Yu-Wei Leu

Loss of Estrogen Receptor Signaling Triggers Epigenetic Silencing of Downstream Targets in Breast Cancer

Alterations in histones, chromatin-related proteins, and DNA methylation contribute to transcriptional silencing in cancer, but the sequence of these molecular events is not well understood. Here we demonstrate that on disruption of estrogen receptor (ER) α signaling by small interfering RNA, polycomb repressors and histone deacetylases are recruited to initiate stable repression of the progesterone receptor (PR) gene, a known ERα target, in breast cancer cells. The event is accompanied by acquired DNA methylation of the PR promoter, leaving a stable mark that can be inherited by cancer cell progeny. Reestablishing ERα signaling alone was not sufficient to reactivate the PR gene; reactivation of the PR gene also requires DNA demethylation. Methylation microarray analysis further showed that progressive DNA methylation occurs in multiple ERα targets in breast cancer genomes. The results imply, for the first time, the significance of epigenetic regulation on ERα target genes, providing new direction for research in this classical signaling pathway.

Mapping Geographic Zones of Cancer Risk with Epigenetic Biomarkers in Normal Breast Tissue

Purpose: Genetic alterations were previously identified in normal epithelia adjacent to invasive cancers. The aim of this study was to determine DNA methylation in histologically normal tissues from multiple geographic zones adjacent to primary breast tumors. Experimental Design: First, methylation status of a 4-kb region of RASSF1A promoter was interrogated using oligonucleotide-based microarray in 144 samples (primary tumors, 47; adjacent normals, 69; reduction mammoplasty tissues, 28). Second, allelic imbalance (AI)/loss of heterozygosity (LOH) surrounding RASSF1A promoter were analyzed in 30 samples (tumors, 8; adjacent normals, 22). Third, global methylation screening of 49 samples (tumors, 12; adjacent normals, 25; reduction mammoplasty, 12) was done by differential methylation hybridization. Real-time quantitative methylation-specific PCR was used to validate the microarray findings. Results: DNA methylation in the core RASSF1A promoter was low in reduction mammoplasty tissues (P = 0.0001) when compared with primary tumors. The adjacent normals had an intermediate level of methylation. The regions surrounding the core were highly methylated in all sample types. Microsatellite markers showed AI/LOH in tumors and some of the adjacent normals. Concurrent AI/LOH and DNA methylation in RASSF1A promoter occurred in two of six tumors. Global methylation screening uncovered genes more methylated in adjacent normals than in reduction mammoplasty tissues. The methylation status of four genes was confirmed by quantitative methylation-specific PCR. Conclusions: Our findings suggest a field of methylation changes extending as far as 4 cm from primary tumors. These frequent alterations may explain why normal tissues are at risk for local recurrence and are useful in disease prognostication.

Epigenetic changes in virus-associated human cancers

ABSTRACTEpigenetics of human cancer becomes an area of emerging research direction due to a growing understanding of specific epigenetic pathways and rapid development of detection technologies. Aberrant promoter hypermethylation is a prevalent phenonmena in human cancers. Tumor suppressor genes are often hypermethylated due to the increased activity or deregulation of DNMTs. Increasing evidence also reveals that viral genes are one of the key players in regulating DNA methylation. In this review, we will focus on hypermethylation and tumor suppressor gene silencing and the signal pathways that are involved, particularly in cancers closely associated with the hepatitis B virus, simian virus 40 (SV40), and Epstein-Barr virus. In addition, we will discuss current technologies for genome-wide detection of epigenetically regulated targets, which allow for systematic DNA hypermethylation analysis. The study of epigenetic changes should provide a global view of gene profile in cancer, and epigenetic markers could be used for early detection, prognosis, and therapy of cancer.

Targeted methylation of two tumor suppressor genes is sufficient to transform mesenchymal stem cells into cancer stem/initiating cells

Although DNA hypermethylation within promoter CpG islands is highly correlated with tumorigenesis, it has not been established whether DNA hypermethylation within a specific tumor suppressor gene (TSG) is sufficient to fully transform a somatic stem cell. In this study, we addressed this question using a novel targeted DNA methylation technique to methylate the promoters of HIC1 and RassF1A, two well-established TSGs, along with a two-component reporter system to visualize successful targeting of human bone marrow-derived mesenchymal stem cells (MSC) as a model cell system. MSCs harboring targeted promoter methylations of HIC1/RassF1A displayed several features of cancer stem/initiating cells including loss of anchorage dependence, increased colony formation capability, drug resistance, and pluripotency. Notably, inoculation of immunodeficient mice with low numbers of targeted MSC resulted in tumor formation, and subsequent serial xenotransplantation and immunohistochemistry confirmed the presence of stem cell markers and MSC lineage in tumor xenografts. Consistent with the expected mechanism of TSG hypermethylation, treatment of the targeted MSC with a DNA methyltransferase inhibitor reversed their tumorigenic phenotype. To our knowledge, this is the first direct demonstration that aberrant TSG hypermethylation is sufficient to transform a somatic stem cell into a fully malignant cell with cancer stem/initiating properties.

Glutamate receptor, ionotropic, kainate 2 silencing by DNA hypermethylation possesses tumor suppressor function in gastric cancer

Aberrant DNA methylation is considered a major mechanism for silencing tumor suppressor genes in gastric cancer. We used CpG microarray and differential methylation hybridization strategies to identify potential tumor suppressor genes and recovered glutamate receptor, ionotropic, kainate 2 (GRIK2) as a novel epigenetic target in gastric cancer. Additional experiments showed that the promoter region of GRIK2 was hypermethylated in 3 of the 4 tested gastric cancer cell lines, and its expression was restored by treatment of cells with the DNA methylation inhibitor, 5′‐aza‐dC. In clinical samples, the GRIK2 promoter was differentially hypermethylated in tumor tissues compared with adjacent normal tissues (p < 0.001), and this methylation was inversely correlated with the expression level of GRIK2 mRNA (r = −0.44). Functional studies further showed that GRIK2‐expressing gastric cancer cell lines showed decreased colony formation and cell migration. Taken together, these results suggest that GRIK2 may play a tumor‐suppressor role in gastric cancer. Future studies are warranted to examine whether DNA hypermethylation of the GRIK2 promoter can be used as a potential tumor marker for gastric cancer.

Epigenetic regulation of the X-linked tumour suppressors BEX1 and LDOC1 in oral squamous cell carcinoma.

The strong associations between oral squamous cell carcinoma (OSCC) and dietary habits such as alcohol consumption (A), betel quid chewing (B) and cigarette smoking (C) and its predominance in men have been well documented; however, systemic analysis of OSCC is limited. Our study applied high‐throughput screening methods to identify causative epigenetic targets in a cohort of men with ABC‐associated OSCC. We identified BEX1 and LDOC1 as two epigenetically silenced X‐linked tumour suppressors and demonstrated a functional link between the transcription of BEX1 and LDOC1 and promoter hypermethylation. Methylation of the BEX1 and LDOC1 promoters was associated significantly (p < 0.0001) with OSCC and were detected in 75% (42/56) and 89% (50/56) of the samples, respectively. We observed concordant increases in the methylation of both genes in 71% (40/56) of the tumours, and potent in vitro and in vivo growth inhibitory effects in OSCC cells ectopically expressing BEX1 and/or LDOC1. Restored expression of BEX1 and LDOC1 suppressed the nuclear factor‐κB (NF‐κB) signalling pathway, which is the most frequently hyperactivated signalling pathway in OSCC. This suppression might result from decreased p50 and p65 expression. These findings suggest that silencing of BEX1 and LDOC1 by promoter hypermethylation might represent a critical event in the molecular pathogenesis of OSCC and account for the oncogenic effects of ABC exposure and the male predominance of OSCC occurrence. Microarray data are available in the Gene Expression Omnibus (GEO; http://www.ncbi.nlm.nih.gov/geo/) Copyright

Expression of T‐cell lymphoma invasion and metastasis 2 (TIAM2) promotes proliferation and invasion of liver cancer

The T‐cell lymphoma invasion and metastasis 2 (TIAM2) gene is the homolog of human TIAM1, a Rac‐specific guanine nucleotide exchange factor that plays important roles in neuron development and human malignancies. Although the role of TIAM1 is well characterized, the physiological and pathological functions of TIAM2 remain unknown. In our study, human cDNA and protein panels were evaluated for endogenous expression of TIAM2. Four hepatocellular carcinoma (HCC) cell lines and 91 HCC samples were used to demonstrate expression of TIAM2S (the short form of TIAM2) in cancer cells. In addition, HepG2 cells stably expressing TIAM2S were used for tumorigenic assays in both cellular and mouse models. We demonstrate that endogenous TIAM2S was induced in several human cancers including HCC. TIAM2S expression was undetectable in normal human liver but was induced in all HCC cell lines and in 86% (78/91) of HCC biopsies. TIAM2S expression was positively associated with TIAM1 expression, hepatitis B virus (HBV) infection and metastatic phenotype. Expression of recombinant TIAM2S in HepG2 cells promoted growth and invasiveness. In vivo study using a xenografted mouse model demonstrated that induced endogenous expression of TIAM2S converted non‐invasive human HCC cells into highly aggressive vascular tumors. Further examination revealed that TIAM2S expression resulted in up‐regulation of N‐cadherin and vimentin, and in redistribution of E‐cadherin. These findings show, for the first time, that human TIAM2S is involved in HCC pathogenesis, and that increased expression of TIAM2S promotes epithelial‐to‐mesenchymal transition and results in proliferation and invasion in liver cancer cells.

Aberrant methylation impairs low density lipoprotein receptor-related protein 1B tumor suppressor function in gastric cancer

DNA methylation plays a significant role in tumor progression. In this study, we used CpG microarray and differential methylation hybridization approaches to identify low density lipoprotein receptor‐related protein 1B (LRP1B) as a novel epigenetic target in gastric cancer. LRP1B was hypermethylated in four gastric cancer cell lines, and low LRP1B mRNA expression was associated with high methylation levels in gastric cancer cell lines. Addition of a DNA methylation inhibitor (5‐Aza‐dC) restored the mRNA expression of LRP1B in these cell lines, indicating that DNA methylation is involved in regulating LRP1B expression. In 45 out of 74 (61%) clinical samples, LRP1B was highly methylated; LRP1B mRNA expression was significantly lower in 15 out of 19 (79%, P < 0.001) gastric tumor tissues than in corresponding adjacent normal tissues. In addition, ectopic expression of mLRP1B4 in gastric cancer cell lines suppressed cell growth, colony formation and tumor formation in nude mice. These results collectively indicate that LRP1B is a functional tumor suppressor gene in gastric cancer and that is regulated by DNA methylation.

Changes in DNA methylation are associated with the development of drug resistance in cervical cancer cells

Background and proposeChanges in DNA methylation are associated with changes in somatic cell fate without the alteration of coding sequences. In addition to its use as a traceable biomarker, reversible DNA methylation could also serve as a therapeutic target. In particular, if the development of drug resistance is associated with changes in DNA methylation, then demethylation might reverse the resistance phenotype. The reversion of the drug-resistance might then be feasible if the association between abnormal DNA methylation and the development of drug-resistance could be identified.MethodsMethylation differences between the drug-resistance cervical cancer cell, SiHa, and its derived oxaliplatin-resistant S3 cells were detected by methylation specific microarray. The drug-resistance cells were treated with demethylation agent to see if the resistance phenotype were reversed. Targeted methylation of one of the identified locus in normal cell is expected to recapitulate the development of resistance and a two-component reporter system is adopted to monitor the increase of DNA methylation in live cells.ResultsIn this report, we identified methylation changes, both genome-wide and within individual loci, in the oxaliplatin-resistant cervical cancer cell S3 compared with its parental cell line SiHa. Treatment of S3 with a demethylation agent reversed increases in methylation and allowed the expression of methylation-silenced genes. Treatment with the demethylation agent also restored the sensitivity of S3 to cisplatin, taxol, and oxaliplatin to the same level as that of SiHa. Finally, we found that methylation of the target gene Casp8AP2 is sufficient to increase drug resistance in different cells.ConclusionsThese results suggest that global methylation is associated with the development of drug resistance and could serve as a biomarker and therapeutic target for drug resistance in cervical cancer.

Excavating relics of DNA methylation changes during the development of neoplasia.

Epigenetic events like DNA methylation are known to regulate gene expression, and dysregulation of these events is associated with neoplastic proliferation. Here, we provide a step-by-step review of the approach that has gradually developed to identify critical DNA methylation during neoplasia. DNA methylation has first been tightly linked to the regulation of gene expression and functions. Next, the clinical importance of such DNA methylation has been probed by inducing loss of the maintenance of normal DNA methylation, which has been found to trigger onset of disease. Methylation changes can be signal-specific and lineage-specific, providing a record what cells have encountered and what they have become. Comparison of methylation associated with normal cellular differentiation and abnormal cell fate changes is expected to uncover critical methylation changes. We also propose a specific scheme that can be used to excavate critical DNA methylation associated with cell evolution.