Smad4 Decreases the Population of Pancreatic Cancer–Initiating Cells through Transcriptional Repression of ALDH1A1
2015; Elsevier BV; Volume: 185; Issue: 5 Linguagem: Inglês
10.1016/j.ajpath.2015.01.011
ISSN1525-2191
AutoresYukari Hoshino, Jun Nishida, Yoko Katsuno, Daizo Koinuma, Taku Aoki, Norihiro Kokudo, Kohei Miyazono, Shogo Ehata,
Tópico(s)Liver physiology and pathology
ResumoCancer progression involves a rare population of undifferentiated cancer-initiating cells that have stem cell–like properties for self-renewal capacity and high tumorigenicity. We investigated how maintenance of pancreatic cancer–initiating cells is influenced by Smad4, which is frequently deleted or mutated in pancreatic cancers cells. Smad4 silencing up-regulated the expression of aldehyde dehydrogenase 1A1 (ALDH1A1) mRNA, whereas forced expression of Smad4 in pancreatic cancer cells down-regulated it. Smad4 and ALDH1 expression inversely correlated in some human clinical pancreatic adenocarcinoma tissues, suggesting that ALDH1 in pancreatic cancer cells was associated with decreased Smad4 expression. We then examined whether ALDH1 served as a marker of pancreatic cancer–initiating cells. Pancreatic cancer cells contained ALDH1hi cells in 3% to 10% of total cells, with high tumorigenic potential. Because Smad4 is a major mediator of transforming growth factor (TGF)-β family signaling, we investigated the regulatory mechanism of ALDH activity by TGF-β and bone morphogenetic proteins. Treatment with TGF-β attenuated ALDH1hi cells in several pancreatic cancer cells, whereas bone morphogenetic protein-4 was not as potent. Biochemical experiments revealed that TGF-β regulated ALDH1A1 mRNA transcription through binding of Smad4 to its regulatory sequence. It appears that TGF-β negatively regulates ALDH1 expression in pancreatic cancer cells in a Smad-dependent manner and in turn impairs the activity of pancreatic cancer–initiating cells. Cancer progression involves a rare population of undifferentiated cancer-initiating cells that have stem cell–like properties for self-renewal capacity and high tumorigenicity. We investigated how maintenance of pancreatic cancer–initiating cells is influenced by Smad4, which is frequently deleted or mutated in pancreatic cancers cells. Smad4 silencing up-regulated the expression of aldehyde dehydrogenase 1A1 (ALDH1A1) mRNA, whereas forced expression of Smad4 in pancreatic cancer cells down-regulated it. Smad4 and ALDH1 expression inversely correlated in some human clinical pancreatic adenocarcinoma tissues, suggesting that ALDH1 in pancreatic cancer cells was associated with decreased Smad4 expression. We then examined whether ALDH1 served as a marker of pancreatic cancer–initiating cells. Pancreatic cancer cells contained ALDH1hi cells in 3% to 10% of total cells, with high tumorigenic potential. Because Smad4 is a major mediator of transforming growth factor (TGF)-β family signaling, we investigated the regulatory mechanism of ALDH activity by TGF-β and bone morphogenetic proteins. Treatment with TGF-β attenuated ALDH1hi cells in several pancreatic cancer cells, whereas bone morphogenetic protein-4 was not as potent. Biochemical experiments revealed that TGF-β regulated ALDH1A1 mRNA transcription through binding of Smad4 to its regulatory sequence. It appears that TGF-β negatively regulates ALDH1 expression in pancreatic cancer cells in a Smad-dependent manner and in turn impairs the activity of pancreatic cancer–initiating cells. Among cancer patients, the major causes of mortality are local recurrences and distant metastases. Recent evidence indicates that these events occur because of a specific population of the so-called cancer-initiating cells (CICs), also known as cancer stem cells, which exist within various types of tumors. Therefore, understanding and targeting CICs are essential for improving cancer treatment. CICs are capable of both self-renewal and forming non-CICs to maintain the heterogeneous lineages of cancer cells that comprise the tumor.1Clarke M.F. Dick J.E. Dirks P.B. Eaves C.J. Jamieson C.H. Jones D.L. Visvader J. Weissman I.L. Wahl G.M. Cancer stem cells–perspectives on current status and future directions: AACR Workshop on cancer stem cells.Cancer Res. 2006; 66: 9339-9344Crossref PubMed Scopus (2503) Google Scholar This hierarchy produces the diversity and heterogeneity of cancer cells found in tumor tissue. CICs can be identified by expression of cell surface markers, such as CD133 and CD44, by expression of the aldehyde dehydrogenase 1 (ALDH1) enzyme, or as a side population using flow cytometry. ALDH1 oxidizes retinal to retinoic acid and is responsible for resistance against anticancer drugs, such as cyclophosphamide.2Russo J.E. Hilton J. Characterization of cytosolic aldehyde dehydrogenase from cyclophosphamide resistant L1210 cells.Cancer Res. 1988; 48: 2963-2968PubMed Google Scholar ALDH1 expression has been detected in normal stem cells and progenitor cells,3Armstrong L. Stojkovic M. Dimmick I. Ahmad S. Stojkovic P. Hole N. Lako M. Phenotypic characterization of murine primitive hematopoietic progenitor cells isolated on basis of aldehyde dehydrogenase activity.Stem Cells. 2004; 22: 1142-1151Crossref PubMed Scopus (210) Google Scholar, 4Hess D.A. Wirthlin L. Craft T.P. Herrbrich P.E. Hohm S.A. Lahey R. Eades W.C. Creer M.H. Nolta J.A. Selection based on CD133 and high aldehyde dehydrogenase activity isolates long-term reconstituting human hematopoietic stem cells.Blood. 2006; 107: 2162-2169Crossref PubMed Scopus (251) Google Scholar, 5Hess D.A. Craft T.P. Wirthlin L. Hohm S. Zhou P. Eades W.C. Creer M.H. Sands M.S. Nolta J.A. Widespread nonhematopoietic tissue distribution by transplanted human progenitor cells with high aldehyde dehydrogenase activity.Stem Cells. 2008; 26: 611-620Crossref PubMed Scopus (69) Google Scholar as well as in several types of cancers, including breast, lung, colon, diffuse-type gastric, and pancreatic cancers.6Jiang F. Qiu Q. Khanna A. Todd N.W. Deepak J. Xing L. Wang H. Liu Z. Su Y. Stass S.A. Katz R.L. Aldehyde dehydrogenase 1 is a tumor stem cell-associated marker in lung cancer.Mol Cancer Res. 2009; 7: 330-338Crossref PubMed Scopus (652) Google Scholar, 7Huang E.H. Hynes M.J. Zhang T. Ginestier C. Dontu G. Appelman H. Fields J.Z. Wicha M.S. Boman B.M. Aldehyde dehydrogenase 1 is a marker for normal and malignant human colonic stem cells (SC) and tracks SC overpopulation during colon tumorigenesis.Cancer Res. 2009; 69: 3382-3389Crossref PubMed Scopus (841) Google Scholar, 8Rasheed Z.A. Yang J. Wang Q. Kowalski J. Freed I. Murter C. Hong S.M. Koorstra J.B. Rajeshkumar N.V. He X. Goggins M. Iacobuzio-Donahue C. Berman D.M. Laheru D. Jimeno A. Hidalgo M. Maitra A. Matsui W. Prognostic significance of tumorigenic cells with mesenchymal features in pancreatic adenocarcinoma.J Natl Cancer Inst. 2010; 102: 340-351Crossref PubMed Scopus (347) Google Scholar, 9Ginestier C. Hur M.H. Charafe-Jauffret E. Monville F. Dutcher J. Brown M. Jacquemier J. Viens P. Kleer C.G. Liu S. Schott A. Hayes D. Birnbaum D. Wicha M.S. Dontu G. ALDH1 is a marker of normal and malignant human mammary stem cells and a predictor of poor clinical outcome.Cell Stem Cell. 2007; 1: 555-567Abstract Full Text Full Text PDF PubMed Scopus (3149) Google Scholar, 10Katsuno Y. Ehata S. Yashiro M. Yanagihara K. Hirakawa K. Miyazono K. Coordinated expression of REG4 and aldehyde dehydrogenase 1 regulating tumourigenic capacity of diffuse-type gastric carcinoma-initiating cells is inhibited by TGF-β.J Pathol. 2012; 228: 391-404Crossref PubMed Scopus (85) Google Scholar High ALDH1 expression correlates with poor prognosis in several types of cancers.8Rasheed Z.A. Yang J. Wang Q. Kowalski J. Freed I. Murter C. Hong S.M. Koorstra J.B. Rajeshkumar N.V. He X. Goggins M. Iacobuzio-Donahue C. Berman D.M. Laheru D. Jimeno A. Hidalgo M. Maitra A. Matsui W. Prognostic significance of tumorigenic cells with mesenchymal features in pancreatic adenocarcinoma.J Natl Cancer Inst. 2010; 102: 340-351Crossref PubMed Scopus (347) Google Scholar, 11Ohi Y. Umekita Y. Yoshioka T. Souda M. Rai Y. Sagara Y. Tanimoto A. Aldehyde dehydrogenase 1 expression predicts poor prognosis in triple-negative breast cancer.Histopathology. 2011; 59: 776-780Crossref PubMed Scopus (68) Google Scholar Transforming growth factor (TGF)-β is the prototypic member of a family of secreted proteins that includes TGF-βs, activins, and bone morphogenetic proteins (BMPs). At the cell surface, TGF-β binds to two different serine-threonine kinase receptors, type I (TβRI) and type II (TβRII), thus exerting many biological functions in both normal and cancer cells.12Heldin C.H. Miyazono K. ten Dijke P. TGF-β signalling from cell membrane to nucleus through SMAD proteins.Nature. 1997; 390: 465-471Crossref PubMed Scopus (3341) Google Scholar On ligand binding, two TβRIIs and two TβRIs form a heteromeric complex, which transduces intracellular signals by phosphorylating receptor-regulated Smads (R-Smads): Smad2 and Smad3 for TGF-β signaling and Smad1 and Smad5 for BMP signaling. These phosphorylated R-Smads form heteromeric Smad complexes with the common-partner Smad Smad4. R-Smad and common-partner Smad complexes associate with various transcription factors and transcriptional co-activators or co-repressors in the nucleus, thereby regulating transcription of a wide spectrum of target genes. TGF-β also activates non-Smad pathways, including various mitogen-activated protein kinase pathways. TGF-β inhibits epithelial cell proliferation through the induction of p15INK4B and p21CIP1/WAF1 or through the reduction or c-Myc; therefore, TGF-β signaling pathways have tumor-suppressive function during cancer progression. Accordingly, perturbation of TGF-β signaling is thought to be involved in many types of cancers. Alterations of genes encoding TβRII, TβRI, Smad4, and Smad2 have been reported to be responsible for progression of various types of cancers.13Bierie B. Moses H. Tumour microenvironment: TGFβ: the molecular Jekyll and Hyde of cancer.Nat Rev Cancer. 2006; 6: 506-520Crossref PubMed Scopus (1097) Google Scholar Pancreatic cancer is one of the most aggressive cancers and is most commonly diagnosed when it is already at an advanced stage of either metastatic or locally advanced cancer. For the past 40 years, the 5-year survival rate of pancreatic cancer patients has remained only 6%. It appears that lesions occurring in the pancreatic ducts, pancreatic intraepithelial neoplasia (PanIN), are a precursor of pancreatic ductal adenocarcinoma. Numerous studies of PanIN and associated adenocarcinomas have identified common mutation patterns, particularly in KRAS and CDKN2A (encoding p16INK4A). Coincident lesions also reportedly show similar patterns of loss of heterozygosity at chromosomes 9q, 17p, and 18q, which harbor CDKN2A, TP53, and SMAD4, respectively, with studies consistently showing an increasing number of gene alterations in higher grade PanINs.14Bardeesy N. DePinho R.A. Pancreatic cancer biology and genetics.Nat Rev Cancer. 2002; 2: 897-909Crossref PubMed Scopus (941) Google Scholar Blackford et al15Blackford A. Serrano O.K. Wolfgang C.L. Parmigiani G. Jones S. Zhang X. Parsons D.W. Lin J.C. Leary R.J. Eshleman J.R. Goggins M. Jaffee E.M. Iacobuzio-Donahue C.A. Maitra A. Cameron J.L. Olino K. Schulick R. Winter J. Herman J.M. Laheru D. Klein A.P. Vogelstein B. Kinzler K.W. Velculescu V.E. Hruban R.H. SMAD4 gene mutations are associated with poor prognosis in pancreatic cancer.Clin Cancer Res. 2009; 15: 4674-4679Crossref PubMed Scopus (295) Google Scholar suggested that inactivation of the SMAD4 gene correlates with metastasis and poor prognosis in patients with surgically resected pancreatic ductal adenocarcinoma. We present evidence that TGF-β negatively regulates ALDH1 expression in pancreatic cancer cells in a Smad-dependent manner and that TGF-β in turn impairs the activity of pancreatic CICs. Our model predicts that this regulatory mechanism might be disrupted by mutations and deletions that occur in SMAD4 in human pancreatic cancer cells. Human pancreatic adenocarcinoma Panc-1 and BxPC-3 cells were cultured as described previously.16Ijichi H. Otsuka M. Tateishi K. Ikenoue T. Kawakami T. Kanai F. Arakawa Y. Seki N. Shimizu K. Miyazono K. Kawabe T. Omata M. Smad4-independent regulation of p21/WAF1 by transforming growth factor-β.Oncogene. 2004; 23: 1043-1051Crossref PubMed Scopus (67) Google Scholar, 17Horiguchi K. Shirakihara T. Nakano A. Imamura T. Miyazono K. Saitoh M. Role of Ras signaling in the induction of snail by transforming growth factor-β.J Biol Chem. 2009; 284: 245-253Crossref PubMed Scopus (180) Google Scholar Human pancreatic adenocarcinoma SUIT-2 cells were cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, 100 U/mL of penicillin, and 100 μg/mL of streptomycin. Cells were grown in a 5% CO2 atmosphere at 37°C. All experiments were performed on heterogenous populations of shRNA-transfected or adenovirus-infected cells. Among three TGF-β isoforms, TGF-β3 (R&D Systems, Minneapolis, MN) was used in this study. Recombinant BMP-4 was purchased from R&D Systems. Lentivirus vectors carrying shRNA were generated as described previously.18Ehata S. Hanyu A. Fujime M. Katsuno Y. Fukunaga E. Goto K. Ishikawa Y. Nomura K. Yokoo H. Shimizu T. Ogata E. Miyazono K. Shimizu K. Imamura T. Ki26894, a novel transforming growth factor-β type I receptor kinase inhibitor, inhibits in vitro invasion and in vivo bone metastasis of a human breast cancer cell line.Cancer Sci. 2007; 98: 127-133Crossref PubMed Scopus (154) Google Scholar pENTR4-H1 was used to insert shRNA specific for human SMAD4 and ALDH1A1 into the lentivirus vectors CSII-RfA-CG and CS-RfA, respectively. Vectors were also generated using control shRNA. The sequences of shRNAs are listed in Table 1. Lentiviruses were concentrated using Lenti-X Concentrator (Clontech, Mountain View, CA).Table 1Sequences of Oligonucleotides Used for Generation of shRNAs and Primers in RT-PCRGeneOligonucleotidesOligonucleotides used for generation of shRNAsSMAD4Forward: 5′-GATCCCCAAGCAATGGAACACCAATACTCAGGGTGTGCTGTCCCCTGAGTATTGGTGTTCCATTGCTTTTTTTGGAAAT-3′Reverse: 5′-CTAGATTTCCAAAAAAAGCAATGGAACACCAATACTCAGGGGACAGCACACCCTGAGTATTGGTGTTCCATTGCTTGGG-3′ALDH1A1Forward: 5′-GATCCCCGTAGCCTTCACAGGATCAAACGTGTGCTGTCCGTTTGATCCTGTGAAGGCTACTTTTTGGAAAT-3′Reverse: 5′-CTAATTTCCAAAAAGTAGCCTTCACAGGATCAAACGGACAGCACACGTTTGATCCTGTGAAGGCTACGGG-3′ControlForward: 5′-GATCCCCGCGCGCTTTGTAGGATTCGGTGTGCTGTCCCGAATCCTACAAAGCGCGCTTTTTGGAAAT-3′Reverse: 5′-CTAGATTTCCAAAAAGCGCGCTTTGTAGGATTCGGGACAGCACACCGAATCCTACAAAGCGCGCGGG-3′Primers used for quantitative real-time PCR in chromatin immunoprecipitation analysisALDH1A1Forward: 5′-ACCCCAGGAGTCACTCAAGG-3′Reverse: 5′-ACTGTGGGCTGGACAAAGTAG-3′ALDH1A3Forward: 5′-TCTCGACAAAGCCCTGAAGT-3′Reverse: 5′-TATTCGGCCAAAGCGTATTC-3′CDKN1AForward: 5′-AGTGGACAGCGAGCAGCTGA-3′Reverse: 5′-CGAAGTTCCATCGCTCACGG-3′CDKN1BForward: 5′-CGGTGGACCACGAAGAGTTAA-3′Reverse: 5′-GGCTCGCCTCTTCCATGTC-3′CDKN2BForward: 5′-CCGCCCACAACGACTTTATT-3′Reverse: 5′-CAGCCTTCATCGAATTAGGTG-3′CDC25AForward: 5′-GCCTGTCACCAACCTGAC-3′Reverse: 5′-CCAGGAGAATCTAGACAGAAACC-3′MYCForward: 5′-CCACACATCAGCACAACTACGC-3′Reverse: 5′-CGGTTGTTGCTGATCTGTCTCA-3′CCNE1Forward: 5′-GCACTTTCTTGAGCAACACCCT-3′Reverse: 5′-GTGTCGCCATATACCGGTCAAA-3′SMAD4Forward: 5′-AAAACGGCCATCTTCAGCAC-3′Reverse: 5′-AGGCCAGTAATGTCCGGGA-3′NANOGForward: 5′-ATTCAGGACAGCCCTGATTCTTC-3′Reverse: 5′-TTTTTGCGACACTCTTCTCTGC-3′POU5F1Forward: 5′-GTGGAGAGCAACTCCGATG-3′Reverse: 5′-TGCTCCAGCTTCTCCTTCTC-3′SOX2Forward: 5′-CGAGTGGAAACTTTTGTCGGA-3′Reverse: 5′-TGTGCAGCGCTCGCAG-3′PROM1Forward: 5′-TGGATGCAGAACTTGACAACGT-3′Reverse: 5′-ATACCTGCTACGACAGTCGTGGT-3′CXCR4Forward: 5′-CCTGCCCTCCTGCTGACTATT-3′Reverse: 5′-TCATCTGCCTCACTGACGTTG-3′EPCAMForward: 5′-CGGCGACGGCGACTTTTG-3′Reverse: 5′-GAGCCATTCATTTCTGCCTTCATC-3′CD44Forward: 5′-TCAGAGGAGTCGGAGAGAGGAAAC-3′Reverse: 5′-GAAAAGTCAAAGTAACAATAACGTGG-3′CD24Forward: 5′-TGTTCTCTTGGGAACTGAACTC-3′Reverse: 5′-ACCTAAAAGGTCAAATGCAATG-3′HPRT1Forward: 5′-TTTGCTTTCCTTGGTCAGGC-3′Reverse: 5′-GCTTGCGACCTTGACCATCT-3′Primers used for quantitative real-time PCR in chromatin immunoprecipitation analysisHPRT1Forward: 5′-TGTTTGGGCTATTTACTAGTTG-3′Reverse: 5′-ATAAAATGACTTAAGCCCAGAG-3′ALDH1A1 +25,060 to +27,715 bpForward: 5′-TGCAACAGGGCATACTCCTT-3′Reverse: 5′-CAGGGCAGAAGAATCACAGA-3′ PromoterForward: 5′-ACTGTGGTGCAAACAGCAACACC-3′Reverse: 5′-TTGGTGTGGTGGTACCCATAAGAGC-3′ Intron11Forward: 5′-CCAAGCAGCTATTAGGTCTGGGACA-3′Reverse: 5′-CACCCCACTGAGGGTCTTGGGA-3′ −124,970 to −123,530 bpForward: 5′-CCATCTGGTGAAACATGCTG-3′Reverse: 5′-TACTGCGAGGCTTTCTGTGA-3′ Open table in a new tab Transient transfection of Smad4 was performed using FuGENE 6 (Roche Diagnostics, Basel, Switzerland) following the manufacturer's instructions. In a 6-well plate, BxPC-3 cells were transfected with 5 μg of control vector or pcDNA3-Smad4 in the presence of 6 μL of FuGENE 6 per well. Knockdown of Smad4 using siRNA was performed as described previously.19Nishimori H. Ehata S. Suzuki H.I. Katsuno Y. Miyazono K. Prostate cancer cells and bone stromal cells mutually interact with each other through bone morphogenetic protein-mediated signals.J Biol Chem. 2012; 287: 20037-20046Crossref PubMed Scopus (37) Google Scholar The siRNA sequence targeting human SMAD4 was 5′-UUACAUUCCAACUGCACACCUUUGC-3′. Panc-1 cells were transfected with 100 nmol/L of either siRNA or control siRNA in the presence of 5 μL of Lipofectamine 2000 (Invitrogen, Carlsbad, CA) per well of a 6-well plate. Total RNA was extracted using Isogen reagent (Nippon Gene, Toyama, Japan). Same cDNA was prepared from each cell, subjected to quantitative real-time RT-PCR analysis.20Hoshino Y. Katsuno Y. Ehata S. Miyazono K. Autocrine TGF-β protects breast cancer cells from apoptosis through reduction of BH3-only protein, Bim.J Biochem. 2011; 149: 55-65Crossref PubMed Scopus (46) Google Scholar All samples were run in duplicate. Values were normalized by hypoxanthine guanine phosphoribosyl transferase 1 mRNA. The primers for quantitative real-time RT-PCR are listed in Table 1. Immunoblotting was performed as described previously.20Hoshino Y. Katsuno Y. Ehata S. Miyazono K. Autocrine TGF-β protects breast cancer cells from apoptosis through reduction of BH3-only protein, Bim.J Biochem. 2011; 149: 55-65Crossref PubMed Scopus (46) Google Scholar Anti–phospho-retinoblastoma protein (Ser 807/811) antibody and anti–poly (ADP-ribose) polymerase antibody were purchased from Cell Signaling Technology (Beverly, MA). Anti-ALDH1 antibody was purchased from BD Biosciences (San Jose, CA), and anti-Smad4 antibody was from Santa Cruz Biotechnology (Santa Cruz, CA). Anti–α-tubulin antibody was purchased from Sigma-Aldrich (St Louis, MO). Cells from each cell stock were seeded in triplicate in 6-well plates (1 × 104 cells per well). At the indicated days after preparation, cells were counted using a hemocytometer as described previously.20Hoshino Y. Katsuno Y. Ehata S. Miyazono K. Autocrine TGF-β protects breast cancer cells from apoptosis through reduction of BH3-only protein, Bim.J Biochem. 2011; 149: 55-65Crossref PubMed Scopus (46) Google Scholar The colony formation assay was performed as described previously.21Ehata S. Johansson E. Katayama R. Koike S. Watanabe A. Hoshino Y. Katsuno Y. Komuro A. Koinuma D. Kano M.R. Yashiro M. Hirakawa K. Aburatani H. Fujita N. Miyazono K. Transforming growth factor-β decreases the cancer-initiating cell population within diffuse-type gastric carcinoma cells.Oncogene. 2011; 30: 1693-1705Crossref PubMed Scopus (64) Google Scholar Cells were seeded in 0.3% agar at 1 × 104 cells per well and cultured for 2 weeks. Diethylaminobenzaldehyde (DEAB) (100 mmol/L; Sigma-Aldrich) was diluted in ethanol and added to the medium of each well at a concentration of 100 μmol/L every 3 days. The colony diameters were measured using Cellsens Standard (Olympus, Tokyo, Japan). Chromatin immunoprecipitation (ChIP) analysis was performed as described previously.22Koinuma D. Tsutsumi S. Kamimura N. Taniguchi H. Miyazawa K. Sunamura M. Imamura T. Miyazono K. Aburatani H. Chromatin immunoprecipitation on microarray analysis of Smad2/3 binding sites reveals roles of ETS1 and TFAP2A in transforming growth factor β signaling.Mol Cell Biol. 2009; 29: 172-186Crossref PubMed Scopus (159) Google Scholar Anti-Smad4 antibody (R&D Systems) and anti-Smad2/3 antibody (BD Biosciences) were used for immunoprecipitation.23Morikawa M. Koinuma D. Miyazono K. Heldin C.H. Genome-wide mechanisms of Smad binding.Oncogene. 2013; 32: 1609-1615Crossref PubMed Scopus (73) Google Scholar Genomic DNA eluted in 100 μL of Tris-EDTA buffer was used for quantitative real-time RT-PCR. The primers for quantitative real-time RT-PCR in ChIP analysis are listed in Table 1. The upstream region (−25,060 to −27,715 bp) and downstream region (+124,970 to +123,530 bp) of human ALDH1A1 were subcloned into the promoter-reporter construct pGL4.10 luc2 (Promega, Madison, WI). Panc-1 cells were seeded in a 24-well plate (5 × 104 cells per well) and transfected with promoter–reporter constructs using FuGENE 6, following the manufacturer's instructions. Twenty-four hours later, cells were treated with TGF-β3 for an additional 24 hours and then lyzed. Luciferase activity was measured with the Dual Luciferase Reporter Assay System (Promega) using Mithras LB 940 (Berthold Technologies, Oak Ridge, TN). Luciferase activity was normalized to that of pGL4.10-TK-hRluc, which was co-transfected. Aldefluor assay was performed using the ALDEFLUOR Kit (StemCell Technologies, Vancouver, BC, Canada) as described previously.10Katsuno Y. Ehata S. Yashiro M. Yanagihara K. Hirakawa K. Miyazono K. Coordinated expression of REG4 and aldehyde dehydrogenase 1 regulating tumourigenic capacity of diffuse-type gastric carcinoma-initiating cells is inhibited by TGF-β.J Pathol. 2012; 228: 391-404Crossref PubMed Scopus (85) Google Scholar MoFlo Astrios (Beckman Coulter, Pasadena, CA) was used for cell sorting, and an Epics-XL flow cytometer (Beckman Coulter) was used for analysis. Male BALB/c nu/nu mice (4 weeks of age) were purchased from the Oriental Yeast Company (Tokyo, Japan). The same stocks of cells were prepared, a total volume of 100 μL of cells in Matrigel (BD Biosciences) was subcutaneously injected into the left flank of each mouse, and tumors were measured as described previously.24Shirai Y.T. Ehata S. Yashiro M. Yanagihara K. Hirakawa K. Miyazono K. Bone morphogenetic protein-2 and -4 play tumor suppressive roles in human diffuse-type gastric carcinoma.Am J Pathol. 2011; 179: 2920-2930Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar All animal experiments were performed in accordance with the policies of the Animal Ethics Committee at The University of Tokyo. Formalin-fixed, paraffin-embedded pancreatic tissues were obtained from patients with pancreatic adenocarcinoma at The University of Tokyo Hospital. All patients gave their informed consent. Hematoxylin and eosin staining of tissues, antigen retrieval, and immunodetection were performed as described previously.25Kawabata K.C. Ehata S. Komuro A. Takeuchi K. Miyazono K. TGF-β-induced apoptosis of B-cell lymphoma Ramos cells through reduction of MS4A1/CD20.Oncogene. 2013; 32: 2096-2106Crossref PubMed Scopus (21) Google Scholar Anti-Smad4 antibody (Santa Cruz Biotechnology) and anti-ALDH1 antibody (BD Biosciences) were the primary antibodies used for section immunostaining. All studies were conducted using protocols approved by Research Ethics Committee at The University of Tokyo, Graduate School of Medicine. Two-sided Student's t-test was used for examining differences in gene expression levels, tumorigenic potentials, and in vitro proliferative ability in multiple groups. Repeated-measures analysis of variance was used for examining differences in the size of tumors. P < 0.05 was considered significant. SMAD4 is frequently mutated in pancreatic cancer cells. We examined Smad4 expression in three human pancreatic cancer cells: Panc-1, BxPC-3, and SUIT-2. Consistent with a previous report,26Carbone C. Melisi D. NF-κB as a target for pancreatic cancer therapy.Expert Opin Ther Targets. 2012; 16: S1-S10Crossref PubMed Scopus (84) Google Scholar quantitative real-time RT-PCR analysis and immunoblotting revealed that SMAD4 mRNA and Smad4 protein were expressed in Panc-1 and SUIT-2 cells but remained undetected in BxPC-3 cells (Figure 1, A and B). On the basis of these findings, Panc-1 and SUIT-2 cells were used as Smad4-expressing pancreatic cancer cells in this study. To determine the role of Smad4 expression in tumorigenesis of pancreatic cancer cells in vivo, we established Panc-1 cells with knockdown of Smad4 using shRNA. SMAD4 mRNA and its protein were successfully silenced in cells introduced with shRNA targeting SMAD4 (Panc-1-shSMAD4 cells) but not in control cells (Panc-1-shNTC cells) (Figure 1, C and D). These cells were then xenografted into nude mice, and tumorigenic potential was monitored (Figure 1E). Tumors generated by Panc-1-shSMAD4 cells were larger than those by Panc-1-shNTC cells, suggesting that Smad4 plays a tumor suppressive role in the formation of pancreatic cancer in vivo. It is assumed that rapid cell growth is critical for the tumor-forming ability of cancer cells; thus, we assessed the proliferation of these cancer cells in vitro (Figure 1F). Panc-1-shSMAD4 and Panc-1-shNTC cells exhibited similar in vitro proliferation rates. Smad4 knockdown did not alter the expression levels of cyclin-dependent kinase (CDK) regulators (CDKN1A, CDKN1B, CDKN2B, CCNE1, MYC, and CDC25A) or phosphorylation of retinoblastoma proteins, suggesting that Smad4 did not influence cell cycle progression of pancreatic cancer cells (Supplemental Figure S1, A and B). Moreover, poly (ADP-ribose) polymerase cleavage, which is an indicator of apoptotic cell death, was not enhanced in Panc-1-shSMAD4 cells (Supplemental Figure S1C). These results suggested that Panc-1-shSMAD4 cells have high tumorigenic activity despite the fact that proliferative activity does not differ between Panc-1-shSMAD4 cells and Panc-1-shNTC cells. In xenograft models, many factors have been implicated in tumor growth, including anchorage-independent growth, escape from various apoptotic stimuli, and interactions between cancer cells and tumor microenvironment. In addition to these mechanisms, we postulated that heterogeneity of pancreatic cancer cells might be altered by the expression of Smad4. Thus, we examined whether certain population of cancer cells (eg, CICs) were enriched in pancreatic cancer cells on knockdown of SMAD4. It is thought that the specific expressions of transcription factors Nanog and Oct4 are important for the characteristics of pancreatic CICs.27Lu Y. Zhu H. Shan H. Lu J. Chang X. Li X. Fan X. Zhu S. Wang Y. Guo Q. Wang L. Huang Y. Zhu M. Wang Z. Knockdown of Oct4 and Nanog expression inhibits the stemness of pancreatic cancer cells.Cancer Lett. 2013; 340: 113-123Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar Smad4 silencing in Panc-1 cells elevated the expressions of these pluripotent stem cell markers, including NANOG, POUF5F1 (encoding Oct4 protein), and SOX2 (Figure 2A). On the other hand, forced expression of Smad4 in BxPC-3 cells uniformly decreased the expression levels of these markers (Figure 2B). These results support the hypothesis that Smad4 expression may negatively regulate the number of CICs in pancreatic cancer cells. To isolate the CIC population in pancreatic cancer cells, we investigated the expression levels of markers that were highly expressed in pancreatic cancer cells with knockdown of SMAD4. CD133 (alias PROM1), CD44, CD24, epithelial specific antigen (alias epithelial cell adhesion molecule), and CXCR4 are reportedly useful for isolation of pancreatic CICs.28Visvader J.E. Lindeman G.J. Cancer stem cells in solid tumours: accumulating evidence and unresolved questions.Nat Rev Cancer. 2008; 8: 755-768Crossref PubMed Scopus (2808) Google Scholar, 29Li C. Heidt D. Dalerba P. Burant C. Zhang L. Adsay V. Wicha M. Clarke M. Simeone D. Identification of pancreatic cancer stem cells.Cancer Res. 2007; 67: 1030-1037Crossref PubMed Scopus (2752) Google Scholar, 30Hermann P. Huber S. Herrler T. Aicher A. Ellwart J. Guba M. Bruns C. Heeschen C. Distinct populations of cancer stem cells determine tumor growth and metastatic activity in human pancreatic cancer.Cell Stem Cell. 2007; 1: 313-323Abstract Full Text Full Text PDF PubMed Scopus (2274) Google Scholar ALDH1 activity is also reported to be able to identify CICs in pancreatic cancers.31Bardeesy N. Cheng K.H. Berger J.H. Chu G.C. Pahler J. Olson P. Hezel A.F. Horner J. Lauwers G.Y. Hanahan D. DePinho R.A. Smad4 is dispensable for normal pancreas development yet critical in progression and tumor biology of pancreas cancer.Genes Dev. 2006; 20: 3130-3146Crossref PubMed Scopus (511) Google Scholar Quantitative real-time RT-PCR analysis revealed much higher expression of ALDH1A1 mRNA in Panc-1-shSMAD4 cells than in control Panc-1-shNTC cells (Figure 3A). The amounts of ALDH1 protein were also remarkably up-regulated in Panc-1-shSMAD4 cells (Figure 3B). Because other ALDH1 isoforms are reportedly expressed in certain types of CICs,32Marcato P. Dean C.A. Giacomantonio C.A. Lee P.W. Aldehyde dehydrogenase: its role as a cancer stem cell marker comes down to the specific isoform.Cell Cycle. 2011; 10: 1378-1384Crossref PubMed Scopus (381) Google Scholar their expressions in Panc-1 cells were also examined (Figure 3A). ALDH1A2 mRNA was not detected in these cells. Expression of ALDH1A3 mRNA was suppressed in Panc-1-shSMAD4 cells, implying that this gene was not important for pancreatic CIC identification. Together, these findings suggested that ALDH1A1-expressing pancreatic cancer cells were enriched in cancer cells with SMAD4 knockdown. Next, we used Aldefluor assay to detect the cell population that possessed high ALDH1 enzymatic activity among these pancreatic cancer cells (Figure 4A). This assay specifically distinguished cells with high enzymatic activity from those with low activity by using DEAB, whi
Referência(s)