Hoi-Hung Cheung

Methylation of an intronic region regulates miR-199a in testicular tumor malignancy.

In the testicular cancer cell line, NT2, we previously demonstrated that differentially methylated regions were located in introns or intergenic regions, and postulated these might regulate non-coding RNAs. Three microRNAs and three small nucleolar RNAs were differentially methylated; one, miR-199a, was associated with the progression and prognosis of gastric and ovarian cancers. In this report we document, by epigenomic profiling of testicular tissue, that miR-199a is transcribed as antisense of dynamin 3 (chromosome 1q24.3), and hypermethylation of this region is correlated with miR-199a-5p/3p repression and tumor malignancy. Re-expression of miR-199a in testicular cancer cells led to suppression of cell growth, cancer migration, invasion and metastasis. The miR-199a-5p, one of two mature miRNA species derived from miR-199a, is associated with tumor malignancy. We further identified the embryonal carcinoma antigen podocalyxin-like protein 1 (PODXL), an anti-adhesive protein expressed in aggressive tumors, as a target of miR-199a-5p. We demonstrated PODXL is overexpressed in malignant testicular tumor, and cellular depletion of PODXL resulted in suppression of cancer invasion. The inverse relationship between PODXL and miR-199a-5p expression suggests PODXL is a downstream effector mediating the action of miR199a-5p. This report identifies DNA methylation, miR-199a dysregulation and PODXL as critical factors in tumor malignancy.

Genome-wide DNA methylation profiling reveals novel epigenetically regulated genes and non-coding RNAs in human testicular cancer

Background:Testicular germ cell tumour (TGCT) is the most common malignant tumour in young males. Although aberrant DNA methylation is implicated in the pathophysiology of many cancers, only a limited number of genes are known to be epigenetically changed in TGCT. This report documents the genome-wide analysis of differential methylation in an in vitro model culture system. Interesting genes were validated in TGCT patient samples.Methods:In this study, we used methylated DNA immunoprecipitation (MeDIP) and whole-genome tiling arrays to identify differentially methylated regions (DMRs).Results:We identified 35 208 DMRs. However, only a small number of DMRs mapped to promoters. A genome-wide analysis of gene expression revealed a group of differentially expressed genes that were regulated by DNA methylation. We identified several candidate genes, including APOLD1, PCDH10 and RGAG1, which were dysregulated in TGCT patient samples. Surprisingly, APOLD1 had previously been mapped to the TGCT susceptibility locus at 12p13.1, suggesting that it may be important in TGCT pathogenesis. We also observed aberrant methylation in the loci of some non-coding RNAs (ncRNAs). One of the ncRNAs, hsa-mir-199a, was downregulated in TGCT patient samples, and also in our in vitro model culture system.Conclusion:This report is the first application of MeDIP-chip for identifying epigenetically regulated genes and ncRNAs in TGCT. We also demonstrated the function of intergenic and intronic DMRs in the regulation of ncRNAs.

DNA methylation of cancer genome

DNA methylation plays an important role in regulating normal development and carcinogenesis. Current understanding of the biological roles of DNA methylation is limited to its role in the regulation of gene transcription, genomic imprinting, genomic stability, and X chromosome inactivation. In the past 2 decades, a large number of changes have been identified in cancer epigenomes when compared with normals. These alterations fall into two main categories, namely, hypermethylation of tumor suppressor genes and hypomethylation of oncogenes or heterochromatin, respectively. Aberrant methylation of genes controlling the cell cycle, proliferation, apoptosis, metastasis, drug resistance, and intracellular signaling has been identified in multiple cancer types. Recent advancements in whole-genome analysis of methylome have yielded numerous differentially methylated regions, the functions of which are largely unknown. With the development of high resolution tiling microarrays and high throughput DNA sequencing, more cancer methylomes will be profiled, facilitating the identification of new candidate genes or ncRNAs that are related to oncogenesis, new prognostic markers, and the discovery of new target genes for cancer therapy.

Telomerase Protects Werner Syndrome Lineage-Specific Stem Cells from Premature Aging

Summary Werner syndrome (WS) patients exhibit premature aging predominantly in mesenchyme-derived tissues, but not in neural lineages, a consequence of telomere dysfunction and accelerated senescence. The cause of this lineage-specific aging remains unknown. Here, we document that reprogramming of WS fibroblasts to pluripotency elongated telomere length and prevented telomere dysfunction. To obtain mechanistic insight into the origin of tissue-specific aging, we differentiated iPSCs to mesenchymal stem cells (MSCs) and neural stem/progenitor cells (NPCs). We observed recurrence of premature senescence associated with accelerated telomere attrition and defective synthesis of the lagging strand telomeres in MSCs, but not in NPCs. We postulate this “aging” discrepancy is regulated by telomerase. Expression of hTERT or p53 knockdown ameliorated the accelerated aging phenotypein MSC, whereas inhibition of telomerase sensitized NPCs to DNA damage. Our findings unveil a role for telomerase in the protection of accelerated aging in a specific lineage of stem cells.

Apoptosis: Reprogramming and the Fate of Mature Cells

Apoptosis is essential for embryogenesis, organ metamorphosis, and tissue homeostasis. In embryonic stem cells, self-renewal is balanced with proliferative potential, inhibition of differentiation, and prevention of senescence and apoptosis. Growing evidence supports the role of apoptosis in self-renewal, differentiation of pluripotent stem cells, and dedifferentiation (reprogramming) of somatic cells. In this paper we discuss the multiple roles of apoptosis in embryonic stem cells (ESCs) and reprogramming of differentiated cells to pluripotency. The role of caspases and p53 as key effectors in controlling the generation of iPSC is emphasized. Remarkably, the complication of apoptosis arising during reprogramming may provide insights into technical improvements for derivation of iPSC from senescent cells as a tool for modeling aging-related diseases.

GermSAGE: a comprehensive SAGE database for transcript discovery on male germ cell development

GermSAGE is a comprehensive web-based database generated by Serial Analysis of Gene Expression (SAGE) representing major stages in mouse male germ cell development, with 150 000 sequence tags in each SAGE library. A total of 452 095 tags derived from type A spermatogonia (Spga), pachytene spermatocytes (Spcy) and round spermatids (Sptd) were included. GermSAGE provides web-based tools for browsing, comparing and searching male germ cell transcriptome data at different stages with customizable searching parameters. The data can be visualized in a tabulated format or further analyzed by aligning with various annotations available in the UCSC genome browser. This flexible platform will be useful for gaining better understanding of the genetic networks that regulate spermatogonial cell renewal and differentiation, and will allow novel gene discovery. GermSAGE is freely available at http://germsage.nichd.nih.gov/

Oncogene AF1q enhances doxorubicin-induced apoptosis through BAD-mediated mitochondrial apoptotic pathway.

AF1q is an oncogenic factor involved in leukemia development, thyroid tumorigenesis, and breast cancer metastasis. In the present study, AF1q was found to be down-regulated in a doxorubicin-resistant subline of human squamous carcinoma A431 cells. Knockdown of AF1q decreased the apoptosis induced by doxorubicin, Taxol, γ-radiation, IFN-α, and IFN-γ in A431 cells. On the other hand, overexpression of AF1q increased the doxorubicin-induced apoptosis in A431 cells as well as in HepG2 and HL60 cells. Both exogenous and ectopic expression of AF1q in A431 cells increased the mRNA and protein levels of BAD, a proapoptotic BCL-2 family protein. Gene silencing of BAD by small interfering RNA suppressed the AF1q enhancement of apoptosis, suggesting that BAD is downstream of AF1q in regulation of apoptosis. Furthermore, AF1q enhanced the mitochondrial membrane depolarization, mitochondrial cytochrome c release, and activation of caspase-9 and caspase-3 on doxorubicin treatment. Collectively, AF1q increases doxorubicin-induced apoptosis in cells through activation of BAD-mediated apoptotic pathway. The study provides the first evidence that AF1q plays a critical role in the regulation of apoptosis and drug resistance. [Mol Cancer Ther 2008;7(10):3160–8]

Molecular mechanisms of regulation and action of microRNA-199a in testicular germ cell tumor and glioblastomas

MicroRNA-199a (miRNA-199a) has been shown to have comprehensive functions and behave differently in different systems and diseases. It is encoded by two loci in the human genome, miR-199a-1 in chromosome 19 and miR-199a-2 in chromosome 1. Both loci give rise to the same miRNAs (miR-199a-5p and miR-199a-3p). The cause of the diverse action of the miRNA in different systems is not clear. However, it is likely due to different regulation of the two genomic loci and variable targets of the miRNA in different cells and tissues. Here we studied promoter methylation of miR-199a in testicular germ cell tumors (TGCTs) and glioblastomas (gliomas) and discovered that hypermethylation in TGCTs of both miR-199a-1 and -2 resulted in its reduced expression, while hypomethylation of miR-199a-2 but not -1 in gliomas may be related to its elevated expression. We also identified a common regulator, REST, which preferentially bound to the methylated promoters of both miR-199a-1 and miR-199a-2. The action of miR-199a is dependent on its downstream targets. We identified MAFB as a putative target of miRNA-199a-5p in TGCTs and confirmed that the tumor suppression activity of the microRNA is mediated by its target MAFB. By studying the mechanisms that control the expressions of miR-199a and its various downstream targets, we hope to use miR-199a as a model to understand the complexity of miRNA biology.

Hypermethylation of genes in testicular embryonal carcinomas

Background:Testicular embryonal carcinoma (EC) is a major subtype of non-seminomatous germ cell tumours in males. Embryonal carcinomas are pluripotent, undifferentiated germ cell tumours believed to originate from primordial germ cells. Epigenetic changes during testicular EC tumorigenesis require better elucidation.Methods:To identify epigenetic changes during testicular neoplastic transformation, we profiled DNA methylation of six ECs. These samples represent different stages (stage I and stage III) of divergent invasiveness. Non-cancerous testicular tissues were included. Expression of a number of hypermethylated genes were examined by quantitative RT-PCR and immunohistochemistry (IHC).Results:A total of 1167 tumour-hypermethylated differentially methylated regions (DMRs) were identified across the genome. Among them, 40 genes/ncRNAs were found to have hypermethylated promoters. Quantitative RT-PCR confirmed downregulation of 8 out of 9 of the genes. Among the confirmed genes, five were sex-linked genes, including X-linked genes STAG2, SPANXD/E and MIR1184, and Y-linked genes RBMY1A1/1B/1D and FAM197Y2P. RBMY1A is a testis-specific gene for spermatogenesis. RNF168 and USP13 are potential tumour suppressors. Expression of RBMY1A was lost in EC and seminoma as documented in the Protein Atlas. We confirmed downregulation of USP13 in EC by IHC.Conclusions:Our genome-wide analysis of testicular EC identified methylation changes in several previously unknown genes. This may provide insight of crosstalk between normal germ cell development and carcinogenesis.

Idiopathic Autism: Cellular and Molecular Phenotypes in Pluripotent Stem Cell-Derived Neurons

Autism spectrum disorder is a complex neurodevelopmental disorder whose pathophysiology remains elusive as a consequence of the unavailability for study of patient brain neurons; this deficit may potentially be circumvented by neural differentiation of induced pluripotent stem cells. Rare syndromes with single gene mutations and autistic symptoms have significantly advanced the molecular and cellular understanding of autism spectrum disorders; however, in aggregate, they only represent a fraction of all cases of autism. In an effort to define the cellular and molecular phenotypes in human neurons of non-syndromic autism, we generated induced pluripotent stem cells (iPSCs) from three male autism spectrum disorder patients who had no identifiable clinical syndromes, and their unaffected male siblings and subsequently differentiated these patient-specific stem cells into electrophysiologically active neurons. iPSC-derived neurons from these autistic patients displayed decreases in the frequency and kinetics of spontaneous excitatory postsynaptic currents relative to controls, as well as significant decreases in Na+ and inactivating K+ voltage-gated currents. Moreover, whole-genome microarray analysis of gene expression identified 161 unique genes that were significantly differentially expressed in autistic patient iPSC-derived neurons (>twofold, FDR < 0.05). These genes were significantly enriched for processes related to synaptic transmission, such as neuroactive ligand-receptor signaling and extracellular matrix interactions, and were enriched for genes previously associated with autism spectrum disorder. Our data demonstrate aberrant voltage-gated currents and underlying molecular changes related to synaptic function in iPSC-derived neurons from individuals with idiopathic autism as compared to unaffected siblings controls.