Breast carcinogenesis is a multistep process involving both genetic and epigenetic changes. Since epigenetic changes like histone modifications are potentially reversible processes, much effort has been directed toward understanding this mechanism with the goal of finding novel therapies as well as more refined diagnostic and prognostic tools in breast cancer. Lysine-specific demethylase 1 (LSD1) plays a key role in the regulation of gene expression by removing the methyl groups from methylated lysine 4 of histone H3 and lysine 9 of histone H3. LSD1 is essential for mammalian development and involved in many biological processes. Considering recent evidence that LSD1 is involved in carcinogenesis, we investigated the role of LSD1 in breast cancer. Therefore, we developed an enzyme-linked immunosorbent assay to determine LSD1 protein levels in tissue specimens of breast cancer and measured very high LSD1 levels in estrogen receptor (ER)-negative tumors. Pharmacological LSD1 inhibition resulted in growth inhibition of breast cancer cells. Knockdown of LSD1 using small interfering RNA approach induced regulation of several proliferation-associated genes like p21, ERBB2 and CCNA2. Additionally, we found that LSD1 is recruited to the promoters of these genes. In summary, our data indicate that LSD1 may provide a predictive marker for aggressive biology and a novel attractive therapeutic target for treatment of ER-negative breast cancers.

This is the first study that reports the long-term outcome of ARPKD patients with defined PKHD1 mutations. The 1- and 10-year survival rates were 85% and 82%, respectively. Chronic renal failure was first detected at a mean age of 4 years. Actuarial renal survival rates [end point defined as start of dialysis/renal transplantation (RTX) or by death due to end-stage renal disease (ESRD)] were 86% at 5 years, 71% at 10 years, and 42% at 20 years. All but six patients (92%) had a kidney length above or on the 97th centile for age. About 75% of the study population developed systemic hypertension. Sequelae of congenital hepatic fibrosis and portal hypertension developed in 44% of patients and were related with age. Positive correlations could further be demonstrated between renal and hepatobiliary-related morbidity suggesting uniform disease progression rather than organ-specific patterns. PKHD1 mutation analysis revealed 193 mutations (70 novel ones; 77% nonconservative missense mutations). No patient carried two truncating mutations corroborating that one missense mutation is indispensable for survival of newborns. We attempted to set up genotype-phenotype correlations and to categorize missense mutations. In 96% of families we identified at least one mutated PKHD1 allele (overall detection rate 76.6%) indicating that PKHD1 mutation screening is a powerful diagnostic tool in patients suspected with ARPKD.

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.

Aberrant epigenetic changes in DNA methylation and histone acetylation are hallmarks of most cancers, whereas histone methylation was previously considered to be irreversible and less versatile. Recently, several histone demethylases were identified catalyzing the removal of methyl groups from histone H3 lysine residues and thereby influencing gene expression. Neuroblastomas continue to remain a clinical challenge despite advances in multimodal therapy. Here, we address the functional significance of the chromatin-modifying enzyme lysine-specific demethylase 1 (LSD1) in neuroblastoma. LSD1 expression correlated with adverse outcome and was inversely correlated with differentiation in neuroblastic tumors. Differentiation of neuroblastoma cells resulted in down-regulation of LSD1. Small interfering RNA-mediated knockdown of LSD1 decreased cellular growth, induced expression of differentiation-associated genes, and increased target gene-specific H3K4 methylation. Moreover, LSD1 inhibition using monoamine oxidase inhibitors resulted in an increase of global H3K4 methylation and growth inhibition of neuroblastoma cells in vitro. Finally, targeting LSD1 reduced neuroblastoma xenograft growth in vivo. Here, we provide the first evidence that a histone demethylase, LSD1, is involved in maintaining the undifferentiated, malignant phenotype of neuroblastoma cells. We show that inhibition of LSD1 reprograms the transcriptome of neuroblastoma cells and inhibits neuroblastoma xenograft growth. Our results suggest that targeting histone demethylases may provide a novel option for cancer therapy.

Charcot-Marie-Tooth disease type 4C (CMT4C) is a childhood-onset demyelinating form of hereditary motor and sensory neuropathy associated with an early-onset scoliosis and a distinct Schwann cell pathology. CMT4C is inherited as an autosomal recessive trait and has been mapped to a 13-cM linkage interval on chromosome 5q23-q33. By homozygosity mapping and allele-sharing analysis, we refined the CMT4C locus to a suggestive critical region of 1.7 Mb. We subsequently identified mutations in an uncharacterized transcript, KIAA1985, in 12 families with autosomal recessive neuropathy. We observed eight distinct protein-truncating mutations and three nonconservative missense mutations affecting amino acids conserved through evolution. In all families, we identified a mutation on each disease allele, either in the homozygous or in the compound heterozygous state. The CMT4C gene is strongly expressed in neural tissues, including peripheral nerve tissue. The translated protein defines a new protein family of unknown function with putative orthologues in vertebrates. Comparative sequence alignments indicate that members of this protein family contain multiple SH3 and TPR domains that are likely involved in the formation of protein complexes.