Ethanol exposure of human pancreatic normal ductal epithelial cells induces EMT phenotype and enhances pancreatic cancer development in KC (Pdx1‐Cre and LSL‐KrasG12D) mice

Abstract Alcohol is a risk factor for pancreatic cancer. However, the molecular mechanism by which chronic alcohol consumption influences pancreatic cancer development is not well understood. We have recently demonstrated that chronic ethanol exposure of pancreatic normal ductal epithelial cells (HPNE) induces cellular transformation by generating cancer stem cells (CSCs). Here, we examined whether chronic ethanol treatment induces epithelial–mesenchymal transition in HPNE cells and promotes pancreatic cancer development in KC (Pdx1‐Cre, and LSL‐KrasG12D) mice. Our data demonstrate that chronic ethanol exposure of HPNE cells induces SATB2 gene and those cells became highly motile. Ethanol treatment of HPNE cells results in downregulation of E‐Cadherin and upregulation of N‐Cadherin, Snail, Slug, Zeb1, Nanog and BMI‐1. Suppression of SATB2 expression in ethanol‐transformed HPNE cells inhibits EMT phenotypes. KC mice fed with an ethanol‐containing diet show enhanced pancreatic cancer growth and development than those fed with a control diet. Pancreas isolated from KC mice fed with an ethanol‐containing diet show higher expression of stem cell markers (CD133, CD44, CD24), pluripotency‐maintaining factors (cMyc, KLF4, SOX‐2, and Oct‐4), N‐Cadherin, EMT‐transcription factors (Snail, Slug, and Zeb1), and lower expression of E‐cadherin than those isolated from mice fed with a control diet. Furthermore, pancreas isolated from KC mice fed with an ethanol‐containing diet show higher expression of inflammatory cytokines (TNF‐α, IL‐6, and IL‐8) and PTGS‐2 (COX‐2) gene than those isolated from mice fed with a control diet. These data suggest that chronic alcohol consumption may contribute to pancreatic cancer development by generating inflammatory signals and CSCs.


| INTRODUC TI ON
Pancreatic cancer is the fourth leading cause of cancer-related deaths in the US. 1 With an overall 5-year survival rate of 8%, 2 pancreatic cancer has one of the poorest prognoses among all cancers. 3 The incidence of pancreatic cancer varies significantly throughout the world, suggesting that several factors may be responsible for this deadly disease. 4 Genetic, race, gender, environmental carcinogen, diet, and lifestyle are the primary factors for pancreatic cancer. 5 Other factors, such as smoking, alcohol, and exposure to organochlorine or hydrocarbon solvents, have been associated with the Kras mutations causing pancreatic ductal adenocarcinoma (PDAC). [6][7][8][9] Metabolic conditions such as obesity, hypertension, dyslipidaemia, insulin resistance, and type 2 diabetes mellitus are also risk factors for pancreatic cancer. 8,10 About 5%-10% of patients with pancreatic cancer have underlying germline mutations or disorders, while the remaining percentage of cancer cases may be due to somatic mutations. 4 Epidemiological data suggest that heavy alcohol drinking increases the risk for pancreatic cancer. [11][12][13][14] Alcohol intake promotes intestinal tumourigenesis and tumour invasion in genetically susceptible mice, increases in polyp-associated mast cells, and mast cellmediated tumour migration in vitro, 15 suggesting mast cell-mediated inflammation could promote carcinogenesis. 15 Heavy alcohol intake is associated with the risk of developing chronic pancreatitis, [16][17][18] which may lead to pancreatic cancer. Alcohol drinking increases the permeability of the gut wall and translocation of lipopolysaccharide, which enhances pancreatic injury. 19,20 The effects of alcohol are modulated by polymorphisms in genes encoding enzymes for ethanol metabolism (e.g., alcohol dehydrogenases, aldehyde dehydrogenases, and cytochrome P450 2E1), folate metabolism, and DNA repair. During metabolism, ethanol is oxidized to acetaldehyde by ADH or CYP2E1. 21,22 Ethanol, acetaldehyde, and reactive oxygen species (ROS) are considered potential human carcinogens.
Chronic ethanol exposure of HPNE cells induced transformation, and those transformed cells gained the phenotypes of cancer stem cells (CSCs). 23 However, the molecular mechanism by which ethanol toxicity exerts its effects on pancreatic carcinogenesis is not well understood.
SATB2 (special AT-rich binding protein-2), a transcription factor and epigenetic regulator that binds DNA 24 to regulate gene expression. [25][26][27] SATB2 gene, although not expressed in healthy adults, is essential for normal mammalian development and proper facial patterning of the embryo and healthy bone development. 27 Hyperactivation/induction of SATB2 gene causes malignant cellular transformation. 23,28-31 SATB2 regulates transcription of pluripotency-maintaining factors (KLF4, Oct-4, SOX-2, and cMyc) which form the core regulatory positive feedback-loop for sustaining self-renewal capacity of stem cells. It has been shown that SATB2 binds to the promoters of Bcl-2, Bsp, Nanog, cMyc, XIAP, KLF4, and Hoxa2, suggesting a role of this gene in the regulation of cell survival, pluripotency, and proliferation. 29 Interestingly, high levels of ROS generated by ethanol exposure can induce SATB2 expression in HPNE cells. 29 Therefore, SATB2 protein may play a critical role in cellular transformation and carcinogenesis.
The primary goal of this paper is to examine the molecular mechanisms by which chronic ethanol exposure of HPNE cells induces EMT characteristics and ethanol feeding of KC mice enhances pancreatic cancer growth and development. To investigate the role of SATB2 at an early step of cell transformation, we utilized HPNE cells as a model to generate stem-like cells through chronic ethanol exposure. Our data demonstrate that chronic ethanol exposure can induce EMT and oral ethanol feeding of KC mice promotes pancreatic cancer growth and development by regulating SATB2, inflammatory cytokines, PTGS-2, stem cell markers, and pluripotency-maintaining factors. These data suggest that excessive alcohol can promote pancreatic carcinogenesis.

| Cell culture conditions and reagents
Human pancreatic normal ductal epithelial (HPNE) cells were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). HPNE cells were grown in well-defined cell culture medium as described. 32

| Lentiviral particle production and transduction
The lentivirus production and transduction were performed as described elsewhere. 33 In brief, lentivirus was produced by triple transfection of HEK 293T cells. Packaging 293T cells were plated in 10-cm plates at a cell density of 5 × 10 6 1 day before transfection in DMEM containing 10% heat-inactivated foetal bovine serum. A total of 293T cells were transfected with 4 µg of plasmid and 4 µg of the lentiviral vector using lipid transfection (Lipofectamine-2000) according to the manufacturer's protocol. Viral supernatants were collected and concentrated by adding PEG-it virus precipitation solution (System Biosciences, Palo Alto, CA) to produce virus stocks with titres of 1 × 10 8 to 1 × 10 9 infectious units per ml. Viral supernatant was collected for 3 days by ultracentrifugation and concentrated 100-fold. Titres were determined on 293T cells. Cells were transduced with lentiviral particles expressing the gene of interest.

| Quantitative real-time PCR
Total RNA in cells was extracted by the TRIzol reagent (Invitrogen) and reverse transcribed into cDNA using High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific). qRT-PCR was conducted using fast SYBR Green Master Mix (Thermo Fisher Scientific). The 2 −ΔΔC t method was used to evaluate relative mRNA expressions compared with controls.

| Motility assay
Assay for cell motility was performed as we described elsewhere. [33][34][35] 2.5 | KC (Pdx1-Cre and LSL-Kras G12D ) mice KC (Pdx1-Cre, and LSL-Kras G12D ) mice were generated as described elsewhere. 36 Mice (4-6 weeks old) were fed either a control diet or ethanolcontaining liquid diet (Dyets, Inc.) as described. 37,38 Mice were fed the control diet for 1 week to acclimate to the liquid diet and then fed either the control or ethanol-containing liquid diet (4%, v/v) for 6 months. At the end of the treatment, mice were sacrificed. Histological examination of the pancreas was performed by H&E staining as we described elsewhere, 29 and the numbers of PanINs and PDAC were quantified. 36

| Statistical analysis
The mean and standard deviation (SD) were calculated for each experimental group with replicates. Differences between groups were analysed by analysis of variance (ANOVA), followed by Bonferroni's multiple comparison tests using PRISM statistical analysis software (GrafPad Software, Inc.). Significant differences among groups were calculated at p < 0.05.

| SATB2 shRNA inhibits cell motility and regulates cadherin expression in EtOHtransformed cells
To examine whether SATB2 in involved in inducing EMT, we knocked down the expression of SATB2 by shRNA in ethanol-transformed HPNE cells, which were exposed to 10 or 100 mM ethanol for

| Ethanol feeding enhances stem cell markers, pluripotency-maintaining factors N-Cadherin, EMTtranscription factors and inflammatory cytokines, and inhibits E-cadherin expression in KC (Pdx1-Cre and LSL-Kras G12D ) mice
Since ethanol feeding of KC mice promotes pancreatic cancer growth and development, we next sought to measure the expression of stem cell markers, pluripotency-maintaining factors

| DISCUSS ION
The present study demonstrates the carcinogenic effects of alcohol on pancreatic cancer. We have recently shown that during ethanolinduced malignant transformation, CSCs/ progenitor cells are developed, which may play a significant role in pancreatic carcinogenesis. 23  The conversion of HPNE cells to cancer stem-like cells by ethanol confirms alcohol as a risk factor for pancreatic cancer.
Epidemiological data suggest that heavy alcohol drinking increases the risk for pancreatic cancer. 6 mice. Similarly, a recent finding has reported that moderate alcohol intake promoted pancreatic ductal adenocarcinoma in Pdx1 Cre ;LSL-Kras G12D mice but not in the control Pdx1 Cre mice. 64 We and others have not observed any induction of pancreatic cancer by alcohol without oncogenic Kras G12D mutations. 64 The reasons for differences observed in gene expressions of pancreatic ductal epithelial cells by chronic ethanol exposure in vitro compared with ethanol feeding of mice are not known. However, it may be due to the fact that mice are capable of detoxifying adverse effects of alcohol which will be absent in vitro studies.
In conclusion, our data demonstrate that chronic ethanol exposure of HPNE cells induces EMT in vitro, and oral ethanol feeding of KC mice promoted pancreatic cancer growth and development by regulating SATB2, inflammatory cytokines, PTGS-2, stem cell markers, and pluripotency-maintaining factors. These data suggest that alcohol is capable of promoting carcinogenesis and metastasis and could harm human health.

ACK N OWLED G EM ENT
The authors acknowledge all the lab members for critical reading, assistance, and suggestions during manuscript preparation.

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