Ramana V. Davuluri

A MicroRNA Signature of Hypoxia

ABSTRACT Recent research has identified critical roles for microRNAs in a large number of cellular processes, including tumorigenic transformation. While significant progress has been made towards understanding the mechanisms of gene regulation by microRNAs, much less is known about factors affecting the expression of these noncoding transcripts. Here, we demonstrate for the first time a functional link between hypoxia, a well-documented tumor microenvironment factor, and microRNA expression. Microarray-based expression profiles revealed that a specific spectrum of microRNAs (including miR-23, -24, -26, -27, -103, -107, -181, -210, and -213) is induced in response to low oxygen, at least some via a hypoxia-inducible-factor-dependent mechanism. Select members of this group (miR-26, -107, and -210) decrease proapoptotic signaling in a hypoxic environment, suggesting an impact of these transcripts on tumor formation. Interestingly, the vast majority of hypoxia-induced microRNAs are also overexpressed in a variety of human tumors.

SINGLE NUCLEOTIDE POLYMORPHISMS INSIDE microRNA TARGET SITES INFLUENCE TUMOR SUSCEPTIBILITY

Single-nucleotide polymorphisms (SNP) associated with polygenetic disorders, such as breast cancer (BC), can create, destroy, or modify microRNA (miRNA) binding sites; however, the extent to which SNPs interfere with miRNA gene regulation and affect cancer susceptibility remains largely unknown. We hypothesize that disruption of miRNA target binding by SNPs is a widespread mechanism relevant to cancer susceptibility. To test this, we analyzed SNPs known to be associated with BC risk, in silico and in vitro, for their ability to modify miRNA binding sites and miRNA gene regulation and referred to these as target SNPs. We identified rs1982073-TGFB1 and rs1799782-XRCC1 as target SNPs, whose alleles could modulate gene expression by differential interaction with miR-187 and miR-138, respectively. Genome-wide bioinformatics analysis predicted approximately 64% of transcribed SNPs as target SNPs that can modify (increase/decrease) the binding energy of putative miRNA::mRNA duplexes by >90%. To assess whether target SNPs are implicated in BC susceptibility, we conducted a case-control population study and observed that germline occurrence of rs799917-BRCA1 and rs334348-TGFR1 significantly varies among populations with different risks of developing BC. Luciferase activity of target SNPs, allelic variants, and protein levels in cancer cell lines with different genotypes showed differential regulation of target genes following overexpression of the two interacting miRNAs (miR-638 and miR-628-5p). Therefore, we propose that transcribed target SNPs alter miRNA gene regulation and, consequently, protein expression, contributing to the likelihood of cancer susceptibility, by a novel mechanism of subtle gene regulation.

The functional consequences of alternative promoter use in mammalian genomes

We are beginning to appreciate the increasing complexity of mammalian gene structure. A phenomenon that adds an important dimension to this complexity is the use of alternative gene promoters that drive widespread cell type, tissue type or developmental gene regulation. Recent annotations of the human genome suggest that almost one half of the protein-coding genes contain alternative promoters, including those of many disease-associated genes. Aberrant use of one promoter over another has been found to be associated with various diseases, including cancer. Here we discuss the functional consequences of use and misuse of alternative promoters in normal and disease genomes and review the molecular mechanisms regulating alternative promoter use in mammalian genomes.

A microRNA component of the hypoxic response

microRNAs participate in a wide variety of physiological and pathological cellular processes. Recent studies have established a link between a specific group of microRNAs and hypoxia, a key feature of the neoplastic microenvironment. A significant proportion of the hypoxia-regulated microRNAs (HRMs) are also overexpressed in human cancers, suggesting a role in tumorigenesis. Preliminary evidence suggests that they could affect important processes such as apoptosis, proliferation and angiogenesis. Several HRMs exhibit induction in response to HIF activation, thus extending its repertoire of targets beyond translated genes. In the present review, we discuss the emerging roles of HRMs in oxygen deprivation in cancer context.

AGRIS and AtRegNet. A Platform to Link cis-Regulatory Elements and Transcription Factors into Regulatory Networks

Gene regulatory pathways converge at the level of transcription, where interactions among regulatory genes and between regulators and target genes result in the establishment of spatiotemporal patterns of gene expression. The growing identification of direct target genes for key transcription factors (TFs) through traditional and high-throughput experimental approaches has facilitated the elucidation of regulatory networks at the genome level. To integrate this information into a Web-based knowledgebase, we have developed the Arabidopsis Gene Regulatory Information Server (AGRIS). AGRIS, which contains all Arabidopsis (Arabidopsis thaliana) promoter sequences, TFs, and their target genes and functions, provides the scientific community with a platform to establish regulatory networks. AGRIS currently houses three linked databases: AtcisDB (Arabidopsis thaliana cis-regulatory database), AtTFDB (Arabidopsis thaliana transcription factor database), and AtRegNet (Arabidopsis thaliana regulatory network). AtTFDB contains 1,690 Arabidopsis TFs and their sequences (protein and DNA) grouped into 50 (October 2005) families with information on available mutants in the corresponding genes. AtcisDB consists of 25,806 (September 2005) promoter sequences of annotated Arabidopsis genes with a description of putative cis-regulatory elements. AtRegNet links, in direct interactions, several hundred genes with the TFs that control their expression. The current release of AtRegNet contains a total of 187 (September 2005) direct targets for 66 TFs. AGRIS can be accessed at http://Arabidopsis.med.ohio-state.edu.

miR-145 participates with TP53 in a death-promoting regulatory loop and targets estrogen receptor- α in human breast cancer cells

Understanding the consequences of miR-145 reintroduction in human breast cancer (BC) could reveal its tumor-suppressive functions and may disclose new aspects of BC biology. Therefore, we characterized the effects of miR-145 re-expression in BC cell lines by using proliferation and apoptosis assays. As a result, we found that miR-145 exhibited a pro-apoptotic effect, which is dependent on TP53 activation, and that TP53 activation can, in turn, stimulate miR-145 expression, thus establishing a death-promoting loop between miR-145 and TP53. We also found that miR-145 can downregulate estrogen receptor-α (ER-α) protein expression through direct interaction with two complementary sites within its coding sequence. In conclusion, we described a tumor suppression function of miR-145 in BC cell lines, and we linked miR-145 to TP53 and ER-α. Moreover, our findings support a view that miR-145 re-expression therapy could be mainly envisioned in the specific group of patients with ER-α-positive and/or TP53 wild-type tumors.

NF-κB Regulation of YY1 Inhibits Skeletal Myogenesis through Transcriptional Silencing of Myofibrillar Genes

ABSTRACT NF-κB signaling is implicated as an important regulator of skeletal muscle homeostasis, but the mechanisms by which this transcription factor contributes to muscle maturation and turnover remain unclear. To gain insight into these mechanisms, gene expression profiling was examined in C2C12 myoblasts devoid of NF-κB activity. Interestingly, even in proliferating myoblasts, the absence of NF-κB caused the pronounced induction of several myofibrillar genes, suggesting that NF-κB functions as a negative regulator of late-stage muscle differentiation. Although several myofibrillar promoters contain predicted NF-κB binding sites, functional analysis using the troponin-I2 gene as a model revealed that NF-κB-mediated repression does not occur through direct DNA binding. In the search for an indirect mediator, the transcriptional repressor YinYang1 (YY1) was identified. While inducers of NF-κB stimulated YY1 expression in multiple cell types, genetic ablation of the RelA/p65 subunit of NF-κB in both cultured cells and adult skeletal muscle correlated with reduced YY1 transcripts and protein. NF-κB regulation of YY1 occurred at the transcriptional level, mediated by direct binding of the p50/p65 heterodimer complex to the YY1 promoter. Furthermore, YY1 was found associated with multiple myofibrillar promoters in C2C12 myoblasts containing NF-κB activity. Based on these results, we propose that NF-κB regulation of YY1 and transcriptional silencing of myofibrillar genes represent a new mechanism by which NF-κB functions in myoblasts to modulate skeletal muscle differentiation.

Epigenetic Profiling in Chronic Lymphocytic Leukemia Reveals Novel Methylation Targets

CpG island methylation is an epigenetic alteration that contributes to tumorigenesis by transcriptional inactivation of genes. Little is known about the overall levels of CpG island methylation in chronic lymphocytic leukemia (CLL). To provide a baseline estimate of global aberrant methylation and identify target sequences for additional investigation, we performed Restriction Landmark Genomic Scanning on 10 CLL samples. Two methylation-sensitive landmark enzymes were used (NotI and AscI), allowing assessment of over 3000 CpG islands in each sample. Tumor-derived Restriction Landmark Genomic Scanning profiles were compared with profiles from CD19-selected B cells from normal volunteers and matched normal neutrophils from 4 CLL patients. We found 2.5–8.1% (mean 4.8%) of the CpG islands in CLL samples were aberrantly methylated compared with controls, and the methylation events had a nonrandom distribution (P < 0.0001). Furthermore, we identified 193 aberrantly methylated sequences, of which 93% have CpG island characteristics and 90% have homology to genes or expressed sequences. One such gene, the G protein-coupled metabotropic glutamate receptor 7 (GRM7), possibly inhibits cyclic AMP signaling in the induction of apoptosis. Bisulfite sequencing of GRM7 confirmed extensive CpG island methylation, and treatment with 5-aza-2′-deoxycytidine (decitabine) resulted in up-regulated expression of several genes in vitro with concurrent cellular depletion of DNMT1 protein. Our dual-enzyme global methylation study shows that CLL is characterized by widespread nonrandom CpG island methylation similar to other tumors and provides a panel of novel methylation targets that can be used in larger studies designed to assess impact on disease progression and survival.

Regulation of microRNA Expression The Hypoxic Component

microRNAs are involved in a wide variety of normal and pathological cellular processes, including tumorigenic transformation. Despite significant progress made towards understanding their mechanisms of action, much less is known about the regulation of expression of specific microRNAs. Recent reports have established a link between hypoxia, a key feature of the tumor microenvironment, and a group of microRNAs. Select members of this group seem to affect apoptotic signaling in a hypoxic environment and are also predicted to target genes of critical importance for tumor biology. Interestingly, most hypoxia-induced microRNAs are also overexpressed in human cancers, suggesting a role in tumorigenesis. We hereby discuss the known and predicted regulators of microRNA expression and approaches for expanding this fledgling research area.

Prognostic DNA Methylation Biomarkers in Ovarian Cancer

Purpose: Aberrant DNA methylation, now recognized as a contributing factor to neoplasia, often shows definitive gene/sequence preferences unique to specific cancer types. Correspondingly, distinct combinations of methylated loci can function as biomarkers for numerous clinical correlates of ovarian and other cancers. Experimental Design: We used a microarray approach to identify methylated loci prognostic for reduced progression-free survival (PFS) in advanced ovarian cancer patients. Two data set classification algorithms, Significance Analysis of Microarray and Prediction Analysis of Microarray, successfully identified 220 candidate PFS-discriminatory methylated loci. Of those, 112 were found capable of predicting PFS with 95% accuracy, by Prediction Analysis of Microarray, using an independent set of 40 advanced ovarian tumors (from 20 short-PFS and 20 long-PFS patients, respectively). Additionally, we showed the use of these predictive loci using two bioinformatics machine-learning algorithms, Support Vector Machine and Multilayer Perceptron. Conclusion: In this report, we show that highly prognostic DNA methylation biomarkers can be successfully identified and characterized, using previously unused, rigorous classifying algorithms. Such ovarian cancer biomarkers represent a promising approach for the assessment and management of this devastating disease.