GA6在乳腺癌曲妥珠单抗耐药中的作用及机制研究

Breast cancer with HER2 gene overexpression is associated with reduced disease-free and overall survival. Now Trastuzumab is a standard therapy targeted against the HER2 positive breast cancer. In fact, the rate of primary resistance and acquired resistance to trastuzumab is 66-88%. And trastuzmab is very expensive. Patients need cost ￥25000 every therapy cycle. At present, HER2 is the only marker for selecting the trastuzumab therapy. Obviously only one marker is not enough. We have found a new protein GA6, which can active the MAPK/ERK and PI3K/Akt signal pathway when GA6 is overexpressing in MCF-7 cell. We found the patients with GA6-positive showed significantly poor disease-free survival(DFS). In addition, we found GA6 could enhance transcriptional activity of HER family and their protein expression. IP test showed there is interaction between GA6 and HER2. These results prompted GA6 might be associated with trastuzumab resistance . Based on these results, we want to investigate the relation of GA6 expression and the breast cancer prognosis. And we want to know if GA6 expression can forecast the sensitivity of trastumab therapy. We plan to use the breast cancer cell model which are transduced activated GA6 or inactivated GA6 by siRNA to discuss. And we plan to use the western-blot, total precipitation technology immune techniques, EMSA and so on.Generally, we hope to find the molecular mechanism of the relation between the GA6 expression and trastuzumab resistance. And we hope to discover the new molecular biomarker to guide the clinical therapy choice.

HER2阳性乳腺癌复发风险较阴性患者高2-3倍。曲妥珠单抗是临床中针对HER2阳性患者的标准治疗。但其原发或继发耐药的发生率高达 66%-88%。且该药价格昂贵。目前HER2是应用曲妥珠单抗的唯一靶标，但是如此高的耐药显示HER2作为唯一靶标显然是不够的。我们前期研究的新分子GA6在MCF-7细胞中过量表达能激活MAPK/ERK和PI3K/Akt信号途径，该通路激活可能参与曲妥珠单抗耐药。随后研究发现GA6过表达乳癌患者无病生存期短，GA6能升高HER家族成员的RNA和蛋白水平，免疫共沉淀示GA6与HER2存在相互作用，这些提示GA6可能参与曲妥珠单抗耐药。在此基础上，我们拟进一步应用临床标本确定GA6与曲妥珠单抗治疗敏感性的临床意义；利用Western blot、免疫共沉淀、EMSA、RNA干扰等技术确定GA6介导耐药的相关分子机制，为减少及避免耐药提供重要的理论依据和潜在的分子靶标。

乳腺癌是严重威胁女性健康的恶性肿瘤。乳腺癌患者约有20%-30% HER2过表达，HER2过表达常预示着肿瘤的强侵袭性和不良预后。曲妥珠单抗是早在1998年全球第一个被批准治疗HER2阳性晚期乳腺癌的药物，目前也是临床指南推荐的HER2阳性乳腺癌的标准治疗。但是单药曲妥珠单抗原发和继发耐药的发生率在66%-88%。这说明仅凭HER2这样一个分子标志物来决定是否使用曲妥珠单抗是不足够的。因此，阐明曲妥珠单抗原发和继发耐药相关机制及调控因子对于改善HER2阳性乳腺癌患者的预后非常重要。.研究中，我们收集了255例乳腺癌患者的石蜡标本和患者的临床资料，进行了GA6免疫组化检测。我们发现GA6阳性患者较阴性患者的无病生存期（DFS）更短； HER2阳性、激素受体阳性患者，GA6高表达患者的DFS也明显短。多变量COX回归分析显示GA6表达是DFS的预后因素。同时我们对应用曲妥珠单抗联合化疗作为新辅助治疗的乳癌患者进行前瞻性研究，结果显示，HER2阳性、激素受体阳性患者中，GA6过表达者的病理学完全缓解（pCR）率更低，Logistic回归分析显示GA6高表达预示曲妥珠单抗治疗的疗效差。.临床研究中我们发现了GA6是乳腺癌的一个新的不良预后因子，并且与抗HER2治疗药物曲妥珠单抗治疗耐药相关。进一步通过体外实验、转录及蛋白水平检测实验、免疫共沉淀实验等分子机制研究，我们发现;第一，GA6过表达的HER2阳性细胞系对曲妥珠单抗耐药；第二，GA6能够上调HER1、HER2、HER3和HER4的转录，并增强HER1、HER2、HER3和HER4的蛋白表达；第三，GA6通过泛素-蛋白酶体途径抑制HER2降解，延长HER2半衰期；第四，GA6能够抵抗曲妥珠单抗对PI3K/AKT和ERK/MAPK通路的抑制，使得两条通路持续激活，产生耐药。.基于我们对GA6的临床和分子机制研究的结果，我们有理由认为GA6可能是一个新的调控HER2表达并介导曲妥珠单抗耐药的新分子。如果把GA6作为抗肿瘤药物新靶点，构建效果良好的GA6小干扰RNA或拮抗GA6的化学小分子，将GA6有效敲低或抑制GA6功能将可能是克服曲妥珠单抗耐药的很有前景的治疗方案。