Clinical applications of microRNAs

MicroRNAs represent a class of small RNAs derived from polymerase II controlled transcriptional regions. The primary transcript forms one or several bulging double stranded hairpins which are processed by Drosha and Dicer into hetero-duplexes. The targeting microRNA strand of the duplex is incorporated into the RNA Induced Silencing Complex from where it silences up to hundreds of mRNA transcript by inducing mRNA degradation or blocking protein translation. Apart from involvement in a variety of biological processes, microRNAs were early recognized for their potential in disease diagnostics and therapeutics. Due to their stability, microRNAs could be used as biomarkers. Currently, there are microRNA panels helping physicians determining the origins of cancer in disseminated tumors. The development of microRNA therapeutics has proved more challenging mainly due to delivery issues. However, one drug is already in clinical trials and several more await entering clinical phases. This review summarizes what has been recognized pre-clinically and clinically on diagnostic microRNAs. In addition, it highlights individual microRNA drugs in running platforms driven by four leading microRNA-therapeutic companies

Not only P-glycoprotein: amplification of the ABCB1-containing chromosome region 7q21 confers multidrug resistance upon cancer cells by coordinated overexpression of an assortment of resistance-related proteins

The development of drug resistance continues to be a dominant hindrance toward curative cancer treatment. Overexpression of a wide-spectrum of ATP-dependent efflux pumps, and in particular of ABCB1 (P-glycoprotein or MDR1) is a well-known resistance mechanism for a plethora of cancer chemotherapeutics including for example taxenes, anthracyclines, Vinca alkaloids, and epipodopyllotoxins, demonstrated by a large array of published papers, both in tumor cell lines and in a variety of tumors, including various solid tumors and hematological malignancies. Upon repeated or even single dose treatment of cultured tumor cells or tumors in vivo with anti-tumor agents such as paclitaxel and doxorubicin, increased ABCB1 copy number has been demonstrated, resulting from chromosomal amplification events at 7q11.2-21 locus, leading to marked P-glycoprotein overexpression, and multidrug resistance (MDR). Clearly however, additional mechanisms such as single nucleotide polymorphisms (SNPs) and epigenetic modifications have shown a role in the overexpression of ABCB1 and of other MDR efflux pumps. However, notwithstanding the design of 4 generations of ABCB1 inhibitors and the wealth of information on the biochemistry and substrate specificity of ABC transporters, translation of this vast knowledge from the bench to the bedside has proven to be unexpectedly difficult. Many studies show that upon repeated treatment schedules of cell cultures or tumors with taxenes and anthracyclines as well as other chemotherapeutic drugs, amplification, and/or overexpression of a series of genes genomically surrounding the ABCB1 locus, is observed. Consequently, altered levels of other proteins may contribute to the establishment of the MDR phenotype, and lead to poor clinical outcome. Thus, the genes contained in this ABCB1 amplicon including ABCB4, SRI, DBF4, TMEM243, and RUNDC3B are overexpressed in many cancers, and especially in MDR tumors, while TP53TG1 and DMTF1 are bona fide tumor suppressors. This review describes the role of these genes in cancer and especially in the acquisition of MDR, elucidates possible connections in transcriptional regulation (co-amplification/repression) of genes belonging to the same ABCB1 amplicon region, and delineates their novel emerging contributions to tumor biology and possible strategies to overcome cancer MDR

GENETIC CHARACTERIZATION OF MICRORNAS AND THEIR POTENTIAL AS BIOMARKERS IN HEPATOCELLULAR CARCINOMA

Ph.DDOCTOR OF PHILOSOPH

The interplay of genetic variation and regulation of long noncoding RNAs in colorectal cancer

Background: Colorectal cancer (CRC) is the third leading cause of death worldwide comprising ~8% of cancer deaths per year. The survival rates of metastatic CRC is 13% because of the lack of successful treatment due to a lack of understanding of the scope and environment of stage IV CRC. Materials and Methods: The transcripts of five normal colon mucosa tissue samples and their matched five stage IV CRC samples were chosen and analyzed from the dataset with the GEO Accession GSE50760. The Tuxedo Suite pipeline was used to determine the differentially expressed genes (DEGs) with a fold change cut off of 0.5 and -0.5 and a p value cut off of 0.05. Using the the DEG list, PANTHER database was used for pathway enrichment. LncRNA2Target database was used to find associated long non coding RNAs (lncRNAs) of the genes of interest. The Integrated Genome Viewer (IGV) was used to visualize any mutations or variations among the genes of interest. DeepSEA was used to functionally predict any potential novel SNPs. Using literature along with the results from PANTHER, lncRNA2Target, and IGV a novel connection was deduced. Results: There were 5,303 DEGs. The Wnt pathway had the greatest portion of DEGs indicating pathway activity. Interestingly, a number of inhibitors of the Wnt pathway were also upregulated including WIF1 and SFRP4. LncRNA2Target analysis showed that HOTAIR, a lncRNA, has a number of target genes and effectively silenced all of its targets except for WIF1 and CD82 in this dataset. WIF1, CD82, and SFRP4 has increased fold change values of 5.165, 1.05, 2.121, respectively. Additionally, lncRNAs, UCA1 and CRNDE, were found to positively regulate WNT5A, WNT2 and WNT3 and were upregulated. Using the integrative genome viewer, 10 SNPs were found in WIF1, SFRP4, CD82, WNT5A, and UCA1 of which one was novel. The potentially novel SNP in CD82 was functionally predicted to create a binding site with ZBTB7A. Additionally, CDKN2A and CDKN2B were found to have decreased expression with a fold change value of -2.266. Discussion: One synonymous SNP was in WIF1 and CD82. The missense SNPs in SFRP4 and CD82 are likely causing protein dysfunction resulting in ill-inhibition of the WNT pathway and metastasis, respectively. The novel SNP was found in CD82 at the location chr11:44,619,242 in the 3’ untranslated region. Functional prediction showed that this SNP may create a binding site with ZBTB7A which may be repressing CD82 function. Although most of the SNPs found were recorded to result in synonymous codons, the prevalence and frequency of these SNPs in these vital genes requires further investigation to confirm whether if these SNPs are coincidental or if they are damaging. Moreover, it is also probable that WIF1 and SFRP4 may be competing with UCA1 to exert their effects on WNT5A. CRNDE may also be competing to ultimately positively regulate WNT2 and WNT3. Together with the SNPs, HOTAIR may not be able to silence WIF1 and CD82, WIF1 and SFRP4 are ineffective in inhibiting the WNT ligands, the missense and the potentially novel SNPs in CD82 may be the cause for the lack of metastasis suppression. Finally, the downreglulation of CDKN2A and CDKN2B may be due to environmental and/or ethnic causes as shown in previous studies with Egyptian and Chinese CRC patients

SND1 mediated downregulation of PTPN23 in HCC

SND1 MEDIATED DOWNREGULATION OF PTPN23 IN HEPATOCELLULAR CARCINOMA By Nidhi Jariwala, MS A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science at Virginia Commonwealth University, 2014. ADVISOR: Dr. Devanand Sarkar Associate Professor, Department of Human and Molecular Genetics Blick Scholar Associate Scientific Director, Cancer Therapeutics VCU Institute of Molecular Medicine Massey Cancer Center ABSTRACT Staphyloccocal nuclease domain containing protein 1 (SND1) is identified as an oncogene in multiple cancers, including hepatocellular carcinoma (HCC). SND1 regulates gene expression at transcriptional as well as post-transcriptional level and mediates molecular pathways that culminate into carcinogenesis. SND1 is a component of RNA-induced silencing complex (RISC) and functions as a nuclease for RNAi-mediated mRNA degradation. On the other hand SND1 also binds to specific mRNAs, increasing their stability and hence expression. The aim of the present study is to identify mRNAs to which SND1 binds and modulates them either by degradation or increasing stability which might facilitate promotion of HCC by SND1. We performed RNA immunoprecipitation followed by RNA sequencing (RIP-Seq) using anti-SND1 antibody and human HCC cell line QGY-7703. More than 350 mRNAs were identified to be interacting with SND1, of which Protein tyrosine phosphatase non-receptor 23 (PTPN23) was of particular interest, since PTPN23 has been identified to be a tumor suppressor and its role in HCC has not been studied. We document that SND1 can bind to PTPN23 mRNA and induce its degradation. There is an inverse correlation between SND1 and PTPN23 levels in human HCC cell lines and PTPN23 level is downregulated in HCC. Our study thus identifies a novel mechanism by which SND1 promotes hepatocarcinogenesis and identifies PTPN23 as a potential tumor suppressor in HCC. Further studies need to be performed to explore the relationship of these two molecules in in vivo models and to develop PTPN23 overexpression as a potential therapeutic approach for HCC

Role of miRNAs in Cancer

MicroRNAs are the best representatives of the non-coding part of the genome and their functions are mostly linked to their target genes. During the process of carcinogenesis, both dysregulation of microRNAs and their target genes can explain the development of the disease. However, most of the target genes of microRNAs have not yet been elucidated. In this book, we add new information related to the functions of microRNAs in various tumors and their associated targetome

Polymorphic micro-RNA targets and risk of colorectal cancer

Recent evidence indicate that small non-coding RNA molecules, called micro-RNAs (miRNA), can bind to the 3’UTRs of mRNAs and interfere with their translation, thereby regulating cell growth, differentiation, apoptosis, and tumorigenesis. Genetic polymorphisms can reside on miRNA binding sites. Thus, it is conceivable that the miRNA regulation may be affected by polymorphisms on the 3’ UTRs. Since gene de-regulation is one of the key mechanisms by which cells can progress to cancer, we hypothesize that common polymorphisms within miRNA target binding sites could play a role in the individual risk of cancer. In the present study, we selected the 3’UTR regions of 129 genes candidate for colorectal cancer (CRC) and we identified putative miRNA binding sites by specialized algorithms (PicTar, DianaMicroT, miRBase, miRanda, TargetScan, and microInspector). We evaluated the SNPs for their ability to affect the binding of the miRNA with its target, by assessing the variation of Gibbs free energy between the two alleles of each SNP. We found 15 common polymorphisms. We added to this list 8 SNPs in miRNA sequences. All the polymorphisms were further investigated by a case-control association studies. The study was carried out on a series of cases and controls from Czech Republic, a population with the highest worldwide incidence of CRC. We found statistically significant associations between risk of CRC and variant alleles of CD86 (OR=2.74 95%CI=1.24-6.04, for the variant homozygotes) and INSR genes (OR=1.94; 95%CI=1.03-3.66, for the variant homozygotes). Then, these two polymorphisms were genotyped in three different populations: Spanish, Italian, and German.The statistical analyses for all the samples (Czech, Spanish, Italian, and German) confirmed the assciation between risk of CRC and the polymorphisms in CD86 and INSR. These results are the first reporting positive association between miRNA-binding SNPs sequences and cancer risk

Targeting HIF-2α in the Tumor Microenvironment: Redefining the Role of HIF-2α for Solid Cancer Therapy.

Inadequate oxygen supply, or hypoxia, is characteristic of the tumor microenvironment and correlates with poor prognosis and therapeutic resistance. Hypoxia leads to the activation of the hypoxia-inducible factor (HIF) signaling pathway and stabilization of the HIF-α subunit, driving tumor progression. The homologous alpha subunits, HIF-1α and HIF-2α, are responsible for mediating the transcription of a multitude of critical proteins that control proliferation, angiogenic signaling, metastasis, and other oncogenic factors, both differentially and sequentially regulating the hypoxic response. Post-translational modifications of HIF play a central role in its behavior as a mediator of transcription, as well as the temporal transition from HIF-1α to HIF-2α that occurs in response to chronic hypoxia. While it is evident that HIF-α is highly dynamic, HIF-2α remains vastly under-considered. HIF-2α can intensify the behaviors of the most aggressive tumors by adapting the cell to oxidative stress, thereby promoting metastasis, tissue remodeling, angiogenesis, and upregulating cancer stem cell factors. The structure, function, hypoxic response, spatiotemporal dynamics, and roles in the progression and persistence of cancer of this HIF-2α molecule and it

Exploring the Impact of Single-Nucleotide Polymorphisms on Translation

Over the past 15 years, sequencing of the human genome and The Cancer Genome Atlas (TCGA) project have led to comprehensive lists of single-nucleotide polymorphisms (SNPs) and gene mutations across a large number of human samples. However, our ability to predict the functional impact of SNPs and mutations on gene expression is still in its infancy. Here, we provide key examples to help understand how mutations present in genes can affect translational output

Bioinformatics Tools for Exploring Regulatory Mechanisms

Gene expression is the fundamental initial step in the flow of genetic information in biological systems and it is controlled by multiple precisely coordinated regulatory mechanisms, such as structural and epigenetic regulations. Dysregulation of gene expression plays important roles in the development of a broad range of diseases. Modern high-throughput technologies provide unprecedented opportunities to investigate these diverse regulatory mechanisms on a genome-wide scale. Here we develop several methods to analyze these omics profiles. First, Hi-C experiments generate genome-wide contact frequencies between pairs of loci by sequencing DNA segments ligated from loci in close spatial proximity. To detect biologically meaningful interactions between loci, we propose a hidden Markov random field (HMRF) based Bayesian method to rigorously model interaction probabilities in the two-dimensional space based on the contact frequency matrix. By borrowing information from neighboring loci pairs, our method demonstrates superior reproducibility and statistical power in both simulation studies and real data analysis. Second, DNA methylation is a key epigenetic mark involved in both normal development and disease progression. To facilitate joint analysis of methylation data from multiple platforms with varying resolution, we propose a penalized functional regression model to impute missing methylation data. By incorporating functional predictors, our model utilizes information from non-local probes to improve imputation quality. We compared the performance of our functional model to linear regression and the best single probe surrogate in real data and via simulations, and our method showed higher imputation accuracy. The simulated association study further demonstrated that our method substantially improves the statistical power to identify trait- associated methylation loci in epigenome-wide association study (EWAS). Finally, we applied an integrative analysis to characterize molecular systems associated with hepatocellular carcinoma (HCC). Dysregulaton of inflammation-related genes plays a pivotal role in the development of HCC. We performed array-based analyses to comprehensively investigate the contributions of DNA methylation and somatic copy number aberration (SCNA) to the aberrant expression of inflammation-related genes in 30 HCCs and paired non-tumor tissues. The results were validated in public datasets and an additional sample set of 47 paired HCCs and non-tumor tissues. We found that DNA methylation and SCNA together contributed to less than 30% aberrant expression of inflammation-related genes, suggesting that other molecular mechanisms might play major role in the dysregulation in HCCs.Doctor of Philosoph