Xenobiotic receptors

Division Of Basic Sciences - Nci

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Abstract

Peroxisome proliferator activated receptor alpha (PPARA) is a ligand activated transcription factor and key mediator of lipid metabolism. PPARA agonists are considered classic nongenotoxic carcinogens, and chronic PPARA activation causes hepatocyte proliferation, hepatomegaly, and, eventually, hepatocellular carcinoma (HCC) in rodents. Studies have shown that the Myc oncogene contributes to agonist induced hepatocyte proliferation. MYC upregulation occurs indirectly through the PPARA mediated down regulation of miR-let7c1, which targets Myc mRNA for degradation. The contribution of MYC to hepatocyte proliferation was confirmed in vivo using liver specific Myc knockout (Myc-dHep) mice treated with the potent PPARA agonist pirinixic acid (Wy14643). Interestingly, agonist treated MycdHep mice were resistant to, but not completely protected from, hepatocyte proliferation and hepatomegaly. Additionally, Myc-dHep mice were only partially protected from N nitrosodiethylamine (DEN) induced HCC. DEN treated Myc-dHep mice had less than half the incidence of preneoplastic foci and adenomas. These data suggest that, in these models, MYC does not initiate cell proliferation but rather potentiates the proliferative effects initiated by alternate mechanisms. A model of MYC action was recently proposed in which MYC functions as a general amplifier of transcription, further elevating expression of transcriptionally active genes. In this model, MYC binding accelerates transcription rates by facilitating the release of RNA polymerase from its paused state, thereby promoting transcript elongation. This model originated from the fact that MYC targets vary greatly between cell types, and the lack of target gene overlap makes it difficult to ascribe a distinct gene signature associated with MYC activation. Studies supporting MYC amplification of gene expression have primarily relied on microarrays and next generation sequencing data derived from cell lines in which MYC levels or activity are artificially modulated. Other studies have suggested that MYC does not universally amplify transcriptionally active genes and instead activates discrete sets of genes in a tumor specific, tissue specific, and cell type specific fashion. A major obstacle for testing MYC amplifier activity in vivo is the need for coordinated control of both a defined set of target genes and MYC expression levels. Pharmacological activation of the transcription factor PPARA in combination with MycdHep mice affords a unique opportunity to test the MYC amplifier model in vivo. Myc ablation only provided partial protection against the proliferative effects of prolonged PPARA activation. This observation could be explained by an underlying mechanism involving the MYC mediated amplification of PPARA target genes. Transcriptional amplification of select genes by MYC may therefore play a major role in agonist induced HCC models. Chronic activation of PPARA promotes MYC linked hepatocellular carcinoma (HCC) in mice. Recent studies have shown that MYC can function as an amplifier of transcription where MYC does not act as an on off switch for gene expression but rather accelerates transcription rates at active promoters by stimulating transcript elongation. Considering the possibility that MYC may amplify the expression of PPARA target genes to potentiate cell proliferation and liver cancer, gene expression was analyzed from livers of wild type and liver specific Myc knockout (MycdHEP) mice treated with the PPARA agonist pirinixic acid. A subset of PPARA target genes was amplified in the presence of MYC, including keratin 23 (Krt23). The induction of Krt23 was significantly attenuated in MycdHEP mice and completely abolished in Ppara null mice. Reporter gene assays and chromatin immunoprecipitation confirmed direct binding of both PPARA and MYC to sites within the Krt23 promoter. Forced expression of KRT23 in primary hepatocytes induced cell cycle related genes. These data indicate that PPARA activation elevates MYC expression, which in turn potentiates the expression of select PPARA target genes involved in cell proliferation. Finally, KRT23 protein is highly elevated in human HCCs indicating that the studies in mice are translatable to humans. PPARA was also found to influence colorectal cancer (CRC) which is the third most common cancer and is the third leading cause of cancer death in the United States according to 2018 American Cancer Society statistics. CRC is associated with diverse etiological components including both genetic and environmental factors. The most commonly found genetic mutations include APC (adenomatous polyposis coli), KRAS, and TP53. Environmental factors, including inflammation, obesity, diabetes, and diet also play important roles in colon cancer. Currently the major treatment option for CRC is a combination of colectomy with chemotherapy employing agents such as 5 fluorouracil (5FU). Unfortunately, the 5 year survival rate for metastatic colon cancer is only about 13.3%. Therefore, it is necessary to further understand the cancer biology driving colon carcinogenesis in order to identify novel drug targets for both the prevention and treatment of this disease. Although significant efforts have been undertaken to understand the pathogenesis of colon cancer, the molecular mechanisms are not fully understood. A comprehensive analysis of PPARA's role in inflammatory diseases and tumorigenesis can only be achieved by considering the tissue-specific context of PPARalpha expression. In the current study, villin-cre/Ppara-floxed (Ppara-dIE) mice were generated and the role of intestinal PPARA in colon carcinogenesis studied. Levels of PPARA mRNA were reduced in colon tumors from mice. Ppara-dIE mice developed more and larger colon tumors than control mice following administration of azoxymethane, with or without DSS. Metabolomic analyses revealed increases in methylation related metabolites in urine and colons from Ppara-dIE mice, compared with control mice, following administration of azoxymethane, with or without DSS. Levels of DNA methyltransferase 1 (DNMT1) and protein arginine methyltransferase 6 (PRMT6) were increased in colon tumors from Ppara-dIE mice, compared with colon tumors from control mice. Depletion of PPARA reduced the expression of retinoblastoma protein (RB1), resulting in increased expression of DNMT1 and PRMT6. DNMT1 and PRMT6 decreased expression of the tumor suppressor genes Cdkn1a (P21) and Cdkn1b (p27) via DNA methylation and histone H3R2 dimethylation-mediated repression of transcription, respectively. Fenofibrate protected human PPARA transgenic mice from azoxymethane and DSS-induced colon cancer. Human colon adenocarcinoma specimens had lower levels of PPARA and RB1 and higher levels of DNMT1 and PRMT6 than normal colon tissues. Loss of PPARA from the intestine promotes colon carcinogenesis by increasing DNMT1 mediated methylation of P21 and PRMT6 mediated methylation of p27 in mice. Human colorectal tumors have lower levels of PPARA mRNA and protein than non-tumor tissues. Agents that activate PPARA might be developed for chemoprevention or treatment of colon cancer.