Different roles of FAT10, FOXO1, and ADRA2A in hepatocellular carcinoma tumorigenesis in patients with alcoholic steatohepatitis (ASH) vs non-alcoholic steatohepatitis (NASH)

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Abstract

Hepatocellular carcinoma (HCC) is the fifth most common cancer and the second leading cause of cancer related deaths worldwide. Among others, non-alcoholic steatohepatitis (NASH) and alcoholic steatohepatitis (ASH) are the two major risk factors as both of them may develop cirrhosis and hepatocellular carcinoma (HCC) if left untreated. However, patients with NASH progress to HCC at a rate around 0.5% annually, while 3–10% ASH patients may progress to HCC annually. The present study is to demonstrate the molecular differences in oncogenesis pathway between NASH and ASH. By using immunofluorescence study and quantitating the fluorescence intensity morphometrically in liver biopsied specimens from NASH and ASH patients, the protein expression of candidate molecules within hepatocytes cytoplasm are studied, including two HCC-related molecules FAT10 and FOXO1, and one GPCR pathway related molecule ADRA2A. Compared with the control group patients, the expression levels of all the molecules were upregulated in the ASH group of patients (p < 0.001 in all molecules), while FAT10 and ADRA2A were upregulated, FOXO1 did not change in the NASH group of patients. The most important finding is that compared with the ASH group of patients, the expression levels of all three molecules were significantly lower than in the NASH group of patients (p < 0.001 in all molecules). These results confirmed our previous finding that there are significant differences of molecules change in ASH compared to NASH. Thus, we conclude that there are significantly different molecules and pathways involved during the pathogenesis of HCC development in ASH compared to NASH which could help explain why the tumorigenic rate is different in ASH and NASH.

Introduction

Liver cancer is the fifth most common cancer and the second leading cause of cancer related deaths worldwide (Ferlay et al., 2012). The major risk factors include hepatitis B (HBV) and hepatitis C (HCV), obesity/metabolic syndrome, and alcoholism which all may progress to hepatocellular carcinoma (HCC). In the last two decades the rising rates of obesity/metabolic syndrome have led to the increased development of the non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH). Experimental and epidemic studies have shown relationships between NASH and HCC development. Alcoholic liver disease (ALD) and ASH are the major causes of HCC in US (Morgan et al., 2004; Ramadori et al., 2017; Testino et al., 2014). NASH has similar histological features with alcoholic hepatitis, such as overt lipid deposition and fat storage in the liver parenchymal cells. Both ASH and NASH may progress to fibrosis, cirrhosis, and ultimately hepatocellular carcinoma (HCC), about 7–10% of NASH may progress to cirrhosis (Scaglioni et al., 2011) and 0.5% to HCC (Lindenmeyer and McCullough, 2018), while 10–20% of ASH progress to cirrhosis (Schwartz and Reinus, 2012; Dam-Larsen et al., 2004) and 7–10% to HCC (Schwartz and Reinus, 2012), annually. Our previous published work showed that there are different molecules protein changes in ASH compared with NASH (Nguyen et al., 2018; Lu et al., 2018)

The mechanisms by which NASH and ASH progress to HCC are not completely understood and there are many theories including chromosomal loss of tumor suppressor genes, oxidative stress, decreased liver retinoic acid level, altered DNA methylation, and genetic susceptibility (Morgan et al., 2004; Stickel et al., 2002; Stickel, 2015). In our previous studies, we showed that in addition to the TLR/NFKB/CXCR4/7 (Liu et al., 2014; French et al., 2012a, French et al., 2012b; Nan et al., 2005; French et al., 2010) and PI3K/AKT/mTORC1 signaling pathways (Afifiyan et al., 2017a, Afifiyan et al., 2017b), Tec kinase signaling pathway connects these two systems together in Mallory-Denk Bodies (MDB) formation both in ASH and NASH (Afifiyan et al., 2017a, Afifiyan et al., 2017b). During these studies we found that HLA-F-adjacent transcript 10 (FAT10), a ubiquitin-like modifier protein which functions as a proteasomal degradation signal (Schmidtke et al., 2009; Rani et al., 2012; Hipp et al., 2005), plays an important role in MDB formation and tumorigenesis (Oliva et al., 2010; Oliva et al., 2008; French et al., 2012a, French et al., 2012b) and we proved that MDB forming cells express preneoplastic phenotypic features (Nan et al., 2006). To confirm the roles of FAT10 and other related molecules in HCC tumorigenesis in NASH and ASH, we studied the changes of FAT10, FOXO1, and ADRA2A in liver biopsy specimens from NASH and ASH patients and control groups.

Section snippets

Methods

Formalin-fixed paraffin-embedded biopsies of ASH liver (n = 39 in FAT10, 50 in FOXO1, 40 in ADRA2A), NASH liver (n = 30), and normal liver (n = 20) were collected from Harbor-UCLA Medical Center and from the Long Beach Veterans Affairs' clinical trial in treatment of alcoholic hepatitis. The study was conducted following the principals of the Declaration of Helsinki and was designated as exempt by our institutional ethics review board and the data was analyzed anonymously. The primary

Results

The protein expression level of several candidate molecules in specimens from patients with ASH, NASH, and normal controls were compared. Representative data are shown in Fig. 1, Fig. 2, Fig. 3.

In ASH patients, levels of all tested candidate proteins including FAT10, FOXO1, and ADRA2A (Fig. 1, Fig. 2, Fig. 3) were markedly increased compared with the control group. In NASH patients, FAT10 and ADRA2A were upregulated when compared to control group, while FOXO1 level was not changed (Fig. 2).

The

Discussion and conclusions

The increasing rates of obesity and the metabolic syndrome become a major health problem worldwide which is emphasized by the relationship from NASH to cirrhosis and even HCC. Wong et al. showed that NASH is the most rapid growing risk for liver transplantation in patients with HCC (Wong et al., 2014, Wong et al., 2015). Chronic (longer than 10 years) alcohol consumption (>80 g/day) increases the risk for HCC approximately 5 fold (French, 2013). ALD is the most common cause of HCC which

Acknowledgements

This study was funded by NIH/AAA grant # UO-21898-05.

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