转录辅激活因子SRC-1调控乳腺癌淋巴管生成的作用及分子机制研究

Lymphatic metastasis is the most common pathway for breast carcinoma metastatic spread,but the mechanisms still remain unclear.Our previous study showed that the expression of transcriptional coactivator SRC-1 was positively correlated with an increased number of lymphatic vessels in human breast cancer samples.We further found that the expression of SRC-1 was positively correlated with lymphangiogenesis factor VEGF-C expression in transgenic mice model of breast cancer.Based on that SRC-1 plays an important role in breast cancer metastasis, and VEGF-C is a key lymphangiogenic factor,we presume that lymphangiogenesis of breast cancer may be regulated by SRC-1 through upregulating the expression of VEGF-C,and then breast cancer lymphatic metastasis was promoted.This study will further validate roles of SRC-1 in breast cancer lymphangiogenesis and lymphatic metastasis using transgenic mice models and gene transfection methods. Effects of SRC-1 on biological behavior of lymphatic endothelial cells will be observed.Another aim of this project is to verify that whether SRC-1 promoted lymphangiogenesis in breast cancer by upregulating VEGF-C expression, and to verify AP-1 is a key transcription factor which was coactivated by SRC-1 and eventually they together activated VEGF-C gene transcription and upregulate VEGF-C expression.The molecular mechanisms of SRC-1 in the regulation of VEGF-C expression will be explored in this project.We do believe that our findings should enhance our understanding of the mechanisms of breast cancer lymphatic metastasis.

淋巴转移是乳腺癌转移最主要的方式，其机制尚待阐明。我们前期研究发现，转录辅激活因子SRC-1在人乳腺癌中的表达与肿瘤淋巴管数量显著相关，进一步在转基因小鼠乳腺癌模型中发现SRC-1表达与促淋巴管形成因子VEGF-C表达呈正相关。基于SRC-1在乳腺癌转移中发挥重要作用，VEGF-C是促进淋巴管新生的关键因子，推测SRC-1可能通过上调VEGF-C表达调控乳腺癌淋巴管形成，促进乳腺癌淋巴转移。本项目拟通过转基因小鼠模型和基因转染方法，研究SRC-1在乳腺癌淋巴管形成及淋巴转移中的作用。观察SRC-1对淋巴管内皮细胞生物学行为的影响；明确SRC-1是否通过上调VEGF-C表达促进乳腺癌淋巴管形成，并证实AP-1作为核转录因子受到SRC-1共激活后启动了VEGF-C基因转录，调控VEGF-C表达，探讨SRC-1在转录水平对VEGF-C的调控作用及分子机制。本项目将丰富对乳腺癌淋巴转移机制的认识。

淋巴转移是乳腺癌转移最主要的方式，其机制尚待阐明，我们推测转录辅激活因子SRC-1可能通过上调血管内皮生长因子C（VEGFc）表达调控乳腺癌淋巴管形成，促进乳腺癌淋巴转移。主要研究内容及重要结果：1.培育转基因及基因敲除小鼠MMTV-PyMT;SRC-1-/-小鼠建立原发性乳腺癌动物模型，研究SRC-1对小鼠原发性乳腺癌淋巴管生成及肺转移的影响。结果发现：SRC-1-/-/PyMT+（敲除组）中，小鼠肺转移灶数目明显低于SRC-1+/+/PyMT+（野生型组），并且SRC-1、VEGFc以及微淋巴管密度均也明显低于野生型组，提示SRC-1基因下调与VEGFc表达降低有关，也与乳腺癌肺转移减少有关。2.初步阐明SRC-1对VEGFc的调控以及在乳腺癌淋巴管形成中的分子机制。在小鼠乳腺癌细胞和人乳腺癌MCF-7细胞中，当SRC-1下调后，VEGFc的mRNA的表达下调，利用ELISA检测小鼠乳腺癌细胞和MCF-7细胞，也发现当SRC-1下调后VEGFc蛋白含量下降，而恢复SRC-1表达后可以上调VEGFc的表达，说明SRC-1对VEGFc有调控作用。通过构建VEGFc启动子区域全长的荧光素酶报告载体（pGL3-VEGFc-Luc），证明SRC-1对乳腺癌细胞中VEGFc的转录翻译起着重要的调节作用，并发现SRC-1可以通过转录因子FOS、Twist2、NF-κB和ETV4参与对VEGFc的转录调控；然后再分别通过EMSA实验（体外）和CHIP-PCR实验（体内）进一步验证了FOS、Twist2、NF-κB 、ETV4均有和VEGFc启动子结合能力；最后通过Co-IP证明SRC-1作为辅助活化因子在乳腺癌细胞内能与FOS、Twist2、NF-κB和ETV4蛋白形成蛋白复合物。3）收集人乳腺癌石蜡标本制成组织芯片，检测人乳腺癌中SRC-1表达和微淋巴管形成的关系及与患者临床预后的关联。发现，SRC-1 高表达的乳腺癌组织中微淋巴管数量也明显增多，结合临床随访资料发现： SRC-1高表达和微淋巴管数量多的患者， 5 年总生存率（OS）均明显降低。总之，上述研究揭示了SRC-1通过共激活转录因子FOS、Twist2、NF-κB和ETV4而启动了VEGFc基因转录，调控VEGFc表达以促进乳腺癌微淋巴管生成的新机制，为乳腺癌转移的防治和新药研制提供新的思路及实验数据。