Paul Howell

Identification of novel epigenetically modified genes in human melanoma via promoter methylation gene profiling

The inactivation of tumor‐related genes through the aberrant methylation of promoter CpG islands is thought to contribute to tumor initiation and progression. We therefore investigated promoter methylation events involved in cutaneous melanoma by screening 30 genes of interest for evidence of promoter hypermethylation, examining 20 melanoma cell lines and 40 freshly procured melanoma samples. Utilizing quantitative methylation‐specific PCR, we identified five genes (SOCS1, SOCS2, RAR‐beta 2, TNFSF10C, and TNFSF10D) with hypermethylation frequencies ranging from 50% to 80% in melanoma cell lines as well as freshly procured tissue samples. Eighteen genes (LOX, RASSF1A, WFDC1, TM, APC, TFPI2, TNFSF10A, CDKN2A, MGMT, TIMP3, ASC, TPM1, IRF8, CIITA‐PIV, CDH1, SYK, HOXB13, and DAPK1) were methylated at lower frequencies (2–30%). Two genes (CDKN1B and PTEN), previously reported as methylated in melanoma, and five other genes (RECK, IRF7, PAWR, TNFSF10B, and Rb) were not methylated in the samples screened here. Daughter melanoma cell lines showed identical methylation patterns when compared with original samples from which they were derived, as did synchronous metastatic lesions from the same patient. We identified four genes (TNFSF10C, TNFSF10D, LOX, and TPM1) that have never before been identified as hypermethylated in melanoma, with an overall methylation frequency of 60, 80, 50, and 10%, respectively, hypothesizing that these genes may play an important role in melanoma progression.

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.

The Impact of Genomics in Understanding Human Melanoma Progression and Metastasis

BACKGROUND Recent technological advances in the analysis of the human genome have opened the door to improving our primitive understanding of the gene expression patterns in cancer. For the first time, we have an overview of the complexities of tumorigenesis and metastatic progression of cancer. The examination of the phenotypic and (epi)genetic changes in cutaneous melanoma has identified several genes deemed central to the development and progression of melanoma. METHODS A review of the recent literature was performed to determine the role of array-based high-throughput gene expression analysis in understanding the specific genes involved as well as the pathways and the comparative gene expression patterns of primary and metastatic melanoma. RESULTS Most studies utilizing gene microarray analysis and other whole genome approaches reveal a wide array of genes and expression patterns in human melanoma. Furthermore, several of the same genes have been found in comparative studies, with some studies attempting correlation with clinical outcome. Several genes have been identified as potential prognostic markers of tumor progression and overall clinical outcome. CONCLUSIONS High-throughput gene expression analysis has had a major impact in melanoma research. Several gene expression platforms have provided insight into the gene expression patterns in melanoma. Such data will provide the foundations for the future development of prognostic markers and improved targeted therapies for patients with melanoma.

A personalized microRNA microarray normalization method using a logistic regression model

MOTIVATION MicroRNA (miRNA) is a set of newly discovered non-coding small RNA molecules. Its significant effects have contributed to a number of critical biological events including cell proliferation, apoptosis development, as well as tumorigenesis. High-dimensional genomic discovery platforms (e.g. microarray) have been employed to evaluate the important roles of miRNAs by analyzing their expression profiling. However, because of the small total number of miRNAs and the absence of well-known endogenous controls, the traditional normalization methods for messenger RNA (mRNA) profiling analysis could not offer a suitable solution for miRNA analysis. The need for the establishment of new adaptive methods has come to the forefront. RESULTS Locked nucleic acid (LNA)-based miRNA array was employed to profile miRNAs using colorectal cancer cell lines under different treatments. The expression pattern of overall miRNA profiling was pre-evaluated by a panel of miRNAs using Taqman-based quantitative real-time polymerase chain reaction (qRT-PCR) miRNA assays. A logistic regression model was built based on qRT-PCR results and then applied to the normalization of miRNA array data. The expression levels of 20 additional miRNAs selected from the normalized list were post-validated. Compared with other popularly used normalization methods, the logistic regression model efficiently calibrates the variance across arrays and improves miRNA microarray discovery accuracy. AVAILABILITY Datasets and R package are available at http://gauss.usouthal.edu/publ/logit/.

Aquaporins mediate the chemoresistance of human melanoma cells to arsenite.

The integral membrane channel protein aquaporin (AQP) is aberrantly expressed with oncogenic characteristics in various human cancers. In this study, we analyzed the expression pattern of all subtypes of AQPs, and found that 8 out of 13 AQPs expressed in melanoma cells. To understand the role of aberrant expression of AQP in this disease, we over‐expressed AQP3 and AQP9 in human melanoma WM266.4 cells and found that both AQPs significantly increased the chemoresistance of WM266.4 cells to arsenite. Functional studies showed that AQP3 and AQP9 can inhibit cell apoptosis induced by arsenite through down‐regulating p53 and up‐regulating Bcl‐2 and XIAP. Our data suggest the implication of APQ in melanoma progression and that the over‐expression of AQP3 and AQP9 contributes to the chemoresistance of melanoma to arsenite.

Expression and functional analysis of the WAP four disulfide core domain 1 gene in human melanoma

The exact cellular and molecular mechanisms involved in melanoma tumorigenesis remain obscure. Previous gene expression profiling analyses performed upon NHEM and human melanoma samples identified WFDC1 as one of the most frequently down-regulated genes. Here we further showed that NHEM readily express WFDC1 but expression is reduced or completely lost in 80% of the patients-derived melanoma cell lines and tissue samples examined. Furthermore, we show that promoter hypermethylation accounts for the silencing of the WFDC1 gene in 20% of the melanoma cell lines examined. The over-expression of WFDC1 in two metastatic melanoma cell lines, A375 and LOX, resulted in a significant delay of tumor growth in a murine xenograft model, despite a non-significant difference in tumor cell growth in vitro. Gene expression microarray analysis and further expression validation suggests that the Dickkopf-1 (Dkk1) gene is up-regulated in WFDC1 over-expressing cell lines, suggesting that the tumor suppressive function of WFDC1 may be partially a result of up-regulated Dkk1 gene expression, which is known to be a potent inhibitor of the Wnt signaling pathway.

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.

Abstract 4042: MiR-200c sensitizes colorectal cancer cells to 5-FU through Bcl-2-involved apoptotic pathway

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Effective clinical management of colorectal cancer is still a major challenge in cancer care. MicroRNA is a set of newly-discovered, non-coding small RNA molecules that significantly contribute to a number of critical biological events including cell proliferation, apoptosis development, as well as tumorigenesis. Our group has previously identified a number of microRNA candidates associated with potential prognostic value in colorectal cancer patients. In this study, we investigated miR-200c and validated its impact on 5-FU chemosensitivity in colorectal cancer. Human colorectal cancer cell lines HCT116 and HCT15 were employed in this study. Pre-miR-200c precursor molecules and negative control (Ambion, Inc.) were transiently transfected into these cell lines and cell viability was examined using WST-1 assay (Roche, Inc.) after 5-FU treatment for 24 hours. The results indicate that miR-200c can potentially sensitize colorectal cancer cells to 5-FU up to 25% compared to controls. Also, cells over-expressing miR-200c have an approximately 50% higher apoptotic rate than controls based on flow cytometry results. Bioinformatics predicts Bcl-2 is a putative mRNA target of miR200c. Bcl-2 is an integral membrane protein located mainly on the outer membrane of mitochondria. Overexpression of Bcl-2 has been demonstrated to prevent cells from undergoing apoptosis in response to a variety of stimuli. In this study, we further experimentally validated the repressive abilities of miR-200c on Bcl-2 expression using Western Blot and Luciferase reporter assay. Our results demonstrates that miR-200c is a potential biomarker for predicting chemotherapeutic response in colorectal cancer and functions by repressing Bcl-2 expression and inducing apoptotic susceptibility to 5-FU. These results hold great promise for moving miRNAs toward future clinical application and contribute to the ultimate goal of developing or improving therapeutic intervention for patients with colorectal cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4042.

Epigenetic Biomarkers in Melanoma

Epigenetics, defined in its most basic sense as a stable heritable change in gene function that is not a result of changes in the actual DNA sequence, has been one of the fastest growing fields of cancer research over the past decade. DNA promoter methylation is known to directly inhibit gene expression and is a common occurrence during tumor formation and progression. This action may lead to the formation of a heterochromatic environment at the promoter or other sites by histone deacetylases to further suppress target genes.

Abstract 3997: MiR-181 promotes megakaryocyte differentiation

MiRNAs have attracted attention due to their key regulatory functions in biological events such as proliferation, differentiation, apoptosis, and tumorigenesis. A few recent studies reported on the existence of a reciprocal regulatory loop between the family of let-7 miRNAs and an RNA-binding protein lin28, both of which play important roles during cell differentiation. Using bipotent K562 human leukemia cells and human CD34+ hematopoietic stem cells as research models, we demonstrate that let-7 and lin28 appear to exhibit inverse expression trends in phorbol ester-induced megakaryocyte differentiation, while miR-181 is up-regulated early during this process. Enforced expression of miR-181a in K562 cells effectively represses lin28 expression, breaking up the lin28-let-7 reciprocal regulatory loop, and sequentially triggers megakaryocyte differentiation as measured by CD41 and CD61 induction. Elevated miR-181 also significantly increases the number of megakaryocyte colonies in CD34+ hematopoietic stem cells. However, miR-181 expression levels only slightly change during hemin-induced erythrocyte differentiation. These results suggest that miR-181 can function as a “molecular switch” during hematopoietic lineage differentiation and can be a potential target for future miRNA-oriented therapeutics for human cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3997. doi:10.1158/1538-7445.AM2011-3997