Therapeutic Target For Malignant Melanoma Research Articles

Ferroptosis as a promising therapeutic strategy for melanoma.

Malignant melanoma (MM) is the most common and deadliest type of skin cancer and is associated with high mortality rates across all races and ethnicities. Although present treatment options combined with surgery provide short-term clinical benefit in patients and early diagnosis of non-metastatic MM significantly increases the probability of survival, no efficacious treatments are available for MM. The etiology and pathogenesis of MM are complex. Acquired drug resistance is associated with a pool prognosis in patients with advanced-stage MM. Thus, these patients require new therapeutic strategies to improve their treatment response and prognosis. Multiple studies have revealed that ferroptosis, a non-apoptotic form of regulated cell death (RCD) characterized by iron dependant lipid peroxidation, can prevent the development of MM. Recent studies have indicated that targeting ferroptosis is a promising treatment strategy for MM. This review article summarizes the core mechanisms underlying the development of ferroptosis in MM cells and its potential role as a therapeutic target in MM. We emphasize the emerging types of small molecules inducing ferroptosis pathways by boosting the antitumor activity of BRAFi and immunotherapy and uncover their beneficial effects to treat MM. We also summarize the application of nanosensitizer-mediated unique dynamic therapeutic strategies and ferroptosis-based nanodrug targeting strategies as therapeutic options for MM. This review suggests that pharmacological induction of ferroptosis may be a potential therapeutic target for MM.

237 (PB117) - A phase II study to evaluate the efficacy of regorafenib in C-KIT mutated metastatic malignant melanoma patients who have progressed on first-line treatment: A Multicenter Trial of Korean Cancer Study Group (UN-14–13)

Background: C-KIT mutation is a therapeutic target for malignant melanoma, and C-KIT inhibitors such as imatinib have previously shown clinical efficacy. Regorafenib is a 3rd generation inhibitor of C-KIT and demonstrates high response rate in C-KIT mutated gastrointestinal stromal tumor. We evaluated the anti-tumor activity and safety of regorafenib in C-KIT mutated metastatic malignant melanoma patients who have progressed on first-line treatment.

EIF3B stabilizes PTGS2 expression by counteracting MDM2-mediated ubiquitination to promote the development and progression of malignant melanoma.

Malignant melanoma (MM) is a neoplasm that develops from human melanocytes. It was reported that eukaryotic translation initiation factor 3 subunit B (EIF3B) is associated with multiple types of cancers, but its role in MM has not been reported. In the present study, we found that EIF3B was abundantly expressed in MM and was strongly related to lymphatic metastasis and pathological stage of MM patients. In addition, EIF3B depletion could block the progression of MM in vitro and in vivo. In contrast, EIF3B overexpression increased cell proliferation and migration in melanoma cells. More importantly, we identified that EIF3B's driver role in MM was mediated by PTGS2. In detail, we found that EIF3B stabilized PTGS2 expression by inhibiting PTGS2 ubiquitination, which is mediated by the E3 ligase MDM2. Moreover, like EIF3B, silencing PTGS2 could suppress MM development, and more interestingly, it could reverse the situation caused by overexpression of EIF3B in vitro and in vivo. Furthermore, the proliferation and migration inhibited by silencing of EIF3B were also partially recovered by overexpression of PTGS2. Overall, our findings revealed the potential of EIF3B as a therapeutic target for MM. Identification of EIF3B's function in MM may pave the way for future development of more specific and more effective targeted therapy strategies against MM.

Abstract 2443: Knocking out SNCA in SKMEL28 melanoma cells suppresses EMT and prevents invasion

Abstract Malignant melanoma is one of the most aggressive and fifth most prevalent cancer among men and women. Clinical and epidemiological studies shows that Parkinson’s disease (PD) patients have higher risk of developing invasive melanoma, and, reciprocally, patients with invasive melanoma have a higher risk of developing PD. Alpha-synuclein (α-syn; SNCA), an intrinsically disordered protein expressed in neurons, is also highly expressed in malignant melanoma and is a common molecular signature between Parkinson’s disease and melanoma. We have previously reported that SK-Mel-28 SNCA-knockout (KO) cells exhibit suppressed tumor growth and proliferation in vivo and in vitro, and that putting back SNCA via lenti virus restored the phenotype to that of the control cells. The present study is designed to investigate the role of α-syn in melanoma progression in SK-MEL-28 cells in the context of metastasis and the epithelial-to-mesenchymal transition (EMT). The SK-Mel-28 SNCA-KO clones showed a significant decrease in the cell-surface protein L1CAM, a major cell adhesion molecule highly expressed in various melanoma and correlates with melanoma progression, whereas the knock in clones resembled the control. Migration and invasion assay were performed using transwell chambers showed a profound migratory and invasive defect in SNCA-KO clones. To explore further whether this is a proliferative or migratory defect, a single cell phagokinetic motility assay was performed with colloidal gold which showed a marked decrease in cell motility in knockout clones compared to control and knock in clones, which was consistent with our migration and invasion experiments. To determine the proteins involved in the inhibition of invasion mediated by α-syn, we immunoblotted for the mesenchymal markers N-cadherin and vimentin, and these were significantly downregulated in the SNCA-KO clones. These data reinforce that knocking out SNCA impedes melanoma cell migration and invasion in SK-Mel- 28 cell line, implying that α-syn might be a novel therapeutic target for malignant melanoma. Citation Format: Nithya Gajendran, Santhanasabapathy Rajasekaran, Nirjhar M. Aloy, Sahar Shekoohi, Stephan N. Witt. Knocking out SNCA in SKMEL28 melanoma cells suppresses EMT and prevents invasion [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 2443.

ZC3H15 Correlates with a Poor Prognosis and Tumor Progression in Melanoma.

Zinc figure CCCH-type containing 15 (ZC3H15), also called developmentally regulated GTP-binding protein 1 (DRG1) family regulatory protein 1 (DFRP1), is a zinc finger containing protein. Despite playing a role in cellular signaling, it is found overexpressed in acute myeloid leukemia and also an independent prognostic marker in hepatocellular carcinoma patients. However, the biological effect of ZC3H15 in malignant melanoma (MM) remains unexplored. The expression of ZC3H15 in patients was analyzed using the R2: Genomics Analysis and Visualization Platform database. Immunohistochemical analysis, western blot, and qRT-PCR were used to detect ZC3H15 expression in melanoma tissues and cell lines. MTT, BrdU, flow cytometry assay, transwell, and western blot were performed to explore the proliferation, cell cycle, invasion, and migration of melanoma cells. We undertaken colony formation assay in vitro and tumor xenograft in vivo to detect the tumorigenicity of melanoma cells. In the present study, ZC3H15 was demonstrated highly expressed in melanoma tissues and cells. Elevated ZC3H15 impairs the survival of melanoma patients. Meanwhile, attenuation of ZC3H15 in melanoma cells inhibited cell proliferation and induced cycle arrest at G0/G1 phase. Consistently, the expression of cell cycle-related proteins cyclin dependent kinase 4 (CDK4), CDK6, and cyclin D1 (CCND1) was decreased while p21 was upregulated. Furthermore, we found the migration and invasion abilities were inhibited in ZC3H15-knockdown melanoma cells. In addition, downregulation of ZC3H15 resulted in inhibition of colony formation abilities in vitro and tumorigenesis in vivo. ZC3H15 promotes proliferation, migration/invasion, and tumorigenicity of melanoma cells. As a promising biomarker and therapeutic target in MM, ZC3H15 is worthy of further exploration.

P2X7 promotes metastatic spreading and triggers release of miRNA-containing exosomes and microvesicles from melanoma cells

Tumor growth and metastatic spreading are heavily affected by the P2X7 receptor as well as microvesicles and exosomes release into the tumor microenvironment. P2X7 receptor stimulation is known to trigger vesicular release from immune and central nervous system cells. However, P2X7 role in microvesicles and exosomes delivery from tumor cells was never analyzed in depth. Here we show that P2X7 is overexpressed in patients affected by metastatic malignant melanoma and that its expression closely correlates with reduced overall survival. Antagonism of melanoma cell-expressed P2X7 receptor inhibited in vitro anchorage-independent growth and migration and in vivo dissemination and lung metastasis formation. P2X7 stimulation triggered the release of miRNA-containing microvesicles and exosomes from melanoma cells, profoundly altering the nature of their miRNA content, as well as their dimensions and quantity. Among the more than 200 miRNAs that we found up-or-down-modulated for each vesicular fraction tested, we identified three miRNAs, miR-495-3p, miR-376c-3p, and miR-6730-3p, that were enriched in both the exosome and microvesicle fraction in a P2X7-dependent fashion. Interestingly, upon transfection, these miRNAs promoted melanoma cell growth or migration, and their vesicular release was minimized by P2X7 antagonism. Our data unveil an exosome/microvesicle and miRNA-dependent mechanism for the pro-metastatic activity of the P2X7 receptor and highlight this receptor as a suitable prognostic biomarker and therapeutic target in malignant melanoma.

529 HHLA2 modulates MMP/NF-kappa B recycling to drive malignant melanoma metastasis

Background: Malignant melanoma (MM) is a highly aggressive tumor. Its treatment has been revolutionized by the development of immune-checkpoint inhibitors which targeted at B7 protein or their putative receptors. HHLA2 was a newly identified member of B7 family, but its expression and function in MM was unknown. Herein, we aimed to study its clinical implications and potential regulatory role in MM. Methods: In this study,we examined HHLA2 expression in tissue-arrays containing nevus, malignant melanoma and metastatic malignant melanoma by immunohistochemistry staining. The intervention of HHLA2 in A375 cell line was performed and its effect on the cellular function was also analyzed. Then we identifed the diferentially expressed genes upon HHLA2 knockdown in A375 cell lines by using KEGG and GO analysis. Results: We found that HHLA2 was overexpressed in mucosal melanoma tissues and metastatic melanoma tissues but absent in nevus and cutaneous melanoma tissues. In vitro analysis, knockdown of HHLA2 in human MM cell line inhibited its proliferation, migration and invasion ability. According to the microarray data, MMP3 and NF-kappa B signaling pathway were involved in HHLA2 mediated progression in A375 cells. We confirmed the down-regulated expression of MMPs and core genes of NF-κB signaling pathway upon the HHLA2 knock-down. In addition, we checked the expression of epithelial-mesenchymal transition (EMT) related markers, and found that HHLA2 knock-down induced a MET process in A375 cells. Conclusion: Our findings indicated a novel role for HHLA2 in regulation of the metastatic capacity, which might be a potential therapeutic target for MM.

Knockdown of DEAD-box RNA helicase 52 (DDX52) suppresses the proliferation of melanoma cells in vitro and of nude mouse xenografts by targeting c-Myc

ABSTRACT The ATP-dependent protein DEAD-box RNA helicase 52 (DDX52) is an important regulator in RNA biology and has been implicated in the development of prostate and lung cancer. However, its biological functions and clinical importance in malignant melanoma (MM) are still unclear. Understanding the potential mechanism underlying the regulation of MM progression by DDX52 might lead to novel therapeutic strategies. The aim of the present study was to investigate the role of DDX52 in the regulation of MM progression and its clinical relevance. DDX52 expression in normal and MM tissues was evaluated by GEO analysis and immunohistochemistry. The effects of DDX52 on cell growth were evaluated in MM cells with downregulated DDX52 expression. In this study, we found that DDX52 was markedly overexpressed in MM tissues compared with nontumor tissues and was associated with shorter overall survival in patients; therefore, DDX52 might be a prognostic marker in MM. Downregulation of DDX52 expression in the MM cell lines A2058 and MV3 markedly inhibited cell proliferation and colony formation. Additionally, knockdown of DDX52 in MM cells caused significant regression of established tumors in nude mice and delayed the onset time. Moreover, downregulation of DDX52 markedly suppressed c-Myc mRNA and protein expression, and an RNA immunoprecipitation assay confirmed the association between DDX52 and c-Myc. Restoration of c-Myc expression partly rescued the effects of DDX52 deficiency in MM cells. In conclusion, our study found that DDX52 mediated oncogenesis by promoting the transcriptional activity of c-Myc and could be a therapeutic target in MM.

Long non-coding RNA SNHG7 promotes malignant melanoma progression through negative modulation of miR-9.

Long non-coding small nucleolar RNA host gene 7 (lncRNA SNHG7) was verified to act as an onco-gene in human cancers. Nevertheless, the role of SNHG7 in malignant melanoma remains elusive. The present study showed an increase of SNHG7 expression in malignant melanoma tissues and cell lines. Besides, SNHG7 knockdown inhibited proliferation and migration in malignant melanoma cells. Bioinformatics analysis demonstrated that SNHG7 functions as a molecular sponge for miR-9 in biological behavior of melanoma cells. And miR-9 could inhibit the expression of PI3KR3 by binding with the 3'-UTR. Furthermore, PI3KR3, pAKT, cyclin D1 and Girdin expression was down-regulated after SNHG7 knockdown by siRNA. In addition, SNHG7 knockdown decreased xenograft growth in vivo. Taken together, this research demonstrated that SNHG7 was an oncogene in malignant melanoma, providing a novel insight for the pathogenesis and new potential therapeutic target for malignant melanoma.

Clinical significance of lncRNA MIR31HG in melanoma.

The aim of this study was to explore the expression of long non-coding RNA (lncRNA) MIR31HG in malignant melanoma (MM), and to investigate its clinical significance. The quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) was used to detect the expression of lncRNA MIR31HG in MM tissues and cells. The relationship between lncRNA MIR31HG expression and the clinicopathological characteristics was analyzed. Furthermore, the cell counting kit-8 (CCK-8) and the transwell assays were performed to assess the effect of MIR31HG on cell proliferation and metastasis in vitro, respectively. The expression of MIR31HG was significantly upregulated in MM tissues and cells. To explore the relationship between MIR31HG expression and clinical features, the patients were divided into two groups according to the mean expression of MIR31HG, including high expression group and low expression group. The subsequent results indicated that MIR31HG expression was correlated with lymph nodes metastasis, distal metastasis, and TNM stage. The multivariate analysis indicated that a high expression of MIR31HG could be used as an independent prognostic factor for MM. MIR31HG low-expression cells were constructed in vitro. Compared with the control cells, the cells with low expression of MIR31HG showed significantly low malignancy, including decreased cell proliferation rate and migration and invasion rates. LncRNA MIR31HG was a novel factor involved in MM progression, which could be used as a potential biomarker and therapeutic target for MM.

The role of vascular mimicry as a biomarker in malignant melanoma: a systematic review and meta-analysis

BackgroundVasculogenic mimicry (VM) a microvascular system consisting of non-endothelial cells that is newly formed by aggressive tumors, has been proposed as an important therapeutic target in malignant melanoma (MM). We performed a systematic literature review to evaluate the diagnostic and prognostic accuracy of VM status for overall survival of MM patients.MethodsThe quality of the included studies was evaluated using the QUADAS-2 tool. Diagnostic capacity of VM variables, including sensitivity, specificity, positive likelihood ratio (PLR), negative likelihood ratio (NLR), diagnostic odds ratio (DOR), and the area under summary receiver operating characteristic (SROC), were pooled using Meta-DiSc software.ResultsA retrospective observational study was conducted based on twelve clinical studies including 978 clinically confirmed melanoma patients with proportion (P). VM+ melanoma cells were associated with poor prognosis in 38% of MM group (P = 0.35, 95% confidence intervals (CI): 0.27–0.42, p < 0.001). The pooled sensitivity and specificity were 0.82 (95% CI: 0.79–0.84) and 0.69 (95% CI: 0.66–0.71), respectively. Furthermore, the pooled PLR, NLR, and DOR were 2.56 (95% CI: 1.94–3.93), 0.17 (95% CI: 0.07–0.42), and 17.75 (95% CI: 5.30–59.44), respectively. Furthermore, the AUC of SROC was 0.63, indicating high reliability of VM status as a biomarker. Importantly, subgroup results suggested that VM+ status is a significantly accurate prognostic biomarker when diagnosed by the CD31−/PAS+ staining methods in Asian MM samples (p < 0.001).ConclusionsOur findings support the potential of VM status of tumors as a promising prognostic biomarker and emphasize an effective adjuvant therapeutic strategy in the prognosis of Asian MM patients.

Abstract LB-230: Quantitative immunohistochemistry for melanocortin 1 receptor using phosphor integrated dots

Abstract Visual scoring of biomarkers by immunohistochemistry (IHC) on formalin-fixed paraffin embedded tissues constitutes a major part of histopathologic diagnosis and staging of several cancers. To improve sensitivity and linearity of IHC methods, we have developed a quantitative IHC technique using streptavidin coated phosphor integrated dot (PID) fluorescent nanoparticles (Gonda et al, Scientific Reports, 2017). In retrospective analysis of clinically established biomarkers (i.e., PD-L1, HER2, CSF-1R), IHC-PID has provided high-sensitivity quantitative detection of target proteins with a broad dynamic range. The present study examines the utility of IHC-PID in the biomarker discovery and validation process by evaluating melanocortin subtype 1 receptor (MC1R) in cell lines and clinical biopsy samples. MC1R is currently under investigation as a potential diagnostic and therapeutic target for malignant melanoma, a lethal cancer with poor long-term prognosis. MC1R is an endocytic cell-surface G-protein coupled receptor that exhibits relatively high expression in malignant melanoma tumors vs. normal tissues. Notably, current melanoma therapies, including MAPKi (e.g., vemurafenib, dabrafenib) increase MC1R expression pharmacologically on melanoma cells (unpublished results). MC1R expression was determined in cell lines by standard radio-ligand binding assay (125I-NDP-α-MSH). The SK-Mel-28 cell line showed 5-6 fold increased expression of endogenous MC1R when compared to the A375 cell line. Formalin fixed pellets of these two cell lines were used for analytical validation of PID-IHC. By utilizing dedicated image-processing algorithms, the readout of average PID score/cell was determined to be 285 and 98, for SK-Mel-28 and A375 cell lines, respectively. To confirm the increase in sensitivity with the PID technology, enabling quantitative IHC and improved detection of MC1R, de-identified melanoma samples from varying sites were collected by surgical biopsy at The University of Iowa Hospitals and Clinics and scored by a pathologist. Sister sections were stained by conventional IHC or IHC-PID. For IHC-PID, sections were counter stained by hematoxylin; tumor regions were hand annotated by a pathologist; and fluorescent images in 5 microscopic fields were captured and analyzed. Background PID threshold was determined by using no-primary antibody as controls. IHC-PID was performed on MS0759 (T1b N0 M0), MS0045 (T3a N1a M0) and MS0868 (T4b N3 M1a) samples and showed average PID score/cell of 217±38, 68±12, and 160±21, respectively. Ongoing studies are expanding on IHC-PID use to assess MC1R expression on a clinical melanoma panel (n=20) in late stage patients undergoing pharmacological interventions including MAPKi. The overall goal of this project is to develop PID-based quantitative IHC to support the development of MC1R as an imaging biomarker for patient stratification and monitoring clinical response to MC1R-targeted radionuclide therapy for malignant melanoma. Note: This abstract was not presented at the meeting. Citation Format: Apollina Goel, Kenji Nishikawa, Etsuko Futaya, Ildiko Poylak, Robert Holt, Mengshi Li, Edwin Sagastum, Brenna Marks, Aaron D. Bossler, Frances L. Johnson, Michael K. Schultz, Hideki Goda, Hisatake Okada, Kelly Orcutt, George Abe, Kenneth Bloom. Quantitative immunohistochemistry for melanocortin 1 receptor using phosphor integrated dots [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-230.

Long noncoding RNA X-inactive specific transcript promotes malignant melanoma progression and oxaliplatin resistance.

Long noncoding RNA X-inactive specific transcript (XIST) was confirmed to participate in the development of many cancers. However, the function of XIST in malignant melanoma (MM) remained largely unknown. In the current study, we found that the XIST expression level was upregulated in MM tissues and cell lines. In addition, the growth rate of MM cells transfected with silencing XIST was significantly decreased compared with that with silencing normal control. XIST knockdown inhibited proliferation and migration in MM cells and increased the oxaliplatin sensitivity of oxaliplatin-resistant MM cells. Bioinformatics analysis showed that XIST acts as a molecular sponge for miR-21 and miR-21 directly targets with 3'-UTR of PI3KR1. Furthermore, XIST knockdown inhibited PI3KRI and AKT expression, and promoted Bcl-2 and Bax expression. In short, the current study showed that XIST was a crucial regulator in progression and oxaliplatin resistance of MM, providing a novel insight into the pathogenesis and underlying therapeutic target for MM.

Phycocyanin Reduces Proliferation of Melanoma Cells through Downregulating GRB2/ERK Signaling.

As a type of functional food additive, phycocyanin is shown to have a potential antineoplastic property. However, its underlying anticancer mechanism in melanoma cells remains unknown. We previously reported a 35S in vivo/vitro labeling analysis for dynamic proteomic (SiLAD) technology. It could exclusively detect protein synthesis rates via pulse labeling of newly expressed proteins by 35S, providing a high time-resolution method for analysis of protein variations. In the present study, we performed a time course analysis in A375 melanoma cells after phycocyanin treatment using SiLAD. Protein expression velocities were specifically visualized and their regulation modes were dynamically traced. Strikingly, novel protein synthesis patterns were discovered in the early phase of phycocyanin treatment, suggesting a possible mechanism of phycocyanin regulation. Furthermore, network analysis and phenotype experiments demonstrated that GRB2-ERK1/2 pathway was involved in phycocyanin-mediated regulation process and responsible for the proliferation suppression of melanoma cell, which could be a therapeutic target for malignant melanoma.

MiR‑590‑5p inhibits tumor growth in malignant melanoma by suppressing YAP1 expression.

The microRNAs (miRNAs/miRs) involved in the carcinogenesis and progression of malignant melanoma (MM) remain unclear. In the present study, miR-590-5p was identified to be upregulated in MM cells compared with human melanocytes using a reverse transcription-quantitative polymerase chain reaction to screen established oncogenic and tumor suppressor miRNAs. miR-590-5p was demonstrated to inhibit the cell proliferation and tumor growth of MM cells in vitro and in vivo by performing Cell Counting Kit-8 and tumour xenograft assays, respectively. In addition, flowcytometry assays indicated that miR-590-5p induced cell apoptosis and cell cycle arrest at the G1 stage in MM cells. Finally, luciferase assays and western blot analysis results confirmed that the transcriptional regulator Yes-associated protein 1 (YAP1) is upregulated and inversely associated with miR-590-5p expression in MM cells, and is the direct target and functional mediator of miR-590-5p in MM. Altogether these results reveal the functional and mechanistic link between miR-590-5p and YAP1 in the progression of MM. Therefore, miR-590-5p is a potential therapeutic target in MM.

Carnosic acid, an inducer of NAD(P)H quinone oxidoreductase 1, enhances the cytotoxicity of β-lapachone in melanoma cell lines.

NAD(P)H quinone oxidoreductase 1 (NQO1)-dependent antitumor drugs such as β-lapachone (β-lap) are attractive candidates for cancer chemotherapy because several tumors exhibit higher expression of NQO1 than adjacent tissues. Although the association between NQO1 and β-lap has been elucidated, the effects of a NQO1-inducer and β-lap used in combination remain to be clarified. It has previously been reported that melanoma cell lines have detectable levels of NQO1 expression and are sensitive to NQO1-dependent drugs such as 17-allylamino-17-demethoxygeldanamycin. The present study was conducted to investigate the involvement of NQO1 in β-lap-mediated toxicity and the utility of combination treatment with a NQO1-inducer and β-lap in malignant melanoma cell lines. Decreased expression or inhibition of NQO1 caused these cell lines to become less sensitive to β-lap, indicating a requirement of NQO1 activity for β-lap-mediated toxicity. Of note was that carnosic acid (CA), a compound extracted from rosemary, was able to induce further expression of NQO1 through NF-E2 related factor 2 (NRF2) stabilization, thus significantly enhancing the cytotoxicity of β-lap in all of the melanoma cell lines tested. Taken together, the data presented in the current study indicated that the NRF2-NQO1 axis may have potential value as a therapeutic target in malignant melanoma to improve the rate of clinical response to NQO1-dependent antitumor drugs.

Lysyl oxidase-like 3 is required for melanoma cell survival by maintaining genomic stability

Lysyl oxidase-like 3 (LOXL3) is a member of the lysyl oxidase family comprising multifunctional enzymes with depicted roles in extracellular matrix maturation, tumorigenesis, and metastasis. In silico expression analyses followed by experimental validation in a comprehensive cohort of human cell lines revealed a significant upregulation of LOXL3 in human melanoma. We show that LOXL3 silencing impairs cell proliferation and triggers apoptosis in various melanoma cell lines. Further supporting a pro-oncogenic role in melanoma, LOXL3 favors tumor growth in vivo and cooperates with oncogenic BRAF in melanocyte transformation. Upon LOXL3 depletion, melanoma cells display a faulty DNA damage response (DDR), characterized by ATM checkpoint activation and inefficient ATR activation leading to the accumulation of double-strand breaks (DSBs) and aberrant mitosis. Consistent with these findings, LOXL3 binds to proteins involved in the maintenance of genome integrity, in particular BRCA2 and MSH2, whose levels dramatically decrease upon LOXL3 depletion. Moreover, LOXL3 is required for efficient DSB repair in melanoma cells. Our results reveal an unexpected role for LOXL3 in the control of genome stability and melanoma progression, exposing its potential as a novel therapeutic target in malignant melanoma, a very aggressive condition yet in need for more effective treatment options.

Regulator of G protein signaling 4 inhibits human melanoma cells proliferation and invasion through the PI3K/AKT signaling pathway.

Melanoma is a tumor produced by skin melanocytes, which has a high metastatic rate and poor prognosis. So far, plenty of work has been done on melanoma, but mechanisms underlying melanoma development have not been fully elucidated. Here we identified regulator of G protein signaling 4(RGS4) as novel therapeutic target for malignant melanoma and its regulating effect on melanoma. We found that endogenous RGS4 expression was much lower in melanoma tissues and cells. In A375 cell line with low endogenous RGS4 expression, the function of RGS4 was detected by up-regulation its expression with pcDNA3.1-RGS4 and knockdown its expression with siRNA. Our results showed that RGS4 could significantly reduce the proliferation, migration and invasion of melanoma cells. RGS4 is an important regulator for the apoptosis of melanocyte, and the apoptosis rate is significantly decreased in low RGS4 enviroment. RGS4 induced non-activation of PI3K/AKT pathway, resulting in decreased expression of E2F1 and Cyclin D1, thus constraining cell proliferation and invasion. These results were further confirmed in M14 cell lines. Collectively, our findings show that RGS4 plays an important role in multiple cellular functions of melanoma development and is valuable to be a therapeutic target.

Multicenter Evaluation of a Novel Automated Rapid Detection System of BRAF Status in Formalin-Fixed, Paraffin-Embedded Tissues

The mutated BRAF oncogene represents a therapeutic target in malignant melanoma. Because BRAF mutations are also involved in the pathogenesis of other human malignancies, the use of specific BRAF inhibitors might also be extended to other diseases in the future. A prerequisite for the clinical application of BRAF inhibitors is the reliable detection of activating BRAF mutations in routine histopathological samples. In a multicenter approach, we evaluated a novel and fully automated PCR-based system (Idylla) capable of detecting BRAF V600 mutations in formalin-fixed, paraffin-embedded tissue within 90 minutes with high sensitivity. We analyzed a total of 436 samples with the Idylla system. Valid results were obtained in 421 cases (96.56%). Its performance was compared with conventional methods (pyrosequencing or Sanger sequencing). Concordant results were obtained in 406 cases (96.90%). Reanalysis of eight discordant samples by next-generation sequencing and/or pyrosequencing with newly extracted DNA and the BRAF RGQ Kit confirmed the Idylla result in seven cases, resulting in an overall agreement of 98.57%. In conclusion, the Idylla system is a highly reliable and sensitive platform for detection of BRAF V600 mutations in formalin-fixed, paraffin-embedded material, providing an efficient alternative to conventional diagnostic methods, particularly for routine diagnostics laboratories with limited experience in molecular pathology.

PRL-3 Promotes the Malignant Progression of Melanoma via Triggering Dephosphorylation and Cytoplasmic Localization of NHERF1

Phosphatase of regenerating liver-3 (PRL-3) has been reported to have a critical role in metastatic progression of cancers. Here, we investigate how PRL-3 increases the malignant degree of melanoma cells. The expression of PRL-3 increased gradually during the malignant progression of melanoma. The phosphorylation of Akt was elevated in highly malignant melanoma cells, which was accompanied by a decrease in nuclear phosphatase and tensin homolog (PTEN). The phosphorylation of NHERF1 in the serine site was regulated by PRL-3 and showed cytoplasmic translocation upon dephosphorylation, which resulted in a decrease in nuclear PTEN. The co-translocation of NHERF1 and PTEN from the nucleus to the cytoplasm was observed during the malignant progression of melanoma cells. Tumor growth was inhibited significantly, and the survival was prolonged upon knockdown of cytoplasmic NHERF1 in B16BL6 cells prior to the inoculation into mice. Taken together, to our knowledge previously unreported, we have identified NHERF1 as a potential substrate of PRL-3. Its phosphorylation status as well as its change in cellular localization and association with PTEN correlated with the malignant progression of melanoma. Our data provide an explanation for how PRL-3 promotes the malignant progression of melanoma, as well as a diagnostic marker or therapeutic target for malignant melanoma.