Promotes Melanoma Cell Migration Research Articles

Insulin-like growth factor 2 mRNA-binding protein 3 enhanced melanoma migration through regulation of AKT1 and RELA expression.

IMP-3 expression is a poor prognostic factor of melanomas and it promotes melanoma cell migration and invasion by a pathway modulating HMGA2 mRNA expression. We tried to identify other putative targets of IMP-3. We identified putative IMP-3-binding RNAs, including AKT1, MAPK3, RB1 and RELA, by RNA immunoprecipitation coupled with next-generation sequencing. IMP-3 overexpression increased AKT and RELA levels in MeWo cells. siRNAs against AKT1 and RELA inhibited MeWo/Full-length IMP-3 cell migration. IMP-3 knockdown of A2058 cells decreased AKT1 and RELA expression and lowered migration ability. Co-transfection of A2058 cells with AKT1- or RELA-expressing plasmids with IMP-3 siRNA restored the inhibitory effects of IMP-3 knockdown on migration. HMGA2 did not influence AKT1 and RELA expression in melanoma cells. Human melanoma samples with high IMP-3 levels also showed high HMGA2, AKT1 and RELA expression. Our results show that IMP-3 enhances melanoma cell migration through the regulation of the AKT1 and RELA axis.

MiR-509-3p Suppresses Migration, Invasion, and Epithelial- Mesenchymal Transition in Melanoma Cells by Targeting Collagen Triple Helix Repeat Containing 1.

Background: microRNAs (miRNAs) are ubiquitously dysregulated in numerous tumor cell types, including melanoma cells. The anti-tumor effect of miR-509-3p was widely evaluated in various cancers.Aims: To determine the functional role of miR-509-3p in melanoma.Study Design: Cell culture study.Methods: Expression of miR-509-3p in melanoma cell models were assessed by qRT-PCR. Cell migration and invasion were analyzed by wound healing and transwell assays, respectively. Expression levels of biomarkers of epithelial–mesenchymal transition were determined by Western blot. Luciferase vectors containing wildtype or mutant miR-509-3p binding site were constructed, and then dual-luciferase reporter assay.Results: Dysregulated miR-509-3p level was found in melanoma cells. Elevated miR-509-3p expression suppressed melanoma cell migration (P < .001) and invasion (P < .001) capacities. Epithelial–mesenchymal transition of melanoma cells was repressed by miR-509-3p, along with increased α-catenin/E-cadherin (P < .001) and decreased vimentin/fibronectin (P < .001). CTHRC1 (collagen triple helix repeat containing 1) contained a potential binding site for miR-509-3p, and miR-509-3p decreased protein expression of CTHRC1 in melanoma cells (P < .001). CTHRC1 promoted melanoma cell migration and invasion (P < .001), as well as contributed to epithelial–mesenchymal transition. Increased CTHRC1 expression attenuated miR-509-3p-induced inhibition of melanoma cell migration (P < .001), invasion, and epithelial–mesenchymal transition.Conclusion: miR-509-3p suppressed the biological function of melanoma cells through negatively regulating CTHRC1, shedding light on miR-509-3p as a potential candidate for melanoma therapeutics and treatments.

The α9 Nicotinic Acetylcholine Receptor Mediates Nicotine-Induced PD-L1 Expression and Regulates Melanoma Cell Proliferation and Migration.

Cigarette smoking is associated with an increased risk of melanoma metastasis. Smokers show higher PD-L1 expression and better responses to PD-1/PD-L1 inhibitors than nonsmokers. Here, we investigate whether nicotine, a primary constituent of tobacco, induces PD-L1 expression and promotes melanoma cell proliferation and migration, which is mediated by the α9 nicotinic acetylcholine receptor (α9-nAChR). α9-nAChR overexpression in melanoma using melanoma cell lines, human melanoma tissues, and assessment of publicly available databases. α9-nAChR expression was significantly correlated with PD-L1 expression, clinical stage, lymph node status, and overall survival (OS). Overexpressing or knocking down α9-nAChR in melanoma cells up- or downregulated PD-L1 expression, respectively, and affected melanoma cell proliferation and migration. Nicotine-induced α9-nAChR activity promoted melanoma cell proliferation through stimulation of the α9-nAChR-mediated AKT and ERK signaling pathways. In addition, nicotine-induced α9-nAchR activity promoted melanoma cell migration via activation of epithelial-mesenchymal transition (EMT). Moreover, PD-L1 expression was upregulated in melanoma cells after nicotine treatment via the transcription factor STAT3 binding to the PD-L1 promoter. These results highlight that nicotine-induced α9-nAChR activity promotes melanoma cell proliferation, migration, and PD-L1 upregulation. This study may reveal important insights into the mechanisms underlying nicotine-induced melanoma growth and metastasis through α9-nAChR-mediated carcinogenic signals and PD-L1 expression.

PO-161 Cancer cell migratory phenotype characterisation on IGDQ-expressing surface

IntroductionUltraviolet radiation (UV) is the major risk factor for malignant melanoma. We have made the novel finding that melanocytes damaged by UVA, but not UVB, show plasma membrane damage that is readily repaired by lysosomal exocytosis. The exocytosis process is immediately followed by shedding of extracellular vesicles (EVs) from the cells. The generated EVs contain lysosomal marker proteins and microRNA that may affect neighbouring cells. The aim of our study is to investigate how isolated UVA-generated EVs affect cell proliferation and invasion of malignant melanoma cells.Material and methodsMelanoma cell cultures were exposed to UV irradiation and EVs were purified from conditioned media. The EVs were added to new cell cultures and gene expression analysis was performed to identify central signalling pathways. The candidate genes and miRNAs in UV-induced intercellular communication and in melanoma progression are verified and function tested in cell culture models of normal skin cells and malignant melanoma.Results and discussionsData analysis show up-regulation of 127 genes (FC >1.5) and in depth bioinformatic analysis identify TGF-β/SMAD signalling and associated microRNAs (mir21, mir24-2, mir200c) as candidate signalling molecules. In accordance, transfection with mir21 mimic induce proliferation in melanocytes, as well as in melanoma cells. Isolated EVs contained TGF-β, which promoted melanoma cell migration and invasion. Interestingly, melanoma cells spontaneously release EVs and mir21 is upregulated in melanoma progression.ConclusionUV-irradiation cause generation of extracellular vesicles with prophecy to stimulate cell proliferation and dissemination. UV-irradiation is an important component not only for the initiation of malignant melanoma but also for its progression.

PO-158 Extracellular vesicles, generated from UVA-exposed melanoma cells, promotes cell proliferation and invasion

IntroductionUltraviolet radiation (UV) is the major risk factor for malignant melanoma. We have made the novel finding that melanocytes damaged by UVA, but not UVB, show plasma membrane damage that is readily repaired by lysosomal exocytosis. The exocytosis process is immediately followed by shedding of extracellular vesicles (EVs) from the cells. The generated EVs contain lysosomal marker proteins and microRNA that may affect neighbouring cells. The aim of our study is to investigate how isolated UVA-generated EVs affect cell proliferation and invasion of malignant melanoma cells.Material and methodsMelanoma cell cultures were exposed to UV irradiation and EVs were purified from conditioned media. The EVs were added to new cell cultures and gene expression analysis was performed to identify central signalling pathways. The candidate genes and miRNAs in UV-induced intercellular communication and in melanoma progression are verified and function tested in cell culture models of normal skin cells and malignant melanoma.Results and discussionsData analysis show up-regulation of 127 genes (FC >1.5) and in depth bioinformatic analysis identify TGF-β/SMAD signalling and associated microRNAs (mir21, mir24-2, mir200c) as candidate signalling molecules. In accordance, transfection with mir21 mimic induce proliferation in melanocytes, as well as in melanoma cells. Isolated EVs contained TGF-β, which promoted melanoma cell migration and invasion. Interestingly, melanoma cells spontaneously release EVs and mir21 is upregulated in melanoma progression.ConclusionUV-irradiation cause generation of extracellular vesicles with prophecy to stimulate cell proliferation and dissemination. UV-irradiation is an important component not only for the initiation of malignant melanoma but also for its progression.

GPR56/ADGRG1 activation induces IL‐6 production in melanoma cell via the Gα12/13‐ RhoA‐ROCK pathway

GPR56/ADGRG1, a member of the adhesion G protein‐coupled receptor family, is important for tumor progression. The role of GPR56 in melanoma development is unresolved. Here, we showed that GPR56 receptor ligation by immobilized CG4 mAb in melanoma cell enhanced the production of IL‐6, which promoted melanoma cell migration and invasion. siRNA‐mediated Gα12/13 knockdown significantly reduced CG4‐induced IL‐6 production in melanoma cell. Likewise, the IL‐6 level was decreased in melanoma cells expressing the dominant negative RhoA N19, treated with exoenzyme C3, or the ROCK inhibitors (Y27632 and H1152). We conclude that GPR56 activation‐induced IL‐6 production is mediated through the Gα12/13‐RhoA‐ROCK signaling pathway. IL‐6 in turn promotes melanoma cell migration and invasion. Our results indicate a critical role of GPR56 in melanoma progression.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Uncovering and deciphering the pro-invasive role of HACE1 in melanoma cells.

HACE1 is an E3 ubiquitin ligase described as a tumour suppressor because HACE1-knockout mice develop multi-organ, late-onset cancers and because HACE1 expression is lost in several neoplasms, such as Wilms' tumours and colorectal cancer. However, a search of public databases indicated that HACE1 expression is maintained in melanomas. We demonstrated that HACE1 promoted melanoma cell migration and adhesion in vitro and was required for mouse lung colonisation by melanoma cells in vivo. Transcriptomic analysis of HACE1-depleted melanoma cells revealed an inhibition of ITGAV and ITGB1 as well changes in other genes involved in cell migration. We revealed that HACE1 promoted the K27 ubiquitination of fibronectin and regulated its secretion. Secreted fibronectin regulated ITGAV and ITGB1 expression, as well as melanoma cell adhesion and migration. Our findings disclose a novel molecular cascade involved in the regulation of fibronectin secretion, integrin expression and melanoma cell adhesion. By controlling this cascade, HACE1 displays pro-tumoural properties and is an important regulator of melanoma cell invasive properties.

Disease-associated extracellular loop mutations in the adhesion G protein-coupled receptor G1 (ADGRG1; GPR56) differentially regulate downstream signaling

Mutations to the adhesion G protein-coupled receptor ADGRG1 (G1; also known as GPR56) underlie the neurological disorder bilateral frontoparietal polymicrogyria. Disease-associated mutations in G1 studied to date are believed to induce complete loss of receptor function through disruption of either receptor trafficking or signaling activity. Given that N-terminal truncation of G1 and other adhesion G protein-coupled receptors has been shown to significantly increase the receptors' constitutive signaling, we examined two different bilateral frontoparietal polymicrogyria-inducing extracellular loop mutations (R565W and L640R) in the context of both full-length and N-terminally truncated (ΔNT) G1. Interestingly, we found that these mutations reduced surface expression of full-length G1 but not G1-ΔNT in HEK-293 cells. Moreover, the mutations ablated receptor-mediated activation of serum response factor luciferase, a classic measure of Gα12/13-mediated signaling, but had no effect on G1-mediated signaling to nuclear factor of activated T cells (NFAT) luciferase. Given these differential signaling results, we sought to further elucidate the pathway by which G1 can activate NFAT luciferase. We found no evidence that ΔNT activation of NFAT is dependent on Gαq/11-mediated or β-arrestin-mediated signaling but rather involves liberation of Gβγ subunits and activation of calcium channels. These findings reveal that disease-associated mutations to the extracellular loops of G1 differentially alter receptor trafficking, depending on the presence of the N terminus, and differentially alter signaling to distinct downstream pathways.

SOX4 promotes melanoma cell migration and invasion though the activation of the NF-κB signaling pathway.

SOX4 has been reported to be abnormally expressed in many types of cancer, including melanoma. However, its role in cell proliferation and metastasis remains controversial. In this study, SOX4 was downregulated or overexpressed in A375, A2058 and A875 melanoma cells by siRNA or lentivirus transfection, respectively. Cell metastasis was observed by Transwell assay. In an aim to elucidate the underlying mechanisms, we determined the expression of nuclear factor-κB (NF-κB) by real-time PCR assay and western blot analysis. Our data indicated that SOX4 knockdown inhibited melanoma cell migration and invasion. In the melanoma cells in which SOX4 was downregulated, the expression levels of NF-κB/p65, matrix metalloproteinase (MMP)2 and MMP9 were suppressed at both the mRNA and protein levels. Conversely, the overexpres-sion of SOX4 promoted melanoma cell migration and invasion. In the melanoma cells in which SOX4 was overexpressed, the expression levels of NF-κB/p65, MMP2 and MMP9 were increased at both the mRNA and protein level. On the whole, our findings indicate that SOX4 promotes melanoma cell migration and invasion through the activation of the NF-κB/p65 signaling pathway. Thus, SOX4 may prove to be a potential therapeutic target for the treatment of melanoma.

FAK regulates E-cadherin expression via p-SrcY416/p-ERK1/2/p-Stat3Y705 and PPARγ/miR-125b/Stat3 signaling pathway in B16F10 melanoma cells.

Focal adhesion kinase (FAK) is involved in tumor cell migration and metastasis. However, the underlying mechanism remains unclear. Here, we present a signaling pathway involved in the regulation of melanoma cell migration by FAK. We found that the interference of FAK expression suppressed B16F10 cell migration/metastasis, and altered the expressions of genes involved in melanoma migration/metastasis. The down-regulation of FAK inhibited the expression of p-SrcY416, p-ERK1/2, Stat3 and p-Stat3Y705, while promoted the expression of PPARγ, miR-125b and E-cadherin. Then we found that FAK inhibited E-cadherin expression via p-SrcY416/p-ERK1/2/ p-Stat3Y705 and PPARγ/miR-125b/Stat3 signaling pathway in B16F10 cell. Moreover, miR-125b inhibited B16F10 cell migration. Furthermore, we repeated the key data with human melanoma cell line A375. The results obtained from A375 cells fell in line with those from B16F10 cells. Using Oncomine database, we found that the mRNA levels of FAK, Src, ERK1/2 and Stat3 increased, while the mRNA levels of PPARγ, C21orf34 (miR-125b host gene) and E-cadherin decreased in human metastatic melanoma. The data from human breast cancer confirmed those from metastatic melanoma.Taken together, our study suggests that down-regulation of FAK promotes E-cadherin expression via p-SrcY416/p-ERK1/2/p-Stat3Y705 and PPARγ/miR-125b/Stat3 signaling pathway. Our findings provide a novel explanation regarding how FAK promotes melanoma cell migration, suggesting that FAK might be a potential target for melanoma therapy.

MiRNA-7-5p inhibits melanoma cell migration and invasion

Aberrant expression of microRNAs (miRNAs), a class of small non-coding regulatory RNAs, has been implicated in the development and progression of melanoma. However, the precise mechanistic role of many of these miRNAs remains unclear. We have investigated the functional role of miR-7-5p in melanoma, and demonstrate that miR-7-5p expression is reduced in metastatic melanoma-derived cell lines compared with primary melanoma cells, and that when ectopically expressed miR-7-5p significantly inhibits melanoma cell migration and invasion. Additionally, we report that insulin receptor substrate-2 (IRS-2) is a target of miR-7-5p in melanoma cells, and using RNA interference (RNAi) we provide evidence that IRS-2 activates protein kinase B (Akt), and promotes melanoma cell migration. Thus, miR-7-5p may represent a novel tumor suppressor miRNA in melanoma, acting at least in part via its inhibition of IRS-2 expression and oncogenic Akt signaling.

Abstract 1515: The role of the Notch/WISP-1 axis in determining the regulatory effect of mesenchymal stem cells on melanoma cell motility

Abstract Purpose. Bone marrow (BM)-derived mesenchymal stem cells (MSC) are known to be recruited to the tumor tissues where they integrate into the reactive tumor stroma and play a critical role in regulating tumor cell behavior. However, the molecular mechanisms underlying the influential role of MSC for melanoma cell behavior remain poorly understood. We sought to identify the “molecular switch” controlling the regulatory effect of MSC on melanoma cell growth and migration. Methods. MSC from BM of Notch1Flox/Flox mice were cultured in MesenCult® media. Deletion of the Notch1 gene was achieved by transduction of MSC with Cre/Lentivirus. The role of Notch1-/– vs. Notch1+/+-MSC in modulating human melanoma cell behavior in vitro was examined by testing the effect of conditioned medium (CM) of MSC on melanoma cell growth and migration using MTT and Transwell® cell migration assays, respectively, and by counting the number of DsRed-melanoma cells in melanoma-MSC co-culture assay using flow cytometry. The downstream targets of the Notch1 signaling in MSC were screened by genome-wide gene microarray analysis. Expression of selected target gene was confirmed by immunoblotting assay. The biological function of the selected target in mediating the regulatory effect of the Notch1-/–MSC on melanoma cell growth and migration was examined through the reconstitution of target gene expression in Notch1-/–MSC and evaluated by testing the direct effect of supplemented recombinant protein encoding the target gene on melanoma cells. The mechanism underlying the regulatory effect of the Notch1 target on melanoma cell mobility was analyzed by PCR-Array specifically focused on exploring cell migration. Results. Notch1-/–MSC, either by CM or in co-culture, had little effect on melanoma cell proliferation, while the CM significantly promoted melanoma cell migration. Global gene expression profiling of Notch1-/– vs. Notch1+/+-MSC by microarray identified Wnt-induced secreted protein-1 (WISP-1) as a Notch1 downstream target gene. The Notch1 signaling-commanded regulatory effect of MSC on melanoma cell migration is partially mediated by modulating WISP-1 expression, since the CM of Notch1-/–MSC promoted melanoma cell motility, while the enhanced motility of melanoma cell was reversed by reconstituted overexpression of WISP-1 in Notch1-/–MSC. Consistently, supplement of γhWISP-1 inhibited melanoma cell migration, presumably via modulating Cdc42/PAK cascade. Conclusions. Our data reveal Notch1 signaling as a molecular switch controlling the regulatory role of MSC in modulating melanoma cell motility, and demonstrate that melanoma cell behavior can be determined by external signals emanating from the MSC. WISP-1 is a molecular relay in the transmission of Notch1 signaling, and may serve as a molecular target for melanoma therapeutic intervention. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1515. doi:1538-7445.AM2012-1515

Recognition of CD146 as an ERM-binding protein offers novel mechanisms for melanoma cell migration

Tumor cell migration is a well-orchestrated multistep process that drives cancer development and metastasis. Previous data indicated that CD146 expression correlates with malignant progression and metastatic potential of human melanoma cells. However, the exact molecular mechanism of how CD146 promotes melanoma cell migration still remains poorly understood. Here, we report that CD146 physically interacts with actin-linking ezrin-radixin-moesin (ERM) proteins and recruits ERM proteins to cell protrusions, promoting the formation and elongation of microvilli. Moreover, CD146-promoted melanoma cell migration is linked to RhoA activation and ERM phosphorylation. CD146 recruits Rho guanine nucleotide dissociation inhibitory factors 1 (RhoGDI1) through ERM proteins and thus sequesters RhoGDI1 from RhoA, which leads to upregulated RhoA activity and increased melanoma cell motility. CD146-activated RhoA also promotes further ERM phosphorylation and activation through Rho-phosphatidylinositol-4-phosphate-5-kinase-phosphatidylinositol 4,5-biphosphate pathway, which reinforces CD146/ERM association. Thus, our results provide a mechanistic basis to understand the role of CD146 in regulating human melanoma cell motility.

Phosphoglucose Isomerase/Autocrine Motility Factor Promotes Melanoma Cell Migration through ERK Activation Dependent on Autocrine Production of Interleukin-8

It is well known that phosphoglucose isomerase/autocrine motility factor (AMF) promotes cell migration in an autocrine manner in various tumor cells. However, it remains unclear whether certain cytokines modulate the effects of AMF on tumor cell migration. Because interleukin (IL)-8, a proinflammatory cytokine, is produced by melanoma cells and has been correlated with melanoma migration, the migratory ability of melanoma cells induced by AMF may also involve induction of IL-8 expression. In the present study, we assessed whether AMF promotes melanoma cell migration through autocrine production of IL-8. We found that AMF stimulation increased IL-8 production through up-regulation of IL-8 mRNA transcription, especially in biologically early stage melanoma cells. AMF-induced migration of these cells was inhibited by a specific neutralizing antibody against IL-8. The IL-8 production induced by AMF was mediated by the ERK1/2 pathways. These findings suggest that melanoma migration induced by AMF is mediated by autocrine production of IL-8 as a novel downstream modulator of the AMF signaling pathway.

Cell surface heparan sulfate released by heparanase promotes melanoma cell migration and angiogenesis

Heparan sulfate (HS) proteoglycans are essential components of the cell-surface and extracellular matrix (ECM) which provide structural integrity and act as storage depots for growth factors and chemokines, through their HS side chains. Heparanase (HPSE) is the only mammalian endoglycosidase known that cleaves HS, thus contributing to matrix degradation and cell invasion. The enzyme acts as an endo-beta-D-glucuronidase resulting in HS fragments of discrete molecular weight size. Cell-surface HS is known to inhibit or stimulate tumorigenesis depending upon size and composition. We hypothesized that HPSE contributes to melanoma metastasis by generating bioactive HS from the cell-surface to facilitate biological activities of tumor cells as well as tumor microenvironment. We removed cell-surface HS from melanoma (B16B15b) by HPSE treatment and resulting fragments were isolated. Purified cell-surface HS stimulated in vitro B16B15b cell migration but not proliferation, and importantly, enhanced in vivo angiogenesis. Furthermore, melanoma cell-surface HS did not affect in vitro endothelioma cell (b.End3) migration. Our results provide direct evidence that, in addition to remodeling ECM and releasing growth factors and chemokines, HPSE contributes to aggressive phenotype of melanoma by releasing bioactive cell-surface HS fragments which can stimulate melanoma cell migration in vitro and angiogenesis in vivo.

SK3 channel promotes melanoma cell migration

SK3 channel promotes melanoma cell migration