Snail1 induces epithelial-to-mesenchymal transition and tumor initiating stem cell characteristics
© Dang et al; licensee BioMed Central Ltd. 2011
Received: 27 June 2011
Accepted: 19 September 2011
Published: 19 September 2011
Tumor initiating stem-like cells (TISCs) are a subset of neoplastic cells that possess distinct survival mechanisms and self-renewal characteristics crucial for tumor maintenance and propagation. The induction of epithelial-mesenchymal-transition (EMT) by TGFβ has been recently linked to the acquisition of TISC characteristics in breast cancer. In HCC, a TISC and EMT phenotype correlates with a worse prognosis. In this work, our aim is to elucidate the underlying mechanism by which cells acquire tumor initiating characteristics after EMT.
Gene and protein expression assays and Nanog-promoter luciferase reporter were utilized in epithelial and mesenchymal phenotype liver cancer cell lines. EMT was analyzed with migration/invasion assays. TISC characteristics were analyzed with tumor-sphere self-renewal and chemotherapy resistance assays. In vivo tumor assay was performed to investigate the role of Snail1 in tumor initiation.
TGFβ induced EMT in epithelial cells through the up-regulation of Snail1 in Smad-dependent signaling. Mesenchymal liver cancer post-EMT demonstrates TISC characteristics such as tumor-sphere formation but are not resistant to cytotoxic therapy. The inhibition of Snail1 in mesenchymal cells results in decreased Nanog promoter luciferase activity and loss of self-renewal characteristics in vitro. These changes confirm the direct role of Snail1 in some TISC traits. In vivo, the down-regulation of Snail1 reduced tumor growth but was not sufficient to eliminate tumor initiation. In summary, TGFβ induces EMT and TISC characteristics through Snail1 and Nanog up-regulation. In mesenchymal cells post-EMT, Snail1 directly regulates Nanog expression, and loss of Snail1 regulates tumor growth without affecting tumor initiation.
Tumor initiating stem-like cells (TISCs), also defined as cancer stem cells, are a subpopulation of neoplastic cells that possess distinct survival and regeneration mechanisms important for chemotherapy resistance and disease progression [1, 2]. By definition, TISCs possess stem cell features including resistance to apoptosis and self-renewal [3–5]. After their initial discovery and characterization within hematological malignancies [6, 7], TISCs have now been described in many different malignancies including hepatocellular carcinoma (HCC) [8, 9]. Further evidence supports that HCC arises as a direct consequence of dysregulated proliferation of hepatic progenitor cells [10, 11]. Transcriptome analysis of HCC demonstrated that a progenitor-based (TISC-phenotype) expression profile is associated with a poor prognosis compared to differentiated tumors (hepatocyte-phenotype) [12–14].
Resistance to therapy and metastatic disease are two factors that correlate a TISC-phenotype HCC with poor survival. TISCs are hypothesized to be the source of metastatic lesions, as a tumor-initiating cell . Although this hypothesis remains controversial, recent work establishes a connection between epithelial-mesenchymal-transition (EMT) and a TISC-phenotype [16, 17]. EMT is a critical developmental process that plays a central role in the formation and differentiation of multiple tissues and organs. During EMT, epithelial cells lose cell-cell adhesion and apical-polarity, and acquire mesenchymal features, such as motility, invasiveness, and resistance to apoptosis .
One of the key hallmarks of EMT is loss of E-cadherin, a cell-adhesion protein that is regulated by multiple transcription factors including Snail, Slug, and Twist. These transcription factors act as E-box repressors and block E-cadherin transcription . In cancer biology, EMT is one mechanism to explain the invasive and migratory capabilities that epithelial carcinomas acquire during metastasis [19, 20]. In HCC, increased expression of the E-cadherin repressors Twist and Snail correlates with poor clinical outcomes . In breast cancer, EMT is associated with the acquisition of a TISC CD44+/CD24low phenotype [17, 22].
One of the major inducer of EMT is transforming growth factor-β (TGFβ), a multifunctional cytokine that regulates cell proliferation, differentiation and apoptosis . In early stages of carcinogenesis, TGFβ serves as a tumor suppressor by inhibiting cell growth, and in later stages of disease, tumor cells escape this growth inhibition. As late stage cancer tends to be resistant to TGFβ-driven growth arrest signals and as TGFβ is a known inducer of EMT, TGFβ is proposed to be a facilitator of cancer progression during late stage disease [24–26]. TGFβ induces EMT by up-regulating Snail1 via the Smad-dependent pathways . Mishra and colleagues have reviewed the complexity of TGFβ signaling during hepatocarcinogenesis, specifically as related to β2-Spectrin loss and stem cell malignant transformation [15, 28–30].
As additional evidence linking EMT to TISCs, TGFβ regulates Nanog expression, a transcription factor that contributes to self-renewal and cell fate determination in embryonic stem cells [31, 32]. In prostate cancer, increased Nanog expression is implicated in tumor progression, and the co-expression of Nanog and Oct4 promotes tumor-sphere formation [4, 33, 34]. In colon cancer, increased Snail1 expression correlates to increased Nanog expression . In human HCC cell lines, TGFβ regulates CD133 expression, a marker of TISCs, through induction of epigenetic modifications of the CD133 promoter [23, 36].
Thus, several studies have demonstrated that TGFβ drives EMT through Snail1 up-regulation, and other studies have correlated EMT to the acquisition of TISC characteristics. What is lacking is an understanding of the mechanism of how liver cancer cells acquire TISC characteristics through EMT. Our hypothesis is that mesenchymal cells acquire TISC traits after EMT through Snail1-dependent mechanisms. In this report, we demonstrate that mesenchymal liver cancer cells (post-EMT) possess several TISC characteristics compared to epithelial cells. TGFβ induces EMT and TISC characteristics in epithelial cells through Snail1. In mesenchymal cells, knock-down of Snail1 results in loss of Nanog and reduction of TISC traits. In vivo studies demonstrate that Snail1 regulates tumor growth but does not fully control tumor initiation.
Epithelial and mesenchymal murine liver cancer cells were cultured in Dulbecco's modified Eagle's medium (DMEM)/F12 (Sigma) supplemented with 10% fetal bovine serum as described . The human HCC cell line Huh7 was provided by Jianming Huh, Penn State College of Medicine and cultured as described [36, 38]. The human HCC The human HCC cell lines MHCC97-L were provided by Xinwei Wang, National Cancer Institute, under agreement with the Liver Cancer Institute, Zhongshan Hospital, Fudan University, Shanghai, China and cultured as described .
For Snail1 transient knockdown, cells were transfected with 100 pM of Snail1 Stealth siRNA (Invitrogen) using Lipofectamine 2000 (Invitrogen). For Smad signaling inhibition, cells were transfected with 2 ug of DNA using Fugene 6 (Roche). To generate Snail1 knockdown stable transfectants, mesenchymal cells were transfected with Snail1 Mission shRNA lentivirus (Sigma) and selected with 2 ug/ml of puromycin.
pCMV5-Smad7-HA (Plasmid 11733), pRK-Smad3ΔC (Plasmid 12626), and Nanog-Luc (Plasmid 16337) were provided by Addgene. Cells were plated in 12 well plates, incubated overnight, and transfected with the Nanog-Luc plasmid and Renilla for 24 hours (4:1 Nanog-Luc:Renilla ratio). Cells were washed with 1 × PBS, serum free starved for 2 hours, and treated with 5 ng/ml of TGFβ for 24 hours. Following cell lysis, luciferase activity was measured using the Dual Luciferase Assay Kit (Promega) and a Sirius Luminometer V3.1 (Zylux). Luciferase reading light units (RLU) were normalized to Renilla RLU and a fold change was calculated.
Trizol (Invitrogen) was used to isolate total RNA from cells according to manufacturer's protocol. Isolated RNA was quantified using the ND-1000 spectrophotometer (NanoDrop) and complementary single strand DNA was synthesized using the Omniscript RT Kit according to the manufacturers protocol (Qiagen). qPCR was performed using Taqman Gene Expression Assays and ABI-Prism 7700 Thermal Cycler (Applied Biosystems). Normalization was performed using β-actin or Gapdh as an endogenous control and relative gene expression was calculated using the comparative 2(-ΔΔCt) method with SDS 2.2.2 software .
Cell Viability Assays
Cell viability was performed using the XTT (2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide) kit (Trevigen) according to the manufacturer's protocol. 5 × 103 cells were plated in 96-well plates, incubated for 24 hours at 37°C, and treated with specified agents at defined time points.
Western Blot Analysis
Cells were washed twice with ice cold 1XPBS and cell lysates were harvested by the addition of lysis buffer (40 nM Tris [pH 7.4], 150 mM NaCl, 10 mM ethylene diamine tetraccetic acid, 10% glycerol, 1% Triton X-100, 10 mM glycerophosphate, 1 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride) supplemented with protease inhibitor cocktail tablets (Roche). BCA protein assay (Thermo Fisher Scientific) was used to determine protein concentration as described . 30 ug of protein lysates were separated on a NuPAGE 4-12% Bis-Tris Gel (Invitrogen) and the separated proteins were transferred onto a polyvinylidene difluoride membrane (Invitrogen). After blocking for 60 min with 5% non-fat dry milk, membranes were incubated with the primary antibody overnight at 4°C followed by incubation with corresponding secondary antibody for 60 min at room temperature. The membranes were developed using enhance chemiluminescence solutions (Thermo Fisher Scientific) .
Cell Migration Assay
The capability of tumor cell migration was assessed using a wound-healing assay. Confluent cell monolayers were manually wounded by scraping the cells with a 1,000 μL pipette tip down the center of the well. The cell culture medium was replaced and migration was assessed at 24 hours .
Matrigel Invasion Assay
Cell invasion was assessed using 6-well Transwell permeable inserts with 8-μm pores (Corning) . In brief, 1 × 105 cells were cultured in a serum-free DMEM/F12 medium in an insert coated with Matrigel (BD). Below the insert, the chamber of 6-well plates contained DMEM/F12 supplemented with 10% FBS. Cells were incubated in a 37°C incubator for 48 hours and the number of cells that invaded across the membranes and fallen onto the bottom of the plate was counted.
Using the cell lines from the liver specific Pten -/- model described  P2E (epithelial) and P2M (mesenchymal) messenger RNA were analyzed using an Illumina mouse gene chip according to the manufacturer's protocol and as described . Housekeeping genes were used as standards to generate expression levels, and data analysis was conducted using 1.4-fold or greater change in expression with p < 0.05 as significant. The full complement of the expression data is available at http://www.ncbi.nlm.nih.gov/geo (Accession number GSE18255).
Spheroid Formation Assay
The capability of self-renewal was assessed using Corning Ultra-Low Attachment Surface (Corning). 5 × 103 cells were seeded and incubated in a cell culture incubator for 1 week in DMEM/F12 supplemented with 10% FBS or serum free medium and phase-contrast images were obtained.
In vivo tumor growth assay
Cells were counted with trypan blue exclusion and suspended in a 1:3 dilution of Matrigel (Matrigel:DMEM/F12 supplemented with 10% FBS) . 1 × 104 and 1 × 105 cells/50 μL were injected subcutaneously into 10-week-old nude mice. Caliper measurements of tumor volume (length × width × height) were conducted every 2 days. After 3 weeks, mice were sacrificed for tumor analysis. All procedures were in compliance with our institution's guidelines for the use of laboratory animals and approved by the Penn State College of Medicine Institutional Animal Care and Use Committee.
Microarray statistical analysis was performed as describe . Student t test was used comparing two groups. One-way ANOVA was used comparing multiple groups followed by Tukeys post-hoc test. All analysis with a p < 0.05 was considered significant.
Mesenchymal cells acquire TISC characteristics post-EMT
Resistance to chemotherapy is linked to cell proliferation
In addition to resistance to genotoxic agents, we assessed whether the mesenchymal cells are resistant to TRAIL-induced and TGFβ-induced apoptosis. Although there was no significant difference in response to TRAIL stimulation (Figure 3C), the mesenchymal cells demonstrate resistance to TGFβ-induced apoptosis (Figure 3D), a characteristic of TISCs .
TGFβ-induced EMT results in TISC characteristics
Inhibition of Snail1 blocks TISC characteristics
TGFβ regulates Snail and Nanog through Smad signaling
TGFβ regulates Nanog promoter activity through Smad signaling in human embryonic stem cells . To confirm that TGFβ can induce Nanog promoter activity in our model, epithelial cells were co-transfected with Nanog-Luc and Smad7 or ΔSmad3 vectors. Following TGFβ stimulation, Nanog-Luc activity was significantly attenuated by inhibitory Smads (Figure 6C & 6D), indicating that TGFβ stimulates Nanog promoter activity through Smad-dependent signaling.
Snail1 directly regulates Nanog promoter
Inhibition of Snail1 results in decreased tumor growth in vivo
As demonstrated, Snail1 is a key regulator of TISC characteristics in vitro. To investigate the role of Snail1 in tumor initiation, we inoculated 1 × 104 mesenchymal-Snail1-shRNA cells into nude mice. The mesenchymal-Snail1-shRNA cells demonstrate reduced in tumor growth compared to control mesenchymal cells. Analysis of tumors demonstrates that Snail1 expression was down-regulated in 1 × 104 cell initiated tumors from mesenchymal-Snail1-siR cells (Figure 7C). However, tumor initiation was not affected by Snail1 suppression, as evidence by all inoculations forming tumors, even in Snail1 inhibited cells.
Epithelial and mesenchymal differences in human HCC
Although liver transplantation has significantly improved survival in patients with early stage HCC, the prognosis for late stage HCC remains poor . Causes of poor prognosis in late stage disease include invasive/metastatic disease and tumor recurrence after treatment. In breast cancer, EMT has been linked to TISC characteristics and resistant disease. Although this link between EMT and TISCs has been established in other cancers, including breast, prostate, nasopharyngeal, and colon cancer, this relationship has yet to be defined in HCC [17, 22, 46]. One potential link between EMT and TISCs in liver cancer is TGFβ.
TGFβ has a dual role in HCC either as a tumor suppressor in early stages or tumor promoter in later stages [15, 43]. One of the mechanisms of early neoplastic transformation is through the evasion of cytostatic effects of TGFβ . During the late stages of HCC tumorgenesis, TGFβ stimulates cellular invasion through the EMT program .
TGFβ induces EMT through Snail1, which represses E-cadherin by binding to E-box promoter elements [18, 19, 47]. In cancer patients, an EMT-phenotype transcriptome profile, with increased Snail1 expression, correlates with invasive tumors [21, 48, 49]. In this report, TGFβ stimulation of epithelial liver cancer cells results in a mesenchymal phenotype with fibroblastoid appearance, loss of E-cadherin, increased invasion and migration, and an up-regulation of Snail1. In addition, TGFβ treatment induces a TISC phenotype in epithelial cells. Although TGFβ-induced EMT generates TISC characteristics [17, 22], the underlying mechanism has not yet been elucidated. Based on our results, we hypothesize that these TISC characteristics are Snail1 dependent. Inhibition of Snail1 causes the down-regulation of Nanog, Bmi-1 and CD44, loss of a migration and self-renewal as evidenced by decreased tumor-sphere formation.
Another key regulatory signaling pathway known to induce EMT in liver cells is the Hedgehog (Hh) signaling pathway. Hh promotes EMT in response to chronic liver injury . In addition, Hh signaling has been suggested to play an important role in the maintenance of TISCs, and BMI-1, the polycomb group protein, may directly mediate Hh signaling in order to confer a self-renewal capacity in TISCs [10, 46, 51]. However, within our system, we were unable to see significant differences of BMI-1 between epithelial and mesenchymal cells.
TGFβ also directly controls Nanog in human embryonic stem cells . Nanog is a key transcription factor that regulates self-renewal in stem cells [4, 52]. Recent studies demonstrate that Nanog promotes TISC characteristics, and the down regulation of Nanog inhibits sphere formation and tumor development [4, 34, 35, 53]. In this report, Nanog is up-regulated by TGFβ through Smad signaling. In addition, Snail1 directly regulates Nanog promoter activity.
TISCs are proposed to initiate tumors. In our model, liver cancer cells with a mesenchymal phenotype demonstrate TISCs characteristics, including tumor-sphere formation and increased expression of CD44 and Nanog. We further investigated epithelial and mesenchymal phenotypes in human HCC, Huh7 and MHCC97-L cells. Accordingly, Huh7 cells follow an epithelial phenotype whereas MHCC97-L cells are more mesenchymal demonstrating increased Snail1, Zeb1, Zeb2 mRNA expression, decreased E-cadherin expression, increased migration/invasion and increased tumorsphere formation .
In our murine system, Snail1 inhibition resulted in loss of tumor-sphere formation, decreased expression of CD44 and Nanog, and decreased tumor growth. According to our in vitro results, Snail1 clearly regulates TISC characteristics. However, the loss of Snail1 is not sufficient to inhibit tumor initiation, as evidenced by in vivo results. These findings are not un-expected in that the proposed TISC-driven tumor initiation is an early event in tumorigenesis, and cells that acquire TISC characteristics after EMT are a late event in tumor progression. In addition, Snail1 is one of many regulators of EMT, and thus manipulation of multiple factors may be required to fully inhibit tumor initiation.
In summary, we demonstrated that TGFβ induces EMT and TISC characteristics through the up-regulation of Snail1 and Nanog. In addition, Snail1 directly regulates Nanog promoter activity. Notably, expression of both SNAIL1 and NANOG is higher in human mesenchymal cells. Inhibition of Snail1 alone is not sufficient to inhibit tumor initiation, but does result in reduction of tumor growth in vivo.
List of abbreviations
Tumor initiating stem-like cells
transforming growth factor-β
fetal bovine serum.
This publication was made possible by generous support from the National Institute of Health, K08DK080928 and R03DK088013 (CBR); the American Cancer Society, Research Scholar Award, RSG-10-073-01-TBG (CBR); and the Children's Miracle Network (CBR).
- Clarke MF, Dick JE, Dirks PB, Eaves CJ, Jamieson CH, Jones DL, Visvader J, Weissman IL, Wahl GM: Cancer stem cells--perspectives on current status and future directions: AACR Workshop on cancer stem cells. Cancer Res. 2006, 66: 9339-9344. 10.1158/0008-5472.CAN-06-3126.View ArticlePubMedGoogle Scholar
- Reya T, Morrison SJ, Clarke MF, Weissman IL: Stem cells, cancer, and cancer stem cells. Nature. 2001, 414: 105-111. 10.1038/35102167.View ArticlePubMedGoogle Scholar
- Lobo NA, Shimono Y, Qian D, Clarke MF: The biology of cancer stem cells. Annu Rev Cell Dev Biol. 2007, 23: 675-699. 10.1146/annurev.cellbio.22.010305.104154.View ArticlePubMedGoogle Scholar
- Jeter CR, Liu B, Liu X, Chen X, Liu C, Calhoun-Davis T, Repass J, Zaehres H, Shen JJ, Tang DG: NANOG promotes cancer stem cell characteristics and prostate cancer resistance to androgen deprivation. Oncogene. 2011Google Scholar
- Hu T, Liu S, Breiter DR, Wang F, Tang Y, Sun S: Octamer 4 small interfering RNA results in cancer stem cell-like cell apoptosis. Cancer Res. 2008, 68: 6533-6540. 10.1158/0008-5472.CAN-07-6642.View ArticlePubMedGoogle Scholar
- Lapidot T, Sirard C, Vormoor J, Murdoch B, Hoang T, Caceres-Cortes J, Minden M, Paterson B, Caligiuri MA, Dick JE: A cell initiating human acute myeloid leukaemia after transplantation into SCID mice. Nature. 1994, 367: 645-648. 10.1038/367645a0.View ArticlePubMedGoogle Scholar
- Bonnet D, Dick JE: Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell. Nat Med. 1997, 3: 730-737. 10.1038/nm0797-730.View ArticlePubMedGoogle Scholar
- Ma S, Chan KW, Hu L, Lee TK, Wo JY, Ng IO, Zheng BJ, Guan XY: Identification and characterization of tumorigenic liver cancer stem/progenitor cells. Gastroenterology. 2007, 132: 2542-2556. 10.1053/j.gastro.2007.04.025.View ArticlePubMedGoogle Scholar
- Yin S, Li J, Hu C, Chen X, Yao M, Yan M, Jiang G, Ge C, Xie H, Wan D, et al: CD133 positive hepatocellular carcinoma cells possess high capacity for tumorigenicity. Int J Cancer. 2007, 120: 1444-1450. 10.1002/ijc.22476.View ArticlePubMedGoogle Scholar
- Rangwala F, Omenetti A, Diehl AM: Cancer stem cells: repair gone awry?. J Oncol. 2011, 2011: 465343-View ArticlePubMedGoogle Scholar
- Tang Y, Kitisin K, Jogunoori W, Li C, Deng CX, Mueller SC, Ressom HW, Rashid A, He AR, Mendelson JS, et al: Progenitor/stem cells give rise to liver cancer due to aberrant TGF-beta and IL-6 signaling. Proc Natl Acad Sci USA. 2008, 105: 2445-2450. 10.1073/pnas.0705395105.View ArticlePubMedPubMed CentralGoogle Scholar
- Yamashita T, Forgues M, Wang W, Kim JW, Ye Q, Jia H, Budhu A, Zanetti KA, Chen Y, Qin LX, et al: EpCAM and alpha-fetoprotein expression defines novel prognostic subtypes of hepatocellular carcinoma. Cancer Res. 2008, 68: 1451-1461. 10.1158/0008-5472.CAN-07-6013.View ArticlePubMedGoogle Scholar
- Thorgeirsson SS, Grisham JW: Molecular pathogenesis of human hepatocellular carcinoma. Nat Genet. 2002, 31: 339-346. 10.1038/ng0802-339.View ArticlePubMedGoogle Scholar
- Lee JS, Heo J, Libbrecht L, Chu IS, Kaposi-Novak P, Calvisi DF, Mikaelyan A, Roberts LR, Demetris AJ, Sun Z, et al: A novel prognostic subtype of human hepatocellular carcinoma derived from hepatic progenitor cells. Nat Med. 2006, 12: 410-416. 10.1038/nm1377.View ArticlePubMedGoogle Scholar
- Yao Z, Mishra L: Cancer stem cells and hepatocellular carcinoma. Cancer Biol Ther. 2009, 8: 1691-1698. 10.4161/cbt.8.18.9843.View ArticlePubMedPubMed CentralGoogle Scholar
- Yang J, Weinberg RA: Epithelial-mesenchymal transition: at the crossroads of development and tumor metastasis. Dev Cell. 2008, 14: 818-829. 10.1016/j.devcel.2008.05.009.View ArticlePubMedGoogle Scholar
- Mani SA, Guo W, Liao MJ, Eaton EN, Ayyanan A, Zhou AY, Brooks M, Reinhard F, Zhang CC, Shipitsin M, et al: The epithelial-mesenchymal transition generates cells with properties of stem cells. Cell. 2008, 133: 704-715. 10.1016/j.cell.2008.03.027.View ArticlePubMedPubMed CentralGoogle Scholar
- Thiery JP, Acloque H, Huang RY, Nieto MA: Epithelial-mesenchymal transitions in development and disease. Cell. 2009, 139: 871-890. 10.1016/j.cell.2009.11.007.View ArticlePubMedGoogle Scholar
- Thiery JP: Epithelial-mesenchymal transitions in tumour progression. Nat Rev Cancer. 2002, 2: 442-454. 10.1038/nrc822.View ArticlePubMedGoogle Scholar
- Polyak K, Weinberg RA: Transitions between epithelial and mesenchymal states: acquisition of malignant and stem cell traits. Nat Rev Cancer. 2009, 9: 265-273. 10.1038/nrc2620.View ArticlePubMedGoogle Scholar
- Yang MH, Chen CL, Chau GY, Chiou SH, Su CW, Chou TY, Peng WL, Wu JC: Comprehensive analysis of the independent effect of twist and snail in promoting metastasis of hepatocellular carcinoma. Hepatology. 2009, 50: 1464-1474. 10.1002/hep.23221.View ArticlePubMedGoogle Scholar
- Morel AP, Lievre M, Thomas C, Hinkal G, Ansieau S, Puisieux A: Generation of breast cancer stem cells through epithelial-mesenchymal transition. PLoS One. 2008, 3: e2888-10.1371/journal.pone.0002888.View ArticlePubMedPubMed CentralGoogle Scholar
- Amin R, Mishra L: Liver stem cells and tgf-Beta in hepatic carcinogenesis. Gastrointest Cancer Res. 2008, 2: S27-30.PubMedPubMed CentralGoogle Scholar
- Roberts AB, Wakefield LM: The two faces of transforming growth factor beta in carcinogenesis. Proc Natl Acad Sci USA. 2003, 100: 8621-8623. 10.1073/pnas.1633291100.View ArticlePubMedPubMed CentralGoogle Scholar
- Abou-Shady M, Baer HU, Friess H, Berberat P, Zimmermann A, Graber H, Gold LI, Korc M, Buchler MW: Transforming growth factor betas and their signaling receptors in human hepatocellular carcinoma. Am J Surg. 1999, 177: 209-215. 10.1016/S0002-9610(99)00012-4.View ArticlePubMedGoogle Scholar
- Mishra L, Shetty K, Tang Y, Stuart A, Byers SW: The role of TGF-beta and Wnt signaling in gastrointestinal stem cells and cancer. Oncogene. 2005, 24: 5775-5789. 10.1038/sj.onc.1208924.View ArticlePubMedGoogle Scholar
- Peinado H, Olmeda D, Cano A: Snail, Zeb and bHLH factors in tumour progression: an alliance against the epithelial phenotype?. Nat Rev Cancer. 2007, 7: 415-428. 10.1038/nrc2131.View ArticlePubMedGoogle Scholar
- Thenappan A, Li Y, Kitisin K, Rashid A, Shetty K, Johnson L, Mishra L: Role of transforming growth factor beta signaling and expansion of progenitor cells in regenerating liver. Hepatology. 2010, 51: 1373-1382. 10.1002/hep.23449.View ArticlePubMedPubMed CentralGoogle Scholar
- Mishra L, Derynck R, Mishra B: Transforming growth factor-beta signaling in stem cells and cancer. Science. 2005, 310: 68-71. 10.1126/science.1118389.View ArticlePubMedGoogle Scholar
- Tang Y, Katuri V, Dillner A, Mishra B, Deng CX, Mishra L: Disruption of transforming growth factor-beta signaling in ELF beta-spectrin-deficient mice. Science. 2003, 299: 574-577. 10.1126/science.1075994.View ArticlePubMedGoogle Scholar
- Xu RH, Sampsell-Barron TL, Gu F, Root S, Peck RM, Pan G, Yu J, Antosiewicz-Bourget J, Tian S, Stewart R, Thomson JA: NANOG is a direct target of TGFbeta/activin-mediated SMAD signaling in human ESCs. Cell Stem Cell. 2008, 3: 196-206. 10.1016/j.stem.2008.07.001.View ArticlePubMedPubMed CentralGoogle Scholar
- Greber B, Lehrach H, Adjaye J: Control of early fate decisions in human ES cells by distinct states of TGFbeta pathway activity. Stem Cells Dev. 2008, 17: 1065-1077. 10.1089/scd.2008.0035.View ArticlePubMedGoogle Scholar
- Chiou SH, Wang ML, Chou YT, Chen CJ, Hong CF, Hsieh WJ, Chang HT, Chen YS, Lin TW, Hsu HS, Wu CW: Coexpression of Oct4 and Nanog enhances malignancy in lung adenocarcinoma by inducing cancer stem cell-like properties and epithelial-mesenchymal transdifferentiation. Cancer Res. 2010, 70: 10433-10444. 10.1158/0008-5472.CAN-10-2638.View ArticlePubMedGoogle Scholar
- Jeter CR, Badeaux M, Choy G, Chandra D, Patrawala L, Liu C, Calhoun-Davis T, Zaehres H, Daley GQ, Tang DG: Functional evidence that the self-renewal gene NANOG regulates human tumor development. Stem Cells. 2009, 27: 993-1005. 10.1002/stem.29.View ArticlePubMedPubMed CentralGoogle Scholar
- Meng HM, Zheng P, Wang XY, Liu C, Sui HM, Wu SJ, Zhou J, Ding YQ, Li JM: Overexpression of nanog predicts tumor progression and poor prognosis in colorectal cancer. Cancer Biol Ther. 2010, 9:Google Scholar
- You H, Ding W, Rountree CB: Epigenetic regulation of cancer stem cell marker CD133 by transforming growth factor-beta. Hepatology. 2010, 51: 1635-1644. 10.1002/hep.23544.View ArticlePubMedPubMed CentralGoogle Scholar
- Ding W, You H, Dang H, Leblanc F, Galicia V, Lu SC, Stiles B, Rountree CB: Epithelial-to-mesenchymal transition of murine liver tumor cells promotes invasion. Hepatology. 2010, 52: 945-953. 10.1002/hep.23748.View ArticlePubMedPubMed CentralGoogle Scholar
- You H, Ding W, Dang H, Jiang Y, Rountree CB: c-Met represents a potential therapeutic target for personalized treatment in hepatocellular carcinoma. Hepatology. 2011Google Scholar
- Li Y, Tang ZY, Ye SL, Liu YK, Chen J, Xue Q, Gao DM, Bao WH: Establishment of cell clones with different metastatic potential from the metastatic hepatocellular carcinoma cell line MHCC97. World J Gastroenterol. 2001, 7: 630-636.PubMedPubMed CentralGoogle Scholar
- Ding W, Mouzaki M, You H, Laird JC, Mato J, Lu SC, Rountree CB: CD133+ liver cancer stem cells from methionine adenosyl transferase 1A-deficient mice demonstrate resistance to transforming growth factor (TGF)-beta-induced apoptosis. Hepatology. 2009, 49: 1277-1286. 10.1002/hep.22743.View ArticlePubMedPubMed CentralGoogle Scholar
- Rountree CB, Van Kirk CA, You H, Ding W, Dang H, Vanguilder HD, Freeman WM: Clinical application for the preservation of phospho-proteins through in-situ tissue stabilization. Proteome Sci. 2010, 8: 61-10.1186/1477-5956-8-61.View ArticlePubMedPubMed CentralGoogle Scholar
- Zen Y, Fujii T, Yoshikawa S, Takamura H, Tani T, Ohta T, Nakanuma Y: Histological and culture studies with respect to ABCG2 expression support the existence of a cancer cell hierarchy in human hepatocellular carcinoma. Am J Pathol. 2007, 170: 1750-1762. 10.2353/ajpath.2007.060798.View ArticlePubMedPubMed CentralGoogle Scholar
- Akhurst RJ, Derynck R: TGF-beta signaling in cancer--a double-edged sword. Trends Cell Biol. 2001, 11: S44-51.PubMedGoogle Scholar
- Xu J, Lamouille S, Derynck R: TGF-beta-induced epithelial to mesenchymal transition. Cell Res. 2009, 19: 156-172. 10.1038/cr.2009.5.View ArticlePubMedPubMed CentralGoogle Scholar
- El-Serag HB: Hepatocellular carcinoma: an epidemiologic view. J Clin Gastroenterol. 2002, 35: S72-78.View ArticlePubMedGoogle Scholar
- Song LB, Li J, Liao WT, Feng Y, Yu CP, Hu LJ, Kong QL, Xu LH, Zhang X, Liu WL, et al: The polycomb group protein Bmi-1 represses the tumor suppressor PTEN and induces epithelial-mesenchymal transition in human nasopharyngeal epithelial cells. J Clin Invest. 2009, 119: 3626-3636. 10.1172/JCI39374.View ArticlePubMedPubMed CentralGoogle Scholar
- Cano A, Perez-Moreno MA, Rodrigo I, Locascio A, Blanco MJ, del Barrio MG, Portillo F, Nieto MA: The transcription factor snail controls epithelial-mesenchymal transitions by repressing E-cadherin expression. Nat Cell Biol. 2000, 2: 76-83. 10.1038/35000025.View ArticlePubMedGoogle Scholar
- Dooley S, Hamzavi J, Ciuclan L, Godoy P, Ilkavets I, Ehnert S, Ueberham E, Gebhardt R, Kanzler S, Geier A, et al: Hepatocyte-specific Smad7 expression attenuates TGF-beta-mediated fibrogenesis and protects against liver damage. Gastroenterology. 2008, 135: 642-659. 10.1053/j.gastro.2008.04.038.View ArticlePubMedGoogle Scholar
- Coulouarn C, Factor VM, Thorgeirsson SS: Transforming growth factor-beta gene expression signature in mouse hepatocytes predicts clinical outcome in human cancer. Hepatology. 2008, 47: 2059-2067. 10.1002/hep.22283.View ArticlePubMedPubMed CentralGoogle Scholar
- Omenetti A, Porrello A, Jung Y, Yang L, Popov Y, Choi SS, Witek RP, Alpini G, Venter J, Vandongen HM, et al: Hedgehog signaling regulates epithelial-mesenchymal transition during biliary fibrosis in rodents and humans. J Clin Invest. 2008, 118: 3331-3342.PubMedPubMed CentralGoogle Scholar
- Liu S, Dontu G, Mantle ID, Patel S, Ahn NS, Jackson KW, Suri P, Wicha MS: Hedgehog signaling and Bmi-1 regulate self-renewal of normal and malignant human mammary stem cells. Cancer Res. 2006, 66: 6063-6071. 10.1158/0008-5472.CAN-06-0054.View ArticlePubMedPubMed CentralGoogle Scholar
- Silva J, Nichols J, Theunissen TW, Guo G, van Oosten AL, Barrandon O, Wray J, Yamanaka S, Chambers I, Smith A: Nanog is the gateway to the pluripotent ground state. Cell. 2009, 138: 722-737. 10.1016/j.cell.2009.07.039.View ArticlePubMedPubMed CentralGoogle Scholar
- Chen C, Wei Y, Hummel M, Hoffmann TK, Gross M, Kaufmann AM, Albers AE: Evidence for epithelial-mesenchymal transition in cancer stem cells of head and neck squamous cell carcinoma. PLoS One. 2011, 6: e16466-10.1371/journal.pone.0016466.View ArticlePubMedPubMed CentralGoogle Scholar
- The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/11/396/prepub
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