Given the increasing incidence of HCCs and absence of effective therapies, the need is urgent to identify biomarkers of inherited risk and disease status as well as treatments that prevent or reverse adverse outcomes. HCCs in humans have remarkably diverse etiologies that presumably converge on a smaller number of down-stream pathogenic pathways. Animal models, which typically involve chemical carcinogenesis, drug exposure or genetic engineering, have been instrumental for dissecting the molecular pathways that contribute to carcinogenesis in the liver (20
). These models test hypotheses about the involvement of specific genes, but do not capture the genetic and environmental complexity of HCC tumorigenesis. Several inbred strains of mice have been used to study gene-diet interactions in metabolic diseases. In particular, C57BL/6J but not A/J inbred mice are susceptible to diet-induced obesity, dyslipidemia and insulin resistance (6
). In these models, multiple genes and gene-environment interactions that control disease susceptibility might more closely reflect the natural circumstances in NASH and HCC pathogenesis.
We found that long-term exposure to a high-fat diet in a genetically susceptible C57BL/6J inbred strain of laboratory mouse led to NASH and to high rate of spontaneous HCCs. This model shows classic histological, biochemical and molecular features of both common subclasses of HCCs in humans. Similarly, despite long-term exposure to a high-fat diet, the A/J strain provides a contrasting model of genetically determined resistance to NASH and HCCs. Together these two strains afford an unprecedented opportunity to study the ways in which a high-fat diet leads to liver damage and malignant transformation of liver cells.
Diet-induced obesity has many comorbidities including pathologies of diverse organ systems as well as cancers of the liver, kidney, pancreas, colon and esophagus in both females and males, and other sites such as breast and uterus in females (27
). In particular, non-alcoholic pancreatic disease has been described in leptin-deficient mice that were fed a 15% high-fat diet, but pancreatic cancer was not observed perhaps because the 8 week exposure period was too short (29
). In the present study, visual and histological examination of the pancreas did not reveal evidence either for lipid accumulation or inflammation, or for pancreatic cancer in either C57BL/6J or A/J males at the end of our ~500 day study. In addition, no other organs showed gross evidence of pathologies. Thus, long-term exposure to saturated high-fat diet induced liver cancer, but not other cancers or other obvious pathologies in C57BL/6J or A/J males.
Although part of many models, the necessity of cirrhosis in HCC pathogenesis is questionable (3
). In fact, cirrhosis is sometimes not found in HCCs (8
). In addition, leptin-deficient mice show steatosis and HCCs but not inflammation or cirrhosis (30
). In the livers with NASH and diet-induced HCCs described here, cirrhosis was never extensive and was in some cases not evident (Fig. ). Thus, cirrhosis does not appear to be an essential step in some classes of HCC tumorigenesis.
HCC associated with NASH is thought to result from a series of steps that begin with lipid accumulation in the liver, either from dietary lipids or from lipolysis, then peroxidation of these lipids triggers inflammation (hepatitis) and ultimately this continuous process induces development of HCCs. Diets that are high in saturated fats as well as genetic variants in key genes in lipid metabolism exacerbate this process (8
). For example, dietary fat increases pro-inflammatory responses, exacerbating liver injury (10
). In addition, PPARα senses excess free fatty acids and up-regulates programs for fatty acid disposal and PPARα variants increase risk for HCCs in mouse models (23
). HCCs can also result from genetic variants affecting cell signaling (Akt, E-cadherin, β-catenin, ERK, MEK, MET, PI3K, Ras, Rat, mTOR and Wnt), cell cycle regulation (p16, p53, INK4, cyclin's and cdk's) and invasiveness (IEMT and TGFβ) (20
). In humans and mouse models, several classes of HCCs have been described, suggesting that these various etiologies and genetic variants activate alternative pathogenic pathways (32
). These classes show epithelial versus mesenchymal characteristics and differ both in their propensity to metastasis and invasiveness as well as response to EGFR inhibitors (35
). In the diet-induced HCCs described here, one class, which corresponds to human HCC subclass B, was characterized by differential expression of genes involved in the Myc, NFκB and TGFβ networks, whereas the other class involved in the NFκB, IGF2/FGF2 and PPARα/PPARγ networks (Fig. ). An important challenge involves building a predictive model that integrates the ways in which these overlapping networks contribute to the alternative classes of HCCs. Another key question concerns whether these HCC subclasses represent different stages along a single pathogenic pathway, or related but alternative paths to tumorigenesis.
miRNAs are emerging as a new class of regulators of cancer development. We found several changes in the miRNA expression that are relevant to diet-induced HCCs. Three miRNAs showed up-regulation (miR-31, -146 and -182) and one showed down-regulation (miR-191) in HCCs versus corresponding livers with NASH. Interestingly, mmu-miR-31 is also up-regulated in colon cancers in humans (37
) and differentially processed in liver cancer cell lines (38
), whereas mmu-miR-146 is up-regulated in chronic lymphocytic leukemia (39
) and papillary thyroid carcinoma (40
). In addition, NFκB activates mmu-miR-146 expression which in turn negatively regulates the IRAK1 and TRAF6 in the TNFα signaling pathway of innate immunity (Fig. ; see also refs. 20
). In contrast, miR-191 is up-regulated in colon cancer in humans (42
) and down-regulated in the diet-induced HCCs described here, but it is unclear whether these contrasting results reflect different functions of miR-191 in colon and liver cancers or differences in its regulation in humans and mice.
A cluster of miRNAs located on mouse chromosome X showed significant increases in expression levels, without amplification of the chromosome segment, in HCCs versus corresponding livers (Fig. ). Although the mRNA targets of this recently reported X-linked miRNA cluster have not yet been identified, analysis of the intersection between computationally predicted mRNA targets for these X-linked miRNAs and mRNAs that showed reduced expression in tumor versus liver revealed a short-list mRNAs that are candidate mRNAs for these miRNAs (Supplementary Material, Tables S2 and S3
). These genes are occurred preferentially in networks related to necrosis, apoptosis and degeneration (Supplementary Material, Table S3
), all of which are relevant to NASH. Thus, a small number of miRNAs appear to be intimately involved in HCC tumorigenesis.
Considerable evidence shows that dietary factors affect occurrence and progression of various cancers (43
). Few examples are known, however, where alternative diets reverse pathology and protect against cancer development. An important challenge is to find ways to reverse or stop progression of premalignant conditions to invasive tumors associated with diet-induced liver pathologies (45
). We showed that simply switching to a diet that has low rather than high levels of saturated fats, even after onset of liver pathologies but before the development of dysplasia or carcinoma, reversed outcome, with switched mice being lean at the end of the study and with no evidence of NASH or HCCs. These studies suggest that diet modification might have important implications for HCC prevention in humans.