酪氨酸磷酸酶SHP-1去磷酸化调控乳腺癌HER家族分子逆转耐药的机制研究

The HER3/PI3K/Akt signaling pathway can not be effectively inhibited for cross-signaling from related HER/erbB receptors and compensatory signaling from receptors outside of the HER/erbB family, including receptors for IGF-I, VEGF, and c-Met, which play an important role in determining resistance to trastuzumab. Our previos studies have found that tyrosine phosphatase SHP-1 down-regulated in advanced breast cancer tissues and breast cancer cell of herceptin resistance. The growth of herceptin resistance cells transfected SHP-1 was obviously suppressed. Combination of SHP-1 and HER family proteins was identified. However the mechanism also is unclear. We therefore will investigate the function relationship of SHP-1 and HER family receptors using GST pull-down, substrate trapping, BiFC and dephosphorylation analysis.Further, the signaling pathways of HER family regulated by SHP-1 should be analyzed by genes transfection, RNA interference, up-regulation or down-regulation of SHP-1 and so on. Reverse resistance to herceptin and tumor suppression should be studied in level of cellular biology and animal model. In order to provide better and more efficacious treatment strategies for trastuzumab-refractory HER2-overexpresssing breast cancer patients through the dephosphorylation of HER family cross-talk by SHP-1.

表皮生长因子受体家族（HER家族）之间以及同其它分子间的交互作用（cross-talk），常使HER3/PI3K/Akt信号通路不能被有效的抑制，导致针对HER2的靶向治疗耐药。我们前期研究发现，酪氨酸磷酸酶SHP-1在晚期和Herceptin耐药的乳腺癌细胞中表达明显下调，耐药乳腺癌细胞转入SHP-1基因后，细胞增殖明显受抑，并证实SHP-1能与HER家族蛋白相结合，但是具体的调控机制不详。我们拟通过GST pull-down、Substrate trapping、BiFC和去磷酸化分析验证SHP-1与HER家族的功能联系。通过基因转染和RNA干扰，上调和下调SHP-1基因的表达，探讨SHP-1去磷酸化调控 HER家族的具体机制，并在细胞水平和动物模型上观察SHP-1表达前后的抑瘤作用和逆转耐药作用。为通过SHP-1去磷酸化作用抑制HER家族交互活化导致的肿瘤耐药，提供新的治疗思路。

为了明确SHP-1 去磷酸化抑制HER 家族交互活化导致的曲妥珠耐药机制。我们首先构建一系列重组质粒pcDNA3.1+-SHP1-C/S质粒、pGEX-SHP1 WT质粒、pGEX-SHP1 C/S质粒、BiFC 质粒等。通过免疫共沉淀及双分子荧光互补技术分析，结果表明SHP-1可与EGFR和HER-2紧密结合，而 SHP-1与HER-3和HER-4结合水平较弱。体内、体外磷酸化活性分析表明，野生型SHP-1同无磷酸酶活性的SHP-1突变体（C453S）相比，其可以明显降低磷酸化EGFR和磷酸化HER-2的水平，但是未检测到SHP-1和SHP1-C/S与HER3和HER4的结合和去磷酸化作用。证实EGFR和HER-2才是SHP-1的作用底物。进一步通过细胞增殖实验、克隆形成实验、EdU实验、细胞周期及细胞凋亡实验分析表明，SHP-1表达上调后，细胞周期停滞、细胞凋亡增多、细胞生长能力降低。Transwell实验、Boyden实验及划痕实验分析表明，SHP-1表达上调后，细胞侵袭、迁移能力下降。而SHP-1过表达可明显抑制曲妥珠耐药MDA-MB-231细胞的增殖、克隆化生长，促进其凋亡。接着在裸鼠皮下成瘤模型上进一步验证SHP-1基因的抑癌功能，结果显示过表达SHP-1的MDA-MB-231细胞，肿瘤生长速度减慢。同时，SHP-1高表达耐药细胞恢复了对曲妥珠单抗的敏感性，细胞生长速度减慢，SHP-1高表达耐药细胞荷瘤小鼠的生存期明显较耐药细胞荷瘤小鼠生存期长。最后通过免疫组织化学染色分析189例癌组织及39例癌旁正常组织，SHP-1蛋白表达在人乳腺癌组织中表达较癌旁低，进一步表明SHP-1的抑癌功能。生存分析显示SHP-1表达高低与肿瘤大小、ER和PR状态、乳腺癌患者生存相关，可作为独立预后指标之一。此外，我们对具体作用机制进行了深入探讨，发现SHP-1表达上调后，其下游增殖相关蛋白Cyclin D1、c-Myc、k-ras, h-ras, p-Erk1/2下调，与侵袭、迁移相关的蛋白β-catenin、N-cadherin、MMP-9下调，而GSK3β和E-cadherin上调。研究表明SHP-1通过Ras/Erk/GSK3β/β-catenin 信号通路和JAK/STAT3通路而不是PI3K/Akt通路发挥主要作用。