ABSTRACT
BACKGROUND: Gap-junctional intercellular communication is crucial for epidermal cellular homeostasis. Inability to establish melanocyte-keratinocyte contact and loss of the intercellular junction's integrity may contribute to melanoma development. Connexins, laminins and desmocollins have been implicated in the control of melanoma growth, where their reduced expression has been reported in metastatic lesions. OBJECTIVES: The aim of this study was to investigate connexin 31·1 (GJB5) expression and identify any association with BRAF mutational status, prognosis of patients with melanoma and mitogen-activated protein kinase (MAPK) inhibitor (MAPKi) treatment. METHODS: GJB5 expression was measured at RNA and protein level in melanoma clinical samples and established cell lines treated (or not) with BRAF and MEK inhibitors (MEKi), as well as in cell lines which developed MAPKi resistance. Findings were further validated and confirmed by analysis of independent datasets. RESULTS: Our analysis reveals significant downregulation of GJB5 expression in metastatic melanoma lesions compared with primary ones and in BRAF-mutated vs. BRAF-wildtype (BRAFWT ) melanomas. Likewise, GJB5 expression is significantly lower in BRAFV600E compared with BRAFWT cell lines and increases on MAPKi treatment. MAPKi-resistant melanoma cells display a similar expression pattern compared with BRAFWT cells, with increased GJB5 expression associated with morphological changes. Enhancement of BRAFV600E expression in BRAFWT melanoma cells significantly upregulates miR-335-5p expression with consequent downregulation of GJB5, one of its targets. Furthermore, overexpression of miR-335-5p in two BRAFWT cell lines confirms specific GJB5 protein downregulation. Reverse transcriptase quantitative polymerase chain reaction analysis also revealed upregulation of miR-335 in BRAFV600E melanoma cells, which is significantly downregulated in cells resistant to MEKi. Our data were further validated using the TCGA_SKCM dataset, where BRAF mutations associate with increased miR-335 expression and inversely correlate with GJB5 expression. In clinical samples, GJB5 underexpression is also associated with patient overall worse survival, especially at early stages. CONCLUSIONS: We identified a significant association between metastases/BRAF mutation and low GJB5 expression in melanoma. Our results identify a novel mechanism of gap-junctional protein regulation, suggesting a prognostic role for GJB5 in cutaneous melanoma.
Subject(s)
Melanoma , MicroRNAs , Skin Neoplasms , Cell Line, Tumor , Connexins , Humans , Melanoma/pathology , MicroRNAs/genetics , MicroRNAs/metabolism , Mutation , Protein Kinase Inhibitors/pharmacology , Proto-Oncogene Proteins B-raf/genetics , Skin Neoplasms/drug therapy , Skin Neoplasms/geneticsABSTRACT
By integrating gene profiling and immunohistochemical data with functional experiments in cell lines in this study we show for the first time that doublecortin (DCX) domain containing 2 (DCDC2), a protein belonging to the DCX family and involved in neuronal cell migration, is aberrantly expressed in prostate tumors whereas absent in normal prostate. Furthermore, in patients treated with radical prostatectomy, high levels of DCDC2 RNA were significantly associated with increased biochemical relapse (LogRank Mantel-Cox=0.012). Mechanistically, we found that the ETS transcription factor ESE3/EHF, which is expressed in normal prostate and frequently lost in prostate tumors, maintained DCDC2 repressed by binding to a novel identified ETS binding site in the gene promoter. Consistently, in prostate tumors and in cellular models of gain and loss of ESE3/EHF, the expression of DCDC2 and ESE3/EHF were inversely correlated. In prostate cancer cells, DCDC2 colocalized with microtubules and promoted cell migration and resistance to the microtubule-targeting drug taxol. Collectively, this study establishes DCDC2 as a novel ESE3/EHF oncogenic target in prostate cancer. These findings may be relevant for the clinical management of prostate cancer as DCDC2 may signal tumors more prone to relapse and resistant to taxol treatment.
Subject(s)
Drug Resistance, Neoplasm/genetics , Microtubule-Associated Proteins/genetics , Prostatic Neoplasms/genetics , Prostatic Neoplasms/pathology , Binding Sites , Cell Movement/genetics , Disease Progression , Gene Expression Regulation, Neoplastic , Humans , Male , Microtubule-Associated Proteins/metabolism , Microtubules/metabolism , Paclitaxel/pharmacology , Promoter Regions, Genetic , Prostatic Neoplasms/drug therapy , Prostatic Neoplasms/mortality , Prostatic Neoplasms/surgery , Reference Values , Transcription Factors/genetics , Transcription Factors/metabolism , Tubulin Modulators/pharmacologyABSTRACT
Epigenetic silencing of tumour suppressor genes is an important mechanism involved in cell transformation and tumour progression. The Set and RING-finger-associated domain-containing protein UHRF1 might be an important link between different epigenetic pathways. Here, we report that UHRF1 is frequently overexpressed in human prostate tumours and has an important role in prostate cancer pathogenesis and progression. Analysis of human prostate cancer samples by microarrays and immunohistochemistry showed increased expression of UHRF1 in about half of the cases. Moreover, UHRF1 expression was associated with reduced overall survival after prostatectomy in patients with organ-confined prostate tumours (P < 0.0001). UHRF1 expression was negatively correlated with several tumour suppressor genes and positively with the histone methyltransferase (HMT) EZH2 both in prostate tumours and cell lines. UHRF1 knockdown reduced proliferation, clonogenic capability and anchorage-independent growth of prostate cancer cells. Depletion of UHRF1 resulted in reactivation of several tumour suppressor genes. Gene reactivation upon UHRF1 depletion was associated with changes in histone H3K9 methylation, acetylation and DNA methylation, and impaired binding of the H3K9 HMT Suv39H1 to the promoter of silenced genes. Co-immunoprecipitation experiments showed direct interaction between UHRF1 and Suv39H1. Our data support the notion that UHRF1, along with Suv39H1 and DNA methyltransferases, contributes to epigenetic gene silencing in prostate tumours. This could represent a parallel and convergent pathway to the H3K27 methylation catalyzed by EZH2 to synergistically promote inactivation of tumour suppressor genes. Deregulated expression of UHRF1 is involved in the prostate cancer pathogenesis and might represent a useful marker to distinguish indolent cancer from those at high risk of lethal progression.
Subject(s)
CCAAT-Enhancer-Binding Proteins/genetics , CCAAT-Enhancer-Binding Proteins/metabolism , Prostatic Neoplasms/genetics , Prostatic Neoplasms/metabolism , Acetylation , Cell Growth Processes/physiology , Cell Line, Tumor , DNA Methylation , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Disease Progression , Enhancer of Zeste Homolog 2 Protein , Epigenesis, Genetic , Gene Expression Regulation, Neoplastic , Gene Silencing , Genes, Tumor Suppressor , HEK293 Cells , Histone Methyltransferases , Histone-Lysine N-Methyltransferase/genetics , Histone-Lysine N-Methyltransferase/metabolism , Histones/genetics , Histones/metabolism , Humans , Immunoprecipitation/methods , Male , Polycomb Repressive Complex 2/genetics , Polycomb Repressive Complex 2/metabolism , Promoter Regions, Genetic , Prostatic Neoplasms/pathology , Ubiquitin-Protein LigasesABSTRACT
Microarray experiments have a large variety of applications and several important achievements have been obtained by means of this technology, especially within the field of whole genome expression profiling, which undoubtedly is the most diffused world-wide. Nevertheless, care must be taken in unconditionally applying such high-throughput techniques and in extracting/interpreting their results. Both the validity and the reproducibility of microarray-based clinical research have recently been challenged. Pitfalls and potentials of the microarray technology for gene expression profiling are critically reviewed in this paper.
Subject(s)
Genome , Oligonucleotide Array Sequence Analysis/methods , Algorithms , Animals , Base Sequence , Gene Amplification , Gene Expression , Gene Expression Profiling , Genetic Variation , Genome, Human , Humans , RNA Processing, Post-Transcriptional , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
Deregulated expression of ETS transcription factors has emerged as an important event in prostate cancer pathogenesis. Here we show that the expression of epithelial-specific ETS (ESE)-3 factor is frequently reduced at the RNA and protein level in prostate cancer clinical samples compared to normal prostate. In PC3 and DU145 cells, ESE-3 was silenced by methylation of an evolutionarily conserved CpG site in its promoter and treatment with 5-aza-2'-deoxycytidine restored its expression. In a prostate epithelial cell transformation model, methylation of this site was inversely correlated with ESE-3 expression and occurred only in Ras-transformed and tumorigenic cells and not in normal and immortalized cells suggesting that ESE-3 silencing was functionally linked to oncogenic transformation. Consistent with a tumor suppressor function, re-expression of ESE-3 in prostate cancer cells inhibited clonogenic survival and induced apoptotic cell death. ESE-3 increased the level of procaspase-3, a key element in the apoptotic cascade. This effect was mediated at the transcriptional level by direct binding of ESE-3 to the caspase-3 promoter. Collectively, our findings implicate ESE-3 as a candidate tumor suppressor in prostate cancer. Decreased expression of ESE-3 may result in loss of important regulatory mechanisms in prostate epithelial cells and contribute to the pathogenesis of prostate cancer.
Subject(s)
Prostatic Neoplasms/metabolism , Transcription Factors/antagonists & inhibitors , Transcription Factors/genetics , Tumor Suppressor Proteins/antagonists & inhibitors , Tumor Suppressor Proteins/biosynthesis , Adult , Aged , Cell Line, Tumor , Gene Silencing/physiology , Humans , Male , Middle Aged , Prostatic Neoplasms/genetics , Transcription Factors/biosynthesis , Tumor Suppressor Proteins/genetics , Tumor Suppressor Proteins/physiologyABSTRACT
The analysis of expression signatures is a powerful tool for the classification of cancer and other tissue samples. Several protocols and platforms are available on the market, and these lead to both confirmatory and complementary results. We review the main processing techniques for cross-platform comparisons and the different tissue sources for cancer profiling. Some examples and the cross-interpretation of bibliographic data related to prostate cancer are also presented.
