Challenges in studying the etiology of breast cancer subtypes

Research that classifies breast tumors into homogenous subgroups could ultimately help to define public health prevention strategies for aggressive breast cancer subtypes. However, etiologic research on molecular breast cancer subtypes must overcome several challenges. Stratifying breast cancers into subgroups can reduce statistical power and, therefore, may require non-traditional analytical methods. Integrating results across studies is hampered by varying definitions of molecular subtypes, with some studies using triple negative status and others using specific markers to define basal-like cancers. In addition, triple negative and basal-like breast cancers appear to show strong associations with race, so the varied racial and ethnic composition of different datasets can make comparison across studies challenging. In spite of these challenges, some strong and consistent associations between triple negative or basal-like breast cancer and demographic variables are emerging, and there are hints that prevention strategies for this aggressive subtype of breast cancer may also be attainable

Genetic Polymorphisms Regulating Dopamine Signaling in the Frontal Cortex Interact to Affect Target Detection under High Working Memory Load

Frontal-dependent task performance is typically modulated by dopamine (DA) according to an inverted-U pattern, whereby intermediate levels of DA signaling optimizes performance. Numerous studies implicate trait differences in DA signaling based on differences in the catechol-O-methyltransferase (COMT) gene in executive function task performance. However, little work has investigated genetic variations in DA signaling downstream from COMT. One candidate is the dopamine- and cAMP-regulated phosphoprotein of molecular weight 32 kDa (DARPP-32), which mediates signaling through the DA D1-type receptor, the dominant DA receptor in the frontal cortex. Using an n-back task, we used signal detection theory to measure performance in a healthy adult population (n=97) genotyped for single nucleotide polymorphisms in the COMT (rs4680) and DARPP-32 (rs907094) genes. Correct target detection (hits), and false alarms were used to calculate d' measures for each working memory load (0-, 2-, and 3-back). At the highest load (3-back) only, we observed a significant COMT×DARPP-32 interaction, such that the DARPP-32 T/T genotype enhanced target detection in COMTValVal individuals, but impaired target detection in COMTMet carriers. These findings suggest that enhanced dopaminergic signaling via the DARPP-32 T allele aids target detection in individuals with presumed low frontal DA (COMTValVal) but impairs target detection in those with putatively higher frontal DA levels (COMTMet carriers). Moreover, these data support an inverted-U model with intermediate levels of DA signaling optimizing performance on tasks requiring maintenance of mental representations in working memory

Comprehensive interrogation of CpG island methylation in the gene encoding COMT, a key estrogen and catecholamine regulator

Abstract Background The catechol-O-methyltransferase (COMT) enzyme has been widely studied due to its multiple roles in neurological functioning, estrogen biology, and methylation metabolic pathways. Numerous studies have investigated variation in the large COMT gene, with the majority focusing on single nucleotide polymorphisms (SNPs). This body of work has linked COMT genetic variation with a vast array of conditions, including several neurobehavioral disorders, pain sensitivity, and multiple human cancers. Based on COMT’s numerous biological roles and recent studies suggesting that methylation of the COMT gene impacts COMT gene expression, we comprehensively interrogated methylation in over 200 CpG dinucleotide sequences spanning the length of the COMT gene. Methods Using saliva-derived DNA from a non-clinical sample of human subjects, we tested for associations between COMT CpG methylation and factors reported to interact with COMT genetic effects, including demographic factors and alcohol use. Finally, we tested associations between COMT CpG methylation state and COMT gene expression in breast cancer cell lines. We interrogated >200 CpGs in 13 amplicons spanning the 5’ UTR to the last exon of the CpG dinucleotide-rich COMT gene in n = 48 subjects, n = 11 cell lines and 1 endogenous 18S rRNA control. Results With the exception of the CpG island in the 5’UTR and 1st exon, all other CpG islands were strongly methylated with typical dynamic ranges between 50-90%. In the saliva samples, methylation of multiple COMT loci was associated with socioeconomic status or ethnicity. We found associations between methylation at numerous loci and genotype at the functional Val 158 Met SNP (rs4680), and most of the correlations between methylation and demographic and alcohol use factors were Val 158 Met allele-specific. Methylation at several of these loci also associated with COMT gene expression in breast cancer cell lines. Conclusions We report the first comprehensive interrogation of COMT methylation. We corroborate previous findings of variation in COMT methylation with gene expression and the Val 158 Met genotype, and also report novel associations with socioeconomic status (SES) and ethnicity at several methylated loci. These results point to novel mechanisms for COMT regulation, which may have broad therapeutic implications

Stable oncogenic silencing in vivo by programmable and targeted de novo DNA methylation in breast cancer

With the recent comprehensive mapping of cancer genomes, there is now a need for functional approaches to edit the aberrant epigenetic state of key cancer drivers to reprogram the epi-pathology of the disease. In this study we utilized a programmable DNA-binding methyltransferase to induce targeted incorporation of DNA methylation (DNAme) in the SOX2 oncogene in breast cancer through a six zinc finger (ZF) protein linked to DNA methyltransferase 3A (ZF-DNMT3A). We demonstrated long-lasting oncogenic repression, which was maintained even after suppression of ZF-DNMT3A expression in tumor cells. The de novo DNAme was faithfully propagated and maintained through cell generations even after the suppression of the expression of the chimeric methyltransferase in the tumor cells. Xenograft studies in NUDE mice demonstrated stable SOX2 repression and long-term breast tumor growth inhibition, which lasted for >100 days post implantation of the tumor cells in mice. This was accompanied with a faithful maintenance of DNAme in the breast cancer implants. In contrast, downregulation of SOX2 by ZF domains engineered with the Krueppel-associated box repressor domain resulted in a transient and reversible suppression of oncogenic gene expression. Our results indicated that targeted de novo DNAme of the SOX2 oncogenic promoter was sufficient to induce long-lasting epigenetic silencing, which was not only maintained during cell division but also significantly delayed the tumorigenic phenotype of cancer cells in vivo, even in the absence of treatment. Here, we outline a genome-based targeting approach to long-lasting tumor growth inhibition with potential applicability to many other oncogenic drivers that are currently refractory to drug design

Waking up dormant tumor suppressor genes with zinc fingers, TALEs and the CRISPR/dCas9 system

The aberrant epigenetic silencing of tumor suppressor genes (TSGs) plays a major role during carcinogenesis and regaining these dormant functions by engineering of sequence-specific epigenome editing tools offers a unique opportunity for targeted therapies. However, effectively normalizing the expression and regaining tumor suppressive functions of silenced TSGs by artificial transcription factors (ATFs) still remains a major challenge. Herein we describe novel combinatorial strategies for the potent reactivation of two class II TSGs, MASPIN and REPRIMO, in cell lines with varying epigenetic states, using the CRISPR/dCas9 associated system linked to a panel of effector domains (VP64, p300, VPR and SAM complex), as well as with protein-based ATFs, Zinc Fingers and TALEs. We found that co-delivery of multiple effector domains using a combination of CRISPR/dCas9 and TALEs or SAM complex maximized activation in highly methylated promoters. In particular, CRISPR/dCas9 VPR with SAM upregulated MASPIN mRNA (22,145-fold change) in H157 lung cancer cells, with accompanying re-expression of MASPIN protein, which led to a concomitant inhibition of cell proliferation and induction of apoptotic cell death. Consistently, CRISPR/dCas9 VP64 with SAM upregulated REPRIMO (680-fold change), which led to phenotypic reprogramming in AGS gastric cancer cells. Altogether, our results outlined novel sequence-specific, combinatorial epigenome editing approaches to reactivate highly methylated TSGs as a promising therapy for cancer and other diseases

Differential methylation relative to breast cancer subtype and matched normal tissue reveals distinct patterns

Due to the heterogeneous nature of breast cancer and the widespread use of single-gene studies, there is limited knowledge of multi-gene, locus-specific DNA methylation patterns in relation to molecular subtype and clinical features. We, therefore, quantified DNA methylation of 70 candidate gene loci in 140 breast tumors and matched normal tissues and determined associations with gene expression and tumor subtype. Using Sequenom’s EpiTYPER platform, approximately 1,200CpGs were interrogated and revealed six DNA methylation patterns in breast tumors relative to matched normal tissue. Differential methylation of several gene loci was observed within all molecular subtypes, while other patterns were subtype-dependent. Methylation of numerous gene loci was inversely correlated with gene expression, and in some cases, this correlation was only observed within specific breast tumor subtypes. Our findings were validated on a larger set of tumors and matched adjacent normal tissue from The Cancer Genome Atlas dataset, which utilized methylation data derived from both Illumina Infinium 27 and 450k arrays. These findings highlight the need to control for subtype when interpreting DNA methylation results, and the importance of interrogating multiple CpGs across varied gene regions.Electronic supplementary materialThe online version of this article (doi:10.1007/s10549-013-2738-0) contains supplementary material, which is available to authorized users

Antimetastatic gene expression profiles mediated by retinoic acid receptor beta 2 in MDA-MB-435 breast cancer cells

BACKGROUND: The retinoic acid receptor beta 2 (RARβ2) gene modulates proliferation and survival of cultured human breast cancer cells. Previously we showed that ectopic expression of RARβ2 in a mouse xenograft model prevented metastasis, even in the absence of the ligand, all-trans retinoic acid. We investigated both cultured cells and xenograft tumors in order to delineate the gene expression profiles responsible for an antimetastatic phenotype. METHODS: RNA from MDA-MB-435 human breast cancer cells transduced with RARβ2 or empty retroviral vector (LXSN) was analyzed using Agilent Human 1A Oligo microarrays. The one hundred probes with the greatest differential intensity (p < 0.004, jointly) were determined by selecting the top median log ratios from eight-paired microarrays. Validation of differences in expression was done using Northern blot analysis and quantitative RT-PCR (qRT-PCR). We determined expression of selected genes in xenograft tumors. RESULTS: RARβ2 cells exhibit gene profiles with overrepresentation of genes from Xq28 (p = 2 × 10(-8)), a cytogenetic region that contains a large portion of the cancer/testis antigen gene family. Other functions or factors impacted by the presence of exogenous RARβ2 include mediators of the immune response and transcriptional regulatory mechanisms. Thirteen of fifteen (87%) of the genes evaluated in xenograft tumors were consistent with differences we found in the cell cultures (p = 0.007). CONCLUSION: Antimetastatic RARβ2 signalling, direct or indirect, results in an elevation of expression for genes such as tumor-cell antigens (CTAG1 and CTAG2), those involved in innate immune response (e.g., RIG-I/DDX58), and tumor suppressor functions (e.g., TYRP1). Genes whose expression is diminished by RARβ2 signalling include cell adhesion functions (e.g, CD164) nutritional or metabolic processes (e.g., FABP6), and the transcription factor, JUN