Human Cutaneous Melanoma Research Articles

Unraveling the Role of JMJD1B in Genome Stability and the Malignancy of Melanomas.

Genome instability relies on preserving the chromatin structure, with any histone imbalances threating DNA integrity. Histone synthesis occurs in the cytoplasm, followed by a maturation process before their nuclear translocation. This maturation involves protein folding and the establishment of post-translational modifications. Disruptions in this pathway hinder chromatin assembly and contribute to genome instability. JMJD1B, a histone demethylase, not only regulates gene expression but also ensures a proper supply of histones H3 and H4 for the chromatin assembly. Reduced JMJD1B levels lead to the cytoplasmic accumulation of histones, causing defects in the chromatin assembly and resulting in DNA damage. To investigate the role of JMJD1B in regulating genome stability and the malignancy of melanoma tumors, we used a JMJD1B/KDM3B knockout in B16F10 mouse melanoma cells to perform tumorigenic and genome instability assays. Additionally, we analyzed the transcriptomic data of human cutaneous melanoma tumors. Our results show the enhanced tumorigenic properties of JMJD1B knockout melanoma cells both in vitro and in vivo. The γH2AX staining, Micrococcal Nuclease sensitivity, and comet assays demonstrated increased DNA damage and genome instability. The JMJD1B expression in human melanoma tumors correlates with a lower mutational burden and fewer oncogenic driver mutations. Our findings highlight JMJD1B's role in maintaining genome integrity by ensuring a proper histone supply to the nucleus, expanding its function beyond gene expression regulation. JMJD1B emerges as a crucial player in preserving genome stability and the development of melanoma, with a potential role as a safeguard against oncogenic mutations.

G protein-coupled estrogen receptor 1 and collagen XVII endodomain expression in human cutaneous melanomas: can they serve as prognostic factors?

Melanoma incidence is increasing globally. Although novel therapies have improved the survival of primary melanoma patients over the past decade, the overall survival rate for metastatic melanoma remains low. In addition to traditional prognostic factors such as Breslow thickness, ulceration, and mitotic rate, novel genetic and molecular markers have been investigated. In our study, we analyzed the expression of G-protein coupled estrogen receptor 1 (GPER1) and the endodomain of collagen XVII (COL17) in relation to clinicopathological factors in primary cutaneous melanomas with known lymph node status in both sexes, using immunohistochemistry. We found, that GPER1 expression correlated with favorable clinicopathological factors, including lower Breslow thickness, lower mitotic rate and absence of ulceration. In contrast, COL17 expression was associated with poor prognostic features, such as higher tumor thickness, higher mitotic rate, presence of ulceration and presence of regression. Melanomas positive for both GPER1 and COL17 had significantly lower mean Breslow thickness and mitotic rate compared to cases positive for COL17 only. Our data indicate that GPER1 and COL17 proteins may be of potential prognostic value in primary cutaneous melanomas.

Rational design of solid lipid-polymer hybrid nanoparticles: An innovative glycoalkaloids-carrier with potential for topical melanoma treatment

Despite the promising potential of Solanum plant glycoalkaloids in combating skin cancer, their clinical trials have been halted due to dose-dependent toxicity and poor water solubility. In this study, we present a rational approach to address these limitations and ensure colloidal stability of the nanoformulation over time by designing solid lipid-polymer hybrid nanoparticles (SLPH). Leveraging the biocompatible and cationic properties of polyaspartamides, we employed a new polyaspartamide derivative (P1) as a raw material for this class of nanostructures. Subsequently, we prepared SLPH through a one-step process involving hot-melt emulsification followed by ultrasonication. The physicochemical properties of the SLPH were thoroughly characterized using dynamic light scattering (DLS), ζ-potential analysis, nanoparticle tracking analysis (NTA), differential scanning calorimetry (DSC), Fourier-transform infrared spectroscopy (FT-IR), and transmission electron microscopy (TEM). The optimized formulation exhibited long-term stability over six months under low temperatures, maintaining a particle size around 200 nm, a polydispersity index (PdI) lower than 0.2, and a ζ-potential between +35–40 mV. Furthermore, we evaluated the cytotoxic effect of the SLPH against human cutaneous melanoma cells (SK-MEL-28) compared to human foreskin fibroblast cells (HFF-1). Encapsulation of glycoalkaloids into the nanoparticles (SLPH-GE) resulted in a two-fold greater selective cytotoxic profile for melanoma cells than glycoalkaloids-free (GE). The nanoparticles disrupted the stratum corneum barrier with a penetration depth of approximately 77 μm. These findings underscore the potential of the developed nanosystem as an effective glycoalkaloid carrier with suitable colloidal and biological properties for further studies in topical treatment strategies for cutaneous melanoma.

Abstract 5938: Development of HH044, an orally bioavailable nNOS inhibitor, for the treatment of cutaneous melanoma

Abstract Background: Human cutaneous melanoma is one of the few cancers in which the incidence rate continues to increase, and despite recent advances in immune and targeted therapies, the 5-year survival rate of metastatic disease remains low. Neuronal nitric oxide synthase (nNOS) has been identified to contribute to melanoma development, stimulating disease progression, and facilitating escape of melanoma cells from the immune response. We developed a novel small molecule, HH044, which specifically inhibits nNOS activity and reduced nitric oxide production in melanoma cells. We aim to conduct a proof-of-concept study using our nNOS inhibitor administered orally for anti-melanoma treatment. Methods: Human melanoma cell lines (A375, Sk-Mel-28, wm3211) were employed to analyze the anti-cancer activities of HH044 in vitro. An immune-competent syngeneic melanoma mouse model was used to determine the anti-tumor activity of HH044 when administered orally (50 mg/kg/day) or intraperitoneally (10 mg/kg/day) for 21 days. A pharmacokinetic study of drug levels in serum and tumor samples was conducted after a single dose given intraperitoneally or orally using LC-MS/MS analysis at different time points. Results: HH044 has high selectivity of nNOS over iNOS (340-fold) and eNOS (540-fold). The IC50 of HH044 in melanoma cell lines was 5.27 ± 3.3 μM, while iNOS inhibitor 1200W did not exhibit any cytotoxicity up to 100 μM concentration. Of note, nNOS blockade by HH044 did not result in reactivation of iNOS, and expression levels of iNOS were not changed after HH044 treatment. In vivo studies demonstrated that HH044 treatment given intraperitoneally exhibited potent anti-tumor activity and reduced tumor weight to 45% of control. When administered orally, HH044 at 50 mg/kg significantly reduced tumor growth to 61% of control and extended mice median survival from 32 days to 53 days. Pharmacokinetic analysis showed that oral administration achieved sufficient drug levels in xenograft tumors, which were well above the Ki value (0.005 μM) despite a wide range of bioavailability. The drug level peaked at 2 hours after a single dose (2.27 ± 0.666 μM in the tumor and 23.68 ± 17.43 μM in the serum). Intraperitoneal injection achieved much higher levels in serum and tumor samples, which also declined slower compared to oral gavage administration. By 24 hours, the drug levels in serum and xenograft tumors remained at 1.55 ± 0.12 μM and 1.77 ± 0.24 μM concentration, respectively. No significant systemic toxicities were observed with HH044 treatments either given via i.p. injection or orally. Further in vivo studies combining nNOS inhibitors with immune checkpoint blockade are currently underway. Conclusions: HH044, as the first-in-class nNOS inhibitor, exhibits potent anti-melanoma activity by oral administration. Developing selective nNOS inhibitors has shown to be a novel therapeutic strategy to improve melanoma treatment. Citation Format: Anika Patel, Shirley Tong, Richard B. Silverman, Sun Yang. Development of HH044, an orally bioavailable nNOS inhibitor, for the treatment of cutaneous melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5938.

Upregulation of IL4-induced gene 1 enzyme by B2 cells during melanoma progression impairs their antitumor properties.

B cells present in human cutaneous melanoma have been associated with protective or detrimental effects on disease progression according to their phenotype. By using the RET model of spontaneous melanoma and adoptive transfer of B16 melanoma cells, we show that immature and follicular B2 (B2-FO) cells exert a protective effect on melanoma progression by promoting the generation of effector memory T cells and limiting the recruitment of polymorphonuclear myeloid-derived suppressor cells. Unfortunately, this beneficial effect progressively wanes as a consequence of enhanced expression of the IL4-induced gene 1 (IL4I1) enzyme by immature B cells and B2-FO cells. Endogenous IL4I1 selectively decreases CXCR5 expression in splenic immature B cells, subverting their trafficking to primary tumors and enhancing the production of IL-10 by B2 cells, thereby promoting an immunosuppressive microenvironment. Accordingly, B2 cells from RET IL4I1KO mice more efficiently controlled B16 melanoma growth than B2 cells from IL4I1-competent RET mice. Collectively, immature B cells and B2-FO cells are key actors in the control of melanoma growth, but their mobility and functions are differently impaired by IL4I1 overexpression during melanoma progression. Thus, our present data strongly urge us to associate an IL4I1 antagonist with current immunotherapy to improve the treatment of metastatic melanoma.

Chemical profile and biological properties of the Piper corcovadense C.DC. essential oil

The essential oil fromPiper corcovadense D.DC. (EOPc), an important plant belonging to the Piperaceae family, which is commonly found in the northern region of Brazil and poorly explored scientifically, was used in this study. Thus, the EOPc was characterized chemically by Gas Chromatography/Mass Spectrometry (GC/MS) and the antioxidant and antimicrobial activities and their potential effects on cutaneous melanoma (SK-MEL-28) and healthy peripheral blood mononuclear (PBMC) cells were determined. The major compounds identified in the EOPc were: trans-sesquisabinene hydrate, trans-caryophyllene, β-pinene, trans-β-farnesene, 14-hydroxycaryophyllene, limonene and p-cymene. The EOPc demonstrated antioxidant activity as evaluated by Folin-Ciocalteu reagent (FC) reducing capacity, DPPH, and ABTS methods. The values found were respectively 5.41 ± 0.17 mg GAE mL−1 (GAE: Gallic acid equivalent), 2.88 ± 0.17 µmol TE mL−1 (TE: Trolox equivalent) and 6.26 ± 0.02 µmol TE mL−1. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were determined for different bacterial strains. The EOPc at a concentration of 2.61 µg mL−1 exhibited both bactericidal and bacteriostatic properties against Escherichia coli. The EOPc showed potential antitumor activity as it reduced the cell viability of human cutaneous melanoma cells SK-MEL-28. Besides, the EOPc did not exhibit cytotoxic activity against healthy PBMCs, indicating that it does not harm healthy cells at the tested concentrations. The EOPc increased the levels of ROS at concentrations of 250 µg mL−1. The EOPc also did not stimulate the mobilization of endogenous antioxidant defenses, as assessed by total thiol (PSH) and non-protein thiols (NPSH). Thus, the study suggests that the EOPc has antioxidant and antimicrobial properties due to the presence of specific compounds. It also exhibits antitumor potential against cutaneous melanoma cells while showing no cytotoxicity to healthy PBMCs. It directly influenced ROS levels at the highest tested concentration in the cells, suggesting an antitumor effect related to the intrinsic apoptosis pathway. Nevertheless, while the study has initial findings, the results are promising and indicate an attractive biological potential of P. corcovadense, mainly in human cutaneous melanoma cells.

Cancer Cell Biomechanical Properties Accompany Tspan8-Dependent Cutaneous Melanoma Invasion.

The intrinsic biomechanical properties of cancer cells remain poorly understood. To decipher whether cell stiffness modulation could increase melanoma cells' invasive capacity, we performed both in vitro and in vivo experiments exploring cell stiffness by atomic force microscopy (AFM). We correlated stiffness properties with cell morphology adaptation and the molecular mechanisms underlying epithelial-to-mesenchymal (EMT)-like phenotype switching. We found that melanoma cell stiffness reduction was systematically associated with the acquisition of invasive properties in cutaneous melanoma cell lines, human skin reconstructs, and Medaka fish developing spontaneous MAP-kinase-induced melanomas. We observed a systematic correlation of stiffness modulation with cell morphological changes towards mesenchymal characteristic gains. We accordingly found that inducing melanoma EMT switching by overexpressing the ZEB1 transcription factor, a major regulator of melanoma cell plasticity, was sufficient to decrease cell stiffness and transcriptionally induce tetraspanin-8-mediated dermal invasion. Moreover, ZEB1 expression correlated with Tspan8 expression in patient melanoma lesions. Our data suggest that intrinsic cell stiffness could be a highly relevant marker for human cutaneous melanoma development.

Quantitative multiplex immunohistochemistry reveals inter-patient lymphovascular and immune heterogeneity in primary cutaneous melanoma.

Quantitative, multiplexed imaging is revealing complex spatial relationships between phenotypically diverse tumor infiltrating leukocyte populations and their prognostic implications. The underlying mechanisms and tissue structures that determine leukocyte distribution within and around tumor nests, however, remain poorly understood. While presumed players in metastatic dissemination, new preclinical data demonstrates that blood and lymphatic vessels (lymphovasculature) also dictate leukocyte trafficking within tumor microenvironments and thereby impact anti-tumor immunity. Here we interrogate these relationships in primary human cutaneous melanoma. We established a quantitative, multiplexed imaging platform to simultaneously detect immune infiltrates and tumor-associated vessels in formalin-fixed paraffin embedded patient samples. We performed a discovery, retrospective analysis of 28 treatment-naïve, primary cutaneous melanomas. Here we find that the lymphvasculature and immune infiltrate is heterogenous across patients in treatment naïve, primary melanoma. We categorized five lymphovascular subtypes that differ by functionality and morphology and mapped their localization in and around primary tumors. Interestingly, the localization of specific vessel subtypes, but not overall vessel density, significantly associated with the presence of lymphoid aggregates, regional progression, and intratumoral T cell infiltrates. We describe a quantitative platform to enable simultaneous lymphovascular and immune infiltrate analysis and map their spatial relationships in primary melanoma. Our data indicate that tumor-associated vessels exist in different states and that their localization may determine potential for metastasis or immune infiltration. This platform will support future efforts to map tumor-associated lymphovascular evolution across stage, assess its prognostic value, and stratify patients for adjuvant therapy.

Abstract IA011: Polymerase-based bypass of atypical UV photoproducts

Abstract UV light is a ubiquitous environmental mutagen that is an etiological cause of skin cancers like melanoma. While the genomes of human cutaneous melanomas have high mutation burdens primarily consisting of C to T substitutions in TC and CC dinucleotides (consistent with cyclobutane pyrimidine dimer formation), many of the driver mutations contributing to disease progression involve other types of substitutions at non-dipyrimidine sequences. Our whole genome sequencing of UVB or UVC irradiated yeast has revealed non-canonical UV induced substitution types. These mutations included TA to TT and AC to TT substitutions that commonly occur as activating BRAF mutations in human melanomas. UV treatment and sequencing of deletion mutants of DNA polymerase eta, the protein mutated in human XPV patients, indicated that this polymerase does not impact TA to TT mutagenesis, but contributes to the formation of AC to TT substitutions. Surprisingly, loss of pol eta revealed additional CA to AA substitutions, highlighting another UV-induced mutation class possibly created by a non-canonical photoproduct. All novel mutation classes in pol eta deficient yeast displayed transcriptional asymmetry, consistent with the repair of the cognate lesion by transcription-coupled nucleotide excision repair. However, UV-induced mutations lacked replication asymmetry in both wild type and rad30Δ yeast, suggesting TLS occurs nearly equally on leading and lagging strands. Both TA to TT and AC to TT substitutions were dependent on DNA polymerase zeta, and altered by a mutant of DNA polymerase epsilon, suggesting the involvement of both TLS and replicative polymerases in UV-induced mutagenesis. These results emphasize the importance of non-canonical UV lesions in driving melanomagenesis, provide mechanisms of how these lesions result in mutations, and suggest the variable accuracy of TLS polymerases across from atypical UV photoproducts may alter the trajectory of skin cancer evolution in XPV patients. Citation Format: Steven A. Roberts. Polymerase-based bypass of atypical UV photoproducts [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: DNA Damage Repair: From Basic Science to Future Clinical Application; 2024 Jan 9-11; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2024;84(1 Suppl):Abstract nr IA011.

Laser capture microdissection provides a novel molecular profile of human primary cutaneous melanoma.

Melanoma accounts for the majority of skin cancer-related mortality, highlighting the need to better understand melanoma initiation and progression. In-depth molecular analysis of neoplastic melanocytes in whole tissue biopsies may be diluted by inflammatory infiltration, which may obscure gene signatures specific to neoplastic cells. Thus, a method is needed to precisely uncover molecular changes specific to tumor cells from a limited sample of primary melanomas. Here, we performed laser capture microdissection (LCM) and gene expression profiling of patient-derived frozen sections of pigmented lesions and primary cutaneous melanoma. Compared to bulk tissue analysis, analysis of LCM-derived samples identified 9528 additional differentially expressed genes (DEGs) including melanocyte-specific genes like PMEL and TYR, with enriched of pathways related to cell proliferation. LCM methodology also identified potentially targetable kinases specific to melanoma cells that were not detected by bulk tissue analysis. Taken together, our data demonstrate that there are marked differences in gene expression profiles depending on the method of sample isolation. We found that LCM captured higher expression of melanoma-related genes while whole tissue biopsy identified a wider range of inflammatory markers. Taken together, our data demonstrate that LCM is a valid approach to identify melanoma-specific changes using a relatively small amount of primary patient-derived melanoma sample.

The spread of interferon-γ in melanomas is highly spatially confined, driving nongenetic variability in tumor cells

Interferon-γ (IFNγ) is a critical antitumor cytokine that has varied effects on different cell types. The global effect of IFNγ in the tumor depends on which cells it acts upon and the spatial extent of its spread. Reported measurements of IFNγ spread vary dramatically in different contexts, ranging from nearest-neighbor signaling to perfusion throughout the entire tumor. Here, we apply theoretical considerations to experiments both in vitro and in vivo to study the spread of IFNγ in melanomas. We observe spatially confined niches of IFNγ signaling in 3-D mouse melanoma cultures and human tumors that generate cellular heterogeneity in gene expression and alter the susceptibility of affected cells to T cell killing. Widespread IFNγ signaling only occurs when niches overlap due to high local densities of IFNγ-producing T cells. We measured length scales of ~30 to 40 μm for IFNγ spread in B16 mouse melanoma cultures and human primary cutaneous melanoma. Our results are consistent with IFNγ spread being governed by a simple diffusion-consumption model and offer insight into how the spatial organization of T cells contributes to intratumor heterogeneity in inflammatory signaling, gene expression, and immune-mediated clearance. Solid tumors are often viewed as collections of diverse cellular "neighborhoods": Our work provides a general explanation for such nongenetic cellular variability due to confinement in the spread of immune mediators.

Suicide-Gene-Modified Extracellular Vesicles of Human Primary Uveal Melanoma in Future Therapies.

Extracellular vesicles secreted from uveal melanoma (UM) cells are involved in the establishment of the premetastatic niche and display transforming potential for the formation of metastases, preferentially in the liver. In this study, we cultivated human primary UM cells and uveal melanoma-associated fibroblasts in vitro to be transduced by infection with a retrovirus containing the suicide gene-fused yeast cytosine deaminase::uracil phospho-ribosyl transferase (yCD::UPRT). A homogenous population of yCD::UPRT-UM cells with the integrated provirus expressed the gene, and we found it to continuously secrete small extracellular vesicles (sEVs) possessing mRNA of the suicide gene. The yCD::UPRT-UM-sEVs were internalized by tumor cells to the intracellular conversion of the prodrug 5-fluorocytosine (5-FC) to the cytotoxic drug 5-fluorouracil (5-FU). The host range of the yCD::UPRT-UM-sEVs was not limited to UMs only. The yCD::UPRT-UM-sEVs inhibited the growth of the human cutaneous melanoma cell line A375 and uveal melanoma cell line MP38, as well as other primary UMs, to various extents in vitro. The yCD::UPRT-UM-sEVs hold the therapeutic and prophylactic potential to become a therapeutic drug for UM. However, the use of yCD::UPRT-UM-sEVs must first be tested in animal preclinical studies.

Deciphering the immune reaction leading to spontaneous melanoma regression: initial role of MHCII+ CD163− macrophages

The human cutaneous metastatic melanoma is the deadliest skin cancer. Partial, or less frequently complete spontaneous regressions could be observed, mainly mediated by T cells. Nevertheless, the underlying mechanisms are not fully unraveled. We investigated the first events of the immune response related to cancer regression in Melanoma-bearing Libechov Minipigs (MeLiM), a unique swine model of cutaneous melanoma that regresses spontaneously. Using a multiparameter flow cytometry strategy and integrating new clinical and histological criteria of the regression, we show that T cells and B cells are present only in the late stages, arguing against their role in the initial destruction of malignant cells. NK cells infiltrate the tumors before T cells and therefore might be involved in the induction of the regression process. Myeloid cells represent the main immune population within the tumor microenvironment regardless of the regression stage. Among those, MHCII+ CD163− macrophages that differ phenotypically and functionally compared to other tumor-associated macrophages, increase in number together with the first signs of regression suggesting their crucial contribution to initiating the regression process. Our study supports the importance of macrophage reprogramming in humans to improve current immunotherapy for metastatic melanoma.

The Pro-Oncogenic Sphingolipid-Metabolizing Enzyme β-Galactosylceramidase Modulates the Proteomic Landscape in BRAF(V600E)-Mutated Human Melanoma Cells.

β-Galactosylceramidase (GALC) is a lysosomal enzyme involved in sphingolipid metabolism by removing β-galactosyl moieties from β-galactosylceramide and β-galactosylsphingosine. Previous observations have shown that GALC may exert pro-oncogenic functions in melanoma and Galc silencing, leading to decreased oncogenic activity in murine B16 melanoma cells. The tumor-driving BRAF(V600E) mutation is present in approximately 50% of human melanomas and represents a major therapeutic target. However, such mutation is missing in melanoma B16 cells. Thus, to assess the impact of GALC in human melanoma in a more relevant BRAF-mutated background, we investigated the effect of GALC overexpression on the proteomic landscape of A2058 and A375 human melanoma cells harboring the BRAF(V600E) mutation. The results obtained by liquid chromatography-tandem mass spectrometry (LC-MS/MS) demonstrate that significant differences exist in the protein landscape expressed under identical cell culture conditions by A2058 and A375 human melanoma cells, both harboring the same BRAF(V600E)-activating mutation. GALC overexpression resulted in a stronger impact on the proteomic profile of A375 cells when compared to A2058 cells (261 upregulated and 184 downregulated proteins versus 36 and 14 proteins for the two cell types, respectively). Among them, 25 proteins appeared to be upregulated in both A2058-upGALC and A375-upGALC cells, whereas two proteins were significantly downregulated in both GALC-overexpressing cell types. These proteins appear to be involved in melanoma biology, tumor invasion and metastatic dissemination, tumor immune escape, mitochondrial antioxidant activity, endoplasmic reticulum stress responses, autophagy, and/or apoptosis. Notably, analysis of the expression of the corresponding genes in human skin cutaneous melanoma samples (TCGA, Firehose Legacy) using the cBioPortal for Cancer Genomics platform demonstrated a positive correlation between GALC expression and the expression levels of 14 out of the 27 genes investigated, thus supporting the proteomic findings. Overall, these data indicate for the first time that the expression of the lysosomal sphingolipid-metabolizing enzyme GALC may exert a pro-oncogenic impact on the proteomic landscape in BRAF-mutated human melanoma.

Immunohistochemical Expression of Programmed Cell Death Ligand 1 (PD-L1) in Human Cutaneous Malignant Melanoma: A Narrative Review with Historical Perspectives.

Programmed death-ligand 1 (PD-L1) is the primary ligand of the receptor programmed death-1 (PD-1) which is constitutively expressed or activated in myeloid, lymphoid (T, B and NK), normal epithelial cells, and cancer. The PD-1/PD-L1 interaction is crucial for the physiological development of immunological tolerance but also in the development of the cancer. Among these, malignant melanoma represents a tumour in which the immunohistochemical expression of PD-L1 is important to guide future therapeutic choices based on the presence/absence of expression. Various clones have been used over time for immunohistochemical determination, and different results and heterogeneity remain among the various studies in the literature. We perform a narrative review of the present studies in order to discuss and take stock of what certain achievements have been made in this field, what challenges remain, and what possible solutions can be found.

Overexpression of c-MYC Promoter Binding Protein-1 Enhances Proliferation and Glucose Metabolism of Melanoma Cells Lines.

c-MYC promoter binding protein (MBP-1) is a product of alternatively translated mRNA encoding alpha-enolase (ENO1). In contrast to ENO1, MBP-1 possesses no enzymatic activity but acts as a transcriptional repressor of c-MYC. Ectopic over-expression of MBP-1 in tumor cells was shown to reduce cell proliferation and tumorigenicity, thus making it an attractive target for anticancer strategies. This study aimed to assess the effects of MBP-1 over-expression on human cutaneous melanoma cell lines. We overexpressed the full-length MBP-1 or its C-terminal truncated variant (MBP-1ΔC), in two human melanoma cell lines (A375, WM9) and assessed their subcellular localization. qPCR was then used to quantitate c-MYC transcription. Further, 5-ethynyl-2'-deoxyuridine incorporation assay was used to measure cell proliferation and a lactate assay was performed to measure the glycolysis rate of cells in normoxia and hypoxia. Finally, an in vitro wound-healing assay was performed to evaluate cell migration. The overexpressed MBP-1 variants predominantly localized in the cytoplasm and barely decreased c-MYC expression. Unexpectedly, the proliferation rate of MBP-1- transduced cells increased in comparison to controls, as did the rate of glucose metabolism in hypoxia. Furthermore, over-expression of MBP-1, but not MBP-1ΔC, led to a substantial decrease in the cell migration capacity of metastatic WM9 cells but not A375 cells from the primary tumor lesion. Misslocalization of over-expressed MBP-1 in the cytoplasm of two melanoma cell lines resulted in an unexpected tumor promoting activity by increasing cell proliferation and glycolysis rates in hypoxia.

Connecting the dots: Melanoma cell of origin, tumor cell plasticity, trans-differentiation, and drug resistance.

Melanoma, a lethal malignancy that arises from melanocytes, exhibits a multiplicity of clinico-pathologically distinct subtypes in sun-exposed and non-sun-exposed areas. Melanocytes are derived from multipotent neural crest cells and are present in diverse anatomical locations, including skin, eyes, and various mucosal membranes. Tissue-resident melanocyte stem cells and melanocyte precursors contribute to melanocyte renewal. Elegant studies using mouse genetic models have shown that melanoma can arise from either melanocyte stem cells or differentiated pigment-producing melanocytes depending on a combination of tissue and anatomical site of origin and activation of oncogenic mutations (or overexpression) and/or the repression in expression or inactivating mutations in tumor suppressors. This variation raises the possibility that different subtypes of human melanomas (even subsets within each subtype) may also be a manifestation of malignancies of distinct cells of origin. Melanoma is known to exhibit phenotypic plasticity and trans-differentiation (defined as a tendency to differentiate into cell lineages other than the original lineage from which the tumor arose) along vascular and neural lineages. Additionally, stem cell-like properties such as pseudo-epithelial-to-mesenchymal (EMT-like) transition and expression of stem cell-related genes have also been associated with the development of melanoma drug resistance. Recent studies that employed reprogramming melanoma cells to induced pluripotent stem cells have uncovered potential relationships between melanoma plasticity, trans-differentiation, and drug resistance and implications for cell or origin of human cutaneous melanoma. This review provides a comprehensive summary of the current state of knowledge on melanoma cell of origin and the relationship between tumor cell plasticity and drug resistance.

Sensory Nerves inhibit the development of protective anti-melanoma immune response and formation of tertiary lymphoid structures

Abstract Peripheral nervous system (PNS) is an important component of the tumor microenvironment (TME). It has been established that autonomic innervation plays an important role in cancer. However, the effect of the afferent (sensory) neurons on tumor progression remains unclear. Utilizing surgical and chemical skin sensory denervation methods, we showed that afferent neurons supported the growth of melanoma tumors in vivo and demonstrated that sensory innervation limited the activation of effective antitumor immune responses. Precisely, sensory ablation enhanced leukocyte recruitment into tumors, with decreased presence of lymphoid and myeloid immunosuppressive cells and increased activation of T-effector cells within the TME. Denervation further increased T-cell clonality and expanded the B-cell repertoire in the TME. Furthermore, Depletion of CD8+ lymphocytes prevented denervation-restricted tumor growth. Importantly, denervated tumors contained higher densities of mature high endothelial venules (HEV) associated with the formation of classic tertiary lymphoid structures (TLS), which associate with a more favorable melanoma prognosis. Transcriptional profiling of human melanomas confirmed our findings in murine melanoma models. Specifically, we observed that gene signatures of inflammation and the content of neuron-associated transcripts inversely correlated in human primary cutaneous melanomas, with the latter representing a negative prognostic marker of patient overall survival.. Our results suggest that tumor-associated sensory neurons negatively regulate the development of protective antitumor immune responses within the TME, thereby providing a new therapeutic target for melanoma treatment.

Photodynamic Activity of TMPyP4/TiO2 Complex under Blue Light in Human Melanoma Cells: Potential for Cancer-Selective Therapy.

The combination of TiO2 nanoparticles (NPs) and photosensitizers (PS) may offer significant advantages in photodynamic therapy (PDT) of melanoma, such as improved cell penetration, enhanced ROS production, and cancer selectivity. In this study, we aimed to investigate the photodynamic effect of 5,10,15,20-(Tetra-N-methyl-4-pyridyl)porphyrin tetratosylate (TMPyP4) complexes with TiO2 NPs on human cutaneous melanoma cells by irradiation with 1 mW/cm2 blue light. The porphyrin conjugation with the NPs was analyzed by absorption and FTIR spectroscopy. The morphological characterization of the complexes was performed by Scanning Electron Microscopy and Dynamic Light Scattering. The singlet oxygen generation was analyzed by phosphorescence at 1270 nm. Our predictions indicated that the non-irradiated investigated porphyrin has a low degree of toxicity. The photodynamic activity of the TMPyP4/TiO2 complex was assessed on the human melanoma Mel-Juso cell line and non-tumor skin CCD-1070Sk cell line treated with various concentrations of the PS and subjected to dark conditions and visible light-irradiation. The tested complexes of TiO2 NPs with TMPyP4 presented cytotoxicity only after activation by blue light (405 nm) mediated by the intracellular production of ROS in a dose-dependent manner. The photodynamic effect observed in this evaluation was higher in melanoma cells than the effect observed in the non-tumor cell line, demonstrating a promising potential for cancer-selectivity in PDT of melanoma.

The inhibitory role of microRNA-141-3p in human cutaneous melanoma growth and metastasis through the fibroblast growth factor 13-mediated mitogen-activated protein kinase axis.

Human cutaneous melanoma (CM) is a highly invasive malignancy arising from melanocytes, and accompanied by ever-increasing incidence and mortality rates worldwide. Interestingly, microRNAs (miRNAs) possess the ability to regulate CM cell biological functions, resulting in the aggressive progression of CM. Nevertheless, a comprehensive understanding of the underlying mechanism remains elusive. Accordingly, the current study sought to elicit the functional role of miR-141-3p in human CM cells in association with fibroblast growth factor 13 (FGF13) and the MAPK pathway. First, miR-141-3p expression patterns were detected in human CM tissues and cell lines, in addition to the validation of the targeting relationship between miR-141-3p and FGF13. Subsequently, loss- and gain-of-function studies of miR-141-3p were performed to elucidate the functional role of miR-141-3p in the malignant features of CM cells. Intriguingly, our findings revealed that FGF13 was highly expressed, whereas miR-141-3p was poorly expressed in the CM tissues and cells. Further analysis highlighted FGF13 as a target gene of miR-141-3p. Meanwhile, overexpression of miR-141-3p inhibited the proliferative, invasive, and migratory abilities of CM cells, while enhancing their apoptosis accompanied by downregulation of FGF13 and the MAPK pathway-related genes. Collectively, our findings highlighted the inhibitory effects of miR-141-3p on CM cell malignant properties via disruption of the FGF13-dependent MAPK pathway, suggesting a potential target for treating human CM.