Michal Mikula

Modeling oncogenic signaling in colon tumors by multidirectional analyses of microarray data directed for maximization of analytical reliability.

Background Clinical progression of colorectal cancers (CRC) may occur in parallel with distinctive signaling alterations. We designed multidirectional analyses integrating microarray-based data with biostatistics and bioinformatics to elucidate the signaling and metabolic alterations underlying CRC development in the adenoma-carcinoma sequence. Methodology/Principal Findings Studies were performed on normal mucosa, adenoma, and carcinoma samples obtained during surgery or colonoscopy. Collections of cryostat sections prepared from the tissue samples were evaluated by a pathologist to control the relative cell type content. The measurements were done using Affymetrix GeneChip HG-U133plus2, and probe set data was generated using two normalization algorithms: MAS5.0 and GCRMA with least-variant set (LVS). The data was evaluated using pair-wise comparisons and data decomposition into singular value decomposition (SVD) modes. The method selected for the functional analysis used the Kolmogorov-Smirnov test. Expressional profiles obtained in 105 samples of whole tissue sections were used to establish oncogenic signaling alterations in progression of CRC, while those representing 40 microdissected specimens were used to select differences in KEGG pathways between epithelium and mucosa. Based on a consensus of the results obtained by two normalization algorithms, and two probe set sorting criteria, we identified 14 and 17 KEGG signaling and metabolic pathways that are significantly altered between normal and tumor samples and between benign and malignant tumors, respectively. Several of them were also selected from the raw microarray data of 2 recently published studies (GSE4183 and GSE8671). Conclusion/Significance Although the proposed strategy is computationally complex and labor–intensive, it may reduce the number of false results.

Integrating proteomic and transcriptomic high-throughput surveys for search of new biomarkers of colon tumors

To the search of new colon tumor biomarkers in the transition from normal colon (NC) mucosa to adenoma (AD) and adenocarcinoma (AC), we integrated microarray data with the results of a high-throughput proteomic workflow. In proteomic study, we used a modified isoelectric focusing protocol on strips with an immobilized pH gradient to separate peptides labeled with iTRAQ (isobaric tags for relative and absolute quantitation) tags followed by liquid chromatography–tandem mass spectrometry analysis. Gene expression measurements were done using Affymetrix GeneChip HG-U133plus2 microarrays and quantitative reverse transcriptase PCR (q-RT-PCR). We identified 3,886 proteins with at least two peptides. Of them, 1,061 proteins were differentially expressed [FC ≥ 1.5; FDR ≤ 0.01] in two pair-wise comparisons: AD vs. NC and AC vs. AD while 15 and 23 proteins were progressively up-regulated and down-regulated in the NC/AD/AC sequence, respectively. The quantitative proteomic information was subsequently correlated with microarray data. For a collection of genes with the same direction of changes of both mRNA and protein levels, we obtained 785/853/795 genes in AD vs. NC/AC vs. NC/AC vs. AD comparison, respectively. Further evaluation of sequentially altered gene expression by q-RT-PCR on individual samples of 24 NCs, 42 ADs, and 26 ACs confirmed progressive expression of six genes: biglycan, calumenin, collagen type XII, alpha 1 (COL12A1), monoamine oxidase A (MAOA), ectonucleoside triphosphate diphosphohydrolase 5 (ENTPD5), and MOCO sulphurase C-terminal domain-containing 2 (MOSC2). Among them, three continuously down-regulated (MAOA, ENTPD5, and MOSC2) and one continuously overexpressed (COL12A1) are reported, to our best knowledge, for the first time in a connection to colon cancer onset.

Quantitative detection for low levels of Helicobacter pylori infection in experimentally infected mice by real-time PCR.

Accurate diagnosis of Helicobacter pylori infection is important in both clinical practice and clinical research. Molecular methods are highly specific and sensitive, and various PCR-based tests have been developed to detect H. pylori in gastric biopsy specimens. We optimized a sensitive and specific quantitative SYBR Green I real-time PCR assay for detection of H. pylori based on amplification of the fragment of a 26-kDa Helicobacter species-specific antigen gene that allows for detection of 5 bacterial cells per PCR sample. Under the assay conditions, SYBR Green I real-time PCR is highly reproducible with a precise log-linear relation in the range of six orders of magnitude of bacterial DNA concentrations. For accurate comparison of H. pylori infection in different tissue samples, the amount of total host DNA in each sample is normalized by TaqMan real-time PCR of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) pseudogenes. The developed method was validated in prophilactically immunized and experimentally infected mice and revealed a level of H. pylori gastric colonisation that was below the limit of detection for a rapid urease test. This new method established for a quantitative analysis of H. pylori in the hosts stomach may be useful in experimental studies evaluating new anti-H. pylori drugs and vaccines.

Molecular defense mechanisms of Barrett’s metaplasia estimated by an integrative genomics

Barrett’s esophagus is characterized by the replacement of squamous epithelium with specialized intestinal metaplastic mucosa. The exact mechanisms of initiation and development of Barrett’s metaplasia remain unknown, but a hypothesis of “successful adaptation” against noxious reflux components has been proposed. To search for the repertoire of adaptation mechanisms of Barrett’s metaplasia, we employed high-throughput functional genomic and proteomic methods that defined the molecular background of metaplastic mucosa resistance to reflux. Transcriptional profiling was established for 23 pairs of esophageal squamous epithelium and Barrett’s metaplasia tissue samples using Affymetrix U133A 2.0 GeneChips and validated by quantitative real-time polymerase chain reaction. Differences in protein composition were assessed by electrophoretic and mass-spectrometry-based methods. Among 2,822 genes differentially expressed between Barrett’s metaplasia and squamous epithelium, we observed significantly overexpressed metaplastic mucosa genes that encode cytokines and growth factors, constituents of extracellular matrix, basement membrane and tight junctions, and proteins involved in prostaglandin and phosphoinositol metabolism, nitric oxide production, and bioenergetics. Their expression likely reflects defense and repair responses of metaplastic mucosa, whereas overexpression of genes encoding heat shock proteins and several protein kinases in squamous epithelium may reflect lower resistance of normal esophageal epithelium than Barrett’s metaplasia to reflux components. Despite the methodological and interpretative difficulties in data analyses discussed in this paper, our studies confirm that Barrett’s metaplasia may be regarded as a specific microevolution allowing for accumulation of mucosal morphological and physiological changes that better protect against reflux injury.

Comprehensive Analysis of the Palindromic Motif TCTCGCGAGA: A Regulatory Element of the HNRNPK Promoter

Definitive identification of promoters, their cis-regulatory motifs, and their trans-acting proteins requires experimental analysis. To define the HNRNPK promoter and its cognate DNA–protein interactions, we performed a comprehensive study combining experimental approaches, including luciferase reporter gene assays, chromatin immunoprecipitations (ChIP), electrophoretic mobility shift assays (EMSA), and mass spectrometry (MS). We discovered that out of the four potential HNRNPK promoters tested, the one containing the palindromic motif TCTCGCGAGA exhibited the highest activity in a reporter system assay. Although further EMSA and MS analyses, performed to uncover the identity of the palindrome-binding transcription factor, did identify a complex of DNA-binding proteins, neither method unambiguously identified the pertinent direct trans-acting protein(s). ChIP revealed similar chromatin states at the promoters with the palindromic motif and at housekeeping gene promoters. A ChIP survey showed significantly higher recruitment of PARP1, a protein identified by MS as ubiquitously attached to DNA probes, within heterochromatin sites. Computational analyses indicated that this palindrome displays features that mark nucleosome boundaries, causing the surrounding DNA landscape to be constitutively open. Our strategy of diverse approaches facilitated the direct characterization of various molecular properties of HNRNPK promoter bearing the palindromic motif TCTCGCGAGA, despite the obstacles that accompany in vitro methods.

Histone H3 lysine 27 acetylation is altered in colon cancer

BackgroundHistone post-translational modifications (PTMs) play an important role in the regulation of the expression of genes, including those involved in cancer development and progression. However, our knowledge of PTM patterns in human tumours is limited.MethodsMS-based analyses were used to quantify global alterations of histone PTMs in colorectal cancer (CRC) samples. Histones isolated from 12 CRCs and their corresponding normal mucosa by acidic extraction were separated by SDS-PAGE and analysed by liquid chromatography-mass spectrometry.ResultsAmong 96 modified peptides, 41 distinct PTM sites were identified, of which 7, 13, 11, and 10 were located within the H2A, H2B, H3, and H4 sequences, respectively, and distributed among the amino-terminal tails and the globular domain of the four histones. Modification intensities were quantified for 33 sites, of which 4 showed significant (p-value ≤ 0.05) differences between CRC tissues and healthy mucosa samples. We identified histone H3 lysine 27 acetylation (H3K27Ac) as a modification upregulated in CRC, which had not been shown previously.ConclusionsThe present results indicate the usefulness of a bottom-up proteomic approach for the detection of histone modifications at a global scale. The differential abundance of H3K27Ac mark in CRC, a PTM associated with active enhancers, suggests its role in regulating genes whose expression changes in CRC.

Direct Recruitment of ERK Cascade Components to Inducible Genes Is Regulated by Heterogeneous Nuclear Ribonucleoprotein (hnRNP) K

Components of the ERK cascade are recruited to genes, but it remains unknown how they are regulated at these sites. The RNA-binding protein heterogeneous nuclear ribonucleoprotein (hnRNP) K interacts with kinases and is found along genes including the mitogen-inducible early response gene EGR-1. Here, we used chromatin immunoprecipitations to study co-recruitment of hnRNP K and ERK cascade activity along the EGR-1 gene. These measurements revealed that the spatiotemporal binding patterns of ERK cascade transducers (GRB2, SOS, B-Raf, MEK, and ERK) at the EGR-1 locus resemble both hnRNP K and RNA polymerase II (Pol II). Inhibition of EGR-1 transcription with either serum-responsive factor knockdown or 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole altered recruitment of all of the above ERK cascade components along this locus that mirrored the changes in Pol II and hnRNP K profiles. siRNA knockdown of hnRNP K decreased the levels of active MEK and ERK at the EGR-1, changes associated with decreased levels of elongating pre-mRNA and less efficient splicing. The hnRNP K dependence and pattern of ERK cascade activation at the c-MYC locus were different from at EGR-1. Ribonucleoprotein immunoprecipitations revealed that hnRNP K was associated with the EGR-1 but not c-MYC mRNAs. These data suggest a model where Pol II transcription-driven recruitment of hnRNP K along the EGR-1 locus compartmentalizes activation of the ERK cascade at these genes, events that regulate synthesis of mature mRNA.

CDK8 kinase--An emerging target in targeted cancer therapy.

Cyclin-dependent kinase (CDK) inhibitors have been developed as potential anticancer therapeutics and several nonselective compounds are currently in advanced clinical trials. This review is focused on the key biological roles of CDK8 kinase, which provide a proof-of-principle for continued efforts toward effective cancer treatment, targeting activity of this CDK family member. Among currently identified kinase inhibitors, several displayed significant selectivity for CDK8 and notably the effectiveness in targeting cancer specific gene expression programs. Structural features of CDK8 and available ligands were discussed from a perspective of the rational drug design process. Current state of the art confirms that further development of CDK8 inhibitors will translate into targeted therapies in oncology. This article is part of a Special Issue entitled:Inhibitors of Protein Kinases.

New recurrent BRCA1/2 mutations in Polish patients with familial breast/ovarian cancer detected by next generation sequencing

BackgroundTargeted PCR-based genetic testing for BRCA1 and BRCA2 can be performed at a lower cost than full gene testing; however, it may overlook mutations responsible for familial breast and/or ovarian cancers. In the present study, we report the utility of next generation sequencing (NGS) to identify new pathogenic variants of BRCA1/2.MethodsBRCA1 and BRCA2 exons were amplified using the Ion AmpliSeq BRCA1/2 Panel and sequenced on the Ion Torrent PGM sequencer in 512 women with familial and/or only early onset breast and/or ovarian cancers who were negative for selected BRCA1/2 mutations.Results146 single nucleotide variants (SNVs) and 32 indels were identified. Of them, 14 SNVs and 17 indels were considered as pathogenic or likely pathogenic. One and 18 pathogenic mutations had been detected previously in the Polish and other populations, respectively, and 12 deleterious mutations were previously unknown. Eight mutations were recurrent; Q563X (BRCA1), N3124I (BRCA2) and c.4516delG (BRCA1) were found in eight, six and four patients, respectively, and two other mutations (c.9118-2A > G and c.7249delCA in BRCA2) were detected in three patients each. Altogether, BRCA1/2 pathogenic mutations were identified in 52 out of 512 (10%) patients.ConclusionsNGS substantially improved the detection rates of a wide spectrum of mutations in Polish patients with familial breast and/or ovarian cancer. Although targeted screening for specific BRCA1 mutations can be offered to all Polish breast or ovarian cancer patients, NGS-based testing is justified in patients with breast or ovarian cancer likely related to BRCA1/2 who test negative for the selected BRCA1/2 pathogenic mutations.

Comparative kinome analysis to identify putative colon tumor biomarkers

Kinase domains are the type of protein domain most commonly found in genes associated with tumorigenesis. Because of this, the human kinome (the protein kinase component of the genome) represents a promising source of cancer biomarkers and potential targets for novel anti-cancer therapies. Alterations in the human colon kinome during the progression from normal colon (NC) through adenoma (AD) to adenocarcinoma (AC) were investigated using integrated transcriptomic and proteomic datasets. Two hundred thirty kinase genes and 42 kinase proteins showed differential expression patterns (fold change ≥ 1.5) in at least one tissue pair-wise comparison (AD vs. NC, AC vs. NC, and/or AC vs. AD). Kinases that exhibited similar trends in expression at both the mRNA and protein levels were further analyzed in individual samples of NC (n = 20), AD (n = 39), and AC (n = 24) by quantitative reverse transcriptase PCR. Individual samples of NC and tumor tissue were distinguishable based on the mRNA levels of a set of 20 kinases. Altered expression of several of these kinases, including chaperone activity of bc1 complex-like (CABC1) kinase, bromodomain adjacent to zinc finger domain protein 1B (BAZ1B) kinase, calcium/calmodulin-dependent protein kinase type II subunit delta (CAMK2D), serine/threonine-protein kinase 24 (STK24), vaccinia-related kinase 3 (VRK3), and TAO kinase 3 (TAOK3), has not been previously reported in tumor tissue. These findings may have diagnostic potential and may lead to the development of novel targeted therapeutic interventions for colorectal cancer.