Snail1 Mediates Hypoxia-Induced Melanoma Progression
2011; Elsevier BV; Volume: 179; Issue: 6 Linguagem: Inglês
10.1016/j.ajpath.2011.08.038
ISSN1525-2191
AutoresShujing Liu, Suresh Kumar, James S. Martin, Ruifeng Yang, Xiaowei Xu,
Tópico(s)Cancer, Hypoxia, and Metabolism
ResumoTumor hypoxia is a known adverse prognostic factor, and the hypoxic dermal microenvironment participates in melanomagenesis. High levels of hypoxia inducible factor (HIF) expression in melanoma cells, particularly HIF-2α, is associated with poor prognosis. The mechanism underlying the effect of hypoxia on melanoma progression, however, is not fully understood. We report evidence that the effects of hypoxia on melanoma cells are mediated through activation of Snail1. Hypoxia increased melanoma cell migration and drug resistance, and these changes were accompanied by increased Snail1 and decreased E-cadherin expression. Snail1 expression was regulated by HIF-2α in melanoma. Snail1 overexpression led to more aggressive tumor phenotypes and features associated with stem-like melanoma cells in vitro and increased metastatic capacity in vivo. In addition, we found that knockdown of endogenous Snail1 reduced melanoma proliferation and migratory capacity. Snail1 knockdown also prevented melanoma metastasis in vivo. In summary, hypoxia up-regulates Snail1 expression and leads to increased metastatic capacity and drug resistance in melanoma cells. Our findings support that the effects of hypoxia on melanoma are mediated through Snail1 gene activation and suggest that Snail1 is a potential therapeutic target for the treatment of melanoma. Tumor hypoxia is a known adverse prognostic factor, and the hypoxic dermal microenvironment participates in melanomagenesis. High levels of hypoxia inducible factor (HIF) expression in melanoma cells, particularly HIF-2α, is associated with poor prognosis. The mechanism underlying the effect of hypoxia on melanoma progression, however, is not fully understood. We report evidence that the effects of hypoxia on melanoma cells are mediated through activation of Snail1. Hypoxia increased melanoma cell migration and drug resistance, and these changes were accompanied by increased Snail1 and decreased E-cadherin expression. Snail1 expression was regulated by HIF-2α in melanoma. Snail1 overexpression led to more aggressive tumor phenotypes and features associated with stem-like melanoma cells in vitro and increased metastatic capacity in vivo. In addition, we found that knockdown of endogenous Snail1 reduced melanoma proliferation and migratory capacity. Snail1 knockdown also prevented melanoma metastasis in vivo. In summary, hypoxia up-regulates Snail1 expression and leads to increased metastatic capacity and drug resistance in melanoma cells. Our findings support that the effects of hypoxia on melanoma are mediated through Snail1 gene activation and suggest that Snail1 is a potential therapeutic target for the treatment of melanoma. 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Poellinger L. Lendahl U. Notch signaling mediates hypoxia-induced tumor cell migration and invasion.Proc Natl Acad Sci USA. 2008; 105: 6392-6397Crossref PubMed Scopus (648) Google Scholar In this report we demonstrate that hypoxia promotes melanoma metastasis and drug resistance through Snail1 activation via HIF-2α. In addition, Snail1 activation in melanoma also leads to melanoma cells acquiring cancer stem cell-like features. Cisplatin was purchased from Ben Venue Laboratories (Bedford, OH); temozolomide from TOCRIS Biosciences (Bristol, UK); monoclonal and polyclonal antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA) or BD Biosciences (San Jose, CA). pWZL-Blast-Snail-ER plasmids (plasmid 18798) were obtained from Addgene Inc (Cambridge, MA); pGIPZ-Snail1 was purchased from Thermo Scientific; tamoxifen was purchased from Sigma (Selma, CA). Human melanoma cell lines (WM35, WM793, WM115A, WM3523A, and 1205Lu) were kind gifts from Meenhard Herlyn (The Wistar Institute, Philadelphia, PA), pCDNA3-HIF-1α was kindly provided by Frank Lee (University of Pennsylvania, Philadelphia, PA), and pGFP-HIF2α plasmid was a kind gift from Volker Haase (Vanderbilt University Medical Center, Nashville, TN). Si-snail1 was purchased from Qiagen (Valencia, CA). Si-HIF1α and Si-HIF2α were purchased from BD Biosciences. Human melanoma cell lines were maintained in 2% MCDB medium.11Kumar S.M. Yu H. Edwards R. Chen L. Kazianis S. Brafford P. Acs G. Herlyn M. Xu X. Mutant V600E BRAF increases hypoxia inducible factor-1alpha expression in melanoma.Cancer Res. 2007; 67: 3177-3184Crossref PubMed Scopus (112) Google Scholar Mouse embryonic fibroblast cells were cultured in DMEM with high glucose and L-Glutamine (Invitrogen, Carlsbad, CA), 10% mouse embryonic fibroblast (MEF)-specific fetal bovine serum (FBS) (Invitrogen), 1% nonessential amino acids, and L-glutamine (200 mmol/L/L) (Invitrogen). 293T cells were maintained in high glucose Dulbecco's modified Eagle's medium with 10% FBS, penicillin/streptomycin (100 units/mL and 100 mg/mL) (Invitrogen). Phoenix-Ampho cells were cultured in high glucose Dulbecco's modified Eagle's medium with 10% FBS, 4 mmol/L L-glutamine, and penicillin/streptomycin. Lentiviral vectors containing short hairpin RNA (shRNA) of Snail1 in pGIPZ vector or non-silencing control shRNA in pGIPZ (Thermo Scientific, Huntsville, AL) was co-transfected into 293T cells with packing vector (pCMV-dR8.2-dupr and pCMV-VSV). Viral supernatants were collected 48 and 72 hours post-transfection, and were used to infect melanoma cells as previously described.38Yu H. McDaid R. Lee J. Possik P. Li L. Kumar S.M. Elder D.E. Van Belle P. Gimotty P. Guerra M. Hammond R. Nathanson K.L. Dalla Palma M. Herlyn M. Xu X. The role of BRAF mutation and p53 inactivation during transformation of a subpopulation of primary human melanocytes.Am J Pathol. 2009; 174: 2367-2377Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar After a 48-hour period, cells were cultured in puromycin (1 μg/mL) selection medium. The pWZL-Blast-Snail-ER plasmids were co-transfected into Phoenix Ampho packaging cells, and viral supernatants were collected 48 and 72 hours post-transfection to infect melanoma cells. After a 48-hour period, cells were cultured in blasticidin (Invitrogen, San Diego, CA) (5 μg/mL) selection medium for 5 days. Infected melanoma cells were then cultured in MCDB media with 2% FBS and 20 nmol/L tamoxifen. Si-Snail1, Si-HIF1α, and Si-HIF2α and the irrelevant Si-control (negative control) were used as instructed by the manufacturer. Briefly, on the day of transfection, 5 × 104 melanoma cells were plated per well in 2 mL 2% FBS MCDB tumor media. Cells were then incubated with siPORT NeoFX Transfectin Agent (Ambion Inc., Austin, TX) (10 μL in 200 μL OPTI-MEM I medium (Invitrogen, San Diego, CA) without serum) for 5 minutes. Then 10 μmol/L Si-Snail1, Si-HIF1α, or Si-HIF2α were added and cells were incubated for 10 minutes at room temperature to allow the formation transfection complexes. The next day, the medium was replaced with 2% MCDB tumor medium, and after 48 hours, cells were harvested and analyzed. The 1 × 104 melanoma cells with pWZL-Blast-Snail-ER (inducible Snail1), pGIPZ-Snail1 (Sh-Snail1), or control vectors were seeded in triplicate in 24 well plates and incubated at 37°C in a regular CO2 incubator for 24 hours. The next day, the media was replaced with 2% MCDB media and the cells were incubated in with 1% O2 conditions at 37°C for 8, 16, and 24 hours. At the end of this period, we counted the viable cells in each well by trypan blue exclusion assays. For cell migration assay, pWZL-Blast-Snail-ER, pGIPZ-Snail1, or control vector infected melanoma cells were grown to confluence and wounded by dragging a 1-mL pipette tip through the monolayer. Cells were washed to remove cellular debris and were allowed to migrate for 20 hours. Images were taken after wounding at various time points under a DMI6000 inverted microscope (Leica, Wetzlar, Germany). Melanoma cells with pWZL-Blast-Snail-ER, pGIPZ-Snail1 or control vectors, as well as hypoxia-treated melanoma cells were washed with PBS and 1 × 105 cells were plated in 6 well plates. The plates were incubated at 37°C for 24 hours and cultured with serum free MCDB medium for another 24 hours in a humidified CO2 incubator. The culture medium was aspirated and 2% tumor media containing different concentrations of cisplatin or temozolomide (1–100 μmol/L) was added to each well. Drug treated and control cells were incubated another 24 hours at 37°C in a humidified CO2 incubator. After 24 hours, the cells were washed with 2% tumor media and allowed to grow another 24 hours before counting viable cells. Experiments were performed in triplicate. Total RNA was isolated using RNeasy Kit (Qiagen) followed by cDNA synthesis using SuperScript First-Strand Synthesis System (Invitrogen). Quantitative PCR was performed using iQ SYBR green supermix (Bio-Rad Laboratories, Hercules, CA) with specific primers listed as follows. cDNA corresponding to 1 μg RNA was added to the iQsyber green supermix and analyzed with icycler (Bio-rad Laboratories), according to the manufacturer's instructions. The thermal profiles were 95°C for 30s and 56°C for 30s. Melting curve analysis was done for each PCR reaction to confirm the specificity of amplification. At the end of each phase, florescence was measured and used for quantitative purpose. Primer sequences are shown in Table 1.Table 1Primers Used for Real-Time PCRGeneForward primerReverse primerE-Cadherin (CDH1)5′-TTCCCTGCGTATACCCTGGT-3′5′-GCGAAGATACCGGGGGACACTCATGAG-3′N-Cadherin (CDH2)5′-CACTGCTCAGGACCCAGAT-3′5′-TAAGCCGAGTGATGGTCC-3′Sox105′-CTTCGGCAACGTGGACATT-3′5′-TCAGCCACATCAAAGGTCTCC-3′α-SMA (ACTA2)5′-ACTGGGACGACATGGAAAAG-3′5′-TAGATGGGGACATTGTGGGT-3′Snail15′-GACTAGAGTCTGAGATGCCC-3′5′-CAGACATTGTTAAATTGGCCG-3′HIF1α5′-CATAAAGTCTGCAACATGGAAGGT-3′5′-ATTTGATGGGTGAGGAATGGGTT-3′HIF2α5′-CACTGCTTCAGTGCCATGACA-3′5′-TGTCCAGGAGGAAGGGACTGT-3′Twist5′-TGTCCGCGTCCCACTAGC-3′5′-TGTCCATTTTCTCCTTCTCTGGA-3′JARID1B5′-CGATAAACTTCATTTCACCCCG-3′5′-ACCCACCTTCTTCTGCGACTAAC-3′p75NGFR5′-TTCAAGGGCTTACACGTGGAGGAA-3′5′-TGTGTGTAAGTTTCAGGAGGGCCA-3′β-Actin (ACTB)5′-TGACTGACTACCTCATGAAGATCC-3′5′-GCCATCTCTTGCTCGAAGTCC-3′HIF, hypoxia inducible factor; SMA, smooth muscle actin. Open table in a new tab HIF, hypoxia inducible factor; SMA, smooth muscle actin. Normoxia- or hypoxia-treated melanoma cells, or melanoma cells with pWZL-Blast-Snail-ER or pGIPZ-Snail1 vectors were seeded on fibronectin pretreated chamber slides. Cells were fixed with glutaraldehyde for 20 minutes at room temperature and then washed three times with 1% bovine serum albumin (BSA) for 5 minutes. Cells were then permeabilized and blocked by 0.3% Triton X-100, 1% BSA, and 10% normal donkey serum in PBS at room temperature for 45 minutes. The cells were then stained with Snail1, E-cadherin, and N-cadherin primary antibodies overnight at 4°C. Stained cells were then washed three times with PBS containing 1% BSA and incubated with the appropriate secondary antibodies conjugated to Alexa Fluor. Nuclei were counterstained with DAPI (Vector Laboratories, Dana Point, CA). Cells were imaged with a Leica Inverted fluorescence microscope with a Leica camera. The images were processed and analyzed using Adobe Photoshop software. We performed Western blot as previously described.39Liu S. Yu M. He Y. Xiao L. Wang F. Song C. Sun S. Ling C. Xu Z. Melittin prevents liver cancer cell metastasis through inhibition of the Rac1-dependent pathway.Hepatology. 2008; 47: 1964-1973Crossref PubMed Scopus (157) Google Scholar Melanoma cells were seeded in 24-well plates at densities of 5 × 104 cells per well, grown for 24 hours, treated with 1% O2 for 16 hours, normoxia cells were used as the control, and then WST-1 cell proliferation assay (Roche Diagnostics GmbH, Mannheim, Germany) was used to quantify proliferative cells. The absorbance at 450 nm was measured using an lQuant Universal Microplate Spectrophotometer (Bio-tek Instruments, Winooski, VT). 1 × 106 melanoma cells with Snail1 knockdown or control cells were harvested, rinsed twice with PBS, and fixed with 70% ethanol overnight at 4°C. Fixed cells were then washed 2 times with PBS and stained with 20 μg/mL propidium iodide (PI). Analysis was performed on a FACS Calibur using CellQuest Pro software. Cell cycle analysis was performed using ModFit software (Verity Software House, Topsham, ME). The chromatin immunoprecipitation (ChIP) assay was conducted by using a Chip assay kit, according to the manufacturer's instruction (Active Motif, Carlsbad, CA), and rabbit antibody against human HIF-1α and HIF-2α protein (Abcam, Cambridge, MA). The immunoprecipitated chromatin was analyzed in triplicate by PCR using the primers (5′-ATCCCTGGAAGCTGCTCTCT-3′ forward and 5′-TCTGGTCCAGTGAGGGAG-3′) for human Snail1 promoter.37Sahlgren C. Gustafsson M.V. Jin S. Poellinger L. Lendahl U. Notch signaling mediates hypoxia-induced tumor cell migration and invasion.Proc Natl Acad Sci USA. 2008; 105: 6392-6397Crossref PubMed Scopus (648) Google Scholar Melanoma cells over-expressing Snail1 or control cells were trypsinized, washed, and re-suspended at a concentration of 100 cells/mL in HECM4 media.40Yu H. Fang D. Kumar S.M. Li L. Nguyen T.K. Acs G. Herlyn M. Xu X. Isolation of a novel population of multipotent adult stem cells from human hair follicles.Am J Pathol. 2006; 168: 1879-1888Abstract Full Text Full Text PDF PubMed Scopus (294) Google Scholar 10 μL of the cell suspension was dispensed into wells of 96-well plates, containing 90 μL of 2% MCDB media. All plates were examined to ensure that that one cell was delivered to each well. The cells were incubated for another 7 days, with media changes every other day. Colonies were counted in single cell seeded wells. The 1 × 104 control or tamoxifen-induced Snail1 infected WM115A cells were re-suspended in 3 mL of 1.8% (w/v) Bacto-Agar solution containing MCDB with 20% FBS. The mixtures were overlaid onto a 3.3% (w/v) BactoAgar solution in 6-well plates. On the following day, 0.5 mL of MCDB supplemented with 2.0% FBS was added. Colonies were counted under a microscope after 15 days. Colony forming efficiency was calculated by the number of colonies times 100, divided by the number of cells plated. Melanoma cells with over-expressing Snail1 or control vectors were washed with PBS, trypsinized, and then re-washed with PBS, and re-suspended in HECM4 media. The 5 × 104 cells were aliquoted into an ultra-low attachment 6-well plate containing 2 mL of HECM4 media. The cells were incubated for another 7 days, and 0.5 mL of new media was added every other day. The number of colonies formed was counted using an inverted microscope. Four- to five-week-old male athymic nu/nu mice were used in this study. The control, pWZL-Blast-Snail1-ER, or pGIPZ-Snail1-infected WM115A cells (2 × 106 cells/animal) were injected subcutaneously into nude mice (10 mice per group). The mice that received pWZL-Blast-Snail1-ER cells continued to receive tamoxifen to maintain Snail1 expression. After 35 days, we sacrificed the mice and performed necropsy for further analysis. The data represent mean ± SEM values. The effect of treatments and differences among experimental groups were assessed using analysis of variance and the appropriate post hoc test. The differences between the two experimental groups were determined using the Student's t-test. A two-tailed value of P < 0.05 was considered statistically significant. All of the analyses were performed using the GraphPad Prism software (GraphPad Software, Inc., San Diego, CA). We first studied the effects of hypoxia on melanoma cells in vitro. WM115A, 1205Lu and 451Lu cells were incubated under hypoxic (1% O2) or normoxic conditions for 8, 16, or 24 hours. Hypoxia significantly inhibited tumor cell proliferation, and only 50% of the WM115A cells survived after 16 or 24 hours of treatment (Figure 1A). We chose 16-hour hypoxic treatment for further studies because there was no significant difference between the cells treated for 16 or 24 hours. To examine drug resistance in hypoxia-treated melanoma cells, we treated the cells that survived hypoxia treatment with various doses of cisplatin and temozolomide. In the absence of hypoxia treatment, cisplatin and temozolomide-induced cell death in a dose-dependent manner, killing most of the melanoma cells at 25 μmol/L cisplatin (Figure 1B). However, tumor cells survived hypoxia treatment showed dramatically increased resistance to cisplatin and temozolomide, with 29.8 ± 3% or 44 ± 5.0% of the cells still alive after exposure to 25 μmol/L cisplatin or temozolomide (Figure 1B). Cell motility (measured by the wound healing assay) was significantly increased after hypoxic treatment (Figure 1C). When hypoxia-treated cells were used in soft agar assays, the resultant colonies were significantly larger and more abundant when compared to control cells (Figure 1D), which suggests that the tumor cells that survived hypoxia treatment had high migratory and proliferation capacity. The results from 1205Lu and 451Lu cells were similar to WM115A cells (data not shown). We cultured WM115A cells under hypoxic or normoxic conditions for 8, 16, or 24 hours and measured gene expression in these cells. E-cadherin levels were significantly decreased under hypoxia, which was accompanied by increased expression of N-cadherin and smooth muscle actin levels. In contrast, Sox10 levels did not significantly change. Similarly, E-cadherin protein levels were reduced after hypoxia treatment, wherea
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