Potential role of gut microbiota, the proto-oncogene PIKE (Agap2) and cytochrome P450 CYP2W1 in promotion of liver cancer by alcoholic and nonalcoholic fatty liver disease and protection by dietary soy protein
Introduction
HCC is the world's second leading cause of cancer mortality. It is found 3-times more frequently in men than women [[1], [2], [3]]. Incidence of HCC has increased in the U.S. since the 1970s from 1.6 to 4.9/100,000 [1]. One-year survival rates remain below 50% [1]. Fatty liver diseases produced by alcohol (EtOH) abuse (alcoholic steatohepatitis, ASH) and associated with obesity, type II diabetes, metabolic syndrome and high levels of dietary fat (nonalcoholic steatohepatitis, NASH) are well-known risk factors for HCC. ASH and NASH account for 36–67% of all HCC cases [4,5]. The mechanisms whereby EtOH/high fat consumption cause HCC remain incompletely understood and there are currently few clinical strategies to treat HCC in patients with ASH/NASH other than by liver transplant. Epidemiological data suggest that consumption of >80 g/d of alcohol over 10 years increases the risk of HCC 5-fold in Western populations [6]. Moreover, obesity rates have also recently risen dramatically with an estimated 5% increase in HCC risk for each unit of body mass index (BMI) [5]. There is evidence that EtOH and dietary fat may initiate tumors as a result of generation of reactive oxygen species and as a result of increased activation of environmental pro-carcinogens such as nitrosamines found in well-cooked meats and cigarette smoke by the major EtOH and dietary fat-inducible cytochrome P450 enzyme CYP2E1 [[6], [7], [8], [9]]. EtOH and FA metabolism by CYP2E1 also produce reactive metabolites including acetaldehyde and lipid peroxides. EtOH disrupts of one-carbon metabolism which may further contribute to initiation [2]. Alcohol use and dietary fat consumption may also synergize with hepatitis C and other initiating factors such as cigarette smoking in development of HCC [10,11]. However, a major role for EtOH/dietary fat appears to be to act as tumor promoters [4,6]. Chronic EtOH consumption in experimental animals results in continuing liver injury with the appearance of inflammation and fibrosis [8,12]. Many groups have reported increased hepatocyte proliferation after chronic alcohol consumption as a repair response [[12], [13], [14]]. A similar pattern of progressive injury accompanied by increased hepatocyte proliferation has been observed in animal models of high fat-driven NASH [[15], [16], [17]]. The hepatic regenerative response associated with fatty liver disease is influenced by dietary EtOH/fat concentration, length of exposure, fat type, nutritional status and hormonal milieu. It has been suggested that the pro-proliferative signals associated with the regenerative response are responsible for tumor promotion [6,[18], [19], [20]].
We have demonstrated that EtOH and dietary fat act as hepatic tumor promoters in mice where carcinogenesis was initiated by treatment with a single dose of diethylnitrosamine (DEN) on postnatal day (PND) 13 [[18], [19], [20], [21]]. Tumor development was coincident with development of steatosis, elevation of ceramide/sphingosine synthesis and development of inflammation and fibrosis [[18], [19], [20]]. Development of ASH was additionally linked to depletion of hepatic retinoids [18]. These models replicate the progression of HCC in fatty liver diseases observed clinically. We have previously shown that tumors in the DEN/EtOH mouse model were β-catenin positive [18]. In addition, increased β-catenin signaling has also been reported in a DEN/high fat mouse model of NASH-driven HCC [20].
There is epidemiological data to suggest that Asians are at lower risk of development of alcoholic HCC. In the U.S. there is a reported 5-fold increased risk of HCC among chronic or excessive drinkers [1]. In contrast, epidemiological studies in Asia report only a 1.6–1.8-fold increase in risk [22,23]. We have published data showing that substitution of soy protein isolate (SPI) for casein as the protein source in high fat liquid diets with EtOH results in inhibition of tumor promotion coincident with blockade of EtOH-induced Wnt-β-catenin activation and of hepatocyte proliferation [18,19]. However, anti-tumor and anti-proliferative effects of SPI appear to involve pathways in addition to Wnt signaling. We observed protective effects of SPI against high fat-induced tumorigenesis in the DEN model even in the absence of changes in nuclear β-catenin expression [20]. SPI blocks development of steatosis in rodent models of both ASH and NASH and anti-cancer effects may simply reflect inhibition of multiple pathways triggered by excess triglyceride accumulation. The anti-steatotic effects of feeding SPI appear to be associated with activation of PPARα and increased FA degradation [20,[24], [25], [26]]. However, it appears that the protective effects of SPI on EtOH-associated tumor promotion are not mediated via the isoflavone genistein which has been implicated in some of the anti-cancer properties of soy [27]. Studies included in the current report were designed to determine if protein/peptide or phytochemical components of SPI other than isoflavones play a role in protection against EtOH-induced steatosis by feeding SPI.
One possible additional factor in development of HCC in ASH and NASH and in protection by SPI may be effects related to the microbiome and the microbiome –associated metabolome. Alterations in the mucosa-associated colonic bacterial microbiota by chronic alcohol consumption was first reported in rats by Mutulu et al. [28]. Bacterial overgrowth and intestinal microbial dysbiosis was subsequently described in the mouse Tsukomoto-French intragastric model after three weeks of alcohol consumption accompanied by decreases in Firmicutes, beneficial bacteria including Lactobacillus, Pediococcus, Leuconostoc and Lactococcus and by increases in Bacteriodetes [29]. One consequence of microbial dysbiosis is a significant alterations in the microbial metabolome. Alcohol consumption has been reported to suppress production of the short chain fatty acid (SFA) butyrate, reduce the levels of taurine-conjugated bile acids and increase levels of more toxic unconjugated and glycine-conjugated bile acids and significantly reduce intestinal amino acid metabolism [[30], [31], [32], [33]]. Changes in the microbial metabolome and microbial metabolism of EtOH to acetaldehyde have been linked to disruption of the intestinal epithelial barrier, intestinal inflammation and changes in intestinal FXR signaling and production of FGF15/19 [[30], [31], [32], [33]]. In addition, increased intestinal permeability results in translocation of pathogen-associated molecular patterns (PAMPs) such as endotoxin, peptidoglycan and bacterial DNA which may contribute to development of hepatic inflammation [29]. Microbial dysbiosis and alterations in microbial metabolism of bile acids have also been suggested to play a role in NASH progression to HCC [34]. Probiotic treatment/fetal transplants have been proposed as potential therapies for ASH and NASH [35,36]. It has also been postulated that protective effects of soy on lipid homeostasis and steatosis are the result of beneficial changes in gut microbiota populations and altered bile acid metabolism and signaling [35,36]. Soy feeding was reported to increase microbial diversity in Golden Syrian hamsters including elevation in Bifidobacteria and Clostridales in comparison to feeding milk protein [37]. This was accompanied by reductions in serum triglycerides [37]. Similar increases in Bifidobacter have been reported in women fed soy bars [38]. Improved body composition and lipid homeostasis were also reported in soy-fed female low-fit rats and in obese OLETF rats accompanied by a lower ratio of Firmicutes to Bacterioides and increases in Lactobacillus [39]. We have analyzed microbiome composition in mice fed EtOH liquid diets with casein or SPI as the protein source or fed high fat diets with differing fat composition and conducted adoptive transfer studies to examine the role of microbial dysbiosis in development of ASH/NASH and the role of altered microbiome composition in the protective effects of SPI.
CYP2W1 is an orphan CYP enzyme, with a well conserved sequence between humans, rats and mice, which was first cloned from the hepatoma HepG2 cell line [40]. Neither the CYP2W1 mRNA nor protein were detected in adult human livers or other adult tissues but was observed in human colon tumors and in fetal intestine and colon [41]. The orthologues of human CYP2W1 were detected in the fetal GI tract of rats and mice [40,41]. Increased rates of tumor growth have been reported in subcutaneous CYP2W1 +ve colon cancer cell xenografts compared to xenografts using cells where CYP2W1 expression was abolished [42]. A variety of potential endogenous CYP2W1 substrates have been identified using recombinant CYP2W1 which might be metabolized to regulate tumor growth. These include retonoids, free fatty acids, lysophospholipids and arachidonic acid [[43], [44], [45]]. CYP2W1 has been found to be inducible in colon cancer cell lines by linoleic acid and its derivatives which is consistent with these compounds being CYP2W1 substrates [41]. Since CYP2W1 appears to be specific to tumors, it has been suggested that it may also serve as both a cancer biomarker and as a therapeutic target for prodrugs which can be converted by CYP2W1 to cytotoxic metabolites [[40], [41], [42]]. A recent series of studies using the chloromethylindoline duocarmycin analogue ICT2706, which is activated by CYP2W1 to a DNA alkylating agent, demonstrated abolition of tumor growth in CYP2W1 positive human colon cancer xenografts whereas CYP2W1 negative xenografts were resistant [46]. One small previous study using an antibody against CYP2W1, suggested that in addition to expression in colon tumors, CYP2W1 is also over-expressed in HCC [47]. We have conducted studies to examine if CYP2W1 is over-expressed in tumors from our DEN/EtOH mouse model and in human HCC compared to surrounding tissues.
Section snippets
In vivo mouse models
Two models of tumor promotion in DEN mouse models were utilized. In the first model, male C57/BL6 mice were injected i.p. with 10 mg/kg DEN on PND13 and were fed standard rodent chow until PND 65. Mice were then pair-fed 35% fat Lieber DeCarli liquid diets (Dyets Inc., Bethlehem, PA) with or without substitution of EtOH for carbohydrate calories up to 28% total calories and with either casein or SPI as the sole protein source for 16 weeks as previously described [18]. In the second model male
Potential role of steatosis, the microbiome and Agap2 in tumor promotion and the protective effects of SPI in ASH/NASH livers
We examined the development of steatosis in the NIAAA binge-on-chronic model of ASH in mice fed Lieber DeCarli diets where casein protein was swapped for SPI or where a phytochemical extract of soy was added to the casein diet. As shown in Fig. 1, both SPI and the extract were effective in suppressing triglyceride accumulation after EtOH feeding and the effects of the extract were dose-dependent (P < 0.05).
Moreover, data from an adoptive transfer experiment utilizing a modified NIAAA model [50
Discussion
We have previously demonstrated that both EtOH and high fat liquid diets significantly promote hepatic tumorigenesis in the male mouse DEN model with EtOH the most potent factor [18]. In addition, we have demonstrated that switch of dietary protein from casein to SPI results in suppression of tumorigenesis in both ASH and NASH models [19,20]. However, whereas inhibition of β-catenin signaling is implicated in SPI protection against EtOH-associated tumor promotion [19], we found that SPI also
Funding
Funded in part by R21 CA169389 (M.J.R.) and start-up funding from LSUHSC – New Orleans (M.J.R.) and United States Department of Agriculture (Agricultural Research Service Project 6026-51000-010-05S) (K.M).
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
References (57)
- et al.
Alcohol and hepatocellular carcinoma
Gastroenterology
(2004) - et al.
Alcohol intake increases the risk of HCC in hepatitis C virus-related compensated cirrhosis: a prospective study
J. Hepatol.
(2016) - et al.
N-acetylcysteine attenuates progression of liver pathology in a rat model of non-alcoholic steatohepatitis
J. Nutr.
(2008) - et al.
Global deletion of glutathione-S-transferase A4 exacerbates developmental nonalcoholic steatohepatitis
Am. J. Pathol.
(2017) - et al.
Dysregulation of serum bile acids and FGF19 in alcoholic hepatitis
J. Hepatol.
(2018) - et al.
Gut microbiota dysbiosis in patients with non-alcoholic fatty liver disease
Hepatobiliary Pancreat. Dis. Int.
(2017) - et al.
Probiotics restore bowel flora and improve liver enzymes in human alcohol-induced liver injury: a pilot study
Alcohol
(2008) - et al.
Soy protein compared with milk protein in a western diet increases gut microbial diversity and reduces serum lipids in golden Syrian hamsters
J. Nutr.
(2016) - et al.
Soy compared with milk protein in a Western diet changes fecal microbiota and decreases hepatic steatosis in obese OLETF rats
J. Nutr. Biochem.
(2017) - et al.
Tumor-specific expression of the novel cytochrome P450 enzyme, CYP2W1
Biochem. Biophys. Res. Commun.
(2006)
Metabolomic analysis and identification of a role for the orphan human cytochrome P450 2W1 in selective oxidation of lysophospholipids
J. Lipid Res.
Infant formula feeding increases hepatic cholesterol 7α hydroxylase (CYP7A1) expression and fecal bile acid loss in neonatal piglets
J. Nutr.
Hepatocellular carcinoma incidence, mortality and survival trends in the United States from 1975 to 2005
J. Clin. Oncol.
Sex differences in substance use disorders: focus on side effects
Addiction Biol.
Liver cancer: connections with obesity, fatty liver, and cirrhosis
Annu. Rev. Med.
Nonalcoholic steatohepatitis is the most rapidly growing indication for liver transplantation in patients with hepatocellular carcinoma in the U.S
Hepatology
Cocarcinogenic effect of alcohol in hepatocarcinogenesis
Gut
The CYP 2E1 family
Alcohol metabolism: role in toxicity and carcinogenesis
Alcohol Clin. Exp. Res.
Role of CYP2E1 in diethylnitrosamine-induced hepatocarcinogenesis in vivo
Canc. Res.
Case-control study on hepatitis C virus (HCV) as a risk factor for hepatocellular carcinoma: the role of HCV genotypes and the synergism with hepatitis B virus and alcohol. Brescia HCC Study
Int. J. Canc.
Cytokine and chemokine expression associated with steatohepatitis and hepatocyte proliferation in rats fed ethanol via total enteral nutrition
Exp. Biol. Med.
Undernutrition enhances alcohol-induced hepatocyte proliferation in the liver of rats fed via total enteral nutrition
Am. J. Physiol.
Knockout of the Gsta4 gene in male mice leads to an altered pattern of hepatic protein carbonylation and enhanced inflammation following chronic consumption of an ethanol diet
Alcohol Clin. Exp. Res.
A new rat model for nonalcoholic steatohepatitis utilizing overfeeding of diets high in polyunsaturated fat by total enteral nutrition
Am. J. Physiol.
Alcohol consumption promotes diethylnitrosamine-induced hepatocarcinogenesis in male mice through activation of the Wnt/β-catenin signaling pathway
Canc. Prev. Res.
Soy protein isolate protects against ethanol-mediated tumor progression in diethylnitrosamine-treated male mice
Canc. Prev. Res.
Soy protein isolate inhibits hepatic tumor promotion in mice fed a high-fat liquid diet
Exp. Biol. Med.
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