Role of microR-122 in Hepatocarcinogenesis using Conditional Knockout Mice

描述(申请人提供)：肝细胞癌(肝细胞癌)是最常见的肝癌，由于治疗无效，在癌症相关死亡率中排名第三。肝细胞癌几乎总是由潜在的肝病并发症引起，例如肝炎、纤维化和肝硬变。确定直接或间接促进肝细胞癌发展的细胞和分子实体将是实现开发有效治疗方法的目标的关键。其中一个对维持正常肝功能至关重要的分子是miR-122，它是最丰富的肝脏特异性microRNA。这种小的(22个核苷酸)非编码调控RNA的下调与预后不良、肿瘤复发和转移有关。我们发现，在小鼠中，miR-122在肝脏中作用的中断会导致高穿透性肝癌的发生。这些肝脏特异的miR-122基因敲除(LKO)小鼠会自发发展为肝炎、纤维化和伴有肺转移的肝癌。更重要的是，其他人和我们发现，通过病毒载体(AAV8)或脂质体纳米粒传递miR-122可以抑制不同小鼠模型中肝癌的发展，这表明miR-122对肝癌患者具有治疗潜力。我们现在建议使用我们的新的小鼠模型来描述肝炎、纤维化和肝癌发生的潜在机制，并开发有效的肝癌治疗方法。为此，我们提出了以下目标。目的1以miR-122KO肝脏为阴性对照，通过无偏HITS-CLIP和RNA-SEQ方法验证在野生型肝转录组中发现的miR-122的功能靶点，以及(B)研究miR-122与这些靶点在人原发性肝癌中的反相关性。我们将重点关注在不同阶段促进肿瘤发生的靶点。AIM 2将测试抗炎(CCL2)的治疗效果 中和抗体(CTGF中和抗体)和抗纤维化药物(CTGF中和抗体)与miR-122结合使用时使用的是肝癌靶向脂质体纳米粒。我们将重点关注CCL2-CCR2轴，因为我们的支持数据表明，阻断这个轴可以减少这些小鼠的炎症、纤维化和肿瘤负担，而CTGF则因为它是HITS-CLIP确定的miR-122的直接靶点。本文提出的研究将阐明CCL2和CTGF与miR-122联合作用抑制肝纤维化和肝癌发生的机制。目的3将使用临床相关的c-Myc和GLS1抑制剂，破译细胞癌基因c-Myc及其靶标谷氨酰胺酶1(GLS1)在LKO小鼠肝癌发展中的作用。谷氨酰胺脱氨酶是癌细胞的关键能量来源。这一目的是基于我们的发现，用小分子药物JQ1阻断c-Myc基因的表达，抑制了培养的人肝癌细胞的生长、克隆存活和促进了细胞凋亡。此外，利用同位素研究，我们发现LKO肝脏和肿瘤显示谷氨酰胺到谷氨酸的转化增加，这与c-Myc调节的GLS1的增加相关，GLS1是一种促进谷氨酰胺作为能源利用的酶。总的来说，拟议的研究将产生临床前结果，这些结果将是设计未来将上述一种或多种化疗药物与miR-122结合用于治疗肝癌患者的临床试验的关键。

DESCRIPTION (provided by applicant): 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 contributeto HCC development will be central to achieve the goal of developing effective therapy. One such molecule that is critical to the maintenance of normal liver function is miR-122, the most abundant liver-specific microRNA. Down regulation of this small (22-nucleotide) non-coding regulatory RNA is associated with poor prognosis, tumor recurrence and metastasis. We found that in mice, disruption of miR-122 action in liver results in development of highly penetrant HCC. These liver-specific miR-122 knockout (LKO) mice spontaneously develop hepatitis, fibrosis and HCC with lung metastasis. More importantly, others and we found that miR-122 delivery via the viral vector (AAV8) 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 (a) validate functional targets of miR-122 that are identified in the wild type liver transcriptome by unbiased HITS-CLIP and RNA-seq approach using miR-122KO livers as a negative control, and (b) investigate anti-correlation between miR-122 and these targets in primary human HCC. We will focus on the targets that promote tumorigenesis at different stages. Aim 2 will test therapeutic efficacy of anti- inflammatory (Ccl2 neutralizing Ab) and anti-fibrotic (Ctgf neutralizing Ab) 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, and on Ctgf because it is a direct target of miR-122 identified by HITS-CLIP. Studies proposed here will elucidate the mechanism of suppression of hepatic fibrosis and hepatocarcinogenesis by blocking functions of Ccl2 and Ctgf in combination with miR-122. 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 c-Myc and Gls1 inhibitors. 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, using isotopologue studies, we found that LKO livers and tumors show increased conversion of glutamine to glutamate that correlated with an increase in c-Myc-regulated 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 mor of the above chemotherapeutic agents in combination with miR-122 for treatment of patients with HCC.