Rui Lan Huang

Identification of novel DNA methylation markers in cervical cancer

Testing for DNA methylation has potential in cancer screening. Most previous studies of DNA methylation in cervical cancer used a candidate gene approach. The aim our study was to identify novel genes that are methylated in cervical cancers and to test their potential in clinical applications. We did a differential methylation hybridization using a CpG island (CGI) microarray containing 8640 CGI tags to uncover methylated genes in squamous cell carcinomas (SCC) of the uterine cervix. Pooled DNA from cancer tissues and normal cervical swabs were used for comparison. Methylation‐specific polymerase chain reaction, bisulfite sequencing and reverse transcription polymerase chain reaction were used to confirm the methylation status in cell lines, normal cervices (n = 45), low‐grade lesions (n = 45), high‐grade lesions (HSIL; n = 58) and invasive squamous cell carcinomas (SCC; n = 22 from swabs and n = 109 from tissues). Human papillomavirus (HPV) was detected using reverse line blots. We reported 6 genes (SOX1, PAX1, LMX1A, NKX6‐1, WT1 and ONECUT1) more frequently methylated in SCC tissues (81.5, 94.4, 89.9, 80.4, 77.8 and 20.4%, respectively) than in their normal controls (2.2, 0, 6.7, 11.9, 11.1 and 0%, respectively; p < 0.0001). Parallel testing of HPV and PAX1 methylation in cervical swabs confers an improved sensitivity than HPV testing alone (80% vs. 66%) without compromising specificity (63% vs. 64%) for HSIL/SCC. Testing PAX1 methylation marker alone, the specificity for HSIL/SCC is 99%. The analysis of these novel DNA methylations may be a promising approach for the screening of cervical cancers.

Hypermethylation of the TGF-β target, ABCA1 is associated with poor prognosis in ovarian cancer patients

BackgroundThe dysregulation of transforming growth factor-β (TGF-β) signaling plays a crucial role in ovarian carcinogenesis and in maintaining cancer stem cell properties. Classified as a member of the ATP-binding cassette (ABC) family, ABCA1 was previously identified by methylated DNA immunoprecipitation microarray (mDIP-Chip) to be methylated in ovarian cancer cell lines, A2780 and CP70. By microarray, it was also found to be upregulated in immortalized ovarian surface epithelial (IOSE) cells following TGF-β treatment. Thus, we hypothesized that ABCA1 may be involved in ovarian cancer and its initiation.ResultsWe first compared the expression level of ABCA1 in IOSE cells and a panel of ovarian cancer cell lines and found that ABCA1 was expressed in HeyC2, SKOV3, MCP3, and MCP2 ovarian cancer cell lines but downregulated in A2780 and CP70 ovarian cancer cell lines. The reduced expression of ABCA1 in A2780 and CP70 cells was associated with promoter hypermethylation, as demonstrated by bisulfite pyro-sequencing. We also found that knockdown of ABCA1 increased the cholesterol level and promoted cell growth in vitro and in vivo. Further analysis of ABCA1 methylation in 76 ovarian cancer patient samples demonstrated that patients with higher ABCA1 methylation are associated with high stage (P = 0.0131) and grade (P = 0.0137). Kaplan-Meier analysis also found that patients with higher levels of methylation of ABCA1 have shorter overall survival (P = 0.019). Furthermore, tissue microarray using 55 ovarian cancer patient samples revealed that patients with a lower level of ABCA1 expression are associated with shorter progress-free survival (P = 0.038).ConclusionsABCA1 may be a tumor suppressor and is hypermethylated in a subset of ovarian cancer patients. Hypermethylation of ABCA1 is associated with poor prognosis in these patients.

CpG island shore methylation regulates caveolin-1 expression in breast cancer

Caveolin-1 (Cav1) is an integral membrane, scaffolding protein found in plasma membrane invaginations (caveolae). Cav1 regulates multiple cancer-associated processes. In breast cancer, a tumor suppressive role for Cav1 has been suggested; however, Cav1 is frequently overexpressed in aggressive breast cancer subtypes, suggesting an oncogenic function in advanced-stage disease. To further delineate Cav1 function in breast cancer progression, we evaluated its expression levels among a panel of cell lines representing a spectrum of breast cancer phenotypes. In basal-like (the most aggressive BC subtype) breast cancer cells, Cav1 was consistently upregulated, and positively correlated with increased cell proliferation, anchorage-independent growth, and migration and invasion. To identify mechanisms of Cav1 gene regulation, we compared DNA methylation levels within promoter ‘CpG islands’ (CGIs) with ‘CGI shores’, recently described regions that flank CGIs with less CG-density. Integration of genome-wide DNA methylation profiles (‘methylomes’) with Cav1 expression in 30 breast cancer cell lines showed that differential methylation of CGI shores, but not CGIs, significantly regulated Cav1 expression. In breast cancer cell lines having low Cav1 expression (despite promoter CGI hypomethylation), we found that treatment with a DNA methyltransferase inhibitor induced Cav1 expression via CGI shore demethylation. In addition, further methylome assessments revealed that breast cancer aggressiveness associated with Cav1 CGI shore methylation levels, with shore hypermethylation in minimally aggressive, luminal breast cancer cells and shore hypomethylation in highly aggressive, basal-like cells. Cav1 CGI shore methylation was also observed in human breast tumors, and overall survival rates of breast cancer patients lacking estrogen receptor α (ERα) negatively correlated with Cav1 expression. Based on this first study of Cav1 (a potential oncogene) CGI shore methylation, we suggest this phenomenon may represent a new prognostic marker for ERα-negative, basal-like breast cancer.

Growth Inhibition of Ovarian Tumor–Initiating Cells by Niclosamide

A recent hypothesis for cancer chemoresistance posits that cytotoxic survival of a subpopulation of tumor progenitors drives the propagation of recurrent disease, underscoring the need for new therapeutics that target such primitive cells. To discover such novel compounds active against drug-resistant ovarian cancer, we identified a subset of chemoresistant ovarian tumor cells fulfilling current definitions of cancer-initiating cells from cell lines and patient tumors using multiple stemness phenotypes, including the expression of stem cell markers, membrane dye efflux, sphere formation, potent tumorigenicity, and serial tumor propagation. We then subjected such stem-like ovarian tumor-initiating cells (OTIC) to high-throughput drug screening using more than 1,200 clinically approved drugs. Of 61 potential compounds preliminarily identified, more stringent assessments showed that the antihelmintic niclosamide selectively targets OTICs in vitro and in vivo. Gene expression arrays following OTIC treatment revealed niclosamide to disrupt multiple metabolic pathways affecting biogenetics, biogenesis, and redox regulation. These studies support niclosamide as a promising therapy for ovarian cancer and warrant further preclinical and clinical evaluation of this safe, clinically proven drug for the management of this devastating gynecologic malignancy. Mol Cancer Ther; 11(8); 1703–12. ©2012 AACR.

Quantitative DNA methylation analysis detects cervical intraepithelial neoplasms type 3 and worse

DNA methylation may be used a potential biomarker for detecting cervical cancer. The authors of this report used quantitative methylation analysis of 4 genes in a full spectrum of cervical lesions to test its potential clinical application.

Downregulation of miR-29 contributes to cisplatin resistance of ovarian cancer cells

Drug resistance is an obstacle to the treatment of ovarian cancer. Using a unique cell model, we have proven previously that a subpopulation of ovarian cancer cells is more resistant to cisplatin than are the original cells. MicroRNAs (miRNAs), small noncoding RNAs, are involved in many biological events in cancer cells. In our study, we explored whether miRNAs are involved in cisplatin resistance of ovarian cancer cells. Cisplatin‐resistant cells expressed a lower level of miR‐29a/b/c. Manipulation of microRNA‐29 (miR‐29) expression modulated cisplatin sensitivity of CP70, HeyC2, SKOV3 and A2780 ovarian cancer cells. Knockdown of miR‐29a/b/c increased the ability of cells to escape cisplatin‐induced cell death partly through upregulation of collagen type I alpha 1 (COL1A1) and increased the activation of extracellular signal‐regulated kinase 1/2 and inactivation of glycogen synthase kinase 3 beta. When combined with cisplatin treatment, knockdown of miR‐29 decreased the amount of the active form of caspase‐9 and caspase‐3. Ectopic expression of miR‐29 alone or in combination with cisplatin treatment efficaciously reduced the tumorigenicity of CP70 cells in vivo. Our data show that downregulation of miR‐29 increases cisplatin resistance in ovarian cancer cells. Taken together, these data suggest that overexpression of miR‐29 is a potential sensitizer to cisplatin treatment that may have therapeutic implications.

Comprehensive methylome analysis of ovarian tumors reveals hedgehog signaling pathway regulators as prognostic DNA methylation biomarkers.

Women with advanced stage ovarian cancer (OC) have a five-year survival rate of less than 25%. OC progression is associated with accumulation of epigenetic alterations and aberrant DNA methylation in gene promoters acts as an inactivating “hit” during OC initiation and progression. Abnormal DNA methylation in OC has been used to predict disease outcome and therapy response. To globally examine DNA methylation in OC, we used next-generation sequencing technology, MethylCap-sequencing, to screen 75 malignant and 26 normal or benign ovarian tissues. Differential DNA methylation regions (DMRs) were identified, and the Kaplan–Meier method and Cox proportional hazard model were used to correlate methylation with clinical endpoints. Functional role of specific genes identified by MethylCap-sequencing was examined in in vitro assays. We identified 577 DMRs that distinguished (p < 0.001) malignant from non-malignant ovarian tissues; of these, 63 DMRs correlated (p < 0.001) with poor progression free survival (PFS). Concordant hypermethylation and corresponding gene silencing of sonic hedgehog pathway members ZIC1 and ZIC4 in OC tumors was confirmed in a panel of OC cell lines, and ZIC1 and ZIC4 repression correlated with increased proliferation, migration and invasion. ZIC1 promoter hypermethylation correlated (p < 0.01) with poor PFS. In summary, we identified functional DNA methylation biomarkers significantly associated with clinical outcome in OC and suggest our comprehensive methylome analysis has significant translational potential for guiding the design of future clinical investigations targeting the OC epigenome. Methylation of ZIC1, a putative tumor suppressor, may be a novel determinant of OC outcome.

Methylomics analysis identifies epigenetically silenced genes and implies an activation of β-catenin signaling in cervical cancer

Using DNA methylation biomarkers in cancer detection is a potential direction in clinical testing. Some methylated genes have been proposed for cervical cancer detection; however, more reliable methylation markers are needed. To identify new hypermethylated genes in the discovery phase, we compared the methylome between a pool of DNA from normal cervical epithelium (n = 19) and a pool of DNA from cervical cancer tissues (n = 38) using a methylation bead array. We integrated the differentially methylated genes with public gene expression databases, which resulted in 91 candidate genes. Based on gene expression after demethylation treatment in cell lines, we confirmed 61 genes for further validation. In the validation phase, quantitative MSP and bisulfite pyrosequencing were used to examine their methylation level in an independent set of clinical samples. Fourteen genes, including ADRA1D, AJAP1, COL6A2, EDN3, EPO, HS3ST2, MAGI2, POU4F3, PTGDR, SOX8, SOX17, ST6GAL2, SYT9, and ZNF614, were significantly hypermethylated in CIN3+ lesions. The sensitivity, specificity, and accuracy of POU4F3 for detecting CIN3+ lesions were 0.88, 0.82, and 0.85, respectively. A bioinformatics function analysis revealed that AJAP1, EDN3, EPO, MAGI2, and SOX17 were potentially implicated in β‐catenin signaling, suggesting the epigenetic dysregulation of this signaling pathway during cervical cancer development. The concurrent methylation of multiple genes in cancers and in subsets of precancerous lesions suggests the presence of a driver of methylation phenotype in cervical carcinogenesis. Further validation of these new genes as biomarkers for cervical cancer screening in a larger population‐based study is warranted.

Epigenetic silencing of PTPRR activates MAPK signaling, promotes metastasis and serves as a biomarker of invasive cervical cancer

Epigenetic modifications are a driving force in carcinogenesis. However, their role in cancer metastasis remains poorly understood. The present study investigated the role of DNA methylation in the cervical cancer metastasis. Here, we report evidence of the overexpression of DNA methyltransferases 3B (DNMT3B) in invasive cervical cancer and of the inhibition of metastasis by DNMT3B interference. Using methyl-DNA immunoprecipitation coupled with microarray analysis, we found that the protein tyrosine phosphatase receptor type R (PTPRR) was silenced through DNMT3B-mediated methylation in the cervical cancer. PTPRR inhibited p44/42 MAPK signaling, the expression of the transcription factor AP1, human papillomavirus (HPV) oncogenes E6/E7 and DNMTs. The methylation status of PTPRR increased in cervical scrapings (n=358) in accordance with disease severity, especially in invasive cancer. Methylation of the PTPRR promoter has an important role in the metastasis and may be a biomarker of invasive cervical cancer.

Type-specific distribution of HPV along the full spectrum of cervical carcinogenesis in Taiwan: An indication of viral oncogenic potential

OBJECTIVE The distributional trend of different types of human papillomavirus (HPV) along the spectrum of cervical diseases from non-neoplastic HPV infection (Normal), cervical intraepithelial neoplasia 1 (CIN1), cervical intraepithelial neoplasia 2/3 (CIN2/3) to invasive cancer (CC) reflects the transformation potential of each HPV type. STUDY DESIGN Type-specific distribution of HPV in four hospital- and population-based HPV surveys in Taiwan was analyzed. RESULTS Among the 1605 (out of 6356) women positive for HPV, the prevalence of HPV infection in Normal, CIN1, CIN2/3, and CC was 10%, 60%, 70%, and 86%, respectively. The order of type-specific prevalence was HPV 52 (20.9%), HPV 16 (16.9%), HPV 58 (9.0%), and HPV 18 (8.5%) in Normal; HPV 52 (16.5%), HPV 16 (10.2%), HPV 51 (8.4%), and HPV 58 (6.5%) in CIN1; HPV 16 (29.8%), HPV 52 (17.1%), HPV 58 (16.4%), and HPV 33 (10.3%) in CIN2/3; and HPV 16 (50.7%), HPV 18 (11.9%), HPV 58 (10.1%), and HPV 33 (8.4%) in CC. We compared the step-wise distributional changes of five major HPV types along the spectrum of cervical neoplasia. The CIN1 vs. Normal distributional ratio of each HPV type varied from 0.41 to 0.97, indicating a relatively similar chance of giving rise to CIN1. In the CIN2/3 vs. CIN1 and CC vs. CIN2/3 comparisons, the distributional changes varied dramatically among different HPV types. Upon progression to CIN2/3, the distributional proportions of HPV 16, 33, and 58 became 2.1- to 5.4-fold higher, and that of HPV 18 became 0.3- to 0.5-fold lower than their CIN1 and normal counterparts. In the CC vs. CIN2/3 comparison, the change in distributional proportion was highest in HPV 18 (5.7-fold), followed by HPV 16 (1.7-fold), HPV 33 (0.8-fold), HPV 58 (0.6-fold), and HPV 52 (0.18-fold). CONCLUSION The distribution-defined progression from subclinical infection to CC was highest for HPV 16, followed by HPV 33, 18, 58, and 52. The differential disease progression potential of different HPV types reflects their transformational capability at different steps of cervical carcinogenesis and warrants the clinical attention of HPV infection type specifically.