Role of MicroRNA-122 in hepatocarcinogenesis using conditional knockout mice

Hepatocellular carcinoma (HCC), the most prevalent liver cancer, ranks third in cancer-related mortality because of ineffective therapy. HCC almost always arises due to complication of underlying liver disease, e.g. hepatitis, fibrosis and cirrhosis. Identification of cellular and molecular entities that directly or indirectly contribute to HCC development will be central to achieve the goal of developing effective therapy. One such molecule that seems critical for the maintenance of normal liver function is miR-122, an abundant liver-specific microRNA. Downregulation of this tiny (22-nucleotide) non-coding regulatory RNA is associated with poor prognosis, tumor recurrence and metastasis in hepatitis C virus negative HCCs. We found that in mice, disruption of miR-122 action in liver results in development of highly penetrant HCC. Both liver-specific (aka LKO) and germ-line (KO) miR-122 knockout mice spontaneously develop steatohepatitis, fibrosis and HCC including lung metastasis. More importantly, we and others found that miR-122 delivery via AAV vector or liposomal nanoparticles inhibited HCC development in different mouse models, suggesting a therapeutic potential of miR-122 in HCC patients. We now propose to use our novel mouse model to delineate the underlying mechanism of hepatitis, fibrosis and hepatocarcinogenesis, and to develop effective therapeutics for liver cancer. To this end, we propose the following aims. Aim 1 will validate novel miR-122 targets in the liver (identified using unbiased Argonaute-HiTS-CLIP and RNA-seq analysis of the wild type and KO livers) that are involved in staetohepatitis, fibrosis, tumorigenesis and metastasis. Aim 2 will test therapeutic efficacy of anti-inflammatory agents in combination with miR-122 delivered using HCC-targeted liposomal nanoparticles. We will focus on the Ccl2-Ccr2 axis since our supporting data showed that blocking this axis reduced inflammation, fibrosis and tumor burden in these mice. Aim 3 will decipher the role of the cellular oncogene c-Myc and its target glutaminase 1 (Gls1) that deamidates glutamine to glutamate, a key energy source to cancer cells, in HCC development in LKO mice using clinically relevant inhibitors of c-Myc and Gls1. This aim draws on our finding that blocking c-Myc gene expression, using the small molecule drug JQ1, inhibited growth, clonogenic survival and promoted apoptosis of cultured human HCC cells. Further, we found that LKO livers and tumors show increased conversion of glutamine to glutamate that correlated with an increase in the c-Myc target Gls1, an enzyme facilitating utilization of glutamine as an energy source. Collectively, the proposed studies will generate pre-clinical results that will be key for designing future clinical trials with one or more of these chemotherapeutic agents in combination with miR-122 for treating a human cancer that is in dire need of new therapy.