Andrew Garazha

Human-specific endogenous retroviral insert serves as an enhancer for the schizophrenia-linked gene PRODH

Significance We identified a human-specific endogenous retroviral insert (hsERV) that acts as an enhancer for human PRODH, hsERV_PRODH. PRODH encodes proline dehydrogenase, which is involved in neuromediator synthesis in the CNS. We show that the hsERV_PRODH enhancer acts synergistically with the CpG island of PRODH and is regulated by methylation. We detected high PRODH expression in the hippocampus, which was correlated with the undermethylated state of this enhancer. PRODH regulatory elements provide neuron-specific transcription in hippocampal cells, and the mechanism of hsERV_PRODH enhancer activity involves the binding of transcriptional factor SOX2. Because PRODH is associated with several neurological disorders, we hypothesize that the human-specific regulation of PRODH by hsERV_PRODH may have played a role in human evolution by upregulating the expression of this important CNS-specific gene. Using a systematic, whole-genome analysis of enhancer activity of human-specific endogenous retroviral inserts (hsERVs), we identified an element, hsERVPRODH, that acts as a tissue-specific enhancer for the PRODH gene, which is required for proper CNS functioning. PRODH is one of the candidate genes for susceptibility to schizophrenia and other neurological disorders. It codes for a proline dehydrogenase enzyme, which catalyses the first step of proline catabolism and most likely is involved in neuromediator synthesis in the CNS. We investigated the mechanisms that regulate hsERVPRODH enhancer activity. We showed that the hsERVPRODH enhancer and the internal CpG island of PRODH synergistically activate its promoter. The enhancer activity of hsERVPRODH is regulated by methylation, and in an undermethylated state it can up-regulate PRODH expression in the hippocampus. The mechanism of hsERVPRODH enhancer activity involves the binding of the transcription factor SOX2, whch is preferentially expressed in hippocampus. We propose that the interaction of hsERVPRODH and PRODH may have contributed to human CNS evolution.

Silencing AML1-ETO gene expression leads to simultaneous activation of both pro-apoptotic and proliferation signaling

The t(8;21)(q22;q22) rearrangement represents the most common chromosomal translocation in acute myeloid leukemia (AML). It results in a transcript encoding for the fusion protein AML1-ETO (AE) with transcription factor activity. AE is considered to be an attractive target for treating t(8;21) leukemia. However, AE expression alone is insufficient to cause transformation, and thus the potential of such therapy remains unclear. Several genes are deregulated in AML cells, including KIT that encodes a tyrosine kinase receptor. Here, we show that AML cells transduced with short hairpin RNA vector targeting AE mRNAs have a dramatic decrease in growth rate that is caused by induction of apoptosis and deregulation of the cell cycle. A reduction in KIT mRNA levels was also observed in AE-silenced cells, but silencing KIT expression reduced cell growth but did not induce apoptosis. Transcription profiling of cells that escape cell death revealed activation of a number of signaling pathways involved in cell survival and proliferation. In particular, we find that the extracellular signal-regulated kinase 2 (ERK2; also known as mitogen-activated protein kinase 1 (MAPK1)) protein could mediate activation of 23 out of 29 (79%) of these upregulated pathways and thus may be regarded as the key player in establishing the t(8;21)-positive leukemic cells resistant to AE suppression.

Characteristic patterns of microrna expression in human bladder cancer

MicroRNAs (miRNAs) are small, non-coding RNAs that post-transcriptionally regulate gene expression. Their altered expression and functional activity have been observed in many human cancers. miRNAs represent promising diagnostic and prognostic molecular biomarkers, and also serve as novel therapeutic targets. We performed a systematic analysis of scientific reports that link differences in miRNA expression with the pathogenesis of bladder cancer (BC). This literature review is the first comprehensive database of miRNA molecules with biased expression profiles in BC. Among the 95 differentially expressed miRNAs that we identified from the literature, we classify 48 as up-regulated in BC, 35 as down-regulated, and 12 as contradictory (contradictory data were reported in one or more studies on the gene). In addition, we discuss the possible roles of differentially expressed miRNAs in the regulation of intracellular signaling pathways in BC.

A systematic experimental evaluation of microRNA markers of human bladder cancer.

Background: MicroRNAs (miRNAs) are a class of small RNAs that regulate gene expression. They are aberrantly expressed in many human cancers and are potential therapeutic targets and molecular biomarkers. Methods: In this study, we for the first time validated the reported data on the entire set of published differential miRNAs (102 in total) through a series of transcriptome-wide experiments. We have conducted genome-wide miRNA profiling in 17 urothelial carcinoma bladder tissues and in nine normal urothelial mucosa samples using three methods: (1) An Illumina HT-12 microarray hybridization (MA) analysis (2) a suppression-subtractive hybridization (SSH) assay followed by deep sequencing (DS) and (3) DS alone. Results: We show that DS data correlate with previously published information in 87% of cases, whereas MA and SSH data have far smaller correlations with the published information (6 and 9% of cases, respectively). qRT-PCR tests confirmed reliability of the DS data. Conclusions: Based on our data, MA and SSH data appear to be inadequate for studying differential miRNA expression in the bladder. Impact: We report the first comprehensive validated database of miRNA markers of human bladder cancer.

In search for geroprotectors: in silico screening and in vitro validation of signalome-level mimetics of young healthy state

Populations in developed nations throughout the world are rapidly aging, and the search for geroprotectors, or anti-aging interventions, has never been more important. Yet while hundreds of geroprotectors have extended lifespan in animal models, none have yet been approved for widespread use in humans. GeroScope is a computational tool that can aid prediction of novel geroprotectors from existing human gene expression data. GeroScope maps expression differences between samples from young and old subjects to aging-related signaling pathways, then profiles pathway activation strength (PAS) for each condition. Known substances are then screened and ranked for those most likely to target differential pathways and mimic the young signalome. Here we used GeroScope and shortlisted ten substances, all of which have lifespan-extending effects in animal models, and tested 6 of them for geroprotective effects in senescent human fibroblast cultures. PD-98059, a highly selective MEK1 inhibitor, showed both life-prolonging and rejuvenating effects. Natural compounds like N-acetyl-L-cysteine, Myricetin and Epigallocatechin gallate also improved several senescence-associated properties and were further investigated with pathway analysis. This work not only highlights several potential geroprotectors for further study, but also serves as a proof-of-concept for GeroScope, Oncofinder and other PAS-based methods in streamlining drug prediction, repurposing and personalized medicine.

New bioinformatic tool for quick identification of functionally relevant endogenous retroviral inserts in human genome

Abstract Endogenous retroviruses (ERVs) and LTR retrotransposons (LRs) occupy ∼8% of human genome. Deep sequencing technologies provide clues to understanding of functional relevance of individual ERVs/LRs by enabling direct identification of transcription factor binding sites (TFBS) and other landmarks of functional genomic elements. Here, we performed the genome-wide identification of human ERVs/LRs containing TFBS according to the ENCODE project. We created the first interactive ERV/LRs database that groups the individual inserts according to their familial nomenclature, number of mapped TFBS and divergence from their consensus sequence. Information on any particular element can be easily extracted by the user. We also created a genome browser tool, which enables quick mapping of any ERV/LR insert according to genomic coordinates, known human genes and TFBS. These tools can be used to easily explore functionally relevant individual ERV/LRs, and for studying their impact on the regulation of human genes. Overall, we identified ∼110,000 ERV/LR genomic elements having TFBS. We propose a hypothesis of “domestication” of ERV/LR TFBS by the genome milieu including subsequent stages of initial epigenetic repression, partial functional release, and further mutation-driven reshaping of TFBS in tight coevolution with the enclosing genomic loci.

Friends-Enemies: Endogenous Retroviruses Are Major Transcriptional Regulators of Human DNA

Endogenous retroviruses are mobile genetic elements hardly distinguishable from infectious, or “exogenous,” retroviruses at the time of insertion in the host DNA. Human endogenous retroviruses (HERVs) are not rare. They gave rise to multiple families of closely related mobile elements that occupy ~8% of the human genome. Together, they shape genomic regulatory landscape by providing at least ~320,000 human transcription factor binding sites (TFBS) located on ~110,000 individual HERV elements. The HERVs host as many as 155,000 mapped DNaseI hypersensitivity sites, which denote loci active in the regulation of gene expression or chromatin structure. The contemporary view of the HERVs evolutionary dynamics suggests that at the early stages after insertion, the HERV is treated by the host cells as a foreign genetic element, and is likely to be suppressed by the targeted methylation and mutations. However, at the later stages, when significant number of mutations has been already accumulated and when the retroviral genes are broken, the regulatory potential of a HERV may be released and recruited to modify the genomic balance of transcription factor binding sites. This process goes together with further accumulation and selection of mutations, which reshape the regulatory landscape of the human DNA. However, developmental reprogramming, stress or pathological conditions like cancer, inflammation and infectious diseases, can remove the blocks limiting expression and HERV-mediated host gene regulation. This, in turn, can dramatically alter the gene expression equilibrium and shift it to a newer state, thus further amplifying instability and exacerbating the stressful situation.

Data aggregation at the level of molecular pathways improves stability of experimental transcriptomic and proteomic data

ABSTRACT High throughput technologies opened a new era in biomedicine by enabling massive analysis of gene expression at both RNA and protein levels. Unfortunately, expression data obtained in different experiments are often poorly compatible, even for the same biologic samples. Here, using experimental and bioinformatic investigation of major experimental platforms, we show that aggregation of gene expression data at the level of molecular pathways helps to diminish cross- and intra-platform bias otherwise clearly seen at the level of individual genes. We created a mathematical model of cumulative suppression of data variation that predicts the ideal parameters and the optimal size of a molecular pathway. We compared the abilities to aggregate experimental molecular data for the 5 alternative methods, also evaluated by their capacity to retain meaningful features of biologic samples. The bioinformatic method OncoFinder showed optimal performance in both tests and should be very useful for future cross-platform data analyses.

Acquired resistance to tyrosine kinase inhibitors may be linked with the decreased sensitivity to X-ray irradiation

Acquired resistance to chemotherapy and radiation therapy is one of the major obstacles decreasing efficiency of treatment of the oncologic diseases. In this study, on the two cell lines (ovarian carcinoma SKOV-3 and neuroblastoma NGP-127), we modeled acquired resistance to five target anticancer drugs. The cells were grown on gradually increasing concentrations of the clinically relevant tyrosine kinase inhibitors (TKIs) Sorafenib, Pazopanib and Sunitinib, and rapalogs Everolimus and Temsirolimus, for 20 weeks. After 20 weeks of culturing, the half-inhibitory concentrations (IC50) increased by 25 – 186% for the particular combinations of the drugs and cell types. We next subjected cells to 10 Gy irradiation, a dose frequently used in clinical radiation therapy. For the SKOV-3, but not NGP-127 cells, for the TKIs Sorafenib, Pazopanib and Sunitinib, we noticed statistically significant increase in capacity to repair radiation-induced DNA double strand breaks compared to naïve control cells not previously treated with TKIs. These peculiarities were linked with the increased activation of ATM DNA repair pathway in the TKI-treated SKOV-3, but not NGP-127 cells. Our results provide a new cell culture model for studying anti-cancer therapy efficiency and evidence that there may be a tissue-specific radioresistance emerging as a side effect of treatment with TKIs.

Gene expression and molecular pathway activation signatures of MYCN-amplified neuroblastomas

Neuroblastoma is a pediatric cancer arising from sympathetic nervous system. Remarkable heterogeneity in outcomes is one of its widely known features. One of the traits strongly associated with the unfavorable subtype is the amplification of oncogene MYCN. Here, we performed cross-platform biomarker detection by comparing gene expression and pathway activation patterns from the two literature reports and from our experimental dataset, combining profiles for the 761 neuroblastoma patients with known MYCN amplification status. We identified 109 / 25 gene expression / pathway activation biomarkers strongly linked with the MYCN amplification. The marker genes/pathways are involved in the processes of purine nucleotide biosynthesis, ATP-binding, tetrahydrofolate metabolism, building mitochondrial matrix, biosynthesis of amino acids, tRNA aminoacylation and NADP-linked oxidation-reduction processes, as well as in the tyrosine phosphatase activity, p53 signaling, cell cycle progression and the G1/S and G2/M checkpoints. To connect molecular functions of the genes involved in MYCN-amplified phenotype, we built a new molecular pathway using known intracellular protein interaction networks. The activation of this pathway was highly selective in discriminating MYCN-amplified neuroblastomas in all three datasets. Our data also suggest that the phosphoinositide 3-kinase (PI3K) inhibitors may provide new opportunities for the treatment of the MYCN-amplified neuroblastoma subtype.