Chronic alcohol exposure induces hepatocyte damage by inducing oxidative stress, SATB2 and stem cell‐like characteristics, and activating lipogenesis

Abstract Alcohol is a risk factor for hepatocellular carcinoma (HCC). However, the molecular mechanism by which chronic alcohol consumption contributes to HCC is not well understood. The purpose of the study was to demonstrate the effects of chronic ethanol exposure on the damage of human normal hepatocytes. Our data showed that chronic exposure of hepatocytes with ethanol induced changes similar to transformed hepatocytes that is, exhibited colonies and anchorage‐independent growth. These damaged hepatocytes contained high levels of reactive oxygen species (ROS) and showed induction of the SATB2 gene. Furthermore, damaged hepatocytes gained the phenotypes of CSCs which expressed stem cell markers (CD133, CD44, CD90, EpCAM, AFP and LGR5), and pluripotency maintaining factors (Sox‐2, POU5F1/Oct4 and KLF‐4). Ethanol exposure also induced Nanog, a pluripotency maintaining transcription factor that functions in concert with Oct4 and SOX‐2. Furthermore, ethanol induced expression of EMT‐related transcription factors (Snail, Slug and Zeb1), N‐Cadherin, and inhibited E‐cadherin expression in damaged hepatocytes. Ethanol enhanced recruitment of SATB2 to promoters of Bcl‐2, Nanog, c‐Myc, Klf4 and Oct4. Ethanol also induced activation of the Wnt/TCF‐LEF1 pathway and its targets (Bcl‐2, Cyclin D1, AXIN2 and Myc). Finally, ethanol induced hepatocellular steatosis, SREBP1 transcription, and modulated the expression of SREBP1c, ACAC, ACLY, FASN, IL‐1β, IL‐6, TNF‐α, GPC3, FLNB and p53. These data suggest that chronic alcohol consumption may contribute towards the development of HCC by damaging normal hepatocytes with the generation of inflammatory environment, induction of SATB2, stem cell‐like characteristics, and cellular steatosis.


| INTRODUC TI ON
Hepatocellular carcinoma (HCC) is one of the primary liver cancers, predicted to be the sixth most commonly diagnosed cancer and the third leading cause of cancer death worldwide. 1 The worldwide HCC incidence is 10.1 cases per 100,000 person-years. 1 The burden of HCC in 2012 was 14 million and is expected to rise to 22 million in the next two decades. 2 The incidence of HCC in the US has tripled over the last four decades. HCC has an average five-year survival of <20%. 2 In the United States, approximately 42,230 adults are expected to be diagnosed with liver cancer, and 30,230 deaths are expected to occur from this disease in 2021. 2 The development of HCC is complex. 3 It involves sustained inflammatory damage leading to hepatocyte necrosis, regeneration and fibrotic deposition. Epidemiological data strongly suggest that heavy drinking increases the risk for liver cancer. [4][5][6] Alcohol consumption is an independent risk factor and a primary cause of HCC. [7][8][9][10] According to the National Institute on Alcohol Abuse and Alcoholism (NIAAA), a standard alcoholic drink in the United States contains 14.0 g (0.6 ounces) of pure alcohol. Heavy alcohol drinking is defined as the consumption of more than three drinks on any day or more than seven drinks per week for women, and more than four drinks on any day or more than 14 drinks per week for men.
As per the recent report from the National Cancer Institutes (USA), the chances of getting liver cancer increase significantly with five or more drinks per day. However, the molecular mechanism(s) by which ethanol (EtOH) induces hepatocyte damage/malignant transformation leading to HCC development is not well understood.
SATB2 (special AT-rich binding protein-2) gene is required for normal mammalian development; 11,12 however, it is not expressed in normal adult tissues, including normal hepatocytes. [13][14][15] SATB2 is a DNA binding protein that specifically binds nuclear matric attachment regions and is involved in transcription regulation and chromatin remodelling, 16 and thus, regulates gene expression. [17][18][19] SATB2 −/− mice are defective in bone development and osteoblast differentiation. 19 In addition, satb2 −/− mice and humans with loss-of-function satb2 mutations develop craniofacial abnormalities, including orofacial clefting. [19][20][21][22] SATB2 is essential for proper facial patterning of the embryo and normal bone development. 19 These defects have been attributed to an increased expression of specific members of the Hox gene (a subset of homeobox genes) clusters and a decreased expression of osteoblastspecific genes, whereby satb2 was shown to regulate these genes at the chromatin level. 19 SATB2 regulates the transcription of those genes, which modulates pluripotency maintaining factors, cell growth and stemness. 11,[23][24][25][26][27] Inappropriate activation of this SATB2 gene may be the cause of malignant cellular transformation. 14,15,28 Generation of stem cells/progenitor cells during cellular transformation is the primary cause of cancer initiation, promotion and metastasis. [29][30][31][32] We have recently demonstrated that chronic exposure of pancreatic ductal epithelial cells to ethanol induces transformation, which causes cells to gain the functions of cancer stem cells (CSCs). SATB2 can directly activate Wnt/β-catenin/TCF-LEF pathway, which regulates stem cell self-renewal and transformation. 28 Our recent data demonstrate that inhibition of SATB2 expression by Crisp/Cas9 technique suppresses epithelial-mesenchymal transition, stem cell markers and pluripotency maintaining factors in CSCs derived from HCC. 13 Liver CSCs are resistant to chemotherapy and radiotherapy. 33 Several markers of liver CSCs such as epithelial cellular adhesion molecule (EpCAM, CD326), CD90, CD44, CD24, CD133 and AFP, either alone or in combination, have been used. 32,34 Moreover, the role of SATB2 in ethanol-induced transformation/damage of hepatocytes is unknown.
The main goal of this paper is to examine the molecular mechanisms by which EtOH induces damage to human normal hepatocytes
EpCAM + /CD44 + /CD133 + human liver CSCs were isolated from primary hepatocellular carcinoma (obtained from Celprogen, Torrance, CA) and grown in a well-defined stem cell culture medium as per the supplier's instructions. All cells were checked for the absence of Mycoplasma using the kit (Lonza).

| Lentiviral particle production and transduction
The protocol for lentivirus production and transduction have been described elsewhere. 35 Briefly, 293T cells were transfected with four µg of plasmid and four µg of the lentiviral vectors using Lipofectamine-3000 according to the manufacturer's protocol (Invitrogen). PEG-it virus precipitation solution (SBI System Biosciences) was added to the supernatant, and ultracentrifugation was performed to collect concentrated viral particles. Hepatocytes were transduced with lentiviral particles with 6 μg/ml polybrene (Invitrogen).

| Colony formation assay
For colony formation assays, hepatocytes were grown in matrigelcoated plates and treated with or without ethanol (100 mm). The culture medium was replaced with fresh medium every three days, and cells were treated with ethanol. The hepatocytes were treated for a total of two weeks. After incubation, colonies were fixed with methanol, stained with 0.5% crystal violet and visualized under a microscope.

| Oil Red O staining
To examine fat accumulation, the hepatocytes were rinsed with cold phosphate-buffered saline (PBS) and fixed in 10% paraformaldehyde for 30 min. After the hepatocytes were washed with 60% isopropanol, they were stained for at least

| Lentiviral particle production and transduction
The protocol for lentivirus production and transduction have been described elsewhere. 36,37 In brief, lentivirus was produced by tri-

| Quantitative real-time PCR
Total RNA was extracted with TRIzol reagent (Invitrogen), and

| TCF/LEF reporter assay
Lentiviral particles expressing cop-GFP and luciferase genes (TCF/ LEF-mCMV-EF1-Neo) were prepared as described elsewhere. 38 Cells were transduced with lentiviral particles. Transduced cells (5-10,000 cells per well) were seeded in 96-well plates for 48 h. At the end of the incubation period, luciferase reporter activity was measured per the manufacturer's instructions (Promega Corp., Madison, WI).

| Statistical analysis
All analyses were performed using GraphPad Prism Software (GrafPad Software, Inc., San Diego, CA). Statistical differences between groups were analysed using the Student t-test or Analysis of Variance (ANOVA). Significant differences among groups were calculated at p < 0.05. The mean ± SD or SE was calculated for each experimental group.

| Ethanol induces hepatocyte damage by generating reactive oxygen species and SATB2 expressions
We first examined the effects of ethanol on the morphological changes of human normal hepatocytes. Human normal hepatocytes were grown on the matrigel-coated dishes with a cell culture medium in the presence or absence of EtOH (100 mM) for two weeks ( Figure 1A). Exposure of hepatocytes to ethanol induced cellular transformation as evident by the formation of colony-like structures, loss of contact inhibition and disoriented growth ( Figure 1A).
High concentrations of ethanol cause the production and release of many free radicals and inflammatory mediators, which leads to liver injury. We, therefore, examine the effects of ethanol on ROS production in hepatocytes ( Figure 1B). Compared to the untreated control group, chronic exposure of human normal hepatocytes with ethanol-induced ROS. These data suggest that ROS production during ethanol exposure of hepatocytes may be responsible for cellular damage.
It has been reported that the SATB2 plays an essential role in malignant transformation and stemness. We, therefore, examined the mechanism of ethanol-induced transformation of normal hepatocytes by comparing the expression of SATB2 in untreated hepatocytes and ethanol-transformed hepatocytes (Hepatocytes/Ethanol). Figure 1C, exposure of normal hepatocytes to ethanol (100 nM) resulted in the induction of the SATB2 gene.

| Ethanol-transformed hepatocytes express stem cell markers and pluripotency maintaining factors
We next examined whether ethanol-transformed hepatocytes gained the phenotypes of CSCs by measuring stem cell markers Ethanol induced the expression of Wnt3a and LEF1 ( Figure 6A). We next examined transcriptional activation of TCF/LEF1 by luciferase assay in hepatocytes treated with or without ethanol ( Figure 6B).
Hepatocytes exposed to ethanol showed enhanced TCF/LEF1 activity.
Since ethanol induced TCF/LEF1 activity in hepatocytes, we sought to examine whether ethanol can induce TCF/LEF target gens such as Bcl-2, Cyclin d1, AXIN2 and Myc. As shown in Figure 6D

| Ethanol induces hepatocellular steatosis, induces SREBP1 target gens, induces inflammation and causes oxidative stress
Oxidative stress is involved in the pathophysiology of many diseases. 8-hydroxy-2′-deoxyguanosine (8-OHdG) is considered the major type of DNA damage and is the commonly used biomarker to evaluate cellular oxidative stress. 42 We, therefore, measured the formation of 8-OHdG in hepatocytes treated with or without ethanol ( Figure 7A). Chronic exposure of hepatocytes produced a significantly higher quantity of 8-OHdG than an untreated control group.
We next examined the effects of ethanol on lipid accumulation.

| N-acetylcysteine (NAC) attenuates the effects of chronic exposure to ethanol in hepatocytes
Oxidative stress is a crucial pathological feature implicated in acute and chronic liver diseases, including drug-induced liver injury. We F I G U R E 5 Binding of SATB2 to promoters of Bcl-2, Bsp, Nanog, c-Myc, XIAP, Klf4 and Hoxa2. Nuclear extracts were prepared from pancreatic CSCs. Chromatin immunoprecipitation (ChIP) assays followed by qRT-PCR were performed to examine the binding of the SATB2 to the promoters of Bcl-2, Nanog, c-Myc, Klf4 and Oct4. Data represent mean ± SD (n = 4). *Significantly different from Hepatocytes/Control, p < 0.   These data suggest that the generation of oxidative stress in hepatocytes by ethanol is responsible for the changes in gene expression.

| DISCUSS ION
Alcohol is a risk factor for HCC, and its effects further accelerate the development of HCC in obesity and diabetes. 45,46 Here we have We hypothesize that exposure of hepatocytes to ethanol generates an inflammatory condition which in turn induces the SATB2 gene.
SATB2 is an emerging transcription factor whose biological significance has recently been recognized. It regulates stemness by controlling the expression of pluripotency and self-renewal factors, and epithelialmesenchymal transition. [13][14][15]28 We have recently demonstrated that   intracellular signal cascade to exacerbate inflammation. 42,58 One of the important mechanisms of ethanol-induced liver injury is the production of oxygen free radicals. 59,60 Excessive production of oxygen free radicals leads to lipid peroxidation. Oxidative stress is activated by fatty acids in cultured human hepatocytes. 61 In the present study,

ACK N OWLED G EM ENT
We thank our lab members for their critical reading of the manuscript.

CO N FLI C T S O F I NTE R E S T
The authors have declared that no conflict of interest exists.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.