Exploring Morphine-Triggered PKC-Targets and Their Interaction with Signaling Pathways Leading to Pain via TrkA

It is well accepted that treatment of chronic pain with morphine leads to μ opioid receptor (MOR) desensitization and the development of morphine tolerance. MOR activation by the selective peptide agonist, D-Ala2, N-MePhe4, Gly-ol]-enkephalin(DAMGO), leads to robust G protein receptor kinase activation, β-arrestin recruitment, and subsequent receptor endocytosis, which does not occur in an activation by morphine. However, MOR activation by morphine induces receptor desensitization, in a Protein kinase C (PKC) dependent manner. PKC inhibitors have been reported to decrease receptor desensitization, reduce opiate tolerance, and increase analgesia. However, the exact role of PKC in these processes is not clearly delineated. The difficulties in establishing a particular role for PKC have been, in part, due to the lack of reagents that allow the selective identification of PKC targets. Recently, we generated a conformation state-specific anti-PKC antibody that preferentially recognizes the active state of this kinase. Using this antibody to selectively isolate PKC substrates and a proteomics strategy to establish the identity of the proteins, we examined the effect of morphine treatment on the PKC targets. We found an enhanced interaction of a number of proteins with active PKC, in the presence of morphine. In this article, we discuss the role of these proteins in PKC-mediated MOR desensitization and analgesia. In addition, we posit a role for some of these proteins in mediating pain by TrKA activation, via the activation of transient receptor potential cation channel subfamily V member 1 (TRPV1). Finally, we discuss how these new PKC interacting proteins and pathways could be targeted for the treatment of pain

Epigenetic signature of differentially methylated genes in cutaneous melanoma

Abstract Background Cutaneous melanoma (CM) is the most aggressive subtype of skin cancer, with increasing incidence over the past several decades. DNA methylation is a key element of several biological processes such as genomic imprinting, cell differentiation and senescence, and deregulation of this mechanism has been implicated in several diseases, including cancer. In order to understand the relationship of DNA methylation in CMs, we searched for an epigenetic signature of cutaneous melanomas by comparing the DNA methylation profiles between tumours and benign melanocytes, the precursor cells of CM. Methods We used 20 primary CMs and three primary cell cultures of melanocytes as a discovery cohort. The tumours mutational background was collected as previously reported. Methylomes were obtained using the HM450K DNA methylation assay, and differential methylation analysis was performed. DNA methylation data of CMs from TCGA were recovered to validate our findings. Results A signature of 514 differentially methylated genes (DMGs) was evident in CMs compared to melanocytes, which was independent of the presence of driver mutations. Pathway analysis of this CM signature revealed an enrichment of proteins involved in the binding of DNA regulatory regions (hypermethylated sites), and related to transmembrane signal transducer activities (hypomethylated sites). The methylation signature was validated in an independent dataset of primary CMs, as well as in lymph node and distant metastases (correlation of DNA methylation level: r > 0,95; Pearson’s test: p < 2.2e-16). Conclusions CMs exhibited a DMGs signature, which was independent of the mutational background and possibly established prior to genetic alterations. This signature provides important insights into how epigenetic deregulation contributes to melanomagenesis in general

DNA Methylation Levels of Melanoma Risk Genes Are Associated with Clinical Characteristics of Melanoma Patients

In melanoma development, oncogenic process is mediated by genetic and epigenetic mutations, and few studies have so far explored the role of DNA methylation either as predisposition factor or biomarker. We tested patient samples for germline CDKN2A methylation status and found no evidence of inactivation by promoter hypermethylation. We have also investigated the association of clinical characteristics of samples with the DNA methylation pattern of twelve genes relevant for melanomagenesis. Five genes (BAP1, MGMT, MITF, PALB2, and POT1) presented statistical association between blood DNA methylation levels and either CDKN2A-mutation status, number of lesions, or Breslow thickness. In tumors, five genes (KIT, MGMT, MITF, TERT, and TNF) exhibited methylation levels significantly different between tumor groups including acral compared to nonacral melanomas and matched primary lesions and metastases. Our data pinpoint that the methylation level of eight melanoma-associated genes could potentially represent markers for this disease both in peripheral blood and in tumor samples

DNA Methylation Levels of Melanoma Risk Genes Are Associated with Clinical Characteristics of Melanoma Patients

In melanoma development, oncogenic process is mediated by genetic and epigenetic mutations, and few studies have so far explored the role of DNA methylation either as predisposition factor or biomarker. We tested patient samples for germline CDKN2A methylation status and found no evidence of inactivation by promoter hypermethylation. We have also investigated the association of clinical characteristics of samples with the DNA methylation pattern of twelve genes relevant for melanomagenesis. Five genes (BAP1, MGMT, MITF, PALB2, and POT1) presented statistical association between blood DNA methylation levels and either CDKN2A-mutation status, number of lesions, or Breslow thickness. In tumors, five genes (KIT, MGMT, MITF, TERT, and TNF) exhibited methylation levels significantly different between tumor groups including acral compared to nonacral melanomas and matched primary lesions and metastases. Our data pinpoint that the methylation level of eight melanoma-associated genes could potentially represent markers for this disease both in peripheral blood and in tumor samples