Metastatic Potential Of Melanoma Cells Research Articles

DLL4/Notch3/WNT5B axis mediates bidirectional prometastatic crosstalk between melanoma and lymphatic endothelial cells.

Despite strong indications that interactions between melanoma and lymphatic vessels actively promote melanoma progression, the molecular mechanisms are not yet completely understood. To characterize molecular factors of this crosstalk, we established human primary lymphatic endothelial cell (LEC) cocultures with human melanoma cell lines. Here, we show that coculture with melanoma cells induced transcriptomic changes in LECs and led to multiple changes in their function. WNT5B, a paracrine signaling molecule upregulated in melanoma cells upon LEC interaction, was found to contribute to the functional changes in LECs. Moreover, WNT5B transcription was regulated by Notch3 in melanoma cells following the coculture with LECs, and Notch3 and WNT5B were coexpressed in melanoma patient primary tumor and metastasis samples. Moreover, melanoma cells derived from LEC coculture escaped efficiently from the primary site to the proximal tumor-draining lymph nodes, which was impaired upon WNT5B depletion. This supported the role of WNT5B in promoting the metastatic potential of melanoma cells through its effects on LECs. Finally, DLL4, a Notch ligand expressed in LECs, was identified as an upstream inducer of the Notch3/WNT5B axis in melanoma. This study elucidated WNT5B as a key molecular factor mediating bidirectional crosstalk between melanoma cells and lymphatic endothelium and promoting melanoma metastasis.

Carbon Ions Suppress Angiogenesis and Lung Metastases in Melanoma by Targeting CXCL10.

Carbon-ion radiotherapy (CIRT) enhanced local control in patients with malignant melanoma. In several in vitro studies, carbon ions (C ions) have been also shown to decrease the metastatic potential of melanoma cells. CXC motif 10 (CXCL10) has been shown to play a crucial role in regulating tumor metastasis and it significantly increase in human embryonic kidney cells after heavy ion irradiations. This study sought to explore the regulatory effect of C ions on melanoma metastasis, emphasizing the role of CXCL10 in this process. To explore the potential regulatory effect of C ions on tumor metastasis in vivo, we developed a lung metastasis mouse model by injecting B16F10 cells into the footpad and subjected all mice to treatment with X rays and C ions. Subsequently, a series of assays, including histopathological analysis, enzyme-linked immunosorbent assay, real-time PCR, and western blotting, were conducted to assess the regulatory effects of C ions on melanoma. Our results showed that mice treated with C ions exhibited significantly less tumor vascularity, enhanced tumor necrosis, alleviated lung metastasis, and experienced longer survival than X-ray irradiated mice. Moreover, VEGF expression in B16F10 cells was significantly reduced by C-ion treatment, which could be alleviated by CXCL10 knockdown in vitro. Further investigations revealed that co-culturing with HUVECs resulted in a significant inhibition of proliferation, migration, and tube formation ability in the C-ion treated group, while the opposite effect was observed in the C-ion treated with si-CXCL10 group. In conclusion, our findings demonstrate that treatment with carbon-ion radiation can suppress angiogenesis and lung metastases in melanoma by specifically targeting CXCL10. These results suggest the potential utility of carbon ions in treating melanoma.

Epithelial–mesenchymal transition inhibition by metformin reduces melanoma lung metastasis in a murine model

Melanoma is an aggressive cancer with fast metastatic spread and reduced survival time. One common event during the neoplastic progression is the epithelial–mesenchymal transition (EMT), which enhances invasiveness, cell migration, and metastasis. In this study, we investigated the effects of metformin at EMT in melanoma cell lines B16-F10 and A-375, in vitro, and the impact of EMT downregulation on melanoma progression in vivo. The metformin cells treatment reduces the migration potential in vitro and reduced the development of pulmonary metastases and the expressions of N-cadherin, vimentin, ZEB1, and ZEB2 at the metastases site, in vivo. These results indicate that metformin can promote EMT downregulation impairing the metastatic potential of melanoma cells.

TRPM2 Oxidation Activates Two Distinct Potassium Channels in Melanoma Cells through Intracellular Calcium Increase.

Tumor microenvironments are often characterized by an increase in oxidative stress levels. We studied the response to oxidative stimulation in human primary (IGR39) or metastatic (IGR37) cell lines obtained from the same patient, performing patch-clamp recordings, intracellular calcium ([Ca2+]i) imaging, and RT-qPCR gene expression analysis. In IGR39 cells, chloramine-T (Chl-T) activated large K+ currents (KROS) that were partially sensitive to tetraethylammonium (TEA). A large fraction of KROS was inhibited by paxilline—a specific inhibitor of large-conductance Ca2+-activated BK channels. The TEA-insensitive component was inhibited by senicapoc—a specific inhibitor of the Ca2+-activated KCa3.1 channel. Both BK and KCa3.1 activation were mediated by an increase in [Ca2+]i induced by Chl-T. Both KROS and [Ca2+]i increase were inhibited by ACA and clotrimazole—two different inhibitors of the calcium-permeable TRPM2 channel. Surprisingly, IGR37 cells did not exhibit current increase upon the application of Chl-T. Expression analysis confirmed that the genes encoding BK, KCa3.1, and TRPM2 are much more expressed in IGR39 than in IGR37. The potassium currents and [Ca2+]i increase observed in response to the oxidizing agent strongly suggest that these three molecular entities play a major role in the progression of melanoma. Pharmacological targeting of either of these ion channels could be a new strategy to reduce the metastatic potential of melanoma cells, and could complement classical radio- or chemotherapeutic treatments.

Oxidative stress in melanogenesis and melanoma development

Melanoma is the most aggressive skin cancer, with a growing number of incidents worldwide and with no effective cure in a metastatic stage so far. There are several pathways and processes engaged in melanoma pathogenesis that have been extensively explored in recent years. The emerging evidence suggests that oxidative stress (OS) is highly involved in melanin synthesis and melanoma formation. Melanoma is particularly susceptible to OS due to the involvement of melanin synthesis and UV radiation in the generation of reactive oxygen species. Oxidative stress influences melanoma immunity, the metastatic potential of melanoma cells and their resistance to therapy. In malignant melanocytes, the process of melanogenesis is frequently upregulated, suggesting possible therapeutic targets. This review describes the role of OS in melanin synthesis in melanocytes and explains how it affects melanoma cells. Better knowledge about the mechanisms involved in cancer progression may result in the development of better treatment strategies.

YAP activation in melanoma contributes to anoikis resistance and metastasis.

Melanoma is inherently heterogeneous, providing resistance to apoptosis. Anoikis resistance is a hallmark feature of metastatic melanoma to escape apoptosis when cells lose contact with adjacent cells or extracellular matrix. The yes-associated protein transcription co-activator is the effector of Hippo pathway. Herein, we investigated the function of yes-associated protein in anoikis resistance of melanoma cells. When melanoma cells were grown under anchorage-independent condition, anoikis-resistant cells displayed higher levels of yes-associated protein activation than the cells that were attached to the basement membrane, as evidenced by downregulated phosphorylated yes-associated protein at Ser127 and higher expression of downstream genes BCL2 and MCL-1. Yes-associated protein overexpression directly enhanced the anoikis resistance and metastatic potential of melanoma cells. Conversely, yes-associated protein inhibitor CA3 exhibited Dose-dependent induction of anoikis in resistant melanoma cells and exerted great inhibition on cell migration. Knockdown of yes-associated protein expression by shRNA also rendered melanoma cells susceptible to anoikis and interrupted cell invasiveness. Yes-associated protein inhibition in anoikis-resistant cells also reduced the number of metastatic nodules in the lung sections of SCID mice. Clinically, higher yes-associated protein level in the lung metastasis tissues correlated with higher BCL2 and MCL1 expressions compared with the non-metastasis tissues. Overall, our finding suggests that the aberrant activation of yes-associated protein exerts important role on anoikis resistance and metastatic capability of melanoma cells.

TRIM59 loss in M2 macrophages promotes melanoma migration and invasion by upregulating MMP-9 and Madcam1.

The culture supernatant from macrophages overexpressing TRIM59 has a cytotoxic effect on melanoma, but the mechanism remains unclear. To investigate whether deletion of TRIM59 in macrophages affects the metastatic potential of melanoma cells, we polarized control and TRIM59-deficient bone marrow-derived macrophages to the M2 phenotype and collected the respective conditioned media (CM). Exposure to CM from TRIM59-/--M2 cultures significantly promoted migration and invasion by B16-F0 and B16-F10 cells. Cytokine profiling indicated a ~15-fold increase in TNF-α production in CM from TRIM59-/--M2 cultures, and neutralizing TNF-α activity abrogated the referred stimulatory effects on cell motility. Transcriptome analysis revealed significant upregulation of MMP-9 and Madcam1 in melanoma cells exposed to TRIM59-/--M2 CM. Inhibitory experiments determined that these changes were also TNF-α-dependent and mediated by activation of ERK signaling. Independent knockdown of MMP9 and Madcam1 in B16-F10 cells impeded epithelial-mesenchymal transition and inhibited subcutaneous tumor growth and formation of metastatic lung nodules in vivo. These data suggest TRIM59 expression attenuates the tumor-promoting effect of tumor-associated macrophages, most of which resemble the M2 phenotype. Moreover, they highlight the relevance of TRIM59 in macrophages as a potential regulator of tumor metastasis and suggest TRIM59 could serve as a novel target for cancer immunotherapy.

The effect of fenofibrate, a PPARα activator on toll-like receptor-4 signal transduction in melanoma both in vitro and in vivo.

The anti-cancer effect of peroxisome proliferator-activated receptor (PPAR) α ligands on growth and metastatic potential of melanoma cells has been shown previously. However, themechanismunderlying these effects remains to be elucidated. Here, we investigated the effects of fenofibrate (PPAR ligand) on Toll-like receptor-4 (TLR-4) signaling in mice melanoma. Mice melanoma cells (B16F10) were treated with fenofibrate or LPS or LPS + fenofibrate or pre-treated with CLI-095 (a TLR4 inhibitor), followed by fenofibrate. In in vivo model, C57BL/6 mice were subcutaneously injected with B16F10 cells (with/without LPS pre-treatment), and fenofibrate was administrated after development of palpable tumors. Cell proliferation, the expression level of Tlr4, Myd88, Nf-κb1 genes, TLR-4 protein expression, TNF-α levels, and tumor volume were measured. Our results indicated that fenofibrate significantly inhibited the Tlr-4, Myd-88, and Nf-kb1 mRNA expression and TNF-α concentration in B16F10 LPS-stimulated cells. In addition, blocking TLR-4 signaling increased the anti-inflammatory potential of fenofibrate. Also fenofibrate can reduce LPS-induced tumor volume, Tlr-4, Myd-88, Nf-kb1 mRNA, and TLR-4 protein expression in tumor tissue and also TNF-α level in tumor tissue lysate. Our data indicate that fenofibrate may exert its anti-melanoma effects via interaction with TLR4-dependent signaling pathway (TLR-4/MyD-88/ NF-kB).

YB-1 Expression and Phosphorylation Regulate Tumorigenicity and Invasiveness in Melanoma by Influencing EMT.

Cutaneous melanoma represents one of the most aggressive human tumor entities possessing a high tendency to metastasize. Cancer cells frequently exploit a highly conserved developmental program, the epithelial-to-mesenchymal transition (EMT), to gain migratory and invasive properties promoting their metastatic spread. Cytoplasmic localization of the oncogenic transcription and translation factor Y-box binding protein 1 (YB-1) is a powerful inducer of EMT in breast carcinoma cells. Interestingly, EMT-like processes have also been observed in cutaneous melanoma despite its neural crest origin. Here, increased expression of YB-1 negatively affects patient survival in malignant melanoma and promotes melanoma cell tumorigenicity both in vitro and in vivo Intriguingly, this effect seems to be mainly mediated by cytoplasmic YB-1 that does not exhibit phosphorylation at serine-102 (S102). Moreover, S102 unphosphorylated YB-1 enhances the migratory and invasive potential of human melanoma cells in two-dimensional (2D) and three-dimensional (3D) culture systems and facilitates acquisition of a mesenchymal-like invasive phenotype in the chick embryo model. Collectively, these data demonstrate that the cytoplasmic activity of YB-1 stimulates tumorigenicity and metastatic potential of melanoma cells by promoting EMT-like properties.Implications: This study reveals for the first time that YB-1 efficiently drives tumorigenicity and invasiveness of melanoma cells in its S102 unphosphorylated cytoplasmic state and that YB-1 expression represents a negative prognostic factor in primary melanoma patients. Mol Cancer Res; 16(7); 1149-60. ©2018 AACR.

Discovery of potential biomarkers in human melanoma cells with different metastatic potential by metabolic and lipidomic profiling

Malignant melanoma, characterized by its ability to metastasize to other organs, is responsible for 90% of skin cancer mortality. To investigate alterations in the cellular metabolome and lipidome related to melanoma metastasis, gas chromatography-mass spectrometry (GC-MS) and direct infusion-mass spectrometry (DI-MS)-based metabolic and lipidomic profiling were performed on extracts of normal human melanocyte (HEMn-LP), low metastatic melanoma (A375, G361), and highly metastatic melanoma (A2058, SK-MEL-28) cell lines. In this study, metabolomic analysis identified aminomalonic acid as a novel potential biomarker to discriminate between different stages of melanoma metastasis. Uptake and release of major metabolites as hallmarks of cancer were also measured between high and low metastatic melanoma cells. Lipid analysis showed a progressive increase in phosphatidylinositol (PI) species with saturated and monounsaturated fatty acyl chains, including 16:0/18:0, 16:0/18:1, 18:0/18:0, and 18:0/18:1, with increasing metastatic potential of melanoma cells, defining these lipids as possible biomarkers. In addition, a partial-least-squares projection to latent structure regression (PLSR) model for the prediction of metastatic properties of melanoma was established, and central metabolic and lipidomic pathways involved in the increased motility and metastatic potential of melanoma cells were identified as therapeutic targets. These results could be used to diagnose and control of melanoma metastasis.

Abstract 4845: Induction of aldolase C expression by NME1 may mediate metastasis suppressor activity in malignant melanoma

Abstract Although the metastasis suppressor activity of NME1 has been demonstrated in a number of settings of human cancer, the underlying molecular mechanisms are not well understood. NME1 has two known enzymatic activities, nucleoside diphosphate kinase (NDPK) and a 3’ to 5’ exonuclease. To determine the relative contribution either of these activities has on the metastasis suppressor function of NME1, our lab previously generated site directed mutants to abrogate one or both of the enzymatic activities (Ma et al., 2004). These studies showed that disrupting the 3’ to 5’ exonuclease activity with the point substitutions glutamate5-to-alanine (E5A) and lysine12-to-glutamine (K12Q) also significantly compromised the metastasis suppressor function of NME1 (Zhang et al., 2011). To identify genes that are regulated by NME1 and potentially modulate its metastasis suppressor activity, we compared the gene expression profile induced by forced expression of wild-type NME to those obtained with the E5A and K12Q mutants. Using the human Exon 1.0 Affymetrix microarray , we identified 13 genes whose expression was modulated by wild-type NME1 expression, but not by the suppressor-deficient mutants, which were designated as metastasis suppressor signature (MSS) genes. We are currently refining the list to genes that have the highest clinical relevance through observing expression profiles of the MSS genes in clinical datasets. Preliminary results of unsupervised hierarchal clustering of GEO melanoma dataset GSE8401 showed that expression of the MSS gene, aldolase C (ALDOC), was significantly lower in a subset of metastatic melanoma lesions compared to primary melanomas. Western blot and qPCR analysis confirm that NME1 induces expression of ALDOC in both WM793 and M14 melanoma cell lines, and that induction of ALDOC is dose-responsive to NME1 expression as observed in the WM1158 metastatic melanoma cell line. Further, NME1 induction of ALDOC is specific compared to the ALDOA and ALDOB isoforms. Recent studies have indicated aldolase isoforms can modulate motile and invasive phenotypes of cancer cells (Li et al., 2015 & Hu et al., 2016), suggesting regulation of ALDOC expression may represent a key mechanism underlying the metastasis suppressor function of NME1. Current work is focused on measuring the impact of NME1-induced expression of ALDOC on the metastatic potential of melanoma cells in culture and in vivo. Citation Format: Nidhi Pamidimukkala, Katie Leonard, Joseph McCorkle, Qingbei Zhang, Anup Mahurkar, Amol Shetty, David Kaetzel. Induction of aldolase C expression by NME1 may mediate metastasis suppressor activity in malignant melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4845. doi:10.1158/1538-7445.AM2017-4845

Modifying microRNAs--another piece of the melanoma puzzle.

Modifying microRNAs--another piece of the melanoma puzzle.

Immunohistochemical study of the expression of N-cadherin in cutaneous melanoma and in dysplastic melanocytic nevi

It has been suggested that the invasive and metastatic potential of melanoma cells reflects their ability to undergo epithelial-mesenchymal transition (EMT)-like phenotypic changes (1). Important hallmarks of EMT include the loss of E-cadherin expression and increased expression of the cell adhesion molecule N-cadherin. This cadherin switch leads melanoma cells to lose contact with keratinocytes in the epi- dermis and interact instead with stromal fibroblasts and endothelial cells, thus promoting dermal and vascular melanoma invasion (2). In melanoma, up-regulation of N-cadherin can be induced by the overexpression of the transmembrane receptor Notch1, thus providing a mechanism that underlines increased melanoma cell adhesion, survival, growth, and tumor progression when Notch signaling is activated (3). In this study, the expression of N-cadherin and Notch1 was evaluated by immuno- histochemical analysis in primary cutaneous melanomas and lymph node metastases. First, we evaluated the prognostic impact of high N-cadherin expression on sur- vival in melanoma patients. Second, we correlated the expression of N-cadherin with the full clinicopathological data of patients. Third, we investigated the relationship between the expression of N-cadherin and Notch1. Moreover, N-cadherin expression was evaluated in dysplastic melanocytic nevi and in normal skin. The results will be discussed.

Abstract B37: Prenylation inhibition induces cell death in NRAS mutant melanoma cells

Abstract While targeted therapeutic approaches are now available for the treatment of BRAF mutant melanomas, non-BRAF mutant melanoma still lacks this modality. In order to explore the potential of prenylation inhibition we investigated the response of NRAS or BRAF mutant and double wild-type melanoma to zoledronic acid treatment. Effect of zoledronic acid on proliferation, apoptosis and migration of melanoma cells as well as the activation of downstream elements of the RAS/RAF pathway were characterized in vitro. In vivo subcutaneous xenograft and spleen to liver colonization models were used to evaluate the influence of zoledronic acid treatment on primary tumor growth and metastatic potential of melanoma cells. Zoledronic acid significantly decreased proliferation in NRAS mutant cells as compared to BRAF mutant and double wild-type cells, in vitro. Similarly, TUNEL assay revealed apoptosis induction in an oncogenic mutation specific manner. In line with this finding, activation of ribosomal protein S6 was decreased. Unexpected activation of the downstream effector Erk1/2 and increase in migration was found in BRAF mutant cells. Zoledronic acid failed to show significant synergism with cytotoxic cisplatin or DTIC treatment in vitro. In vivo zoledronic acid did not inhibit the subcutaneous growth of melanoma cells irrespective of oncogenic mutations. In contrast, the lower dose of zoledronic acid resulted in significantly higher liver metastasis in double wild-type cells. Altogether our data demonstrates that prenylation inhibition may be a novel therapeutic approach in NRAS mutant melanoma. However, further investigations are needed to identify drugs that have appropriate pharmacological properties to efficiently target melanoma cells. Citation Format: Tamas Garay, Istvan Kenessey, Viktoria Laszlo, Jozsef Tovari, Walter Berger, Laszlo Kovacs, Jozsef Timar, Balazs Hegedus. Prenylation inhibition induces cell death in NRAS mutant melanoma cells. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr B37. doi: 10.1158/1557-3125.RASONC14-B37

Abstract 136: PTEN phosphatase inhibits metastasis through negatively affecting Entpd5/IGF1R pathway

Abstract Migration of cancer cell is the key event in metastasis. Recent data suggest that PTEN phosphatase activity may be required for inhibiting cell migration; however, little in vivo experimental evidence exists directly demonstrating the efficacy of PTEN phosphatase in tumor metastasis. We showed here that PTEN loss was correlated with progression to metastatic state using tissue array analysis and UV-irradiated HGF/SF transgenic-PTEN deficient mouse melanoma model. Analysis of various human melanoma cell lines demonstrated that PTEN expression was related to metastatic behavior. To further dissect the role of PTEN activity in metastasis, we have established a model system containing mutant PTEN with various phosphatase activities. We found that wild type PTEN could inhibit cell motility and invasiveness. Moreover, wild type PTEN was able to reduce metastatic potential of melanoma cells in a dose-dependent manner; in contrast, PTEN phosphatase dead mutant promoted metastatic behavior of melanoma cell. To gain more insight into the regulation of tumor cell migration, invasion and metastasis by PTEN, we performed a microarray analysis of cells with various PTEN phosphatase mutants. We found that a protein N-glycosylation and folding promoter, Entpd5 was negatively regulated by PTEN phosphatase. Down regulation of Entpd5 by shRNA in cells with PTEN phosphatase dead mutant blocked PTEN phosphatase dead mutant mediated tumor metastasis in vivo, promoted cell ER stress and growth inhibition as well as reduction of IGF1R. In contrast, overexpression of Entpd5 in cell with PTEN wild-type form enhanced PTEN wild-type-inhibited tumor metastasis in vivo, inhibited cell ER stress, promoted cell growth and abundance of IGF1R. We provide mechanistic insight toward regulation of metastasis by PTEN phosphatase and demonstrate a link between PTEN and Entpd5/IGF1R activation in metastatic process. Citation Format: Yanlin Yu. PTEN phosphatase inhibits metastasis through negatively affecting Entpd5/IGF1R pathway. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 136. doi:10.1158/1538-7445.AM2014-136

Clinical implications of distinct metastasizing preferences of different melanoma subtypes

The incidence and mortality of malignant melanoma have been rising during the past decades, the latter being due to the high invasion capacity and the metastatic potential of melanoma cells to distant organs. We investigated the distribution pattern of melanoma metastases taking into account different clinicopathological subtypes of melanoma. We studied 310 stage IV (AJCC 2009) melanoma patients retrospectively with regard to potential correlations between frequency and occurrence of metastasis and the genetic background and pathological/clinical melanoma subtypes. For all patients, the time to distant metastasis (TTDM) and the distribution patterns of metastases were analyzed and correlated to the median survival time. Superficially Spreading (SSM) and Nodular melanomas (NMM) spread to the brain more frequently than Acrolentiginous (ALM) and Mucosal (MM) melanomas (p = 0.0012). The preference to affect the skeleton was significantly higher for ALM and MM in comparison to SSM and NMM (p = 0.0049). Lentigo maligna (LMM) tumors showed a significantly lower metastatic spread to distant lymph nodes (p = 0.0159). BRAF mutant versus wildtype tumors showed no significant differences concerning localization of metastasis but patients with BRAF mutant tumors were significantly younger at primary diagnosis and had a significantly shorter stage IV survival (p = 0.0106). This study shows a clear distinction of melanoma subtypes with regard to metastasizing preferences. Further knowledge about melanoma subtype specific characteristics, including molecular markers predictive of homing preferences, may help to understand and manage this heterogeneous disease in terms of prognosis and follow-up procedures.

CD147 Promotes Melanoma Progression Through Hypoxia-Induced MMP2 Activation

Hypoxia enhances MMP2 expression and the invasion and metastatic potential of melanoma cells. CD147 has been shown to induce MMP2 in multiple cancers. To investigate the role of CD147 in hypoxiainduced MMP2 activation, we performed immunohistochemistry (IHC) staining in 206 normal and melanoma tissue samples, and analyzed the correlation between HIF1α and CD147. ChIP (chromosome Immunoprecipitation) in melanoma cell lines supports that HIF1α directly binds to CD147 promoter. Moreover, we made a series of deletion mutants of CD147 promoter, and identified a conserved HIF1α binding site. Point mutation in this site significantly decreased CD147 response to hypoxia. Importantly, knocking down CD147 attenuates MMP2 response to hypoxia in melanoma cell lines. MMP2 could not be efficiently activated by hypoxia in CD147 depletion cells. ELISA data showed that MMP2 secretion was reduced in CD147 depletion cells than control under hypoxia condition. To verify the data from cell culture model, we performed in vivo mouse xenograft experiment. IHC staining showed reduced MMP2 level in CD147 depleted xenografts compared to the control group, with the HIF1α level being comparable. Our study demonstrates a novel pathway mediated by CD147 to promote the MMP2 activation induced by hypoxia, and helps to understand the interplay between hypoxia and melanoma progression.

Suppression of tumorigenicity and metastatic potential of melanoma cells by transduction of interferon gene

Suppression of tumorigenicity and metastatic potential of melanoma cells by transduction of interferon gene

Flipping the phenotypic switch on a novel antimelanoma differentiation strategy

Flipping the phenotypic switch on a novel antimelanoma differentiation strategy

Enhancement role of host 12/15-lipoxygenase in melanoma progression

12/15-Lipoxygenase (12/15-LOX) is a non-haeme iron-containing dioxygenase that forms 12(S)-hydroxyeicosatetraenoic acid (12(S)-HETE) or 15(S)-HETE. Several biological mediators including cytokines, growth factors and lipid metabolites released during tumour cell-endothelial cell adhesion are associated with malignant tumour progression. Here we found that HETEs released from the host organ played a critical role in tumour metastasis. Intravenous injection of B16F10 melanoma cells caused lung nodule formation, which was markedly attenuated in 12/15-LOX null mice. Co-injection of melanoma cells with 12(S)-HETE increased the lung homing activity of B16F10 melanoma cells. In vitro studies showed that 12(S)-HETE and 15(S)-HETE treatment resulted in a concentration-dependent increase of adhesion of B16F10 cells on collagen or fibronectin. The melanoma cell adhesion was then evaluated in pulmonary primary cell culture isolated from wild-type (WT) and 12/15-LOX knockout (KO) mice. It was found that the adhesion of melanoma cells on the epithelial cells isolated from 12/15-LOX null mice was reduced in comparison with those isolated from WT mice. Treatment of 12(S)-HETE increased the pFAK in melanoma cells adhering on collagen-coated slide. The enhancement of adherence elicited by 12(S)-HETE in B16F10 cells could be antagonised by focal adhesion kinase (FAK) inhibitor 14 (FAK inhibitor) or PD98059 (extracellular signal-regulated kinase (ERK) inhibitor). 12(S)-HETE increased the phosphorylation of FAK and ERK in adhering melanoma cells. The FAK phosphorylation induced by 12(S)-HETE was further inhibited by PD98059, indicating that FAK is the downstream target of ERK. The adhesion and lung metastasis of human melanoma cells of C32 in NOD/SCID mice were also potentiated by co-treatment with 12(S)-HETE. These results demonstrate that 12(S)-HETE/15(S)-HETE activates ERK and FAK signalling pathways, thereby upregulates the adhesion and metastatic potential of melanoma cells. The endogenous release of 12(S)-HETE/15(S)-HETE in the host organ may affect the metastatic potential of melanoma.