Chronic ethanol exposure of human pancreatic normal ductal epithelial cells induces cancer stem cell phenotype through SATB2

Abstract The incidence of pancreatic cancer is on the rise. Risk factors for pancreatic cancer include alcohol toxicity and metabolic conditions such as obesity, hypertension, dyslipidaemia, insulin resistance and type 2 diabetes. However, the molecular mechanism by which chronic alcohol consumption contributes to pancreatic cancer is not well understood. The purpose of the study was to demonstrate the effects of long‐term chronic ethanol exposure on the transformation of human pancreatic normal ductal epithelial (HPNE) cells. Our data showed that ethanol‐transformed HPNE cells were more progressively transformed exhibiting spheroids and colonies, and anchorage‐independent growth. These transformed cells contained high levels of reactive oxygen species and induced SATB2 expression. Furthermore, during ethanol‐induced cellular transformation, cells gained the phenotypes of cancer stem cells (CSCs) by expressing pluripotency maintaining factors (Oct4, Sox2, cMyc and KLF4) and stem cell markers (CD24, CD44 and CD133). Ethanol‐induced SATB2 can bind to the promoters of KLF4, Oct4, cMyc, Sox2, Bcl‐2 and XIAP genes. Suppression of SATB2 expression in ethanol‐transformed HPNE cells inhibited cell proliferation, colony formation and markers of CSCs and pluripotency. These data suggest that chronic alcohol consumption may contribute toward the development of pancreatic cancer by converting HPNE cells to cancer stem‐like cells.

Epidemiological data strongly suggest that the heavy alcohol drinking increases the risk for pancreatic cancer. [11][12][13][14] A recent study has demonstrated that chronic alcohol intake promotes intestinal tumorigenesis and tumour invasion in genetically susceptible mice, increases in polyp-associated mast cells, and mast-cell-mediated tumour migration in vitro, 15 suggesting mast-cell-mediated inflammation could promote carcinogenesis. 15 These data confirm that ethanol and its metabolites are the potential human carcinogen.
However, the molecular mechanism by which ethanol toxicity induces malignant transformation of human pancreatic normal ductal epithelial (HPNE) cells is not known. SATB2 (special AT-rich binding protein-2), a transcription factor and epigenetic regulator that binds DNA 16 to regulate gene expression. [17][18][19] SATB2 gene is required for normal mammalian development; however, it is not expressed in healthy adult cells. SATB2 is essential for proper facial patterning of the embryo and healthy bone development. 19 Inappropriate activation of this gene may be the cause of malignant cellular transformation. SATB2 regulates transcription of pluripotency maintaining factors (Sox2, cMyc, KLF4 and Oct4) which form the core regulatory positive feedback-loop to sustain self-renewal capacity of stem cells. Using chromatin immunoprecipitation assay, we have shown that SATB2 can directly bind to the promoters of Bcl-2, Bsp, Nanog, cMyc, XIAP, KLF4 and Hoxa2, suggesting a role of SATB2 in the regulation of cell survival, pluripotency and proliferation. 20 Therefore, we reasoned to believe that SATB2 proteins may play a critical role during chronic ethanol exposure of human pancreatic ductal epithelial cells.
The primary goal of this paper was to examine the molecular mechanisms by which chronic ethanol exposure induces cellular transformation of HPNE cells which may lead to pancreatic carcinogenesis. To investigate the role of SATB2 at an early step of cell transformation, we utilized HPNE cells as a model to generate progenitor cells by chronic ethanol exposure. Our studies have established a novel link between exposure to ethanol and SATB2regulated transformation of HPNE cells.

| Cell culture conditions and reagents
Human pancreatic normal ductal epithelial cells were purchased from American Type Culture Collection, Manassas, VA. HPNE cells were grown in well-defined cell medium as described. 21 Antibodies against SATB2 and b-actin were purchased from Abcam (Cambridge, MA).
Enhanced chemiluminescence (ECL) Western blot detection reagents were purchased from Amersham Life Sciences Inc. (Arlington Heights, IL).

| Cell proliferation assay
Cells (1.5 9 10 4 ) were incubated for various time points in 1 mL of culture medium. Cell viability was determined by trypan blue assay using Countess TM Automated Cell Counter (Invitrogen).

| Colony formation assay
Colony formation assays were performed as described elsewhere. 22 In brief, cells were seeded into 6-well plates at a low density (200 cells per well). Cell culture medium was renewed every 3 days. After 21 days, colonies were fixed with cold methanol and then stained with 0.5% crystal violet. The colonies were imaged with a microscope.

| Spheroid formation
Spheroids formation assays were performed as described elsewhere. 22 In brief, cells were plated in ultra-low attachment plates at a density of 100-500 cells/mL. The spheroid formation in suspension was measured after 10 days of culture using a Nikon Eclipse microscope (Nikon).

| Lentiviral particle production and transduction
The lentivirus production and transduction were performed as described elsewhere. 22 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 9 10 6 1 day before transfection in DMEM containing 10% heat-inactivated foetal bovine serum.

| Western blot analysis
The Western blot analysis was performed as we described earlier. 23 In brief, cell lysates were subjected to SDS-PAGE, and gels were

| Chromatin immunoprecipitation assay
Chromatin immunoprecipitation (ChIP) assays were performed as we described elsewhere. 24,25 In brief, chromatin was immunoprecipitated using anti-SATB2 antibody. Normal rabbit IgG (Abcam) was used as a YU ET AL.

| Statistical analysis
The mean and SD were calculated for each experimental group with replicates. Differences between groups were analysed by ANOVA, followed by Bonferroni's multiple comparison tests using PRISM statistical analysis software (GrafPad Software, Inc., San Diego, CA). Significant differences among groups were calculated at P < .05.

| Ethanol induces transformation of HPNE cells by up-regulating SATB2 expression
We have used HPNE cells as a model to assess whether chronic ethanol exposure induces malignant transformation. HPNE cells were grown in culture medium in the presence or absence of ethanol (10 and 100 mmol/L) for 6 months. Long-term chronic exposure of HPNE cells to ethanol-induced cellular transformation as evident by the formation of clumps, loss of contact inhibition, and disoriented growth ( Figure 1A). HPNE cell transformation efficiency was significantly higher with the higher dose of ethanol (100 mmol/L) compared to 10 mmol/L ethanol exposure ( Figure 1B).

Oct4, cMyc, Sox2, Bcl-2 and XIAP in ethanoltransformed HPNE cells
SATB2 is a transcription factor and thus can regulate various cellular functions by directly binding to target genes. 29 Some of the target genes of SATB2 regulate pluripotency, self-renewal, and cell survival.
ChIP assay is commonly used to examine the binding of a transcription factor to the promoter regions of the target genes. We, therefore, investigated the binding of SATB2 to the promoters of potential gene targets in HPNE-transformed cells. SATB2 can directly bind to promoters of KLF4, Oct4, cMyc, Sox2, Bcl-2 and XIAP ( Figure 3). These data suggest that SATB2 can directly bind to these genes and regulate their expression in ethanol-transformed cells.

| Knockdown of SATB2 by shRNA in ethanoltransformed cells inhibits cell proliferation and colony formation
To examine whether SATB2 in involved in in vitro cellular transformation, we knocked-down the expression of SATB2 by shRNA in ethanol-transformed HPNE cells which were exposed to 10 or 100 mmol/ L ethanol for 6 months. Ethanol-transformed HPNE cells were transduced with either scrambled or SATB2 shRNA lentiviral particles, and cell growth and colony formation were measured (Figure 4) HPNE cells were grown in the well-defined culture medium as per American Type Culture Collection recommendations. HPNE cells were cultured for 6 mo with 2 different concentrations of ethanol (10 and 100 mmol/L). Photographs were taken under phase contrast microscope. B, HPNE cell transformation efficiency. Data represent mean AE SD. *, #Significantly different from control, P < .05. C, Expression of SATB2 by immunohistochemistry (IHC). IHC was performed to examine the nuclear expression of SATB2 in HPNE/Control and HPNE/Ethanol cells as we described elsewhere. 22 Red colour = nucleus. Yellow colour = red (nucleus) + green (SATB2) = merged picture (expression of SATB2 in nucleus). D-F, SATB2 expression in HPNE/Control and HPNE/Ethanol transformed cells was measured by PCR, Western blot analysis, and qRT-PCR, respectively. qRT-PCR data represent mean AE SD. *, #Significantly different from HPNE/Control cells, P < .05 expression of SATB2 proteins and mRNA compared to that of HPNE/ Ethanol (10 or 100 mmol/L)/Scrambled cells ( Figure 4A and B).

| SATB2 inhibits the expression of stem cell markers in ethanol-transformed cells
Our data demonstrate that chronic exposure of HPNE cells with ethanol induces stem cell markers CD133, CD44 and CD24. We, therefore, sought to examine whether inhibition of SATB2 by shRNA attenuates the expression of these markers in ethanol-

F I G U R E 3
Binding of SATB2 to promoters of KLF4, Oct4, cMyc, Sox2, Bcl-2 and XIAP. A-B, Chromatin immunoprecipitation (ChIP) assays were performed to examine the binding of the SATB2 to the promoters of KLF4, Oct4, cMyc, Sox2, Bcl-2 and XIAP in HPNE/control and HPNE/Ethanol cells as described elsewhere. 47,50 ChIP-derived DNA was quantified by 2% agarose gel electrophoresis F I G U R E 4 SATB2 shRNA inhibits cell proliferation and colony formation. A, Ethanol-transformed HPNE cells (10 or 100 mmol/L ethanol for 6 mo) were transduced with either scrambled or SATB2 shRNA. The expression of SATB2 was confirmed by the Western blotting analysis. b-actin was used as a loading control. B, RNA was isolated and q-RT-PCR was performed to measured the expression of SATB2 mRNA. Data represent mean AE SD. *, or # = significantly different from scrambled control group, P < .05. C, Cell proliferation was measured over 8 d. Data represent mean AE SD. *, #Significantly different from scrambled control group, P < .05. D, Colony formation. Ethanol-transformed cells were seeded in Petri dishes. The number of colonies formed in 21 d was photographed. proliferation. 24,30 Epidemiological data strongly suggest that the heavy drinking increases the risk for pancreatic cancer. 6,10,31,32 High alcohol intake was associated with a higher risk of pancreatitis. 33 production of ROS and nitrogen species, and changes in folate metabolism have been implicated. Daily consumption of more than 80 g alcohol (more than five to six drinks) with smoking increases the risk of developing cancers by a factor of 50 or more. 40,41 In support of our study, it was demonstrated that chronic alcohol consumption promotes intestinal tumorigenesis and tumour invasion in genetically susceptible mice. 15 During ethanol metabolism, ethanol is oxidized to acetaldehyde by ADH or CYP2E1. 42,43 If the patients consume alcohol, acetaldehyde concentrations in the stomach increase 6.5-fold. 44 In addition to the acetaldehyde generated by cellular enzymes or gastrointestinal bacteria, considerable amounts of acetaldehyde are present in certain alcoholic beverages and cigarette smoke. 44 Based on these findings it appears that ethanol and its metabolites are the potential human carcinogens.

| DISCUSSION
SATB2 may act as a master regulator of pluripotency and selfrenewal because SATB2 binding sites are present in the promoter regions of KLF4, Oct4, cMyc and Sox2. [45][46][47][48] Similar to pancreatic CSCs, we have found that SATB2 is highly expressed in colorectal and breast CSCs. 24,30 Interestingly, the expression of SATB2 was absent or very low in HPNE cells, mammary epithelial cells and colorectal epithelial cells. 20,24,30 Furthermore, overexpression of SATB2 in normal epithelial cells resulted in malignant transformation, suggesting an oncogenic role of SATB2 in various cancers. In the