Treatment Of Melanoma Cells Research Articles

Treatment of Melanoma Cells with Chloroquine and Everolimus Activates the Apoptosis Process and Alters Lipid Redistribution

Treatment of Melanoma Cells with Chloroquine and Everolimus Activates the Apoptosis Process and Alters Lipid Redistribution

Enhanced Anti-Melanoma Activity of Nutlin-3a Delivered via Ethosomes: Targeting p53-Mediated Apoptosis in HT144 Cells.

This study evaluated ethosomes as a novel nanodelivery system for nutlin-3a, a known MDM2 inhibitor and activator of the p53 pathway, to improve nutlin-3a's poor solubility, limiting its bio-distribution and therapeutic efficacy. The potential of nutlin-3a-loaded ethosomes was investigated on two in vitro models of melanoma: the HT144 cell line p53wild-type and the SK-MEL-28 cell line p53mutated. Nutlin-3a-loaded ethosomes were characterized for their physicochemical properties and used to treat melanoma cells at different concentrations, considering nutlin-3a solution and empty ethosomes as controls. The biological effects on cells were evaluated 24 and 48 h after treatment by analyzing the cell morphology and viability, cell cycle, and apoptosis rate using flow cytometry and the p53 pathway's activation via Western blotting. The results indicate that ethosomes are delivery systems able to maintain nutlin-3a's functionality and specific biological action, as evidenced by the molecular activation of the p53 pathway and the biological events leading to cell cycle block and apoptosis in p53wild-type cells. Nutlin-3a-loaded ethosomes induced morphological changes in the HT144 cell line, with evident apoptotic cells and a reduction in the number of viable cells of over 80%. Furthermore, nutlin-3a-loaded ethosomes successfully modulated two p53-regulated proteins involved in survival/apoptosis, with up to a 2.5-fold increase in membrane TRAIL-R2 and up to an 8.2-fold decrease in Notch-1 (Notch intracellular domain, NICD) protein expression. The expression of these molecules is known to be altered or dysfunctional in a large percentage of melanoma tumors. Notably, ethosomes, regardless of their nutlin-3a loading, exhibited the ability to reduce HT144 melanoma cellular migration, as assessed in real time using xCELLigence, likely due to the modification of lipid rafts, suggesting their potential antimetastatic properties. Overall, nutlin-3a delivery using ethosomes appears to be a significantly effective means for upregulating the p53 pathway and downregulating active Notch-1, while also taking advantage of their unexpected ability to reduce cellular migration. The findings of this study could pave the way for the development of specific nutlin-3a-loaded ethosome-based medicinal products for cutaneous use.

Anticancer Activity of Vitamin D, Lumisterol and Selected Derivatives against Human Malignant Melanoma Cell Lines.

Despite the recent development of improved methods of treating melanoma such as targeted therapy, immunotherapy or combined treatment, the number of new cases worldwide is increasing. It is well known that active metabolites of vitamin D3 and lumisterol (L3) exert photoprotective and antiproliferative effects on the skin, while UV radiation is a major environmental risk factor for melanoma. Thus, many natural metabolites and synthetic analogs of steroidal and secosteroidal molecules have been tested on various cancer cells and in animal models. In this study, we tested the anti-melanoma properties of several natural derivatives of vitamin D3 and L3 in comparison to 1,25-dihydroxyvitamin D3 (1,25(OH)2D3). A significant decrease in melanoma cell proliferation and cell mobility was observed for selected derivatives, with (25R)-27-hydroxyL3 showing the highest potency (lowest IC50) in A375 cells but lower potency in SK-MEL-28 cells, whereas the parent L3 failed to inhibit proliferation. The efficacy (% inhibition) by 1,24,25(OH)3D3 and 1,25(OH)2D3 were similar in both cell types. 1,25(OH)2D3 showed higher potency than 1,24,25(OH)3D3 in SK-MEL-28 cells, but lower potency in A375 cells for the inhibition of proliferation. As for 1,25(OH)2D3, but not the other derivatives tested, treatment of melanoma cells with 1,24,25(OH)3D3 markedly increased the expression of CYP24A1, enhanced translocation of the vitamin D receptor (VDR) from the cytoplasm to the nucleus and also decreased the expression of the proliferation marker Ki67. The effects of the other compounds tested were weaker and occurred only under certain conditions. Our data indicate that 1,24,25(OH)3D3, which has undergone the first step in 1,25(OH)2D3 inactivation by being hydroxylated at C24, still shows anti-melanoma properties, displaying higher potency than 1,25(OH)2D3 in SK-MEL-28 cells. Furthermore, hydroxylation increases the potency of some of the lumisterol hydroxy-derivatives, as in contrast to L3, (25R)-27(OH)L3 effectively inhibits proliferation and migration of the human malignant melanoma cell line A375.

Bacterial Cellulose-Chitosan Composite for Prolonged-Action L-Asparaginase in Treatment of Melanoma Cells.

A significant challenge associated with the therapeutic use of L-ASP for treatment of tumors is its rapid clearance from plasma. Effectiveness of L-ASP is limited by the dose-dependent toxicity. Therefore, new approaches are being developed for L-ASP to improve its therapeutic properties. One of the approaches to improve properties of the enzymes, including L-ASP, is immobilization on various types of biocompatible polymers. Immobilization of enzymes on a carrier could improve stability of the enzyme and change duration of its enzymatic activity. Bacterial cellulose(BC) is a promising carrier for various drugs due to its biocompatibility, non-toxicity, high porosity, and high drug loading capacity. Therefore, this material has high potential for application in biomedicine. Native BC is known to have a number of disadvantages related to structural stability, which has led to consideration of the modified BC as a potential carrier for immobilization of various proteins, including L-ASP. In our study, a BC-chitosan composite in which chitosan is cross-linked with glutaraldehyde was proposed for immobilization of L-ASP. Physicochemical characteristics of the BC-chitosan films were found to be superior to those of native BC films, resulting in increase in the release time of L-ASP invitro from 8 to24h. These films exhibited prolonged toxicity (up to 10h) against the melanoma cell line. The suggested strategy for A-ASP immobilization on the BC-chitosan films could be potentially used for developing therapeutics for treatment of surface types of cancers including melanomas.

Light exposure of tetra-cationic porphyrins containing peripheral Pd(II)-bipyridyl complexes and the induced effects on purified DNA molecule, fibroblast and melanoma cell lines.

Photodynamic therapy (PDT) combines a light source, oxygen, and a photosensitizer (PS) to generate reactive oxygen species (ROS) for treating diseases. In this study, we evaluated two meso-tetra-pyridyl porphyrins with [Pd(bpy)Cl]+, namely 3-PdTPyP and 4-PdTPyP, as PS for PDT application. DNA interaction was assessed by spectroscopic measurements (UV-Vis and fluorescence emission), viscosity analysis, and molecular docking simulations. The results indicate that Pd(II)-porphyrins do not intercalate into DNA, suggesting that the minor groove is the primary interaction site, mainly through van der Waals forces. These metalloporphyrins effectively induced nitrogenous bases oxidation, particularly in purines, after white light irradiation. The induced DNA lesions were able to inactivate plasmid DNA metabolism (DNA replication and transcription) in a bacterial model. 3-PdTPyP and 4-PdTPyP significantly decreased the viability of treated melanoma cell lines (A375 and B16-F10), demonstrating that melanoma cell lines were more sensitive to these Pd(II)-porphyrins than the fibroblast cell line (L929). Moreover, 3-PdTPyP was more photototoxic to A375 cells (IC50 = 0.43 μM), whereas 4-PdTPyP was more photototoxic to B16-F10 cells (IC50 = 0.51 μM). These findings suggest that these porphyrins are promising PS for future PDT research focused on skin cancer.

Type I interferon signaling induces melanoma cell-intrinsic PD-1 and its inhibition antagonizes immune checkpoint blockade

Programmed cell death 1 (PD-1) is a premier cancer drug target for immune checkpoint blockade (ICB). Because PD-1 receptor inhibition activates tumor-specific T-cell immunity, research has predominantly focused on T-cell-PD-1 expression and its immunobiology. In contrast, cancer cell-intrinsic PD-1 functional regulation is not well understood. Here, we demonstrate induction of PD-1 in melanoma cells via type I interferon receptor (IFNAR) signaling and reversal of ICB efficacy through IFNAR pathway inhibition. Treatment of melanoma cells with IFN-α or IFN-β triggers IFNAR-mediated Janus kinase-signal transducer and activator of transcription (JAK/STAT) signaling, increases chromatin accessibility and resultant STAT1/2 and IFN regulatory factor 9 (IRF9) binding within a PD-1 gene enhancer, and leads to PD-1 induction. IFNAR1 or JAK/STAT inhibition suppresses melanoma-PD-1 expression and disrupts ICB efficacy in preclinical models. Our results uncover type I IFN-dependent regulation of cancer cell-PD-1 and provide mechanistic insight into the potential unintended ICB-neutralizing effects of widely used IFNAR1 and JAK inhibitors.

SIRT7 promotes mitochondrial biogenesis to render the adaptive resistance to MAPK inhibition in melanoma

Melanoma, arising from the malignant transformation of melanocytes, stands as the most lethal type of skin cancer. While significant strides have been made in targeted therapy and immunotherapy, substantially enhancing therapeutic efficacy, the prognosis for melanoma patients remains unoptimistic. SIRT7, a nuclear-localized deacetylase, plays a pivotal role in maintaining cellular homeostasis and adapting to external stressors in melanoma, with its activity closely tied to intracellular nicotinamide adenine dinucleotide (NAD+). However, its involvement in adaptive resistance to targeted therapy remains unclear. Herein, we unveil that up-regulated SIRT7 promotes mitochondrial biogenesis to render the adaptive resistance to MAPK inhibition in melanoma. Initially, we observed a significant increase of SIRT7 expression in publicly available datasets following targeted therapy within a short duration. In consistent, we found elevated SIRT7 expression in melanoma cells subjected to BRAF or MEK inhibitors in vitro. The up-regulation of SIRT7 expression was also confirmed in xenograft tumors in mice after targeted therapy in vivo. Furthermore, we proved that SIRT7 deficiency led to decreased cell viability upon prolonged exposure to BRAF or MEK inhibitors, accompanied by an increase in cell apoptosis. Mechanistically, SIRT7 deficiency restrained the upregulation of genes associated with mitochondrial biogenesis and intracellular ATP levels in response to targeted therapy treatment in melanoma cells. Ultimately, we proved that SIRT7 deficieny could sensitize BRAF-mutant melanoma cells to MAPK inhibition targeted therapy in vivo. In conclusion, our findings underscore the role of SIRT7 in fostering adaptive resistance to targeted therapy through the facilitation of mitochondrial biogenesis. Targeting SIRT7 emerges as a promising strategy to overcome MAPK inhibitor adaptive resistance in melanoma.

The role of GAPDH in the selective toxicity of CNP in melanoma cells.

Malignant melanoma is the most aggressive form of skin cancer with a rather poor prognosis. Standard chemotherapy often results in severe side effects on normal (healthy) cells finally being difficult to tolerate for the patients. Shown by us earlier, cerium oxide nanoparticles (CNP, nanoceria) selectively killed A375 melanoma cells while not being cytotoxic at identical concentrations on non-cancerous cells. In conclusion, the redox-active CNP exhibited both prooxidative as well as antioxidative properties. In that context, CNP induced mitochondrial dysfunction in the studied melanoma cells via generation of reactive oxygene species (primarily hydrogen peroxide (H2O2)), but that does not account for 100% of the toxicity. Cancer cells often show an increased glycolytic rate (Warburg effect), therefore we focused on CNP mediated changes of the glucose metabolism. It has been shown before that glyceraldehyde 3-phosphate dehydrogenase (GAPDH) activity is regulated via oxidation of a cysteine in the active center of the enzyme with a subsequent loss of activity. Upon CNP treatment, formation of cellular lactate and GAPDH activity were significantly lowered. The treatment of melanoma cells and melanocytes with the GAPDH inhibitor heptelidic acid (HA) decreased viability to a much higher extent in the cancer cells than in the studied normal (healthy) cells, highlighting and supporting the important role of GAPDH in cancer cells. We identified glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as a target protein for CNP mediated thiol oxidation.

Molecular Basis of <i>Sida cordifolia</i> (L.) Induced Apoptosis in Melanoma Cell Line

Sida cordifolia of the family Malvaceae is widely used in traditional medicine for treating inflammation, respiratory and neurological ailments and wound healing. Its extract was found to possess effective antitumor activity in hepatocellular carcinoma and HeLa cell lines. This study was aimed at screening the anticancer activity of S. cordifolia and to investigate its mechanism of action. Aerial parts of the plant were subjected to hot continuous extraction by Soxhlet apparatus with ethanol as solvent. Cytotoxicity of the extract was assessed in various cancer cell lines viz. breast, ovarian, colon, skin, and liver cancer by MTT assay. For each cell line, the IC50 value was calculated. The mechanism of anticancer activity of the extract was studied in melanoma cells by exposing them to 12.5 and 25 μg/ml extract and comparing results with the control. Gel electrophoresis was used to analyse DNA laddering. Expression of TP53, Bcl and Caspase gene family proteins were determined by SDS-PAGE. Mitochondrial membrane potential was studied by the JC-1 kit. Cell cycle analysis was performed by using a flow cytometer. Statistical analysis was done by ANNOVA, and significant values were further analysed by Tucky post-hoc analysis. P value less than 0.05 was considered statistically significant. MTT assay revealed maximum cytotoxicity of the extract against melanoma with an IC50 value of 16.51μg/ml. Melanoma cells treated with the extract demonstrated dose-dependent DNA laddering. The extract also exhibited a dose-dependent increase in the level of Bax, Caspase 3, Caspase 9 and p53 proteins. Expression of Bcl2 protein was significantly reduced. Treatment of melanoma cells with the extract showed significant loss of mitochondrial membrane potential. Melanoma cell population in subG0 and G2/S was significantly elevated. From these results, we conclude that ethanol extract of S. cordifolia is cytotoxic to melanoma cells. It acts by inducing apoptosis via an intrinsic mechanism. The extract also arrests melanoma cells in the G2/M phase.

Combined BET and MEK Inhibition synergistically suppresses melanoma by targeting YAP1.

Rationale: The response rate to the MEK inhibitor trametinib in BRAF-mutated melanoma patients is less than 30%, and drug resistance develops rapidly, but the mechanism is still unclear. Yes1-associated transcriptional regulator (YAP1) is highly expressed in melanoma and may be related to MEK inhibitor resistance. The purpose of this study was to investigate the mechanism of YAP1 in MEK inhibitor resistance in melanoma and to screen YAP1 inhibitors to further determine whether YAP1 inhibition reverses MEK inhibitor resistance. Methods: On the one hand, we analyzed paired melanoma and adjacent tissue samples using RNA-seq and found that the Hippo-YAP1 signaling pathway was the top upregulated pathway. On the other hand, we evaluated the transcriptomes of melanoma samples from patients before and after trametinib treatment and investigated the correlation between YAP1 expression and trametinib resistance. Then, we screened for inhibitors that repress YAP1 expression and investigated the mechanisms. Finally, we investigated the antitumor effect of YAP1 inhibition combined with MEK inhibition both in vitro and in vivo. Results: We found that YAP1 expression levels upon trametinib treatment in melanoma patients were correlated with resistance to trametinib. YAP1 was translocated into the nucleus after trametinib treatment in melanoma cells, which could render resistance to MEK inhibition. Thus, we screened for inhibitors that repress YAP1 expression and identified multiple bromodomain and extra-terminal (BET) inhibitors, including NHWD-870, as hits. BET inhibition repressed YAP1 expression by decreasing BRD4 binding to the YAP1 promoter. Consistently, YAP1 overexpression was sufficient to reverse the proliferation defect caused by BRD4 depletion. In addition, the BET inhibitor NHWD-870 acted synergistically with trametinib to suppress melanoma growth in vitro and in vivo. Conclusions: We identified a new vulnerability for MEK inhibitor-resistant melanomas, which activated Hippo pathway due to elevated YAP1 activity. Inhibition of BRD4 using BET inhibitors suppressed YAP1 expression and led to blunted melanoma growth when combined with treatment with the MEK inhibitor trametinib.

Enzymatic depletion of l-Met using an engineered human enzyme as a novel therapeutic strategy for melanoma.

Many cancers, including melanoma, have a higher requirement for l-methionine in comparison with noncancerous cells. In this study, we show that administration of an engineered human methionine-γ-lyase (hMGL) significantly reduced the survival of both human and mouse melanoma cells in vitro. A multiomics approach was utilized to identify global changes in gene expression and in metabolite levels with hMGL treatment in melanoma cells. There was considerable overlap in the perturbed pathways identified in the two data sets. Common pathways were flagged for further investigation to understand their mechanistic importance. In this regard, hMGL treatment induced S and G2 phase cell cycle arrest, decreased nucleotide levels, and increased DNA double-strand breaks suggesting an important role for replication stress in the mechanism of hMGL effects on melanoma cells. Further, hMGL treatment resulted in increased cellular reactive oxygen specieslevels and increased apoptosis as well as uncharged transfer RNApathway upregulation. Finally, treatment with hMGL significantly inhibited the growth of both mouse and human melanoma cells in orthotopic tumor models in vivo. Overall, the results of this study provide a strong rationale for further mechanistic evaluation and clinical development of hMGL for the treatment of melanoma skin cancer and other cancers.

Abstract 674: CRISPR and drug screens identify ERK as the mediator of IFNg-induced melanoma growth inhibition

Abstract Interferon-gamma (IFNg) is critical for the success of immune checkpoint blockade therapy. Disruption of IFNg signaling makes tumors resistant to anti-PD-1/L1 treatment primarily by evasion of the growth inhibition (GI) induced by this cytokine. However, the pathways mediating IFNg-induced GI are largely unknown. We used two independent approaches to delineate the IFNg GI pathway, a chemical genomics screen and a whole genome targeting CRISPR/Cas9 screen in an IFNg GI-sensitive patient-derived melanoma (PDM) cell line. Drug screening revealed that treatment with RAF and ERK inhibitors disrupted IFNg GI in PDM cells. For the CRISPR screen, gene-level analysis showed that the magnitude of enrichment for guide RNAs targeting ERK2 in IFNg-treated cells was comparable to proximal IFNg signaling proteins like JAK1, STAT1, IFNGR2, and JAK2 (>26-fold enrichment for ERK2, p = 2.28e-07). Thus, results from both these experiments converged upon ERK as the primary downstream target of IFNg essential for GI induction. We validated the involvement of ERK by detecting a sustained increase in phospho-ERK1/2 levels following IFNg treatment. In live imaging experiments, we found that blocking ERK activity with the ERK inhibitor Ulixertinib blocks the induction of cell death after IFNg treatment in 17 of 23 (~74%) IFNg-sensitive PDM lines covering all the MAPK mutant and triple wildtype molecular subtypes of melanoma. Decreasing and increasing ERK activity levels using shRNA and overexpression approaches also led to a decrease and increase in IFNg-mediated GI, respectively. We have identified NOXA and DR5 as likely mediators of cell death through the IFNg-ERK GI pathway by comparing gene expression profiles of control and IFNg-treated PDM cells. Finally, phorbol 12-myristate 13-acetate (PMA), a known ERK activator, synergizes with IFNg treatment to induce increased GI in PDM cells (Bliss synergy score 16.87, p = 1.83e-123). In summary, the IFNg-ERK signaling axis mediates cell death downstream of IFNg treatment in melanoma cells. This pathway is active in all melanoma subtypes, making it an attractive target to enhance IFNg GI in tumor cells. Our results provide a new understanding of the IFNg GI pathway that will also be crucial to define mechanisms of GI resistance in tumor cells. Citation Format: Ameya S. Champhekar, Rachel Heymans, Cynthia Gonzalez, Guillem Turon Font, Daniel Karin, June Lee, Robert Damoiseaux, Antoni Ribas. CRISPR and drug screens identify ERK as the mediator of IFNg-induced melanoma growth inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 674.

Toxicity of Persian Gulf shell-less marine mollusc (Peronia peronii) methanolic extract on melanoma tumor mitochondria

Introduction Melanoma is known as an aggressive and highly lethal cancer. The poor prognosis and resistance to treatment are characteristics of melanoma. In melanoma cells, apoptosis signaling which relies heavily on the acute activity of mitochondria and reactive oxygen species (ROS) formation is suppressed. Studies have shown that compounds isolated from marine herbs and animals, have been shown to have cytotoxic consequences on cancerous cells in prior research. This study was designed to evaluate the apoptotic effect of methanolic extract of Persian Gulf shell-less marine mollusc (Peronia peronii) on skin mitochondria isolated from animal model of melanoma. Purpose Melanoma mitochondria obtained from skin of melanoma animal model are studied in this research to see whether extracts from Persian Gulf shell-less marine mollusc (Peronia peronii), has a cytotoxic impact on them. Material and method In this study, the mitochondria were isolated from melanoma cells via differential centrifugation were treated with various concentrations (650, 1300 and 2600 µg/ml) of methanolic extract of Peronia peronii. Then MTT(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) viability assay, Reactive oxygen species (ROS) determination, Mitochondrial Membrane Potential (MMP) decline assay, mitochondrial swelling and cytochrome c release determination were performed. Flow cytometry assay of % apoptotic vs necrotic phenotypes was also performed on extract treated melanoma cells. Results The results of MTT assay showed that different concentrations of Peronia peronii extract significantly (P < 0.05) decreased the SDH activity in cancerous skin mitochondria with the IC50(1300 μg/ml). The ROS results also showed that all concentrations of Peronia peronii extracts significantly increased ROS production, MMP decline and the release of cytochrome c in cancer groups mitochondria. The swelling of mitochondria was significantly increased compared to the control group. In addition, the results of apoptosis assay showed that addition of root extract of Peronia peronii on melanoma cells increased apoptosis, while it had no effect on control non tumour cells. Discussion and conclusion Based on these results, the presence of potentially bioactive compounds in Peronia peronii make this Persian Gulf coastal herb a strong candidate for further molecular studies and clinical research in the field of melanoma cancer therapy.

Pancancer transcriptomic profiling identifies key PANoptosis markers as therapeutic targets for oncology.

Resistance to programmed cell death (PCD) is a hallmark of cancer. While some PCD components are prognostic in cancer, the roles of many molecules can bemasked by redundancies and crosstalks between PCD pathways, impeding the development of targeted therapeutics. Recent studies characterizing these redundancies have identified PANoptosis, a unique innate immune-mediated inflammatory PCD pathway that integrates components from other PCD pathways. Here, we designed a systematic computational framework to determine the pancancer clinical significance of PANoptosis and identify targetable biomarkers. We found that high expression of PANoptosis genes was detrimental in low grade glioma (LGG) and kidney renal cell carcinoma (KIRC). ZBP1, ADAR, CASP2, CASP3, CASP4, CASP8 and GSDMD expression consistently had negative effects on prognosis in LGG across multiple survival models, while AIM2, CASP3, CASP4 and TNFRSF10 expression had negative effects for KIRC. Conversely, high expression of PANoptosis genes was beneficial in skin cutaneous melanoma (SKCM), with ZBP1, NLRP1, CASP8 and GSDMD expression consistently having positive prognostic effects. As a therapeutic proof-of-concept, we treated melanoma cells with combination therapy that activates ZBP1 and showed that this treatment induced PANoptosis. Overall, through our systematic framework, we identified and validated key innate immune biomarkers from PANoptosis which can be targeted to improve patient outcomes in cancers.

Role of dual specificity phosphatases (DUSPs) in melanoma cellular plasticity and drug resistance

Melanoma cells exhibit phenotypic plasticity that allows transition from a proliferative and differentiated phenotype to a more invasive and undifferentiated or transdifferentiated phenotype often associated with drug resistance. The mechanisms that control melanoma phenotype plasticity and its role in drug resistance are not fully understood. We previously demonstrated that emergence of MAPK inhibitor (MAPKi)-resistance phenotype is associated with decreased expression of stem cell proliferation genes and increased expression of MAPK inactivation genes, including dual specificity phosphatases (DUSPs). Several members of the DUSP family genes, specifically DUSP1, -3, -8 and -9, are expressed in primary and metastatic melanoma cell lines and pre-and post BRAFi treated melanoma cells. Here, we show that knockdown of DUSP1 or DUSP8 or treatment with BCI, a pharmacological inhibitor of DUSP1/6 decrease the survival of MAPKi-resistant cells and sensitizes them to BRAFi and MEKi. Pharmacological inhibition of DUSP1/6 upregulated nestin, a neural crest stem cell marker, in both MAPKi-sensitive cells and cells with acquired MAPKi-resistance. In contrast, treatment with BCI resulted in upregulation of MAP2, a neuronal differentiation marker, only in MAPKi-sensitive cells but caused downregulation of both MAP2 and GFAP, a glial marker, in all MAPKi-resistant cell lines. These data suggest that DUSP proteins are involved in the regulation of cellular plasticity cells and melanoma drug resistance and are potential targets for treatment of MAPKi-resistant melanoma.

Iron oxide nanoparticles coated with polydopamine as a potential nano-photothermal agent for treatment of melanoma cancer: an in vivo study.

Melanoma is a metastatic cancer resistant to a wide range of therapies, including standard chemotherapy and radiation therapy, and cannot be treated with existing treatments owing to its intrinsic drug resistance. In terms of convenience and cheap cost of fabrication, one of the novel treatments is using polydopamine-coated iron oxide nanoparticles (IONs@PDA). Iron oxide nanoparticles (IONs) were synthesized (7.36nm) and coated with polydopamine (15-20nm). To examine the effect of photothermal ablation in melanoma cells (B16-F10), a Q-switched ruby laser (λ = 694nm, spot size = 4mm, output power = 5J/s) was used. The prepared nanoprobe was applied to mice, and their survival after treatment was evaluated. Then histopathological studies were done on the livers and skins of the treated mice. The nanoparticles absorb the laser, raising the temperature and initiating photothermal treatment, with significant apoptosis (74%) after the 4th time of treatment. Photothermal therapy (PTT) by using IONs@PDA proved to be effective in the treatment of melanoma cells (tumor size of < 2mm) without side effects. The lifespan of mice was significantly increased in a group of mice post-administered IONs@PDA and laser ablation. The fabricated nanoprobe (IONs@PDA) enhanced the melanoma cell apoptosis in the mice model, and it has promise for the treatment of melanoma (B16-F10) cells using photothermal therapy.

LB1001 Effectiveness and differentially expressed genes analysis of melanoma cells treated with cold atmospheric plasma

Cold atmospheric plasma (CAP) has attracted increasing attention due to its anti-bacterial and anti-tumor effects. Melanoma is an aggressive malignancy that is increasingly common and with a poor prognosis. By evaluating cell viability, detecting cells apoptosis, this study explored the differences of the biological effect between melanoma cells and human keratinocyte cell lines (HaCaT) treated with CAP, and combining the reactive species injection efficiency and tumor cell physiological characteristics system analyzed the biological safety of CAP. The results showed that under the same injection condition, CAP has a more significant biological effect on melanoma in inhibiting proliferation and apoptosis. Based on the effect study, RNA-sequencing was performed to further elucidate the differential gene expression of CAP treated melanoma cells. The results showed that besides MAPK and p53 apoptosis pathways, autophagy and necroptosis also had important roles in CAP-induced melanoma cells death. Our results provided genomic resources for the study of the apoptosis mechanism of CAP treated melanoma cells, and in particular, provided new insights into the molecular mechanisms in autophagy and necroptosis.

Abstract 3014: PHGDH- A therapeutic vulnerability in melanoma

Abstract Recurrently amplified oncogenes may represent melanoma vulnerabilities that can be exploited for therapy. The genomic locus containing the Phosphoglycerate Dehydrogenase (PHGDH) gene at chromosome 1p12 is frequently amplified in melanoma. PHGDH is the rate limiting enzyme that converts the glycolytic intermediate 3-Phospho Glycerate (3-PG) to serine through the serine synthesis pathway (SSP). Serine is utilized to make glycine, glutathione, one carbon metabolites, proteins and lipids. PHGDH inhibition suppresses proliferation of 1p12 amplified melanoma cells. However, the role of PHGDH in chromosome 1p12 non-amplified melanoma is poorly understood. We observed that 1p12 non-amplified melanoma cell lines have enhanced expression of the SSP enzymes as compared to melanocytes cultured in tetradecanoyl-phorbol acetate (TPA, a growth factor that stimulates MAPK pathway). Moreover, TPA starvation in melanocytes increased PHGDH expression. This led us to hypothesize that activation of oncogenic MAPK signaling leads to high expression of PHGDH which can serve as therapeutic vulnerability in melanomagenesis. Acute expression of oncogenic BRAFV600E in melanocytes resulted in upregulation of SSP enzymes, while treatment of melanoma cells with either BRAF or MEK inhibitor resulted in downregulation of SSP enzymes. We confirmed that PHGDH is regulated by BRAF-ERK-mTOR axis. Downregulation of PHGDH in 1p12 non-amplified melanoma cell lines decreased their cell proliferation and colony formation ability in vitro. We also observed that Phgdh knockdown in a BrafV600E/WT and Pten-/- (BPP) melanoma mouse model significantly increased survival. Furthermore, serine-glycine starvation (external source of Serine-glycine) in BPP chimeras did not affect the survival of these mice while on the other end, it suppressed the growth of the xenograft tumors injected in the NSG mice, which warrants further investigation. These results suggest the importance of PHGDH for melanoma cell proliferation and tumor growth irrespective of its amplification status. The exact mechanism of PHGDH regulation by BRAFV600E and the therapeutic effect of PHGDH inhibition in melanoma initiation and progression are the focus of our studies. Citation Format: Neel N. Jasani, Florian Karreth. PHGDH- A therapeutic vulnerability in melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3014.

Abstract 1004: Growth hormone upregulates tumor-derived exosomal distribution of EMT mediators and multi-drug efflux pumps in melanoma

Abstract Melanoma is the most aggressive form of skin cancer with a dismal 5-year survival rate of 16% at the metastatic stage of the disease. Exosomes have recently emerged as a critical mediator of cancer therapy resistance by regulating epithelial to mesenchymal transition (EMT) and drug efflux. Exosomes are extracellular vesicles (diameters 30-150nm) released from almost all types of cells including tumor cells which package and transfer proteins, RNA, and lipids (often called cargo) from one cell to another. This intercellular communication via exosomes has been reported to induce a drug-resistant phenotype across the tumor by transporting ABC multidrug transporters and mediators of EMT. Multiple studies have established that pituitary-, or tumor- or tumor microenvironment-derived growth hormone (GH) promotes EMT and drug resistance in cancer by binding to GHR-expressing tumor cells. A question arises: Does GH exert this effect across the tumor population via modulating tumor-derived exosomal cargo? No study has yet directly assessed the effect of GH on exosomes in terms of modulating EMT or drug resistance. We isolated exosomes from human melanoma cells with differential levels of GHR expression, following treatment with combinations of GH, anti-cancer drugs, and the GHR antagonist, pegvisomant. We observed no change in the quantity of tumor-derived exosomes with changes in GH action. However, we found significant upregulation of multidrug efflux pumps ABCB1, ABCC1, ABCC2, and ABCG2 in GH-treated tumor exosomes. Importantly, treatment of naïve melanoma cells with these exosomes increased their drug efflux rate and reduced drug retention. Exosomes derived from pegvisomant treated melanoma cells showed markedly reduced ABC transporter contents and did not increase the efflux rate of naïve tumor cells. A similar trend in matrix metalloproteinases (MMP2, MMP9) and TGFb levels in isolated exosomes as well as TGFb signaling and invasion capacity in naïve tumor cells treated with isolated exosomes were observed. These results present a unique effect of GH on tumor-derived exosomal content modulation towards propagating a drug-resistant tumor phenotype in naïve cancer cells. Acknowledgment: This work was supported in part by the State of Ohio’s Eminent Scholar Program that includes a gift from Milton and Lawrence Goll, by the AMVETS, and Ohio University’s Student Enhancement Award and Edison Biotechnology Institute. Citation Format: Prateek Kulkarni, Reetobrata Basu, John J. Kopchick. Growth hormone upregulates tumor-derived exosomal distribution of EMT mediators and multi-drug efflux pumps in melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1004.

5‑Aza‑dC suppresses melanoma progression by inhibiting GAS5 hypermethylation.

The in-depth study of melanoma pathogenesis has revealed that epigenetic modifications, particularly DNA methylation, is a universal inherent feature of the development and progression of melanoma. In the present study, the analysis of the tumor suppressor gene growth arrest-specific transcript 5 (GAS5) demonstrated that its expression was downregulated in melanoma, and its expression level had a certain negative association with its methylation modification level. The promoter of GAS5 presented with detectable CpG islands, and methylation-specific polymerase chain reaction analysis demonstrated that GAS5 was actually modified by methylation in melanoma tissues and cells; however, no methylation modification of GAS5 was detected in normal tissues. Following the treatment of melanoma cells with 5-Aza-2′-deoxycytidine (5-Aza-dC), GAS5 methylation was significantly reversed. The analysis of melanoma cell proliferation revealed that 5-Aza-dC inhibited A375 and SK-MEL-110 cell proliferation in a time-dependent manner. Further analysis of apoptosis demonstrated that 5-Aza-dC significantly increased the apoptosis level of the two cell lines. Moreover, migration analysis of melanoma cells revealed that 5-Aza-dC significantly reduced cell migration. Furthermore, 5-Aza-dC significantly decreased the invasive ability of the two cell lines. However, when the expression of GAS5 was silenced, the effects of 5-Aza-dC on cell proliferation, apoptosis, invasion and migration were not significant. Furthermore, the subcutaneous injection of A375 cells in nude mice successfully resulted in xenograft tumor formation. However, following an intraperitoneal injection of 5-Aza-dC, the volume and weight of xenograft tumors and Ki-67 expression were significantly reduced, and caspase-3 activity and GAS5 expression were enhanced; following the silencing of GAS5, the antitumor effect of 5-Aza-dC was significantly blocked. On the whole, the present study demonstrates that 5-Aza-dC inhibits the growth of melanoma, and its function may be related to the methylation modification of GAS5.