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Loss of FOXA1/2 Is Essential for the Epithelial-To- Mesenchymal Transition in Pancreatic Cancer

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Loss of FOXA1/2 Is Essential for the Epithelial-To- Mesenchymal Transition in Pancreatic Cancer Published OnlineFirst February 16, 2010; DOI: 10.1158/0008-5472.CAN-09-2979 Published Online First on February 16, 2010 as 10.1158/0008-5472.CAN-09-2979 Tumor and Stem Cell Biology Cancer Research Loss of FOXA1/2 Is Essential for the Epithelial-to- Mesenchymal Transition in Pancreatic Cancer Yan Song1, M. Kay Washington2, and Howard C. Crawford1 Abstract FOXA1andFOXA2,membersoftheforkheadtranscriptionfactorfamily,arecriticalforepithelialdifferentiation in many endoderm-derived organs, including the pancreas. However, their role in tumor progression is largely un- known. Here, we identified FOXA1 and FOXA2 as important antagonists of the epithelial-to-mesenchymal transi- tion (EMT) in pancreatic ductal adenocarcinoma (PDA) through their positive regulation of E-cadherin and maintenance of the epithelial phenotype. In human PDA samples, FOXA1/2 are expressed in all epithelia from normal to well-differentiated cancer cells, but are lost in undifferentiated cancer cells. In PDA cell lines, FOXA1/2 expression is consistently suppressed in experimental EMT models and RNAi silencing of FOXA1/2 alone is sufficient to induce EMT. Conversely, ectopic FOXA1/2 expression can potently neutralize several EMT-related E-cadherin repressive mechanisms. Finally, ectopic FOXA2 expression could reactivate E-cadherin expression in a PDA cell line with extensive promoter hypermethylation. In fact, demethylation- mediated reactivation of E-cadherin expression in these cells required concurrent reactivation of endogenous FOXA2 expression. We conclude that suppression of FOXA1/2 expression is both necessary and sufficient for EMT during PDA malignant progression. Cancer Res; 70(5); 2115–25. ©2010 AACR. Introduction mice causes postnatal or embryonic lethality, respectively (8–11). Individual or compound deletions of Foxa1 and Foxa2 The epithelial-to-mesenchymal transition (EMT) is a phys- show that both Foxa1 and Foxa2 are essential for terminal iologic process, originally described in embryonic develop- differentiation and maturation of many endoderm-derived ment, in which cells lose epithelial characteristics and gain cells, including α-cells in the endocrine pancreas (12) and mesenchymal properties. EMT is accompanied by loss of liver (13), lung alveolar (14), and prostate luminal ductal cell-to-cell contact and increased cell motility. It is also im- epithelia (15). These findings suggest that Foxa1/2 proteins plicated in late-stage tumor progression as a prelude to can- are critical for both early embryonic development and late- cer metastasis (1, 2). As a gatekeeper of the epithelial or end-stage epithelial differentiation. phenotype (2), E-cadherin regulation has been extensively Little is known about the role of FOXA1/2 in cancer even studied during EMT, with most research focusing on its tran- though their expression is observed in many human cancers scriptional repression by E12/47, TWIST (3), and members of including prostate (16), breast (17, 18), lung, and esophagus the Snail (4, 5) and ZEB (6) protein families. In contrast, little (19). The involvement of FOXA proteins in pancreatic ductal attention has been paid to the involvement of E-cadherin adenocarcinoma (PDA) has yet to be described. Foxa1 and transcriptional activators in EMT because repressive factors Foxa2 are important transcription factors for endocrine pan- are thought to be dominant. creas development and function (7) and are expressed in exo- f ox FOXA proteins belong to subclass A of the orkhead b crine pancreas where their function is unclear (20, 21). containing transcription factor family (Fox proteins). FOXA1 PDA has a 5-year survival rate of less than 4%, a statistic and FOXA2 share 92% homology within their forkhead DNA- largely attributable to its aggressive invasive and metastasic Foxa1 Foxa2 binding domains (7). Genetic ablation of or in behavior. We found that both FOXA1 and FOXA2 were ex- pressed in most stages of PDA progression, but were lost in undifferentiated cancer cells. In PDA cell lines, FOXA1/2 Authors' Affiliations: 1Department of Pharmacology, Stony Brook expression was suppressed in experimental EMT models, University, Stony Brook, New York and 2Department of Pathology, and forced inhibition of FOXA1/2 factors was sufficient to Vanderbilt University Medical Center, Nashville, Tennessee induce EMT. As activators of E-cadherin transcription, Note: Supplementary data for this article are available at Cancer FOXA1/2 overexpression could overcome several repressive Research Online (http://cancerres.aacrjournals.org/). signals of E-cadherin expression, including Snail1 overexpres- Corresponding Author: Howard C. Crawford, Department of Pharmacol- sion,transforminggrowthfactor-β (TGFβ) treatment, and ogy, Stony Brook University, 1 Circle Road, BST 8-140, Stony Brook, NY 11794-8651. Phone: 631-444-3085; Fax: 631-444-9749; E-mail: E-cadherin promoter hypermethylation. Interestingly, the [email protected]. FOXA2 gene itself was suppressed by DNA methylation, doi: 10.1158/0008-5472.CAN-09-2979 and this repression was critical for methylation-mediated ©2010 American Association for Cancer Research. silencing of E-cadherin gene expression. Taken together, www.aacrjournals.org 2115 Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 2010 American Association for Cancer Research. Published OnlineFirst February 16, 2010; DOI: 10.1158/0008-5472.CAN-09-2979 Song et al. our study shows that loss of FOXA1/2 is a critical event in Medium containing 10% FBS was added to the lower cham- EMT during pancreatic cancer progression. ber. After 6-h incubation at 37°C, cells that migrated to the lower surface of the membrane were fixed in 4% paraformal- Materials and Methods dehyde and stained with 0.5% crystal violet. When siRNA- mediated FOXA1/2 silencing was used, cells were assessed Human tissue and immunohistochemistry. Sections of 48 h after transfection. For each membrane, five random archival formalin-fixed, paraffin-embedded human PDA and fields were counted at ×10 magnification. The mean was pancreatitis samples were obtained from the Tissue Core, calculated and data were presented as mean ± SEM from Vanderbilt University Medical Center. Primary antibodies three independent experiments done in triplicate. used for immunohistochemical analysis were anti-FOXA1 Statistics. Data were presented as mean ± SEM. Student's (Santa Cruz Biotechnology, Inc.) and anti-FOXA2 (Seven Hills t test was used in two-group comparisons. P <0.05was Bioreagents). Hemotoxylin was used at 1:10 dilution as coun- considered statistically significant. Expression of FOXA1/2 terstain to avoid interference with nuclear FOXA1/2 staining. in PDA was analyzed by χ2 test. Imagines were captured on an Olympus U-DO3 microscope. Cell culture and treatments. Human PDA cell lines were Results purchased from the American Type Culture Collection (ATCC) and maintained at 37°C in 5% CO2 in ATCC recom- Expression of FOXA1/2 is lost in undifferentiated PDA mended medium supplemented with 10% fetal bovine serum cells. The critical function of FOXA1/2 in pancreas develop- (FBS) and 0.5 μg/mL gentamicin. Phase-contrast images ment and in several types of cancer led us to examine their were captured on a Zeiss Axiovert 200M microscope. Immu- expression in human PDA using immunohistochemistry. nofluorescence images were captured on a Zeiss LSM-510 Twenty-five human PDA and five chronic pancreatitis sam- Meta confocal microscope. In indicated experiments, ples contained multiple epithelial pathologies, including cellsweretreatedwith5ng/mLTGFβ (R&D Systems), metaplasia, pancreatic intraepithelial neoplasia (PanIN), dif- 20 ng/mL hepatocyte growth factor (HGF; R&D Systems), ferentiated invasive PDA, and undifferentiated invasive PDA. 5′-aza-deoxycytidine (5′-aza-dC; Sigma-Aldrich), or their Results are summarized in Supplementary Table S1, divided corresponding vehicle controls. See Supplementary Data for by pathology, as found in each sample. antibodies, primers, and other methods. In nearby normal tissue, FOXA1 and FOXA2 were expressed Plasmids, siRNA, shRNA, and generation of stable cells. in all islet cell types and at lower levels in acinar and ductal Expression vectors for mouse Foxa1 and rat Foxa2 were gifts cells, primarily localized to the nucleus (Fig. 1A), a pattern of Dr. Robert Matusik (Vanderbilt University, Nashville, TN). consistent with that found in mouse pancreas (20, 21). Among Expression vector of Snail1 (pCDNA3.1/GS-Snail1) was pur- the 25 PDA specimens, 24 contained invasive cancer and 12 chased from Invitrogen. FOXA1-specific or FOXA2-specific contained various stages of PanIN. FOXA1 and FOXA2 local- siRNA (ON-TARGETplus, Dharmacon; see Supplementary ized primarily to the nucleus and were expressed in invasive Table S2 for sequences) was transfected using TransIT-TKO adenocarcinoma cells in 79% (19 of 24) and 75% (18 of 24) of (Mirus). A scrambled siRNA sequence (BLOCK-iT Alexa Fluor PDA samples (Fig. 1B), respectively, although strong cytoplas- fluorescent, Invitrogen) was used as control siRNA and as an mic staining was observed occasionally for FOXA1/2 (3 of 24) indicator of transfection efficiency. Lentiviral-based shRNA in poorly differentiated cancers. Both FOXA1 and FOXA2 constructs (pLKO.1-puro) targeting either the FOXA1 or the were detected in 83% (10 of 12) of PanINs at all stages (Fig. FOXA2 gene were purchased from Sigma (see Supplementary 1A). FOXA1 and FOXA2 were also expressed in all identifiable Table S2 for sequences). Control pLKO.1 vector was
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