Hepatocellular carcinoma (HCC) is among the top four leading causes of cancer-related death worldwide with nearly 700,000 deaths per year, and has increased in recent years in developed nations. Long noncoding RNA (lncRNA) may regulate nuclear gene/protein function of liver tumor-initiating stem-like cells (TICs), which have self-renewal properties and are refractory to chemotherapy, leading to relapse and metastasis. MSI2 and MYC are upregulated in 70% of HCC patients. MYC is a pioneer pluripotent transcription factor that reprograms gene expression in differentiated cells. From preliminary studies: (1) Our MSI2 RIP-seq analysis established that MSI2 binds to c-MYC mRNA and long non-coding RNA (lncRNAs), including microRNA-22 host gene (miR-22hg) and MALAT1. (2) MSI2 inhibited processing of miR22hg and enhanced c-MYC translation via binding to its 5'-IRES (Internal Ribosome Entry Site). (3) MSI2 induction in liver promoted HCC development in inducible-MSI2 mice (ROSA26rtTA; Alb-TetO-Msi2 mice). Our hypothesis is that MSI2-binds to lncRNAs and c-MYC primary mRNA transcripts to change 3D chromatin structure and long-range chromatin interactions resulting in the genesis of TICs through c-MYC/super-enhancer-mediated reprogramming with lncRNAs. Thus, the goal of this proposal is to understand better how the MSI2 pathway and super-enhancer cooperatively mediate the genesis of TICs in alcohol-associated HCC patients. Targeting of the TIC subpopulation and super-enhancer function should effectively eliminate the TIC-derived HCCs. To test this hypothesis, the following two aims are proposed;
In Aim 1, we will test if MSI2 promotes stability and increased translation of c-MYC mRNA by binding to lncRNAs, including miR22hg and MALAT1. We will test in a cell culture model whether MSI2-binding to c-MYC mRNA and lncRNA (including miR-22hg/pri-miR22, MALAT1 and enhancer RNA) stabilizes c-MYC mRNA resulting in increased translation. In a subaim, we will delineate the super-enhancer regions and transcriptionally active genomic loci containing MSI2 and NANOG by integrated analyses of Global Run-On sequence (GRO-seq) assay with Chromatin Interaction Analysis by Paired-End Tag Sequencing (ChIA-PET) assay under collaborative effort with Dr. Rosenfeld (HHMI, UCSD).
In Aim 2, we will test the therapeutic effects of selective TIC inhibitor plus super-enhancer inhibitor to counter the increased expression of c-MYC resulting from the super-enhancer complex in alcohol-associated HCC mouse models: In vivo experiments will be performed by treatment with TIC inhibitor and/or super-enhancer-inhibitor (BET-bromodomain inhibitor JQ1) in alcohol-fed, FRG-hu-hep-HSC humanized mouse models. We will determine if lncRNAs found in primary liver tumors regulate tumor formation at distant metastatic sites. The proposed research will have a broader impact by identifying the mechanism by which MSI2 inhibits miR22hg processing and will validate how alcohol induces MSI2 for liver cancer.
Liver cancer is the fourth leading killer of cancer patients worldwide and is amongst the most intractable tumor types, rarely responding to chemotherapy, and is frequently incurable. Knowledge gained from these studies can be rapidly translated to the development of novel therapies to identify therapeutics to improve clinical care and outcomes of human liver cancer. These results will have a broader impact by identifying the mechanism of MSI2-mediated activation oncogene MYC and inhibition of processing of long non-coding RNA (LncRNA) for cancer.
