Elucidating the Altered Transcriptional Programs in Breast Cancer using Independent Component Analysis

The quantity of mRNA transcripts in a cell is determined by a complex interplay of cooperative and counteracting biological processes. Independent Component Analysis (ICA) is one of a few number of unsupervised algorithms that have been applied to microarray gene expression data in an attempt to understand phenotype differences in terms of changes in the activation/inhibition patterns of biological pathways. While the ICA model has been shown to outperform other linear representations of the data such as Principal Components Analysis (PCA), a validation using explicit pathway and regulatory element information has not yet been performed. We apply a range of popular ICA algorithms to six of the largest microarray cancer datasets and use pathway-knowledge and regulatory-element databases for validation. We show that ICA outperforms PCA and clustering-based methods in that ICA components map closer to known cancer-related pathways, regulatory modules, and cancer phenotypes. Furthermore, we identify cancer signalling and oncogenic pathways and regulatory modules that play a prominent role in breast cancer and relate the differential activation patterns of these to breast cancer phenotypes. Importantly, we find novel associations linking immune response and epithelial–mesenchymal transition pathways with estrogen receptor status and histological grade, respectively. In addition, we find associations linking the activity levels of biological pathways and transcription factors (NF1 and NFAT) with clinical outcome in breast cancer. ICA provides a framework for a more biologically relevant interpretation of genomewide transcriptomic data. Adopting ICA as the analysis tool of choice will help understand the phenotype–pathway relationship and thus help elucidate the molecular taxonomy of heterogeneous cancers and of other complex genetic diseases

ATM in focus:a damage sensor and cancer target

The ability of a cell to conserve and maintain its native DNA sequence is fundamental for the survival and normal functioning of the whole organism and protection from cancer development. Here we review recently obtained results and current topics concerning the role of the ataxia-telangiectasia mutated (ATM) protein kinase as a damage sensor and its potential as therapeutic target for treating cancer. This monograph discusses DNA repair mechanisms activated after DNA double-strand breaks (DSBs), i.e. non-homologous end joining, homologous recombination and single strand annealing and the role of ATM in the above types of repair. In addition to DNA repair, ATM participates in a diverse set of physiological processes involving metabolic regulation, oxidative stress, transcriptional modulation, protein degradation and cell proliferation. Full understanding of the complexity of ATM functions and the design of therapeutics that modulate its activity to combat diseases such as cancer necessitates parallel theoretical and experimental efforts. This could be best addressed by employing a systems biology approach, involving mathematical modelling of cell signalling pathways

Characterization of a tumour suppressor function of RanBPM

Ran-binding protein M (RanBPM) is an evolutionarily conserved nucleocytosolic protein that has been proposed to regulate various cellular processes, including protein stability, gene expression, receptor-mediated signalling pathways, cell adhesion, development, and apoptosis. Despite the multitude of functions attributed to RanBPM however, little is known regarding the precise mechanisms by which RanBPM executes these cellular roles. In this work, we seek to address this matter by describing functions for RanBPM in the regulation of apoptotic and pro-survival signalling pathways, and in cellular transformation. We first identify RanBPM as a pro-apoptotic protein that regulates the activation of the intrinsic apoptotic signalling pathway in response to DNA damage. We show that RanBPM executes its pro-apoptotic functions by modulating the expression and localization of Bcl-2 family proteins. Next, we demonstrate that RanBPM functions as a novel inhibitor of the ERK1/2 signalling cascade, and that RanBPM regulates the expression of Bcl-2 factors through repression of this pathway. We also extend these analyses to show that RanBPM forms a complex with c-Raf, and that it prevents aberrant ERK1/2 signalling by destabilizing the c-Raf-Hsp90 complex, thus maintaining low cellular c-Raf expression. Our studies also implicate an important function for RanBPM in the regulation of gene expression programs. We find that disruption of RanBPM expression affects transcriptional networks involved in the regulation of organism development and tumourigenesis, and that decreased RanBPM levels alter the expression of factors involved in signal transduction through the Notch, Wnt, PI3K, and ERK1/2 pathways. Importantly, our work also reveals that the down-regulation of RanBPM expression is associated with the acquisition of markers of cellular transformation, specifically evasion from apoptosis, sustained proliferative signalling, and increased cellular migration and invasion, suggesting a novel tumour suppressor function for RanBPM. Taken together, our studies provide insight into the molecular mechanisms by which may RanBPM mediate its diverse biological functions, and reveal that altered RanBPM expression may have important ramifications in the regulation of organism development and disease pathogenesis

Epithelial to mesenchymal transition and breast cancer

Epithelial-mesenchymal plasticity in breast carcinoma encompasses the phenotypic spectrum whereby epithelial carcinoma cells within a primary tumor acquire mesenchymal features and re-epithelialize to form a cohesive secondary mass at a metastatic site. Such plasticity has implications in progression of breast carcinoma to metastasis, and will likely influence response to therapy. The transcriptional and epigenetic regulation of molecular and cellular processes that underlie breast cancer and result in characteristic changes in cell behavior can be monitored using an increasing array of marker proteins. Amongst these markers exists the potential for emergent prognostic, predictive and therapeutic targeting

Connexins: synthesis, post-translational modifications, and trafficking in health and disease

Connexins are tetraspan transmembrane proteins that form gap junctions and facilitate direct intercellular communication, a critical feature for the development, function, and homeostasis of tissues and organs. In addition, a growing number of gap junction-independent functions are being ascribed to these proteins. The connexin gene family is under extensive regulation at the transcriptional and post-transcriptional level, and undergoes numerous modifications at the protein level, including phosphorylation, which ultimately affects their trafficking, stability, and function. Here, we summarize these key regulatory events, with emphasis on how these affect connexin multifunctionality in health and disease

The role of the arginine methyltransferase CARM1 in global transcriptional regulation.

Arginine methylation is a prevalent post-translational modification that is found on many nuclear and cytoplasmic proteins, and has been implicated in the regulation of gene expression. CARM1 is a member of the protein arginine methyltransferase (PRMT) family of proteins, and is a key protein responsible for arginine methylation of a subset of proteins involved in transcription. In this thesis I examine some of the mechanisms through which CARM1 contributes to global transcriptional regulation. Using a ChIP-DSL approach, we show that the p/CIP/CARM1 complex is recruited to 204 proximal promoters following 17β-estradiol (E2) treatment in MCF-7 cells. Many of the target genes have been previously implicated in signaling pathways related to oncogenesis. JAK2, a member of the Jak/Stat signaling cascade, is one of the direct E2-dependent targets of the p/CIP/CARM1 complex. Following E2-treatment, histone modifications at the JAK2 promoter are reflective of a transcriptionally permissive gene, and we observed modest increases in RNA and protein expression. Notably, E2-induced expression of Jak2 was diminished when p/CIP or CARM1 were depleted, suggesting that the p/CIP/CARM1 complex is required for the observed transcriptional response. Collectively, these results suggest that E2-dependent recruitment of the p/CIP/CARM1 complex causes JAK2 to become ‘poised’ for transcription, a finding that may be extendable to other target genes and signalling pathways. Furthermore, bioinformatic examination of p/CIP/CARM1 target promoters suggests that transcription factor crosstalk is the favored mechanism of E2-dependent p/CIP/CARM1 complex recruitment. Using ChIP-Seq, we identified genomic regions to which CARM1 is recruited. Subsequent characterization of binding events suggest a role for CARM1 in transcriptional elongation, and implicate the transcription factor PAX1 as a novel mechanism through which CARM1 can be recruited to the genome. Identification of CARM1-dependent differentially expressed genes revealed that direct recruitment of CARM1 is not essential for the majority of its transcriptional effects in MEFs. However, CARM1 does play a critical role in cellular growth and proliferation, and in the absence of CARM1, the expression of many cell cycle regulators is dramatically affected. Collectively, this work provides insight into some of the mechanisms through which CARM1 modulates transcription, and highlights its importance in diverse cellular processes

Identification and Characterization of the P53-Induced Long Noncoding RNA Isoform Pvt1b and Its Role in Stress-Specific Growth Inhibition via Myc Repression

The tumor suppressor p53 and proto-oncogenic Myc transcription factors are frequently deregulated in cancer, with common loss-of-function and gain-of-function mutations observed in the p53 and Myc networks, respectively. Referred to as the ‘guardian of the genome,’ p53 regulates genes important for curtailing cellular proliferation and tumorigenesis under conditions of stress, while the proto-oncogene Myc induces genes that, in contrast, promote cellular growth and can, in overcoming growth inhibitory signals, support cancer development. While previous literature has documented decreased Myc expression in response to cellular stress, researchers have long puzzled over identifying the specific regulatory lever responsible. The work presented here identifies a novel regulatory axis positioned at the intersection of the p53 and Myc pathways, which represses Myc and restricts cellular proliferation downstream of p53 activation. Long noncoding RNAs (lncRNAs) are a diverse class of transcripts lacking protein-coding potential and implicated in gene expression regulation. Here I present my work on the identification of an isoform of the lncRNA Plasmacytoma variant translocation 1 (Pvt1) and the characterization of its role in the p53-mediated response to stress. I found that the stress-specific Pvt1b, expressed 50 Kb downstream of the Myc locus, is induced by p53 in response to oncogenic and genotoxic stress and accumulates at its site of transcription. I demonstrated that production of the Pvt1b RNA is necessary and sufficient to repress Myc transcription in cis without altering the chromatin organization of the locus. I investigated the functional outputs of Pvt1b-mediated Myc downregulation and found that inhibition of Pvt1b increased both Myc levels and transcriptional activity and promoted cellular proliferation. Notably, Pvt1b loss accelerated tumor growth, but not tumor progression, in an autochthonous mouse model of lung cancer. Further examination of the Pvt1b mechanism of action failed to identify Pvt1b-specific sequences required for its function, but uncovered a potential role for histone deacetylation in Pvt1b regulation of Myc. Finally, I initiated development of a suite of genetically engineered Pvt1 mouse models, the characterization of which will shed light on Pvt1 function in vivo and benefit future mechanistic studies. Taken together, this work conceptually advances our understanding of stress-induced growth inhibition orchestrated by p53. Specifically, I identify Pvt1b as the primary mediator of stress-specific Myc repression, providing insight into the long-standing question of how p53 activation triggers Myc downregulation. As such, this work has far-reaching implications not only for our understanding of cis-acting lncRNAs, which can fine-tune local gene expression downstream of broadly active transcription programs, but also for the exciting therapeutic possibility of restricting Myc levels in cancer via Pvt1b modulation

Genome-wide characterization of cytosine-specific 5-hydroxymethylation in normal breast tissue.

Despite recent evidence that 5-hydroxymethylcytosine (5hmC) possesses roles in gene regulation distinct from 5-methylcytosine (5mC), relatively little is known regarding the functions of 5hmC in mammalian tissues. To address this issue, we utilized an approach combining both paired bisulfite (BS) and oxidative bisulfite (oxBS) DNA treatment, to resolve genome-wide patterns of 5hmC and 5mC in normal breast tissue from disease-free women. Although less abundant than 5mC, 5hmC was differentially distributed, and consistently enriched among breast-specific enhancers and transcriptionally active chromatin. In contrast, regulatory regions associated with transcriptional inactivity, such as heterochromatin and repressed Polycomb regions, were relatively depleted of 5hmC. Gene regions containing abundant 5hmC were significantly associated with lactate oxidation, immune cell function, and prolactin signaling pathways. Furthermore, genes containing abundant 5hmC were enriched among those actively transcribed in normal breast tissue. Finally, in independent data sets, normal breast tissue 5hmC was significantly enriched among CpG loci demonstrated to have altered methylation in pre-invasive breast cancer and invasive breast tumors. Primarily, our findings identify genomic loci containing abundant 5hmC in breast tissues and provide a genome-wide map of nucleotide-level 5hmC in normal breast tissue. Additionally, these data suggest 5hmC may participate in gene regulatory programs that are dysregulated during breast-related carcinogenesis