Chronic Ethanol Exposure of Human Pancreatic Normal Ductal Epithelial Cells Induces Cancer Stem Cell Phenotype through SATB2

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 SATB 2 expression. Furthermore, during ethanol‐induced cellular transformation, cells gained the phenotypes of cancer stem cells (CSC s) by expressing pluripotency maintaining factors (Oct4, Sox2, cM yc and KLF 4) and stem cell markers (CD 24, CD 44 and CD 133). Ethanol‐induced SATB 2 can bind to the promoters of KLF 4, Oct4, cM yc, Sox2, Bcl‐2 and XIAP genes. Suppression of SATB 2 expression in ethanol‐transformed HPNE cells inhibited cell proliferation, colony formation and markers of CSC s 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.

INTRODUCTION

Pancreatic cancer has an exceptionally high mortality rate and is the fourth leading cause of cancer‐related death in the United States.1 With an overall 5‐year survival rate of 6%,2 pancreatic cancer has one of the poorest prognoses among all cancers.3 The incidence of pancreatic cancer varies significantly across regions, which suggests that several factors may be responsible for this deadly disease.4 The genetic, race, gender, environmental carcinogen, diet and lifestyle appears to be the primary factors for pancreatic cancer.5 Other factors such as smoking, alcohol, coffee consumption and exposure to organochlorine or hydrocarbon solvent have been associated with the frequency and spectrum of K‐ras mutation in pancreatic tumours.6–9 Metabolic conditions such as obesity, hypertension, dyslipidaemia, insulin resistance, and type 2 diabetes are also the risk factors for pancreatic cancer.8, 10 About 5%‐10% of patients with pancreatic cancer have underlying germline mutations or disorders, whereas the remaining percentage of cancer cases may be because of somatic mutations.4

Epidemiological data strongly suggest that the heavy alcohol drinking increases the risk for pancreatic cancer.11–14 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.17–19 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.

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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 SATB2‐regulated transformation of HPNE cells.

MATERIALS AND METHODS

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 β‐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 × 10⁴) were incubated for various time points in 1 mL of culture medium. Cell viability was determined by trypan blue assay using Countess™ 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 × 10⁶ 1 day before transfection in DMEM containing 10% heat‐inactivated foetal bovine serum. 293T cells were transfected with 4 μg of plasmid and 4 μg of the lentiviral vector using lipid transfection (Lipofectamine‐2000/Plus reagent; Invitrogen) according to the manufacturer’s protocol. Viral supernatants were collected and concentrated by adding PEG‐it virus precipitation solution (SBI System Biosciences) to produce virus stocks with titres of 1 × 10⁸‐1 × 10⁹ 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.

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 blotted onto nitrocellulose membrane (Amersham Biosciences, Piscataway, NJ, USA). The membranes were blocked with 5% BSA in Tris‐Tween buffered saline at 37°C for 2 hours and then incubated with primary antibody diluted in Tris‐buffered saline (1:1000 dilutions) overnight at 4°C, with gentle shaking. The membranes were then washed 3 times with Tris‐buffered saline‐T (TBS‐T) and incubated with the secondary antibody linked to horseradish peroxidase (1:5000) for 1 hour. After incubation with secondary antibody, the membranes were rewashed 3 times with TBS‐T. Finally, protein‐antibody complexes were detected by the addition of ECL substrate (Thermo Fisher Scientific, Rockford, IL).

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 negative control. ChIP‐derived DNA was measured by 2% agarose gel electrophoresis.

• Bcl‐2 promoterChIP‐F, TTTCAGCATCACAGAGGAAG
• Bcl‐2 promoterChIP‐R, CAATCACGCGGAACACTTGATT
• Oct4‐ChIP‐F, ATGACCACTGCGCCCGGACTGC
• Oct4‐ChIP‐R, ACTTGGATCTCTTCCAAGTGC
• KLF4‐Chip‐F, ACCGGACCTACTTACTCGCC
• KLF4‐Chip‐R, TCGGCAGCCCGAAGCAGCTGG
• cMyc‐Chip‐F, AATTAATGCCTGGAAGGCAGCC
• cMyc‐ChIP‐R, AGTCAGCAGAGACCCTTGTG
• XIAP‐Chip‐F,TCCAAGAGAGATGCACTAGGGTC
• XIAP‐Chip‐R, TTATGGCAAGATCTATGTGGAACTC

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^−ΔΔCt method was used to evaluate relative mRNA expressions compared with controls. The following gene‐specific primers were used:

• Sox2 (5′‐AAC CCC AAG ATG CAC AAC TC‐3′, 5′‐GCT TAG CCT CGT CGA TGA AC‐3′)
• cMyc (5′‐CGA CGA GAC CTT CAT CAA AA‐3′, 5′‐TGC TGT CGT TGA GAG GGT AG‐3′)
• Oct4 (5′‐GGA CCA GTG TCC TTT CCT CT‐3′, 5′‐CCA GGT TTT CTT TCC CTA GC‐3′)
• CD24 (5′‐ATG GGA ACA AAC AGA TCG AA‐3′, 5′‐TTT GCT CTT TCA GCC ATT TC‐3′)
• CD44 (5′‐ACT TCA CCC CAC AAT CTT GA‐3′, 5′‐GTG GCT TGT TGC TTT TCA GT‐3′)
• CD133 (5′‐CCT CTG GTG GGG TAT TTC TT‐3′, 5′‐CCT CTG GTG GGG TAT TTC TT‐3′)
• HK‐GAPD (5′‐GAG TCA ACG GAT TTG GTC GT‐3′, 5′‐TTG ATT TTG GAG GGA TCT CG‐3′)

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.

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