Sensitivity Of Melanoma Cells Research Articles

Ubiquitination and degradation of MGMT by TRIM72 increases the sensitivity of uveal melanoma cells to Dacarbazine treatment.

Uveal melanoma (UM) is the most common primary intraocular malignancy in adults with high metastasis rates. The O6-methylguanine DNA methyl transferase (MGMT) is involved in chemoresistance of Dacarbazine (DTIC) treatment. Our previous study found that the combination of oncolytic adenovirus H101 and DTIC in the treatment of UM cells shows a synergistic antitumor effect mainly though down-regulation of MGMT. MGMT knockdown by shRNAs increases the sensitivity of uveal melanoma cells to DTIC treatment. The protein hemostasis of MGMT is important for the antitumor effect of DTIC. Tripartite motif-containing protein 72 (TRIM72) belongs to the tripartite motif (TRIM) proteins family and was identified as a novel E3 ligase for MGMT, which interacts with and mediates the ubiquitination of MGMT. TRIM72 knockdown increases the protein levels of MGMT, while reduces the ubiquitination of MGMT. Further study indicated that MGMT is highly expressed in UM cells, and the protein levels of MGMT and TRIM72 shows a negative correlation. UM cells that ectopically expressing TRIM72 shows increased sensitivity to DTIC treatment, which is consistent with the antitumor affect exhibited by H101. These results suggest that TRIM72 is a promising therapeutic target for UM treatment.

The PIK3CA H1047R Mutation Confers Resistance to BRAF and MEK Inhibitors in A375 Melanoma Cells through the Cross-Activation of MAPK and PI3K-Akt Pathways.

The targeting of the Mitogen-Activated Protein Kinase (MAPK) signalling pathway in melanoma improves the prognosis of patients harbouring the V-Raf Murine Sarcoma Viral Oncogene Homolog B1 (BRAF) mutation. However, a fraction of these patients may experience tumour progression due to resistance to targeted therapy. Mutations affecting the Phosphoinositol-3-Kinase (PI3K)–Akt pathway may favour the onset of drug resistance, suggesting the existence of a crosstalk between the MAPK and PI3K–Akt pathways. We hypothesized that the inhibition of both pathways may be a therapeutic option in resistant melanoma. However, conflicting data have been generated in this context. In this study, three different A375 cell melanoma models either overexpressing or not expressing the wild-type or mutated form of the PhosphatidylInositol-4,5-bisphosphate 3-Kinase Catalytic Subunit Alpha (PIK3CA) gene were used to clarify the therapeutic response of melanoma to BRAF, Mitogen-Activated Protein Kinase Kinase 1 (MEK), and PI3K inhibitors in the presence of the PIK3CA H1047R mutation. Our data strongly support the notion that the crosstalk between the MAPK and PI3K–Akt pathways is one of the main mechanisms associated with melanoma development and progression and that the combination of MAPK and PI3K inhibitors may sensitize melanoma cells to therapy.

Phyto-sesquiterpene lactones DET and DETD-35 induce ferroptosis in vemurafenib sensitive and resistant melanoma via GPX4 inhibition and metabolic reprogramming

Acquired resistance to vemurafenib (PLX4032) is a thorny issue in BRAFV600E mutant melanoma therapy. Ferroptotic programmed cell death is a potential strategy for combating therapy-resistant cancers. This study uncovers the adaptation and abnormal upregulation of PUFAs and bioactive oxylipin metabolism in PLX4032 resistant melanoma cells. Phyto-sesquiterpene lactone, DET, and its derivative, DETD-35, induced lipid ROS accumulation and triggered ferroptotic cell death in PLX4032 sensitive (A375) and resistant (A375-R) BRAFV600E melanoma cells by reprogramming glutathione and primary metabolisms, lipid/oxylipin metabolism, and causing mitochondrial damage in which DETD-35 showed superior efficiency to DET. We discovered that DET and DETD-35 are a new type of GPX4 enzyme inhibitor through non-covalent binding. This study provides new insight into the therapeutic mechanisms of both DET and DETD-35 to combat PLX4032 sensitive/resistant BRAFV600E mutant melanomas via targeting GPX4 and ferroptosis.

Non-Steroidal Anti-Inflammatory Drugs (NSAIDs) sensitize melanoma cells to MEK inhibition and inhibit metastasis and relapse by inducing degradation of AXL.

Melanoma is highly heterogeneous with diverse genomic alterations and partial therapeutic responses. The emergence of drug-resistant tumor cell clones accompanied by a high AXL expression level is one of the major challenges for anti-tumor clinical care. Recent studies have demonstrated that high AXL expression in melanoma cells mediated drug resistance, epithelial-mesenchymal transition (EMT), and elevated survival of cancer stem cells (CSCs). Given that we have identified several non-steroidal anti-inflammatory drugs (NSAIDs) including aspirin potently induce the degradation of AXL, we questioned whether NSAIDs could counteract the AXL-mediated neoplastic phenotypes. In this study, we found that NSAIDs downregulate PKA activity via the PGE2 /EP2/cAMP/PKA signaling pathway and interrupt the PKA-dependent interaction between CDC37 and HSP90, resulting in an incorrect AXL protein folding and finally AXL degradation through the ubiquitination-proteasome system (UPS) pathway. Furthermore, NSAIDs not only sensitized the MEK inhibitor treatment but also reduced EMT and relapse mediated by AXL in tumor tissue. Our findings suggest that the combination of inhibitors and NSAIDs, especially aspirin, could be a simple but efficient modality to treat melanoma in which AXL is a key factor for drug resistance, metastasis, and relapse.

Metformin Increases Sensitivity of Melanoma Cells to Cisplatin by Blocking Exosomal-Mediated miR-34a Secretion.

Melanoma, also known as malignant melanoma, is a type of cancer derived from the pigment-containing cells known as melanocytes. Cisplatin (CDDP) is widely used in the treatment of different types of tumors with high response rates, but it generally has low efficiency in melanoma. This study aimed to investigate whether metformin could sensitize the melanoma cell line A375 to cisplatin. Our results for the first time indicated that CDDP increased the miR-34a secretion by exosomes in melanoma A375 cells, which was, at least partially, related to the cisplatin resistance of melanoma cells. Moreover, metformin significantly sensitized A375 cells to cisplatin. Mechanistically, metformin significantly blocked the exosome-mediated miR-34a secretion induced by cisplatin. Our study not only reveals a novel mechanism that exosomal secretion of miR-34a is involved in the cisplatin resistance of melanoma cells but also provides a promising therapeutic strategy by synergistic addition of metformin.

A Possible Role for PAI-1 Blockade in Melanoma Immunotherapy

In their new article in the Journal of Investigative Dermatology, Tseng etal. (2021) confirm that the sensitivity of melanoma cells to anti‒PD-L1 checkpoint inhibitor therapy is correlated with high PD-L1 surface expression. By blocking PD-L1 membrane clearing, controlled by LRP1 and PAI-1, the expression of high-cell-surface levels of PD-L1 was maintained.

Road to Metastasis: The TWEAK Pathway as a Discriminant between Metastasizing and Non-Metastasizing Thick Melanomas

Cutaneous melanoma (CM) is the most aggressive form of skin cancer, and its worldwide incidence is rapidly increasing. Early stages can be successfully treated by surgery, but once metastasis has occurred, the prognosis is poor. However, some 5–10% of thick (≥2 mm) melanomas do not follow this scenario and run an unpredictable course. Little is known about the factors that contribute to metastasis in some patient with thick melanomas and the lack thereof in thick melanoma patients who never develop metastatic disease. We were therefore interested to study differential gene expression and pathway analysis and compare non-metastatic and metastatic thick melanomas. We found that the TNF-like weak inducer of apoptosis (TWEAK) pathway was upregulated in thick non-metastasizing melanomas. MAP3K14 (NIK1), BIRC2 (cIAP1), RIPK1, CASP7, CASP8, and TNF play an important role in inhibiting proliferation and invasion of tumor cells via the activation of the non-canonical NF-κB signaling pathway. In particular, this pathway sensitizes melanoma cells to TNF-alpha and activates the apoptosis module of the TWEAK pathway in thick non-metastasizing melanomas. Hence, our study suggests a potential role of the TWEAK pathway in inhibiting thick melanoma from metastasis. Exploitation of these genes and the pathway they control may open future therapeutic avenues.

Fibroblasts Influence Metastatic Melanoma Cell Sensitivity to Combined BRAF and MEK Inhibition.

Simple SummaryPreclinical 3D in vitro coculture models are known to be more complex systems than monolayer cell culture and mimic the physiological environment more closely. Three-dimensional dermal equivalents provide a relevant environment for cutaneous metastatic melanoma cells and are capable of modulating a cancer cell’s response to drugs. We showed that a combined targeted therapy (vemurafenib and cobimetinib) efficiently inhibits cell proliferation and induces apoptosis, especially in the 3D coculture model. A cancer-associated fibroblast population isolated from a cutaneous melanoma was also sensitive to the treatment but with no detectable induction of apoptosis. To better understand the complex crosstalk between melanoma cells and their microenvironment, we compared the influence of conditioned media obtained from healthy or cancer-associated fibroblasts on the response of metastatic melanomas to the drugs. Our data indicate that normal fibroblast supernatants potentialize the therapy’s efficiency, whereas cancer-associated fibroblast secretomes favor melanoma cell survival.The sensitivity of melanoma cells to targeted therapy compounds depends on the tumor microenvironment. Three-dimensional (3D) in vitro coculture systems better reflect the native structural architecture of tissues and are ideal for investigating cellular interactions modulating cell sensitivity to drugs. Metastatic melanoma (MM) cells (SK-MEL-28 BRAF V600E mutant and SK-MEL-2 BRAF wt) were cultured as a monolayer (2D) or cocultured on 3D dermal equivalents (with fibroblasts) and treated with a BRAFi (vemurafenib) combined with a MEK inhibitor (MEKi, cobimetinib). The drug combination efficiently inhibited 2D and 3D MM cell proliferation and survival regardless of their BRAF status. Two-dimensional and three-dimensional cancer-associated fibroblasts (CAFs), isolated from a cutaneous MM biopsy, were also sensitive to the targeted therapy. Conditioned media obtained from healthy dermal fibroblasts or CAFs modulated the MM cell’s response differently to the treatment: while supernatants from healthy fibroblasts potentialized the efficiency of drugs on MM, those from CAFs tended to increase cell survival. Our data indicate that the secretory profiles of fibroblasts influence MM sensitivity to the combined vemurafenib and cobimetinib treatment and highlight the need for 3D in vitro cocultures representing the complex crosstalk between melanoma and CAFs during preclinical studies of drugs.

MFN2 Stabilization: A Bridge for Endoplasmic Reticulum Stress Sensitivity in Melanoma

In a new article in the Journal of Investigative Dermatology, Wang etal. (2021) report that mitochondrial quality control modulates responses to endoplasmic reticulum (ER) stress in melanoma. They implicate a linear pathway of XBP1, MARCH5, and MFN2 that act together to regulate mitochondrial fission and mitophagy and ultimately mediate melanoma cell sensitivity to ER stress. This work informs therapeutic combinations and biomarker strategies for targeting melanoma organellar homeostasis as well as for life‒death decisions.

Protein Signatures of NK Cell-Mediated Melanoma Killing Predict Response to Immunotherapies.

Despite impressive advances in melanoma-directed immunotherapies, resistance is common and many patients still succumb to metastatic disease. In this context, harnessing natural killer (NK) cells, which have thus far been sidelined in the development of melanoma immunotherapy, could provide therapeutic benefits for cancer treatment. To identify molecular determinants of NK cell-mediated melanoma killing (NKmK), we quantified NK-cell cytotoxicity against a panel of genetically diverse melanoma cell lines and observed highly heterogeneous susceptibility. Melanoma protein microarrays revealed a correlation between NKmK and the abundance and activity of a subset of proteins, including several metabolic factors. Oxidative phoshorylation, measured by oxygen consumption rate, negatively correlated with melanoma cell sensitivity toward NKmK, and proteins involved in mitochondrial metabolism and epithelial-mesenchymal transition were confirmed to regulate NKmK. Two- and three-dimensional killing assays and melanoma xenografts established that the PI3K/AKT/mTOR signaling axis controls NKmK via regulation of NK cell-relevant surface proteins. A "protein-killing-signature" based on the protein analysis predicted NKmK of additional melanoma cell lines and the response of patients with melanoma to anti-PD-1 checkpoint therapy. Collectively, these findings identify novel NK cell-related prognostic biomarkers and may contribute to improved and personalized melanoma-directed immunotherapies. SIGNIFICANCE: NK-cell cytotoxicity assays and protein microarrays reveal novel biomarkers of NK cell-mediated melanoma killing and enable development of signatures to predict melanoma patient responsiveness to immunotherapies.

FKBP51 Affects TNF-Related Apoptosis Inducing Ligand Response in Melanoma.

Melanoma is one of the most immunogenic tumors and has the highest potential to elicit specific adaptive antitumor immune responses. Immune cells induce apoptosis of cancer cells either by soluble factors or by triggering cell-death pathways. Melanoma cells exploit multiple mechanisms to escape immune system tumoricidal control. FKBP51 is a relevant pro-oncogenic factor of melanoma cells supporting NF-κB-mediated resistance and cancer stemness/invasion epigenetic programs. Herein, we show that FKBP51-silencing increases TNF-related apoptosis-inducing ligand (TRAIL)-R2 (DR5) expression and sensitizes melanoma cells to TRAIL-induced apoptosis. Consistent with the general increase in histone deacetylases, as by the proteomic profile, the immune precipitation assay showed decreased acetyl-Yin Yang 1 (YY1) after FKBP51 depletion, suggesting an impaired repressor activity of this transcription factor. ChIP assay supported this hypothesis. Compared with non-silenced cells, a reduced acetyl-YY1 was found on the DR5 promoter, resulting in increased DR5 transcript levels. Using Crispr/Cas9 knockout (KO) melanoma cells, we confirmed the negative regulation of DR5 by FKBP51. We also show that KO cells displayed reduced levels of acetyl-EP300 responsible for YY1 acetylation, along with reduced acetyl-YY1. Reconstituting FKBP51 levels contrasted the effects of KO on DR5, acetyl-YY1, and acetyl-EP300 levels. In conclusion, our finding shows that FKBP51 reduces DR5 expression at the transcriptional level by promoting YY1 repressor activity. Our study supports the conclusion that targeting FKBP51 increases the expression level of DR5 and sensitivity to TRAIL-induced cell death, which can improve the tumoricidal action of immune cells.

The Downregulation of eIF3a Contributes to Vemurafenib Resistance in Melanoma by Activating ERK via PPP2R1B.

Vemurafenib, a BRAF V600E inhibitor, provides therapeutic benefits for patients with melanoma, but the frequent emergence of drug resistance remains a challenge. An understanding of the mechanisms underlying vemurafenib resistance may generate novel therapeutic strategies for patients with melanoma. Here, we showed that eIF3a, a translational regulatory protein, was an important mediator involved in vemurafenib resistance. eIF3a was expressed at significantly lower levels in vemurafenib-resistant A375 melanoma cells (A375R) than in parental A375 cells. Overexpression of eIF3a enhanced the sensitivity to BRAF inhibitors by reducing p-ERK levels. Furthermore, eIF3a controlled ERK activity by regulating the expression of the phosphatase PPP2R1B via a translation mechanism, thus determining the sensitivity of melanoma cells to vemurafenib. In addition, a positive correlation between eIF3a and PPP2R1B expression was also observed in tumor samples from the Human Protein Atlas and TCGA databases. In conclusion, our studies reveal a previously unknown molecular mechanism of BRAF inhibitor resistance, which may provide a new strategy for predicting vemurafenib responses in clinical treatment.

CAMKK2 Defines Ferroptosis Sensitivity of Melanoma Cells by Regulating AMPK‒NRF2 Pathway

Melanoma is the most lethal skin cancer caused by the malignant transformation of epidermal melanocytes. Recent progress in targeted therapy and immunotherapy has significantly improved the treatment outcome, but the survival of patients with advanced melanoma remains suboptimal. Ferroptosis, a cell death modality triggered by iron-dependent lipid peroxidation, reportedly participates in cancer pathogenesis and can mediate the effect of anti-PD-1 immunotherapy in melanoma. However, the detailed regulatory mechanism of ferroptosis remains far from being understood. In this study, we report that CAMKK2 defines the ferroptosis sensitivity of melanoma cells by regulating the AMPK‒NRF2 pathway. We first found that CAMKK2 was prominently activated in ferroptosis. Then we proved that CAMKK2 negatively regulated ferroptosis through the activation of NRF2 and the suppression of lipid peroxidation. Subsequent mechanistic studies revealed that AMPK connected CAMKK2 upregulation to NRF2-dependent antioxidative machinery in ferroptosis. In addition, the suppression of CAMKK2 increased the efficacy of ferroptosis inducer and anti-PD-1 immunotherapy in the preclinical xenograft tumor model by inhibiting the AMPK‒NRF2 pathway and promoting ferroptosis. Taken together, CAMKK2 plays a protective role in ferroptosis by activating the AMPK‒NRF2 pathway. Targeting CAMKK2 could be a potential approach to increase the efficacy of ferroptosis inducers and immunotherapy for melanoma treatment.

Abstract 1116: HDAC8 activity regulates stress induced phenotype switching in melanoma

Abstract Background: Melanoma cells show great plasticity and can switch from a proliferative, drug-sensitive state to an invasive, drug-resistant phenotype through mechanisms that remain poorly defined. Hypothesis: Transcriptional and chromatin regulatory factors alter the gene expression states of cell in response to targeted therapy treatment and allow melanoma cells to persist and grow in the face of initially effective therapy. These factors/pathways may represent additional targets for anti-melanoma therapy. Methods: BRAF mutant melanoma cells were exposed to the BRAF inhibitor vemurafenib to generate cells that persisted and grew in the face of continued drug treatment. RNA-Seq, CHIP seq, ATAC-Seq and mass spectroscopy analyses were performed on persistent/resistant cells in comparison to parental cells. Additionally, histone deacetylase (HDAC) 8 and EP300 expression was enforced in drug naïve melanoma cell lines and the effects determined. Results: HDAC8 (but not other HDACs) was consistently up-regulated in BRAFi resistant melanoma cells. Increased expression of HDAC8 reduced cell growth, increased p53 activation and suppressed microphthalmia-associated transcription factor MITF, suppressing the melanocyte lineage program driven, leading to increased drug resistance. Introduction of HDAC8 or its active mutant (S39A) increased the tolerance of melanoma cells to multiples stresses (UV-irradiation, hypoxia, and BRAF-MEK inhibitor therapy), reduced their proliferation and increased their invasive potential in in vitro and in vivo models. Gene expression analyses indicate genes associated with a proliferative, drug-sensitive phenotype (e.g. DCT, MITF, MLANA, SOX10) are downregulated in HDAC8 active cells while genes correlative with an invasive, drug-resistant phenotype are upregulated (e.g. AXL, EGFR, TGFB1, WNT5A). ATAC-Seq analysis showed that HDAC8 regulates chromatin structure and decreased accessibility in the DNA binding sites of MITF and Sox10 while increasing accessibility in the DNA binding sites of c-Jun and TEADs (e.g. YAP1). Additionally, ChIP-Seq showed an increase in HDAC8 activity decreased H3K27 acetylation in the promoter region of MITF and MITF regulated genes (e.g. DCT, MLANA, PMEL, TYR). Mass spectrometry-based acetylomics and co-immunoprecipitation experiments demonstrated that active HDAC8 associated with and deacetylated the histone acetyltransferase EP300, a known coactivator of MITF in melanocytes. Increased expression of EP300 increased the sensitivity of melanoma cells to UV-irradiation, hypoxia and BRAF-MEK inhibitor therapy. Conclusions: HDAC8 associated with EP300 and mediated the switch to an invasive, stress resistant state in melanoma cells by suppressing MITF and increasing c-Jun driven transcriptional programs. Targeting HDAC8 could be a strategy to limit plasticity in advanced melanoma, increasing the efficacy of current therapies. Citation Format: Michael Emmons, Richard Bennett, Alberto Riva, Bin Fang, Edward Seto, john koomen, Jonathan Licht, Keiran Smalley. HDAC8 activity regulates stress induced phenotype switching in melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1116.

Development and in vivo evaluation of fused benzazole analogs of anti-melanoma agent HA15.

Background: In line with our recent discovery of an efficient anticancer thiazolebenzenesulfonamide framework HA15 (1) based on a remarkable endoplasmic reticulumstress inducement mode of action, we report herein a series of innovative constrained HA15 analogs, featuring four types of bicylic derivatives. Results: The structure-activity relationship analysis, using acell lineassay, led us to identify a novel version of HA15: a new benzothiazole derivative (10b) exhibiting important anti-melanoma effect against sensitive and resistant melanomacells. Meanwhile, compound 10b induced a significant tumor growth inhibition in vivo with no apparent signs of toxicity. Conclusion: These results consistently open new directions to improve and develop more powerful anticancer therapeutics harboring this type of fused framework.

PARP Inhibitors Talazoparib and Niraparib Sensitize Melanoma Cells to Ionizing Radiation.

(1) Background: Niraparib and Talazoparib are poly (ADP-ribose) polymerase (PARP) 1/2 inhibitors. It is assumed that combining PARP inhibitors with radiotherapy could be beneficial for cancer treatment. In this study, melanoma cells were treated with Niraparib and Talazoparib in combination with ionizing radiation (IR). (2) Methods: The effects of Talazoparib and Niraparib in combination with IR on cell death, clonogenicity and cell cycle arrest were studied in healthy primary fibroblasts and primary melanoma cells. (3) Results: The melanoma cells had a higher PARP1 and PARP2 content than the healthy fibroblasts, and further increased their PARP2 content after the combination therapy. PARP inhibitors both sensitized fibroblasts and melanoma cells to IR. A clear supra-additive effect of KI+IR treatment was detected in two melanoma cell lines analyzing the surviving fraction. The cell death rate increased in the healthy fibroblasts, but to a larger extent in melanoma cells after combined treatment. Finally, a lower percentage of cells in the radiosensitive G2/M phase is present in the healthy fibroblasts compared to the melanoma cells. (4) Conclusions: Both PARP inhibitors sensitize melanoma cells to IR. Healthy tissue seems to be less affected than melanoma cells. However, the great heterogeneity of the results suggests prior testing of the tumor cells in order to personalize the treatment.

Pigmentation Levels Affect Melanoma Responses to Coriolus versicolor Extract and Play a Crucial Role in Melanoma-Mononuclear Cell Crosstalk.

Melanoma, the malignancy originating from pigment-producing melanocytes, is the most aggressive form of skin cancer and has a poor prognosis once the disease starts to metastasize. The process of melanin synthesis generates an immunosuppressive and mutagenic environment, and can increase melanoma cell resistance to different treatment modalities, including chemo-, radio- or photodynamic therapy. Recently, we have shown that the presence of melanin pigment inhibits the melanoma cell response to bioactive components of Coriolus versicolor (CV) Chinese fungus. Herein, using the same human melanoma cell line in which the level of pigmentation can be controlled by the L-tyrosine concentration in culture medium, we tested the effect of suppression of melanogenesis on the melanoma cell response to CV extract and investigated the cell death pathway induced by fungus extract in sensitized melanoma cells. Our data showed that susceptibility to CV-induced melanoma cell death is significantly increased after cell depigmentation. To the best of our knowledge, we are the first to demonstrate that CV extract can induce RIPK1/RIPK3/MLKL-mediated necroptosis in depigmented melanoma cells. Moreover, using the co-culture system, we showed that inhibition of the tyrosinase activity in melanoma cells modulates cytokine expression in co-cultured mononuclear cells, indicating that depigmentation of melanoma cells may activate immune cells and thereby influence a host anticancer response.

Targeting the p300/NONO axis sensitizes melanoma cells to BRAF inhibitors

BRAF inhibitors (BRAFi) that target BRAF V600E kinase, a driver mutation found in 50% of melanomas, show a significant antitumor response, but the common emergence of acquired resistance remains a challenge. Abnormal expression of RAF isoforms CRAF and ARAF reactivates pERK1/2, which plays crucial roles in the acquisition of resistance of melanoma cells. However, the mechanisms of dysregulation of RAF isoforms in resistant melanoma cells remain unknown. Here, we identified NONO interacted with and stabilized both CRAF and ARAF in melanoma cells, and that NONO was acetylated at 198K by p300 acetyltransferase, which stabilized NONO via antagonizing its ubiquitination/degradation mediated by RNF8. The upregulation of both p300 and NONO promoted the rebound of pERK1/2 and the subsequent resistance of melanoma cells to BRAFi, and the activation of ERK1/2 in turn induced p300 to form a positive feedback loop in resistant melanoma cells. There was a positive correlation between p300 and NONO in resistant melanoma cells and clinical samples, and p300 inhibitor C646 overcame the resistance of resistant melanoma cells to BRAF inhibitors in vitro and in vivo. Our findings reveal that targeting the positive feedback loop of p300-NONO-CRAF/ARAF-pERK1/2 may be excellent strategies to overcome the resistance of BRAF inhibitors for melanoma patients.

Targeting SIRT2 Sensitizes Melanoma Cells to Cisplatin via an EGFR-Dependent Mechanism.

Melanoma cells are resistant to most anticancer chemotherapeutics. Despite poor response rates and short-term efficacy, chemotherapy remains the main approach to treating this cancer. The underlying mechanisms of the intrinsic chemoresistance of melanoma remain unclear, but elucidating these mechanisms is important to improve the efficacy of chemotherapy regimens. Increasing evidence suggests that sirtuin 2 (SIRT2) plays a key role in the response of melanoma cells to chemotherapeutics; thus, in the present study, we evaluated the impact of shRNA-mediated and pharmacological inhibition of SIRT2 on the sensitivity of melanoma cells to cisplatin, which is used in several regimens to treat melanoma patients. We found that cells with SIRT2 inhibition revealed increased sensitivity to cisplatin and exhibited increased accumulation of γ-H2AX and reduced EGFR-AKT-RAF-ERK1/2 (epidermal growth factor receptor-protein B kinase–RAF kinase-extracellular signal-regulated kinase 1/2) pathway signaling compared to control cells. Thus, our results show that sirtuin 2 inhibition increased the in vitro efficacy of cisplatin against melanoma cells.

Nuclear Receptor Coactivator NCOA3 Regulates UV Radiation-Induced DNA Damage and Melanoma Susceptibility.

Melanoma occurs as a consequence of inherited susceptibility to the disease and exposure to UV radiation (UVR) and is characterized by uncontrolled cellular proliferation and a high mutational load. The precise mechanisms by which UVR contributes to the development of melanoma remain poorly understood. Here we show that activation of nuclear receptor coactivator 3 (NCOA3) promotes melanomagenesis through regulation of UVR sensitivity, cell-cycle progression, and circumvention of the DNA damage response (DDR). Downregulation of NCOA3 expression, either by genetic silencing or small-molecule inhibition, significantly suppressed melanoma proliferation in melanoma cell lines and patient-derived xenografts. NCOA3 silencing suppressed expression of xeroderma pigmentosum C and increased melanoma cell sensitivity to UVR. Suppression of NCOA3 expression led to activation of DDR effectors and reduced expression of cyclin B1, resulting in G2-M arrest and mitotic catastrophe. A SNP in NCOA3 (T960T) reduced NCOA3 protein expression and was associated with decreased melanoma risk, given a significantly lower prevalence in a familial melanoma cohort than in a control cohort without cancer. Overexpression of wild-type NCOA3 promoted melanocyte survival following UVR and was accompanied by increased levels of UVR-induced DNA damage, both of which were attenuated by overexpression of NCOA3 (T960T). These results describe NCOA3-regulated pathways by which melanoma can develop, with germline NCOA3 polymorphisms enabling enhanced melanocyte survival in the setting of UVR exposure, despite an increased mutational burden. They also identify NCOA3 as a novel therapeutic target for melanoma. SIGNIFICANCE: This study explores NCOA3 as a regulator of the DDR and a therapeutic target in melanoma, where activation of NCOA3 contributes to melanoma development following exposure to ultraviolet light.