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RAC1 is a GTPase member of the RAS superfamily, and RAC1P29S was recently identified as the third most common recurrent mutation in melanomas, affecting 4-7% of the patients. This is an oncogenic mutation, because the mutant protein remains mostly in its active GTP-bound form, and its ectopic expression increases the rate of normal melanocytes proliferation and migration. There is limited information regarding the functional role of RAC1P29S as a "driver" in human melanogenesis and as a cause for drug resistance. This commentary describes the latest data and provides evidence that supports the notion that RAC1 is activated even in melanoma cells that do not carry the mutation rendering it a good target for therapy. On the other hand, its role in conferring resistance to BRAF or MEK inhibitors is still in question. [ABSTRACT FROM AUTHOR]

BRAFV600E/K is a frequent mutationally active tumor-specific kinase in melanomas that is currently targeted for therapy by the specific inhibitor PLX4032. Our studies with melanoma tumor cells that are BRAFV600E/K and BRAFWT showed that, paradoxically, while PLX4032 inhibited ERK1/2 in the highly sensitive BRAFV600E/K, it activated the pathway in the resistant BRAFWT cells, via RAF1 activation, regardless of the status of mutations in NRAS or PTEN. The persistently active ERK1/2 triggered downstream effectors in BRAFWT melanoma cells and induced changes in the expression of a wide-spectrum of genes associated with cell cycle control. Furthermore, PLX4032 increased the rate of proliferation of growth factor-dependent NRAS Q61L mutant primary melanoma cells, reduced cell adherence and increased mobility of cells from advanced lesions. The results suggest that the drug can confer an advantage to BRAFWT primary and metastatic tumor cells in vivo and provide markers for monitoring clinical responses. [ABSTRACT FROM AUTHOR]

Decitabine, an epigenetic modifier that reactivates genes otherwise suppressed by DNA promoter methylation, is effective for some, but not all cancer patients, especially those with solid tumors. It is commonly recognized that to overcome resistance and improve outcome, treatment should be guided by tumor biology, which includes genotype, epigenotype, and gene expression profile. We therefore took an integrative approach to better understand melanoma cell response to clinically relevant dose of decitabine and identify complementary targets for combined therapy. We employed eight different melanoma cell strains, determined their growth, apoptotic and DNA damage responses to increasing doses of decitabine, and chose a low, clinically relevant drug dose to perform whole-genome differential gene expression, bioinformatic analysis, and protein validation studies. The data ruled out the DNA damage response, demonstrated the involvement of p21Cip1 in a p53-independent manner, identified the TGFb pathway genes CLU and TGFBI as markers of sensitivity to decitabine and revealed an effect on histone modification as part of decitabine-induced gene expression. Mutation analysis and knockdown by siRNA implicated activated b-catenin/MITF, but not BRAF, NRAS or PTEN mutations as a source for resistance. The importance of protein stability predicted from the results was validated by the synergistic effect of Bortezomib, a proteasome inhibitor, in enhancing the growth arrest of decitabine in otherwise resistant melanoma cells. Our integrative analysis show that improved therapy can be achieved by comprehensive analysis of cancer cells, identified biomarkers for patient's selection and monitoring response, as well as targets for improved combination therapy. [ABSTRACT FROM AUTHOR]

Tyrosinase is a type I membrane glycoprotein whose activity is essential for melanin synthesis. Loss of function mutations in tyrosinase is the cause of oculocutaneous albinism 1. In the milder oculocutaneous albinism 1B form in which mutant proteins retain residual activity, the severity of albinism depends on the type of mutations expressed in the melanocyte. In this study, we show that coexpression of wild-type protein with temperature-sensitive tyrosinase mutants corrects the mutant conformation defect in an activity-dependent manner. Exit from the endoplasmic reticulum and complex carbohydrate processing in the Golgi was promoted when temperature-sensitive tyrosinase mutants were ectopically expressed in host melanocytes carrying wild-type protein even at the nonpermissive temperature. Incubation of transfected melanocytes with DOPA (the cofactor and substrate for tyrosinase), or tyrosine (the substrate), further enhanced processing of ectopic mutant proteins. The analysis of glycosylation-deficient mutants revealed regions in tyrosinase with high, low, and intermediate dependency on glycans for maturation. We concluded that the presence of tyrosinase activity enhances the maturation of temperature-sensitive and glycosylation-deficient forms of tyrosinase. The results may explain the variation in pigmentation and the development of pigment later in life in patients carrying different mutant alleles of oculocutaneous albinism 1B. [ABSTRACT FROM AUTHOR]

Presents information on a study which explored the possibility that trafficking of albino tyrosinase from the endoplasmic reticulum (ER) to the Golgi apparatus and beyond is disrupted. Analysis of the effect of ER processing and intracellular localization of four mutations associated with oculocutaneous albinism; Materials and methods of the study; Results and discussion.

Reviews the regulation of normal melanocyte proliferation. Synergism among mitogens in vitro; Overview of mitogens in tissues and cells; Description of melanocyte mitogens during development.

Focuses on the loss of tyrosinase, the key enzyme in melanin synthesis, in malignant melanocytes. Investigation of proteolytic degradation as a basis for the loss of tyrosinase; Methods and materials used in the study; Results and discussion.

Describes how ultraviolet (UV)-radiation and other DNA damaging agents induce a novel covalent modification of phosphorylated large subunit of RNA polymerase II (Pol II) molecules. Transcription-coupled repair (TCR) system; Deficiency of UV-induced ubiquitination of Pol II LS in fibroblasts from individuals with two forms of Cockayne syndrome CS-A and CS-B.

Expression of basic fibroblast growth factor cDNA or dominantly acting oncogenes, e.g., E1A, in immortalized mouse melanocytes leads to autonomous growth in vitro, depigmentation, and in the case of the oncogenes, tumorigenesis. Because downregulation of pigmentation is a common event in human metastatic melanoma cells grown in culture, we determined the molecular basis of depigmentation in a mouse melanocyte model system. We tested the effect of E1A mutants deficient in their ability to neutralize several regulatory proteins and determined changes in melanogenic gene expression. We identified Microphthalmia as the affected, downregulated transcription factor in melanocytes rendered amelanotic by E1A, basic fibroblast growth factor, or the oncogenes ras or neu, and in an amelanotic cell variant of Cloudman S91 mouse melanoma. Against expectations, sequestration of p300, a transcriptional adaptor that mediates responses to cyclic adenosine monophosphate, was not required for the full transforming effects of E1A. Our results suggest that in addition to controlling tyrosinase (albino locus) and tyrosinase-related protein 1 (TRP1/gp75/brown locus), both known to possess the DNA consensus site for binding the Microphthalmia protein, this transcription factor also controls other melanocyte-specific genes such as pink-eyed dilution and Pmel 17 (silver), but not tyrosinase-related protein 2 (slaty locus). Furthermore, these findings show that microphthalmia is downregulated not only by experimentally introduced dominantly acting oncogenes but also by the aberrant expression of basic fibroblast growth factor and by spontaneous tumorigenic transformation. [ABSTRACT FROM AUTHOR]

In this article we describe the rapid advances made in the molecular genetics of three inherited pigmentation disorders: albinism, piebaldism, and vitiligo, all of which throw light on normal pigment cell function. The focus is on studies in mice, with comparison of data in humans. The critical role of tyrosinase (c-locus or human tyrosinase protein) in normal pigmentation and albinism has been reinforced by the cloning and identification of mutations in tyrosinase and two other melanocyte-specific oxidoreductases structurally related to but functionally different from tyrosinase: the (b) brown-locus protein/gp7S/catalase B and dopachrome tautomerase. Each possesses a distinct enzyme activity and yet the three share homology in strategic regions. Most of the point mutations that reduce or abrogate the respective enzyme activities are located in those regions. Tyrosinase-negative albinism is caused only by defects in tyrosinase. A locus for human tyrosinase-positive albinism has been recently mapped to chromosome 15q11.2 → q12, at a gene identified in mice as pink-eyed dilution. On the other hand, several genes encoding proteins critical for the proliferation of melanocytes are known to control the piebald phenotype. So far identified are two membrane-receptor tyrosine kinases, c-Kit and PDGF-R/α, and the ligand for c-kit, MGF (mast-cell growth factor, also known as stem-cell factor, c-Kit-ligand, or steel factor). Mutations in W/c-kit (white spotting), Ph/ Pdgfr/a (patch), and Sl/MGF (steel), lead to a reduction in receptor kinase activity and failure of melanocytes to thrive and reach the skin during embryogenesis. Finally, mouse mutant models suggest at least two possible causes for vitiligo, a progressive loss of pigmentation that occurs after birth. In one mutant, the Blt (light) mouse, the cyclic death of hair melanocytes may be due to the toxicity of intermediates and byproducts of melanogenesis in the presence of a dysfunctional b-locus protein. In the other model, the "vitiligo mouse," in which the allele vit has been assigned to the microphthalmia (mi) locus, the loss of melanocytes may be caused by defective signal transduction, because in addition to vitiligo mivit/mivit mice have extensive piebaldism. [ABSTRACT FROM AUTHOR]

Recent advances in the culturing of pigment cells from human beings have made it possible to begin the transplantation of autologous melanocytes into areas of skin that are hypopigmented. In a patient with piebaldism we were able to take pigment cells from a shave biopsy of the normally pigmented skin of the back, expand the cells in culture, and return them to an area devoid of pigment cells and get a perfect take. To grow the cells in culture we used 12-O-tetradecanoyl-phorbol-13-acetate (TPA) as well as cholera toxin and isobutylmethyl xanthine. At this time, one can substitute basic fibroblast growth factor for TPA. The procedure of using autologous pigment cell cultures opens the door for further advances in the treatment of patients who do not have melanocytes in certain areas of the skin, as seen in patients with vitiligo or piebaldism, or as a consequence of severe mechanical or thermal trauma. [ABSTRACT FROM AUTHOR]

The requirements for growth factors of human melanocytes in culture may be dependent on the stage of malignant transformation. Several factors synergistically promote the viability and proliferation of human neonatal melanocytes in culture. They are TPA (12-O-tetradecanoyl-phorbol-13- acetate), isobutylmethyl xanthine, cholera toxin, and as yet unidentified factors from extracts derived from several cell lines and human placenta. Neonatal melanocytes can maintain at least 50 population doublings during a period of 6 months, whereas melanocytes from adult skin proliferate only for 1 month and at less than 1% of the proliferative rate of melanocytes derived from newborn foreskins. In contrast, melanocytes from dysplastic and congenital nevi proliferate well in the presence of mitogens during the initial 4-6 weeks of culture, but then become quiescent. Melanocytes from primary melanomas are the most difficult to grow in culture. They need the mitogens, but their rate of proliferation is slow. Most of the metastatic melanocyte strains that do not need the mitogens in order to proliferate, are strongly inhibited by TPA, and to a lesser extent by WI-38 cell extract. We conclude that the acquisition of independence from mitogens in culture is a late event in the transformation of melanocytcs to melanomas. [ABSTRACT FROM AUTHOR]

In order to obtain pure cultures of chicken melanocytes, neural tubes were excised from 22-somite stage embryos and placed in culture dishes to allow melanoblasts to migrate out and proliferate. The growth of contaminating cells was inhibited by maintaining the primary cultures in low-calcium and low-magnesium medium supplemented with 32 nM 12-O-tetradecanoylphorbol-13-acetate (TPA). Subsequently the pure cultures of melanocytes were maintained in Ham's F-10 medium supplemented with TPA. The population doubling time was approximately 12 h. The cell density at confluency in medium containing 32 nM TPA, 80 nM TPA, or 32 nM TPA plus 1 nM cholera toxin was 3.4, 5.6, or 8.3 x 104 cells/cm2, respectively. The melanocytes were highly pigmented and had tyrosinase activities ranging from 0.7-5.0 mU/mg protein. [ABSTRACT FROM AUTHOR]

Summary: Immune‐related toxicities, otherwise known as immune‐related adverse events (irAEs), occur in a substantial fraction of cancer patients treated with immune checkpoint inhibitors (ICIs). Ranging from asymptomatic to life‐threatening, ICI‐induced irAEs can result in hospital admission, high‐dose corticosteroid treatment, ICI discontinuation, and in some cases, death. A deeper understanding of the factors underpinning severe irAE development will be essential for improved irAE prediction and prevention, toward maximizing the benefits and safety profiles of ICIs. In recent work, we applied mass cytometry, single‐cell RNA sequencing, single‐cell V(D)J sequencing, bulk RNA sequencing, and bulk T‐cell receptor (TCR) sequencing to identify pretreatment determinants of severe irAE development in patients with advanced melanoma. Across 71 patients separated into three cohorts, we found that two baseline features in circulation—elevated activated CD4 effector memory T‐cell abundance and TCR diversity—are associated with severe irAE development, independent of the affected organ system within 3 months of ICI treatment initiation. Here, we provide an extended perspective on this work, synthesize and discuss related literature, and summarize practical considerations for clinical translation. Collectively, these findings lay a foundation for data‐driven and mechanistic insights into irAE development, with the potential to reduce ICI morbidity and mortality in the future. [ABSTRACT FROM AUTHOR]

• A joint model incorporating longitudinal mixed effects and time-event survival was applied. • The death risk of melanoma increased by 31% with one unit change of soluble PD-L1 at a time-point. • Approximately 30% reduction of death risk was found in melanoma patients with one unit change of soluble PD-1/PD-L1 ratio at a time-point. • Patients with a low ratio of PD-1/PD-L1 at baseline had significantly inferior OS compared to patients with a high ratio. • Dynamic change of soluble PD-1 and PD-L1 levels could be used to monitor patient response to ICBs. Immune checkpoint PD-1 and its ligand PD-L1 lead to T cell exhaustion, and a high level of circulating soluble PD-L1 at baseline indicates a poor prognosis in melanoma and other solid tumor types. Here we show that the dynamic changes of circulating soluble PD-1 and PD-L1 across the course of immune checkpoint blockades (ICBs) and their changes associate with patient survival in melanoma in a retrospective study. A high change of soluble PD-L1 level at a time-point but not PD-1 significantly increased the mortality, whereas a high change of soluble PD-1/PD-L1 ratio significantly reduced the mortality. After the initial immunotherapy, both soluble PD-1 and PD-L1 increased. However, the change pattern of soluble PD-L1 level was particularly dependent on patients' survival status. These findings indicate that the magnitudes of circulating soluble PD-L1 and PD-1/PD-L1 ratio changes over the time may reflect the patients' response to ICBs or the progression of the disease and predict the survival in melanoma patients treated with ICBs. [ABSTRACT FROM AUTHOR]

In this article, the author focuses on the professional life of Melanoma Research Foundation acting chair Meenhard Herlyn. He commends Herlyn's contribution to the melanoma field which includes the establishment of the Society for Melanoma Research in 2004. He relates that Herlyn grew up on a farm in Northern Germany and studied in the Veterinary Medical School in Hannover, Germany. He believes that Herlyn represents the voice of melanoma research.

We report on whole-exome sequencing (WES) of 213 melanomas. Our analysis established NF1, encoding a negative regulator of RAS, as the third most frequently mutated gene in melanoma, after BRAF and NRAS. Inactivating NF1 mutations were present in 46% of melanomas expressing wild-type BRAF and RAS, occurred in older patients and showed a distinct pattern of co-mutation with other RASopathy genes, particularly RASA2. Functional studies showed that NF1 suppression led to increased RAS activation in most, but not all, melanoma cases. In addition, loss of NF1 did not predict sensitivity to MEK or ERK inhibitors. The rebound pathway, as seen by the induction of phosphorylated MEK, occurred in cells both sensitive and resistant to the studied drugs. We conclude that NF1 is a key tumor suppressor lost in melanomas, and that concurrent RASopathy gene mutations may enhance its role in melanomagenesis. [ABSTRACT FROM AUTHOR]

The endoplasmic reticulum (ER) quality-control machinery maintains the fidelity of the maturation process by sorting aberrant proteins for ER-associated protein degradation (ERAD), a process requiring retrotranslocation from the ER lumen to the cytosol and degradation by the proteasome. Here, we assessed the role of N-linked glycans in ERAD by monitoring the degradation of wild-type (Tyr) and albino mutant (Tyr(C85S)) tyrosinase. Initially, mutant tyrosinase was established as a genuine ERAD substrate using intact melanocyte and semi-permeabilized cell systems. Inhibiting mannose trimming or accumulating Tyr(C85S) in a monoglucosylated form led to its stabilization, supporting a role for lectin chaperones in ER retention and proteasomal degradation. In contrast, ablating the lectin chaperone interactions by preventing glucose trimming caused a rapid disappearance of tyrosinase, initially due to the formation of protein aggregates, which were subsequently degraded by the proteasome. The colocalization of aggregated tyrosinase with protein disulfide isomerase and BiP, but not calnexin, supports an ER organization, which aids in protein maturation and degradation. Based on these studies, we propose a model of tyrosinase degradation in which interactions between N-linked glycans and lectin chaperones help to minimize tyrosinase aggregation and also target non-native substrates for retro-translocation and subsequent degradation. [ABSTRACT FROM AUTHOR]

Presents abstracts of studies on pigment cells. Genetics and genomics of pigment type-switching; Expression and regulatory elements of the human melanocortin 1 receptor; Maturation and degradation of tyrosinase in the early secretory pathway.

Despite the remarkable success of anti-cancer immunotherapy, its effectiveness remains confined to a subset of patients—emphasizing the importance of predictive biomarkers in clinical decision-making and further mechanistic understanding of treatment response. Current biomarkers, however, lack the power required to accurately stratify patients. Here, we identify interferon-stimulated, Ly6Ehi neutrophils as a blood-borne biomarker of anti-PD1 response in mice at baseline. Ly6Ehi neutrophils are induced by tumor-intrinsic activation of the STING (stimulator of interferon genes) signaling pathway and possess the ability to directly sensitize otherwise non-responsive tumors to anti-PD1 therapy, in part through IL12b-dependent activation of cytotoxic T cells. By translating our pre-clinical findings to a cohort of patients with non-small cell lung cancer and melanoma (n = 109), and to public data (n = 1440), we demonstrate the ability of Ly6Ehi neutrophils to predict immunotherapy response in humans with high accuracy (average AUC ≈ 0.9). Overall, our study identifies a functionally active biomarker for use in both mice and humans. [Display omitted] • Ly6Ehi neutrophils are interferon-stimulated cells present in both mouse and human • Ly6Ehi neutrophils accurately predict immunotherapy outcomes in different cancer types • Activation of the STING pathway in tumors accounts for Ly6Ehi neutrophil enrichment • Ly6Ehi neutrophils sensitize otherwise resistant tumors to immunotherapy in mice A lack of reliable, highly predictive biomarkers remains a major obstacle in immuno-oncology. In this study, Benguigui et al. discover a promising new biomarker: interferon-stimulated, Ly6Ehi neutrophils—whose frequency in the blood of both mice and patients strongly correlates with immunotherapy outcomes across cancer types. [ABSTRACT FROM AUTHOR]

Two forms of cutaneous albinism in the chicken were investigated for the presence and distribution of tyrosinase and acid phosphatase in melanocytes in situ and in culture. In sex-linked recessive tyrosinase-positive albinism, Sal, melanocytes in regenerating feathers and neural tube-derived cultures contained morphologically normal and abnormal premelanosomes. Tyrosinase was localized primarily to the abnormal premelanosomes and probably not to the normal ones. The cells possessed, in addition, vacuoles with membranous inclusions, located in the dendrites, and capped by dopa-positive vesicles (capping yes-ides). Acid phosphatase colocalized with tyrosinase in the abnormal primelanosomes and capping vesicles. Tyrosinase activity in extracts of cultured Sal melanocytes equalled that of e+ control melanocytes. A tyrosinase antiserum, raised against hamster tyrosinase (Pomerantz), precipitated 2 proteins, 68 kD and 82 kD, which had a precursor-product relationship. The amount immunoprecipitate was the same in Sal and control extracts, but in Sal extracts the lower-molecular-weight protein was twice as abundant as the higher-molecular-weight protein. Melanocytes in regenerating feathers from an autosomal recessive, tyrosinase-negative albino, ca, also contained morphologically normal and abnormal premelanosomes. In culture, ca melanocytes had no formal premelanosomes but only dopa-negative multivesicular bodies with wispy filamentous material. Tyrosinase activity and immunoprecipitable tyrosinase were absent. These results suggest that: (1) the tyrosinase-positive albino, Sal, has an aberration in both its tyrosinase and acid phosphatase profiles and (2) the tyrosinase-negative albino, ca, lacks functionally and antigenically normal tyrosinase. [ABSTRACT FROM AUTHOR]

In earlier studies, it was shown that the human plasma-spreading glycoprotein, epibolin (the 65 kD species of serum-spreading factor or vitronectin), requires a second plasma component, termed coepibolin, in order to support maximal dissociated epidermal cell spreading in tissue culture. Whereas epibolin alone in defined medium supports some cell spreading, the purified plasma coepibolin preparations do not effect spreading in the absence of epibolin. Although not yet entirely purified, coepibolin associates with some plasma fractions but not with others; it is certainly not a property of all proteins, e.g., while bovine serum albumin (BSA) has coepibolin activity, ovalbumin does not. The data presented here show that the phorbol ester, 12-tetra-decanoyl-1-phorbol-13-acetate (TPA) can act as a potent coepibolin and support maximal spreading over a concentration range of 10-100 ng/ml. In the absence of epibolin TPA does not stimulate the spreading of epidermal cells when given alone or in the presence of BSA or ovalbumin. Coepibolin activity appears to associate with tumor-promoting activity in that the phorbol derivative, phorbol- 12,13-didecanoate, shows coepibolin activity, while its inactive non-tumor-promoting isomer, phorbol-4α-phorbol-12,13-didecanoate, does not. These data suggest that the proteinaceous plasma-derived cofactor acts in a fashion similar to TPA and that this as yet unexplained mechanism of TPA action is important to the full expression of epibolin and to the early phase of epidermal cell spreading. [ABSTRACT FROM AUTHOR]

Combination checkpoint blockade (CCB) targeting inhibitory CTLA4 and PD1 receptors holds promise for cancer therapy. Immune-related adverse events (IRAEs) remain a major obstacle for the optimal application of CCB in cancer. Here, we analyzed B cell changes in patients with melanoma following treatment with either anti-CTLA4 or anti-PD1, or in combination. CCB therapy led to changes in circulating B cells that were detectable after the first cycle of therapy and characterized by a decline in circulating B cells and an increase in CD21lo B cells and plasmablasts. PD1 expression was higher in the CD21lo B cells, and B cell receptor sequencing of these cells demonstrated greater clonality and a higher frequency of clones compared with CD21hi cells. CCB induced proliferation in the CD21lo compartment, and single-cell RNA sequencing identified B cell activation in cells with genomic profiles of CD21lo B cells in vivo. Increased clonality of circulating B cells following CCB occurred in some patients. Treatment-induced changes in B cells preceded and correlated with both the frequency and timing of IRAEs. Patients with early B cell changes experienced higher rates of grade 3 or higher IRAEs 6 months after CCB. Thus, early changes in B cells following CCB may identify patients who are at increased risk of IRAEs, and preemptive strategies targeting B cells may reduce toxicities in these patients. [ABSTRACT FROM AUTHOR]

Major advances have been made in the treatment of cancer with targeted therapy and immunotherapy; several FDA-approved agents with associated improvement of 1-year survival rates became available for stage IV melanoma patients. Before 2010, the 1-year survival were quite low, at 30%; in 2011, the rise to nearly 50% in the setting of treatment with Ipilimumab, and rise to 70% with BRAF inhibitor monotherapy in 2013 was observed. Even more impressive are 1-year survival rates considering combination strategies with both targeted therapy and immunotherapy, now exceeding 80%. Can we improve response rates even further, and bring these therapies to more patients? In fact, despite these advances, responses are heterogeneous and are not always durable. There is a critical need to better understand who will benefit from therapy, as well as proper timing, sequence and combination of different therapeutic agents. How can we better understand responses to therapy and optimize treatment regimens? The key to better understanding therapy and to optimizing responses is with insights gained from responses to targeted therapy and immunotherapy through translational research in human samples. Combination therapies including chemotherapy, radiotherapy, targeted therapy, electrochemotherapy with immunotherapy agents such as Immune Checkpoint Blockers are under investigation but there is much room for improvement. Adoptive T cell therapy including tumor infiltrating lymphocytes and chimeric antigen receptor modified T cells therapy is also efficacious in metastatic melanoma and outcome enhancement seem likely by improved homing capacity of chemokine receptor transduced T cells. Tumor infiltrating lymphocytes therapy is also efficacious in metastatic melanoma and outcome enhancement seem likely by improved homing capacity of chemokine receptor transduced T cells. Understanding the mechanisms behind the development of acquired resistance and tests for biomarkers for treatment decisions are also under study and will offer new opportunities for more efficient combination therapies. Knowledge of immunologic features of the tumor microenvironment associated with response and resistance will improve the identification of patients who will derive the most benefit from monotherapy and might reveal additional immunologic determinants that could be targeted in combination with checkpoint blockade. The future of advanced melanoma needs to involve education and trials, biobanks with a focus on primary tumors, bioinformatics and empowerment of patients and clinicians. [ABSTRACT FROM AUTHOR]

Vascular tumors are among the most common neoplasms in infants and children; 5%–10% of newborns present with or develop lesions within the first 3 months of life. Most are benign infantile hemangiomas that typically regress by 5 years of age; other vascular tumors include congenital tufted angiomas (TAs), kaposiform hemangioendotheliomas (KHEs), and childhood lobular capillary hemangiomas (LCHs). Some of these lesions can become locally invasive and unresponsive to pharmacologic intervention, leading to significant complications. Recent investigation has revealed that activating mutations in HRAS , KRAS , NRAS , GNAQ , and GNA11 can cause certain types of rare childhood vascular tumors, and we have now identified causal recurrent somatic activating mutations in GNA14 by whole-exome and targeted sequencing. We found somatic activating GNA14 c.614A>T (p.Gln205Leu) mutations in one KHE, one TA, and one LCH and a GNA11 c.547C>T (p.Arg183Cys) mutation in two LCH lesions. We examined mutation pathobiology via expression of mutant GNA14 or GNA11 in primary human endothelial cells and melanocytes. GNA14 and GNA11 mutations induced changes in cellular morphology and rendered cells growth-factor independent by upregulating the MAPK pathway. Our findings identify GNA14 mutations as a cause of childhood vascular tumors, offer insight into mechanisms of oncogenic transformation by mutations affecting Gaq family members, and identify potential targets for therapeutic intervention. [ABSTRACT FROM AUTHOR]

Melanoma development involves members of the AGC kinase family, including AKT, PKC, and, most recently, PDK1, as elucidated recently in studies of Braf::Pten mutantmelanomas. Here, we report that PDK1 contributes functionally to skin pigmentation and to the development of melanomas harboring a wild-type PTEN genotype, which occurs in about 70% of human melanomas. ThePDK1 substrate SGK3was determined to be an important mediator of PDK1 activities in melanoma cells. Genetic or pharmacologic inhibition of PDK1 and SGK3 attenuated melanoma growth by inducing G1 phase cell-cycle arrest. In a synthetic lethal screen, pan-PI3K inhibition synergized with PDK1 inhibition to suppress melanoma growth, suggesting that focused blockade of PDK1/PI3K signaling might offer a new therapeutic modality for wild-type PTEN tumors. We also noted that responsiveness to PDK1 inhibition associated with decreased expression of pigmentation genes and increased expression of cytokines and inflammatory genes, suggesting a method to stratify patients with melanoma for PDK1-based therapies. Overall, our work highlights the potential significance of PDK1 as a therapeutic target to improve melanoma treatment. [ABSTRACT FROM AUTHOR]

Mutations in sunlight-induced melanoma arise from cyclobutane pyrimidine dimers (CPDs), DNA photoproducts that are typically created picoseconds after an ultraviolet (UV) photon is absorbed at thymine or cytosine. We found that in melanocytes, CPDs are generated for >3 hours after exposure to UVA, a major component of the radiation in sunlight and in tanning beds. These "dark CPDs" constitute the majority of CPDs and include the cytosine-containing CPDs that initiate UV-signature C→T mutations. Dark CPDs arise when UV-induced reactive oxygen and nitrogen species combine to excite an electron in fragments of the pigment melanin. This creates a quantum triplet state that has the energy of a UV photon but induces CPDs by energy transfer to DNA in a radiation-independent manner. Melanin may thus be carcinogenic as well as protective against cancer. These findings also validate the long-standing suggestion that chemically generated excited electronic states are relevant to mammalian biology. [ABSTRACT FROM AUTHOR]

Combination therapy concurrently targeting PD-1 and CTLA-4 immune checkpoints leads to remarkable antitumor effects. Although both PD-1 and CTLA-4 dampen the T cell activation, the in vivo effects of these drugs in humans remain to be clearly defined. To better understand biologic effects of therapy, we analyzed blood/tumor tissue from 45 patients undergoing single or combination immune checkpoint blockade. We show that blockade of CTLA-4, PD-1, or combination of the two leads to distinct genomic and functional signatures in vivo in purified human T cells and monocytes. Therapy-induced changes are more prominent in T cells than in monocytes and involve largely nonoverlapping changes in coding genes, including alternatively spliced transcripts and noncoding RNAs. Pathway analysis revealed that CTLA-4 blockade induces a proliferative signature predominantly in a subset of transitional memory T cells, whereas PD-1 blockade instead leads to changes in genes implicated in cytolysis and NK cell function. Combination blockade leads to nonoverlapping changes in gene expression, including proliferation-associated and chemokine genes. These therapies also have differential effects on plasma levels of CXCL10, soluble IL-2R, and IL-la. Importantly, PD-1 receptor occupancy following anti-PD-1 therapy may be incomplete in the tumor T cells even in the setting of complete receptor occupancy in circulating T cells. These data demonstrate that, despite shared property of checkpoint blockade, Abs against PD-1, CTLA-4 alone, or in combination have distinct immunologic effects in vivo. Improved understanding of pharmacodynamic effects of these agents in patients will support rational development of immune-based combinations against cancer. [ABSTRACT FROM AUTHOR]

The fourth ''Melanoma Bridge Meeting'' took place in Naples, December 5 to 8th, 2013. The four topics discussed at this meeting were: Diagnosis and New Procedures, Molecular Advances and Combination Therapies, News in Immunotherapy, and Tumor Microenvironment and Biomarkers. Until recently systemic therapy for metastatic melanoma patients was ineffective, but recent research in tumor biology and immunology has led to the development of new targeted and immunotherapeutic agents that prolong progression-free survival (PFS) and overall survival (OS). New therapies, such as mitogen-activated protein kinase (MAPK) pathway inhibitors, like BRAF and MEK inhibitors, as well as other signaling pathways inhibitors, are being tested in metastatic melanoma either as monotherapy or in combination, and have yielded promising results. Improved survival rates have also been observed with immune therapy for patients with metastatic melanoma. Immune-modulating antibodies came to the forefront with anti-CTLA- 4, programmed cell death-1 (PD-1) and PD-1 ligand 1 (PD-L1) pathway blocking antibodies that result in durable responses in a subset of melanoma patients. Agents targeting other immune inhibitory (e.g., Tim-3) or immune stimulating (e.g., CD137) receptors and other approaches such as adoptive cell transfer demonstrate clinical benefit in melanoma as well. This meeting's specific focus was on advances in targeted therapy and immunotherapy. Both combination targeted therapy approaches and different immunotherapies were discussed. Similarly to the previous meetings, the importance of biomarkers for clinical application as markers for diagnosis, prognosis and prediction of treatment response was an integral part of the meeting. Significant consideration was given to issues surrounding the development of novel therapeutic targets as further study of patterns of resistance to both immunologic and targeted drugs are paramount to future drug development to guide existing and future therapies. The overall emphasis on biomarkers supports novel concepts toward integrating biomarkers into contemporary clinical management of patients with melanoma across the entire spectrum of disease stage. Translation of the knowledge gained from the biology of tumor microenvironment across different tumors represents a bridge to impact on prognosis and response to therapy in melanoma. [ABSTRACT FROM AUTHOR]

BRAF inhibitors improve melanoma patient survival, but resistance invariably develops. Here we report the discovery of a novel BRAF mutation that confers resistance to PLX4032 employing whole-exome sequencing of drug-resistant BRAF V600K melanoma cells. We further describe a new screening approach, a genome-wide piggy Bac mutagenesis screen that revealed clinically relevant aberrations ( N-terminal BRAF truncations and CRAF overexpression). The novel BRAF mutation, a Leu505 to His substitution ( BRAF L505H), is the first resistance-conferring second-site mutation identified in BRAF mutant cells. The mutation replaces a small nonpolar amino acid at the BRAF- PLX4032 interface with a larger polar residue. Moreover, we show that BRAF L505H, found in human prostate cancer, is itself a MAPK-activating, PLX4032-resistant oncogenic mutation. Lastly, we demonstrate that the PLX4032-resistant melanoma cells are sensitive to novel, next-generation BRAF inhibitors, especially the 'paradox-blocker' PLX8394, supporting its use in clinical trials for treatment of melanoma patients with BRAF-mutations. [ABSTRACT FROM AUTHOR]

A letter to the editor is presented related to the growth of clone cells of human melanocytes from single cells.

RAC1 is a small, Ras-related GTPase that was recently reported to harbor a recurrent UV-induced signature mutation in melanoma, resulting in substitution of P29 to serine (RAC1P29S), ranking this the third most frequently occurring gain-of-function mutation in melanoma. Although the Ras family GTPases are mutated in about 30% of all cancers, mutations in the Rho family GTPases have rarely been observed. In this study, we demonstrate that unlike oncogenic Ras proteins, which are primarily activated by mutations that eliminate GTPase activity, the activated melanoma RAC1P29S protein maintains intrinsic GTP hydrolysis and is spontaneously activated by substantially increased inherent GDP/GTP nucleotide exchange. Determination and comparison of crystal structures for activated RAC1 GTPases suggest that RAC1F28L—a known spontaneously activated RAC1 mutant—and RAC1P29S are self-activated in distinct fashions. Moreover, the mechanism of RAC1P29S and RAC1F28L activation differs from the common oncogenic mutations found in Ras-like GTPases that abrogate GTP hydrolysis. The melanoma RAC1P29S gain-of-function point mutation therefore represents a previously undescribed class of cancer-related GTPase activity. [ABSTRACT FROM AUTHOR]

The type II p21-activated kinases (PAKs) are key effectors of RHO-family GTPases involved in cell motility, survival, and proliferation. Using a structure-guided approach, we discovered that type II PAKs are regulated by an N-terminal autoinhibitory pseudosubstrate motif centered on a critical proline residue, and that this regulation occurs independently of activation loop phosphorylation. We determined six X-ray crystal structures of either full-length PAK4 or its catalytic domain, that demonstrate the molecular basis for pseudosubstrate binding to the active state with phosphorylated activation loop. We show that full-length PAK4 is constitutively autoinhibited, but mutation of the pseudosubstrate releases this inhibition and causes increased phosphorylation of the apoptotic regulation protein Bcl-2/Bcl-XL antagonist causing cell death and cellular morphological changes. We also find that PAK6 is regulated by the pseudosubstrate region, indicating a common type II PAK autoregulatory mechanism. Finally, we find Src SH3, but not ß-PIX SH3, can activate PAK4. We provide a unique understanding for type II PAK regulation. [ABSTRACT FROM AUTHOR]

Summary: We recently proposed that competitive endogenous RNAs (ceRNAs) sequester microRNAs to regulate mRNA transcripts containing common microRNA recognition elements (MREs). However, the functional role of ceRNAs in cancer remains unknown. Loss of PTEN, a tumor suppressor regulated by ceRNA activity, frequently occurs in melanoma. Here, we report the discovery of significant enrichment of putative PTEN ceRNAs among genes whose loss accelerates tumorigenesis following Sleeping Beauty insertional mutagenesis in a mouse model of melanoma. We validated several putative PTEN ceRNAs and further characterized one, the ZEB2 transcript. We show that ZEB2 modulates PTEN protein levels in a microRNA-dependent, protein coding-independent manner. Attenuation of ZEB2 expression activates the PI3K/AKT pathway, enhances cell transformation, and commonly occurs in human melanomas and other cancers expressing low PTEN levels. Our study genetically identifies multiple putative microRNA decoys for PTEN, validates ZEB2 mRNA as a bona fide PTEN ceRNA, and demonstrates that abrogated ZEB2 expression cooperates with BRAFV600E to promote melanomagenesis. [ABSTRACT FROM AUTHOR]

Ipilimumab, a monoclonal antibody against cytotoxic T lymphocyte antigen 4 (CTLA-4), has been shown to improve survival in patients with advanced metastatic melanoma. It also enhances immunity to NY-ESO-1, a cancer/testis antigen expressed in a subset of patients with melanoma. To characterize the association between immune response and clinical outcome, we first analyzed NY-ESO-1 serum antibody by ELISA in 144 ipilimumab-treated patients with melanoma and found 22 of 140 (16%) seropositive at baseline and 31 of 144 (22%) seropositive following treatment. These NY-ESO-1-seropositive patients had a greater likelihood of experiencing clinical benefit 24 wk after ipilimumab treatment than NY-ESO-1-seronegative patients (P = 0.02, relative risk = 1.8, two-tailed Fisher test). To understand why some patients with NY-ESO-1 antibody failed to experience clinical benefit, we analyzed NY-ESO-1-specific CD4+ and CD8+ T-cell responses by intracellular multicytokine staining in 20 NY-ESO-1-seropositive patients and found a surprising dissociation between NY-ESO-1 antibody and CD8 responses in some patients. NY-ESO-1-seropositive patients with associated CD8+ T cells experienced more frequent clinical benefit (10 of 13; 77%) than those with undetectable CD8+ T-cell response (one of seven; 14%; P = 0.02; relative risk = 5.4, two-tailed Fisher test), as well as a significant survival advantage (P = 0.01; hazard ratio = 0.2, time-dependent Cox model). Together, our data suggest that integrated NY-ESO-1 immune responses may have predictive value for ipilimumab treatment and argue for prospective studies in patients with established NY-ESO-1 immunity. The current findings provide a strong rationale for the clinical use of modulators of immunosuppression with concurrent approaches to favor tumor antigen-specific immune responses, such as vaccines or adoptive transfer, in patients with cancer. [ABSTRACT FROM AUTHOR]

Background: Genomic aberrations can be used to determine cancer diagnosis and prognosis. Clinically relevant novel aberrations can be discovered using high-throughput assays such as Single Nucleotide Polymorphism (SNP) arrays and next-generation sequencing, which typically provide aggregate signals of many cells at once. However, heterogeneity of tumor subclones dramatically complicates the task of detecting aberrations. Results: The aggregate signal of a population of subclones can be described as a linear system of equations. We employed a measure of allelic imbalance and total amount of DNA to characterize each locus by the copy number status (gain, loss or neither) of the strongest subclonal component. We designed simulated data to compare our measure to existing approaches and we analyzed SNP-arrays from 30 melanoma samples and transcriptome sequencing (RNA-Seq) from one melanoma sample. We showed that any system describing aggregate subclonal signals is underdetermined, leading to non-unique solutions for the exact copy number profile of subclones. For this reason, our illustrative measure was more robust than existing Hidden Markov Model (HMM) based tools in inferring the aberration status, as indicated by tests on simulated data. This higher robustness contributed in identifying numerous aberrations in several loci of melanoma samples. We validated the heterogeneity and aberration status within single biopsies by fluorescent in situ hybridization of four affected and transcriptionally up-regulated genes E2F8, ETV4, EZH2 and FAM84B in 11 melanoma cell lines. Heterogeneity was further demonstrated in the analysis of allelic imbalance changes along single exons from melanoma RNA-Seq. Conclusions: These studies demonstrate how subclonal heterogeneity, prevalent in tumor samples, is reflected in aggregate signals measured by high-throughput techniques. Our proposed approach yields high robustness in detecting copy number alterations using high-throughput technologies and has the potential to identify specific subclonal markers from next-generation sequencing data. [ABSTRACT FROM AUTHOR]

Progress in understanding the molecular basis of melanoma has made possible the identification of molecular targets with important implications in clinical practice. In fact, new therapeutic approaches are emerging from basic science and it will be important to implement their rapid translation into clinical practice by active clinical investigation. The first meeting of Melanoma Research: a bridge Naples-USA, organized by Paolo A. Ascierto (INT, Naples, Italy) and Francesco Marincola (NIH, Bethesda, USA) took place in Naples, on 6-7 December 2010. This international congress gathered more than 30 international and Italian faculty members and was focused on recent advances in melanoma molecular biology, immunology and therapy, and created an interactive discussion across Institutions belonging to Government, Academy and Pharmaceutical Industry, in order to stimulate new approaches in basic, translational and clinical research. Four topics of discussion were identified: New pathways in Melanoma, Biomarkers, Clinical Trials and New Molecules and Strategies. [ABSTRACT FROM AUTHOR]

Background: Genomic aberrations can be used to determine cancer diagnosis and prognosis. Clinically relevant novel aberrations can be discovered using high-throughput assays such as Single Nucleotide Polymorphism (SNP) arrays and next-generation sequencing, which typically provide aggregate signals of many cells at once. However, heterogeneity of tumor subclones dramatically complicates the task of detecting aberrations. Results: The aggregate signal of a population of subclones can be described as a linear system of equations. We employed a measure of allelic imbalance and total amount of DNA to characterize each locus by the copy number status (gain, loss or neither) of the strongest subclonal component. We designed simulated data to compare our measure to existing approaches and we analyzed SNP-arrays from 30 melanoma samples and transcriptome sequencing (RNA-Seq) from one melanoma sample. We showed that any system describing aggregate subclonal signals is underdetermined, leading to non-unique solutions for the exact copy number profile of subclones. For this reason, our illustrative measure was more robust than existing Hidden Markov Model (HMM) based tools in inferring the aberration status, as indicated by tests on simulated data. This higher robustness contributed in identifying numerous aberrations in several loci of melanoma samples. We validated the heterogeneity and aberration status within single biopsies by fluorescent in situ hybridization of four affected and transcriptionally up-regulated genes E2F8, ETV4, EZH2 and FAM84B in 11 melanoma cell lines. Heterogeneity was further demonstrated in the analysis of allelic imbalance changes along single exons from melanoma RNA-Seq. Conclusions: These studies demonstrate how subclonal heterogeneity, prevalent in tumor samples, is reflected in aggregate signals measured by high-throughput techniques. Our proposed approach yields high robustness in detecting copy number alterations using high-throughput technologies and has the potential to identify specific subclonal markers from next-generation sequencing data. [ABSTRACT FROM AUTHOR]

Background Abberant DNA methylation at CpG dinucleotides represents a common mechanism of transcriptional silencing in cancer. Since CpG methylation is a reversible event, tumor supressor genes that have undergone silencing through this mechanism represent promising targets for epigenetically active anti-cancer therapy. The cytosine analog 5-aza-2'-deoxycytidine (decitabine) induces genomic hypomethylation by inhibiting DNA methyltransferase, and is an example of an epigenetic agent that is thought to act by up-regulating silenced genes. Methods It is unclear why decitabine causes some silenced loci to re-express, while others remain inactive. By applying data-mining techniques to large-scale datasets, we attempted to elucidate the qualities of promoter regions that define susceptibility to the drug's action. Our experimental data, derived from melanoma cell strains, consist of genome-wide gene expression data before and after treatment with decitabine, as well as genome-wide data on un-treated promoter methylation status, and validation of specific genes by bisulfite sequencing. Results We show that the combination of promoter CpG content and methylation level informs the ability of decitabine treatment to up-regulate gene expression. Promoters with high methylation levels and intermediate CpG content appear most susceptible to up-regulation by decitabine, whereas few of those highly methylated promoters with high CpG content are up-regulated. For promoters with low methylation levels, those with high CpG content are more likely to be up-regulated, whereas those with low CpG content are underrepresented among up-regulated genes. Conclusions Clinically, elucidating the patterns of action of decitabine could aid in predicting the likelihood of up-regulating epigenetically silenced tumor suppressor genes and others from pathways involved with tumor biology. As a first step toward an eventual translational application, we build a classifier to predict gene up-regulation based on promoter methylation and CpG content, which achieves a performance of 0.77 AUC. [ABSTRACT FROM AUTHOR]

Tumor necrosis factor-α (TNF-α) and Fas ligand induce apoptosis by interacting with their corresponding membrane-bound death receptors and activating caspases. Since both systems share several components of the intracellular apoptotic cascade and are expressed by first trimester trophoblasts, it is unknown how these cells remain resistant to Fas ligand while sensitive to TNF-α. XAF1 (X-linked inhibitor of apoptosis (XIAP)-associated factor 1) is a proapoptotic protein that antagonizes the caspase-inhibitory activity of XIAP. Here, we demonstrated that XAF1 functions as an alternative pathway for TNF-α-induced apoptosis by translocating to the mitochondria and promoting XIAP inactivation. In addition, we showed that the overexpression of XAF1 sensitized first trimester trophoblast cells to Fas-mediated apoptosis. Furthermore, we also determined that the differential expression of XAF1 in first and third trimester trophoblast cells was due to changes in XAF1 gene methylation. Our results establish a novel regulatory pathway controlling trophoblast cell survival and provide a molecular mechanism to explain trophoblast sensitivity to TNF-α and the increased number of apoptotic trophoblast cells observed near term. Aberrant XAF1 expression and/or localization may have consequences for normal pregnancy outcome. [ABSTRACT FROM AUTHOR]

Rab33A, a member of the small GTPase superfamily, is an X-linked gene that is expressed in brain, lymphocytes, and normal melanocytes, but is downregulated in melanoma cells. We demonstrate that in normal melanocytes Rab33A colocalizes with melanosomal proteins and that a constitutively active GTPase mutant suppresses their transport to the melanosomes. In the brain, Rab33A is present throughout the cortex, as well as in the hippocampal CA fields. A survey of melanocytic lesions demonstrated that aberrant downregulation of Rab33A is an early event that is already prevalent in melanocytes of giant congenital nevi. Analyses of bisulfite-modified DNA revealed that Rab33A is regulated by DNA methylation of a specific promoter region proximal to the transcription initiation site, and that suppression of Rab33A in melanoma cells recapitulates normal processes that control silencing of X-linked genes, but not tissue specific gene expression. This information is important for understanding carcinogenesis as well as other aberrant processes because Rab33A may have an important role in disorders involving X-chromosome-linked genes associated with vesicular transport.Journal of Investigative Dermatology (2006) 126, 2257–2271. doi:10.1038/sj.jid.5700386; published online 29 June 2006 [ABSTRACT FROM AUTHOR]

Melanocytes and keratinocytes were analyzed for potential roles of p53, p73, and p63 tumor suppressor family proteins and of malignancy-specific gene expression changes in the etiology of multi-step cancer. Melanocytes expressed ΔNp73α, two p63 isoforms and p53. Although p21 and Noxa mRNA levels increased following DNA damage, p53 family member binding to p21 and Noxa DNA probes was undetectable, suggesting p53 family- independent responses. In contrast, keratinocytes expressed multiple isoforms each of p73 and p63 that were induced to bind p21 and Noxa DNA probes after ionizing (IR) or after ultraviolet B (UVB) irradiation, correlating with p21 and Noxa mRNA induction and with apoptosis. Interestingly, IR-resistant malignant melanocytes and keratinocytes both exhibited Noxa mRNA induction after UVB treatment, correlating with DNA binding of p53 family proteins to the Noxa probe only in keratinocytes. To uncover other malignancy-specific events, we queried mouse initiated keratinocyte clones for early changes that were exacerbated in malignant derivatives and also differentially expressed in human advanced melanoma versus normal melanocytes. Using a new method for ranking and normalization of microarray data for 5000 probe sets, 27 upregulated and 13 downregulated genes satisfied our query. Of these, the majority was associated with late-stage human cancers and six were novel genes. Thus, clonal lineage mouse models representing early through late cancer progression stages may inform the focus on early, potentially causal events from microarray studies of human cancers, facilitating prognosis and molecular therapy. [ABSTRACT FROM AUTHOR]

Discusses the importance of a glycoprotein called tyrosinase in the synthesis of melanin in melanocytes. Identification of mutations in tyrosinase; Sensitivity of human tyrosinase (TYR) to trypsin digestion; Result of the translocation of TYR into semipermeabilized melanocytes.

Basic Fibroblast Growth Factor (bFGF/FGF2) is thought to play a decisive role in malignant progression. Aberrant expression of bFGF causes constitutive autocrine activation of its cognate receptor and autonomous growth of human melanoma cells or bFGF transformed fibroblasts in culture. It remains to be determined, however, whether the endogenous bFGF confers growth advantage to tumors and what are the downstream targets of the activated FGF receptor critical for its transforming capacity. We therefore transfected metastatic melanoma cells and bFGF transformed mouse fibroblasts with a dominant-negative mutant of the murine FGF receptor 1 (fgfr1/flg), comprising the extracellular and transmembrane domains but lacking the intracellular kinase domain (dnflg). Reverse transcriptase-PCR, 125I-bFGF binding and affinity labeling analyses show that the truncated receptor is targeted to the membrane and is expressed at much higher levels than the endogenous receptor in all of the selected clones. Expression of the dnflg dramatically reduces the basal as well as bFGF induced growth of these cells in vitro and also suppresses their tumorigenic potential in nude mice. The expression of the dnflg does not significantly alter the general level of tyrosyl-phosphorylated proteins in the trunsduced melanoma cells. Rather, a major downstream affected target is a Src-family kinase, whose activity, determined by an in vitro immune kinase assay, is stimulated in normal melanocytes by exogenous bFGF, and is markedly reduced in the dnflg-expressing melanoma cells. The present study demonstrates that direct interference with the activity of FGF receptors has a deleterious effect on cell proliferation and survival in vitro and in vivo leading to the suppression of melanoma tumor progression possibly through the inactivation of a Src-family kinase. [ABSTRACT FROM AUTHOR]

The macrophage colony stimulating factor receptor (CSF-1R), the product of the c-fms proto-oncogene, plays an important role in regulating the normal proliferation and differentiation of macrophages and trophoblasts. However, the abnormal expression of CSF-1R transcripts and protein by human breast carcinomas has been shown to correlate with advanced stage and poor prognosis. Ligand activated CSF-1R dimers transphosphorylate several tyrosines in their cytoplasmic domains which provide recognition sites for various effector proteins in multiple signal transduction pathways. In cells transformed by the c-fms oncogene, one of the major CSF-1R phosphotyrosines, pTyr723 is important for phenotypic expression of anchorage-independent growth and metastasis. In order to investigate the relationship between receptor activation/phosphorylation and cellular phenotypes in vitro and in vivo, we prepared a CSF-1R phosphorylation-state specific antibody raised against a specific phosphopeptide of CSF-1R, which included phosphorylated tyrosine 723. On immunoblots of lysates from cells expressing CSF-1R, this antibody recognizes phosphorylated CSF-1R in CSF-1 stimulated cells but not in unstimulated cells. As an immunohistochemical reagent, this antibody stained 52% of invasive human breast tumors (72% of CSF-1R positive cases) in a sample of 114 cases and 38% of carcinoma in situ. This data represents the first direct evidence of in vivo phosphorylation of CSF-1R in human breast carcinomas. [ABSTRACT FROM AUTHOR]

PmeI 17 is preferentially expressed in pigment cells in a manner suggestive of involvement in melanin bio- synthesis. The gene is identical to the silver (si) pigmentation locus in mice. We now have produced a recombinant glutathione-S-transferase-human Pmel 17 fusion protein and raised polyclonal antibodies against it to confirm the ultrastructural location and presumed site of action predicted by the deduced primary structure of Pmel 17/silver, and to authenticate the specificity of the DHICA converting function as inherent to the silver-locus protein. Full- length Pmel 17 cDNA also was produced in insect cells in a baculovirus expression vector to ensure that activity did not originate from a co-precipitated protein. Natural hPmel 17 from human melanoma cells has an approximate molecular size of 100 kDa. By immunoperoxidase electron microscopic cytochemistry, the antigen was localized to the limiting membranes of premelanosomes and presumed premelaflo- genic cytosolic vesicles and, to a minor extent, in the premelanosomal matrix. In an in vitro assay, both the natural and the recombinant Pmel 17 accelerated the conversion of DHICA to melanin. This activity was inhibited by the anti-Pmel 17 polyclonal antibodies, indicating that the acceleration of DHICA conversion by the natural protein is genuine and cannot be due to contaminating complexed proteins. We suggest that in situ Pmel 17/silver is a component of a postulated premelanosomal/melanosomal complex of membrane-bound melanogenic oxidoreductive enzymes and cofactors, in analogy to the electron transfer chain in mitochondria. [ABSTRACT FROM AUTHOR]

Basic fibroblast growth factor (bFGF) is a mitogen for normal human melanocytes and keratinocytes in culture. Experiments in vitro suggest that keratinocytes supply bFGF to melanocytes through a paracrine mechanism and that the aberrant expression of bFGF in melanomas confers growth independence from bFGF-producing cells. To determine whether bFGF is expressed in vivo, we examined a series of benign and malignant melanocytic lesions in situ using bFGF riboprobes on tissue sections, and correlated bFGF expression with histologic phenotype. Seventeen melanocytic neoplasms were studied, including four common acquired nevi, four dysplastic nevi, four primary malignant melanomas, and five metastatic melanomas. Nevic cells in benign intradermal nevi showed low signal intensity (1+), whereas compound and dysplastic nevi showed 2+ to 3+ expression in the junctional nevic cell population and 1+ expression in the dermal nevic cell population. Melanocytes in primary melanomas had intermediate (2+) and those in metastatic melanomas had low (1+) levels of bFGF gene transcripts. Fibroblasts expressed high levels (3+) and epidermal and adnexal keratinocytes moderate (2+) levels of bFGF in all cases studied. Basic PGF expression in endothelial cells, known to produce and respond to this growth factor in vitro, was lower than that in the fibroblast and keratinocyte cell population and, in 10 of 17 cases, no bFGF mRNA was detectable. This study shows that bFGF is expressed in nevomelanocytes in vivo in all melanocytic lesions studied and thus cannot be used as a marker for transformation. The presence of bFGF gene transcripts in the various dermal cell types and in keratinocytes suggests that it may act as an autocrine and paracrine growth factor in regulating cellular proliferation in the skin. [ABSTRACT FROM AUTHOR]

Using a human tyrosinase cDNA probe, we have isolated mouse tyrosinase genomic clones and used them to map the mouse tyrosinase locus and to analyze tile promoter sequence of the tyrosinase gene. Southern blot analyses of DNA from somatic cell hybrids, interspecies backcross mice, and albino deletion mice have revealed that the locus for mouse tyrosinase resides at or near the albino locus on mouse chromosome 7. There were three TATA-elements, but only one CAT-element, and the CAT-demerit appeared to be paired with the third TATA-element, located at the position farthest upstream. Mouse tyrosinase mRNA is approximately 2.4 Kb in size. The amount of tyrosinase mRNA reflects the levels of tyrosinase activity in normal melanocytes and Cloudman S-91 melanoma cell line. [ABSTRACT FROM AUTHOR]

Activating mutations in BRAF kinase are common in melanomas. Clinical trials with PLX4032, the mutant-BRAF inhibitor, show promising preliminary results in patients selected for the presence of V600E mutation. However, activating V600K mutation is the other most common mutation, yet patients with this variant are currently excluded from the PLX4032 trials. Here we present evidence that a patient bearing the BRAF V600K mutation responded remarkably to PLX4032, suggesting that clinical trials should include all patients with activating BRAF V600E/K mutations. [ABSTRACT FROM AUTHOR]