肝细胞生长因子（HGF）调控结肠癌Mcl-1表达的分子机制

Yearly, about 1.2 million cases of colorectal cancer (CRC) occur worldwide. It is one of the most common malignancies and the third leading cause of cancer-related mortality in both men and women. The general 5-year survival rate is estimated to be 65%, declining to 11% if distant metastases are present. Within three years after the diagnosis of CRC, 29% of the patients will develop liver metastases. This condition will be fatal if left untreated, with a median survival measured in months. It is imperative to dissect what mechanism behind CRC progressing in order to find a way to treat the disease..The TGF-β signaling pathway is involved in the control of several biological processes, including cell proliferation, differentiation, migration and apoptosis. It is one of the most commonly altered cellular signaling pathways in human cancers. The overall incidence of TGFBR2 mutations is close to 30% in colorectal cancer and is the most common mechanism identified so far that results in TGF-β signaling alteration; SMAD4, originally identified as a tumor-suppressor gene lost in pancreatic cancers called DPC4 (deleted in pancreatic cancer 4), is mutated in 16-30% of colorectal cancer. Recently, we have reported the dosage decreased expression of Tgfbr1 in mice (Cancer Research, 2009) and allele specific expression (ASE) of TGFBR1 in human (Science, 2008) is associated with development of CRC. Our (Cancer Cell, 2005; JBCs, 2002) and others results showed that HGF/c-Met signaling often up-regulated in various types of solid cancers including head and neck cancer and CRC. Subsequent reports have largely confirmed that MET mRNA/protein is up-regulated in greater than two-thirds of CRC. Thus, available data indicate that MET overexpression is important in CRC progression. Our preliminary data showed that HGF could only induce Mcl-1 expression in SMAD4 mutant SW620 cells and not in the SMAD4 re-stored SW620 cells; TGF-β could not induce Mcl-1 expression in SW620 cells with or without SMAD4. Based on ours and others findings, we hypothesized that HGF confer CRC cells the abilities to proliferate, anti-apoptosis, and metastasis which may promote CRC progressing. It is the purpose of the studies outlined in this application to unravel the molecular mechanisms of how HGF promote colorectal cancer progression by induction of anti-apoptotic protein Mcl-1 in altered TGF-β/SMAD cells. We will use Lentivirus technique to re-store SMAD4 in the mutant CRC cells and to knock SMAD4 in the SMAD4 intact CRC cells to dissect detail signaling pathway how HGF execute its significant role for CRC progression in altered TGF-β/SMAD signaling rather than in the TGF-β/SMAD signaling intact cells. Also, we will investigate clinical CRC samples to make a comparison study with laboratory finding. We expect our proposed study will generate some helpful information for individualized management of CRC in future.

癌细胞转移是癌症预后不良的重要原因。我们的前期工作证实了TGF-β信号传导通路的表达异常与结直肠癌（CRC）发生、发展存在相关性（Science, 2009; Cancer Res, 2010）；同时证实HGF通过激活PI3K/AKT和MAPK/ERK传导通路使癌细胞获得抗"失巢凋亡"的能力；HGF激活传统的Notch传导通路促进肿瘤血管生成进而导致肿瘤恶化（Cancer Cell, 2005, JBCs, 2002）。我们的预实验结果显示HGF在诱导抗凋亡蛋白Mcl-1在结肠癌细胞中的表达受细胞内SMAD4存在状况的制约。本项目拟运用SMAD4突变和野生型CRC细胞系通过各种分子生物学手段和方法干扰SMAD4表达，探索HGF在TGF-β/SMAD传导通路变更的条件下调控Mc-1蛋白表达的机制及其在结直肠癌转移过程中的作用，为临床治疗和延缓结直肠癌转移提供新思路。

我们前期工作中发现HGF 在诱导抗凋亡蛋白 Mcl-1 在结肠癌细胞中 的表达受细胞内SMAD4存在状况的制约。本项目旨在利用慢病毒转染从建SMAD4 表达的SW620和HT-116 结肠癌细胞以及 “短发夹RNA”敲除SMAD4 的DLD1 和HCT-15结肠癌细胞，探讨HGF对Mcl-1表达是否受细胞内SMAD4存在的影响，并结合临床病人标本和小鼠肿瘤模型进行验证。本研究中，HGF对SMAD4缺失结肠癌细胞中Mcl-1表达的诱导作用明显高于对SMAD4野生型的细胞；反之，HGF诱导Mcl-1在SMAD4 的重建结肠癌细胞中的表达明显低于其SMAD4缺失的细胞， 证实结肠癌细胞中MCL-1的表达与SMAD4存在状态有关；采用SMADs抑制剂SB431542阻断SMAD4的表达进一步证实了上述结果； MTT法测定SMAD4重建与敲除对细胞增殖试验的结果表明SMAD4存在状态对结肠癌细胞的增殖没有明显影响；Matrigel肿瘤细胞侵润实验结果表明缺失SMAD4的结肠癌细胞对HGF诱导浸润的细胞明显多于SMAD4野生型的细胞。利用构建含有Mcl-1启动子的荧光素酶报告基因载体对不同SMAD4背景的结肠癌细胞株测定HGF对相应Mcl‐1 基因启动子活性的结果显示SMAD4对Mcl-1基因表达有抑制作用。临床结肠癌患者的血样，癌组织，和癌旁组织，分别进行了生化和免疫组化研究结果显示，HGF血浆浓度在中晚期结肠癌患者中明显高于早期患者；免疫组化结果显示Mcl-1在肿瘤组织中表达高于其在癌旁组织对表达；恶性肿瘤高于良性肿瘤；病人血浆HGF含量于其肿瘤的恶性程度成正相关。小鼠肿瘤实验模型结果显示SMAD敲除的结肠癌细胞成瘤率和生长速度明显高于其对照组。小鼠肺转移模型结果显示SMAD4敲除的结肠癌细胞在小鼠肺中形成克隆的数目较SMAD4野生型的细胞明显增多。本研究从分子水平阐明了HGF对抗凋亡蛋白Mcl-1的调控受细胞内SMAD4状态影响，从而较新地揭示了结肠癌的发生和转移机制，不仅为有效地控制结肠癌的转移及治疗提供实验依据，二期为抗肿瘤药物开发提供新的靶点。