HGF介导的EMT在肠癌抗血管生成治疗耐药中的作用及其调控机制

Currently, anti-angiogenesis therapy has been widely adopted in clinical practice of many common malignant tumors. Bevacizumab combined with chemotherapy considerably improved the outcome of metastatic colorectal cancer (mCRC) patients. However, reliable predictors of the benefit gained from anti-angiogenesis therapy for the treatment of mCRC was lacking.. Our previous study found that mCRC patients with higher baseline plasma levels of Hepatocyte growth factor (HGF) had poor response to bevacizumab. Further study showed that HGF could induce epithelial mesenchymal transition (EMT) in CRC cell lines. Furthermore, plasma HGF level was positively correlated with the expression of EMT markers in tumor tissues and was associated with bevacizumab resistance. In vivo experiments showed that combining bevacizumab and c-Met inhibitor could reverse EMT, therefore exerted the best anti-tumor and anti-angiogenesis effect. . The magnitude of benefit from anti-angiogenesis treatment was limited by the development of drug resistance. Our prior study also found that HGF could upregulate the expression of TWIST1, which was reported to be strongly associated with the process of EMT and Vasculogenic mimicry (VM).VM is a blood supply modality, in which tumor cells mimic endothelial cells by undergoing transendothelial differentiation. VM channels could provide an alternate microcirculation for nutrient supply in many common malignant tumors, and was reported to be induced by EMT. VM was resistant to classic anti-angiogenesis drugs and may act as a potential resistance mechanism for bevacizumab.. Therefore, our study will examine the regulation mechanism of TWIST1 in the process of EMT and VM, aiming at revealing the effect of EMT and VM on bevacizumab resistance through activation of HGF/Met-TWIST1 signaling pathway.Our study will provide a better understanding of the underlying cellular and molecular mechanisms of bevacizumab resistance, which may help to look for new target and optimize the use of anti-angiogenesis therapy.

对于晚期结直肠癌治疗的抗血管生成治疗，目前尚缺乏有效生物标志物来甄选敏感获益人群。我们前期研究发现血浆中肝细胞生长因子（HGF）高表达的肠癌患者贝伐珠单抗疗效不佳。进一步实验证实：HGF可以诱导肠癌细胞的上皮-间充质转化（EMT），介导贝伐珠单抗耐药，体内实验证实c-Met抑制剂可以抑制EMT转化，增强贝伐珠单抗的疗效。抗血管生成治疗耐药性的产生是临床上亟待解决的重要问题。血管生成拟态（VM）是指肿瘤细胞分化成“血管内皮样细胞”构成微循环通道，可能参与抗血管生成治疗的耐药，然而机制未明。我们研究发现HGF可能通过上调TWIST1表达，介导EMT和VM的形成。因此，本课题拟进一步探索TWIST1在EMT转化及VM形成中的调控机制，揭示HGF/c-Met/TWIST1通路介导的EMT转化及VM形成对贝伐珠单抗敏感性的影响及临床意义，为克服抗血管生成治疗耐药、优化抗血管生成个体化治疗提供新方向。

对于晚期结直肠癌（mCRC）治疗的抗血管生成治疗，目前尚缺乏有效生物标志物来甄选敏感获益人群。我们的前期研究发现，血浆中肝细胞生长因子（HGF）高表达的mCRC患者一线贝伐珠单抗疗效不佳。为了探索HGF/c-Met通路介导的上皮-间充质转化（EMT）对贝伐珠单抗治疗敏感性的影响，我们开展了进一步的免疫组织化学染色及ELISA实验。结果显示，血浆中及肿瘤组织中的HGF表达水平与肿瘤组织中c-MET蛋白的表达水平呈高度正相关，与EMT蛋白E-cadherin的表达呈高度负相关。随后，我们利用CD31/PAS双染观察50名mCRC患者一线治疗前肿瘤组织中血管生成拟态（Vasculogenic mimicry，VM）结构的数量（形成VM的肿瘤细胞标记为CD34/PAS双阳性），分析其与含贝伐单抗治疗方案无进展生存期（PFS）、总生存期（OS）及客观有效率（ORR）的关系。结果显示，VM的中位数量为3个/每高倍视野（范围：0-20/每高倍视野）；VM的数量与血浆中HGF水平及其他临床病理指标没有相关性。VM的数量与PFS（HR=0.91，P=0.755）、OS（HR=0.92，P=0.830）以及ORR亦无相关性。我们随后调取134例接受一线含贝伐珠单抗方案治疗的mCRC患者的组织标本，分析肿瘤组织中结肠癌转移相关基因-1（metastasis-associated in colon cancer-1，MACC1）表达水平与含贝伐珠单抗方案疗效的相关性。我们发现，肿瘤组织中的MACC1的高表达与患者年龄大于60岁以及治疗前外周血CEA < 20 ng/mL相关。COX单因素及多因素回归模型分析显示，治疗前CEA水平以及MACC1表达水平是PFS的独立预测因子（HR =1.10，P =0.002）。MACC1免疫组化评分＞8的患者PFS显著短于评分≤8分的患者（8.0月 vs.12.0月，HR=2.02；P=0.003）。评分＞8的患者对比评分≤8分的患者，OS有缩短的趋势（23.0月 vs. 34.8月，HR=1.51；P=0.172）。本研究在转移性疾病的背景下首次报道了MACC1是晚期结直肠癌一线含贝伐珠单抗方案的疗效预测因子，并且MACC1的预后价值独立于其他常见的临床病理指标。本课题将为甄选抗血管生成治疗的敏感获益人群提供基础，为优化抗血管生成的个体化治疗提供新方向。