DNMT3b overexpression contributes to a hypermethylator phenotype in human breast cancer cell lines

<p>Abstract</p> <p>Background</p> <p>DNA hypermethylation events and other epimutations occur in many neoplasms, producing gene expression changes that contribute to neoplastic transformation, tumorigenesis, and tumor behavior. Some human cancers exhibit a hypermethylator phenotype, characterized by concurrent DNA methylation-dependent silencing of multiple genes. To determine if a hypermethylation defect occurs in breast cancer, the expression profile and promoter methylation status of methylation-sensitive genes were evaluated among breast cancer cell lines.</p> <p>Results</p> <p>The relationship between gene expression (assessed by RT-PCR and quantitative real-time PCR), promoter methylation (assessed by methylation-specific PCR, bisulfite sequencing, and 5-aza-2'deoxycytidine treatment), and the DNA methyltransferase machinery (total DNMT activity and expression of DNMT1, DNMT3a, and DNMT3b proteins) were examined in 12 breast cancer cell lines. Unsupervised cluster analysis of the expression of 64 methylation-sensitive genes revealed two groups of cell lines that possess distinct methylation signatures: (i) hypermethylator cell lines, and (ii) low-frequency methylator cell lines. The hypermethylator cell lines are characterized by high rates of concurrent methylation of six genes (<it>CDH1, CEACAM6, CST6, ESR1, LCN2, SCNN1A</it>), whereas the low-frequency methylator cell lines do not methylate these genes. Hypermethylator cell lines coordinately overexpress total DNMT activity and DNMT3b protein levels compared to normal breast epithelial cells. In contrast, most low-frequency methylator cell lines possess DNMT activity and protein levels that are indistinguishable from normal. Microarray data mining identified a strong cluster of primary breast tumors that express the hypermethylation signature defined by <it>CDH1</it>, <it>CEACAM6, CST6, ESR1, LCN2</it>, and <it>SCNN1A</it>. This subset of breast cancers represents 18/88 (20%) tumors in the dataset analyzed, and 100% of these tumors were classified as basal-like, suggesting that the hypermethylator defect cosegregates with poor prognosis breast cancers.</p> <p>Conclusion</p> <p>These observations combine to strongly suggest that: (a) a subset of breast cancer cell lines express a hypermethylator phenotype, (b) the hypermethylation defect in these breast cancer cell lines is related to aberrant overexpression of DNMT activity, (c) overexpression of DNMT3b protein significantly contributes to the elevated DNMT activity observed in tumor cells expressing this phenotype, and (d) the six-gene hypermethylator signature characterized in breast cancer cell lines defines a distinct cluster of primary basal-like breast cancers.</p

Loss of post-transcriptional regulation of DNMT3b by microRNAs: A possible molecular mechanism for the hypermethylation defect observed in a subset of breast cancer cell lines

A hypermethylation defect associated with DNMT hyperactivity and DNMT3b overexpression characterizes a subset of breast cancers and breast cancer cell lines. We analyzed breast cancer cell lines for differential expression of regulatory miRs to determine if loss of miR-mediated post-transcriptional regulation of DNMT3b represents the molecular mechanism that governs the overexpression of DNMT3b that drives the hypermethylation defect in breast cancer. MicroRNAs (miRs) that regulate (miR-29a, miR-29b, miR-29c, miR-148a, miR-148b) or are predicted (miR-26a, miR-26b, miR-203, miR-222) to regulate DNMT3b were examined among 10 hypermethylator and 6 non-hypermethylator breast cancer cell lines. Hypermethylator cell lines express diminished levels of miR-29c, miR-148a, miR-148b, miR-26a, miR-26b, and miR-203 compared to non-hypermethylator cell lines. miR expression patterns correlate inversely with methylation-sensitive gene expression (r=−0.66, p=0.0056) and directly with the methylation status of these genes (r=0.72, p=0.002). To determine the mechanistic role of specific miRs in the dysregulation of DNMT3b among breast cancer cell lines, miR levels were modulated by transfection of pre-miR precursors for miR-148b, miR-26b, and miR-29c into hypermethylator cell lines (Hs578T, HCC1937, SUM185) and transfection of antagomirs directed against miR-148b, miR-26b, and miR-29c into non-hypermethylator cell lines (BT20, MDA-MB-415, MDA-MB-468). Antagomir-mediated knock-down of miR-148b, miR-29c, and miR-26b significantly increased DNMT3b mRNA in non-hypermethylator cell lines, and re-expression of miR-148b, miR-29c, and miR-26b following transfection of pre-miR precursors significantly reduced DNMT3b mRNA in hypermethylator cell lines. These findings strongly suggest that: i) post-transcriptional regulation of DNMT3b is combinatorial, ii) diminished expression of regulatory miRs contributes to DNMT3b overexpression, iii) re-expression of regulatory miRs reduces DNMT3b mRNA levels in hypermethylator breast cancer cell lines, and iv) down-regulation of regulatory miRs increases DNMT3b mRNA levels in non-hypermethylator breast cancer cell lines. In conlcusion, the molecular mechanism governing the DNMT3b-mediated hypermethylation defect in breast cancer cell lines involves the loss of post-transcriptional regulation of DNMT3b by regulatory miRs

Molecular and cellular heterogeneity in breast cancer: Challenges for personalized medicine

Breast cancer is noted for disparate clinical behaviors and patient outcomes, despite common histopathological features at diagnosis. Molecular pathogenesis studies suggest that breast cancer is a collection of diseases with variable molecular underpinnings that modulate therapeutic responses, disease-free intervals, and long-term survival. Traditional therapeutic strategies for individual patients are guided by the expression status of the estrogen and progesterone receptors (ER and PR) and human epidermal growth factor receptor 2 (HER2). Although such methods for clinical classification have utility in selection of targeted therapies, short-term patient responses and long-term survival remain difficult to predict. Molecular signatures of breast cancer based on complex gene expression patterns have utility in prediction of long-term patient outcomes, but are not yet used for guiding therapy. Examination of the correspondence between these methods for breast cancer classification reveals a lack of agreement affecting a significant percentage of cases. To realize true personalized breast cancer therapy, a more complete analysis and evaluation of the molecular characteristics of the disease in the individual patient is required, together with an understanding of the contributions of specific genetic and epigenetic alterations (and their combinations) to management of the patient. Here, we discuss the molecular and cellular heterogeneity of breast cancer, the impact of this heterogeneity on practical breast cancer classification, and the challenges for personalized breast cancer treatment

Methylation-dependent silencing of CST6 in primary human breast tumors and metastatic lesions

CST6 is a breast tumor suppressor gene that is expressed in normal breast epithelium, but is epigenetically silenced as a consequence of promoter hypermethylation in metastatic breast cancer cell lines. In the current study, we investigated the expression and methylation status of CST6 in primary breast tumors and lymph node metastases. 25/45 (56%) primary tumors and 17/20 (85%) lymph node metastases expressed significantly lower levels of cystatin M compared to normal breast tissue. Bisulfite sequencing demonstrated CST6 promoter hypermethylation in 11/23 (48%) neoplastic lesions analyzed, including 3/11 (27%) primary tumors and 8/12 (67%) lymph node metastases. In most cases (12/23, 52%), the expression of cystatin M directly reflected CST6 promoter methylation status. In remaining lesions (8/23, 35%) loss of cystatin M was not associated with CST6 promoter hypermethylation, indicating that other mechanisms can account for loss of CST6 expression. These results show that methylation-dependent silencing of CST6 occurs in a subset of primary breast cancers, but more frequently in metastatic lesions, possibly reflecting progression-related genomic events. To examine this possibility, primary breast tumors and matched lymph node metastases were analyzed. In 2/3 (67%) patients, primary tumors were positive for cystatin M and negative for CST6 promoter methylation, and matched metastatic lesions lacked cystatin M expression and CST6 was hypermethylated. This observation suggests that progression-related epigenetic alterations in CST6 gene expression can accompany metastatic spread from a primary tumor site. Overall, the results of the current investigation suggest that methylation-dependent epigenetic silencing of CST6 represents an important mechanism for loss of CST6 during breast tumorigenesis and/or progression to metastasis

Dysregulation of microRNA expression drives aberrant DNA hypermethylation in basal-like breast cancer

Basal-like breast cancers frequently express aberrant DNA hypermethylation associated with concurrent silencing of specific genes secondary to DNMT3b overexpression and DNMT hyperactivity. DNMT3b is known to be post-transcriptionally regulated by microRNAs. The objective of the current study was to determine the role of microRNA dysregulation in the molecular mechanism governing DNMT3b overexpression in primary breast cancers that express aberrant DNA hypermethylation. The expression of microRNAs (miRs) that regulate (miR-29a, miR-29b, miR-29c, miR-148a and miR-148b) or are predicted to regulate DNMT3b (miR-26a, miR-26b, miR-203 and miR-222) were evaluated among 70 primary breast cancers (36 luminal A-like, 13 luminal B-like, 5 HER2-enriched, 16 basal-like) and 18 normal mammoplasty tissues. Significantly reduced expression of miR-29c distinguished basal-like breast cancers from other breast cancer molecular subtypes. The expression of aberrant DNA hypermethylation was determined in a subset of 33 breast cancers (6 luminal A-like, 6 luminal B-like, 5 HER2-enriched and 16 basal-like) through examination of methylation-sensitive biomarker gene expression (CEACAM6, CDH1, CST6, ESR1, GNA11, MUC1, MYB, TFF3 and SCNN1A), 11/33 (33%) cancers exhibited aberrant DNA hypermethylation including 9/16 (56%) basal-like cancers, but only 2/17 (12%) non-basal-like cancers (luminal A-like, n=1; HER2-enriched, n=1). Breast cancers with aberrant DNA hypermethylation express diminished levels of miR-29a, miR-29b, miR-26a, miR-26b, miR-148a and miR-148b compared to cancers lacking aberrant DNA hypermethylation. A total of 7/9 (78%) basal-like breast cancers with aberrant DNA hypermethylation exhibit diminished levels of ≥6 regulatory miRs. The results show that i) reduced expression of miR-29c is characteristic of basal-like breast cancers, ii) miR and methylation-sensitive gene expression patterns identify two subsets of basal-like breast cancers, and iii) the subset of basal-like breast cancers with reduced expression of multiple regulatory miRs express aberrant DNA hypermethylation. Together, these findings strongly suggest that the molecular mechanism governing the DNMT3b-mediated aberrant DNA hypermethylation in primary breast cancer involves the loss of post-transcriptional regulation of DNMT3b by regulatory miRs

Epstein-Barr virus-specific methylation of human genes in gastric cancer cells

<p>Abstract</p> <p>Background</p> <p>Epstein-Barr Virus (EBV) is found in 10% of all gastric adenocarcinomas but its role in tumor development and maintenance remains unclear. The objective of this study was to examine EBV-mediated dysregulation of cellular factors implicated in gastric carcinogenesis.</p> <p>Methods</p> <p>Gene expression patterns were examined in EBV-negative and EBV-positive AGS gastric epithelial cells using a low density microarray, reverse transcription PCR, histochemical stains, and methylation-specific DNA sequencing. Expression of PTGS2 (COX2) was measured in AGS cells and in primary gastric adenocarcinoma tissues.</p> <p>Results</p> <p>In array studies, nearly half of the 96 human genes tested, representing 15 different cancer-related signal transduction pathways, were dysregulated after EBV infection. Reverse transcription PCR confirmed significant impact on factors having diverse functions such as cell cycle regulation (<it>IGFBP3</it>, <it>CDKN2A, CCND1, HSP70, ID2, ID4)</it>, DNA repair <it>(BRCA1, TFF1</it>), cell adhesion (<it>ICAM1</it>), inflammation (<it>COX2</it>), and angiogenesis (<it>HIF1A</it>). Demethylation using 5-aza-2'-deoxycytidine reversed the EBV-mediated dysregulation for all 11 genes listed here. For some promoter sequences, CpG island methylation and demethylation occurred in an EBV-specific pattern as shown by bisulfite DNA sequencing. Immunohistochemistry was less sensitive than was western blot for detecting downregulation of COX2 upon EBV infection. Virus-related dysregulation of COX2 levels <it>in vitro </it>was not recapitulated <it>in vivo </it>among naturally infected gastric cancer tissues.</p> <p>Conclusions</p> <p>EBV alters human gene expression in ways that could contribute to the unique pathobiology of virus-associated cancer. Furthermore, the frequency and reversability of methylation-related transcriptional alterations suggest that demethylating agents have therapeutic potential for managing EBV-related carcinoma.</p

Targeted silencing of the oncogenic transcription factor SOX2 in breast cancer

The transcription factor (TF) SOX2 is essential for the maintenance of pluripotency and self-renewal in embryonic stem cells. In addition to its normal stem cell function, SOX2 over-expression is associated with cancer development. The ability to selectively target this and other oncogenic TFs in cells, however, remains a significant challenge due to the 'undruggable' characteristics of these molecules. Here, we employ a zinc finger (ZF)-based artificial TF (ATF) approach to selectively suppress SOX2 gene expression in cancer cells. We engineered four different proteins each composed of 6ZF arrays designed to bind 18 bp sites in the SOX2 promoter and enhancer region, which controls SOX2 methylation. The 6ZF domains were linked to the Kruppel Associated Box (SKD) repressor domain. Three engineered proteins were able to bind their endogenous target sites and effectively suppress SOX2 expression (up to 95% repression efficiencies) in breast cancer cells. Targeted down-regulation of SOX2 expression resulted in decreased tumor cell proliferation and colony formation in these cells. Furthermore, induced expression of an ATF in a mouse model inhibited breast cancer cell growth. Collectively, these findings demonstrate the effectiveness and therapeutic potential of engineered ATFs to mediate potent and long-lasting down-regulation of oncogenic TF expression in cancer cells

Generation of tumor-initiating cells by exogenous delivery of OCT4 transcription factor

Abstract Introduction Tumor-initiating cells (TIC) are being extensively studied for their role in tumor etiology, maintenance and resistance to treatment. The isolation of TICs has been limited by the scarcity of this population in the tissue of origin and because the molecular signatures that characterize these cells are not well understood. Herein, we describe the generation of TIC-like cell lines by ectopic expression of the OCT4 transcription factor (TF) in primary breast cell preparations. Methods OCT4 cDNA was over-expressed in four different primary human mammary epithelial (HMEC) breast cell preparations from reduction mammoplasty donors. OCT4-transduced breast cells (OTBCs) generated colonies (frequency ~0.01%) in self-renewal conditions (feeder cultures in human embryonic stem cell media). Differentiation assays, immunofluorescence, immunohistochemistry, and flow cytometry were performed to investigate the cell of origin of OTBCs. Serial dilutions of OTBCs were injected in nude mice to address their tumorigenic capabilities. Gene expression microarrays were performed in OTBCs, and the role of downstream targets of OCT4 in maintaining self-renewal was investigated by knock-down experiments. Results OTBCs overcame senescence, overexpressed telomerase, and down-regulated p16INK4A . In differentiation conditions, OTBCs generated populations of both myoepithelial and luminal cells at low frequency, suggesting that the cell of origin of some OTBCs was a bi-potent stem cell. Injection of OTBCs in nude mice generated poorly differentiated breast carcinomas with colonization capabilities. Gene expression microarrays of OTBC lines revealed a gene signature that was over-represented in the claudin-low molecular subtype of breast cancer. Lastly, siRNA-mediated knockdown of OCT4 or downstream embryonic targets of OCT4, such as NANOG and ZIC1, suppressed the ability of OTBCs to self-renew. Conclusions Transduction of OCT4 in normal breast preparations led to the generation of cell lines possessing tumor-initiating and colonization capabilities. These cells developed high-grade, poorly differentiated breast carcinomas in nude mice. Genome-wide analysis of OTBCs outlined an embryonic TF circuitry that could be operative in TICs, resulting in up-regulation of oncogenes and loss of tumor suppressive functions. These OTBCs represent a patient-specific model system for the discovery of novel oncogenic targets in claudin-low tumors