Apoptosis In Melanoma Research Articles

Abstract 2327: Apoptosis of melanoma induced by non-thermal atmospheric pressure bio-compatible plasma activated media

Abstract Melanoma is the most dangerous type of skin cancer, which mostly related with the exposure of ultraviolet (UV), and especially happened to those who have low skin pigment levels. Epidemiological studies showed Europeans and North Americans have a high incidence of melanoma, while it is less common in Asia and Africa. Therefore, the new and more effective methods to treat melanoma are still necessarily needed. Non-thermal atmospheric pressure bio-compatible plasma (NBP) is defined as a partially ionized gas with electrically charged particle, which has been reported have cytotoxicity on various cancer cells induce DNA damage and apoptosis other than normal cells. Reactive oxygen and/or nitrogen species (RONS) were proved to play the most important role during this process, by inducing oxidative stress and depolarization of mitochondria membrane potential with the consequence of various cancer cells apoptosis. The plasma activated media (PAM), which the media treated directly by NBP with different time, containing mostly long-live secondary species such as hydrogen peroxide (H2O2) which have been confirmed anti-microorganism and cytotoxic activity. Researches already investigated that PAM also effective in cancer therapy via inducing apoptosis. However, the underlying molecular mechanisms of apoptosis are remaining elusive. The purpose of this study was to evaluate the potential of PAM as an effective tool to induce apoptosis in melanoma cells. Our results showed that PAM has a killing effect to melanoma cells in a time-dependent manner, for 3minutes treatment could induce almost 40% cells entry death, after treated by 5 minutes more than 60%, and 10 minutes treatment reduced the cell viability to approximately 30%. Annexin-V/PI staining demonstrated that PAM kills these cells via apoptosis pathway. We also found that with a manner of treatment time PAM significantly increased the concentration of intracellular NO and H2O2, reflecting an influx of extracellular RONS may result in melanoma cells apoptosis. Besides, western blot assay showed that P53 and caspase 3 increased after PAM treatment. Taken together, PAM is effective to induce the apoptosis in melanoma cells in vitro, further in vivo experiments will be performed to investigate the functional effects in the animals. Citation Format: Ihn Han, Ying Li, Eun Ha Choi. Apoptosis of melanoma induced by non-thermal atmospheric pressure bio-compatible plasma activated media [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2327. doi:10.1158/1538-7445.AM2017-2327

Ascorbate induces apoptosis in melanoma cells by suppressing Clusterin expression

Pharmacological levels of ascorbate have long been suggested as a potential treatment of cancer. However, we observed that EC50 of ascorbate was at a similar level for cultured healthy melanocytes and melanoma cells, suggesting a limit of pharmacological ascorbate in treating cancer. Loss of 5-hydroxymethylcytosine (5 hmC) is an epigenetic hallmark of cancer and ascorbate promotes 5 hmC generation by serving as a cofactor for TET methylcytosine dioxygenases. Our previous work demonstrated that ascorbate treatment at physiological level (100 μM) increased 5 hmC content in melanoma cells toward the level of healthy melanocytes. Here we show that 100 µM of ascorbate induced apoptosis in A2058 melanoma cells. RNA-seq analysis revealed that expression of the Clusterin (CLU) gene, which is related to apoptosis, was downregulated by ascorbate. The suppression of CLU was verified at transcript level in different melanoma cell lines, and at protein level in A2058 cells. The anti-apoptotic cytoplasmic CLU was decreased, while the pro-apoptotic nuclear CLU was largely maintained, after ascorbate treatment. These changes in CLU subcellular localization were also associated with Bax and caspases activation, Bcl-xL sequestration, and cytochrome c release. Taken together, this study establishes an impending therapeutic role of physiological ascorbate to potentiate apoptosis in melanoma.

Identification of ACA‐28, a 1′‐acetoxychavicol acetate analogue compound, as a novel modulator of ERK MAPK signaling, which preferentially kills human melanoma cells

The extracellular signal-regulated kinase (ERK) signaling pathway is essential for cell proliferation and is frequently deregulated in human tumors such as melanoma. Melanoma remains incurable despite the use of conventional chemotherapy; consequently, development of new therapeutic agents for melanoma is highly desirable. Here, we carried out a chemical genetic screen using a fission yeast phenotypic assay and showed that ACA-28, a synthetic derivative of 1'-acetoxychavicol acetate (ACA), which is a natural ginger compound, effectively inhibited the growth of melanoma cancer cells wherein ERK MAPK signaling is hyperactivated due to mutations in the upstream activating regulators. ACA-28 more potently inhibited the growth of melanoma cells than did the parental compound ACA. Importantly, the growth of normal human epidermal melanocytes (NHEM) was less affected by ACA-28 at the same 50% inhibitory concentration. In addition, ACA-28 specifically induced apoptosis in NIH/3T3 cells which were oncogenically transformed with human epidermal growth factor receptor-2 (HER2/ErbB2), but not in the parental cells. Notably, the ACA-28-induced apoptosis in melanoma and HER2-transformed cells was abrogated when ERK activation was blocked with a specific MEK inhibitor U0126. Consistently, ACA-28 more strongly stimulated ERK phosphorylation in melanoma cells, as compared in NHEM. ACA-28 might serve as a promising seed compound for melanoma treatment.

807 LRP1 is a tumor promotor by inhibiting apoptosis in human melanoma

807 LRP1 is a tumor promotor by inhibiting apoptosis in human melanoma

P53 targets TSPAN8 to prevent invasion in melanoma cells

Cutaneous melanoma is a very deadly cancer because of its proclivity to metastasize. Despite the recent development of targeted and immune therapies, patient survival remains low. It is therefore crucial to enhance understanding of the molecular mechanisms underlying invasion. We previously identified tetraspanin 8 (TSPAN8) as an important modulator of melanoma invasiveness, and several of its transcriptional regulators, which affect TSPAN8 expression during melanoma progression toward an invasive stage. This study found that TSPAN8 promoter contains consensus-binding sites for p53 transcription factor. We demonstrated that p53 silencing was sufficient to turn on Tspan8 expression in non-invasive melanoma cells and that p53 acts as a direct transcriptional repressor of TSPAN8. We also showed that p53 modulated matrigel invasion in melanoma cells in a TSPAN8-dependent manner. In conclusion, this study reveals p53 as a negative regulator of Tspan8 expression. As TP53 gene is rarely mutated in melanoma, it was hitherto poorly studied but its role in apoptosis and growth suppression in melanoma is increasingly becoming clear. The study highlights the importance of p53 as a regulator of melanoma invasion and the concept that reactivating p53 could provide a strategy for modulating not only proliferative but also invasive capacity in melanoma treatment.

Overexpression of the anti-apoptotic protein BAG3 in human choroidal melanoma: A case report.

Bcl-2-associated athanogene 3 (BAG3), a co-chaperone of heat shock protein 70 (HSP70), exerts anti-apoptotic effects in various malignant tumors. However, relationships between choroidal melanoma and BAG3 are poorly studied. This study investigated the expression of BAG3 in a case of human choroidal melanoma. Funduscopy, computed tomography, and single-photon emission computed tomography with the intravenous injection of N-isopropyl-p-[123I] iodoamphetamine strongly indicated choroidal melanoma in a 68-year-old woman. Accordingly, we carried out an enucleation and pathological diagnosis. Proteins and total RNA were extracted from normal retinochoroidal and tumor tissues. Proteins were also extracted from ocular nevus tissues of other patients. We examined the expression of BAG3 protein and mRNA using Western blotting and the real-time quantitative polymerase chain reaction, respectively. Immunohistochemical stains were positive for melan-A, HMB-45, and S-100. Histopathology confirmed a choroidal melanoma. The expression of BAG3 protein and mRNA in the choroidal melanoma tissue was upregulated with respect to both normal retinochoroidal tissue and ocular nevus tissues from other patients. Because BAG3 may inhibit apoptosis of choroidal melanoma and facilitate its survival, overexpression of this gene product may be a prognostic marker and therapeutic target.

Effects of miR-145-5p through NRAS on the cell proliferation, apoptosis, migration, and invasion in melanoma by inhibiting MAPK and PI3K/AKT pathways.

We aimed to detect the effects of miR‐145‐5p on the cell proliferation, apoptosis, migration, and invasion in NRAS‐mutant, BRAF‐mutant, and wild‐type melanoma cells, in order to figure out the potential mechanisms and provide a novel therapeutic target of melanoma. RT‐qPCR and western blot were used to detect the expression of miR‐145‐5p and NRAS in melanoma tumor tissues and cells, respectively. Luciferase assay was performed to determine whether miR‐145‐5p directly targeted NRAS. After transfecting miR‐145‐5p mimics, miR‐145‐5p inhibitors, NRAS cDNA and NRAS siRNA into CHL‐1, VMM917 and SK‐mel‐28 cells, functional assays were used to detect the proliferation, apoptosis, invasion and migration, including MTT, flow cytometry, Transwell and wound healing assays. In addition, xenograft models in nude mice were also conducted to verify the role of miR‐145‐5p in vivo. MiR‐145‐5p was able to suppress proliferation, invasion, and migration of VMM917 and CHL‐1 cells and induce apoptosis by inhibiting MAPK and PI3K/AKT pathways. However, aberrant expression of miR‐145‐5p and NRAS has little impact on the viability and metastasis of BRAF‐mutant melanoma. The higher expression of miR‐145‐5p in xenograft models repressed the VMM917‐induced and CHL‐1‐induced tumor growth observably and has little effect on SK‐mel‐28‐induced tumor growth which was consistent with the results in vitro. Through targeting NRAS, miR‐145‐5p could suppress cell proliferation, invasion, and migration and induce apoptosis of CHL‐1 and VMM917 melanoma cells by inhibiting MAPK and PI3K/AKT pathways.

NLRP1 promotes tumor growth by enhancing inflammasome activation and suppressing apoptosis in metastatic melanoma.

Inflammasomes are mediators of inflammation, and constitutively activated NLRP3 inflammasomes have been linked to IL-1β-mediated tumorigenesis in human melanoma. Whereas NLRP3 regulation of caspase-1 activation requires the adaptor protein ASC, caspase-1 activation by another danger-signaling sensor NLRP1 does not require ASC because NLRP1 contains a C-terminal CARD domain that facilitates direct caspase-1 activation via CARD-CARD interaction. We hypothesized that NLRP1 has additional biological activities besides IL-1β maturation and investigated its role in melanoma tumorigenesis. NLRP1 expression in melanoma was confirmed by analysis of 216 melanoma tumors and 13 human melanoma cell lines. Unlike monocytic THP-1 cells with prominent nuclear localization of NLRP1, melanoma cells expressed NLRP1 mainly in the cytoplasm. Knocking down NLRP1 revealed a tumor promoting property of NLRP1 both in vitro and in vivo. Mechanistic studies showed that caspase-1 activity, IL-1β production, IL-1β secretion, and NF-kB activity were reduced by knocking down of NLRP1 in human metastatic melanoma cell lines 1205Lu and HS294T, indicating that NLRP1 inflammasomes are active in metastatic melanoma. However, unlike previous reports showing that NLRP1 enhances pyroptosis in macrophages, NLRP1 in melanoma behaved differently in the context of cell death. Knocking down NLRP1 increased caspase-2, -9, and -3/7 activities and promoted apoptosis in human melanoma cells. Immunoprecipitation revealed interaction of NLRP1 with CARD-containing caspase-2 and -9, whereas NLRP3 lacking a CARD motif did not interact with the caspases. Consistent with these findings, NLRP1 activation but not NLRP3 activation reduced caspase-2, -9, and -3/7 activities and provided protection against apoptosis in human melanoma cells, suggesting a suppressive role of NLRP1 in caspase-3/7 activation and apoptosis via interaction with caspase-2 and -9. In summary, we showed that NLRP1 promotes melanoma growth by enhancing inflammasome activation and suppressing apoptotic pathways. Our study demonstrates a tumor-promoting role of NLRP1 in cancer cells.

A novel ECG analog 4-(S)-(2,4,6-trimethylthiobenzyl)-epigallocatechin gallate selectively induces apoptosis of B16-F10 melanoma via activation of autophagy and ROS

Autophagy-induced cancer cell death has become a novel strategy for the development of cancer therapeutic drugs. Numerous studies have indicated that green tea polyphenols induce both autophagy and apoptosis in a variety of cancer cells. Here, we synthesized a series of green tea polyphenol analogues, among which JP8 was shown to potently activate autophagy. JP8 treatment had a stronger effect on apoptosis in B16-F10 melanoma cells than that in normal AML-12 hepatocytes. JP8 selectively resulted in reactive oxygen species (ROS) accumulation in B16-F10 cells, and this effect was associated with corresponding increases in key components of the ER stress-mediated apoptosis pathway. Pharmacological inhibition of ROS by N-acetyl-L-cysteine (NAC) attenuated JP8-induced autophagy and apoptosis, indicating an upstream role of ROS in JP8-induced autophagy. An in vivo study showed that JP8 had significant antitumor effects in a B16-F10 xenograft mouse model. Our results indicate that JP8 is a novel anticancer candidate with both autophagy and ROS induction activities.

MiR-204-5p acts as a tumor suppressor by targeting matrix metalloproteinases-9 and B-cell lymphoma-2 in malignant melanoma.

An increasing number of microRNAs have been found to be involved in tumorigenesis, including melanoma tumorigenesis. miR-204-5p is down-regulated and functions as a tumor suppressor in many human malignant tumors. miR-204-5p expression is also decreased in melanoma tissues, but its biological roles and molecular mechanisms in malignant melanoma remain unclear. In this study, the aberrant down-regulation of miR-204-5p was detected in melanoma, especially in metastatic melanoma. miR-204-5p also served as a protective factor for the prognosis of melanoma patients. We determined that miR-204-5p suppresses cell proliferation, migration and invasion, and promotes cell apoptosis in melanoma. Matrix metalloproteinases-9 and B-cell lymphoma-2 are the functional targets of miR-204-5p, through which it plays an important biological role in malignant melanoma. The effect of miR-204-5p on malignant melanoma is verified using a xenograft model. We also determined that miR-204-5p increases 5-fluorouracil and cisplatin (DDP) chemosensitivity in malignant melanoma cells. This finding elucidates new functions and mechanisms for miR-204-5p in melanoma development, and provides potential therapeutic targets for the treatment of melanoma.

Antiproliferative and Pro-Apoptotic Effects of MiR-4286 Inhibition in Melanoma Cells.

IntroductionMicroRNAs are essential regulators of gene expression at the post-transcriptional level. Their expression is altered in cancer tissues, and evaluation of these alterations is considered a promising tool used to diagnose and identify prognostic markers.Materials and methodsThe microRNA expression profiles of formalin-fixed, paraffin-embedded melanoma and melanocytic nevi samples were estimated with a microarray and subsequently validated by real-time PCR. Melanoma cells were transfected with miR-4286 inhibitor to evaluate the influence of this microRNA on the viability, proliferation, apoptosis, migration, and invasion of melanoma cells.ResultsThe microarray revealed that the expression of 1,171 microRNAs was altered in melanoma samples compared to melanocytic nevi. Real-time PCR validation experiments found the microRNA expression levels to correspond to the melanoma/melanocytic nevi microarray results. The pathway analysis identified 52 modulated pathways in melanoma. Moreover, the application of miR-4286 inhibitor to BRO melanoma cells resulted in a 2.6-fold increase in the apoptosis rate and a 1.7-fold decrease in the cell proliferation/viability but did not affect the invasiveness and migration of these cells. Furthermore, the use of miR-4286 inhibitor altered the mRNA expression of several miR-4286 gene targets: folylpolyglutamate synthase, RNA polymerase I-specific transcription initiation factor, apelin, G-protein-coupled receptor 55, and high-mobility group A1 protein, which have been implicated in cell proliferation/apoptosis regulation. Lastly, the transiently transfected SK-MEL-1 cells with miR-4286 inhibitor decreased proliferation rate and modulated folylpolyglutamate synthase rates of these cells.ConclusionOur results demonstrate that miR-4286 mediates proliferation and apoptosis in melanoma cells, these findings may represent a novel mechanism underlying these processes.

MYO5A Gene Is a Target of MITF in Melanocytes

MYO5A Gene Is a Target of MITF in Melanocytes

HvTRA, a novel TRAIL receptor agonist, induces apoptosis and sustained growth retardation in melanoma.

In recent years, new treatment options for malignant melanoma patients have enhanced the overall survival for selected patients. Despite new hope, most melanoma patients still relapse with drug-resistant tumors or experience intrinsic resistance to the therapy. Therefore, novel treatment modalities beneficial for subgroups of patients are needed. TRAIL receptor agonists have been suggested as promising candidates for use in cancer treatment as they preferentially induce apoptosis in cancer cells. Unfortunately, the first generation of TRAIL receptor agonists showed poor clinical efficacy. hvTRA is a second-generation TRAIL receptor agonist with improved composition giving increased potency, and in the present study, we showed hvTRA-induced activation of apoptosis leading to an efficient and sustained reduction in melanoma cell growth in cell lines and xenograft models. Furthermore, the potential of hvTRA in a clinical setting was demonstrated by showing efficacy on tumor cells harvested from melanoma patients with lymph node metastasis in an ex vivo drug sensitivity assay. Inhibition of mutated BRAF has been shown to regulate proteins in the intrinsic apoptotic pathway, making the cells more susceptible for apoptosis induction. In an attempt to increase the efficacy of hvTRA, combination treatment with the mutated BRAF inhibitor vemurafenib was investigated. A synergistic effect by the combination was observed for several cell lines in vitro, and an initial cytotoxic effect was observed in vivo. Unfortunately, the initial increased reduction in tumor growth compared with hvTRA mono treatment was not sustained, and this was related to downregulation of the DR5 level by vemurafenib. Altogether, the presented data imply that hvTRA efficiently induce apoptosis and growth delay in melanoma models and patient material, and the potential of this TRAIL receptor agonist should be further evaluated for treatment of subgroups of melanoma patients.

(-)-Oleocanthal exerts anti-melanoma activities and inhibits STAT3 signaling pathway.

Tumor angiogenesis, growth and metastasis are three closely related processes. We therefore explored the effects of (-)-oleocanthal(OC) on the three processes in melanoma and investigated underlying mechanisms. Invitro, OC suppressed proliferation, migration, invasion and induced apoptosis in melanoma cells. In addition, OC inhibited proliferation, migration, invasion and tube formation in human umbilical vascular endothelial cells. Invivo, it exhibited potent activity in suppressing tumor growth in a subcutaneous xenograft model. Furthermore, OC suppressed proliferation and angiogenesis as measured by immunohistochemical staining of Ki-67 and CD31. In addition, OC was found to inhibit metastasis of melanoma in a lung metastasis model. Mechanistically, OC significantly suppressed signal transducer and activator of transcription3 (STAT3) phosphorylation, decreased STAT3 nuclear localization and inhibited STAT3 transcriptional activity. OC also downregulated STAT3 target genes, including Mcl-1, Bcl-xL, MMP-2, MMP-9, VEGF, which are involved in apoptosis, invasion and angiogenesis of melanoma. These results support further investigation of OC as a potential anti-melanoma drug.

P63 is required beside p53 for PERP-mediated apoptosis in uveal melanoma.

Background:PERP (p53 apoptosis effector related to PMP-22), a transcriptional target of p53, is downregulated and contributes to the impairment of apoptosis in uveal melanoma (UM). Intriguingly, PERP is not induced in UM despite functional p53. p63, located on chromosome 3, which is characteristically altered in high-risk UM, can transactivate PERP. Here, we determine the functional role of p63 expression in the initiation of p53/PERP-mediated apoptosis in UM.Methods:PERP expression was monitored by quantitative PCR (qPCR) and immunoblotting in UM cell lines treated with DNA-damaging agents. The functional role of p63 was assessed by transient expression of p63-turbo GFP (p63-tGFP) in the apoptosis- resistant, 3q-deficient OCM-1 cells. Expression and localisation of p63, PERP and p53, and induction of apoptosis were characterised by qPCR, immunoblotting and live cell confocal microscopy.Results:PERP expression was significantly downregulated in all UM cell lines. DNA-damaging treatments failed to induce apoptosis and activate PERP in OCM-1 cells, which displayed non-functional levels of p63. Expression of p63-tGFP induced apoptosis with marked increase in PERP expression and associated p53 accumulation.Conclusions:Lack of p63 contributes to reduced PERP levels and impaired p53-mediated apoptosis in UM. p63 expression is required for PERP-mediated apoptosis in UM.

Casein kinase 1α has a non-redundant and dominant role within the CK1 family in melanoma progression.

BackgroundWe previously identified CK1α as a novel tumor suppressor in melanoma and reported that the loss of CK1α leads to increased proliferation and invasive growth of melanoma cells by strong activation of the Wnt/β-catenin signaling pathway.MethodsIn this study we analyzed expression and the functional effects of the dominantly expressed CK1- isoforms α, δ and ε in melanoma cells by quantitative real-time PCR, western blot and immunohistochemistry. We down-regulated CK1 kinase activity with isoform specific siRNAs and small molecule inhibitors. Vice versa we overexpressed the CK1 isoforms α, δ and ε using viral vectors and tested the biological effects on melanoma cell proliferation, migration and invasion.ResultsWe show that protein expression of all three CK1-isoforms is downregulated in metastatic melanoma cells compared to benign melanocytic cells. Furthermore, the CK1δ and ε isoforms are able to negatively regulate expression of each other, whereas CK1α expression is independently regulated in melanoma cells. Inhibition of the expression and activity of CK1δ or CK1ε by specific inhibitors or siRNAs had no significant effect on the growth and survival of metastatic melanoma cells. Moreover, the over-expression of CK1δ or CK1ε in melanoma cells failed to induce cell death and cell cycle arrest although p53 signaling was activated. This is in contrast to the effects of CK1α where up-regulated expression induces cell death and apoptosis in metastatic melanoma cells.ConclusionThese data indicate that CK1α has a dominant and non-redundant function in melanoma cells and that the CK1δ and ε isoforms are not substantially involved in melanoma progression.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-016-2643-0) contains supplementary material, which is available to authorized users.

Knockdown of USP39 induces cell cycle arrest and apoptosis in melanoma.

The spliceosome machinery composed of multimeric protein complexes guides precursor messenger RNAs (mRNAs) (pre-mRNAs) splicing in eukaryotic cells. Spliceosome components have been shown to be downregulated in cancer and could be a promising molecular target for anticancer therapy. The ubiquitin-specific protease 39 (USP39) is essential for pre-mRNA splicing, and upregulated USP39 expression is noted in a variety of cancers. However, the role of USP39 in the development and progression of melanoma remains unclear. In the present study, USP39 expression was found to be increased in melanoma tissues compared with that in nevus tissues. USP39 silencing via lentivirus-mediated short hairpin RNA (shRNA) significantly suppressed melanoma cell proliferation, induced G0/G1 cell cycle phase arrest, and increased apoptosis in vitro. Moreover, USP39 knockdown suppressed melanoma tumor growth in a xenograft model. In addition, USP39 silencing was associated with the increased expressions of p21, p27, and Bax. Furthermore, the inhibition of USP39 expression decreased the phosphorylation of extracellular signal-regulated kinase (ERK)1/2, indicating that ERK signaling pathways might be involved in the regulation of melanoma cell proliferation by USP39. Our findings suggest that USP39 may play crucial roles in the development and pathogenesis of melanoma, and it may serve as a potential therapeutic target for melanoma.

Lentivirus-mediated downregulation of MAT2B inhibits cell proliferation and induces apoptosis in melanoma.

Malignant melanoma is the most lethal of skin cancers and its pathogenesis is complex and heterogeneous. The efficacy of conventional therapeutic regimens for melanoma remains limited. Thus, it is important to explore novel effective therapeutic targets in the treatment of melanoma. The MAT2B gene encodes for the regulatory subunit of methionine adenosyltransferase (MAT). Recent studies have suggested that MAT2B may have functional roles other than modulating catalytic activity of MAT. In order to identify the roles of MAT2B in the tumorigenesis of malignant melanoma, we compared MAT2B expression profile in melanoma tissues with that in benign nevus samples. We employed lentivirus-mediated RNAi to downregulate the expression of MAT2B in malignant melanoma cell lines (A375 and Mel-RM), and investigated the effects of MAT2B on cell growth, colony-formation ability and apoptosis invitro, as well as tumor growth of a xenograft model invivo. The expression levels of BCL2 and XAF1 proteins, which were closely related to tumor cell apoptosis, were analyzed by western blot analysis. Our data showed that MAT2B was elevated in both primary and metastatic melanoma tissues compared with benign nevus samples. Lentivirus-mediated downregulation of MAT2B suppressed cell growth, colony formation and induced apoptosis in A375 and Mel-RM cell lines invitro, affected protein expression of BCL2 and XAF1, extended the transplanted tumor growth invivo. These results indicated that MAT2B was critical in the proliferation of melanoma cells and tumorigenicity. It may be considered as a potential anti-melanoma therapeutic target.

563 - Combined CDK and HDAC inhibition induces apoptosis in both uveal and cutaneous melanoma

563 - Combined CDK and HDAC inhibition induces apoptosis in both uveal and cutaneous melanoma

The deubiquitinase Usp27x stabilizes the BH3-only protein Bim and enhances apoptosis.

Bim is a pro-apoptotic Bcl-2 family member of the BH3-only protein subgroup. Expression levels of Bim determine apoptosis susceptibility in non-malignant and in tumour cells. Bim protein expression is downregulated by proteasomal degradation following ERK-dependent phosphorylation and ubiquitination. Here, we report the identification of a deubiquitinase, Usp27x, that binds Bim upon its ERK-dependent phosphorylation and can upregulate its expression levels. Overexpression of Usp27x reduces ERK-dependent Bim ubiquitination, stabilizes phosphorylated Bim, and induces apoptosis in PMA-stimulated cells, as well as in tumour cells with a constitutively active Raf/ERK pathway. Loss of endogenous Usp27x enhances the Bim-degrading activity of oncogenic Raf. Overexpression of Usp27x induces low levels of apoptosis in melanoma and non-small cell lung cancer (NSCLC) cells and substantially enhances apoptosis induced in these cells by the inhibition of ERK signalling. Finally, deletion of Usp27x reduces apoptosis in NSCLC cells treated with an EGFR inhibitor. Thus, Usp27x can trigger via its proteolytic activity the deubiquitination of Bim and enhance its levels, counteracting the anti-apoptotic effects of ERK activity, and therefore acts as a tumour suppressor.