Up-regulation of miR-182 expression after epigenetic modulation of human melanoma cells.

PURPOSE: We sought to investigate the epigenetic regulation of microRNAs (miRNAs) in melanoma. METHODS: We treated two highly metastatic human melanoma cell lines, C8161.9 and WM266-4, with the demethylating agents DAC (5-aza-2'-deoxycytidine) and trichostatin A. Locked nucleic acid-based miRNA expression profiling was utilized to examine the differential expression of miRNAs before and after treatment. RESULTS: We found that miR-182, a miRNA with oncogenic properties, was significantly up-regulated in human melanoma cells after epigenetic modulation. Genome sequence analysis revealed the presence of a prominent CpG island 8-10 kb upstream of mature miR-182. Methylation analysis showed that this genomic region was exclusively methylated in melanoma cells but not in human melanocytes, skin, or peripheral blood mononuclear cells. DISCUSSION: These results indicate that an epigenetic mechanism is likely involved in modulating the expression level of miR-182 in melanoma, and increased expression of oncogenic-like miR-182 could be a concern for melanoma patients after epigenetic therapy.

Overexpression of the progestagen-associated endometrial protein gene is associated with microphthalmia-associated transcription factor in human melanoma.

BACKGROUND: We recently reported that the progestagen-associated endometrial protein (PAEP) gene is overexpressed and promotes tumor proliferation and metastasis in human melanoma. METHODS: To identify the molecules that regulate its expression and oncogenic properties, we analyzed the gene microarray profiling of melanoma samples of serial clinical stage. RESULTS: We found that the expression profile of the PAEP gene parallels that of microphthalmia-associated transcription factor (MITF, r â€Š=  0.86), a master regulator of melanocyte development and melanoma progression. This parallelism was further confirmed with semiquantitative reverse transcriptase polymerase chain reaction analysis of melanoma-derived daughter cells. Transfection of melanoma cells with MITF small interfering RNA (siRNA) specifically diminishes PAEP gene expression, whereas PAEP siRNA transfection has no effect on MITF. Furthermore, knockdown of either the MITF or PAEP gene reveals a significant inhibition of tumor cell migration. CONCLUSIONS: Our data indicate that PAEP expression is regulated in part by MITF and may thus play a role in MITF-mediated cell migration in human melanoma.

Demethylating Agents in the Treatment of Cancer.

Gene silencing resulting from aberrant DNA methylation can lead to tumorigenesis. Therefore, drugs that inhibit or interfere with DNA methylation have been used to reactivate and induce silenced gene re-expression in malignancies. Two demethylating agents, azacitidine and decitabine, are approved for the treatment of myelodysplastic syndromes (MDS) by the U.S. Food and Drug Administration (FDA), and are now considered the standard of care in MDS. In this review, we discuss clinical data, including clinical benefits and toxicities, which led to the approval of azacitidine and decitabine. We also summarize findings from clinical trials that used these two demethylating agents in the treatment of solid tumors. Lastly, we discuss some limitations in the use of azacitidine and decitabine in cancer therapy.

Functional characterization of the progestagen-associated endometrial protein gene in human melanoma.

Utilizing gene microarray profiling of melanoma samples, we have recently identified a novel gene overexpressed in both thick primary and metastatic melanomas. This gene, progestagen-associated endometrial protein (PAEP), has never before been implicated in the oncogenic processes of melanoma, with its true function in oncogenesis and tumour progression relatively unknown. Overexpression of the PAEP gene in freshly procured thick primary and metastatic melanoma samples (58%) and daughter cell lines (77%) is confirmed by quantitative RT-PCR, immunohistochemistry, Western blotting and mass spectrometric analysis. We suggest that PAEP gene overexpression is involved with melanoma tumour progression as well as an aggressive phenotype. Transfection of melanoma cells with PAEP small interfering RNA (siRNA) reveals a significant decrease in soft agar colony formation and a marked inhibition of both cell migration and cell invasion. Furthermore, we establish stable melanoma transfectants via PAEP lentiviral small hairpin RNA (shRNA), examine their growth characteristics in a murine xenograft model and reveal that tumour growth is significantly inhibited in two separate melanoma cell lines. Our data strongly implicate the PAEP gene as a tumour growth promoter with oncogenic properties and a potential therapeutic target for patients with advanced melanoma.

MicroRNA in Melanoma.

Melanoma is a highly aggressive and deadly skin cancer. Early intervention correlates with nearly 100% patient survival, but greater than 80% mortality is associated with advanced disease. Currently, few treatment options are available for patients with metastatic melanoma, and the global incidence of melanoma is increasing faster than that of other cancers. Therefore, it is vitally important to uncover and use genetic and epigenetic regulatory mechanisms at work during the development and progression of melanoma for better prevention, diagnosis, and clinical management. MicroRNA (miRNA) is a set of small, single-stranded, noncoding RNAs that target the 3'-untranslated region of an estimated 30% of all human genes to inhibit their expression. Our understanding of miRNA-mediated regulation of cancers has grown immensely over the past decade. Here we review currently available data on melanoma-associated miRNAs, highlighting those deregulated miRNAs targeting important genes and signaling pathways involved in the progression of melanocytes to primary and metastatic melanoma. Understanding the important roles of miRNAs in melanoma progression and metastasis development will contribute to the development of miRNA-targeted therapy in the future.

Epigenetics in human melanoma.

BACKGROUND: Recent technological advances have allowed us to examine the human genome in greater detail than ever before. This has opened the door to an improved understanding of the gene expression patterns involved with cancer. METHODS: A review of the literature was performed to determine the role of epigenetic modifications in human melanoma. We focused the search on histone deacetylation, methylation of gene promoter regions, demethylation of CpG islands, and the role of microRNA. We examined the relationship between human melanoma epigenetics and their importance in tumorigenesis, tumor progression, and inhibition of metastasis. The development and clinical application of select pharmacologic agents are also discussed. RESULTS: We identified several articles that have extensively studied the role of epigenetics in melanoma, further elucidating the complex processes involved in gene regulation and expression. Several new agents directly affect epigenetic mechanisms in melanoma, with divergent affects on the metastatic potential of melanoma. CONCLUSIONS: Epigenetic mechanisms have emerged as having a central role in gene regulation of human melanoma, including the identification of several putative tumor suppressor genes and oncogenes. Further research will focus on the development of novel therapeutics that will likely target and alter such epigenetic changes.