Pathogenesis Of Melanoma Research Articles

BRAF Mutations in Melanoma: Biological Aspects, Therapeutic Implications, and Circulating Biomarkers.

Melanoma is an aggressive form of skin cancer resulting from the malignant transformation of melanocytes. Recent therapeutic approaches, including targeted therapy and immunotherapy, have improved the prognosis and outcome of melanoma patients. BRAF is one of the most frequently mutated oncogenes recognised in melanoma. The most frequent oncogenic BRAF mutations consist of a single point mutation at codon 600 (mostly V600E) that leads to constitutive activation of the BRAF/MEK/ERK (MAPK) signalling pathway. Therefore, mutated BRAF has become a useful target for molecular therapy and the use of BRAF kinase inhibitors has shown promising results. However, several resistance mechanisms invariably develop leading to therapeutic failure. The aim of this manuscript is to review the role of BRAF mutational status in the pathogenesis of melanoma and its impact on differentiation and inflammation. Moreover, this review focuses on the mechanisms responsible for resistance to targeted therapies in BRAF-mutated melanoma and provides an overview of circulating biomarkers including circulating tumour cells, circulating tumour DNA, and non-coding RNAs.

SPI1 involvement in malignant melanoma pathogenesis by regulation of HK2 through the AKT1/mTOR pathway.

Spi-1 proto-oncogene (SPI1) plays a vital role in carcinogenesis. Our work aimed to investigate the potential regulatory mechanism of SPI1 in melanoma. The mRNA and protein levels were measured via qRT-PCR and Western blotting. Cell viability was assessed by CCK-8 assay. The target relationship between SPI1 and hexokinase 2 (HK2) was determined using dual-luciferase reporter detection. ChIP was conducted to confirm the targeted relationship between SPI1 and the HK2 promoter. Immunohistochemistry analysis was conducted to measure the positive cell number of SPI1 and HK2 in melanoma tissues. The cell migration abilities were determined using a wound healing assay. Glucose consumption, pyruvate dehydrogenase activity, lactate production and ATP levels were measured to assess glycolysis. SPI1 transcription in melanoma cells and tissues was dramatically higher than that in adjacent normal tissues and epidermal melanocyte HEMa-LP, respectively. Knockdown of SPI1 restrained cell viability, metastasis and glycolysis in melanoma cells. SPI1 directly targeted HK2, and knockdown of SPI1 repressed HK2 expression. Overexpression of HK2 weakened the inhibitory effects of SPI1 knockdown on the viability, metastasis and glycolysis of melanoma cells. The serine-threonine kinase 1 (AKT1)/mammalian target of rapamycin (mTOR) axis is involved in melanoma progression. SPI1 knockdown restrained melanoma cell proliferation, metastasis and glycolysis by regulating the AKT1/mTOR pathway.

Darovasertib, a novel treatment for metastatic uveal melanoma.

The FDA granted orphan drug designation to darovasertib, a first-in-class oral, small molecular inhibitor of protein kinase C (PKC), for the treatment of uveal melanoma, on 2 May 2022. Primary uveal melanoma has a high risk of progressing to metastatic uveal melanoma, with a poor prognosis. The activation of the PKC and mitogen-activated protein kinase pathways play an essential role in the pathogenesis of uveal melanoma, and mutations in the G protein subunit alpha q (GNAQ), and G protein subunit alpha11 (GNA11) genes are considered early events in the development of uveal melanoma. Compared to other PKC inhibitors, such as sotrastaurin and enzastaurin, darovasertib is significantly more potent in inhibiting conventional (α, β) and novel (δ, ϵ, η, θ) PKC proteins and has a better tolerability and safety profile. Current Phase I/II clinical trials indicated that darovasertib, combined with the Mitogen-activated protein kinase/Extracellular (MEK) inhibitors, binimetinib or crizotinib, produced a synergistic effect of uveal melanoma. In this article, we summarize the development of drugs for treating uveal melanomas and discuss problems associated with current treatments. We also discuss the mechanism of action, pharmacokinetic profile, adverse effects, and clinical trial for darovasertib, and future research directions for treating uveal melanoma.

Anorectal melanoma.

Anorectal melanoma is an aggressive mucosal melanoma subtype with a poor prognosis. Although recent advancements have been seen for cutaneous melanoma, the optimal treatment paradigm for management of anorectal melanoma is evolving. In this review, we highlight differences in the pathogenesis of mucosal versus cutaneous melanoma, new concepts of staging for mucosal melanoma, updates to surgical management of anorectal melanoma, and current data for adjuvant radiation and systemic therapy in this unique patient population.

BCKDHA contributes to melanoma progression by promoting the expressions of lipogenic enzymes FASN and ACLY.

The dysregulation of branched-chain amino acid (BCAA) metabolism and related enzymes has been greatly implicated in the progression of multiple types of cancer, whereas remains far from understood in melanoma. Here, we explored the role of the BCAA metabolism enzyme BCKDHA in melanoma pathogenesis and elucidated the underlying mechanisms. In vitro cell biology experiments and in vivo pre-clinical mice model experiments were performed to investigate the role of BCKDHA in melanoma progression. RNA sequencing, immunohistochemical/immunofluorescence staining and bioinformatics analysis were used to examine the underlying mechanism. BCKDHA expression was prominently increased in both melanoma tissues and cell lines. The up-regulation of BCKDHA promoted long-term tumour cell proliferation, invasion and migration in vitro and tumour growth in vivo. Through RNA-sequencing technology, it was found that BCKDHA regulated the expressions of lipogenic fatty acid synthase (FASN) and ATP-citrate lyase (ACLY), which was thereafter proved to mediate the oncogenic role of BCKDHA in melanoma. Our results demonstrate that BCKDHA promotes melanoma progression by regulating FASN and ACLY expressions. Targeting BCKDHA could be exploited as a promising strategy to restrain tumour progression in melanoma.

DP03 (P23) Primary penile melanoma and penile lichen sclerosus

Abstract The association between squamous cell malignancies and genital lichen sclerosus (GLSc) is well established. There is an increasing literature regarding the relationship of GLSc with genital malignant melanoma (MM). While there have been several reported cases of vulvar melanoma associated with lichen sclerosus (LSc), there have been fewer cases published with regard to male genital (M)GLSc and penile (Pe)MM. The aims of this study were to explore the relationship between PeMM and MGLSc by reviewing all cases managed by our multidisciplinary service over a period of 11 years. All patients known to our tertiary urology and male genital dermatology service with a diagnosis of penile MM and where histology was available for review were identified over an 11-year period (2011–2022). Two independent dermatologists reviewed the clinical records and photographs where available. Two independent mutually blinded histopathologists reviewed the histology and recorded features of GLSc where present. In cases of discordance between histopathologists, a third independent histopathologist reviewed the cases. Eleven patients were identified for inclusion in the study. Overall, GLSc was present on histopathology in nine of 11 cases (82%). Findings were concordantly positive in five cases and concordantly negative in one case. In cases of discordant findings (n = 5), the third histopathologist determined GLSc to be present in four cases and absent in one case. Clinical photographs were available for three cases, and clinical features of GLSc were seen in all of them; they also all had positive histopathological findings. Clinical notes, including electronic notes and letters, were available for review in eight cases. Symptoms in keeping with GLSc were mentioned in only one case, and signs suggestive of GLSc were documented in three cases. Penile MM is a rare entity accounting for 0.7% of penile cancer and 0.1% of MM cases. Like other malignancies, MM is a multifactorial disease caused by a combination of environmental factors and altered host responses contributing to an alteration of regulatory processes. Although ultraviolet exposure is important in the pathogenesis of extragenital MM, this cannot be a factor for genital disease. However, specific risk factors for genital MM have not yet been elucidated. To our knowledge, there have only been three previously documented cases (all from our group) of PeMM coexisting with GLSc. In this present study, finding of GLSc in nine of 11 men with PeMM is suggestive of a causative relationship. We postulate that this is related to chronic melanocytic distress created by chronic inflammation secondary to LSc.

P23 Primary penile melanoma and penile lichen sclerosus

Abstract The association between squamous cell malignancies and genital lichen sclerosus (GLSc) is well established. There is an increasing literature regarding the relationship of GLSc with genital malignant melanoma (MM). While there have been several reported cases of vulvar melanoma associated with lichen sclerosus (LSc), there have been fewer cases published with regard to male genital (M)GLSc and penile (Pe)MM. The aims of this study were to explore the relationship between PeMM and MGLSc by reviewing all cases managed by our multidisciplinary service over a period of 11 years. All patients known to our tertiary urology and male genital dermatology service with a diagnosis of penile MM and where histology was available for review were identified over an 11-year period (2011–2022). Two independent dermatologists reviewed the clinical records and photographs where available. Two independent mutually blinded histopathologists reviewed the histology and recorded features of GLSc where present. In cases of discordance between histopathologists, a third independent histopathologist reviewed the cases. Eleven patients were identified for inclusion in the study. Overall, GLSc was present on histopathology in nine of 11 cases (82%). Findings were concordantly positive in five cases and concordantly negative in one case. In cases with discordant findings (n = 5), the third histopathologist determined GLSc to be present in four cases and absent in one case. Clinical photographs were available for three cases and clinical features of GLSc were seen in all of them; they also all had positive histopathological findings. Clinical notes, including electronic notes and letters, were available for review in eight cases. Symptoms in keeping with GLSc were mentioned in only one case, and signs suggestive of GLSc were documented in three cases. Penile MM is a rare entity accounting for 0.7% of penile cancer and 0.1% of MM cases. Like other malignancies, MM is a multifactorial disease caused by a combination of environmental factors and altered host responses contributing to an alteration of regulatory processes. Although ultraviolet exposure is important in the pathogenesis of extragenital MM, this cannot be a factor for genital disease. However, specific risk factors for genital MM have not yet been elucidated. To our knowledge, there have only been three previously documented cases (all from our group) of PeMM coexisting with GLSc. This present study finding of GLSc in 9 of 11 men with PeMM, is suggestive of a causative relationship. We postulate that this is related to chronic melanocytic distress created by chronic inflammation secondary to LSc.

Management of Advanced Invasive Melanoma: New Strategies.

The incidence of cutaneous melanoma (CM) is increasing. CM is defined as melanoma in situ when limited within the epidermis and invasive when atypical melanocytes progressively invade the dermis. Treatment of CM is challenging. On one hand, melanoma in situ does not require further treatment except for a limited secondary excision with reduced margins to minimize the risk of local recurrences; on the other, invasive melanoma requires a personalized approach based on tumor staging. Consequently, an association of surgical and medical treatments is often necessary for invasive forms of the disease. In this scenario, new knowledge on melanoma pathogenesis has led to the development of safe and effective treatments, and several drugs are currently under investigation. However, extensive knowledge is required to offer patients a tailored-tail approach. The aim of our article was to review current literature to provide an overview of treatment options for invasive melanoma, highlighting strategical approaches that can be used in patients with these forms of disease.

Knocking out alpha-synuclein in melanoma cells downregulates L1CAM and decreases motility

The Parkinson’s disease (PD) associated protein, alpha-synuclein (α-syn/SNCA), is highly expressed in aggressive melanomas. The goal of this study was to reveal possible mechanism(s) of α-syn involvement in melanoma pathogenesis. Herein, we asked whether α-syn modulates the expression of the pro-oncogenic adhesion molecules L1CAM and N-cadherin. We used two human melanoma cell lines (SK-MEL-28, SK-MEL-29), SNCA-knockout (KO) clones, and two human SH-SY5Y neuroblastoma cell lines. In the melanoma lines, loss of α-syn expression resulted in significant decreases in the expression of L1CAM and N-cadherin and concomitant significant decreases in motility. On average, there was a 75% reduction in motility in the four SNCA-KOs tested compared to control cells. Strikingly, comparing neuroblastoma SH-SY5Y cells that have no detectable α-syn to SH-SY5Y cells that stably express α-syn (SH/+αS), we found that expressing α-syn increased L1CAM and single-cell motility by 54% and 597%, respectively. The reduction in L1CAM level in SNCA-KO clones was not due to a transcriptional effect, rather we found that L1CAM is more efficiently degraded in the lysosome in SNCA-KO clones than in control cells. We propose that α-syn is pro-survival to melanoma (and possibly neuroblastoma) because it promotes the intracellular trafficking of L1CAM to the plasma membrane.

Targeting ferroptosis as a cell death pathway in Melanoma: From molecular mechanisms to skin cancer treatment.

Melanoma, the most aggressive form of human skin cancer, has been under investigation to reach the most efficient treatment. Surgical resection for early-diagnosed primary melanoma, targeted therapies, and immune checkpoint inhibitors for advanced/metastatic melanoma is the best clinical approach. Ferroptosis, a newly identified iron-dependent cell death pathway, which is morphologically and biochemically different from apoptosis and necrosis, has been reported to be involved in several cancers. Ferroptosis inducers could provide therapeutic options in case of resistance to conventional therapies for advanced/metastatic melanoma. Recently developed ferroptosis inducers, MEK and BRAF inhibitors, miRNAs such as miR-137 and miR-9, and novel strategies for targeting major histocompatibility complex (MHC) class II in melanoma can provide new opportunities for melanoma treatment. Combining ferroptosis inducers with targeted therapies or immune checkpoint inhibitors increases patient response rates. Here we review the mechanisms of ferroptosis and its environmental triggers. We also discuss the pathogenesis and current treatments of melanoma. Moreover, we aim to elucidate the relationship between ferroptosis and melanoma and ferroptosis implications to develop new therapeutic strategies against melanoma.

In vitro and in silico study on the effect of carvedilol and sorafenib alone and in combination on the growth and inflammatory response of melanoma cells

Melanoma is an aggressive skin cancer. Increasing evidence has shown the role of β-adrenergic receptors in the pathogenesis of melanoma. Carvedilol is a widely used non-selective β-AR antagonist with potential anticancer activity. The purpose of the study was to estimate the influence of carvedilol and sorafenib alone and in combination on the growth and inflammatory response of C32 and A2058 melanoma cells. Furthermore, this study also aimed to predict the probable interaction of carvedilol and sorafenib when administered together. Predictive study of the interaction of carvedilol and sorafenib was performed using the ChemDIS-Mixture system. Carvedilol and sorafenib alone and in combination showed a growth inhibitory effect on cells. The greatest synergistic antiproliferative effect on both cell lines was observed at Car 5 μM combined with Sor 5 μM. Analysis in silico identified diseases, proteins, and metabolic pathways that can be affected by the interaction of carvedilol and sorafenib. The results obtained demonstrated that carvedilol and sorafenib modulated the secretion of IL-8 by IL-1β-stimulated by melanoma cell lines but the use of a combination of both drugs did not intensify the effect. In summary, the results presented indicate that the combination of carvedilol and sorafenib may have a promising anticancer effect on melanoma cells.

A comprehensive review on novel targeted therapy methods and nanotechnology-based gene delivery systems in melanoma

Melanoma, a malignant form of skin cancer, has been swiftly increasing in recent years. Although there have been significant advancements in clinical treatment underlying a well-understanding of melanoma-susceptible genes and the molecular basis of melanoma pathogenesis, the permanency of response to therapy is frequently constrained by the emergence of acquired resistance and systemic toxicity. Conventional therapies, including surgical resection, chemotherapy, radiotherapy, and immunotherapy, have already been used to treat melanoma and are dependent on the cancer stage. Nevertheless, ineffective side effects and the heterogeneity of tumors pose major obstacles to the therapeutic treatment of malignant melanoma through such strategies. In light of this, advanced therapies including nucleic acid therapies (ncRNA, aptamers), suicide gene therapies, and gene therapy using tumor suppressor genes, have lately gained immense attention in the field of cancer treatment. Furthermore, nanomedicine and targeted therapy based on gene editing tools have been applied to the treatment of melanoma as potential cancer treatment approaches nowadays. Indeed, nanovectors enable delivery of the therapeutic agents into the tumor sites by passive or active targeting, improving therapeutic efficiency and minimizing adverse effects. Accordingly, in this review, we summarized the recent findings related to novel targeted therapy methods as well as nanotechnology-based gene systems in melanoma. We also discussed current issues along with potential directions for future research, paving the way for the next-generation of melanoma treatments.

The Cross Talk between Cellular Senescence and Melanoma: From Molecular Pathogenesis to Target Therapies.

Melanoma is a malignant skin tumor that originates from melanocytes. The pathogenesis of melanoma involves a complex interaction that occurs between environmental factors, ultraviolet (UV)-light damage, and genetic alterations. UV light is the primary driver of the skin aging process and development of melanoma, which can induce reactive oxygen species (ROS) production and the presence of DNA damage in the cells, and results in cell senescence. As cellular senescence plays an important role in the relationship that exists between the skin aging process and the development of melanoma, the present study provides insight into the literature concerning the topic at present and discusses the relationship between skin aging and melanoma, including the mechanisms of cellular senescence that drive melanoma progression, the microenvironment in relation to skin aging and melanoma factors, and the therapeutics concerning melanoma. This review focuses on defining the role of cellular senescence in the process of melanoma carcinogenesis and discusses the targeting of senescent cells through therapeutic approaches, highlighting the areas that require more extensive research in the field.

Signaling Switching from Hedgehog-GLI to MAPK Signaling Potentially Serves as a Compensatory Mechanism in Melanoma Cell Lines Resistant to GANT-61.

Melanoma represents the deadliest skin cancer due to its cell plasticity which results in high metastatic potential and chemoresistance. Melanomas frequently develop resistance to targeted therapy; therefore, new combination therapy strategies are required. Non-canonical signaling interactions between HH-GLI and RAS/RAF/ERK signaling were identified as one of the drivers of melanoma pathogenesis. Therefore, we decided to investigate the importance of these non-canonical interactions in chemoresistance, and examine the potential for HH-GLI and RAS/RAF/ERK combined therapy. We established two melanoma cell lines resistant to the GLI inhibitor, GANT-61, and characterized their response to other HH-GLI and RAS/RAF/ERK inhibitors. We successfully established two melanoma cell lines resistant to GANT-61. Both cell lines showed HH-GLI signaling downregulation and increased invasive cell properties like migration potential, colony forming capacity, and EMT. However, they differed in MAPK signaling activity, cell cycle regulation, and primary cilia formation, suggesting different potential mechanisms responsible for resistance occurrence. Our study provides the first ever insights into cell lines resistant to GANT-61 and shows potential mechanisms connected to HH-GLI and MAPK signaling which may represent new hot spots for noncanonical signaling interactions.

A BRAF mutation-associated gene risk model for predicting the prognosis of melanoma

BRAF mutation plays an important role in the pathogenesis and progression of melanoma and is correlated to the prognosis of melanoma patients. However, fewer studies have attempted to develop a BRAF mutation-associated gene risk model for predicting the prognosis of melanoma. The current research explores BRAF mutation-related biological features in melanoma and establishes a prognostic signature. First, we identified three significantly enriched KEGG pathways (glycosphingolipid biosynthesis - ganglio series, ether lipid metabolism, and glycosaminoglycan biosynthesis - keratan sulfate) and corresponding genes in the BRAF mutant group by gene set enrichment analysis. We then developed a prognostic signature based on 7 BRAF-associated genes (PLA2G2D, FUT8, PLA2G4E, PLA2G5, PLA2G1B, B3GNT2, and ST3GAL5) and assessed its prediction accuracy using ROC curve analysis. Finally, the nomogram was established according to the prognostic signature and independent clinical characteristics to predict the survival of melanoma patients. Furthermore, we found higher proportions of naive B cells, plasma cells, CD8 T cells, CD4 memory-activated T cells, and regulatory T cells in the low-risk group. Whereas lower proportions of M0, M1, and M2 macrophages and resting NK cells were observed in the high-risk group. The analysis also showed a significantly higher expression of immune checkpoint molecules (PD-1, PD-L1, CTLA4, BTLA, CD28, CD80, CD86, HAVCR2, ICOS, LAG3, and TIGIT) in the low-risk group. Our results provide novel insights into the effect of BRAF mutation on melanoma growth and indicate a promising direction toward immunotherapy and precision medicine in melanoma patients.

Identification of CRKL as an oncogenic biomarker for prognosis and immunotherapy in melanoma, and its potential molecular mechanism

CRKL (CRK Like Proto-Oncogene) belongs to the Crk family and is a 39-kDa adapter protein that encodes SH2 and SH3 (src homologs) domains. To identify its oncogenic role in malignant melanoma, we investigated the association between CRKL and mutation, prognosis, tumor mutation burden, immune cell infiltration of melanoma, and explored the associations between CRKL and immunotherapy response. Our results showed that abnormal CRKL expression is associated with poor prognosis in melanoma and is significantly correlated with immune-activated pathways and processes, immune cell infiltrations, and expression of immunoregulators. Importantly, we found that CRKL expression is a predictive biomarker for anti-PD1 therapy response in melanoma patients. Furthermore, inhibiting CRKL expression in melanoma cell lines suppressed their proliferation and metastasis, as well as activated the pyroptosis-related pathway. Our study provides potential mechanisms of melanoma pathogenesis, which may suggest new avenues for targeted therapy in this disease.

Differential Expression of Autophagy-Related Long Non-Coding RNA in Melanoma

To explore the role of autophagy-related differential long non-coding RNA (lncRNA) in the pathogenesis of melanoma, we established a prognostic prediction model for patients with melanoma based on the expression profiles of autophagy-related gene. Based on The Cancer Genome Atlas and GeneCard database, we used single-sample gene set enrichment analysis (ssGSEA), weighted gene co-expression network analysis (WGCNA), uniCOX in R software for COX proportional hazard regression analysis, and enrichment analysis to get an idea of biological processes with autophagy-related genes, which evaluates the relationship between autophagy-related genes and immune cell infiltration in patients with melanoma. The roles of identified lncRNA were evaluated by the risk score based on the results of single factor regression analysis for each lncRNA and on the prognosis for patients obtained from the database. Then, the whole sample was divided into high- and low-risk groups. Survival curve analysis showed that low-risk group had a better prognosis. Enrichment analysis revealed multiple key pathways enriched with lncRNA-associated genes. Analysis of immune cell infiltration revealed differences between high- and low-risk groups. Finally, 3 datasets verified the effect of our model on prognosis. There are important autophagy-related lncRNA in patients with melanoma. Top 6 lncRNA are significantly related to the overall survival rate of patients with melanoma and provide the basis for predicting the prognostic survival of patients.

Current Insights into the Role of BRAF Inhibitors in Treatment of Melanoma.

Melanomas represent only 4% of all skin cancers, but their mortality rate is more than 50 % of any other skin cancer. Alteration in genetic and environmental factors are the risk factors for melanoma development. The RAS/RAF/MEK/ERK or Mitogen-activated protein kinase (MAPK) pathway is activated in melanoma. BRAF activation is necessary to govern differentiation, proliferation, and survival. Mutations in BRAF were found in 80-90% of all melanomas. Over 90% of BRAF mutations occur at codon 600, and over 90% of them are BRAFV600E other common mutations are BRAFV600K, BRAFV600R, BRAF V600'E2', and BRAF V600D. Based on αC-helix and DFG motif (αC-helix-IN/DFG-IN), (αC-helix-IN/DFG-OUT), (αC-helix-OUT/DFG-IN) and (αC-helix-OUT/ DFG-OUT) are four structural types of inhibitors for targeting BRAF. Sorafenib, Vemurafenib, Dabrafenib, and Encorafenib are FDAapproved for the treatment of BRAF. Understanding melanoma pathogenesis, RAS/RAF/MEK/ERK or MAPK pathway, and BRAF conformations, mutations, the problems with FDA approved BRAF inhibitors will be important for new drug discovery, modification of existing BRAF barriers to improve target specific action, and prevent increasing response levels while minimizing toxicity.

IFNα and 5-Aza-2'-deoxycytidine combined with a dendritic-cell targeting DNA vaccine alter tumor immune cell infiltration in the B16F10 melanoma model.

DNA vaccines containing a fusion of the gene encoding chemokine MIP-3α (CCL20), the ligand for CCR6 on immature dendritic cells (DCs), to melanoma-associated antigen genes have enhanced anti-tumor immunity and efficacy compared to those lacking the chemokine gene. Previous work has shown that type-I interferon (IFNα or IFN) and 5-Aza-2'-deoxycytidine (5Aza) significantly enhance the therapeutic benefit of DNA vaccines as measured by reduced tumor burden and improved mouse survival. Here, we explored mouse intratumoral immune correlates underlying the therapeutic benefit of this combination regimen (vaccine, IFN, and 5Aza) as compared to vaccine alone and IFN and 5Aza without vaccine, focusing on chemokine mRNA expression by qRT-PCR and inflammatory cellular infiltration into the tumor microenvironment (TME) by flow cytometry and immunohistochemistry (IHC). The combination group significantly upregulated intratumoral mRNA expression of key immune infiltration chemokines XCL1 and CXCL10. Flow cytometric analyses of tumor suspensions exhibited greater tumor infiltration of CD8+ DCs, CCR7+ DCs, and NK cells in the combination group, as well as reduced levels of myeloid-derived suppressor cells (MDSCs) in vaccinated groups. The mice receiving combination therapy also had greater proportions of effector/memory T-cells (Tem), in addition to showing an enhanced infiltration of Tem and central memory CD8+ T-cells, (Tcm). Tem and Tcm populations both correlated with smaller tumor size. Immunohistochemical analysis of tumors confirmed that CD8+ cells were more abundant overall and especially in the tumor parenchyma with combination therapy. Efficient targeting of antigen to immature DCs with a chemokine-fusion vaccine offers a potential alternative approach to classic and dendritic cell-based vaccines. Combining this approach with IFNα and 5Aza treatments significantly improved vaccine efficacy. This treatment creates an environment of increased inflammatory chemokines that facilitates the trafficking of CD8+ DCs, NK cells, and CD8+ T-cells, especially memory cells, while reducing the number of MDSCs. Importantly, in the combination group, CD8+ cells were more able to penetrate the tumor mass in addition to being more numerous. Further analysis of the pathways engaged by our combination therapy is expected to provide additional insights into melanoma pathogenesis and facilitate the development of novel treatment strategies.

PCSK9 facilitates melanoma pathogenesis via a network regulating tumor immunity

BackgroundPCSK9 regulates cholesterol homeostasis and promotes tumorigenesis. However, the relevance of these two actions and the mechanisms underlying PCSK9’s oncogenic roles in melanoma and other cancers remain unclear.MethodsPCSK9’s association with melanoma was analysed using the TCGA dataset. Empty vector (EV), PCSK9, gain-of-function (D374Y), and loss-of-function (Q152H) PCSK9 mutant were stably-expressed in murine melanoma B16 cells and studied for impact on B16 cell-derived oncogenesis in vitro and in vivo using syngeneic C57BL/6 and Pcsk9−/− mice. Intratumoral accumulation of cholesterol was determined. RNA-seq was performed on individual tumor types. Differentially-expressed genes (DEGs) were derived from the comparisons of B16 PCSK9, B16 D374Y, or B16 Q152H tumors to B16 EV allografts and analysed for pathway alterations.ResultsPCSK9 expression and its network negatively correlated with the survival probability of patients with melanoma. PCSK9 promoted B16 cell proliferation, migration, and growth in soft agar in vitro, formation of tumors in C57BL/6 mice in vivo, and accumulation of intratumoral cholesterol in a manner reflecting its regulation of the low-density lipoprotein receptor (LDLR): Q152H, EV, PCSK9, and D374Y. Tumor-associated T cells, CD8 + T cells, and NK cells were significantly increased in D374Y tumors along with upregulations of multiple immune checkpoints, IFNγ, and 143 genes associated with T cell dysfunction. Overlap of 36 genes between the D374Y DEGs and the PCSK9 DEGs predicted poor prognosis of melanoma and resistance to immune checkpoint blockade (ICB) therapy. CYTH4, DENND1C, AOAH, TBC1D10C, EPSTI1, GIMAP7, and FASL (FAS ligand) were novel predictors of ICB therapy and displayed high level of correlations with multiple immune checkpoints in melanoma and across 30 human cancers. We observed FAS ligand being among the most robust biomarkers of ICB treatment and constructed two novel and effective multigene panels predicting response to ICB therapy. The profiles of allografts produced by B16 EV, PCSK9, D374Y, and Q152H remained comparable in C57BL/6 and Pcsk9−/− mice.ConclusionsTumor-derived PCSK9 plays a critical role in melanoma pathogenesis. PCSK9’s oncogenic actions are associated with intratumoral cholesterol accumulation. PCSK9 systemically affects the immune system, contributing to melanoma immune evasion. Novel biomarkers derived from the PCSK9-network effectively predicted ICB therapy responses.