SUMMARY We describe the landscape of genomic alterations in cutaneous melanomas through DNA, RNA, and protein-based analysis of 333 primary and/or metastatic melanomas from 331 patients. We establish a framework for genomic classification into one of four subtypes based on the pattern of the most prevalent significantly mutated genes: mutant BRAF, mutant RAS, mutant NF1, and Triple-WT (wild-type). Integrative analysis reveals enrichment of KIT mutations and focal amplifications and complex structural rearrangements as a feature of the Triple-WT subtype. We found no significant outcome correlation with genomic classification, but samples assigned a transcriptomic subclass enriched for immune gene expression associated with lymphocyte infiltrate on pathology review and high LCK protein expression, a T cell marker, were associated with improved patient survival. This clinicopathological and multidimensional analysis suggests that the prognosis of melanoma patients with regional metastases is influenced by tumor stroma immunobiology, offering insights to further personalize therapeutic decision-making.

Purpose The prevalence of BRAF, NRAS, and p16CDKN2A mutations during melanoma progression remains inconclusive. We investigated the prevalence and distribution of mutations in these genes in different melanoma tissues. Patients and Methods In all, 291 tumor tissues from 132 patients with melanoma were screened. Paired samples of primary melanomas (n = 102) and synchronous or asynchronous metastases from the same patients (n = 165) were included. Tissue samples underwent mutation analysis (automated DNA sequencing). Secondary lesions included lymph nodes (n = 84), and skin (n = 36), visceral (n = 25), and brain (n = 44) sites. Results BRAF/NRAS mutations were identified in 58% of primary melanomas (43% BRAF; 15% NRAS); 62% in lymph nodes, 61% subcutaneous, 56% visceral, and 70% in brain sites. Mutations were observed in 63% of metastases (48% BRAF; 15% NRAS), a nonsignificant increase in mutation frequency after progression from primary melanoma. Of the paired samples, lymph nodes (93% consistency) and visceral metastases (96% consistency) presented a highly similar distribution of BRAF/NRAS mutations versus primary melanomas, with a significantly less consistent pattern in brain (80%) and skin metastases (75%). This suggests that independent subclones are generated in some patients. p16CDKN2A mutations were identified in 7% and 14% of primary melanomas and metastases, with a low consistency (31%) between secondary and primary tumor samples. Conclusion In the era of targeted therapies, assessment of the spectrum and distribution of alterations in molecular targets among patients with melanoma is needed. Our findings about the prevalence of BRAF/NRAS/p16CDKN2A mutations in paired tumor lesions from patients with melanoma may be useful in the management of this disease.

Postreplicative mismatch repair plays a major role in mediating the cytotoxicity of agents generating O6-methylguanine in DNA. We previously showed that a methylating antitumor triazene compound, temozolomide, induces apoptosis and that the persistence of O6-methylguanine in DNA is required to trigger the process. We wanted to test whether the latter apoptotic signal is dependent on a functional mismatch repair system. To this end, we used two human lymphoblastoid cell lines (i.e., the mismatch repair-proficient TK6 line and its mismatch repair-deficient subline MT1) that are both deficient in O6-methylguanine repair. Temozolomide treatment of TK6 cells brought about efficient cell growth inhibition, G2/M arrest, and apoptosis, as indicated by the results of cytofluorimetric analysis of 5-bromo-2'-deoxyuridine incorporation and DNA content and evaluation of DNA fragmentation. The drug treatment resulted also in the induction of p53 and p21/waf-1 protein expression. In contrast, MT1 cells were highly resistant to the drug and no p53 and p21/waf-1 induction was observed. Importantly, we could show that MT1 cells are not deficient in the p53-dependent apoptosis pathway; treatment with etoposide, a topoisomerase II inhibitor, resulted in p53 and p21/waf-1 protein expression and apoptosis in both cell lines. In conclusion, we demonstrate the existence of a link between a functional mismatch repair system and the trigger of apoptosis in cells exposed to clinically relevant concentrations of temozolomide. The results also suggest that p53 induction in response to O6-guanine methylation involves the mismatch repair system.

The vascular endothelial growth factor is produced by a large variety of human tumors, including melanoma, in which it appears to play an important role in the process of tumor-induced angiogenesis. Little information is available on the role of placenta growth factor, a member of the vascular endothelial growth factor family of cytokines, in tumor angiogenesis, even though placenta growth factor/vascular endothelial growth factor heterodimers have been recently isolated from tumor cells. To investigate the role of placenta growth factor and vascular endothelial growth factor homodimers and heterodimers in melanoma angiogenesis and growth, 19 human melanoma cell lines derived from primary or metastatic tumors were characterized for the expression of these cytokines and their receptors. Release of placenta growth factor and vascular endothelial growth factor polypeptides into the supernatant of human melanoma cells was demonstrated. Reverse transcriptase polymerase chain reaction analysis showed the presence of mRNAs encoding at least three different vascular endothelial growth factor isoforms (VEGF(121), VEGF(165), and VEGF(189)) and transcripts for two placenta growth factor isoforms (PlGF-1 and PlGF-2) in human melanoma cells. In addition, placenta growth factor expression in human melanoma in vivo was detected by immunohistochemical staining of tumor specimens. Both primary and metastatic melanoma cells were found to express the mRNAs encoding for vascular endothelial growth factor and placenta growth factor receptors (KDR, Flt-1, neuropilin-1, and neuropilin-2), and exposure of melanoma cells to these cytokines resulted in a specific proliferative response, supporting the hypothesis of a role of these angiogenic factors in melanoma growth. J Invest Dermatol 115:1000-1007 2000

BackgroundGrowing evidence is showing that metastatic cell populations are able to transfer their characteristics to less malignant cells. Exosomes (EXOs) are membrane vesicles of endocytic origin able to convey their cargo of mRNAs, microRNAs (miRs), proteins and lipids from donors to proximal as well as distant acceptor cells. Our previous results indicated that miR-221&222 are key factors for melanoma development and dissemination. The aim of this study was to verify whether the tumorigenic properties associated with miR-222 overexpression can be also propagated by miR-222-containing EXOs.MethodsEXOs were isolated by UltraCentrifugation or Exoquick-TC® methods. Preparations of melanoma-derived vesicles were characterized by using the Nanosight™ technology and the expression of exosome markers analyzed by western blot. The expression levels of endogenous and exosomal miRNAs were examined by real time PCR. Confocal microscopy was used to evaluate transfer and uptake of microvesicles from donor to recipient cells. The functional significance of exosomal miR-222 was estimated by analyzing the vessel-like process formation, as well as cell cycle rates, invasive and chemotactic capabilities.ResultsBesides microvesicle marker characterization, we evidenced that miR-222 exosomal expression mostly reflected its abundance in the cells of origin, correctly paralleled by repression of its target genes, such as p27Kip1, and induction of the PI3K/AKT pathway, thus confirming its functional implication in cancer. The possible differential significance of PI3K/AKT blockade was assessed by using the BKM120 inhibitor in miR-222-transduced cell lines. In addition, in vitro cultures showed that vesicles released by miR-222-overexpressing cells were able to transfer miR-222-dependent malignancy when taken-up by recipient primary melanomas. Results were confirmed by antagomiR-221&222 treatments and by functional observations after internalization of EXOs devoid of these miRs.ConclusionAll together these data, besides generally confirming the role of miR-222 in melanoma tumorigenesis, supported its responsibility in the exosome-associated melanoma properties, thus further indicating this miR as potential diagnostic and prognostic biomarker and its abrogation as a future therapeutic option.Electronic supplementary materialThe online version of this article (doi:10.1186/s12967-016-0811-2) contains supplementary material, which is available to authorized users.

Sézary syndrome (SS) is an incurable leukemic variant of cutaneous T-cell lymphoma and its pathogenesis is still unknown. Diagnosis/prognosis may strongly ameliorate the management of SS individuals. Here, we profiled the expression of 470 microRNAs (miRNAs) in a cohort of 22 SS patients, and we identified 45 miRNAs differentially expressed between SS and controls. Using predictive analysis, a list of 19 miRNAs, including miR-21, miR-214, miR-486, miR-18a, miR-342, miR-31 and let-7 members were also found. Moreover, we defined a signature of 14 miRNAs including again miR-21, potentially able to discriminate patients with unfavorable and favorable outcome. We validated our data for miR-21, miR-214 and miR-486 by qRT-PCR, including an additional set of array-independent SS cases. In addition, we also provide an in vitro evidence for a contribution of miR-214, miR-486 and miR-21 to apoptotic resistance of CTCL cell line.

The prognosis of advanced melanoma is generally poor, because this tumor commonly exhibits intrinsic or acquired resistance to chemotherapy. In an attempt to identify the underlying causes of this resistance, we studied the roles played by the DNA repair enzyme O 6 -alkylguanine-DNA alkyltransferase (OGAT) and the mismatch repair (MMR) system in the sensitivity of melanoma cells to temozolomide (TMZ), 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), or cis-diamminedichloroplatinum(II) (CDDP). To this end, OGAT levels and MMR efficiency of extracts of nine melanoma cell lines and selected clones derived from four of these lines were determined and correlated with the sensitivity of the respective cells to these drugs. The effectiveness of O 6 -benzylguanine (BG), a specific OGAT inhibitor, in potentiating TMZ-or BCNU-mediated cytotoxicity was also evaluated. Our results demonstrate that MMR efficiency and OGAT levels strongly affect melanoma cell sensitivity to TMZ. In MMR-proficient cells, a direct correlation between OGAT levels and TMZ IC 50 values was found. When OGAT activity was inhibited with BG, the sensitivity of these cells to TMZ increased and was then dictated largely by their MMR efficiency. MMR-deficient cells were highly resistant to the drug irrespective of their OGAT levels. Although OGAT activity and MMR status seemed to be the major determinants of melanoma sensitivity to TMZ, this was not the case for BCNU and CDDP; resistance to the latter drugs clearly involves processes other than the two DNA repair pathways analyzed in this study.

The SKI protein is a transcriptional coregulator over-expressed in melanoma. Experimentally induced down-regulation of SKI inhibits melanoma cell growth in vitro and in vivo. MicroRNAs (miRNAs) negatively modulate gene expression and have been implicated in oncogenesis. We previously showed that microRNA-155 (miR-155) is down-regulated in melanoma cells as compared with normal melanocytes and that its ectopic expression impairs proliferation and induces apoptosis. Here, we investigated whether miR-155 could mediate melanoma growth inhibition via SKI gene silencing. Luciferase reporter assays demonstrated that miR-155 interacted with SKI 3'UTR and impaired gene expression. Transfection of melanoma cells with miR-155 reduced SKI levels, while inhibition of endogenous miR-155 up-regulated SKI expression. Specifically designed small interfering RNAs reduced SKI expression and inhibited proliferation. However, melanoma cells over-expressing a 3'UTR-deleted SKI were still susceptible to the antiproliferative effect of miR-155. Our data demonstrate for the first time that SKI is a target of miR-155 in melanoma. However, impairment of SKI expression is not the leading mechanism involved in the growth-suppressive effect of miR-155 found in this malignancy.