Prostate cancer biology varies from locally confined tumors with low risk for relapse to tumors with high risk for progression even after radical prostatectomy. Currently, there are no reliable biomarkers to predict tumor relapse and poor clinical outcome. In this study, we correlated expression patterns of the androgen receptor (AR) coactivators lysinespecific histone demethylase 1 (LSD1) and four and a half LIM-domain protein 2 (FHL2), AR, Gleason score, Gleason grade, and p53 expression in clinically organ confined prostate cancers with relapse after radical prostatectomy. Our data reveal that high levels of LSD1, nuclear expression of the FHL2 coactivator, high Gleason score and grade, and very strong staining of nuclear p53 correlate significantly with relapse during follow-up. No correlation exists with relapse and the expression of AR and cytoplasmic expression of FHL2. To confirm these data, we did quantitative reverse transcription-PCR and Western blot analyses in a subset of tumor specimens. Consistently, both LSD1 mRNA and protein levels were significantly up-regulated in high-risk tumors. We previously identified LSD1 and FHL2 as nuclear cofactors interacting specifically with the AR in prostate cells and showed that both stimulate androgen-dependent gene transcription. Our present study suggests that LSD1 and nuclear FHL2 may serve as novel biomarkers predictive for prostate cancer with aggressive biology and point to a role of LSD1 and FHL2 in constitutive activation of AR-mediated growth signals. (Cancer Res 2006; 66(23): 11341-7)

Demethylation at distinct lysine residues in histone H3 by lysine-specific demethylase 1 (LSD1) causes either gene repression or activation. As a component of co-repressor complexes, LSD1 contributes to target gene repression by removing mono- and dimethyl marks from lysine 4 of histone H3 (H3K4). In contrast, during androgen receptor (AR)-activated gene expression, LSD1 removes mono- and dimethyl marks from lysine 9 of histone H3 (H3K9). Yet, the mechanisms that control this dual specificity of demethylation are unknown. Here we show that phosphorylation of histone H3 at threonine 6 (H3T6) by protein kinase C beta I (PKCbeta(I), also known as PRKCbeta) is the key event that prevents LSD1 from demethylating H3K4 during AR-dependent gene activation. In vitro, histone H3 peptides methylated at lysine 4 and phosphorylated at threonine 6 are no longer LSD1 substrates. In vivo, PKCbeta(I) co-localizes with AR and LSD1 on target gene promoters and phosphorylates H3T6 after androgen-induced gene expression. RNA interference (RNAi)-mediated knockdown of PKCbeta(I) abrogates H3T6 phosphorylation, enhances demethylation at H3K4, and inhibits AR-dependent transcription. Activation of PKCbeta(I) requires androgen-dependent recruitment of the gatekeeper kinase protein kinase C (PKC)-related kinase 1 (PRK1). Notably, increased levels of PKCbeta(I) and phosphorylated H3T6 (H3T6ph) positively correlate with high Gleason scores of prostate carcinomas, and inhibition of PKCbeta(I) blocks AR-induced tumour cell proliferation in vitro and cancer progression of tumour xenografts in vivo. Together, our data establish that androgen-dependent kinase signalling leads to the writing of the new chromatin mark H3T6ph, which in consequence prevents removal of active methyl marks from H3K4 during AR-stimulated gene expression.

Resistance to chemotherapy is a major complication during treatment of cancer patients. Hypermethylation of the MGMT gene alters DNA repair and is associated with longer survival of glioblastoma patients treated with alkylating agents. Therefore, MGMT promoter methylation plays an important role as a predictive biomarker for chemotherapy resistance. To adopt this established correlation into a molecular diagnosis procedure, we compared and optimized three experimental techniques [combined bisulfite restriction analysis, a primer extension-and denaturing high-performance liquid chromatography-based method named SIRPH (SNuPE ion pairreverse phase high-performance liquid chromatography), and pyrosequencing] with regard to their accuracy of detecting MGMT promoter methylation. Initially, bisulfite sequencing was used to obtain a comprehensive methylation profile of the MGMT promoter region in 22 glioblastoma samples and in three normal brain controls. Next, we statistically identified CpG sites that best discriminate between methylated and unmethylated MGMT promoters. These results were then used to design optimal combined bisulfite restriction analysis, SIRPH, and pyrosequencing assays for accurate and cost-efficient assessment of MGMT promoter methylation. We compared all three techniques with regard to their reliability and reproducibility on well-characterized tumor samples. The optimized pyrosequencing assay performed best and provides a sensitive, robust, and easy-to-use method for quantitative assessment of MGMT methylation, for both snap-frozen and paraffin-embedded specimens. (J Mol Diagn 2007, 9:368 -381;

Recently, we identified 3' end deletions in the EPCAM gene as a novel cause of Lynch syndrome. These truncating EPCAM deletions cause allele-specific epigenetic silencing of the neighboring DNA mismatch repair gene MSH2 in tissues expressing EPCAM. Here we screened a cohort of unexplained Lynch-like families for the presence of EPCAM deletions. We identified 27 novel independent MSH2-deficient families from multiple geographical origins with varying deletions all encompassing the 3' end of EPCAM, but leaving the MSH2 gene intact. Within The Netherlands and Germany, EPCAM deletions appeared to represent at least 2.8% and 1.1% of the confirmed Lynch syndrome families, respectively. MSH2 promoter methylation was observed in epithelial tissues of all deletion carriers tested, thus confirming silencing of MSH2 as the causative defect. In a total of 45 families, 19 different deletions were found, all including the last two exons and the transcription termination signal of EPCAM. All deletions appeared to originate from Alu-repeat mediated recombination events. In 17 cases regions of microhomology around the breakpoints were found, suggesting nonallelic homologous recombination as the most likely mechanism. We conclude that 3' end EPCAM deletions are a recurrent cause of Lynch syndrome, which should be implemented in routine Lynch syndrome diagnostics.

Clinical classification of AK lesions using the system of Olsen does not accurately match histological classification of the same lesions using the system of Roewert-Huber. Consequently, it is not possible to draw conclusions about the histology of AK lesions from their clinical appearance. This finding reinforces the need to treat all AK lesions as well as field cancerization.

Global histone modification levels may help to identify patients with adverse prognosis, and represent a target for the future therapy of PCA.

No abstract

Epigenetic alterations play an important role in carcinogenesis. Recent studies suggested that global histone modifications are predictors of cancer recurrence in various tumor entities. Our study was performed to evaluate histone H3 lysine 4 monomethyl (H3K4me1), -di-methyl (H3K4me2) and -trimethyl (H3K4me3) patterns in renal cell carcinoma (RCC) using a tissue microarray with 193 RCC (including 142 clear cell, 31 papillary, 10 chromophobe and 10 sarcomatoid RCC) and 10 oncocytoma specimens: H3K4me3 staining was more intense in papillary RCC, whereas H3K4me1 and H3K4me2 were similar in the diverse RCC subtypes. H3K4me2 and H3K4me3 levels were increased in oncocytoma. H3K4me1-3 levels were inversely correlated with Fuhrman grading, pT stage, lymph node involvement and distant metastasis. Progression-free survival and cancer-specific survival were shorter in patients with low levels of H3K4me1-3 in the univariate analysis, but we did not observe a significant correlation of a single modification in a multivariate model, which also included the established prognostic parameters TNM-stage and Fuhrman grade. In comparison, the H3K4me score, which combined staining levels of the H3K4 modifications, was an independent predictor of RCC progression-free survival. Our study on H3K4 methylation supports the concept of global histone modifications as potential cancer prognosis markers.In 2009, 57,760 new cases and 12,980 deaths of kidney cancer were estimated in the United States. 1 Renal cell carcinoma (RCC) is a heterogeneous group of histological subtypes, of which clear cell RCC (ccRCC) is the most common, comprising more than 85% of all cases; papillary (pRCC, 10%), chromophobe (chRCC, 3%) and sarcomatoid renal cell carcinoma (sRCC) are less common. 2 The clinical outcome of patients with RCC is mainly depending on TNM stage, Fuhrman grade and the Eastern Cooperative Oncology Group (ECOG) performance status. 2 However, RCC patients with similar tumor characteristics still show heterogeneity in the course and outcome of cancer. Therefore, a subclassification of patients with similar clinical and pathological variables is necessary, and may also allow the development of novel therapies.Epigenetic alterations are common in solid tumors, and they lead to silencing of various tumor suppressor genes. 3 DNA methylation has been widely studied, and tumor suppressor gene CpG island hypermethylation was identified as an important step during renal cell carcinogenesis. 4 Histone modifications are also important regulators of transcriptional activity; so far, they have been less comprehensively studied in RCC despite the fact that changes in DNA methylation are closely related to histone alterations. 5 Post-translational modifications (i.e., acetylation, methylation, phosphorylation, ubiquitination, sumoylation, ADP-ribosylation and deimination) occur on the N-terminal tail of the histone. 6 The modifications function either by disrupting chromatin contacts or by affecting the recruitment of various proteins to the chromatin, and...