Gene Expression In Melanoma Research Articles

Comprehensive in silico prioritization of pathogenic nsSNPs in human β-adducin gene towards finding its relation with cancer

The adducin family consists of three closely related proteins ADD1 (α-isoform), ADD2 (β-isoform) and ADD3 (γ-isoform) with similar domain structures. Adducins exist as heterodimers or tetramers composed of either α/β or α/γ heterodimers. Experimental evidences implicate the involvement of ADD2 variants in hypertension, renal dysfunction, impaired locomotor function and learning abilities. Non-synonymous SNPs (nsSNPs) are potentially damaging in nature as they results in amino acid substitutions. In human, a total 476 nsSNPs of ADD2 gene are reported in Ensembl and in the present work, computational prioritization has revealed 27 nsSNPs as highly deleterious and destabilizing in nature. Pan-cancer transcriptomic analyses reveal differential ADD2 expression in adrenal, breast, colon, lung, ovary, pancreas, testis, thyroid, and uterine cancer. In several cancers, overexpression of ADD2 is also found to be associated with lower overall survival rate and more patients at risk. It was also found that several nsSNPs of ADD2 had direct, proximal, distal and network rewriting PTM effects in different cancers and thereby account for potential structural and functional alterations. The coding mutations in ADD2 can affect overall gene expression in colon adenocarcinoma, melanoma, gastric cancer, and uterine cancer. The hub proteins, from the PPI network of these differentially expressed genes, are involved in positive regulation of acute inflammatory response to antigenic stimulus, acute phase response, G protein-coupled receptor signaling pathway. The present study is hypothetically driven and performed using public databases. Further experimental works are necessary to understand the role of ADD2 nsSNPs in tumor development and metastatic transition.

Innovative GenExpA software for selecting suitable reference genes for reliable normalization of gene expression in melanoma

Innovative GenExpA software for selecting suitable reference genes for reliable normalization of gene expression in melanoma

Innovative GenExpA software for selecting suitable reference genes for reliable normalization of gene expression in melanoma

The algorithms commonly used to select the best stable reference gene in RT-qPCR data analysis have their limitations. We showed that simple selection of the reference gene or pair of genes with the lowest stability value from the pool of potential reference genes—a commonly used approach—is not sufficient to accurately and reliably normalize the target gene transcript and can lead to biologically incorrect conclusions. For reliable assessment of changes in a target gene expression level, we propose our innovative GenExpA software, which works in a manner independent of the experimental model and the normalizer used. GenExpA software selects the best reference by combining the NormFinder algorithm with progressive removal of the least stable gene from the candidate genes in a given experimental model and in the set of daughter models assigned to it. The reliability of references is validated based on the consistency of the statistical analyses of normalized target gene expression levels through all models, described by the coherence score (CS). The use of the CS value imparts a new quality to qPCR analysis because it clarifies how low the stability value of reference must be in order for biologically correct conclusions to be drawn. We tested our method on qPCR data for the B4GALT genes family in melanoma, which is characterized by a high mutation rate, and in melanocytes. GenExpA is available at https://github.com/DorotaHojaLukowicz/GenExpA or https://www.sciencemarket.pl/baza-programow-open-source#oferty.

The Prognostic and Predictive Role of Xeroderma Pigmentosum Gene Expression in Melanoma.

BackgroundAssessment of immune-specific markers is a well-established approach for predicting the response to immune checkpoint inhibitors (ICIs). Promising candidates as ICI predictive biomarkers are the DNA damage response pathway genes. One of those pathways, which are mainly responsible for the repair of DNA damage caused by ultraviolet radiation, is the nucleotide excision repair (NER) pathway. Xeroderma pigmentosum (XP) is a hereditary disease caused by mutations of eight different genes of the NER pathway, or POLH, here together named the nine XP genes. Anecdotal evidence indicated that XP patients with melanoma or other skin tumors responded impressively well to anti-PD-1 ICIs. Hence, we analyzed the expression of the nine XP genes as prognostic and anti-PD-1 ICI predictive biomarkers in melanoma.MethodsWe assessed mRNA gene expression in the TCGA-SKCM dataset (n = 445) and two pooled clinical melanoma cohorts of anti-PD-1 ICI (n = 75). In TCGA-SKCM, we applied hierarchical clustering on XP genes to reveal clusters, further utilized as XP cluster scores. In addition, out of 18 predefined genes representative of a T cell inflamed tumor microenvironment, the TIS score was calculated. Besides these scores, the XP genes, immune-specific single genes (CD8A, CXCL9, CD274, and CXCL13) and tumor mutational burden (TMB) were cross-correlated. Survival analysis in TCGA-SKCM was conducted for the selected parameters. Lastly, the XP response prediction value was calculated for the two pooled anti-PD-1 cohorts by classification models.ResultsIn TCGA-SKCM, expression of the XP genes was divided into two clusters, inversely correlated with immune-specific markers. A higher ERCC3 expression was associated with improved survival, particularly in younger patients. The constructed models utilizing XP genes, and the XP cluster scores outperformed the immune-specific gene-based models in predicting response to anti-PD-1 ICI in the pooled clinical cohorts. However, the best prediction was achieved by combining the immune-specific gene CD274 with three XP genes from both clusters.ConclusionOur results suggest pre-therapeutic XP gene expression as a potential marker to improve the prediction of anti-PD-1 response in melanoma.

Δ40p53 isoform up-regulates netrin-1/UNC5B expression and potentiates netrin-1 pro-oncogenic activity

Netrin-1, a secreted protein recently characterized as a relevant cancer therapeutic target, is the antiapoptotic ligand of the dependence receptors deleted in colorectal carcinoma and members of the UNC5H family. Netrin-1 is overexpressed in several aggressive cancers where it promotes cancer progression by inhibiting cell death induced by its receptors. Interference of its binding to its receptors has been shown, through the development of a monoclonal neutralizing antinetrin-1 antibody (currently in phase II of clinical trial), to actively induce apoptosis and tumor growth inhibition. The transcription factor p53 was shown to positively regulate netrin-1 gene expression. We show here that netrin-1 could be a target gene of the N-terminal p53 isoform Δ40p53, independent of full-length p53 activity. Using stable cell lines, harboring wild-type or null-p53, in which Δ40p53 expression could be finely tuned, we prove that Δ40p53 binds to and activates the netrin-1 promoter. In addition, we show that forcing immortalized human skeletal myoblasts to produce the Δ40p53 isoform, instead of full-length p53, leads to the up-regulation of netrin-1 and its receptor UNC5B and promotes cell survival. Indeed, we demonstrate that netrin-1 interference, in the presence of Δ40p53, triggers apoptosis in cancer and primary cells, leading to tumor growth inhibition in preclinical in vivo models. Finally, we show a positive correlation between netrin-1 and Δ40p53 gene expression in human melanoma and colorectal cancer biopsies. Hence, we propose that inhibition of netrin-1 binding to its receptors should be a promising therapeutic strategy in human tumors expressing high levels of Δ40p53.

LB735 Epidermal growth factor modifies immune gene expression in keratinocytes and Melanoma

Epidermal growth factor (EGF) ligands play a key role in cellular proliferation and cancer progression via activation of the EGF receptor (EGFR). However, EGFR inhibitors (EGFRIs) frequently induce a rash in patients that is associated with improved cancer survival, suggesting immunomodulatory properties that may be therapeutically beneficial. Human keratinocytes (HaCaT) were treated with IFN-γ (a potent pro-inflammatory cytokine), EGF, both, or neither (control) and differentially expressed genes (DEGs) were analyzed using RNA-seq and gene-set enrichment analyses.

Retraction Note to: Differential modulatory effects of GSK-3b and HDM2 on sorafenib-induced AIF nuclear translocation (programmed necrosis) in melanoma

GSK-3β phosphorylates numerous substrates that govern cell survival. It phosphorylates p53, for example, and induces its nuclear export, HDM2-dependent ubiquitination, and proteasomal degradation. GSK-3β can either enhance or inhibit programmed cell death, depending on the nature of the pro-apoptotic stimulus. We previously showed that the multikinase inhibitor sorafenib activated GSK-3β and that this activation attenuated the cytotoxic effects of the drug in various BRAF-mutant melanoma cell lines. In this report, we describe the results of studies exploring the effects of GSK-3β on the cytotoxicity and antitumor activity of sorafenib combined with the HDM2 antagonist MI-319. MI-319 alone increased p53 levels and p53-dependent gene expression in melanoma cells but did not induce programmed cell death. Its cytotoxicity, however, was augmented in some melanoma cell lines by the addition of sorafenib. In responsive cell lines, the MI-319/sorafenib combination induced the disappearance of p53 from the nucleus, the down modulation of Bcl-2 and Bcl-xL, the translocation of p53 to the mitochondria and that of AIF to the nuclei. These events were all GSK-3β-dependent in that they were blocked with a GSK-3β shRNA and facilitated in otherwise unresponsive melanoma cell lines by the introduction of a constitutively active form of the kinase (GSK-3β-S9A). These modulatory effects of GSK-3β on the activities of the sorafenib/MI-319 combination were the exact reverse of its effects on the activities of sorafenib alone, which induced the down modulation of Bcl-2 and Bcl-xL and the nuclear translocation of AIF only in cells in which GSK-3β activity was either down modulated or constitutively low. In A375 xenografts, the antitumor effects of sorafenib and MI-319 were additive and associated with the down modulation of Bcl-2 and Bcl-xL, the nuclear translocation of AIF, and increased suppression of tumor angiogenesis. Our data demonstrate a complex partnership between GSK-3β and HDM2 in the regulation of p53 function in the nucleus and mitochondria. The data suggest that the ability of sorafenib to activate GSK-3β and alter the intracellular distribution of p53 may be exploitable as an adjunct to agents that prevent the HDM2-dependent degradation of p53 in the treatment of melanoma.

Enhanced expression of cyclooxygenase-2 related multi-drug resistance gene in melanoma and osteosarcoma cell lines by TSG-6 secreted from canine adipose-derived mesenchymal stem/stromal cells.

BackgroundMultiple drug resistance (MDR) of cancer cells is the main cause of intrinsic or acquired desensitization to chemotherapy in many cancers. A number of studies have found high expression of COX‐2 to be a factor for expression of MDR gene in several cancer. Furthermore, adipose tissue derived mesenchymal stem/stromal cells (ADSC) have been found to increase cyclo‐oxygenase‐2 (COX‐2) expression in some tumour cells. The mechanism for this, however, is not yet clear and needs further study.ObjectiveThe purpose of this study was to determine whether tumour necrosis factor‐alpha stimulated gene/protein 6 (TSG‐6) secreted from ADSCs is associated with an increase in MDR genes by inducing COX‐2 gene expression in melanoma and osteosarcoma cell lines.MethodsADSCs were transfected with TSG‐6 siRNA or Control RNA respected, and cancer cell line were transfected with COX‐2 siRNA or Control RNA respected. Using trans well coculture system, the interactions of ADSCs with tumour cells were investigated.ResultsIncreased COX‐2 expression was observed in cancer cell co‐cultured with ADSCs. Additionally, we identified that COX‐2 expression was related to drug resistance genes (P‐glycoprotein, multidrug resistance‐associated protein). Transfecting canine ADSCs with small interfering RNA, TSG‐6 secreted from ADSCs was found to be a major factor in the regulation of COX‐2 expression and drug resistance genes in osteosarcoma and melanoma cell lines.ConclusionTSG‐6 mediated COX‐2 up‐regulation is a possible mechanism of chemoresistance development induced by ADSCs. These findings provide better understanding about the mechanism associated with ADSC‐induced chemoresistance in cancer.

Deuterium Oxide (D2O) Induces Early Stress Response Gene Expression and Impairs Growth and Metastasis of Experimental Malignant Melanoma.

Simple SummarySystemic administration of deuterium oxide (‘heavy water’) has shown promise in suppressing tumor growth and metastasis in mammalian cancer models, but no detailed molecular studies have revealed specific molecular pathways mediating cancer-cell-directed activities. Here, for the first time, transcriptomic analysis complemented by in vivo efficacy experiments have addressed this unresolved topic.There are two stable isotopes of hydrogen, protium (1H) and deuterium (2H; D). Cellular stress response dysregulation in cancer represents both a major pathological driving force and a promising therapeutic target, but the molecular consequences and potential therapeutic impact of deuterium (2H)-stress on cancer cells remain largely unexplored. We have examined the anti-proliferative and apoptogenic effects of deuterium oxide (D2O; ‘heavy water’) together with stress response gene expression profiling in panels of malignant melanoma (A375V600E, A375NRAS, G361, LOX-IMVI), and pancreatic ductal adenocarcinoma (PANC-1, Capan-2, or MIA PaCa-2) cells with inclusion of human diploid Hs27 skin fibroblasts. Moreover, we have examined the efficacy of D2O-based pharmacological intervention in murine models of human melanoma tumor growth and metastasis. D2O-induction of apoptosis was substantiated by AV-PI flow cytometry, immunodetection of PARP-1, and pro-caspase 3 cleavage, and rescue by pan-caspase inhibition. Differential array analysis revealed early modulation of stress response gene expression in both A375 melanoma and PANC-1 adenocarcinoma cells elicited by D2O (90%; ≤6 h) (upregulated: CDKN1A, DDIT3, EGR1, GADD45A, HMOX1, NFKBIA, or SOD2 (up to 9-fold; p < 0.01)) confirmed by independent RT-qPCR analysis. Immunoblot analysis revealed rapid onset of D2O-induced stress response phospho-protein activation (p-ERK, p-JNK, p-eIF2α, or p-H2AX) or attenuation (p-AKT). Feasibility of D2O-based chemotherapeutic intervention (drinking water (30% w/w)) was demonstrated in a severe combined immunodeficiency (SCID) mouse melanoma metastasis model using luciferase-expressing A375-Luc2 cells. Lung tumor burden (visualized by bioluminescence imaging) was attenuated by D2O, and inhibition of invasiveness was also confirmed in an in vitro Matrigel transwell invasion assay. D2O supplementation also suppressed tumor growth in a murine xenograft model of human melanoma, and median survival was significantly increased without causing adverse effects. These data demonstrate for the first time that systemic D2O administration impairs growth and metastasis of malignant melanoma through the pharmacological induction of deuterium (2H)-stress.

Gene expression analysis in formalin fixed paraffin embedded melanomas is associated with density of corresponding immune cells in those tissues

Immune cell infiltrates in melanoma have important prognostic value. Gene expression analysis may simultaneously quantify numbers and function of multiple immune cell subtypes in formalin-fixed paraffin-embedded (FFPE) tissues. Prior studies report single gene expression can represent individual immune cell subtypes, but this has not been shown in FFPE melanomas. We hypothesized that gene expression profiling of human melanomas using a new RNA expression technology in FFPE tissue would correlate with the same immune cells identified by immunohistochemistry (IHC). This retrospective study included melanoma specimens analyzed by IHC on tumor tissue microarray (TMA) cores and by gene expression profiling with EdgeSeq Immuno-Oncology Assay using qNPA technology on the corresponding tumors. Standardized gene expression levels were analyzed relative to enumerated cells by IHC using Spearman rank test to calculate r-values. Multivariate analysis was performed by Kruskal–Wallis test. 119 melanoma specimens had both IHC and gene expression information available. There were significant associations between the level of gene expression and its quantified IHC cell marker for CD45+, CD3+, CD8+, CD4+, and CD20+ cells (all p < 0.001). There were also significant associations with exhaustion markers FoxP3+, PD-1+, and PD-L1+ (all p ≤ 0.0001). This new qNPA technology is useful to quantify intratumoral immune cells on FFPE specimens through RNA gene expression in metastatic melanoma. As previous studies have shown on other solid human tumors, we also confirm that the expression level of a single gene may be used to represent a single IHC immune cell marker in melanoma.

FoxP3, CTLA-4, and IDO in Canine Melanocytic Tumors.

Despite promising immunotherapy strategies in human melanoma, there are few studies on the immune environment of canine melanocytic tumors. In humans, the activation of immunosuppressive cell subpopulations, such as regulatory T cells (Tregs) that express forkhead box protein P3 (FoxP3), the engagement of immunosuppressive surface receptors like cytotoxic T lymphocyte antigen (CTLA-4), and the secretion of molecules inhibiting lymphocyte activation, such as indoleamine-pyrrole 2,3-dioxygenase (IDO), are recognized as immunoescape mechanisms that allow tumor growth and progression. The aim of our study was to investigate the expression of these immunosuppression markers in canine melanocytic tumors and to postulate their possible role in melanoma biology and progression. Fifty-five formalin-fixed, paraffin-embedded canine melanocytic tumors (25 oral melanomas; 20 cutaneous melanomas; 10 cutaneous melanocytomas) were selected to investigate the expression of FoxP3, CTLA-4, and IDO by immunohistochemistry and RT-qPCR (real-time quantitative polymerase chain reaction). All of the tested markers showed high gene and protein expression in oral melanomas and were differently expressed in cutaneous melanomas when compared to their benign counterpart. IDO expression was associated with an increased hazard of death both in univariable and multivariable analyses (P < .05). FoxP3 protein expression >6.9 cells/HPF (high-power field) was an independent predictor of death (P < .05). CTLA-4 gene and protein expressions were associated with a worse prognosis, but only in the univariable analysis (P < .05). FoxP3, CTLA-4, and IDO likely play a role in canine melanoma immunoescape. Their expression, if supported by future studies, could represent a prognostic tool in canine melanoma and pave the way to future immunotherapeutic approaches in dogs.

Differential association of CD68+ and CD163+ macrophages with macrophage enzymes, whole tumour gene expression and overall survival in advanced melanoma

BackgroundThe density and phenotype of tumour-associated macrophages have been linked with prognosis in a range of solid tumours. While there is strong preclinical evidence that tumour-associated macrophages promote aspects of tumour progression, it can be challenging to infer clinical activity from surface markers and ex vivo behaviour. We investigated the association of macrophage infiltration with prognosis and functional changes in the tumour microenvironment in primary human melanoma.MethodsFifty-seven formalin-fixed, paraffin-embedded primary melanomas were analysed by immunohistochemical analysis of CD68, CD163, inducible nitric oxide synthase (iNOS) and arginase expression. RNA sequencing was performed on serial sections of 20 of the stained tumours to determine the influence of macrophage infiltration on gene expression.ResultsCD68+ cells are a functionally active subset of macrophages that are associated with increased iNOS and arginase staining and altered gene expression. In comparison, while there is a greater accumulation of CD163+ macrophages in larger tumours, these cells are comparatively inactive, with no association with the level of iNOS or arginase staining, and no effect on gene expression within the tumour. The infiltration of either subset of macrophages did not correlate to overall survival.ConclusionsThus, melanomas contain distinct macrophage populations with diverse phenotypes, but with no observable prognostic role.

Comparison of SOX10 gene expression in melanoma and melanocytic nevus samples using Real-time PCR and immunohistochemistry

Melanoma is a malignant skin cancer and the most deadly skin tumor which is treatable if diagnosed early. The SOX10 protein is a nuclear transcription factor that regulates genes essential for melanin synthesis.The aim of this study was to compare the SOX10 gene expression in melanoma tumors and benign melanocyte lesions as a possible biological indicator in the diagnosis and prognosis and better understanding of the molecular process involved in melanoma cancer.In this study, 25 samples of melanoma, 25 samples of melanocytic nevus and 10 samples of normal skin as control were examined. The RNA was extracted from paraffin block tissues. After cDNA synthesis, Real-time PCR was performed to evaluate the expression of the SOX10 gene. The SOX10 protein level was assessed by immunohistochemistry.Increase in the SOX10 gene expression and significant increase in its protein levels in melanoma samples compared to the nevus and normal samples was confirmed by both Real-time PCR and immunohistochemistry. No correlation was observed between SOX10 gene expression and other factors such as the Clark and Breslow scales, mitotic counts and demographic characteristics including age and gender.Given the high expression of the target gene in the melanoma group, more research is needed on SOX10 as a molecular marker in the diagnosis of melanoma cancer. As SOX10 staining intensity varies in both nevus and melanoma groups, this biological marker can be used for differentiation.

Potential Prognostic Predictors and Molecular Targets for Skin Melanoma Screened by Weighted Gene Co-expression Network Analysis.

Among skin cancers, malignant skin melanoma is the leading cause of death. Identification of gene markers of malignant skin melanoma associated with survival may provide new clues for prognosis prediction and treatment. This research aimed to screen out potential prognostic predictors and molecular targets for malignant skin melanoma. Information regarding gene expression in skin melanoma and patients' clinical traits was obtained from the Gene Expression Omnibus database. Weighted gene co-expression network analysis (WGCNA) was applied to build co-expression modules and investigate the association between the modules and clinical traits. Moreover, functional enrichment analysis was performed for clinically significant co-expression modules. Hub genes of these modules were validated via Gene Expression Profiling Interactive Analysis (GEPIA) and the Human Protein Atlas (http:// www.proteinatlas.org). First, using WGCNA, 9 co-expression modules were constructed by the top 25% differentially expressed genes (4406 genes) from 77 human melanoma samples. Two co-expression modules (magenta and blue modules) were significantly correlated with survival months (r = -0.27, p = 0.02; r = 0.27, p = 0.02, respectively). The results of functional enrichment analysis demonstrated that the magenta module was mainly enriched in the cell cycle process and the blue module was mainly enriched in the immune response process. Additionally, the GEPIA and Human Protein Atlas results suggested that the hub genes CCNB2, ARHGAP30, and SEMA4D were associated with relapse-free survival and overall survival (all p-values < 0.05) and were differentially expressed in melanoma tumors and normal skin. The results provided the framework of co-expression gene modules of skin melanoma and screened out CCNB2, ARHGAP30, and SEMA4D associated with survival as potential prognostic predictors and molecular targets of treatment.

Characterization of glycolysis-related gene expression in malignant melanoma

Malignant melanoma exhibits a distinct metabolic phenotype with high glycolytic activity. Previously, we have shown that glucose transporter isoform 1 (GLUT1) favors growth and metastasis of malignant melanoma. In this study, we investigated the expression of GLUT1 and the further glycolysis-related genes hexokinase 1 and 2 (HK1, HK2), lactate dehydrogenase A (LDH-A) and monocarboxylate transporters 1 and 4 (MCT1, MCT4) in eleven human melanoma cell lines under normoxic and hypoxic conditions. Furthermore, a set of 25 human malignant melanoma tissue samples was analyzed.Under hypoxic conditions, we could observe a significant upregulation of hypoxia-inducible factor 1 alpha (HIF-1a) target genes GLUT1, HK2 and LDH-A, but not MCT4. While under normoxic conditions the expression of glycolysis-related genes showed no correlation with origin or BRAF mutation status, GLUT1 expression was significantly elevated in metastatic and BRAF-V600E mutated melanoma cell lines under hypoxic conditions. Furthermore, GLUT1 expression in human melanoma tissue samples correlated significantly with HK1, LDH-A and MCT1 expression, confirming a glycolytic phenotype. Notably, Cyclin D1 expression, which is used as a prognostic marker for the outcome of melanoma patients, as it is associated with proliferation and invasiveness of melanoma, significantly correlated with GLUT1, HK1, LDH-A and MCT1 expression. In summary, our findings provide further evidence that enhanced glycolytic activity in melanoma favors disease progression and is an attractive therapeutic target for this highly aggressive tumor.

Identification of robust reference genes for studies of gene expression in FFPE melanoma samples and melanoma cell lines.

There is an urgent need for novel diagnostic melanoma biomarkers that can predict increased risk of metastasis at an early stage. Relative quantification of gene expression is the preferred method for quantitative validation of potential biomarkers. However, this approach relies on robust tissue-specific reference genes. In the melanoma field, this has been an obstacle due to lack of validated reference genes. Accordingly, we aimed to identify robust reference genes for normalization of gene expression in melanoma. The robustness of 24 candidate reference genes was evaluated across 80 formalin-fixed paraffin-embedded melanomas of different thickness, -/+ ulceration, -/+ reported cases of metastases and of different BRAF mutation status using quantitative real-time PCR. The expression of the same genes and their robustness as normalizers was furthermore evaluated across a number of melanoma cell lines. We show that housekeeping genes like GAPDH do not qualify as stand-alone normalizers of genes expression in melanoma. Instead, we have as the first identified a panel of robust reference genes for normalization of gene expression in melanoma tumors and cultured melanoma cells. We recommend using a geometric mean of the expression of CLTA, MRPL19 and ACTB for normalization of gene expression in melanomas and a geometric mean of the expression of CASC3 and RPS2 for normalization of gene expression in melanoma cell lines. Normalization, according to our recommendation will allow for quantitative validation of potential novel melanoma biomarkers by quantitative real-time PCR.

Abstract 5204: ICAM-1 and IL-17E upregulate FOXO1 to suppress the expression of oncogenic PKC-ι in human melanoma

Abstract Throughout the span of the last 15 years, the number of cases of melanoma has been on a steady increase. Approximately, 92,000 new melanoma cases will be diagnosed in the United States in 2018. Our previous studies demonstrated that protein kinase C-iota (PKC-ι) is crusial for melanoma progression, which promotes the activation of nuclear factor (NF)-κB through a PI3K/AKT manner, thereby supporting survival and progression. In addition, PKC-ι induced the metastasis of melanoma cells by stimulating ephithilial-mesenchmal transition. Results also showed that PKC- specific inhibitors diminished the expression of both PKC-ι and phospo-PKC-ι, suggesting that PKC-ι plays a role in regulating its own expression in melanoma cells in-vitro {Ratnayake, W.S., et al. Cell Adhes. Migr. (2018) https://doi.org/10.1080/19336918.2018.1471323}. In this study, we report the transcriptional regulation mechanisms of PRKCI (PKC-ι gene) expression in melanoma. PROMO and Genomatix Matinspector methods were used to identify c-Jun, interferon-stimulated gene factor 3 (ISGF3), paired box gene 3 (PAX3), early growth response protein 1 (EGR1) and forkhead box protein O1 (FOXO1), which bind on or near the promoter sequence of the PRKCI and they were analyzed for their roles in PKC-ι expression. siRNAs were used to silence selected transcription factors, and the results revealed that, silencing of FOXO1 and c-Jun significantly altered the expression of PRKCI. In addtion, we knocked down NF-κB in order to demonstrate changes on activities of c-Jun and FOXO1. Both phosphorylated and total PKC-ι levels increased upon FOXO1 silencing and decreased upon c-Jun silencing, suggesting that c-Jun turns is an upregulator, while FOXO1 acts as a downregulator of PRKCI expression. Multiplex ELISA assays were used to analyze multiple pathways and the expression of cytokines with respect to crosstalk between signaling pathways affected by specific inhibiton PKC-ι. Western blot data for all siRNA treatments of PKC-ι, NF-κB, FOXO1 and c-Jun were supported by real-time qPCR. Taken together, results suggested that the regulation of PKC-ι expression was strongly associated with FOXO1 and c-Jun through the trascriptional deactivation/activation. In addition, we observed a significant decrease in the mRNA levels of both interleukin (IL)-6 and IL-8, with a significant increase in the levels of IL-17E and intercellular adhesion molecule 1 (ICAM-1) upon the knockdown of expression of PKC-ι in both cell lines. This suggested that PKC-ι expression was affected by said cytokines in an autocrine modus. Overall, the findings suggest that PKC-ι inhibition suppresses its own expression, diminishing oncogenic signaling such as NF-κB, PI3K/AKT and JNK/c-Jun while upregulating anti-tumor signaling such as ICAM-1/FOXO1, thus rendering it an effective novel biomarker for use in the design of novel targeted therapeutics for melanoma metastasis. Citation Format: Wishrawana Sarathi Ratnayake, Christopher Apostolatos, Sloan Breedy, André Apostolatos, Mildred Acevedo-Duncan. ICAM-1 and IL-17E upregulate FOXO1 to suppress the expression of oncogenic PKC-ι in human melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5204.

The X-Linked DDX3X RNA Helicase Dictates Translation Reprogramming and Metastasis in Melanoma

The X-linked DDX3X gene encodes an ATP-dependent DEAD-box RNA helicase frequently altered in various human cancers, including melanomas. Despite its important roles in translation and splicing, how DDX3X dysfunction specifically rewires gene expression in melanoma remains completely unknown. Here, we uncover a DDX3X-driven post-transcriptional program that dictates melanoma phenotype and poor disease prognosis. Through an unbiased analysis of translating ribosomes, we identified the microphthalmia-associated transcription factor, MITF, as a key DDX3X translational target that directs a proliferative-to-metastatic phenotypic switch in melanoma cells. Mechanistically, DDX3X controls MITF mRNA translation via an internal ribosome entry site (IRES) embedded within the 5' UTR. Through this exquisite translation-based regulatory mechanism, DDX3X steers MITF protein levels dictating melanoma metastatic potential invivo and response to targeted therapy. Together, these findings unravel a post-transcriptional layer of gene regulation that may provide a unique therapeutic vulnerability in aggressive male melanomas.

Gene Expression and Methylation Analysis in Melanomas and Melanocytes From the Same Patient: Loss of NPM2 Expression Is a Potential Immunohistochemical Marker for Melanoma.

DNA methylation is considered the primary epigenetic mechanism underlying the development of malignant melanoma. Since DNA methylation can be influenced by environmental factors, it is preferable to compare cancer and normal cells from the same patient. In order to compare the methylation status in melanoma tissues and melanocytes from the same individuals, we employed a novel epidermal sheet cultivation technique to isolate normal melanocytes from unaffected sites of melanoma patients. We also analyzed primary and metastatic melanoma samples, three commercially available melanocytes, and four melanoma cell lines. Cluster analysis of DNA methylation data classified freshly isolated melanomas and melanocytes into the same group, whereas the four melanoma cell lines were clustered together in a distant clade. Moreover, our analysis discovered methylation at several novel loci (KRTCAP3, AGAP2, ZNF490), in addition to those identified in previous studies (COL1A2, GPX3); however, the latter two were not observed in fresh melanoma samples. Subsequent studies revealed that NPM2 was hypermethylated and downregulated in melanomas, which was consistent with previous reports. In many normal melanocytes, NPM2 showed distinct immunohistochemical staining, while its expression was lost in malignant melanoma cells. In particular, intraepithelial lesions of malignant melanoma, an important challenge in clinical practice, could be distinguished from benign nevi. The present findings indicate the importance of using fresh melanoma samples, not melanoma cell lines and melanocytes in epigenetic studies. In addition, NPM2 immunoreactivity could be used to differentiate melanomas from normal melanocytes or benign disease.

PTGS2 and ITGB1 gene expression in canine melanoma

PTGS2 and ITGB1 gene expression in canine melanoma