The identification of human hepatocellular carcinoma metastasis genes

我们正在使用微阵列技术对与人类肝细胞癌转移和不良预后相关的临床标本进行全球基因表达谱分析。在基于基因表达阵列的原发性肝肿瘤伴或不伴肝内转移的比较中，我们发现骨桥蛋白是一种分泌的多功能糖蛋白，是能够区分这一表型的先导基因。骨桥蛋白在转移性肝细胞癌的前缘和肝细胞癌的空泡区均有表达，但在正常肝组织中不表达。我们还证明了骨桥蛋白的中和抗体可以减少裸鼠的肺转移和抑制肿瘤细胞的侵袭，突出了骨桥蛋白在肝细胞癌转移中的重要作用。我们还观察到骨桥蛋白和基质金属蛋白酶-9在原发性转移性肝细胞癌中的表达一致升高，并利用体外系统显示，基质金属蛋白酶-9引导骨桥蛋白切割成三个特定的片段。一个小的5千道长的骨桥蛋白片段可以通过CD44受体诱导细胞侵袭，并可以通过在5千道尔顿的骨桥蛋白区域内加入小肽而被有效地阻断。此外，可溶性骨桥蛋白剪接变异体的表达增加与临床肝细胞癌转移、增强的细胞侵袭性和较高的骨桥蛋白5千道尔顿水平有关。因此，骨桥蛋白的一个独特区域被证明是肝细胞癌细胞侵袭最重要的区域，并且似乎与转移潜能相关。我们的数据还表明，发生了另一种剪接事件，促进了基质金属蛋白酶-9对骨桥蛋白的细胞外切割，从而释放了骨桥蛋白5千道尔顿的片段。这项研究的发现可能有助于改善晚期肝癌的预后，并提示小肽用于新的治疗方法。由于肝细胞癌通常在慢性肝硬变和/或病毒介导性肝硬变的炎症微环境中发展。在另一项研究中，我们分析了有或没有转移的人肝细胞癌患者的基因表达谱。我们已经证明，在有转移的患者样本中发生了向抗炎Th2细胞因子的转变。我们证明集落刺激因子1可能与转移性肝细胞癌患者肝脏微环境中存在的独特信号有关。我们还总结了用集落刺激因子1或骨桥蛋白处理的丰富的人类淋巴细胞群体中的这种转变。此外，我们已经证明在转移患者中观察到的Th2细胞因子的转变涉及T细胞群体。这些结果表明，转移患者肝脏微环境中免疫相关基因发生了显着改变，这可能是通过基质产生的集落刺激因子1的活性或肿瘤产生的骨桥蛋白来实现淋巴细胞的差异性启动。我们最近已经证明，在肝细胞癌转移过程中，某些被称为microRNAs的小RNA的表达水平发生了变化。在一项后续研究中，验证了这一20微RNA签名，并确定了特定的微RNA let-7g在肝癌进展中的作用。我们证实，与非转移性肝细胞癌相比，转移性肝细胞癌的let-7g水平显著降低，并预示着预后不良。功能研究表明，let-7G通过靶向可溶性胶原蛋白，显著抑制细胞迁移和生长。这些结果表明，let-7g可能通过靶向胶原蛋白抑制肝细胞癌的转移，let-7g可以作为预测预后的工具。

We are using a microarray technology to perform global gene expression profiling of clinical specimens that are associated with human hepatocellular carcinoma metastasis and poor outcome. In a gene expression array-based comparison of primary liver tumors with or without accompanying intrahepatic metastasis, we found that osteopontin, a secreted multifunctional glycoprotein was a lead gene capable of differentiating this phenotype. Osteopontin, whose expression is elevated in many tumor types, was found at the leading edge of metastatic hepatocellular carcinoma and in vacularized regions of hepatocellular carcinoma, but was absent in normal liver. We also demonstrated that a neutralizing antibody to osteopontin could decrease lung metastases in nude mice and inhibit tumor cell invasion, highlighting an essential role of osteopontin in hepatocellular carcinoma metastasis. We have also observed a concordant elevated expression of osteopontin and matrix metalloproteinase-9 in primary metastatic hepatocellular carcinoma and using an in-vitro system, showed that matrix metalloproteinase-9 directs the cleavage of osteopontin into three specific fragments. A small 5-kilodalton osteopontin fragment could induce cellular invasion via CD44 receptors and could be effectively blocked by the addition of small peptides within the 5-kilodalton region of osteopontin. Furthermore, increased expression of a soluble osteopontin splice variant was associated with clinical hepatocellular carcinoma metastasis, enhanced cellular invasion and higher osteopontin 5-kilodalton levels. Thus, a distinct region of osteopontin was shown to be most essential for hepatocellular carcinoma cellular invasion and appeared to correlate with metastatic potential. Our data also suggests that an alternative splicing event occurs to promote extracellular cleavage of osteopontin by matrix metalloproteinase-9 to release an osteopontin 5-kilodalton fragment. The findings of this study may help to improve advanced stage HCC prognosis and suggests a utility of small peptides for novel therapies. Since hepatocellular carcinoma usually develops in an inflamed microenvironment due to chronic cirrhosis and/or viral mediated cirrhosis. In another study, we analyzed gene expression profiles of human hepatocellular carcinoma patients with or without metastasis. We have shown that a shift towards anti-inflammatory Th2 cytokines occurs in patient samples with metastasis. We demonstrated that colony stimulating factor 1 may be responsible for the unique signature present in the liver microenvironment of metastatic hepatocellular carcinoma patients. We have also recapitulated this shift in an enriched human lymphocyte population treated with colony stimulating factor 1 or osteopontin. Furthermore, we have shown that the Th2 cytokine shift observed in metastasis patients involves a T cell population. These results show that a significant alteration of immune related genes occurs in the liver microenvironment of patients with metastasis that seems to involve differential priming of lymphocytes possibly through the activity of stroma-produced colony stimulating factor 1 or tumor produced osteopontin. We have recently demonstrated that the expression levels of certain small RNAs, termed microRNAs, are altered in hepatocellular carcinoma metastasis. In a follow-up study, this 20-microRNA signature was validated and the role of a particular microRNA, let-7g in HCC progression, was determined. We confirmed the that the level of let-7g was significantly lower in metastatic compared to non-metastatic hepatocellular carcinoma and was predictive of poor survival. Functional studies indicated that let-7g could significantly inhibit cell migration and cell growth through targeting of soluble collagens. These results suggest that let-7g may suppress hepatocellular carcinoma metastasis through targeting collagen and that let-7g could be used as a tool to predict poor survival.