Expression In Melanoma Cells Research Articles

MicroRNA expression in melanocytes and melanoma cells

Diagnostic biomarkers cancer diagnostics at preclinical stage seem to be a very promising strategy to increase effectiveness of anti-cancer treatment. Currently there are no such biomarkers available for daily routine practice. However, there are some candidate molecules in research that possibly can be used as biomarkers for early diagnosis, one of them is microRNA. MicroRNA is a small, 20–25 bp, non-coding RNA that is highly involved into epigenetic regulation of gene expression. These molecules participate in malignant transformation of normal cells into cancer cells including melanoma. And moreover, definite expression level of some microRNAs are essential for normal differentiation and function of human cells. Changes in microRNA profile are one of the reasons for malignant tumor development. Identification of these changes may help to develop diagnostic systems to start anti-cancer treatment at early stages.

Inhibition of mTORC2/RICTOR Impairs Melanoma Hepatic Metastasis

Mammalian target of rapamycin complex 2 (mTORC2) with its pivotal component rapamycin-insensitive companion of mTOR (RICTOR) is the major regulator of AKT phosphorylation and is increasingly implicated in tumor growth and progression. In cutaneous melanoma, an extremely aggressive and highly metastatic disease, RICTOR overexpression is involved in tumor development and invasiveness. Therefore, we investigated the impact of RICTOR inhibition in melanoma cells in vitro and in vivo with special emphasis on hepatic metastasis. Moreover, our study focused on the interaction of tumor cells and hepatic stellate cells (HSC) which play a crucial role in the hepatic microenvironment. In silico analysis revealed increased RICTOR expression in melanoma cells and tissues and indicated higher expression in advanced melanoma stages and metastases. In vitro, transient RICTOR knock-down via siRNA caused a significant reduction of tumor cell motility. Using a syngeneic murine splenic injection model, a significant decrease in liver metastasis burden was detected in vivo. Moreover, stimulation of melanoma cells with conditioned medium (CM) from activated HSC or hepatocyte growth factor (HGF) led to a significant induction of AKT phosphorylation and tumor cell motility. Blocking of RICTOR expression in cancer cells diminished constitutive and HGF-induced AKT phosphorylation as well as cell motility. Interestingly, RICTOR blockade also led to an abrogation of CM-induced effects on AKT phosphorylation and motility in melanoma cells. In conclusion, these results provide first evidence for a critical role of mTORC2/RICTOR in melanoma liver metastasis via cancer cell/HSC interactions.

Apigenin suppresses PD-L1 expression in melanoma and host dendritic cells to elicit synergistic therapeutic effects

BackgroundThe PD-L1/PD-1 pathway blockade-mediated immune therapy has shown promising efficacy in the treatment of multiple cancers including melanoma. The present study investigated the effects of the flavonoid apigenin on the PD-L1 expression and the tumorigenesis of melanoma.MethodsThe influence of flavonoids on melanoma cell growth and apoptosis was investigated using cell proliferation and flow cytometric analyses. The differential IFN-γ-induced PD-L1 expression and STAT1 activation were examined in curcumin and apigenin-treated melanoma cells using immunoblotting or immunofluorescence assays. The effects of flavonoid treatment on melanoma sensitivity towards T cells were investigated using Jurkat cell killing, cytotoxicity, cell viability, and IL-2 secretion assays. Melanoma xenograft mouse model was used to assess the impact of flavonoids on tumorigenesis in vivo. Human peripheral blood mononuclear cells were used to examine the influence of flavonoids on PD-L1 expression in dendritic cells and cytotoxicity of cocultured cytokine-induced killer cells by cell killing assays.ResultsCurcumin and apigenin showed growth-suppressive and pro-apoptotic effects on melanoma cells. The IFN-γ-induced PD-L1 upregulation was significantly inhibited by flavonoids, especially apigenin, with correlated reductions in STAT1 phosphorylation. Apigenin-treated A375 cells exhibited increased sensitivity towards T cell-mediated killing. Apigenin also strongly inhibited A375 melanoma xenograft growth in vivo, with enhanced T cell infiltration into tumor tissues. PD-L1 expression in dendritic cells was reduced by apigenin, which potentiated the cytotoxicity of cocultured cytokine-induced killer cells against melanoma cells.ConclusionsApigenin restricted melanoma growth through multiple mechanisms, among which its suppression of PD-L1 expression exerted a dual effect via regulating both tumor and antigen presenting cells. Our findings provide novel insights into the anticancer effects of apigenin and might have potential clinical implications.

Hyaluronic acid-CD44 interactions promote BMP4/7-dependent Id1/3 expression in melanoma cells

BMP4/7-dependent expression of inhibitor of differentiation/DNA binding (Id) proteins 1 and 3 has been implicated in tumor progression and poor prognosis of malignant melanoma patients. Hyaluronic acid (HA), a pericellular matrix component, supports BMP7 signalling in murine chondrocytes through its receptor CD44. However, its role in regulating BMP signalling in melanoma is not clear. In this study we found that depletion of endogenously-produced HA by hyaluronidase treatment or by inhibition of HA synthesis by 4-methylumbelliferone (4-MU) resulted in reduced BMP4/7-dependent Id1/3 protein expression in mouse melanoma B16-F10 and Ret cells. Conversely, exogenous HA treatment increased BMP4/7-dependent Id1/3 protein expression. Knockdown of CD44 reduced BMP4/7-dependent Id1/3 protein expression, and attenuated the ability of exogenous HA to stimulate Id1 and Id3 expression in response to BMP. Co-IP experiments demonstrated that CD44 can physically associate with the BMP type II receptor (BMPR) ACVR2B. Importantly, we found that coordinate expression of Id1 or Id3 with HA synthases HAS2, HAS3, and CD44 is associated with reduced overall survival of cutaneous melanoma patients. Our results suggest that HA-CD44 interactions with BMPR promote BMP4/7-dependent Id1/3 protein expression in melanoma, contributing to reduced survival in melanoma patients.

Cold atmospheric plasma induces apoptosis of melanoma cells via Sestrin2-mediated nitric oxide synthase signaling.

Cold atmospheric plasma (CAP) represents a promising therapy for selectively cancer killing. However, the mechanism of CAP-induced cancer cell death remains unclear. Here, we identified the tumor necrosis factor-family members, especially Fas, and overloaded intracellular nitric oxide participated in CAP induced apoptosis in A375 and A875 melanoma cell lines, which was known as extrinsic apoptosis pathway. This progress was mediated by antagonistic protein of reactive oxygen species, Sestrin2. The over expression of Sestrin2 induced by plasma treatment resulted in phosphorylation of p38 mitogen-activated protein kinase (MAPK), followed by increased expression of nitric oxide synthase (iNOS), Fas and Fas ligand. Depletion of Sestrin2 reduced iNOS and Fas expression, which was associated with reduction of plasma-induced apoptosis. In contrast, inhibition of iNOS activity and phosphorylation of p38 did not alter Sestrin2 expression in plasma-treated melanoma cells. Taken together, cold atmospheric plasma increases Sestrin2 expression and further activates downstream iNOS, Fas and p38 MAPK signaling to induce apoptosis of melanoma cell lines. These findings suggest a previously unrecognized mechanism in melanoma cells response to cold atmospheric plasma therapy.

Antitumorigenic effect of damnacanthal on melanoma cell viability through p53 and NF-κB/caspase-3 signaling pathways.

Melanoma is highly malignant, particularly prone to metastasizing to the skin. The incidence of melanoma varies markedly between countries, and is relatively low in China. The aim of the present study was to investigate the antitumorigenic effect of damnacanthal on melanoma cells, and its molecular mechanism. MUM-2B cells were treated with 0-20 µM damnacanthal for 12, 24 and 48 h. In vitro, it was demonstrated that damnacanthal inhibited proliferation and promoted apoptosis of melanoma cells in a dose- and time-dependent manner. Damnacanthal treatment increased caspase-3/8 and 9 activity, and promoted B-cell lymphoma 2-associated X protein, tumor protein p53 (p53) and p21 protein expression levels in melanoma cells. Damnacanthal treatment also resulted in downregulated nuclear factor-κB (NF-κB), cyclin D and cyclin E protein expression in melanoma cells. In conclusion, the results of the present study demonstrated that the antitumorigenic activity of damnacanthal on melanoma cells is executed via the p53/p21 and NF-κB/cyclin/ caspase-3 signaling pathways.

Melanoma LAMP-2C Modulates Tumor Growth and Autophagy.

Autophagy plays critical but diverse roles in cellular quality control and homeostasis potentially checking tumor development by removing mutated or damaged macromolecules, while conversely fostering tumor survival by supplying essential nutrients during cancer progression. This report documents a novel inhibitory role for a lysosome-associated membrane protein, LAMP-2C in modulating autophagy and melanoma cell growth in vitro and in vivo. Solid tumors such as melanomas encounter a variety of stresses in vivo including inflammatory cytokines produced by infiltrating lymphocytes directed at limiting tumor growth and spread. Here, we report that in response to the anti-tumor, pro-inflammatory cytokine interferon-gamma, melanoma cell expression of LAMP2C mRNA significantly increased. These results prompted an investigation of whether increased melanoma cell expression of LAMP-2C might represent a mechanism to control or limit human melanoma growth and survival. In this study, enhanced expression of human LAMP-2C in melanoma cells perturbed macroautophagy and chaperone-mediated autophagy in several human melanoma lines. In vitro analysis showed increasing LAMP-2C expression in a melanoma cell line, triggered reduced cellular LAMP-2A and LAMP-2B protein expression. Melanoma cells with enhanced LAMP-2C expression displayed increased cell cycle arrest, increased expression of the cell cycle regulators Chk1 and p21, and greater apoptosis and necrosis in several cell lines tested. The increased abundance of Chk1 protein in melanoma cells with increased LAMP-2C expression was not due to higher CHEK1 mRNA levels, but rather an increase in Chk1 protein abundance including Chk1 molecules phosphorylated at Ser345. Human melanoma cell xenografts with increased LAMP-2C expression, displayed reduced growth in immune compromised murine hosts. Melanomas with high LAMP-2C expression showed increased necrosis and reduced cell density upon histological analysis. These results reveal a novel role for LAMP-2C in negatively regulating melanoma growth and survival.

Suppression of IGF1R in Melanoma Cells by an Adenovirus-Mediated One-Step Knockdown System

Abnormal activation of the IGF1R signaling pathway accelerates melanoma development and metastases. RNAi systems with complex cloning procedures and unsatisfactory efficiency in suppressing gene expression have become the technical difficulties that hinder their utility when studying gene knockdown. Here we established a simplified adenovirus-mediated gene knockdown system by which a single adenoviral vector carries multiple siRNA fragments that can effectively suppress IGF1R expression in melanoma cells. We first generated the adenovirus that simultaneously expresses three human or mouse siRNAs targeting IGF1R (AdRIGF1R-OK). qRT-PCR and immunofluorescence staining revealed that IGF1R expression was significantly decreased in the melanoma cells that were infected with AdRIGF1R-OK. Bioluminescence imaging showed that the size of the tumor formed by the xenografts infected with AdRIGF1R-OK was significantly smaller than that of the controls. Annexin V-FITC flow cytometry assay, immunofluorescence staining for cleaved caspase-3, and Hoechst staining showed that more cells underwent apoptosis after infection with AdRIGF1R-OK. Luciferase reporter assay, crystal violet cell viability assay, and cell-cycle analysis showed that the proliferation of melanoma cells infected with AdRIGF1R-OK was significantly decreased compared to the controls. This study demonstrates that the OK system is effective in silencing gene expression, with promising potential to treat melanoma and other diseases.

Abstract 993: Wnt-er is coming: WNT5A promotes a slow cycling phenotype via p53 in conditions of stress

Abstract Resistance to targeted therapy and chemotherapy increases with age. Previously, we found that aged skin fibroblasts secrete sFRP2, which decreases ß-catenin, MITF, and APE1 in melanoma cells, and promotes invasion. Loss of APE1, a key redox effector, increased DNA damage in melanoma cells and caused the cells to become resistant to targeted therapy. Here we show that the aging tumor microenvironment drives a slow cycling phenotype in melanoma cells via WNT5A and p53. We found WNT5A expression in melanoma cells correlates with p53 expression and a higher percent of slow cycling cells. Knocking down WNT5A decreases both p53 expression and the percent of slow cycling cells. When we isolated slow cycling cells by flow cytometry, we found that they have increased WNT5A, p53, and p21 expression. Analysis of patient tumors in the TCGA data base revealed that patients with higher than average p53 expression, also express WNT5A. To determine if WNT5A and p53 may be promoting the survival of metastatic melanoma in an aged microenvironment, we examined tumors from young and aged C57/BL6 mice generated using Yumm1.7 cells. We found that tumors in aged mice have a higher percentage of cells expressing p53 and increased WNT5A expression compared to tumors in young mice. Treatment of human melanoma cells with conditioned media from aged dermal fibroblasts decreases proliferation and increases the percent of slow cycling. Inhibition of p53 re-sensitizes human melanoma cells in conditioned media from aged fibroblasts to combination BRAFi/MEKi. These data suggest, it may be these slow cycling cells driven by WNT5A and p53, which are enriched in an aging microenvironment, that are driving resistance to targeted therapy in aged patients. Citation Format: Marie R. Webster, Mitchell Fane, Amanpreet Kaur, Gretchen Alicea, Brett L. Ecker, Abibatou Ndoye, Curtis Kugel, Subhasree Basu, Alexander Valiga, Jessica L. Appleton, Maureen E. Murphy, Ashani Weeraratna. Wnt-er is coming: WNT5A promotes a slow cycling phenotype via p53 in conditions of stress [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 993.

PO-116 Increase in prothymosin alpha expression with histological aggressiveness of glioma

IntroductionWhile much progress has been made in melanoma treatment in the recent years, melanoma still remains a generally lethal disease at the metastatic stage. Therefore, discovery of additional critical target genes and pathways, and the development of novel therapeutic approaches remain a priority. Like most cancers, abnormal gene regulatory and epigenetic mechanisms are also linked to melanomas.The transcription factor interferon regulatory factor 4 (IRF4) was discovered as a factor implicated in immunoglobulin expression in B-cells, and was later shown to perform central roles in immune cell development and function. We have previously demonstrated the critical role of IRF4 in a variety of B-cell cancer cells, and identified its mechanisms of action. These and related work suggested IRF4 pathways as therapy targets in cancer. Several studies implicate IRF4 expression and function also in non-immune cells, such as melanocytes. For instance, a number of genome-wide genetic studies associated variation at the IRF4 gene with pigmentation phenotypes and skin cancers. However, despite the observed genetic links and generally high expression, the role of IRF4 in melanoma remains under-studied and poorly understood.Material and methodsWe set out to identify the functions of IRF4 in melanoma cells using genome-wide and cell and molecular biological approaches. We have taken a candidate approach to discover the upstream modulators of IRF4 expression in melanoma cells. In parallel, we have performed localization (ChIP-seq) and transcriptomic (RNA-seq) assays in order to identify the genome-wide targets of IRF4 in melanoma cell lines.Results and discussionsAs upstream regulators of IRF4 expression, we identified a known melanoma master transcription factor and a major signalling pathway with therapeutic targeting options. For downstream targets of IRF4, together with The Cancer Genome Atlas (TCGA) data analyses, our data point to a role of IRF4 in epigenetic regulation of melanoma cells, among other cancer- and development-related pathways. In addition, our preliminary studies implicate IRF4 as a critical factor in melanoma cell proliferation and survival.ConclusionOverall these studies on IRF4 in melanoma cells are complementing mechanistically the available genetic findings in melanoma patient samples, and describing a novel pathway in melanoma cells, with the potential to point to new therapy approaches for melanoma.

Abstract 2367: Identification of p-AKT as a PD marker for MER kinase in human G361 cells

Abstract Abnormalities in receptor tyrosine kinases (RTKs) have been shown to be involved in a wide range of important biological activities in cancer, including cell proliferation, differentiation and drug resistance. The TAM family of RTKs includes three family members: TYRO3, AXL, and MER. Binding of ligands to the these receptors was reported to lead to activation of the TAM kinases, as well as PI3K/AKT, RAS/RAF/MAPK and JAK/STAT signaling pathways in human cancer cells. Among these three TAM kinases, the MER signaling pathway represents an attractive target for the treatment of human cancers. MER phosphorylation is dependent on the ligand GAS6, however, ligand activated phospho-MER is often not stable and is very difficult to detect without pervanadate pretreatment in human cancer cells and this has been a hurdle for developing a selective MER kinase inhibitor. Therefore, it is important to identify a specific pharmacodynamic marker to monitor MER kinase activity in human cancer cells. Here, we report the development of a phospho-AKT assay for characterization of MER kinase inhibitors in human G361 cells. We profiled the expression of MER among multiple human cancer cells and demonstrated that MER and TYRO3, but not AXL show high levels of protein expression in human G361 melanoma cells. In G361 cells, phospho-AKT is induced by GAS6 treatment and the induction of phospho-AKT can be reversed by AXL/MER tool compounds. To determine the role MER and TYRO3 play in the activation of phospho-AKT, either MER or TYRO3 was selectively depleted by siRNA knockdown. We demonstrate that GAS6 induced phospho-AKT is only dependent on MER kinase, but not TYRO3 in human G361 melanoma cells. In addition, using phospho-AKT as readout, a high-throughput cell-based assay was established in the G361 human melanoma cells for evaluation of compound potency in inhibiting MER pathway activation. Further, we observed a correlation in compound potency between inhibition of phospho-AKT in G-361 cells and phospho-MER in MER-overexpressing BAF3 cells. In summary, we have demonstrated that GAS6 induced phospho-AKT can be a potential pharmacodynamic marker for inhibition of MER kinase and have successfully developed a cell-based functional assay for screening small molecule inhibitors of MER kinase for potential therapeutic utility in treating GAS6/MER-deregulated human cancers. Citation Format: Yaoyu Chen. Identification of p-AKT as a PD marker for MER kinase in human G361 cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2367.

Abstract 5493: Targeted inhibition of glutaminase in GRM1-expressing melanoma cells inhibits cell proliferation by reducing glutamate bioavailability

Abstract Aberrant glutamatergic signaling has been implicated in many cancer types. Our laboratory has previously illustrated the role of metabotropic glutamate receptor 1 (GRM1) in neoplastic transformation of melanocytes in vitro and spontaneous metastatic melanoma in vivo. Glutamate, the natural ligand of GRM1 is also the predominant excitatory neurotransmitter in the central nervous system. We have demonstrated significant upregulation of glutaminase (GLS) expression in GRM1 expressing melanoma cells, resulting in excess glutamate production and the establishment of autocrine loops in vitro. GLS catalyzes the first step in glutamine metabolism–the conversion of glutamine to glutamate. Comparison of glutamate levels in circulating blood plasma between melanoma prone and wild type mice showed elevated glutamate levels in melanoma prone GRM1-transgenic mice, suggesting that aberrant GRM1 expression also promotes an increase in circulating glutamate, to ensure constitutive activation of the GRM1 receptor and its downstream signaling affecters in vivo. Inclusion of CB-839, a potent, selective, and orally bioavailable inhibitor of GLS, resulted in significant inhibition of GRM1+ melanoma cell proliferation compared to GRM1- ones. We also demonstrated that treatment with Riluzole, an inhibitor of glutamatergic signaling by decreasing the available ligand, glutamate, led to significantly reduced melanoma cell proliferation in vitro and tumor progression in vivo. We found that simultaneous targeting of glutamate production (CB-839) and release (Riluzole) in GRM1+ melanoma cells led to synergistic suppression of cell proliferation in vitro. Furthermore, we found that CB-839 altered the function not the expression of GLS in melanoma cells. Additionally, we also discovered that manipulation of GRM1 expression by genetic means led to robust parallel changes in gene expression levels and intracellular glutamate concentrations by mass spectrometry analysis, as well as the endogenous levels of GLS in melanoma cells, suggesting association between GRM1 and GLS. The sensitivity of GRM1+ melanoma cells to modulation of GLS point to the dependency of these cells to glutamate amounts via GLS activity. We are continuing to investigate the mechanisms and regulation of GLS in our experimental system, with the goal of developing a rational approach for the treatment of GRM1 expressing tumors. Citation Format: Raj Shah, Mengying Zhu, Andrew Boreland, Fabian Filipp, Suzie Chen. Targeted inhibition of glutaminase in GRM1-expressing melanoma cells inhibits cell proliferation by reducing glutamate bioavailability [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5493.

Depigmenting effect of argan press-cake extract through the down-regulation of Mitf and melanogenic enzymes expression in B16 murine melanoma cells

Oil extraction from the kernels of Argania spinosa (L.) Skeels (Sapotaceae), an endemic tree of Morocco, produces argan press-cake (APC) used as a shampoo and to treat sprains, scabies, and for healing wounds. We have previously reported that argan oil has antimelanogenesis effect. Here, we determined if the by-product, APC, has melanogenesis regulatory effect using B16 murine melanoma cells. The effect of APC ethanol extract on cell proliferation and melanin content of B16 cells were measured, and to elucidate the mechanism involved, the expression level of melanogenic enzymes tyrosinase (TYR), dopachrome tautomerase (DCT), and tyrosinase-related protein 1 (TRP1) were determined and mRNA expression level of microphthalmia- associated transcription factor (Mitf) and Tyr genes were quantified. APC ethanol extract showed a significant melanin biosynthesis inhibitory effect on B16 cells in a time-dependent manner without cytotoxicity, which could be due to the decreased expression of TYR, TRP1, and DCT in a time-dependent manner. APC extract down regulated Mitf and Tyr. Decreased TRP1 and DCT levels could be due to post-translational modifications. These results suggest that APC extract may be used as a new source of natural whitening products and may be introduced as an important pharmacological agent for the treatment of hyperpigmentation disorders.

Codon-specific translation reprogramming promotes resistance to targeted therapy.

Reprogramming of mRNA translation has a key role in cancer development and drug resistance 1 . However, the molecular mechanisms that are involved in this process remain poorly understood. Wobble tRNA modifications are required for specific codon decoding during translation2,3. Here we show, in humans, that the enzymes that catalyse modifications of wobble uridine 34 (U34) tRNA (U34 enzymes) are key players of the protein synthesis rewiring that is induced by the transformation driven by the BRAF V600E oncogene and by resistance to targeted therapy in melanoma. We show that BRAF V600E -expressing melanoma cells are dependent on U34 enzymes for survival, and that concurrent inhibition of MAPK signalling and ELP3 or CTU1 and/or CTU2 synergizes to kill melanoma cells. Activation of the PI3K signalling pathway, one of the most common mechanisms of acquired resistance to MAPK therapeutic agents, markedly increases the expression of U34 enzymes. Mechanistically, U34 enzymes promote glycolysis in melanoma cells through the direct, codon-dependent, regulation of the translation of HIF1A mRNA and the maintenance of high levels of HIF1α protein. Therefore, the acquired resistance to anti-BRAF therapy is associated with high levels of U34 enzymes and HIF1α. Together, these results demonstrate that U34 enzymes promote the survival and resistance to therapy of melanoma cells by regulating specific mRNA translation.

PO-263 Epigenetic modulation of cell metabolism and its effects on cell survival in melanoma

Introduction Melanoma is an aggressive tumour with a median survival of only 6–12 month after occurrence of metastasis. In detail, one key factor of drug resistance mechanism in melanoma pathogenesis is cell heterogeneity. A defined cell subpopulation of slow cycling cells marked by a high histone demethylase JARID1B expression are therapy resistant and are most likely responsible for tumour repopulation. Moreover, cells with a slow cycling phenotype are described to modulate metabolism under targeted therapy. Therefore, the aim of my thesis is to characterise metabolic ‘hubs’ within metabolic landscaping analysis of melanoma cells. Material and methods We induced JARID1Blow and JARID1Bhigh phenotypes in WM3734 melanoma cells via shRNA-mediated knockdown and Doxycyclin-inducible Tet3G-Overexpression and prepared for a subpopulation-specific LC-HRMS based metabolomics landscaping analysis. Additionally, we investigated if chemical H3K4 demethylase inhibition could be used to specifically target the JARID1B-driven malignant metabolic phenotype in melanoma cells. Results and discussions JARID1Bhigh expressing melanoma cells show upregulated metabolites of glutathione-redox-system and pentose phosphate pathway. Moreover, several upregulated metabolites in JARID1Bhigh melanoma cells confirm existing data stating that those tumour cells do not follow the Warburg effect, but rather depend on mitochondrial OXPHOS to gain ATP and maintain a multi-resistant phenotype. Furthermore, a treatment with H3K4 demethylase inhibitors modulates the detected active metabolic pathways in melanoma cells functionally and also impairs cell proliferation and invasion capacity. Conclusion Our present metabolic landscaping analysis unravelled heterogeneous metabolic profiles in melanoma pointing to a possible relevance for therapy resistance of distinct melanoma cell subpopulations. In detail, we could profile the metabolome of multi-drug resistant JARID1Bhigh melanoma cells including their underlying enzymatic hubs. To overcome drug resistance in future, such hubs could be either directly targeted or indirectly by inhibiting their epigenetic regulators.

PO-264 Mitochondrial metabolism: a key factor of myeloid leukemic cell response upon exposure to tyrosine kinase inhibitors

IntroductionMelanoma is an aggressive tumour with a median survival of only 6–12 month after occurrence of metastasis. In detail, one key factor of drug resistance mechanism in melanoma pathogenesis is cell heterogeneity. A defined cell subpopulation of slow cycling cells marked by a high histone demethylase JARID1B expression are therapy resistant and are most likely responsible for tumour repopulation. Moreover, cells with a slow cycling phenotype are described to modulate metabolism under targeted therapy. Therefore, the aim of my thesis is to characterise metabolic ‘hubs’ within metabolic landscaping analysis of melanoma cells.Material and methodsWe induced JARID1Blow and JARID1Bhigh phenotypes in WM3734 melanoma cells via shRNA-mediated knockdown and Doxycyclin-inducible Tet3G-Overexpression and prepared for a subpopulation-specific LC-HRMS based metabolomics landscaping analysis. Additionally, we investigated if chemical H3K4 demethylase inhibition could be used to specifically target the JARID1B-driven malignant metabolic phenotype in melanoma cells. PO-263 Figure 1Results and discussionsJARID1Bhigh expressing melanoma cells show upregulated metabolites of glutathione-redox-system and pentose phosphate pathway. Moreover, several upregulated metabolites in JARID1Bhigh melanoma cells confirm existing data stating that those tumour cells do not follow the Warburg effect, but rather depend on mitochondrial OXPHOS to gain ATP and maintain a multi-resistant phenotype. Furthermore, a treatment with H3K4 demethylase inhibitors modulates the detected active metabolic pathways in melanoma cells functionally and also impairs cell proliferation and invasion capacity.ConclusionOur present metabolic landscaping analysis unravelled heterogeneous metabolic profiles in melanoma pointing to a possible relevance for therapy resistance of distinct melanoma cell subpopulations. In detail, we could profile the metabolome of multi-drug resistant JARID1Bhigh melanoma cells including their underlying enzymatic hubs. To overcome drug resistance in future, such hubs could be either directly targeted or indirectly by inhibiting their epigenetic regulators.

Migration and invasion in B16-F10 mouse melanoma cells are regulated by Nrf2 inhibition during treatment with ionizing radiation.

Nuclear factor erythroid 2-related factor 2 (Nrf2) serves a critical role in carcinogenesis. The present study examined the effect of Nrf2 on the proliferation and invasion of melanoma cells that were treated with ionizing radiation. B16-F10 mouse melanoma cells were exposed to various doses of ionizing radiation for different time periods. Small interfering (si)RNAs targeting Nrf2 were transfected into B16-F10 cells, and cell proliferation, invasion and apoptosis were detected by Transwell, MTT or western blot assays. The expression of Nrf2 and its downstream heme oxygenase 1 (HO-1) was analyzed by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blotting. HO-1 activity was also examined. Ionizing radiation stimulated Nrf2 expression, increased caspase-3 expression, and reduced the viability, migration and invasion of B16-F10 mouse melanoma cells. Transfection with Nrf2 siRNA was able to inhibit Nrf2 and HO-1 expression in B16-F10 mouse melanoma cells that were treated by ionizing radiation. Inhibition of Nrf2 further reduced cell viability, invasion and migration, and elevated caspase-3 expression in B16-F10 mice melanoma cells that were treated by ionizing radiation. In summary, treatment with ionizing radiation was able to stimulate Nrf2 expression and regulate cell viability, invasion and migration of B16-F10 cells. A combination of Nrf2 knockdown and ionizing radiation treatment exerted a synergistic effect on migration, invasion and apoptosis in B16-F10 murine melanoma cells.

105 Assessing CEACAM1 blockade as a therapeutic strategy using a CRISPR-Cas9-based mouse melanoma model

Current standard of care for advanced-stage melanoma includes immune checkpoint blockade (ICB) and kinase inhibitors. Unfortunately, melanomas treated with kinase inhibitors typically develop resistance; therefore, ICB has become first line therapy due to more durable responses. However, currently available ICBs, such as anti-CTLA-4 and anti-PD-1/PD-L1, engender durable responses in only a subset of patients, demanding a better understanding of other immune checkpoints as potential therapeutic targets. Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), which is expressed on the surface of activated (but not naïve) T cells and on many types of tumor cells, has been found to contribute to tumor progression and immune evasion. We examine the relevance of CEACAM1 in a genetically engineered mouse melanoma model harboring BRAFV600E mutation, which represents 40-60% of human melanomas. In this model, melanomas are induced by activation of mutant BRAF and neonatal exposure to UV irradiation. We have detected high expression of CEACAM1 at RNA and protein levels in UV-BRAF melanomas. We hypothesize CEACAM1 is involved in immune evasion, and its inhibition alone or in combination with PD-1/PD-L1 and CTLA-4 blockade could be a new strategy to treat melanoma. To test our hypothesis, we developed an inducible CRISPR-Cas9 system to knockout CEACAM1 expression in UV-BRAF melanoma cells. We are injecting these cells into syngeneic immunocompetent mice to evaluate the effects of CEACAM1 knockdown on tumor growth, and will use immunodeficient mice to determine the implication of the adaptive immune system in our findings. Future preclinical studies will include the combination of anti-PD-L1 and anti-CTLA-4 treatments with CEACAM1 inhibition to elucidate potential synergistic effects of these ICBs. Overall, we have validated CEACAM1 expression in a mouse model representing a large cohort of human melanoma and created CEACAM1 knockout mouse melanoma cells for mechanistic studies. Amid current immunotherapies, blockade of CEACAM1 may offer a promising alternative treatment strategy for advanced melanoma.

MiR-30a-5p confers cisplatin resistance by regulating IGF1R expression in melanoma cells

BackgroundMelanoma is notoriously resistant to all current modalities of cancer therapies including chemotherapy. In recent years, microRNAs (miRNAs) have emerged as molecular regulators in the development and progression of melanoma. However, the relationship between microRNA and chemo-resistance of melanoma is little known. In present study, we aimed to investigate the miRNAs related to cisplatin-resistance in melanoma cells.MethodsAfter cisplatin (DDP) resistant melanoma cells (M8/DDP and SK-Mel-19/DDP) were established in-vitro, high-throughput screening of differentially expressed miRNAs between resistant cells and parental cells were performed.ResultsIt was found that a cancer-related miRNA, miR-30a-5p, was highly over-expressed in resistant cells. Transfection of miR-30a-5p mimic or inhibitor could alter the sensitivity of melanoma cells to cisplatin. Next, we showed that Insulin Like Growth Factor 1 Receptor (IGF1R) gene turned out to be a direct target of miR-30a-5p. Knockdown of IGF1R in melanoma cells could not only reduce the sensitivity to cisplatin but also lead to cell cycle arrest by regulating phosphorylation of Serine-Threonine Protein Kinase (P-AKT (Ser473)) and Tumor Protein P53 (P53).ConclusionTaken together, our study demonstrated that miR-30a-5p could influence chemo-resistance by targeting IGF1R gene in melanoma cells, which might provide a potential target for the therapy of chemo-resistant melanoma cells.

Immunohistochemical expression of vitamin D receptor in melanocytic naevi and cutaneous melanoma: a case-control study.

Vitamin D deficiency is associated with higher risk of cancer, possibly due to its antiproliferative, antiangiogenic, proapoptotic, cell-differentiating and anti-invasive effects. The anticarcinogenic role of vitamin D in melanoma is still a matter of debate. Loss of nuclear and cytoplasmic vitamin D receptor (VDR) expression in melanoma cells has been reported. To analyse VDR immunohistochemical expression in benign dermal naevi (DN) and malignant melanoma (MM). A case-control study evaluated nuclear and cytoplasmic VDR immunohistochemical staining in 54 DN and 55 MM tissue samples. There was significantly higher cytoplasmic VDR positivity in DN compared with MM (59% vs. 16%, P < 0·001). The mean VDR cytoplasmic expression was also higher in DN vs. MM (P < 0·001). No differences in nuclear VDR positivity were observed between groups, but mean nuclear VDR expression was significantly lower in DN vs. MM (P = 0·02). The loss of cytoplasmic VDR in MM was associated with Clark level, tumour staging and American Joint Committee on Cancer pTNM staging (P=0·004, 0·009 and 0·02, respectively). Alterations in VDR expression and localization are found in MM compared with DN. Loss of cytoplasmic VDR was associated with melanoma tumour size, suggesting that loss of cytoplasmic VDR may be a prognostic factor.