Mechanisms In Melanoma Research Articles

Oxidative Stress-Related Mechanisms in Melanoma and in the Acquired Resistance to Targeted Therapies.

Melanoma is a highly aggressive cancer with the poorest prognosis, representing the deadliest form of skin cancer. Activating mutations in BRAF are the most frequent genetic alterations, present in approximately 50% of all melanoma cases. The use of specific inhibitors towards mutant BRAF variants and MEK, a downstream signaling target of BRAF in the MAPK pathway, has significantly improved progression-free and overall survival in advanced melanoma patients carrying BRAF mutations. Nevertheless, despite these improvements, resistance still develops within the first year of therapy in around 50% of patients, which is a significant problem in managing BRAF-mutated advanced melanoma. Understanding these mechanisms is one of the mainstreams of the research on BRAFi/MEKi acquired resistance. Both genetic and epigenetic mechanisms have been described. Moreover, in recent years, oxidative stress has emerged as another major force involved in all the phases of melanoma development, from initiation to progression until the onsets of the metastatic phenotype and chemoresistance, and has thus become a target for therapy. In the present review, we discuss the current knowledge on oxidative stress and its signaling in melanoma, as well as the oxidative stress-related mechanisms in the acquired resistance to targeted therapies.

IRF4-activated TEX41 promotes the malignant behaviors of melanoma cells by targeting miR-103a-3p/C1QB axis

BackgroundMalignant melanoma is an aggressive skin cancer and a tumor of melanocytic origin. Recent studies have suggested that long non-coding RNAs (lncRNAs) play crucial regulatory roles in multiple malignancies, including melanoma. Testis expressed 41 (TEX41) is a relatively new lncRNA whose mechanism in melanoma remains vague.AimsThis study aimed to explore the role and specific mechanism of TEX41 in melanoma.MethodsThe expression of genes involved in this study was determined by qRT-PCR. Functional assays were conducted to analyze the role of relevant genes in melanoma cells. The interaction between TEX41 promoter and IRF4 as well as the relationship among TEX41, miR-103a-3p and C1QB was verified by mechanism assays.ResultsIRF4 up-regulated TEX41 at the transcriptional level in melanoma cells. TEX41 knockdown hindered melanoma cell proliferation, migration and invasion while promoting cell apoptosis. TEX41 bound to miR-103a-3p and regulated C1QB. The suppressive impact of TEX41 depletion on melanoma cell malignant behaviors could be counteracted by miR-103a-3p inhibition or C1QB overexpression. Moreover, IRF4 could facilitate melanoma cell growth via up-regulating C1QB.ConclusionsIRF4-activated TEX41 sequestered miR-103a-3p and modulated C1QB to promote melanoma cell malignant behaviors, for which TEX41 might be regarded as a potential therapeutic target for melanoma.

Cell type identification from single-cell transcriptomes in melanoma

BackgroundSingle-cell sequencing approaches allow gene expression to be measured at the single-cell level, providing opportunities and challenges to study the aetiology of complex diseases, including cancer.MethodsBased on single-cell gene and lncRNA expression levels, we proposed a computational framework for cell type identification that fully considers cell dropout characteristics. First, we defined the dropout features of the cells and identified the dropout clusters. Second, we constructed a differential co-expression network and identified differential modules. Finally, we identified cell types based on the differential modules.ResultsThe method was applied to single-cell melanoma data, and eight cell types were identified. Enrichment analysis of the candidate cell marker genes for the two key cell types showed that both key cell types were closely related to the physiological activities of the major histocompatibility complex (MHC); one key cell type was associated with mitosis-related activities, and the other with pathways related to ten diseases.ConclusionsThrough identification and analysis of key melanoma-related cell types, we explored the molecular mechanism of melanoma, providing insight into melanoma research. Moreover, the candidate cell markers for the two key cell types are potential therapeutic targets for melanoma.

Abstract 1674: WNT signal pathway activation correlates with innate resistant to immune checkpoint therapies in melanoma

Abstract Background: Immune checkpoint inhibitors targeting the programmed cell death-1 receptor (PD-1) improve survival in a subset of patients with melanoma. There are still lots of patients could not reach the clinical benefit, even though with the positive expression of the programmed cell death 1 ligand 1 (PD-L1). Here, we aimed to research the immune resistance mechanism in melanoma. Methods: The genomic data and clinical data of the discovery cohort was obtained from The Cancer Genome Atlas (TCGA). The clinical data of validation cohort in melanoma treated by immunotherapy was retrospective collected. And tissue from patients with melanoma were performed to whole exome sequencing in a College of American Pathologists-certified and Clinical Laboratory Improvement Amendments-accredited lab. Results: Activation of WNT signaling was inferred that somatic mutations or somatic copy number alterations in WNT signaling elements including APC, WNT1, WNT4, WNT6, WNT16, CTNNB1, BCL9L, SMAD3 and SMAD4. Two cohort have been enrolled in this study, the discovery cohort from TCGA, and the validation cohort from the Chinese patients with melanoma. The frequency of WNT pathway alterations from the TCGA cohort was 28.4%, and which was represented mutually exclusive molecular subsets. In TCGA cohort, activating alteration WNT signaling were associated with shorter median OS (22.5 vs 13.6 months, p=0.047, HR=0.61 (0.37-0.89)). Conclusions: Mutations predicted to activate the WNT pathway were associated with innate resistance to immune checkpoint blockade in melanoma. Citation Format: Fuyu Gong, Yuezong Bai, wenzhuan Xie, Chuanliang Cui. WNT signal pathway activation correlates with innate resistant to immune checkpoint therapies in melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1674.

Abstract LB261: BRAF inhibitor reprograms cancer-associated fibroblasts to elicit matrix remodeling and therapeutic escape in melanoma

Abstract The tumor stroma and its cellular components are known to play important roles in tumor resilience towards treatment but remain to be urgently characterized in melanoma targeted therapy. By performing a paired-analysis of melanoma tissue samples collected from the patients before and after combined BRAF inhibitors (BRAFi)/MEK inhibitors (MEKi) treatment, we discovered that the number of intratumoral cancer-associated fibroblasts (CAFs) increased significantly in BRAFi/MEKi-treated melanoma stroma comparing with the samples before the therapy. Those intratumoral CAFs exhibit increased amounts of nuclear β-catenin under BRAFi/MEKi therapy. Further studies show that BRAFi not only leads to increased β-catenin nuclear accumulation in CAFs and enhances their biological properties, suggesting BRAFi induces a new resistant phenotype in CAFs. In both in vitro and in vivo models, depleting β-catenin in CAFs abolishes their ability to elicit melanoma cell growth and drug resistance. BRAFi induces BRAF and CRAF dimerization and subsequently the activation of MEK/ERK signaling in CAFs, leading to the inactivation of the β-catenin destruction complex. RNA-Seq data reveals that nuclear β-catenin is a major regulator of CAF phenotype. We identify matricellular protein periostin (POSTN) as a downstream effector of β-catenin. In melanoma, POSTN is uniquely expressed in CAFs, and BRAFi can specifically upregulate POSTN production in CAFs but not in melanoma cells. Recombinant POSTN contributes to melanoma cell BRAFi/MEKi resistance and can compensate for the loss of β-catenin in CAFs. By reactivating ERK pathway in melanoma cells through PI3K/AKT signaling, POSTN compensates for the loss of β-catenin in CAFs in conferring melanoma cell BRAFi/MEKi resistance. In summary, our data reveal a novel resistance mechanism in melanoma that is elicited by BRAFi-induced non-canonical nuclear β-catenin signaling in CAFs and reveal POSTN as an important matrix target to eliminate BRAFi/MEKi resistance in melanoma. Citation Format: Tianyi Liu, Linli Zhou, Yuhang Zhang. BRAF inhibitor reprograms cancer-associated fibroblasts to elicit matrix remodeling and therapeutic escape in melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB261.

ITIH5, a p53-responsive gene, inhibits the growth and metastasis of melanoma cells by downregulating the transcriptional activity of KLF4

ITIH5, a member of the inter-α-trypsin inhibitory (ITI) gene family, acts as a putative tumour-suppressor gene in many cancers. However, its role and the regulatory mechanism in melanoma are still unclear. Here, we found that the expression of ITIH5 was decreased in melanoma tissues compared with normal skin tissues. Decreased expression of ITIH5 was correlated with clinicopathological features and predicted poor prognosis in patients with melanoma. Forced expression of ITIH5 significantly inhibited melanoma cell proliferation and metastasis in vitro and ex vivo while knockdown of ITIH5 expression enhanced the malignant behaviour of melanoma cells. In further mechanistic studies, we showed that p53 can directly bind to the promoter of ITIH5 and thus promotes transcription of ITIH5 in melanoma cells. Additionally, we found that ITIH5 interacted with Krüppel-like factor 4 (KLF4) and inhibited its transcriptional activity. Collectively, our data not only identified a tumour-suppressive role of ITIH5 in melanoma but also revealed that upregulation of ITIH5 by p53 suppressed melanoma cell growth and migration likely by downmodulating the transcriptional activity of KLF4.

New Insights into the Link between Melanoma and Thyroid Cancer: Role of Nucleocytoplasmic Trafficking.

Cancer remains a major public health concern, mainly because of the incompletely understood dynamics of molecular mechanisms for progression and resistance to treatments. The link between melanoma and thyroid cancer (TC) has been noted in numerous patients. Nucleocytoplasmic transport of oncogenes and tumor suppressor proteins is a common mechanism in melanoma and TC that promotes tumorigenesis and tumor aggressiveness. However, this mechanism remains poorly understood. Papillary TC (PTC) patients have a 1.8-fold higher risk for developing cutaneous malignant melanoma than healthy patients. Our group and others showed that patients with melanoma have a 2.15 to 2.3-fold increased risk of being diagnosed with PTC. The BRAF V600E mutation has been reported as a biological marker for aggressiveness and a potential genetic link between malignant melanoma and TC. The main mechanistic factor in the connection between these two cancer types is the alteration of the RAS-RAF-MEK-ERK signaling pathway activation and translocation. The mechanisms of nucleocytoplasmic trafficking associated with RAS, RAF, and Wnt signaling pathways in melanoma and TC are reviewed. In addition, we discuss the roles of tumor suppressor proteins such as p53, p27, forkhead O transcription factors (FOXO), and NF-KB within the nuclear and cytoplasmic cellular compartments and their association with tumor aggressiveness. A meticulous English-language literature analysis was performed using the PubMed Central database. Search parameters included articles published up to 2021 with keyword search terms melanoma and thyroid cancer, BRAF mutation, and nucleocytoplasmic transport in cancer.

Comprehensive upstream and downstream regulatory analyses identify miR-675-3p as a potential prognostic biomarker in melanoma

This study assessed miR-675-3p-related regulatory mechanisms in melanoma and the clinical relevance of such regulatory activities. We downloaded miRNA mature strand expression RNA-Seq, phenotypic, and DNA methylation data pertaining to the TCGA Melanoma cohort. Differentially expressed miRNAs (DEMs) between metastatic and primary melanoma patient tissues were then identified, and miR-675-3p expression in melanoma patient peripheral blood was confirmed using the GSE20994 GEO dataset, while its expression in melanoma cell lines was evaluated via qRT-RCR. The clinical and prognostic implications of miR-675-3p in melanoma were assessed, and miR-675-3p target genes were identified using bioinformatics tools. Functional roles of this miRNA were explored via Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses. We identified 3 and 22 miRNAs that were up- and downregulated, respectively, in metastatic melanoma samples relative to primary melanoma samples. Upregulation of miR-675-3p was associated with poorer overall patient survival, tumor histologic grade, and Clark's level. Consistently, miR-675-3p was also overexpressed in the peripheral blood of melanoma patients relative to healthy controls, and in melanoma cell lines relative to control cells. Gene regulatory networks indicated that 32 transcription factors control miR-675-3p expression, and that it, in turn, regulates 10 target genes. KEGG analyses indicated that these genes were associated with cell cycle, transcriptional misregulation in cancer, TGF-beta signaling, and HIF-1 signaling pathways. Gain-of-function assays revealed that miR-675-3p could promote cell proliferation via accelerating cell cycle progression. Western blotting results indicated that miR-675-3p could active TGF-beta and HIF-1 signaling. Through upstream and downstream analyses of miR-675-3p-related regulatory activity, we confirmed that this miRNA participates in key melanoma-related processes and offers value as a prognostic biomarker in melanoma patients.

Identification of tumor mutation burden-related hub genes and the underlying mechanism in melanoma.

Background: Tumor mutation burden (TMB) has emerged as an important predictive factor for drug resistance in cancers; however, the specific mechanism underlying TMB function in melanoma remains elusive.Methods: Data on somatic mutations, RNA sequencing (RNA-seq), miRNA sequencing (miRNA-seq), and clinical characteristics for 472 melanoma patients were extracted from the TCGA cohort. RNA-seq data of melanoma cell lines were obtained from the Cancer Cell Line Encyclopedia, and sensitivity of cell lines to therapeutic agents is available in the Cancer Therapeutics Response Portal. TMB was calculated based on somatic mutation data. Differentially expressed gene analysis, weighted gene co-expression network analysis, protein-protein interaction networks, Minimal Common Oncology Data Elements, and survival analysis were leveraged to determine TMB-related hub genes. Competing endogenous RNA (ceRNA) networks were constructed to explore the molecular mechanisms underlying hub gene function. The influence of key genes on drug sensitivity was analyzed to investigate their clinical significance.Results: Elevated TMB levels were significantly correlated with improved survival outcomes. In addition, six tumor-infiltrating immune cells, including naive B cells, regulatory T cells, memory resting CD4 T cells, memory B cells, activated mast cells, and resting NK cells, were significantly overexpressed in the low-TMB group relative to the high-TMB group. Furthermore, we identified FLNC, NEXN, and TNNT3 as TMB-related hub genes, and constructed their ceRNA networks, including five miRNAs (has-miR-590-3p, has-miR-374b-5p, has-miR-3127-5p, has-miR-1913, and has-miR-1291) and 31 lncRNAs (FAM66C, MIAT, NR2F2AS1, etc.). Finally, we observed that TMB-related genes were associated with distinct therapeutic responses to AKT/mTOR pathway inhibitors.Conclusions: We identified three TMB-associated key genes, established their ceRNA networks, and investigated their influence on therapeutic responses, which could provide insights into future precision medicine.

CSN6 promotes melanoma proliferation and metastasis by controlling the UBR5-mediated ubiquitination and degradation of CDK9

As a critical subunit of the constitutive photomorphogenesis 9 (COP9) signalosome (CSN), CSN6 is upregulated in some human cancers and plays critical roles in tumorigenesis and progression, but its biological functions and molecular mechanisms in melanoma remain unknown. Our study showed that CSN6 expression was upregulated in melanoma patients and cells, and correlated with poor survival in melanoma patients. In melanoma cells, CSN6 knockdown remarkably inhibited cell proliferation, tumorigenicity, migration, and invasion, whereas CSN6 recovery rescued the proliferative and metastatic abilities. Notably, we identified that CSN6 stabilized CDK9 expression by reducing CDK9 ubiquitination levels, thereby activating CDK9-mediated signaling pathways. In addition, our study described a novel CSN6-interacting E3 ligase UBR5, which was negatively regulated by CSN6 and could regulate the ubiquitination and degradation of CDK9 in melanoma cells. Furthermore, in CSN6-knockdown melanoma cells, UBR5 knockdown abrogated the effects caused by CSN6 silencing, suggesting that CSN6 activates the UBR5/CDK9 pathway to promote melanoma cell proliferation and metastasis. Thus, this study illustrates the mechanism by which the CSN6-UBR5-CDK9 axis promotes melanoma development, and demonstrate that CSN6 may be a potential biomarker and anticancer target in melanoma.

Abstract 6585: Systems biology for investigating drug resistance mechanism of melanoma

Abstract Integrated proteomic and metabolic single-cell assays reveal multiple independent adaptive responses to drug tolerance in a BRAF-mutant melanoma cell line Cancers commonly develop resistance against chemotherapeutics or targeted therapies through various types of genetic or non-genetic mechanisms. Non-genetic mechanisms have been shown to occur early on and can provide a latent reservoir of cells for the emergence of various different type of mechanisms, yet very limited understanding of process were resolve main from bulk analysis. Considering the heterogeneous nature of the tumor cells, a single-cell level characterization of the process worth detailed further investigation. Using MAPK inhibition of BRAF-mutant melanomas as a model system, we resolved that cells take different paths to go from drug-sensitive to drug-resistant state. Using a microfludic-based single-cell integrated proteomic and metabolic assay, we assayed for a panel of signaling, phenotypic, and metabolic regulators at four time points during the first five days of drug treatment. Dimensional reduction of the resultant data set, coupled with information theoretic analysis, uncovered a complex cell state landscape and identified two distinct paths connecting drug-naïve and drug-tolerant states. Cells are shown to exclusively traverse one of the two pathways depending on the level of the lineage restricted transcription factor MITF in the drug-naïve cells. The two trajectories are associated with distinct signaling and metabolic susceptibilities, and are independently druggable. Our results update the paradigm of adaptive resistance development in an isogenic cell population and offer insight into the design of more effective combination therapies. Citation Format: Yapeng Su, Guideng Li, Melissa Ko, Hanjun Cheng, Ronghui Zhu, Min Xue, Lidia Robert, Raphael Levine, Antoni Ribas, Garry Nolan, Wei Wei, Sylvia Plevritis, David Baltimore, James R. Heath. Systems biology for investigating drug resistance mechanism of melanoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6585.

Arginase-II promotes melanoma migration and adhesion through enhancing hydrogen peroxide production and STAT3 signaling.

Elevated arginase type II (Arg-II) associates with higher grade tumors. Its function and underlying molecular mechanisms in melanoma remain elusive. In the present study, we observed a significantly higher frequency of Arg-II expression in melanoma of patients with metastasis than those without metastasis. Silencing Arg-II in two human melanoma cell lines slowed down the cell growth, while overexpression of native but not a catalytically inactive Arg-II promoted cell proliferation without affecting cell death. Treatment of cells with arginase inhibitor also reduced melanoma cell number, demonstrating that Arg-II promotes melanoma cell proliferation dependently of its enzymatic activity. However, results from silencing Arg-II or overexpressing native or the inactive Arg-II as well as treatment with arginase inhibitor showed that Arg-II promotes melanoma metastasis-related processes, such as melanoma cell migration and adhesion on endothelial cells, independently of its enzymatic activity. Moreover, the treatment of the cells with STAT3 inhibitor suppressed Arg-II-promoted melanoma cell migration and adhesion. Furthermore, catalase, but not superoxide dismutase, prevented STAT3 activation as well as increased melanoma cell migration and adhesion induced by overexpressing native or the inactive Arg-II. Taken together, our study uncovers both activity-dependent and independent mechanisms of Arg-II in promoting melanoma progression. While Arg-II enhances melanoma cell proliferation through polyamine dependently of its enzymatic activity, it promotes metastasis-related processes, that is, migration and adhesion onto endothelial cell, through mitochondrial H2 O2 -STAT3 pathway independently of the enzymatic activity. Suppressing Arg-II expression rather than inhibiting its enzymatic activity may, therefore, represent a novel strategy for the treatment of melanoma.

Long non-coding RNA AFAP1-AS1 accelerates the progression of melanoma by targeting miR-653-5p/RAI14 axis

BackgroundMelanoma is the most aggressive skin cancer that derived from pigment cells, accounting for the majority of the skin-cancer-related deaths. Despite great development and evolution have been made in surgery, radiotherapy and adjuvant chemotherapy, the prognosis of melanoma patients exhibited no significant improvement. Long noncoding RNAs (lncRNAs) are frequently dysregulated and involved in the development of cancers. LncRNA AFAP1-AS1 has been explored in various cancers, whereas its role and regulatory mechanism in melanoma are not well understood.MethodsThe expression of AFAP1-AS1 was detected by qRT-PCR. CCK-8, colony formation, transwell and western blot assays were performed to investigate the biological role of AFAP1-AS1 in melanoma. Male BALB/c nude mice were applied for in vivo experiments. The interaction among AFAP1-AS1, miR-653-5p and RAI14 was investigated by RNA pull down, RIP and luciferase reporter assays.ResultsAFAP1-AS1 was highly expressed in melanoma cell lines. Suppression of AFAP1-AS1 impaired cell proliferation, migration, invasion and EMT in melanoma. Moreover, AFAP1-AS1 was a ceRNA of RAI14 by competitively binding with miR-653-5p. Besides, miR-653-5p overexpression or RAI14 inhibition could repress tumor growth. Eventually, rescue assays indicated that the function of AFAP1-AS1 in the cellular process of melanoma was dependent on miR-653-5p and RAI14.ConclusionsAFAP1-AS1 exerts its oncogenic function in melanoma by targeting miR-653-5p/RAI14 axis.

BET inhibitor suppresses melanoma progression via the noncanonical NF-κB/SPP1 pathway.

Background: Bromodomain and extra-terminal domain (BET) inhibitors have shown profound efficacy against hematologic malignancies and solid tumors in preclinical studies. However, the underlying molecular mechanism in melanoma is not well understood. Here we identified secreted phosphoprotein 1 (SPP1) as a melanoma driver and a crucial target of BET inhibitors in melanoma. Methods: Bioinformatics analysis and meta-analysis were used to evaluate the SPP1 expression in normal tissues, primary melanoma, and metastatic melanoma. Real-time PCR (RT-PCR) and Western blotting were employed to quantify SPP1 expression in melanoma cells and tissues. Cell proliferation, wound healing, and Transwell assays were carried out to evaluate the effects of SPP1 and BET inhibitors in melanoma cells in vitro. A xenograft mouse model was used to investigate the effect of SPP1 and BET inhibitors on melanoma in vivo. Chromatin immunoprecipitation (ChIP) assay was performed to evaluate the regulatory mechanism of BET inhibitors on SPP1. Results: SPP1 was identified as a melanoma driver by bioinformatics analysis, and meta-analysis determined it to be a diagnostic and prognostic biomarker for melanoma. SPP1 overexpression was associated with poor melanoma prognosis, and silencing SPP1 suppressed melanoma cell proliferation, migration, and invasion. Through a pilot drug screen, we identified BET inhibitors as ideal therapeutic agents that suppressed SPP1 expression. Also, SPP1 overexpression could partially reverse the suppressive effect of BET inhibitors on melanoma. We further demonstrated that bromodomain-containing 4 (BRD4) regulated SPP1 expression. Notably, BRD4 did not bind directly to the SPP1 promoter but regulated SPP1 expression through NFKB2. Silencing of NFKB2 resembled the phenotype of BET inhibitors treatment and SPP1 silencing in melanoma. Conclusion: Our findings highlight SPP1 as an essential target of BET inhibitors and provide a novel mechanism by which BET inhibitors suppress melanoma progression via the noncanonical NF-κB/SPP1 pathway.

Long noncoding RNA LINC00518 acts as a competing endogenous RNA to promote the metastasis of malignant melanoma via miR-204-5p/AP1S2 axis

Long intergenic nonprotein coding RNA 518 (LINC00518) has been shown to promote cancer cell growth and metastasis in some human tumors. Although it has been reported that LINC00518 is dysregulated in melanoma, its exact role and molecular mechanism in melanoma remain unclear. RNA-seq analysis and qRT-PCR was used to detect the expression of LINC00518 in melanoma tissues. Melanoma cases from The Cancer Genome Atlas (TCGA), GEO#GSE15605 and GEO#GSE24469 were included in this study. 3D migration, transwell and scratch wound assay were used to explore the role of LINC00518 in melanoma cells. Bioinformatics, luciferase reporter assays, MS2-RIP assay, RNA pull-down assay and RNA-ChIP assay were used to demonstrate the mechanism of LINC00518 in melanoma. We found that LICN00518 was significantly upregulated in melanoma tissue, and high LICN00518 level was an independent risk factor for melanoma patients. LICN00518 promoted the invasion and migration of melanoma cells. LICN00518 exerted its role by decoying miR-204-5p to upregulate Adaptor Related Protein Complex 1 Sigma 2 Subunit (AP1S2) expression. We also demonstrated that LICN00518 promoted melanoma metastasis in vivo through pulmonary metastasis assay. This result elucidates a new mechanism for LICN00518 in the metastasis of melanoma. LICN00518 may serve as a survival indicator and potential therapeutic target in melanoma patients.

784 A new immune evasion mechanism in melanoma

We present a novel and highly effective immune evasion mechanism in melanoma through the downregulation of man1a1, which codes for mannosidase I. This enzyme modifies glycosylation of cell-surface proteins. Using mass spectrometry, the glycocalyx of four human melanoma cell lines and normal human melanocytes (NHEM) were analyzed. All cancer lines expressed abnormal surface carbohydrates, specifically, an elevated ratio of high mannose sugars. RNA arrays of the human cancer cells showed downregulation of man1a1 compared to NHEM. To determine if this resulted in an altered glycocalyx, mouse melanoma cell line (B16) was treated with Kifunensine (Kif), a mannosidase I inhibitor. Kif-treated cells showed more high mannose structures, similar to human melanoma cell lines, compared to untreated cells. NK cells express receptors against various carbohydrates, providing targeting mechanisms for cell-mediated killing of tumor cells. Cytotoxicity assays using isolated mouse NK cells led to a 5-fold decrease in cell-mediated lysis of Kif-treated B16 cells compared to untreated cells. B16 cells overexpressing man1a1(B16+man1a1) were generated by stable transfection of a mammalian vector containing man1a1. The glycocalyx of these cells appeared more normal by mass spectrometry. Cytotoxicity assay of B16+man1a1 resulted in a 2-fold increase in cell lysis, compared to B16 transfected with empty vector. To test our hypothesis in vivo, a control mouse group and an experimental mouse group were subcutaneously and intravenously injected with either B16+man1a1 or B16 cells carrying empty vector, respectively. After two weeks, mouse skin and lungs were harvested. Compared to control mice, skin tumor volume was 10-fold smaller in mice injected with B16+man1a1 cells. There were 7-fold (p < 0.005) fewer metastatic colonies in lung parenchyma of mice injected intravenously with B16+man1a1 cells compared to control mice. Our results show glycosylation is a major factor in cancer immune evasion and/or suppression and can be reversed by overexpressing a single gene.

Size Matters: The Functional Role of the CEACAM1 Isoform Signature and Its Impact for NK Cell-Mediated Killing in Melanoma.

Malignant melanoma is the most aggressive and treatment resistant type of skin cancer. It is characterized by continuously rising incidence and high mortality rate due to its high metastatic potential. Various types of cell adhesion molecules have been implicated in tumor progression in melanoma. One of these, the carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), is a multi-functional receptor protein potentially expressed in epithelia, endothelia, and leukocytes. CEACAM1 often appears in four isoforms differing in the length of their extracellular and intracellular domains. Both the CEACAM1 expression in general, and the ratio of the expressed CEACAM1 splice variants appear very dynamic. They depend on both the cell activation stage and the cell growth phase. Interestingly, normal melanocytes are negative for CEACAM1, while melanomas often show high expression. As a cell–cell communication molecule, CEACAM1 mediates the direct interaction between tumor and immune cells. In the tumor cell this interaction leads to functional inhibitions, and indirectly to decreased cancer cell immunogenicity by down-regulation of ligands of the NKG2D receptor. On natural killer (NK) cells it inhibits NKG2D-mediated cytolysis and signaling. This review focuses on novel mechanistic insights into CEACAM1 isoforms for NK cell-mediated immune escape mechanisms in melanoma, and their clinical relevance in patients suffering from malignant melanoma.

Resistance mechanisms in melanoma to immuneoncologic therapy with checkpoint inhibitors.

Checkpoint inhibitors act by blocking physiologic mechanisms coopted by tumor cells to evade immune surveillance, restoring the immune system’s ability to identify and kill malignant cells. These therapies have dramatically improved outcomes in multiple tumor types with durable responses in many patients, leading to FDA approval first in advanced melanoma, then in many other malignancies. However, as experience with checkpoint inhibitors has grown, populations of patients who are primary nonresponders or develop secondary resistance have been the majority of cases, even in melanoma. Mechanisms of resistance include those inherent to the tumor microenvironment, the tumor cells themselves, and the function of the patient’s native immune cells. This review will discuss resistance to checkpoint inhibitors in melanoma as well as possible methods to restore sensitivity.

Targeting of TRX2 by miR-330-3p in melanoma inhibits proliferation

ObjectiveThis study is intended to identify the key gene from gene expression profile and validate its role and regulatory mechanism in melanoma. MethodsGene expression profile of GSE3189 from GEO database was selected among which 7 are normal skin samples, 18 are benign skin lesion samples, and 45 are melanoma samples. The present study examined the 7 normal skin samples and the 45 melanoma samples. Differentially expressed genes (DEGs) between melanoma patients and health people were performed using Morpheus online tool. The 100 most differentially expressed genes (50 upregulated genes and 50 downregulated genes) were selected as hub genes. Then, expression levels and survival analysis of hub genes were conducted via GEPIA tool to choose target gene. The expression of target gene in melanoma cell lines was examined by RT-qPCR and western blotting. The biological function of target gene on cell proliferation in melanoma was measured in vitro. The predicted target of target gene was validated by dual-luciferase reporter assay and rescue experiment. The gene expression in clinical samples were determined by RT-qPCR, immunohistochemistry (IHC) and in situ hybridization (ISH). The tumor formation study was conducted in vivo. ResultsTargeting protein for Xklp2 (TPX2) was identified as key gene in melanoma. TPX2 could promote the proliferation of melanoma cells. The dual luciferase reporter assay confirmed that miR-330-3p targets TPX2. In rescue experiment, it was proved that miR-330-3p inhibits the proliferation of melanoma cells by negatively regulating the expression of TPX2. The results in vitro were also confirmed in vivo. miR-330-3p/TPX2 pathway expressed differently between melanoma patients and health people. These differences were statistically significant (P < 0.05). ConclusionInhibiting TPX2 by miR-330-3p suppresses the proliferation of melanoma cell lines. miR-330-3p/TPX2 pathway could be a potential therapeutic target in melanoma.

Abstract 2530: An analysis of correlative interactions between Plk1, Notch1 and FoxM1 in melanoma

Abstract Melanoma is one of the most aggressive forms of skin cancer, and existing therapies have not been effective in managing this neoplasm. Therefore, a deeper understanding of the mechanisms of melanoma development/progression is required. The goal of this study was to define the potential interactions between several key melanoma-linked proteins, namely Polo-like kinase 1 (Plk1; a cell cycle regulator involved in G2/M transition), Notch (Notch1; a type I transmembrane protein that decides cell fate during development), and forkhead box M1 (FoxM1; a transcription factor regulating S and G2/M phases of cell cycle) in human melanoma. Previously, we have shown that Plk1 is overexpressed in melanoma, and its inhibition results in G2/M phase arrest and apoptosis in multiple melanoma cells. Recent studies have shown that both Notch1 and FoxM1 may be involved in the pathogenesis of certain cancers, including melanoma. In this study, employing an in-house human tissue microarray (TMA) coupled with high-throughput, multispectral Vectra scanning and inForm analysis, we analyzed 126 clinical tissue specimens of normal skin and localized cutaneous melanoma. The TMA was co-immunostained for Plk1, Notch1, FoxM1, and the melanoma biomarker S100, followed by Vectra scanning and analyses with inForm software. Using this system, we determined the levels of Plk1, Notch1, and FoxM1 in both the nucleus and cytoplasm of melanoma cells which were positive for S100 staining. By applying statistical tools of Wilcoxon Signed-Rank Test and Pearson's Correlation Coefficient in R programming, we found that while all the proteins are detected in both nucleus and cytoplasm, FoxM1 and Notch1 are enriched in the nucleus, and Plk1 is enriched in the cytoplasm. Visualization of the correlation coefficients on a heat-map showed that all 3 sets of proteins (Plk1:Notch1, Plk1:FoxM1, and Notch1:FoxM1) are positively correlated, with Plk1:Notch1 having the strongest correlation, and FoxM1 having a weaker correlation with the other two proteins. The associative relationship between Plk1 and Notch1 leads to the hypothesis that Plk1 and Notch1 may be positively correlated in melanoma. We further studied how Breslow thickness, a measure of melanoma aggressiveness, is related to Plk1, Notch1 and FoxM1. By analyzing the TMA cores with available clinical data, we found a positive correlation between Breslow thickness and Notch1, as well as Plk1. Interestingly, although Breslow thickness was significantly correlated with FoxM1 in the nucleus, no correlation was found in the cytoplasm, suggesting that FoxM1 levels may be independent of Plk1 and Notch1. Overall, our study found novel correlative interactions between Plk1 and Notch1 in melanoma, which may have relevance to melanoma progression. Additional studies are needed to validate our findings and dissect the possible interaction between Plk1 and Notch1 during melanoma development and progression. Citation Format: Shengqin Su, Chandra K. Singh, Mary A. Ndiaye, Vijayasaradhi Setaluri, Nihal Ahmad. An analysis of correlative interactions between Plk1, Notch1 and FoxM1 in melanoma [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 2530.