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.
