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• W1966973894 abstract "Malignant melanoma-initiating cells (MMIC) are a subpopulation of cells responsible for melanoma tumor growth and progression. They are defined by the expression of the ATP-binding cassette (ABC) subfamily B member 5 (ABCB5). Here, we identified a critical role for the DEAD-box helicase antigen (HAGE) in ABCB5+ MMIC-dependent tumorigenesis and show that HAGE-specific inactivation inhibits melanoma tumor growth mediated by this tumor-initiating population. Knockdown of HAGE led to a significant decrease in RAS protein expression with a concomitant decrease in activation of the AKT and ERK signaling pathways implicated to play an important role in melanoma progression. To confirm that the reduction in NRAS (Neuroblastoma RAS) expression was dependent on the HAGE helicase activity, we showed that NRAS, effectively silenced by siRNA, could be rescued by reintroduction of HAGE in cells lacking HAGE. Furthermore, we provide a mechanism by which HAGE promotes NRAS unwinding in vitro. We also observed using tumor transplantation in Non-obese diabetic/severe combined immunodeficiency mice that the HAGE knockdown in a ABCB5+ melanoma cell line displayed a significant decrease in tumor growth and compared with the control. Our results suggest that the helicase HAGE is required for ABCB5+ MMIC-dependent tumor growth through promoting RAS protein expression and that cancer therapies targeting HAGE helicase may have broad applications for treating malignant melanoma and potentially other cancer types. Malignant melanoma-initiating cells (MMIC) are a subpopulation of cells responsible for melanoma tumor growth and progression. They are defined by the expression of the ATP-binding cassette (ABC) subfamily B member 5 (ABCB5). Here, we identified a critical role for the DEAD-box helicase antigen (HAGE) in ABCB5+ MMIC-dependent tumorigenesis and show that HAGE-specific inactivation inhibits melanoma tumor growth mediated by this tumor-initiating population. Knockdown of HAGE led to a significant decrease in RAS protein expression with a concomitant decrease in activation of the AKT and ERK signaling pathways implicated to play an important role in melanoma progression. To confirm that the reduction in NRAS (Neuroblastoma RAS) expression was dependent on the HAGE helicase activity, we showed that NRAS, effectively silenced by siRNA, could be rescued by reintroduction of HAGE in cells lacking HAGE. Furthermore, we provide a mechanism by which HAGE promotes NRAS unwinding in vitro. We also observed using tumor transplantation in Non-obese diabetic/severe combined immunodeficiency mice that the HAGE knockdown in a ABCB5+ melanoma cell line displayed a significant decrease in tumor growth and compared with the control. Our results suggest that the helicase HAGE is required for ABCB5+ MMIC-dependent tumor growth through promoting RAS protein expression and that cancer therapies targeting HAGE helicase may have broad applications for treating malignant melanoma and potentially other cancer types. The helicase antigen (HAGE), a non-X-linked cancer testis (CT) antigen of 648 amino acids (73 kDa) was originally identified in a rhabdomyosarcoma cell line (1Martelange V. De Smet C. De Plaen E. Lurquin C. Boon T. Identification on a human sarcoma of two new genes with tumor-specific expression.Cancer Res. 2000; 60: 3848-3855PubMed Google Scholar). HAGE, also known as DDX43 or CT13, belongs to the DEAD-box family of ATP-dependent RNA helicases characterized by the presence of nine conserved motifs grouped into two domains connected by a polylinker SAT region (2Caruthers J.M. McKay D.B. Helicase structure and mechanism.Curr. Opin. Struct. Biol. 2002; 12: 123-133Crossref PubMed Scopus (453) Google Scholar). The Q motif, motifs I, II (also called the D-E-A-D-box as a single-letter code for Asp-Glu-Ala-Asp), and III, together with motif IV, bind and hydrolyze ATP molecules. The remaining motifs (V, VI, Ia, and Ib) are thought to interact with the RNA substrate. It has been suggested that RNA helicases browse RNA molecules in a bidirectional fashion using the energy gained from ATP hydrolysis until they encounter ribonucleoprotein-RNA complexes (3Silverman E. Edwalds-Gilbert G. Lin R.J. DExD/H-box proteins and their partners. Helping RNA helicases unwind.Gene. 2003; 312: 1-16Crossref PubMed Scopus (164) Google Scholar). The helicase activity then allows the dissociation of RNA from the ribonucleoprotein to which they have high affinity (4Fuller-Pace F.V. DExD/H box RNA helicases. Multifunctional proteins with important roles in transcriptional regulation.Nucleic Acids Res. 2006; 34: 4206-4215Crossref PubMed Scopus (323) Google Scholar). RNA helicases are often described as the driving forces behind RNA metabolism in processes such as transcription, pre-mRNA splicing, ribosome biogenesis, cytoplasmic transport, translation initiation/elongation, and RNA decay (4Fuller-Pace F.V. DExD/H box RNA helicases. Multifunctional proteins with important roles in transcriptional regulation.Nucleic Acids Res. 2006; 34: 4206-4215Crossref PubMed Scopus (323) Google Scholar, 5Cordin O. Banroques J. Tanner N.K. Linder P. The DEAD-box protein family of RNA helicases.Gene. 2006; 367: 17-37Crossref PubMed Scopus (733) Google Scholar, 6Linder P. Dead-box proteins. A family affair–active and passive players in RNP-remodeling.Nucleic Acids Res. 2006; 34: 4168-4180Crossref PubMed Scopus (353) Google Scholar, 7Jankowsky A. Guenther U.P. Jankowsky E. Nucleic Acids Res. 2011; 39: 338-341Crossref PubMed Scopus (52) Google Scholar, 8Linder P. Jankowsky E. From unwinding to clamping. The DEAD box RNA helicase family.Nat. Rev. Mol. Cell Biol. 2011; 12: 505-516Crossref PubMed Scopus (693) Google Scholar). Previous studies have suggested that several RNA helicases, such as DDX1 (9Amler L.C. Schürmann J. Schwab M. The DDX1 gene maps within 400 kbp 5′ to MYCN and is frequently coamplified in human neuroblastoma.Genes Chromosomes Cancer. 1996; 15: 134-137Crossref PubMed Google Scholar, 10Godbout R. Packer M. Bie W. Overexpression of a DEAD box protein (DDX1) in neuroblastoma and retinoblastoma cell lines.J. Biol. Chem. 1998; 273: 21161-21168Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar), DDX2 (11Eberle J. Fecker L.F. Bittner J.U. Orfanos C.E. Geilen C.C. Decreased proliferation of human melanoma cell lines caused by antisense RNA against translation factor eIF-4A1.Br. J. Cancer. 2002; 86: 1957-1962Crossref PubMed Scopus (42) Google Scholar), DDX6 (12Akao Y. Marukawa O. Morikawa H. Nakao K. Kamei M. Hachiya T. Tsujimoto Y. The rck/p54 candidate proto-oncogene product is a 54-kilodalton D-E-A-D box protein differentially expressed in human and mouse tissues.Cancer Res. 1995; 55: 3444-3449PubMed Google Scholar), and DDX53 (13Por E. Byun H.J. Lee E.J. Lim J.H. Jung S.Y. Park I. Kim Y.M. Jeoung D.I. Lee H. The cancer/testis antigen CAGE with oncogenic potential stimulates cell proliferation by up-regulating cyclins D1 and E in an AP-1- and E2F-dependent manner.J. Biol. Chem. 2010; 285: 14475-14485Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar) play an important role in tumor cell development and proliferation in a variety of cancers. In this study, we looked at the role of HAGE in melanoma cancer initiation and growth associated with malignant melanoma-initiating cells (MMIC) 3The abbreviations used are: MMICmalignant melanoma-initiating cellABCATP-binding cassetteABCB5ATP-binding cassette subfamily B member 5IBimmunoblottingIFimmunofluorescenceHAGEhelicase antigenNRASneuroblastoma RASpAKTphosphorylated AKTPCNAproliferating cell nuclear antigenFOXOForkhead box O., a subpopulation of chemoresistant cells capable of self-renewal and differentiation and responsible for melanoma tumor initiation, growth, and progression (14Frank N.Y. Margaryan A. Huang Y. Schatton T. Waaga-Gasser A.M. Gasser M. Sayegh M.H. Sadee W. Frank M.H. ABCB5-mediated doxorubicin transport and chemoresistance in human malignant melanoma.Cancer Res. 2005; 65: 4320-4333Crossref PubMed Scopus (463) Google Scholar). This population of cells express the ATP-binding cassette (ABC) subfamily B member 5 (ABCB5), a protein that renders malignant melanoma drug-resistant and refractory to therapy. As a consequence, this population is thought to be responsible for a highly aggressive type of cancer with a poor prognosis and outcome (15Frank N.Y. Schatton T. Kim S. Zhan Q. Wilson B.J. Ma J. Saab K.R. Osherov V. Widlund H.R. Gasser M. Waaga-Gasser A.M. Kupper T.S. Murphy G.F. Frank M.H. VEGFR-1 expressed by malignant melanoma-initiating cells is required for tumor growth.Cancer Res. 2011; 71: 1474-1485Crossref PubMed Scopus (129) Google Scholar, 16Wilson B.J. Schatton T. Zhan Q. Gasser M. Ma J. Saab K.R. Schanche R. Waaga-Gasser A.M. Gold J.S. Huang Q. Murphy G.F. Frank M.H. Frank N.Y. ABCB5 identifies a therapy-refractory tumor cell population in colorectal cancer patients.Cancer Res. 2011; 71: 5307-5316Crossref PubMed Scopus (104) Google Scholar). Although the mechanisms by which ABCB5+ MMIC resist systemic therapy have been well studied, their role in promoting tumor growth and progression is still under investigation. Here, we reveal a previously unknown role of the helicase HAGE in ABCB5+ MMIC-dependent tumor growth and progression. Using a stem cell proliferation assay, we show that HAGE is required for tumor growth initiated by this population of cells. HAGE knockdown in a melanoma cell line expressing ABCB5 caused a decrease of RAS protein expression, subsequently leading to a significant decrease in the activation of its downstream effectors, AKT and ERK. Silencing of NRAS in the HAGE knockdown melanoma cell line using NRAS-specific siRNA was rescued successfully after reintroduction of HAGE in these cells. We took this further and provide a mechanistic insight by demonstrating the capacity of the helicase HAGE to unwind NRAS RNA complexes using an in vitro unwinding assay. Finally, tumor transplantation assays clearly demonstrated the in vivo role of HAGE in promoting ABCB5+ MMIC-dependent tumor progression. Collectively, these findings clearly implicate the helicase HAGE in the promotion of tumor growth in melanoma through RAS/AKT and RAS/ERK pathways and shed new light on our understanding of the mechanisms underlying ABCB5+ MMIC-dependent tumorigenesis. malignant melanoma-initiating cell ATP-binding cassette ATP-binding cassette subfamily B member 5 immunoblotting immunofluorescence helicase antigen neuroblastoma RAS phosphorylated AKT proliferating cell nuclear antigen Forkhead box O. The human melanoma cell lines FM82 and FM55 were a kind gift from Professor D. Schadendorff of the Deutsches Krebsforschungszentrum (Heidelberg, Germany). All cells were cultured in RPMI 1640 medium (Lonza) supplemented with 10% FCS and 1% (w/v) l-glutamine (Lonza) and incubated at 37 °C supplied with a 5% CO2 humid environment. A stable knockdown cell line was created using shRNA-bearing plasmids specific for HAGE (SureSilencing shRNA Plasmid for Human DDX43, SABiosciences). FM82 and FM55 cells were seeded at 5 × 104 cells/well into a 24-well plate and grown overnight. 1.2 μg of SureSilencing shRNA was transfected into cells using Lipofectamine 2000 (Invitrogen) as described by the manufacturer. 48 h later, the cells were seeded into a 96-well plate and grown in media treated with 500 μg/ml G418 antibiotic to allow selection of plasmid-transfected cells. HAGE expression status was monitored using real-time quantitative PCR, Western blotting, and immunofluorescence. The transient knockdown of NRAS was carried out using an NRAS-specific siRNA molecule (Eurogentec) (sense, 5′-CAGCAGUGAUGAUGGGACUTT-3′ and antisense, 5′-AGUCCCAUCAUCACUGCUGTT-3′) and Interferin transfection reagent (Polyplus) following the recommendations of the manufacturer. To investigate if the introduction of HAGE into cells results in increased NRAS protein expression, FM82 stable transfectants were firstly cultured and then transfected with NRAS siRNA as described above. 24 h following transfection, a second transfection was carried out using a HAGE-specific cDNA vector, pcDNA3.1-HAGE, to ectopically express HAGE. Transfection was performed using the Lipofectamine 2000 protocol and 12 μg of the vector to transfect a T25 flask as described by the manufacturer. At 48 hours post-transfection, cells were harvested for protein extraction. FM82 and FM55 control and FM82 and FM55 shRNAs cells were grown to 80% confluence as described above. Total RNA was extracted from cells using RNA-STAT 60 reagent (AMS Biotechnology) as described by the manufacturer. Extracted RNAs were quantified using a Nanodrop spectrophotometer (Thermo Scientific). Muloney murine leukemia virus reverse transcriptase (Promega), oligo(dT) primers (Promega), and 2 μg of total RNA were subsequently used for cDNA synthesis following the protocol of the manufacturer. Real-time quantitative PCR was then carried out using primers (all MWG Eurofins) specific for HAGE (forward, 5′-GGAGATCGGCCATTGATAGA-3′ and reverse, 5′-GGATTGGGGATAGGTCGTTT-3′), NRAS (forward, 5′-AAACCTCAGCCAAGACCAGA-3′ and reverse, 5′-CCCTGAGTCCCATCATCACT-3′) and the housekeeping genes TBP1 (forward, 5′-TGCACAGGAGCCAAGAGTGAA-3′ and reverse, 5′-CACATCACAGCTCCCCACCA-3′) and HPRT1 (forward, 5′-TGACACTGGCAAAACAATGCA-3′ and reverse, 5′-GGTCCTTTTCACCAGCAAGCT-3′) using a Rotor-Gene 6000 real-time PCR cycler (Qiagen). Forty cycles were performed, and melt curves were ratified following each analysis. Expression of the genes of interest was normalized using averaged results for the housekeeping genes, and 2ΔCT calculations were performed. For this study, we used a monoclonal HAGE antibody (1:250 for immunoblotting (IB), 1:50 immunofluorescence (IF), SAB1400618, Sigma), ABCB5 (1:500 for IB, 1:100 for IF, HPA026975, Sigma), β-actin (1:1000, 4967, Cell Signaling Technologies), AKT (1:250 for IB, 9272, Cell Signaling Technologies), p-AKT (Ser-473) (1:250 for IB, 4058, Cell Signaling Technologies), GSK3β (1:250 for IB, 9315, Cell Signaling Technologies), p-GSK3β (Ser-9) (1:250 for IB, 9336, Cell Signaling Technologies), NRAS (1:100 for IB, 1:50 for IF, SC-31, Santa Cruz Biotechnology), Kirsten RAS (1:100 for IB, SC-30, Santa Cruz Biotechnology), Harvey RAS (1:100 for IB, SC-29, Santa Cruz Biotechnology), p21CIP1 (1:250 for IB, ab80633, Abcam), c-SRC (cellular-SRC) (1:250 for IB, SC-5266, Santa Cruz Biotechnology), GRB2 (1:250 for IB, 3972, Cell Signaling Technologies), SOS1 (1:250 for IB, SC-55528, Santa Cruz Biotechnology), ERK1/2 (1:250 for IB, 4695, Cell Signaling Technologies), p-ERK1/2 (Thr-202/Tyr-204) (1:250 for IB, 9101, Cell Signaling Technologies), p-FOXO1 (Thr-24)/FOXO3a (Thr-32)/FOXO4 (Thr-28) (1:250 for IB, 2599, Cell Signaling Technology), and PCNA (1:100 for IF, ab29, Abcam). HRP-conjugated antibodies were used as secondary antibodies for IB (1:500, Dako). Alexa Fluor 488- and Alexa Fluor 568-conjugated antibodies were used as secondary antibodies for IF (1:500, Invitrogen). Paraffin-embedded human melanoma tissue microarrays were purchased from US Biomax. The sections were dewaxed, rehydrated in graded alcohols, rinsed in double distilled H2O, and then antigen retrieval was performed for 10 min (0.01 m citrate phosphate buffer (pH 6.0)) in a microwave at 1000 Watt. The tumors excised from non-obese diabetic/severe combined immunodeficiency mice were embedded in OCT (optimal cutting temperature) media, fixed in 2-butanol (Sigma) that had been super-cooled in liquid nitrogen, and sectioned using a cryostat CM1900 (Leica). Melanoma cells were fixed in 4% (w/v) paraformaldehyde and then treated as follows. The sections or cancer cells were washed three times in 1× PBS for 10 min each, blocked and permeabilized in 10% (w/v) BSA in 0.1% (v/v) PBS-Tween, incubated overnight with primary antibody (in blocking solution), washed three times for 10 min each with 1× PBS, incubated for 1 h with secondary antibody (in blocking solution), and washed three times with 1× PBS. Sections and melanoma cells were counterstained and mounted with DAPI fluorescent medium (Vector Laboratories) for IF microscopy. For IB, cancer cells were collected, washed with 1× PBS, lysed in 1× solution containing 50 mm Tris-HCl (pH 6.8), 100 mm dithiothreitol, 2% (w/v) SDS, 0.1% (w/v) bromphenol blue, and 10% (v/v) glycerol and loaded on Tris/glycine SDS-polyacrylamide gels. Proteins were separated alongside a molecular weight marker (Bio-Rad). Protein bands were transferred onto Amersham Biosciences Hybond-P PVDF membranes (GE Healthcare). Membranes were blocked with 10% Marvel milk/TBS solution with 0.05% Tween 20 containing sodium orthovanadate and sodium fluoride. Following TBS solution with 0.05% Tween 20 washes, membranes were incubated with primary antibodies (in blocking solution) at 4 °C overnight followed by washing and incubation with secondary antibodies for 1 h at room temperature ahead of visualization with Rapid Step ECL reagent (Calbiochem) and viewed using a charge coupled device camera (Fujifilm). For immuno-dot-blot analysis of signaling phosphoproteins, Proteome Profiler arrays (R&D Systems) specific for the PI3K and MAPK pathways were acquired and used in accordance with the guidelines of the manufacturer using protein lysates extracted from FM82 control and FM82 shRNA1 cells. Blots were visualized in the same manner as described above, and densitometry readings of each dot were carried out using densitometry software (Aida v. 5.2). All cells were grown in 24-well plates (Sarstedt) at 5 × 104 cells/well. All necessary treatments were performed in duplicate. Assessment of the effect of HAGE knockdown on cell proliferation was performed on day 1 and day 3 following plating (n = three independent experiments). The day prior to testing, cells were treated with complete RPMI 1640 medium containing 3H and allowed to absorb the isotope during overnight incubation. On the day of testing, medium was removed, and cells were lysed with ddH20, leading to the release of incorporated 3H. The contents of the plate were subsequently harvested onto a Unifilter 96-well plate using a Filtermate Harvester (Packard), which was allowed to dry using a drying cabinet (SLS). Each test well was treated with 40 μl of Microscint-O media (PerkinElmer). Plate reading was carried out using a Top-Count NXT microplate scintillation counter (Packard). Non-obese diabetic/severe combined immunodeficiency mice were acquired (Harlan Laboratories, UK) and kept in accordance with UK Home Office regulations. Mice were kept in individual ventilated cages supplied with laminar air flow and autoclaved food and water taken ad libitum. Mice were separated into two groups and injected subcutaneously into the right flank with 1 × 106 FM82 control or FM82 shRNA cells per 100 μl of serum-free medium. Tumor development was observed twice weekly using caliper measurements. Once one of the tumors reached a diameter of 1 cm, the experiment was stopped, and mice were euthanized prior to tumors being extracted, snap-frozen in liquid nitrogen-cooled 2-butanol (Sigma), and stored at −80 °C. For the culture of melanospheres, cells from FM82 and FM55 controls and FM82 and FM55 shRNAs were plated at clonal density (10 × 103 cells/ml in 24-well plates) in a DMEM/F12 medium (Invitrogen, 32500) supplemented with N-2 Max Media (R&D Systems, AR009), and daily addition of fibroblast growth factor basic 100 ng/ml and EGF 100 ng/ml (both from Sigma) for a 10-day culture period (17Na Y.R. Seok S.H. Kim D.J. Han J.H. Kim T.H. Jung H. Park J.H. Zebrafish embryo extracts promote sphere-forming abilities of human melanoma cell line.Cancer Sci. 2009; 100: 1429-1433Crossref PubMed Scopus (7) Google Scholar). The differentiation of melanospheres was induced upon FGF basic and EGF withdrawal. The cells were stained by immunofluorescence as described above. The plasmid pDNR-LIB containing NRAS was purchased from Open Biosystems and used for in vitro transcription using the T7 promoter to generate the NRAS transcript (Ambion). NRAS transcripts were labeled using the Pierce RNA 3′ end biotinylation kit (Thermo Scientific). The labeling efficiency was determined by dot blotting following the recommendations of the manufacturer. The biotinylated transcripts (100 pmol) were transfected in 6-well plates containing FM82 control and FM82 shRNA cells using Lipofectamine 2000 (Invitrogen). The transcripts were detected by immunofluorescence using Streptavidin Alexa Fluor 488 conjugate (Invitrogen). Biotinylated RNA (2 pmol, labeling efficiency higher than 75%, 5′-GACUCAGGGUUGUAUGGGAUUGCCAUGUGUGGUGAUGUAA-3′) was placed to wind with unlabeled NRAS RNA in a solution containing 3.5 mm equimolar of ATP/MgCl2 for 5 min at 95 °C followed by 1 h at 37 °C. HAGE recombinant protein was added at two different concentrations (0.6 μg and 1.2 μg) after the RNA winding assay, followed by 1 h of incubation at 37 °C. The reactions were stopped by the addition of 50% (v/v) glycerol, 20 mm EDTA, 2% (w/v) SDS, and 0.025% (w/v) bromophenol blue. Samples were analyzed on 12% (w/v) native acrylamide gels in 1× Tris/Borate/EDTA buffer followed by blot transfer and visualization using the chemiluminescent detection module (Thermo Scientific). A number of studies have demonstrated that ATP-dependent RNA helicases belonging to the family of DEAD-box proteins could be involved in the process of tumorigenesis (9Amler L.C. Schürmann J. Schwab M. The DDX1 gene maps within 400 kbp 5′ to MYCN and is frequently coamplified in human neuroblastoma.Genes Chromosomes Cancer. 1996; 15: 134-137Crossref PubMed Google Scholar, 10Godbout R. Packer M. Bie W. Overexpression of a DEAD box protein (DDX1) in neuroblastoma and retinoblastoma cell lines.J. Biol. Chem. 1998; 273: 21161-21168Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 11Eberle J. Fecker L.F. Bittner J.U. Orfanos C.E. Geilen C.C. Decreased proliferation of human melanoma cell lines caused by antisense RNA against translation factor eIF-4A1.Br. J. Cancer. 2002; 86: 1957-1962Crossref PubMed Scopus (42) Google Scholar, 12Akao Y. Marukawa O. Morikawa H. Nakao K. Kamei M. Hachiya T. Tsujimoto Y. The rck/p54 candidate proto-oncogene product is a 54-kilodalton D-E-A-D box protein differentially expressed in human and mouse tissues.Cancer Res. 1995; 55: 3444-3449PubMed Google Scholar, 13Por E. Byun H.J. Lee E.J. Lim J.H. Jung S.Y. Park I. Kim Y.M. Jeoung D.I. Lee H. The cancer/testis antigen CAGE with oncogenic potential stimulates cell proliferation by up-regulating cyclins D1 and E in an AP-1- and E2F-dependent manner.J. Biol. Chem. 2010; 285: 14475-14485Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 18Camats M. Guil S. Kokolo M. Bach-Elias M. p68 RNA helicase (DDX5) alters activity of cis- and trans-acting factors of the alternative splicing of H-Ras.PLoS ONE. 2008; 3: 2926Crossref PubMed Scopus (50) Google Scholar, 19Janknecht R. Multi-talented DEAD-box proteins and potential tumor promoters. p68 RNA helicase (DDX5) and its paralog, p72 RNA helicase (DDX17).Am. J. Transl. Res. 2010; 2: 223-234PubMed Google Scholar). Because the role of HAGE in tumor growth has yet to be described, we wanted to determine its role in melanoma growth. We examined HAGE expression in tumor tissue microarrays containing malignant melanoma patient samples by performing immunostaining using a HAGE-specific polyclonal antibody. As a control, we tested normal skin. HAGE expression was observed in 6 of 10 melanoma samples (Fig. 1 and supplemental Fig. 1). At the cellular level, HAGE was demonstrated to be cytoplasmic, with few cells exhibiting a nucleocytoplasmic localization. HAGE expression was confined to cytoplasmic aggregates in the FM82 and FM55 melanoma cell lines (FM82 control and FM55 control) as detected by immunofluorescence, which was markedly decreased when HAGE was stably knocked down using shRNAs (Fig. 2, A and B, and supplemental Fig. 2, A and C). To determine the effect of HAGE expression on cell proliferation, we performed a thymidine incorporation assay to measure the proliferation rate of FM82 and FM55 shRNAs cells compared with FM82 and FM55 control cells. A significant reduction in proliferation was observed in the FM82 and FM55 shRNA cell lines after days 1 and 3 (Fig. 2C and supplemental Fig. 2B).FIGURE 2HAGE silencing results in a decrease of proliferation in vitro and in vivo. A, immunofluorescence staining showing HAGE expression in the FM82 control and FM82 shRNA1 melanoma cell lines. HAGE aggregates are shown in the cytoplasm by arrows. Scale bars = 20 μm. Also shown is immunoblotting using an antibody to HAGE in FM82 control and FM82 shRNA1 extracts. β-actin was measured as a loading control. B, immunofluorescence staining showing HAGE expression in FM82 shRNA2 and FM82 shRNA3. Scale bars = 20 μm. Also shown is immunoblotting using an antibody to HAGE in FM82 shRNA2 and FM82 shRNA3 extracts. β-actin was measured as a loading control. C, standard 3H incorporation assays of stable knockdown and control FM82 cells carried out one day and three days post-plating. Mann-Whitney U test: **, p = < 0.01; ***, p = < 0.001. D, measurement of tumor growth in mice injected with either FM82 control cells (n = 12) or FM82 shRNA1 cells (n = 12). Student's t test: **, p = < 0.01; ***, p = < 0.001. E, immunostaining of FM82 control and FM82 shRNA1 tumors using antibody to HAGE (green) and the proliferative marker PCNA (red). Scale bars = 20 μm. The graph illustrates the percentage of HAGE- and PCNA-positive cells from 10 random counting microscopic fields. Student's t test: *, p = < 0.05; ***, p = < 0.001.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The effect of HAGE expression on tumor growth was investigated using a tumor transplantation assay. FM82 control and FM82 shRNA1 were injected subcutaneously into two groups of immunodeficient mice (non-obese diabetic/severe combined immunodeficiency). Mice injected with FM82 shRNA1 cells developed tumors at a significantly slower rate compared with the FM82 control group (Fig. 2D). The tumors that developed in FM82 control- and FM82 shRNA1-injected mice were excised and histologically prepared for analysis by immunofluorescence using antibodies against HAGE and the proliferation marker PCNA. HAGE and PCNA protein expression was significantly reduced in FM82 shRNA tumors compared with FM82 control tumors (Fig. 2E). Taken together, these results demonstrate that HAGE promotes proliferation and tumor growth of malignant melanoma cells. To determine whether HAGE is involved in tumor growth via regulation of downstream kinases involved in promoting cell proliferation, FM82 control and FM82 shRNA1 cell lysates were diluted and incubated with nitrocellulose membranes spotted with phospho-kinase antibodies. Densitometry analysis of the different spots revealed a significant decrease of intensity of phospho-AKT and phospho-ERK spots when incubated with FM82 shRNA1 cell lysates compared with controls (Fig. 3, A and B). Immunoblotting using phospho-AKT (Ser-473) and phospho-ERK1/2 (Thr-202/Tyr-204) antibodies confirmed that both p-AKT and p-ERK levels were significantly decreased in FM82 and FM55 shRNAs cells lacking HAGE (Fig. 3, C and E, and supplemental Fig. 2C). This result suggests that HAGE promotes predominantly AKT and ERK phosphorylation. The total level of AKT protein appeared to increase following HAGE silencing, whereas ERK1/2 total protein levels were slightly decreased in FM82 shRNAs, suggesting that HAGE may play a role in ERK1/2 protein expression (Fig. 3, C and E). Phosphorylated AKT and ERK promote proliferation and survival by inducing the phosphorylation of FOXO transcription factors and GSK3β, thereby inactivating their function in regulating apoptosis and proliferation (20Manoukian A.S. Woodgett J.R. Role of glycogen synthase kinase-3 in cancer. Regulation by Wnts and other signaling pathways.Adv. Cancer Res. 2002; 84: 203-229Crossref PubMed Scopus (130) Google Scholar, 21Ding Q. Xia W. Liu J.C. Yang J.Y. Lee D.F. Xia J. Bartholomeusz G. Li Y. Pan Y. Li Z. Bargou R.C. Qin J. Lai C.C. Tsai F.J. Tsai C.H. Hung M.C. Erk associates with and primes GSK-3β for its inactivation resulting in up-regulation of β-catenin.Mol. Cell. 2005; 19: 159-170Abstract Full Text Full Text PDF PubMed Scopus (492) Google Scholar, 22Asada S. Daitoku H. Matsuzaki H. Saito T. Sudo T. Mukai H. Iwashita S. Kako K. Kishi T. Kasuya Y. Fukamizu A. Mitogen-activated protein kinases, Erk and p38, phosphorylate and regulate Foxo1.Cell. Signal. 2007; 19: 519-527Crossref PubMed Scopus (187) Google Scholar, 23Huang H. Tindall D.J. Dynamic FoxO transcription factors.J. Cell Sci. 2007; 120: 2479-2487Crossref PubMed Scopus (879) Google Scholar) (Fig. 3B). Immunoblotting experiments using antibodies against the phosphorylated forms of FOXO transcription factors (p-FOXO1 (Thr-24)/p-FOXO3a (Thr-32)/p-FOXO4 (Thr-28)) and phospho-GSK3β (Ser-9) revealed a decrease in phosphorylation of these factors upon HAGE stable knockdown (Fig. 3C). Furthermore, the decreased phosphorylation of FOXO factors correlated with an increase of p21CIP1 protein expression (Fig. 3C), a known transcriptional target of FOXO transcription factors. Together these results suggest that HAGE expression promotes the phosphorylation of AKT and ERK proteins, with concomitant effects on their downstream targets. AKT and ERK are well known downstream targets of the proto-oncogene RAS (22Asada S. Daitoku H. Matsuzaki H. Saito T. Sudo T. Mukai H. Iwashita S. Kako K. Kishi T. Kasuya Y. Fukamizu A. Mitogen-activated protein kinases, Erk and p38, phosphorylate and regulate Foxo1.Cell. Signal. 2007; 19: 519-527Crossref Pu" @default.
• W1966973894 created "2016-06-24" @default.
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• W1966973894 title "The Helicase HAGE Expressed by Malignant Melanoma-Initiating Cells Is Required for Tumor Cell Proliferation in Vivo" @default.
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