Chung-Hsing Chen

FLJ10540-elicited cell transformation is through the activation of PI3-kinase/AKT pathway.

A significant challenge in the post-genomic era is how to prioritize differentially expressed and uncharacterized novel genes found in hepatocellular carcinoma (HCC) microarray profiling. One such category is cell cycle regulated genes that have only evolved in higher organisms but not in lower eukaryotic cells. Characterization of these genes may reveal some novel human cancer-specific abnormalities. A novel transcript, FLJ10540 was identified. FLJ10540 is overexpressed in HCC as examined by quantitative reverse transcription–polymerase chain reaction and immunohistochemistry. The patients with higher FLJ10540 expression had a poor survival than those with lower FLJ10540 expression. Functional characterization indicates that FLJ10540 displays a number of characteristics associated with an oncogene, including anchorage-independent growth, enhanced cell growth at low serum levels and induction of tumorigenesis in nude mice. FLJ10540-elicited cell transformation is mediated by activation of the phosphatidylinositol 3′-kinase (PI3K)/AKT pathway. Moreover, FLJ10540 forms a complex with PI3K and can activate PI3K activity, which provides a mechanistic basis for FLJ10540-mediated oncogenesis. Together, using a combination of bioinformatics searches and empirical data, we have identified a novel oncogene, FLJ10540, which is conserved only in higher organisms. The finding raises the possibility that FLJ10540 is a potential new therapeutic target for HCC treatment. These findings may contribute to the development of new therapeutic strategies that are able to block the PI3K/AKT pathway in cancer cells.

Disease-related growth factor and embryonic signaling pathways modulate an enhancer of TCF21 expression at the 6q23.2 coronary heart disease locus

Coronary heart disease (CHD) is the leading cause of mortality in both developed and developing countries worldwide. Genome-wide association studies (GWAS) have now identified 46 independent susceptibility loci for CHD, however, the biological and disease-relevant mechanisms for these associations remain elusive. The large-scale meta-analysis of GWAS recently identified in Caucasians a CHD-associated locus at chromosome 6q23.2, a region containing the transcription factor TCF21 gene. TCF21 (Capsulin/Pod1/Epicardin) is a member of the basic-helix-loop-helix (bHLH) transcription factor family, and regulates cell fate decisions and differentiation in the developing coronary vasculature. Herein, we characterize a cis-regulatory mechanism by which the lead polymorphism rs12190287 disrupts an atypical activator protein 1 (AP-1) element, as demonstrated by allele-specific transcriptional regulation, transcription factor binding, and chromatin organization, leading to altered TCF21 expression. Further, this element is shown to mediate signaling through platelet-derived growth factor receptor beta (PDGFR-β) and Wilms tumor 1 (WT1) pathways. A second disease allele identified in East Asians also appears to disrupt an AP-1-like element. Thus, both disease-related growth factor and embryonic signaling pathways may regulate CHD risk through two independent alleles at TCF21.

Downregulation of a putative tumor suppressor BMP4 by SOX2 promotes growth of lung squamous cell carcinoma

SOX2 is a transcription factor essential for self‐renewal and pluripotency of embryonic stem cells. Recently, SOX2 was found overexpressed in the majority of the lung squamous cell carcinoma (SQC), in which it acts as a lineage‐survival oncogene. However, downstream targets/pathways of SOX2 in lung SQC cells remain to be identified. Here, we show that BMP4 is a downstream target of SOX2 in lung SQC. We found that SOX2‐silencing‐mediated inhibition of cell growth was accompanied by upregulation of BMP4 mRNA and its protein expression. Meta‐analysis with 293 samples and qRT‐PCR validation with 73 clinical samples revealed an inversely correlated relationship between levels of SOX2 and BMP4 mRNA, and significantly lower mRNA levels in tumor than in adjacent normal tissues. This was corroborated by immunohistochemistry analysis of 35 lung SQC samples showing lower BMP4 protein expression in tumor tissues. Cell‐based experiments including siRNA transfection, growth assay and flow cytometry assay, further combined with a xenograft tumor model in mice, revealed that reactivation of BMP4 signaling could partially account for growth inhibition and cell cycle arrest in lung SQC cells upon silencing SOX2. Finally, chromatin immunoprecipitation analysis and luciferase reporter assay revealed that SOX2 could negatively regulate BMP4 promoter activity, possibly through binding to the promoter located in the first intron region of BMP4. Collectively, our findings suggest that BMP4 could act as a tumor suppressor and its downregulation by elevated SOX2 resulting in enhanced growth of lung SQC cells.

Lysine demethylase 2A promotes stemness and angiogenesis of breast cancer by upregulating Jagged1

Alterations of histone methylation dynamically regulated by methyltransferases and demethylases are frequently found in human cancers. Here, we showed that expression of lysine demethylase 2A (KDM2A) is markedly increased in human breast cancer and its overexpression is associated with tumor progression and poor prognosis. Knockdown of KDM2A in breast cancer cells reduced proliferation but not viability. Gene set enrichment analysis revealed that inhibition of KDM2A down-regulates angiogenic genes with concurrent reduction of Jagged1 (JAG1), NOTCH1 and HEY1 in the NOTCH signaling. Chromatin immunoprecipitation- quantitative polymerase chain reaction (ChIP-qPCR) demonstrated the binding of KDM2A to the JAG1 promoter and the increase of methylation of Lys-36 of histone H3 (H3K36) in KDM2A-depleted MDA-MB-231 cells. Tumorsphere formation was significantly reduced in KDM2A-depleted cells which could be reversed by ectopic expression of JAG1. A selective KDM2A inhibitor daminozide also decreased the number of tumorsphere and the number of CD24−/CD44hi cells. In addition, daminozide acted synergistically with cisplatin in cell killing. We identified SOX2 as a direct transcriptional target of KDM2A to promote cancer stemness. Depletion of KDM2A in MDA-MB-231 cells attenuated NOTCH activation and tube formation in co-cultured endothelial cells. Two pro-angiogenic factors JAG1 and PDGFA are key mediators for KDM2A to enhance angiogenesis. Finally, inhibition of KDM2A significantly decreased tumor growth and angiogenesis in orthotopic animal experiments. Collectively, we conclude that KDM2A functions as an oncogene in breast cancer by upregulating JAG1 to promote stemness, chemoresistance and angiogenesis.

Upregulation of CISD2 augments ROS homeostasis and contributes to tumorigenesis and poor prognosis of lung adenocarcinoma

CISD2 is a redox-sensitive gene critical for normal development and mitochondrial integrity. CISD2 was known to have aberrant expression in several types of human cancers. However, its relation with lung cancer is still not clear. In this study we found CISD2 mRNA was significantly upregulated in lung adenocarcinoma (ADC) samples, compared with their adjacent normal counterparts, and was correlated with tumor stage, grade, and prognosis based on analysis of clinical specimens-derived expression data in public domain and our validation assay. Cell based assay indicated that CISD2 expression regulated accumulation of reactive oxygen species (ROS), polarization of mitochondrial membrane potential, as well as cell viability, apoptosis, invasiveness, and tumorigenicity. In addition, CISD2 expression was found significantly correlated with stress response/redox signaling genes such as EGR1 and GPX3, while such correlations were also found valid in many public domain data. Taken together, upregulation of CISD2 is involved in an increased antioxidant capacity in response to elevated ROS levels during the formation and progression of lung ADC. The molecular mechanism underlying how CISD2 regulates ROS homeostasis and augments malignancy of lung cancer warrants further investigations.

Genetic modifiers of progression-free survival in never-smoking lung adenocarcinoma patients treated with first-line tyrosine kinase inhibitors

Rationale: Patients with non‐small cell lung cancer (NSCLC) with mutated epidermal growth factor receptor (EGFR) are relatively sensitive to EGFR‐tyrosine kinase inhibitor (TKI) treatment and have longer progression‐free survival (PFS) when treated with EGFR‐TKI compared with platinum‐based chemotherapy. However, many patients with advanced NSCLC who have mutated EGFR do not respond to first‐line EGFR‐TKI treatment and still have shorter PFS. Objectives: The aim of this study was to identify genetic variants associated with PFS among patients with lung adenocarcinoma who were treated with first‐line EGFR‐TKIs. Methods: A genome‐wide association study on PFS was performed in never‐smoking women diagnosed with lung adenocarcinoma and who were treated with first‐line EGFR‐TKIs (n = 128). Significant single‐nucleotide polymorphisms (SNPs) were selected for follow‐up association analysis (n = 198) and for replication assay in another independent cohort (n = 153). Measurements and Main Results: We identified SNPs at 4q12 associated with PFS at genome‐wide significance (P < 10‐8) and with an estimated hazard ratio of more than 4. This association was also replicated in a larger but similar cohort and in an independent NSCLC cohort. Follow‐up functional analyses showed that these SNPs were associated with the expression of EGFR, which encodes the TKI target, and with a nearby gene neuromedin‐U, which encodes a G protein‐coupled receptor ligand known to be involved in the progression of NSCLC. Considering these as possible prognostic biomarkers for the treatment of patients with late‐stage lung cancer, we found that these SNPs were not associated with EGFR mutation status or with polymorphism of the Bcl2‐interacting mediator of cell death gene. Conclusions: Genetic variants in 4q12 merit further investigation to assess their potential as pharmacogenomic predictors for and to understand the biology underlying its influence on PFS in patients treated with TKI therapy.

DNA Methylation Identifies Loci Distinguishing Hereditary Nonpolyposis Colorectal Cancer Without Germ-Line MLH1/MSH2 Mutation from Sporadic Colorectal Cancer.

Objectives:Roughly half of hereditary nonpolyposis colorectal cancer (HNPCC) cases are Lynch syndrome and exhibit germ-line mutations in DNA mismatch repair (MMR) genes; the other half are familial colorectal cancer (CRC) type X (FCCTX) and are MMR proficient. About 70% of Lynch syndrome tumors have germ-line MLH1 or MSH2 mutations. The clinical presentation, histopathological features, and carcinogenesis of FCCTX resemble those of sporadic MMR-proficient colorectal tumors. It is of interest to obtain biomarkers that distinguish FCCTX from sporadic microsatellite stable (MSS) CRC, to develop preventive strategies.Methods:The tumors and adjacent normal tissues of 40 patients with HNPCC were assayed using the Illumina Infinium HumanMethylation27 (HM27) BeadChip to assess the DNA methylation level at about 27,000 loci. The germ-line mutation status of MLH1 and MSH2 and the microsatellite instability status in these patients were obtained. Genome-wide DNA methylation measurements of three groups of patients with general CRC were downloaded from public domain databases. Probes with DNA methylation levels that differed significantly between patients with sporadic MSS CRC and FCCTX were examined, to explore their potential as biomarkers.Results:We found that MSS HNPCC tumors were overwhelmingly hypomethylated compared with those from patient groups with other types of CRC, including germ-line MLH1/MSH2-mutated HNPCC and sporadic MSS CRC. Five gene-marker panels that exhibited a sensitivity of 100% and a specificity higher than 90% in both discovery and validation cohorts were proposed to distinguish MSS HNPCC tumors from sporadic MSS CRC.Conclusions:Our results warrant further investigation and validation. The loci identified here may become useful biomarkers for distinguishing between FCCTX and sporadic MSS CRC tumors.

Association between fetal exposure to phthalate endocrine disruptor and genome-wide DNA methylation at birth

Background Phthalic acid esters are ubiquitous and antiandrogenic, and may cause systemic effects in humans, particularly with in utero exposure. Epigenetic modification, such as DNA methylation, has been hypothesized to be an important mechanism that mediates certain biological processes and pathogenic effects of in utero phthalate exposure. Objective The aim of this study was to examine the association between genome‐wide DNA methylation at birth and prenatal exposure to phthalate. Methods We studied 64 infant–mother pairs included in TMICS (Taiwan Maternal and Infant Cohort Study), a long‐term follow‐up birth cohort from the general population. DNA methylation levels at more than 450,000 CpG sites were measured in cord blood samples using Illumina Infinium HumanMethylation450 BeadChips. The concentrations of three metabolites of di‐(2‐ethylhexyl) phthalate (DEHP) were measured using liquid chromatography tandem–mass spectrometry (LC–MS/MS) in urine samples collected from the pregnant women during 28–36 weeks gestation. Results We identified 25 CpG sites whose methylation levels in cord blood were significantly correlated with prenatal DEHP exposure using a false discovery rate (FDR) of 5% (q‐value < 0.05). Via gene‐set enrichment analysis (GSEA), we also found that there was significant enrichment of genes involved in the androgen response, estrogen response, and spermatogenesis within those genes showing DNA methylation changes in response to exposure. Specifically, PA2G4, HMGCR, and XRCC6 genes were involved in genes in response to androgen. Conclusions Phthalate exposure in utero may cause significant alterations in the DNA methylation in cord blood. These changes in DNA methylation might serve as biomarkers of maternal exposure to phthalate in infancy and potential candidates for studying mechanisms via which phthalate may impact on health in later life. Future investigations are warranted. Graphical abstract Figure. No caption available. HighlightsHumans are ubiquitously exposed to phthalates through digestion, inhalation, or dermal absorption.DNA methylation has been hypothesized to be an important mechanism in in utero phthalate exposure.DNA methylation changes are involved in androgen response, estrogen response, protein secretion, and spermatogenesis.Three CpG sites (cg00935782, cg07732421, and cg00764560) are involved in genes of response to androgen.

Comparison of annual percentage change in breast cancer incidence rate between Taiwan and the United States—A smoothed Lexis diagram approach

Recent studies compared the age effects and birth cohort effects on female invasive breast cancer (FIBC) incidence in Asian populations with those in the US white population. They were based on age–period–cohort model extrapolation and estimated annual percentage change (EAPC) in the age‐standardized incidence rates (ASR). It is of interest to examine these results based on cohort‐specific annual percentage change in rate (APCR) by age and without age–period–cohort model extrapolation. FIBC data (1991–2010) were obtained from the Taiwan Cancer Registry and the U.S. SEER 9 registries. APCR based on smoothed Lexis diagrams were constructed to study the age, period, and cohort effects on FIBC incidence. The patterns of age‐specific rates by birth cohort are similar between Taiwan and the US. Given any age‐at‐diagnosis group, cohort‐specific rates increased overtime in Taiwan but not in the US; cohort‐specific APCR by age decreased with birth year in both Taiwan and the US but was always positive and large in Taiwan. Given a diagnosis year, APCR decreased as birth year increased in Taiwan but not in the US. In Taiwan, the proportion of APCR attributable to cohort effect was substantial and that due to case ascertainment was becoming smaller. Although our study shows that incidence rates of FIBC have increased rapidly in Taiwan, thereby confirming previous results, the rate of increase over time is slowing. Continued monitoring of APCR and further investigation of the cause of the APCR decrease in Taiwan are warranted.

Wild-type p53 upregulates an early onset breast cancer-associated gene GAS7 to suppress metastasis via GAS7–CYFIP1-mediated signaling pathway

The early onset breast cancer patients (age ≤ 40) often display higher incidence of axillary lymph node metastasis, and poorer five-year survival than the late-onset patients. To identify the genes and molecules associated with poor prognosis of early onset breast cancer, we examined gene expression profiles from paired breast normal/tumor tissues, and coupled with Gene Ontology and public data base analysis. Our data showed that the expression of GAS7b gene was lower in the early onset breast cancer patients as compared to the elder patients. We found that GAS7 was associated with CYFIP1 and WAVE2 complex to suppress breast cancer metastasis via blocking CYFIP1 and Rac1 protein interaction, actin polymerization, and β1-integrin/FAK/Src signaling. We further demonstrated that p53 directly regulated GAS7 gene expression, which was inversely correlated with p53 mutations in breast cancer specimens. Our study uncover a novel regulatory mechanism of p53 in early onset breast cancer progression through GAS7–CYFIP1-mediated signaling pathways.