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预后不良相关的临床标本进行全局基因表达谱分析。在一项基于基因表达阵列的比较中，我们发现骨桥蛋白（一种分泌的多功能糖蛋白）是能够区分这种表型的主要基因。骨桥蛋白在许多肿瘤类型中表达升高，在转移性肝细胞癌的边缘和肝细胞癌的空化区发现，但在正常肝脏中不存在。我们还证明骨桥蛋白中和抗体可以减少裸鼠肺转移，抑制肿瘤细胞侵袭，强调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一致升高表达，并使用体外系统显示基质金属蛋白酶-9指导骨桥蛋白切割成三个特定片段。一个5千道尔顿的小骨桥蛋白片段可以通过CD44受体诱导细胞入侵，并且可以通过在骨桥蛋白5千道尔顿区域内添加小肽有效阻断。此外，可溶性骨桥蛋白剪接变体的表达增加与临床肝细胞癌转移、细胞侵袭增强和更高的骨桥蛋白5千道尔水平相关。因此，骨桥蛋白的一个独特区域被证明对肝细胞癌的细胞侵袭是最重要的，并且似乎与转移潜力相关。我们的数据还表明，发生了另一种剪接事件，以促进基质金属蛋白酶-9对骨桥蛋白的细胞外切割，从而释放一个5千道尔的骨桥蛋白片段。这项研究的发现可能有助于改善晚期HCC的预后，并提示小肽在新疗法中的应用。由于慢性肝硬化和/或病毒介导的肝硬化，肝细胞癌通常在炎症微环境中发展。在另一项研究中，我们分析了有或无转移的人肝细胞癌患者的基因表达谱。我们已经证明，在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可能是转移性肝细胞癌患者肝脏微环境中存在的独特特征的原因。我们也在集落刺激因子1或骨桥蛋白治疗的丰富的人淋巴细胞群中概括了这种转变。此外，我们已经证明在转移患者中观察到的Th2细胞因子转移涉及T细胞群。这些结果表明，在转移患者的肝脏微环境中，免疫相关基因发生了显著的改变，这似乎与淋巴细胞的差异启动有关，可能是通过基质产生的集落刺激因子1或肿瘤产生的骨桥蛋白的活性。我们最近证明，在肝细胞癌转移过程中，某些小rna（称为microRNAs）的表达水平发生了改变。在一项后续研究中，验证了这一20-microRNA特征，并确定了特定microRNA let-7g在HCC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，并可预测生存率低。功能研究表明，let-7g可通过靶向可溶性胶原，显著抑制细胞迁移和细胞生长。这些结果表明，let-7g可能通过靶向胶原蛋白抑制肝细胞癌转移，并且let-7g可以作为预测预后不良的工具。

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.