通过构建一系列PKCe及MTDH/AEG1的突变体，采用免疫共沉淀方法确定二者相互作用的氨基酸序列，并结合功能分析阐明二者相互作用参与乳腺癌转移及化疗耐药的机制。

乳腺癌是全世界范围内妇女最常见的恶性肿瘤，其远处转移是引起死亡和化疗耐药的主要原因。目前普遍认为各种原因导致的细胞间及细胞内的信号转导紊乱与乳腺癌的发生直接相关。PKC家族激酶（PKCs）作为信号转导通路中的关键效应分子，自上世纪80年代被确定为自然致癌物佛波酯（TPA/PMA）的细胞内受体以来，其与各种肿瘤的发生、演进、转移及预后的关系一直是研究的热点。我们的工作表明PKCe在乳腺癌细胞系中的表达水平明显高于其在永生化正常乳腺上皮中的表达水平，PKCe在乳腺癌细胞中过量表达不仅与其mRNA的增高有关，而且与其蛋白质的降解减缓密不可分。为了进一步研究PKCe参与乳腺癌转移及化疗耐药的机制，我们采用免疫共沉淀并结合蛋白质质谱分析，发现MTDH/AEG1癌基因与PKCe在乳腺上皮细胞内以复合体形式存在。MTDH/AEG1的过量表达与多种恶性肿瘤的发生、转移及化疗药物耐药有关，因而被认定为是一种癌基因。本研究的主要内容是：（1）.采用组织微矩阵免疫组化染色方法，检测PKCe及MTDH/AEG1在乳腺导管上皮增生、乳腺纤维腺瘤及乳腺癌中的表达水平、分布及PPKCe、MTDH/AEG1与乳腺癌临床病理特征与预后的关系，验证细胞学实验的结果; （2）.采用免疫共沉淀方法进一步验证质谱分析的的结果，确定PKCe及MTDH/AEG1是否在细胞内复合体的形式存在；（3）.探讨PKCe、MTDH/AEG1相互作用参与乳腺癌转移及化疗耐药的机制。结果表明：（1）. PKCe及MTDH/AEG1在乳腺癌中的表达均明显高于二者在乳腺纤维腺瘤及乳腺导管上皮普通型增生中的表达；（2）. PKCe及MTDH/AEG1在细胞内以复合体的形式存在；（3）. PKCe及MTDH/AEG1的相互作用导致Akt信号通路活化。PKCe及MTDH/AEG1的相互作用对于进一步阐明乳腺癌转移及化疗耐药的机制有一定意义。