NEFL mRNA expression level is a prognostic factor for early-stage breast cancer patients

Neurofilament, light polypeptide (NEFL) was demonstrated to be ectopically expressed in breast cancer tissues and decreased in lymph node metastases compared to the paired primary breast cancers in our previous study. Moreover, in several studies, NEFL was regarded as a tumor suppressor gene, and its loss of heterozygosity (LOH) was related to carcinogenesis and metastasis in several types of cancer. To explore the role of NEFL in the progression of breast cancer and to evaluate its clinical significance, we detected the NEFL mRNA level in normal breast tissues, primary breast cancer samples and lymph node metastases, and then analyzed the association between the NEFL expression level and several clinicopathological parameters and disease-free survival (DFS). NEFL mRNA was found to be expressed in 92.3% of breast malignancies and down-regulated in lymph node metastases compared to the paired primary tumors. NEFL mRNA level was lower in primary breast cancers with positive lymph nodes than in cancers with negative lymph nodes. Moreover, a low expression level of NEFL mRNA indicated a poor five-year DFS for early-stage breast cancer patients. Thus, NEFL mRNA is ectopically expressed in breast malignancies and could be a potential prognostic factor for early-stage breast cancer patients.     

Neurofilament Heavy Polypeptide Regulates the Akt-β-Catenin Pathway in Human Esophageal Squamous Cell Carcinoma

Aerobic glycolysis and mitochondrial dysfunction are common features of aggressive cancer growth. We observed promoter methylation and loss of expression in neurofilament heavy polypeptide (NEFH) in a significant proportion of primary esophageal squamous cell carcinoma (ESCC) samples that were of a high tumor grade and advanced stage. RNA interference-mediated knockdown of NEFH accelerated ESCC cell growth in culture and increased tumorigenicity in vivo, whereas forced expression of NEFH significantly inhibited cell growth and colony formation. Loss of NEFH caused up-regulation of pyruvate kinase-M2 type and down-regulation of pyruvate dehydrogenase, via activation of the Akt/β-catenin pathway, resulting in enhanced aerobic glycolysis and mitochondrial dysfunction. The acceleration of glycolysis and mitochondrial dysfunction in NEFH-knockdown cells was suppressed in the absence of β-catenin expression, and was decreased by the treatment of 2-Deoxyglucose, a glycolytic inhibitor, or API-2, an Akt inhibitor. Loss of NEFH activates the Akt/β-catenin pathway and increases glycolysis and mitochondrial dysfunction. Cancer cells with methylated NEFH can be targeted for destruction with specific inhibitors of deregulated downstream pathways.     

Role of neurofilament light polypeptide in head and neck cancer chemoresistance

Resistance to cisplatin-based chemotherapy is responsible for therapeutic failure of many common human cancers including cancer of head and neck (HNC). Mechanisms underlying cisplatin resistance remain unclear. In this study, we identified neurofilament light polypeptide (NEFL) as a novel hypermethylated gene associated with resistance to cisplatin-based chemotherapy in HNC. Analysis of 14 HNC cell lines revealed that downregulation of NEFL expression significantly correlated with increased resistance to cisplatin. Hypermethylation of NEFL promoter CpG islands was observed in cell lines as examined by bisulfite DNA sequencing and methylation-specific PCR (MSP) and tightly correlated with reduced NEFL mRNA and protein expression. Furthermore, in patient samples with HNC (n = 51) analyzed by quantitative MSP, NEFL promoter hypermethylation was associated with resistance to cisplatin-based chemotherapy [relative risk (RR), 3.045; 95% confidence interval (CI), 1.459-6.355; P = 0.007] and predicted diminished overall and disease-free survival for patients treated with cisplatin-based chemotherapy. Knockdown of NEFL by siRNA in the highly cisplatin-sensitive cell line PCI13 increased (P < 0.01) resistance to cisplatin. In cisplatin-resistant O11 and SCC25cp cells, restored expression of NEFL significantly increased sensitivity to the drug. Furthermore, NEFL physically associated with tuberous sclerosis complex 1 (TSC1), a known inhibitor of the mTOR pathway, and NEFL downregulation led to functional activation of mTOR pathway and consequentially conferred cisplatin resistance. This is the first study to show a role for NEFL in HNC chemoresistance. Our findings suggest that NEFL methylation is a novel mechanism for HNC chemoresistance and may represent a candidate biomarker predictive of chemotherapeutic response and survival in patients with HNC.     

Functions of TET Proteins in Hematopoietic Transformation

DNA methylation is a well-characterized epigenetic modification that plays central roles in mammalian development, genomic imprinting, X-chromosome inactivation and silencing of retrotransposon elements. Aberrant DNA methylation pattern is a characteristic feature of cancers and associated with abnormal expression of oncogenes, tumor suppressor genes or repair genes. Ten-eleven-translocation (TET) proteins are recently characterized dioxygenases that catalyze progressive oxidation of 5-methylcytosine to produce 5-hydroxymethylcytosine and further oxidized derivatives. These oxidized methylcytosines not only potentiate DNA demethylation but also behave as independent epigenetic modifications per se. The expression or activity of TET proteins and DNA hydroxymethylation are highly dysregulated in a wide range of cancers including hematologic and non-hematologic malignancies, and accumulating evidence points TET proteins as a novel tumor suppressor in cancers. Here we review DNA demethylation-dependent and -independent functions of TET proteins. We also describe diverse TET loss-of-function mutations that are recurrently found in myeloid and lymphoid malignancies and their potential roles in hematopoietic transformation. We discuss consequences of the deficiency of individual Tet genes and potential compensation between different Tet members in mice. Possible mechanisms underlying facilitated oncogenic transformation of TET-deficient hematopoietic cells are also described. Lastly, we address non-mutational mechanisms that lead to suppression or inactivation of TET proteins in cancers. Strategies to restore normal 5mC oxidation status in cancers by targeting TET proteins may provide new avenues to expedite the development of promising anti-cancer agents.     

DNA methylation changes in murine breast adenocarcinomas allow the identification of candidate genes for human breast carcinogenesis

Epigenetic inactivation due to aberrant promoter methylation is a key process in breast tumorigenesis. Murine models for human breast cancer have been established for nearly every important human oncogene or tumor suppressor gene. Mouse-to-human comparative gene expression and cytogenetic profiling have been widely investigated for these models; however, little is known about the conservation of epigenetic alterations during tumorigenesis. To determine if this key process in human breast tumorigenesis is also mirrored in a murine breast cancer model, we mapped cytosine methylation changes in primary adenocarcinomas and paired lung metastases derived from the polyomavirus middle T antigen mouse model. Global changes in methylcytosine levels were observed in all tumors when compared to the normal mammary gland. Aberrant methylation and associated gene silencing was observed for Hoxa7, a gene that is differentially methylated in human breast tumors, and Gata2, a novel candidate gene. Analysis of HOXA7 and GATA2 expression in a bank of human primary tumors confirms that the expression of these genes is also reduced in human breast cancer. In addition, HOXA7 hypermethylation is observed in breast cancer tissues when compared to adjacent tumor-free tissue. Based on these studies, we present a model in which comparative epigenetic techniques can be used to identify novel candidate genes important for human breast tumorigenesis, in both primary and metastatic tumors.     

BRCA1 epigenetic inactivation predicts sensitivity to platinum-based chemotherapy in breast and ovarian cancer

Germline mutations in the BRCA1 or BRCA2 genes are associated with an increased risk of breast and ovarian cancer development. Both genes are involved in DNA repair, and tumors harboring genetic defects in them are thought to be more sensitive to DNA-damaging agents used in chemotherapy. However, as only a minority of breast and ovarian cancer patients carry BRCA1 or BRCA2 mutations, few patients are likely to benefit from these pharmacogenetic biomarkers. Herein, we show that, in cancer cell lines and xenografted tumors, BRCA1 CpG island promoter hypermethylation-associated silencing also predicts enhanced sensitivity to platinum-derived drugs to the same extent as BRCA1 mutations. Most importantly, BRCA1 hypermethylation proves to be a predictor of longer time to relapse and improved overall survival in ovarian cancer patients undergoing chemotherapy with cisplatin.     

Altered Functional Protein Networks in the Prefrontal Cortex and Amygdala of Victims of Suicide

Probing molecular brain mechanisms related to increased suicide risk is an important issue in biological psychiatry research. Gene expression studies on post mortem brains indicate extensive changes prior to a successful suicide attempt; however, proteomic studies are scarce. Thus, we performed a DIGE proteomic analysis of post mortem tissue samples from the prefrontal cortex and amygdala of suicide victims to identify protein changes and biomarker candidates of suicide. Among our matched spots we found 46 and 16 significant differences in the prefrontal cortex and amygdala, respectively; by using the industry standard t test and 1.3 fold change as cut off for significance. Because of the risk of false discoveries (FDR) in these data, we also made FDR adjustment by calculating the q-values for all the t tests performed and by using 0.06 and 0.4 as alpha thresholds we reduced the number of significant spots to 27 and 9 respectively. From these we identified 59 proteins in the cortex and 11 proteins in the amygdala. These proteins are related to biological functions and structures such as metabolism, the redox system, the cytoskeleton, synaptic function, and proteolysis. Thirteen of these proteins (CBR1, DPYSL2, EFHD2, FKBP4, GFAP, GLUL, HSPA8, NEFL, NEFM, PGAM1, PRDX6, SELENBP1 and VIM,) have already been suggested to be biomarkers of psychiatric disorders at protein or genome level. We also pointed out 9 proteins that changed in both the amygdala and the cortex, and from these, GFAP, INA, NEFL, NEFM and TUBA1 are interacting cytoskeletal proteins that have a functional connection to glutamate, GABA, and serotonin receptors. Moreover, ACTB, CTSD and GFAP displayed opposite changes in the two examined brain structures that might be a suitable characteristic for brain imaging studies. The opposite changes of ACTB, CTSD and GFAP in the two brain structures were validated by western blot analysis.     

Hierarchical Clustering of Breast Cancer Methylomes Revealed Differentially Methylated and Expressed Breast Cancer Genes

Oncogenic transformation of normal cells often involves epigenetic alterations, including histone modification and DNA methylation. We conducted whole-genome bisulfite sequencing to determine the DNA methylomes of normal breast, fibroadenoma, invasive ductal carcinomas and MCF7. The emergence, disappearance, expansion and contraction of kilobase-sized hypomethylated regions (HMRs) and the hypomethylation of the megabase-sized partially methylated domains (PMDs) are the major forms of methylation changes observed in breast tumor samples. Hierarchical clustering of HMR revealed tumor-specific hypermethylated clusters and differential methylated enhancers specific to normal or breast cancer cell lines. Joint analysis of gene expression and DNA methylation data of normal breast and breast cancer cells identified differentially methylated and expressed genes associated with breast and/or ovarian cancers in cancer-specific HMR clusters. Furthermore, aberrant patterns of X-chromosome inactivation (XCI) was found in breast cancer cell lines as well as breast tumor samples in the TCGA BRCA (breast invasive carcinoma) dataset. They were characterized with differentially hypermethylated XIST promoter, reduced expression of XIST, and over-expression of hypomethylated X-linked genes. High expressions of these genes were significantly associated with lower survival rates in breast cancer patients. Comprehensive analysis of the normal and breast tumor methylomes suggests selective targeting of DNA methylation changes during breast cancer progression. The weak causal relationship between DNA methylation and gene expression observed in this study is evident of more complex role of DNA methylation in the regulation of gene expression in human epigenetics that deserves further investigation.     

Genome-wide DNA methylation indicates silencing of tumor suppressor genes in uterine leiomyoma

Background

Uterine leiomyomas, or fibroids, represent the most common benign tumor of the female reproductive tract. Fibroids become symptomatic in 30% of all women and up to 70% of African American women of reproductive age. Epigenetic dysregulation of individual genes has been demonstrated in leiomyoma cells; however, the in vivo genome-wide distribution of such epigenetic abnormalities remains unknown.

Principal Findings

We characterized and compared genome-wide DNA methylation and mRNA expression profiles in uterine leiomyoma and matched adjacent normal myometrial tissues from 18 African American women. We found 55 genes with differential promoter methylation and concominant differences in mRNA expression in uterine leiomyoma versus normal myometrium. Eighty percent of the identified genes showed an inverse relationship between DNA methylation status and mRNA expression in uterine leiomyoma tissues, and the majority of genes (62%) displayed hypermethylation associated with gene silencing. We selected three genes, the known tumor suppressors KLF11, DLEC1, and KRT19 and verified promoter hypermethylation, mRNA repression and protein expression using bisulfite sequencing, real-time PCR and western blot. Incubation of primary leiomyoma smooth muscle cells with a DNA methyltransferase inhibitor restored KLF11, DLEC1 and KRT19 mRNA levels.

Conclusions

These results suggest a possible functional role of promoter DNA methylation-mediated gene silencing in the pathogenesis of uterine leiomyoma in African American women.     

Array-Based Approaches for the Identification of Epigenetic Silenced Tumor Suppressor Genes

Carcinogenesis involves the inactivation or inhibition of genes that function as tumor suppressors. Deletions, mutations, or epigenetic silencing of tumor suppressor genes can lead to altered growth, differentiation, and apoptosis. DNA methylation and histone modifications are important epigenetic mechanisms of gene regulation and play essential roles both independently and cooperatively in tumor initiation and progression. Realization that many tumor suppressor genes are silenced by epigenetic mechanisms has stimulated discovery of novel tumor suppressor genes. One of the most useful of these approaches is an epigenetic reactivation screening strategy that combines treatment of cancer cells in vitro with DNA methyltransferase and/or histone deacetylase (HDAC) inhibitors, followed by global gene expression analysis using microarrays, to identify upregulated genes. This approach is most effective when complemented by microarray analyses to identify genes repressed in primary tumors. Recently, using cancer cell lines treated with a DNA methylation inhibitor and/or a HDAC inhibitor in conjunction with cDNA microarray analysis, candidate tumor suppressor genes, which are subject to epigenetic silencing, have been identified in endometrial, colorectal, esophageal, and pancreatic cancers. An increasing number of studies have utilized epigenetic reactivation screening to discover novel tumor suppressor genes in cancer. The results of some of the most recent studies are highlighted in this review.     

Targeting of >1.5 Mb of Human DNA into the Mouse X Chromosome Reveals Presence of cis-Acting Regulators of Epigenetic Silencing

Regulatory sequences can influence the expression of flanking genes over long distances, and X chromosome inactivation is a classic example of cis-acting epigenetic gene regulation. Knock-ins directed to the Mus musculus Hprt locus offer a unique opportunity to analyze the spread of silencing into different human DNA sequences in the identical genomic environment. X chromosome inactivation of four knock-in constructs, including bacterial artificial chromosome (BAC) integrations of over 195 kb, was demonstrated by both the lack of expression from the inactive X chromosome in females with nonrandom X chromosome inactivation and promoter DNA methylation of the human transgene in females. We further utilized promoter DNA methylation to assess the inactivation status of 74 human reporter constructs comprising >1.5 Mb of DNA. Of the 47 genes examined, only the PHB gene showed female DNA hypomethylation approaching the level seen in males, and escape from X chromosome inactivation was verified by demonstration of expression from the inactive X chromosome. Integration of PHB resulted in lower DNA methylation of the flanking HPRT promoter in females, suggesting the action of a dominant cis-acting escape element. Female-specific DNA hypermethylation of CpG islands not associated with promoters implies a widespread imposition of DNA methylation during X chromosome inactivation; yet transgenes demonstrated differential capacities to accumulate DNA methylation when integrated into the identical location on the inactive X chromosome, suggesting additional cis-acting sequence effects. As only one of the human transgenes analyzed escaped X chromosome inactivation, we conclude that elements permitting ongoing expression from the inactive X are rare in the human genome.     

NEFLb impairs early nervous system development via regulation of neuron apoptosis in zebrafish

Neurofilament light chain (NEFL), a subunit of neurofilament, has been shown to play an important role in pathogenic neurodegenerative disease and in radial axonal growth. However, information remains largely lacking regarding the function of NEFL in early development to date. In this study, we demonstrated the presence of two nefl genes, nefla and neflb, in zebrafish, generated by fish-specific third round genome duplication. These duplicated nefl genes were predominantly expressed in the nervous system with an overlapping and distinct expression pattern. Both gene knockdown and rescue experiments show that it was neflb rather than nefla that played an indispensable role in nervous system development. It was also found that neflb knockdown resulted in striking apoptosis of the neurons in the brain and spinal cord, leading to morphological defects such as brain structure disorder and trunk bending. Thus, we report a previously uncharacterized role of NEFL that NEFLb impairs the early development of zebrafish nervous system via regulation of the neuron apoptosis in the brain and spinal cord.     

Homologous recombination is reduced in female embryonic stem cells by two active X chromosomes

The reactivation of X‐linked genes is observed in some primary breast tumors. Two active X chromosomes are also observed in female embryonic stem cells (ESCs), but whether double doses of X‐linked genes affect DNA repair efficiency remains unclear. Here, we establish isogenic female/male ESCs and show that the female ESCs are more sensitive to camptothecin and have lower gene targeting efficiency than male ESCs, suggesting that homologous recombination (HR) efficiency is reduced in female ESCs. We also generate Xist‐inducible female ESCs and show that the lower HR efficiency is restored when X chromosome inactivation is induced. Finally, we assess the X‐linked genes with a role in DNA repair and find that Brcc3 is one of the genes involved in a network promoting proper HR. Our findings link the double doses of X‐linked genes with lower DNA repair activity, and this may have relevance for common diseases in female patients, such as breast cancer.