Kyu Young Song

Spitz and Spitzoid lesions represent one of the most challenging melanocytic neoplasms in dermatopathology. Nosologic classification has been more recently improved by the discovery of novel molecular drivers, particularly translocations. In the current study, we aimed to use an unbiased approach to explore the gene expression profile of a group of melanocytic Spitz and Spitzoid melanocytic lesions ranging from benign lesions to melanoma, including intermediate lesions such as SPARK nevi and atypical Spitz tumors/melanocytomas. Using unsupervised analysis of gene expression data, we found some distinct hierarchical clusters of lesions, including groups characterized by ALK and NTRK translocations. Few non-ALK translocated tumors demonstrated increased ALK expression, confirmed by immunohistochemistry. Spitz tumors with overlapping features of dysplastic nevi, so-called SPARK nevi, appear to have a common gene expression profile by hierarchical clustering. Finally, weighted gene correlation network analysis identified gene modules variably regulated in subtypes of these cases. Thus, gene expression profiling of Spitz and Spitzoid lesions represents a viable instrument for the characterization of these lesions.

Melanoma is the leading cause of death from cutaneous malignancy. While targeted therapy and immunotherapy with checkpoint inhibitors have significantly decreased the mortality rate of this disease, advanced melanoma remains a therapeutic challenge. Here, we confirmed that interferon-gamma (IFN-γ)-induced PD-L1 expression in melanoma cell lines. This increased expression was down-regulated by the reduction in phosphorylated STAT3 signaling via MET tyrosine kinase inhibitor treatment. Furthermore, immunoprecipitation and confocal immunofluorescence microscopy analysis reveals MET and PD-L1 protein–protein interaction and colocalization on the cell surface membrane of melanoma cells. Together, these findings demonstrate that the IFN-γ-induced PD-L1 expression in melanoma cells is negatively regulated by MET inhibition through the JAK/STAT3 signaling pathway and establish the colocalization and interaction between an RTK and a checkpoint protein in melanoma cells.

The overexpression and activation of the MET receptor tyrosine kinase has been identified in many human malignancies, but its role in canine cancer has only been minimally investigated. In this study we evaluated the expression of MET in two canine malignant melanoma (CMM) cell lines as well as in 30 CMM tissue samples that were collected from the clinical service at our institution. We were able to confirm the expression of the MET protein in both melanoma cell lines, and we demonstrated MET activation by its ligand, HGF, through phosphorylation, in Western blot analysis. We were also able to demonstrate, by immunohistochemistry, the expression of MET in 63% of the tumor tissue samples analyzed, with the majority demonstrating a relatively low expression profile. We then evaluated the association of MET expression scores with histologic parameters, metastasis, and survival. While statistically significant associations were not found across these parameters, an inverse relationship between MET expression levels and time to lymph node versus distant metastasis was suggested in our cohort. These findings may require assessment in a larger group of specimens to further evaluate the role of MET expression in the homing of metastasis in lymph nodes versus that in distant organs.

BAP-1 (BRCA1-associated protein 1) inactivated melanocytic lesions are a group of familial or sporadic lesions with unique histology and molecular features. They are of great clinical interest, at least in part due to the potential for malignant transformation and association with a familial cancer predisposition syndrome. Here, we describe a patient with multiple spatially and temporally distinct melanocytic lesions with loss of BAP1 expression by immunohistochemistry. RNA sequencing was performed on three independent lesions spanning the morphologic spectrum: a benign nevus, an atypical tumor, and a melanoma arising from a pre-existing BAP1-inactivated nevus. The three lesions demonstrated largely distinct gene expression and mutational profiles. Gene expression analysis revealed that genes involved in receptor protein kinase pathways were progressively upregulated from nevus to melanoma. Moreover, a clear enrichment of genes regulated in response to UV radiation was found in the melanoma from this patient, as well as upregulation of MAPK pathway-related genes and several transcription factors related to melanomagenesis.

Tumor heterogeneity is a relevant hallmark of melanoma due to the high mutation burden and immunogenicity commonly encountered. Heterogeneity at the histologic level frequently corresponds to heterogeneity at the molecular level. A better understanding of this feature of malignancy can help refine the development of predictive biomarkers and to define more effective targeted therapies. Here, we describe a case of melanoma displaying a dual phenotype: a DPN‐like/plexiform portion in conjunction with a conventional epithelioid morphology. Molecular studies revealed shared BRAF and PTEN mutations in both components but a CTNNB1 mutation was exclusively found in the DPN‐like area of the tumor, consistent with the distinct morphology observed. There was considerable heterogeneity in sequence variants identified in the two regions. Gene expression analysis highlighted differentially regulated genes between the two histologies, including a relevant cluster of genes in the receptor tyrosine kinase (RTK) family and related signaling pathways upregulated in the DPN‐like/plexiform area.

The proto-oncogene MET, the hepatocyte growth factor (HGF) receptor, is a transmembrane receptor tyrosine kinase (RTK) with a prominent role in tumor metastasis and resistance to anti-cancer therapies. Melanoma demonstrates relatively frequent MET aberrations, including MET gene amplification. Concurrently, programmed death-ligand 1 (PD-L1), with its ability to evade anti-tumor immune responses, has emerged as a prominent therapeutic target in melanoma and other malignancies and its expression is used as a predictive biomarker of response to immunotherapy. We performed immunohistochemistry analysis of MET and PD-L1 in 18 human melanoma cell lines derived from both primary and metastatic lesions, and in a human melanoma tissue microarray containing one hundreds melanocytic lesions, including primary cutaneous melanomas, primary mucosal melanomas, metastatic melanomas and benign melanocytic nevi as controls. After color deconvolution, each core was segmented to isolate staining and calculate the percentage of positive cells. Overall, MET expression was higher in tumors with increased PD-L1 expression. Moreover, a robust correlation between MET and PD-L1 expression was found in samples from metastatic melanoma and not in primary cutaneous or mucosal melanoma. These data suggest that relative expression levels of these proteins in combination is a marker of advanced disease and testing for expression of these markers should be considered in patients with melanoma.

Opiate withdrawal/negative reinforcement has been implicated as one of the mechanisms for the progression from impulsive to compulsive drug use. Increase in the intracellular cAMP level and protein kinase A (PKA) activities within the neurocircuitry of addiction has been a leading hypothesis for opiate addiction. This increase requires the phosphorylation of μ‐opioid receptor (MOR) at Tyr³³⁶ by Src after prolonged opiate treatment in vitro. Here, we report that the Src‐mediated MOR phosphorylation at Tyr³³⁶ is a prerequisite for opiate withdrawal in mice. We observed the recruitment of Src in the vicinity of MOR and an increase in phosphorylated Tyr³³⁶ (pY336) levels during naloxone‐precipitated withdrawal. The intracerebroventricular or stereotaxic injection of a Src inhibitor (AZD0530), or Src shRNA viruses attenuated pY336 levels, and several somatic withdrawal signs. This was also observed in Fyn^−/− mice. The stereotaxic injection of wild‐type MOR, but not mutant (Y336F) MOR, lentiviruses into the locus coeruleus of MOR^−/− mice restored somatic withdrawal jumping. Regulating pY336 levels during withdrawal might be a future target for drug development to prevent opiate addictive behaviors. Synopsis The μ‐opioid receptor (MOR) non‐canonical signaling pathway involves its phosphorylation at Tyr³³⁶ by Src kinase within the MOR signal complex. The focal event of MOR^Y336 phosphorylation may be the key for adenylyl cyclase superactivation during the withdrawal/negative stage of opiate addiction. The phosphorylation levels of MOR^Y336 and Src^Y416 increased during naloxone‐precipitated withdrawal in wild‐type mice. The intracerebroventricular or stereotaxic injection of a Src inhibitor (AZD0530), or Src shRNA viruses reduced pMOR^Y336 and pSrc^Y416 levels, and several somatic withdrawal signs. Increases of pMOR^Y336 levels as well as some somatic withdrawal signs after naloxone‐precipitated withdrawal were not observed in Fyn^−/− mice. The stereotaxic injection of wild‐type MOR lentiviruses into the locus coeruleus of MOR^−/− mice restored somatic withdrawal jumping and wet dog shaking. The stereotaxic injection of the phosphorylation‐deficient mutant (Y336F) MOR, in which the mutation of Tyr³³⁶ to Phe blocks Src‐mediated phosphorylation, attenuated naloxone‐precipitated withdrawal.

The Mu opioid receptor (MOR) mediates various functions of opioid-induced analgesia, euphoria and respiratory depression, and is a major target of opioid analgesics. Understanding of MOR gene expression among species is important for understanding its analgesic function in humans. In the current study, the polypyrimidine/polypurine (PPy/u) region, a key element of MOR gene expression, was compared in humans and mice. The mouse PPy/u element is highly homologous to its human element (84%), and the mouse MOR (mMOR) reporter drives luciferase activity 35-fold more effectively than the human MOR (hMOR) reporter. The structural study of reporter plasmids using S1 nuclease indicates that the mouse PPy/u element has a particular conformational structure, namely a single-stranded DNA (ssDNA) region that promotes strong promoter activity. DNA electrophoretic mobility shift assays demonstrated that the recombinant α-complex protein 1 (α-CP1) is capable of binding to a single-stranded mouse PPy/u sequence. Furthermore, plasmid-expressing α-CP1 activated the expression of a luciferase reporter when cotransfected with a single-stranded (p336/306) construct. In addition, the α-CP1 gene induced the mMOR gene in mouse neuronal cells and did not induce the human neuronal MOR gene. The current study demonstrates that α-CP1 functions as a transcriptional activator in the mMOR gene, but does not function in the hMOR gene due to species-specific structural differences. The differences in human and mouse MOR gene expression are based on α-CP1 and the ssDNA structure of the MOR promoter. The MOR gene is species-specifically regulated, as the PPy/u element adopts a unique species-specific conformation and α-CP1 recruitment.

Expression of the mu‐opioid receptor (MOR) protein is controlled by extensive transcriptional and post‐transcriptional processing. MOR gene expression has previously been shown to be altered by a post‐transcriptional mechanism involving the MOR mRNA untranslated region (UTR). Here, we demonstrate for the first time the role of heterogeneous nuclear ribonucleic acids (hnRNA)‐binding protein (hnRNP) K and poly(C)‐binding protein 1 (PCBP1) as post‐transcriptional inducers in MOR gene regulation. In the absence of morphine, a significant level of MOR mRNA is sustained in its resting state and partitions in the translationally inactive polysomal fraction. Morphine stimulation activates the downstream targets hnRNP K and PCPB1 and induces partitioning of the MOR mRNA to the translationally active fraction. Using reporter and ligand binding assays, as well as RNA EMSA, we reveal potential RNP binding sites located in the 5′‐untranslated region of human MOR mRNA. In addition, we also found that morphine‐induced RNPs could regulate MOR expression. Our results establish the role of hnRNP K and PCPB1 in the translational control of morphine‐induced MOR expression in human neuroblastoma (NMB) cells as well as cells stably expressing MOR (NMB1). J. Cell. Physiol. 232: 576–584, 2017.