- Open Access
Hedgehog signaling regulates hypoxia induced epithelial to mesenchymal transition and invasion in pancreatic cancer cells via a ligand-independent manner
© Lei et al.; licensee BioMed Central Ltd. 2013
- Received: 28 March 2013
- Accepted: 18 June 2013
- Published: 20 June 2013
Hypoxia plays a vital role in cancer epithelial to mesenchymal transition (EMT) and invasion. However, it is not quite clear how hypoxia may contribute to these events. Here we investigate the role of Hedgehog (Hh) signaling in hypoxia induced pancreatic cancer EMT and invasion.
Pancreatic cancer cells were cultured under controlled hypoxia conditions (3% O2) or normoxic conditions. HIF-1α siRNA, cyclopamine (a SMO antagonist) and GLI1 siRNA were used to inhibit HIF-1α transcription or Hh signaling activation. The effect of hypoxia and Hh signaling on cancer cell EMT and invasion were evaluated by Quantitative real-time PCR analysis, Western blot analysis and invasion assay.
Here, we show that non-canonical Hh signaling is required as an important role to switch on hypoxia-induced EMT and invasion in pancreatic cancer cells. Moreover, our data demonstrate hypoxia induces EMT process as well as invasion, and activates the non-canonical Hh pathway without affecting sonic hedgehog homolog (SHH) expression. Moreover, these effects are reversible upon HIF-1α siRNA interference with unchanged SHH and patched1 (PTCH1) level. Furthermore, our data demonstrate that hypoxia induced invasion and EMT process are effectively inhibited by Smoothened (SMO) antagonist cyclopamine and GLI1 siRNA. In addition, GLI1 interference inhibited EMT progress with significantly suppressed vimentin expression, whereas inhibition of SMO through cyclopamine could not reduce vimentin level. This data indicate that hypoxia could trigger other factors (such as TGF-β, KRAS or RTK) bypassing SMO to activate GLI1 directly.
Our findings suggest that Hh signaling modulates hypoxia induced pancreatic cancer EMT and invasion in a ligand-independent manner. Thus, Hh signaling may represent a promising therapeutic target for preventing pancreatic cancer progression.
Accompanying with a 5-year survival rate less than 5% and more than 37, 000 deaths per year, pancreatic ductal adenocarcinoma represents one of the most lethal human cancers and is the fourth leading cause of cancer-related deaths in the United States [1, 2]. Its high tendency to metastasize is considered to partially account for the extremely poor clinical prognosis of pancreatic cancer . However, the underlying molecular mechanisms of the invasion and metastasis of pancreatic cancer remain poorly understood.
Epithelial to mesenchymal transition (EMT) is a process defining the progression that cells lose their polarized epithelial character and acquire a migratory mesenchymal phenotype . EMT plays a pivotal role in normal physiological development and enables the cancer cells to gain migratory and invasive properties consequently lead to tumor metastasis . An important hallmark of EMT is the loss of the homophilic cell adhesion molecule E-cadherin, which is considered as a main determinant of epithelial cell-cell adhesion and cell polarity . This crucial event has found to be resulted from transcriptional repression of E-cadherin through overexpression of several different EMT-inducing factors, such as Snail, a zinc-finger transcription repressor .
Solid tumors often experience low oxygen tension environments, which is predominantly caused by abnormal vasculature formation of the rapidly growing tumor mass. Tumor hypoxia is associated with enhanced tumor invasiveness, angiogenesis, and distant metastasis [8–10]. The adaptation of tumor cells to hypoxia leads to tumor heterogeneity and the selection of resistant clones, consequently evolving into a more malignant phenotype . A transcription factor hypoxia inducible factor-1α (HIF-1α), which mediates hypoxia responses, is overexpressed in many solid tumors, including pancreatic cancer . Stabilization and activation of HIF-1α/HIF-1β transcription complex trigger its target genes related to cell proliferation and metastasis, which correlates with many different cellular processes, such as proliferation, angiogenesis, and EMT [12–15], and poor prognosis and tumor metastasis in cancer patients [13, 16, 17]. HIF-1α consists of a bHLH domain close to the amino (N) terminal, which is required for DNA binding to hypoxia-response elements to activate the HIF target genes such as endothelin-1, vascular endothelial growth factor (VEGF), and erythropoietin .
The Hedgehog (Hh) signaling pathway, which is normally quiescent in adult pancreas, has been shown to be very active in pancreatic cancer where it promotes stromal hyperplasia, myofibroblast differentiation, and production of extracellular matrix (ECM) [19, 20], which may promote cancer cells to undergo EMT process to further facilitate the strong propensity of pancreatic cancer for invasion and metastasis. Without binding to Hh ligands, patched1 (PTCH1) holds Smoothened (SMO), a seven transmembrane spanning protein, in an inactive state and thus prohibits signaling to downstream genes. Upon binding to Hh ligands, SMO dissociates from PTCH1 and the signaling is transduced, leading to the activation of target genes, including PTCH1, by transcription factor GLI1 [21–24]. Therefore, expression of SMO and GLI1 is presumed to be the markers of the Hh pathway activation. Another study demonstrates that Hh signaling activation is a very common event in pancreatic cancer, evidenced by the expression of PTCH1 and GLI1 in seven available pancreatic cancer cell lines and 54 pancreatic cancer surgical specimens . In pancreatic cancer, the activation of the Hh pathway could induce an EMT, which leads to invasion and metastasis through down-regulating E-cadherin expression and up-regulating vimentin expression [26, 27]. Moreover, a number of signal transduction pathways, including Hh signaling, could be activated in human pulmonary arterial smooth muscle cells under hypoxia conditions  or in ischemia tissues .
In this study, we focused on elucidating the regulation of EMT and invasion processes in hypoxia condition via Hh signaling, in a panel of pancreatic cancer cell lines. We found that non-canonical Hh signaling in pancreatic cancer cells is a critical mechanism for hypoxia in regulating the process of EMT and invasion.
GLI1 and HIF-1α are expressed in pancreatic cancer cell lines
Hypoxia accumulates HIF-1α and potentiates Hh signaling in PANC-1 and BxPC-3 cells
Hypoxia induces an EMT phenotype and promotes invasiveness in pancreatic cancer cells
Silencing of HIF-1α reverses the effects of hypoxia on Hh signaling, EMT process and invasion in pancreatic cancer cells
We further delineated the link between hypoxia induced HIF-1α expression and EMT progress. Silencing of HIF-1α resulted in marked decrease in the expression of N-cadherin, vimentin and Snail, but a significant increase in the expression of E-cadherin (Figure 4E), consistent with the reversion to an epithelial phenotype.
To determine the role of HIF-1α in the enhanced invasive capacity of pancreatic cancer cells as a result of exposure to hypoxia, cells were treated with HIF-1α siRNA for 48 h in hypoxia condition prior to the test for invasion. A significantly decreased invasion was observed from HIF-1α silenced hypoxic cells, compared to control cells (Figure 4F). These results demonstrate that the increased invasive ability of cancer cell lines observed in hypoxia was dependent of HIF-1α.
Hypoxia mediates pancreatic cancer EMT progress and invasion through increasing the expression of SMO
Since hypoxia simultaneously induces tumor cell EMT, invasion and Hh signaling activation without affecting SHH expression, we hypothesized that hypoxia contributes to increased pancreatic cancer cell EMT and invasion through a SMO-dependent manner Hh signaling. To test our hypothesis, pancreatic cancer cells incubated in hypoxia condition were treated with or without either cyclopamine (a SMO antagonist) or GLI1 siRNA to inhibit Hh signaling, and then compared the resulting phenotype with control-treated cells.
Epithelial to mesenchymal transition is described as a dynamic and reversible biological process. In recent years, it has become increasingly clear that EMT plays important roles in the progression of cancer . Several factors, including hypoxia could induce this phenomenon via mediating snail transcription [14, 33]. A hypoxic microenvironment is commonly found in the central region of solid tumors, including pancreatic cancer. The correlation between hypoxia and EMT has been previously reported, and HIF-1a has been found to mediate this phenomenon. However, the molecular mechanisms of how HIF-1a mediates EMT process have been largely undefined, although evidence in support of the ability of HIF-1a to activate Nuclear Factor-kB and Notch signaling to induce EMT process has been recently described in several human epithelial cancer cells [12, 34].
Previous study showed that hypoxia could activate canonical Hh signaling through accumulation of HIF-1α in vitro and in vivo[28, 29]. Here, we show that accumulated HIF-1α could also trigger non-canonical Hh signaling to facilitate hypoxia induced EMT and invasion processes. A recent report showed that high expression of VEGF, a HIF-1α target gene, facilitates EMT through promoting Snail nuclear localization in prostate cancer . In this study, our data also show that mRNA level of VEGF was significantly up-regulated by hypoxia in pancreatic cancer cells. Furthermore, we demonstrate that the EMT program attributable to hypoxia is largely driven by activation of the Hh signaling pathway. This EMT program is characterized by vimentin and Snail expression and E-cadherin suppression, a highly invasive and mesenchymal phenotype. A previous study showed that knockdown of GLI1 abrogates characteristics of epithelial differentiation, enhances cell motility, and synergizes with TGF-β to induce EMT progress . Intriguingly, EMT conversion of pancreatic cancer cells occurred without up-regulation of Snail or Slug, two canonical inducers of EMT in many other settings, and GLI1 directly regulates E-cadherin transcription, a vital determinant of epithelial tissue feature . In this study, we show that RNAi-mediated GLI1 interference inhibits the hypoxia-induced EMT and decreases cell invasion. Moreover, Snail expression is dramatically reduced, whereas both E-cadherin mRNA and protein levels are notably increased. This difference might be resulted from the distinct culture conditions used: it is possible that pancreatic cancer cells under hypoxia exposure produce enough cofactors interacting with Hh signaling to mediate the EMT progress and invasion.
The Hh signaling is affiliated with EMT, invasion and metastasis in both non-neoplastic and cancer cells [36–39], probably via directly participating in cell migration and angiogenesis . Recently, it is reported that Hh paracrine signaling is required for epithelial tumor cells conducting signals to the stroma in pancreatic cancer [27, 40]. However, under conditions of ligand blocking, how Hh signaling is activated in pancreatic cancer cells themselves is undefined, even though paracrine Hh signaling plays a vital role in triggering tumor-associated stroma relying on a ligand-dependent manner in pancreatic cancer. The results here provide noteworthy evidences that the Hh signaling is potentiated through a ligand-independent manner leading to cancer cell EMT and invasion.
Although our data support the hypothesis that Hh signaling pathway is critical for hypoxia-induced EMT and invasion of pancreatic cancer cells, we cannot rule out the possibility that other factors are also involved in hypoxia-induced EMT and invasion. This is because inhibition of SMO by cyclopamine could not reduce vimentin levels, Thus, we speculate that hypoxia enhances EMT and invasion of pancreatic cancer cells through activating a multifaceted factors in which the Hh signaling pathway is a part of an essential network.
EMT is a key driving force for tumor growth and recurrence. And hypoxia is often experienced by solid tumors, and has been closely linked to EMT and invasion of cancers. Using two pancreatic cancer cell lines, we have demonstrated that non-canonical Hh signaling is required as an important role to switch on hypoxia-induced EMT and invasion in pancreatic cancer cells. Thus, hypoxia mediated-Hh signaling may play an important role in the initiation of EMT and represent a promising therapeutic target for preventing pancreatic cancer progression. Especially, the development of HIF-1α, SMO or GLI1 inhibitor may provide a new class of potent and selectively anticancer agents.
Cell culture and reagents
Pancreatic cancer cell lines were purchased from ATCC (Manassas, VA, USA) and were cultured at 37°C, 5% CO2 and 95% air in Dulbecco’s modified Eagle’s medium (DMEM) (high Glucose) (HyClone, Logan, USA) containing 10% heat-inactivated fetal bovine serum (FBS) plus 100 μg/ml ampicillin and 100 μg/ml streptomycin. In experiments designed to assess the role of hypoxia, cells were first cultured in normoxic conditions to obtain the desired subconfluence level (65–70%) and then were incubated in strictly controlled hypoxic conditions (3% O2), as previously detailed elsewhere [30, 31] for up to 48 h. Cyclopamine, an antagonist of SMO, was obtained from Selleck Chemicals (Houston, USA). Pancreatic cancer cells at exponential phase were cultured in six orifice plates in DMEM supplemented with 1% FBS for 24 h. The drugs (or solvent only) at given concentrations were then added in medium containing 1% FBS, and cells were incubated for another 48 h before a matrigel invasion assay. Antibodies were obtained from the following resources: anti-HIF-1α antibody (Bioworld, Atlanta, GA, USA), anti-SMO antibody (Bioworld), anti-GLI1 antibody (Santa Cruz Biotechnology, Santa Cruz, USA), anti-E-cadherin antibody (Santa Cruz Biotechnology), anti-vimentin antibody (Bioworld), anti-Snail antibody (Santa Cruz Biotechnology), anti-N-cadherin antibody (Santa Cruz Biotechnology), and anti-β-actin antibody (Santa Cruz Biotechnology).
Cell invasion assay
A chamber based invasion assay (Millipore co., Billerica, USA) was performed to evaluate pancreatic cancer cell invasion. Briefly, the upper surface of the membrane was coated with matrigel (BD Biosciences, Franklin Lakes, USA). Pancreatic cancer cells (1 × 105) were resuspended in upper chamber in serum-free media and allowed to migrate towards a serum gradient (10%) in the lower chamber. The media was aspirated from the inside of the insert and the non-invasive cells on the upper side were removed by scraping with a cotton swab. The membrane was fixed with 4% paraformaldehyde and stained with crystal violet. The number of migrating cells was counted in 10 random fields on each membrane and photographed at ×100 magnification. Values reported here are the averages of triplicate experiments.
Western blot analysis
Pancreatic cancer cells were washed with ice-cold PBS and were lysed in situ with a buffer containing Tris (40 mM, pH 7.4), 10% glycerol, b-glycerophosphate (50 mM), ethylenediaminetetraacetic (5 mM), ethylenediaminetetraa- cetic acid (2 mM), vanadate (0.35 mM), NaF (10 mM), 0.3% Triton X-100, and protease inhibitors (Roche, Penzberg, Germany). After incubation on ice for 30 min, with vortexing every 10 min, cell lysates were centrifuged at 12 000 r.p.m. for 15 min at 4°C. 100 μg of cellular proteins were separated on a 10% SDS-PAGE gel, and the proteins were transferred to the PVDF membranes (Roche). Membranes were blocked with 5% non-fat dry milk in TBST (10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.05% Tween 20) and were then incubated with primary antibodies overnight at 4°C. After washing five times for 10 min each in TBST, membranes were incubated with HRP-conjugated secondary antibodies for 2 h, washed again and the peroxidase reaction was performed by an enhanced chemiluminescence detection system to visualize the immunoreactive bands.
Quantitative real-time PCR assay (qRT-PCR)
Total RNAs were extracted from pancreatic cancer cells using TRIzol reagent (Invitrogen, CA, USA), and the reverse transcription was developed using a PrimeScript RT reagent Kit (TaKaRa, Dalian, China) according to the manufacturer's instruction. The real-time experiments were carried out using the iQ5 Multicolor Real-Time PCR Detection System (Bio-Rad, Hercules, CA) and a SYBR Green PCR Kit (TaKaRa). Following program was used: denaturation at 95°C for 30 sec and 40 cycles consisting of denaturation at 95°C for 5 sec, annealing at 60°C for 30 sec, and extension at 72°C for 30 sec. A melting curve analysis was applied to assess the specificity of the amplified PCR products. The PCR primer sequences for HIF-1α, SHH, PTCH1, SMO, GLI1, E-cadherin, vimentin, Snail, VEGF and GAPDH are shown in Additional file 1: Table S1. The amount of each target gene was quantitated by the comparative C (T) method using GAPDH as the normalization control .
siRNA for HIF-1α (HIF-1α-Homo-2258: 5′-CCACCACUGAUGAAUUAAATT-3′, 5′-UUUAAUUCAUCAGUGGUGGTT-3′), siRNA for GLI1 (GLI1-Homo-2758: 5′-GGCUCAGCUUGUGUGUAAUTT-3′, 5′-AUUACACACAAGCUGAGCCTT-3′) and a negative control siRNA (NC: 5′-UUCUCCGAACGUGUCACGUTT-3′, 5′-ACGUGACACGUUCGGAGAATT-3′) were purchased from GenePharm (Shanghai, China). Cells (2 × 105 per well) seeded in six-well plates were transfected with 100 nM siRNA using Lipofectamine RNAi MAX Reagent (Invitrogen, CA, USA) according to the manufacturer’s instructions. The cells were used for further experiments at 48 h after transfection.
After designated treatment, pancreatic cancer cells were fixed with 4% paraformaldehyde for 10 min at room temperature, permeabilized in 0.5% Triton X-100 for 10 min, and blocked in 1% BSA for 1 h. Fixed cells were then incubated with Rabbit anti-human-GLI1 antibodies at 1:100 dilution at 4°C overnight. Cells were washed and incubated with Goat anti-rabbit FITC (green) IgG antibody (ZSGB-BIO Inc., Beijing, China) at 1:100 dilution for 60 min. Nuclei were stained with DAPI for 5 min. The cells were visualized by a fluorescent microscope (Nikon, Japan) using appropriate excitation and emission spectra at ×400 magnification.
Data are presented as the mean ± standard error. Differences were evaluated using one-way ANOVA with the LSD post hoc test for multiple comparisons with SPSS (version 13.0; SPSS, Chicago, IL, USA). P-values below 0.05 were considered statistically significant. In all figures, (*) denotes P < 0.05. All experiments were repeated independently at least three times.
This work was supported by grants from the National Natural Science Foundation of China (NSFC) (No. 81172360, 81201824), the Fundamental Research Funds for the Central Universities in Xi’an Jiaotong University, and Pilot Project Grants of the Program Project grants from the National Center for Research Resources (NCRR; P20 RR020151) and the National Institute of General Medical Sciences (NIGMS; P20 GM103505 and P30GM103332-01) from the National Institutes of Health (NIH). The contents of this report are solely the responsibility of the authors and do not necessarily reflect the official views of the NSFC, NIH, NCRR, or NIGMS.
- Hidalgo M: Pancreatic cancer. N Engl J Med. 2010, 362: 1605-1617. 10.1056/NEJMra0901557View ArticlePubMedGoogle Scholar
- Jimeno A, Rubio-Viqueira B, Rajeshkumar NV, Chan A, Solomon A, Hidalgo M: A fine-needle aspirate-based vulnerability assay identifies polo-like kinase 1 as a mediator of gemcitabine resistance in pancreatic cancer. Mol Cancer Ther. 2010, 9: 311-318.View ArticlePubMedGoogle Scholar
- Hezel AF, Kimmelman AC, Stanger BZ, Bardeesy N, Depinho RA: Genetics and biology of pancreatic ductal adenocarcinoma. Genes Dev. 2006, 20: 1218-1249. 10.1101/gad.1415606View ArticlePubMedGoogle 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.2009.11.007View ArticlePubMedGoogle Scholar
- Yilmaz M, Christofori G: EMT, the cytoskeleton, and cancer cell invasion. Cancer Metastasis Rev. 2009, 28: 15-33. 10.1007/s10555-008-9169-0View ArticlePubMedGoogle 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.2008.05.009View ArticlePubMedGoogle Scholar
- Barrallo-Gimeno A, Nieto MA: The Snail genes as inducers of cell movement and survival: implications in development and cancer. Development. 2005, 132: 3151-3161. 10.1242/dev.01907View ArticlePubMedGoogle Scholar
- Michieli P: Hypoxia, angiogenesis and cancer therapy: to breathe or not to breathe?. Cell Cycle. 2009, 8: 3291-3296. 10.4161/cc.8.20.9741View ArticlePubMedGoogle Scholar
- Pennacchietti S, Michieli P, Galluzzo M, Mazzone M, Giordano S, Comoglio PM: Hypoxia promotes invasive growth by transcriptional activation of the met protooncogene. Cancer Cell. 2003, 3: 347-361. 10.1016/S1535-6108(03)00085-0View ArticlePubMedGoogle Scholar
- Esteban MA, Tran MGB, Harten SK, Hill P, Castellanos MC, Chandra A, Raval R, O'Brien TS, Maxwell PH: Regulation of E-cadherin expression by VHL and hypoxia-inducible factor. Cancer Research. 2006, 66: 3567-3575. 10.1158/0008-5472.CAN-05-2670View ArticlePubMedGoogle Scholar
- Semenza GL: Targeting HIF-1 for cancer therapy. Nat Rev Cancer. 2003, 3: 721-732.View ArticlePubMedGoogle Scholar
- Cheng ZX, Sun B, Wang SJ, Gao Y, Zhang YM, Zhou HX, Jia G, Wang YW, Kong R, Pan SH: Nuclear factor-kappaB-dependent epithelial to mesenchymal transition induced by HIF-1alpha activation in pancreatic cancer cells under hypoxic conditions. PLoS One. 2011, 6: e23752- 10.1371/journal.pone.0023752PubMed CentralView ArticlePubMedGoogle Scholar
- Semenza GL: HIF-1 and tumor progression: pathophysiology and therapeutics. Trends Mol Med. 2002, 8: S62-S67. 10.1016/S1471-4914(02)02317-1View ArticlePubMedGoogle Scholar
- Imai T, Horiuchi A, Wang C, Oka K, Ohira S, Nikaido T, Konishi I: Hypoxia attenuates the expression of E-cadherin via up-regulation of SNAIL in ovarian carcinoma cells. Am J Pathol. 2003, 163: 1437-1447. 10.1016/S0002-9440(10)63501-8PubMed CentralView ArticlePubMedGoogle Scholar
- Yang MH, Wu KJ: TWIST activation by hypoxia inducible factor-1 (HIF-1): implications in metastasis and development. Cell Cycle. 2008, 7: 2090-2096. 10.4161/cc.7.14.6324View ArticlePubMedGoogle Scholar
- Harris AL: Hypoxia–a key regulatory factor in tumour growth. Nat Rev Cancer. 2002, 2: 38-47. 10.1038/nrc704View ArticlePubMedGoogle Scholar
- Gupta GP, Massague J: Cancer metastasis: building a framework. Cell. 2006, 127: 679-695. 10.1016/j.cell.2006.11.001View ArticlePubMedGoogle Scholar
- Sharp FR, Bernaudin M: HIF1 and oxygen sensing in the brain. Nat Rev Neurosci. 2004, 5: 437-448. 10.1038/nrn1408View ArticlePubMedGoogle Scholar
- Olive KP, Jacobetz MA, Davidson CJ, Gopinathan A, McIntyre D, Honess D, Madhu B, Goldgraben MA, Caldwell ME, Allard D: Inhibition of Hedgehog signaling enhances delivery of chemotherapy in a mouse model of pancreatic cancer. Science. 2009, 324: 1457-1461. 10.1126/science.1171362PubMed CentralView ArticlePubMedGoogle Scholar
- Bailey JM, Swanson BJ, Hamada T, Eggers JP, Singh PK, Caffery T, Ouellette MM, Hollingsworth MA: Sonic hedgehog promotes desmoplasia in pancreatic cancer. Clin Cancer Res. 2008, 14: 5995-6004. 10.1158/1078-0432.CCR-08-0291PubMed CentralView ArticlePubMedGoogle Scholar
- Porter JA, Young KE, Beachy PA: Cholesterol modification of hedgehog signaling proteins in animal development. Science. 1996, 274: 255-259. 10.1126/science.274.5285.255View ArticlePubMedGoogle Scholar
- Chen Y, Struhl G: Dual roles for patched in sequestering and transducing Hedgehog. Cell. 1996, 87: 553-563. 10.1016/S0092-8674(00)81374-4View ArticlePubMedGoogle Scholar
- Stone DM, Hynes M, Armanini M, Swanson TA, Gu Q, Johnson RL, Scott MP, Pennica D, Goddard A, Phillips H: The tumour-suppressor gene patched encodes a candidate receptor for Sonic hedgehog. Nature. 1996, 384: 129-134. 10.1038/384129a0View ArticlePubMedGoogle Scholar
- Chuang PT, McMahon AP: Vertebrate Hedgehog signalling modulated by induction of a Hedgehog-binding protein. Nature. 1999, 397: 617-621. 10.1038/17611View ArticlePubMedGoogle Scholar
- Villavicencio EH, Walterhouse DO, Iannaccone PM: The sonic hedgehog-patched-gli pathway in human development and disease. Am J Hum Genet. 2000, 67: 1047-1054.PubMed CentralView ArticlePubMedGoogle Scholar
- Inaguma S, Kasai K, Ikeda H: GLI1 facilitates the migration and invasion of pancreatic cancer cells through MUC5AC-mediated attenuation of E-cadherin. Oncogene. 2011, 30: 714-723. 10.1038/onc.2010.459View ArticlePubMedGoogle Scholar
- Bailey JM, Mohr AM, Hollingsworth MA: Sonic hedgehog paracrine signaling regulates metastasis and lymphangiogenesis in pancreatic cancer. Oncogene. 2009, 28: 3513-3525. 10.1038/onc.2009.220PubMed CentralView ArticlePubMedGoogle Scholar
- Wang G, Zhang Z, Xu Z, Yin H, Bai L, Ma Z, Decoster MA, Qian G, Wu G: Activation of the sonic hedgehog signaling controls human pulmonary arterial smooth muscle cell proliferation in response to hypoxia. Biochim Biophys Acta. 1803, 2010: 1359-1367.Google Scholar
- Bijlsma MF, Groot AP, Oduro JP, Franken RJ, Schoenmakers SH, Peppelenbosch MP, Spek CA: Hypoxia induces a hedgehog response mediated by HIF-1alpha. J Cell Mol Med. 2009, 13: 2053-2060. 10.1111/j.1582-4934.2008.00491.xView ArticlePubMedGoogle Scholar
- Aleffi S, Petrai I, Bertolani C, Parola M, Colombatto S, Novo E, Vizzutti F, Anania FA, Milani S, Rombouts K: Upregulation of proinflammatory and proangiogenic cytokines by leptin in human hepatic stellate cells. Hepatology. 2005, 42: 1339-1348. 10.1002/hep.20965View ArticlePubMedGoogle Scholar
- Novo E, Cannito S, Zamara E, Valfre di Bonzo L, Caligiuri A, Cravanzola C, Compagnone A, Colombatto S, Marra F, Pinzani M, Parola M: Proangiogenic cytokines as hypoxia-dependent factors stimulating migration of human hepatic stellate cells. Am J Pathol. 2007, 170: 1942-1953. 10.2353/ajpath.2007.060887PubMed CentralView ArticlePubMedGoogle Scholar
- Huber MA, Kraut N, Beug H: Molecular requirements for epithelial-mesenchymal transition during tumor progression. Curr Opin Cell Biol. 2005, 17: 548-558. 10.1016/j.ceb.2005.08.001View 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/nrc2620View ArticlePubMedGoogle Scholar
- Chen J, Imanaka N, Griffin JD: Hypoxia potentiates Notch signaling in breast cancer leading to decreased E-cadherin expression and increased cell migration and invasion. Br J Cancer. 2010, 102: 351-360. 10.1038/sj.bjc.6605486PubMed CentralView ArticlePubMedGoogle Scholar
- Mak P, Leav I, Pursell B, Bae D, Yang X, Taglienti CA, Gouvin LM, Sharma VM, Mercurio AM: ERbeta impedes prostate cancer EMT by destabilizing HIF-1alpha and inhibiting VEGF-mediated snail nuclear localization: implications for Gleason grading. Cancer Cell. 2010, 17: 319-332. 10.1016/j.ccr.2010.02.030PubMed CentralView ArticlePubMedGoogle Scholar
- Joost S, Almada LL, Rohnalter V, Holz PS, Vrabel AM, Fernandez-Barrena MG, McWilliams RR, Krause M, Fernandez-Zapico ME, Lauth M: GLI1 inhibition promotes epithelial-to-mesenchy -mal transition in pancreatic cancer cells. Cancer Res. 2012, 72: 88-99. 10.1158/0008-5472.CAN-10-4621PubMed CentralView ArticlePubMedGoogle Scholar
- Yang L, Xie G, Fan Q, Xie J: Activation of the hedgehog-signaling pathway in human cancer and the clinical implications. Oncogene. 2010, 29: 469-481. 10.1038/onc.2009.392View ArticlePubMedGoogle Scholar
- Syn WK, Jung Y, Omenetti A, Abdelmalek M, Guy CD, Yang L, Wang J, Witek RP, Fearing CM, Pereira TA: Hedgehog-mediated epithelial-to-mesenchymal transition and fibrogenic repair in nonalcoholic fatty liver disease. Gastroenterology. 2009, 137: 478-1488. e1478View ArticleGoogle Scholar
- Li X, Ma Q, Xu Q, Liu H, Lei J, Duan W, Bhat K, Wang F, Wu E, Wang Z:SDF-1/CXCR4 signaling induces pancreatic cancer cell invasion and epithelial-mesenchymal transition in vitro through non-canonical activation of Hedgehog pathway. Cancer Letters. 2012, 322: 169-176. 10.1016/j.canlet.2012.02.035PubMed CentralView ArticlePubMedGoogle Scholar
- Tian H, Callahan CA, DuPree KJ, Darbonne WC, Ahn CP, Scales SJ, de Sauvage FJ:Hedgehog signaling is restricted to the stromal compartment during pancreatic carcinogenesis. Proc Natl Acad Sci U S A. 2009, 106: 4254-4259. 10.1073/pnas.0813203106PubMed CentralView ArticlePubMedGoogle Scholar
- Onishi H, Kai M, Odate S, Iwasaki H, Morifuji Y, Ogino T, Morisaki T, Nakashima Y, Katano M:Hypoxia activates the hedgehog signaling pathway in a ligand-independent manner by upregulation of Smo transcription in pancreatic cancer. Cancer Sci. 2011, 102: 1144-1150. 10.1111/j.1349-7006.2011.01912.xView ArticlePubMedGoogle Scholar
- Nolan-Stevaux O, Lau J, Truitt ML, Chu GC, Hebrok M, Fernandez-Zapico ME, Hanahan D:GLI1 is regulated through Smoothened-independent mechanisms in neoplastic pancreatic ducts and mediates PDAC cell survival and transformation. Genes Dev. 2009, 23: 24-36. 10.1101/gad.1753809PubMed CentralView ArticlePubMedGoogle Scholar
- Dennler S, Andre J, Alexaki I, Li A, Magnaldo T, ten Dijke P, Wang XJ, Verrecchia F, Mauviel A:Induction of sonic hedgehog mediators by transforming growth factor-beta: Smad3-dependent activation of Gli2 and Gli1 expression in vitro and in vivo. Cancer Res. 2007, 67: 6981-6986. 10.1158/0008-5472.CAN-07-0491View ArticlePubMedGoogle Scholar
- Katoh Y, Katoh M:Integrative genomic analyses on GLI1: positive regulation of GLI1 by Hedgehog-GLI, TGFbeta-Smads, and RTK-PI3K-AKT signals, and negative regulation of GLI1 by Notch-CSL-HES/HEY, and GPCR-Gs-PKA signals. Int J Oncol. 2009, 35: 187-192.View ArticlePubMedGoogle Scholar
- Schmittgen TD, Livak KJ:Analyzing real-time PCR data by the comparative C(T) methodcpa. Nat Protoc. 2008, 3: 1101-1108. 10.1038/nprot.2008.73View ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.