Epigenetically Downregulated Breast Cancer Gene 2 through Acetyltransferase Lysine Acetyltransferase 2B Increases the Sensitivity of Colorectal Cancer to Olaparib.

Olaparib suppresses DNA damage repair by inhibiting the poly ADP ribose polymerase (PARP), especially in cancers with BRCA1/2 mutations or the BRCA-ness phenotype. However, the first trials showed that some patients with defective DNA damage repair are still resistant to olaparib. The recovery of the wildtype BRCA is a prominent mechanism of PARP inhibitor (PARPi) resistance in BRCA-deficient tumors, but additional molecular features of olaparib resistance remain poorly understood. The objective of our study was to find molecular parameters that contribute to olaparib response or resistance in CRC. We report that histone acetyltransferase KAT2B decreases BRCA2 expression by reducing the acetylation of the 27th amino acid in histone H3 (H3K27) at the promoter of the BRCA2 gene in colorectal cancer (CRC). This increases the sensitivity of CRC cells toward olaparib treatment. The H3K27ac binding domain of BRCA2 may be required for its transcription. Low endogenous KAT2B expression, which we identify in a subset of cultured BRCA2-expressing CRC cells, leads to an accumulation of γH2AX (more DNA damage), resulting in low PARPi resistance in BRCA-expressing cells. Our results reveal KAT2B and histone acetylation as regulators of BRCA2 expression and PARPi responses in BRCA2-expressing CRC cells, providing further insights into molecular prerequisites for targeting BRCA-functional tumors.

JAK1 Is a Novel Target of Tumor- and Invasion-Suppressive microRNA 494-5p in Colorectal Cancer.

MiR-494-5p expression has been suggested to be associated with colorectal cancer (CRC) and its metastases in our previous studies. However, functional investigations on the molecule-mediating actions of this miR in CRC are lacking. In silico analysis in the present study revealed a putative binding sequence within the 3'UTR of JAK1. Overexpression of miR-494-5p in cultured CRC significantly reduced the luciferase activity of a reporter plasmid containing the wild-type JAK1-3'UTR, which was abolished by seed sequence mutation. Furthermore, the overexpression of miR-494-5p in CRC cell lines led to a significant reduction in JAK1 expression, proliferation, in vitro migration, and invasion. These effects were abolished by co-transfection with a specific double-stranded RNA that inhibits endogenous miR-494-5p. Moreover, IL-4-induced migration, invasion, and phosphorylation of JAK1, STAT6, and AKT proteins were reduced after an overexpression of this miR, suggesting that this miR affects one of the most essential pathways in CRC. A Kaplan-Meier plotter analysis revealed that patients with high JAK1 expression show reduced survival. Together, these data suggest that miR-494-5p physically inhibits the expression of JAK1 at the translational level as well as in migration and invasion, supporting the hypothesis of miR-494-5p as an early tumor suppressor and inhibitor of early steps of metastasis in CRC.

Expression Status of Rap1 Pathway-Related Genes in Liver Metastases Compared with Corresponding Primary Colorectal Cancer.

Understanding molecular networks of CRLM is an ongoing area of research. In this study, paired CRC tissue and adjacent noncancerous tissue from 15 non-metastatic CRC patients and paired CRC tissue and matched liver metastatic tissues from 15 CRLM patients along with their adjacent noncancerous tissues were evaluated. We assessed Rap1 pathway-related genes including NRAS, FGF-1, NGF, and KDR expression by qRT-PCR and their protein status by Western blot. In CRLM patients, NRAS, FGF1, and KDR mRNA and protein were expressed at higher levels in metastatic than in CRC primary tumor and adjacent noncancerous tissue (p < 0.05). In non-metastatic patients, NRAS, FGF1, KDR, and NGF gene expression did not differ between CRC primary tumor-and adjacent noncancerous tissue (p > 0.05). ROC curve analysis showed a reasonable diagnostic accuracy of NRAS, FGF1, KDR, and FGF for the discrimination of metastatic patients from non- metastatic ones on analysis of their primary tumors. The data suggest that further functional studies on Rap1-related genes' role in CRLM are needed. In conclusion, the present data broaden our knowledge about specific molecular characteristics of CRLM. An increased understanding of the molecular features of metastasis has the potential to create more successful treatment, or prevention, of metastasis, especially in multimodal primary tumor treatment.

Involvement of HHV-4 (Epstein-Barr Virus) and HHV-5 (Cytomegalovirus) in Inflammatory Bowel Disease and Colorectal Cancer: A Meta-Analysis.

Gastrointestinal diseases (GDs) include colorectal cancer (CRC), gastric cancer (GC), and inflammatory bowel disease (IBD). CRC and GC are typically diagnosed at later stages of development, reducing patients' chances of survival. IBD is characterized by chronic intestinal inflammation and is a significant risk factor for the development of CRC. Chronic bacterial infections have been shown to promote some GDs, but the role of viruses in the etiology of these diseases is less clear. The present meta-analysis retrieved literature on the viral prevalence in GD patients, measuring the GD risk in odd ratios. By quantifying the study heterogeneity, the literature bias was fundamentally included in the analysis. The analysis also included 11 metagenomic studies. Our meta-analysis retrieved 11,413 studies, with 196 suitable for analysis. HHV-4 (Epstein-Barr virus) was identified as a significant risk factor for the development of IBD, and HHV-5 (cytomegalovirus) as a risk factor for both CRC and IBD. Polyomaviruses and the Hepatitis B virus were also, less strongly, involved in the risk of CRC and IBD. No relations withstanding the literature bias were identified for GC. The study discusses these findings, as well as the role of other viruses in the etiology of CRC and IBD.

An Editorial View on the Special Issue "Colorectal Cancers: From Present Problems to Future Solutions".

Colorectal cancer (CRC) represents one of the most frequent human cancer entities and is still amongst the "top killers" in human cancer, although fundamental progress has been made in recent years in CRC prevention, early diagnosis, basic and translational research, and (targeted) therapy [...].

Viruses in colorectal cancer.

Increasing evidence suggests that microorganisms might represent at least highly interesting cofactors in colorectal cancer (CRC) oncogenesis and progression. Still, associated mechanisms, specifically in colonocytes and their microenvironmental interactions, are still poorly understood. Although, currently, at least seven viruses are being recognized as human carcinogens, only three of these - Epstein-Barr virus (EBV), human papillomavirus (HPV) and John Cunningham virus (JCV) - have been described, with varying levels of evidence, in CRC. In addition, cytomegalovirus (CMV) has been associated with CRC in some publications, albeit not being a fully acknowledged oncovirus. Moreover, recent microbiome studies set increasing grounds for new hypotheses on bacteriophages as interesting additional modulators in CRC carcinogenesis and progression. The present Review summarizes how particular groups of viruses, including bacteriophages, affect cells and the cellular and microbial microenvironment, thereby putatively contributing to foster CRC. This could be achieved, for example, by promoting several processes - such as DNA damage, chromosomal instability, or molecular aspects of cell proliferation, CRC progression and metastasis - not necessarily by direct infection of epithelial cells only, but also by interaction with the microenvironment of infected cells. In this context, there are striking common features of EBV, CMV, HPV and JCV that are able to promote oncogenesis, in terms of establishing latent infections and affecting p53-/pRb-driven, epithelial-mesenchymal transition (EMT)-/EGFR-associated and especially Wnt/ß-catenin-driven pathways. We speculate that, at least in part, such viral impacts on particular pathways might be reflected in lasting (e.g. mutational or further genomic) fingerprints of viruses in cells. Also, the complex interplay between several species within the intestinal microbiome, involving a direct or indirect impact on colorectal and microenvironmental cells but also between, for example, phages and bacterial and viral pathogens, and further novel species certainly might, in part, explain ongoing difficulties to establish unequivocal monocausal links between specific viral infections and CRC.

Changes in Methylation across Structural and MicroRNA Genes Relevant for Progression and Metastasis in Colorectal Cancer.

MiRs are important players in cancer and primarily genetic/transcriptional means of regulating their gene expression are known. However, epigenetic changes modify gene expression significantly. Here, we evaluated genome-wide methylation changes focusing on miR genes from primary CRC and corresponding normal tissues. Differentially methylated CpGs spanning CpG islands, open seas, and north and south shore regions were evaluated, with the largest number of changes observed within open seas and islands. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis revealed several of these miRs to act in important cancer-related pathways, including phosphatidylinositol 3-kinase (PI3K)-protein kinase B (Akt) and mitogen-activated protein kinase (MAPK) pathways. We found 18 miR genes to be significantly differentially methylated, with MIR124-2, MIR124-3, MIR129-2, MIR137, MIR34B, MIR34C, MIR548G, MIR762, and MIR9-3 hypermethylated and MIR1204, MIR17, MIR17HG, MIR18A, MIR19A, MIR19B1, MIR20A, MIR548F5, and MIR548I4 hypomethylated in CRC tumor compared with normal tissue, most of these miRs having been shown to regulate steps of metastasis. Generally, methylation changes were distributed evenly across all chromosomes with predominance for chromosomes 1/2 and protein-coding genes. Interestingly, chromosomes abundantly affected by methylation changes globally were rarely affected by methylation changes within miR genes. Our findings support additional mechanisms of methylation changes affecting (miR) genes that orchestrate CRC progression and metastasis.

Tackling Tumour Cell Heterogeneity at the Super-Resolution Level in Human Colorectal Cancer Tissue.

Tumour cell heterogeneity, and its early individual diagnosis, is one of the most fundamental problems in cancer diagnosis and therapy. Single molecule localisation microscopy (SMLM) resolves subcellular features but has been limited to cultured cell lines only. Since nuclear chromatin architecture and microRNAs are critical in metastasis, we introduce a first-in-field approach for quantitative SMLM-analysis of chromatin nanostructure in individual cells in resected, routine-pathology colorectal carcinoma (CRC) patient tissue sections. Chromatin density profiles proved to differ for cells in normal and carcinoma colorectal tissues. In tumour sections, nuclear size and chromatin compaction percentages were significantly different in carcinoma versus normal epithelial and other cells of colorectal tissue. SMLM analysis in nuclei from normal colorectal tissue revealed abrupt changes in chromatin density profiles at the nanoscale, features not detected by conventional widefield microscopy. SMLM for microRNAs relevant for metastasis was achieved in colorectal cancer tissue at the nuclear level. Super-resolution microscopy with quantitative image evaluation algorithms provide powerful tools to analyse chromatin nanostructure and microRNAs of individual cells from normal and tumour tissue at the nanoscale. Our new perspectives improve the differential diagnosis of normal and (metastatically relevant) tumour cells at the single-cell level within the heterogeneity of primary tumours of patients.

Metagenomic analysis of primary colorectal carcinomas and their metastases identifies potential microbial risk factors.

The paucity of microbiome studies at intestinal tissues has contributed to a yet limited understanding of potential viral and bacterial cofactors of colorectal cancer (CRC) carcinogenesis or progression. We analysed whole-genome sequences of CRC primary tumours, their corresponding metastases and matched normal tissue for sequences of viral, phage and bacterial species. Bacteriome analysis showed Fusobacterium nucleatum, Streptococcus sanguinis, F. Hwasookii, Anaerococcus mediterraneensis and further species enriched in primary CRCs. The primary CRC of one patient was enriched for F. alocis, S. anginosus, Parvimonas micra and Gemella sp. 948. Enrichment of Escherichia coli strains IAI1, SE11, K-12 and M8 was observed in metastases together with coliphages enterobacteria phage φ80 and Escherichia phage VT2φ_272. Virome analysis showed that phages were the most preponderant viral species (46%), the main families being Myoviridae, Siphoviridae and Podoviridae. Primary CRCs were enriched for bacteriophages, showing five phages (Enterobacteria, Bacillus, Proteus, Streptococcus phages) together with their pathogenic hosts in contrast to normal tissues. The most frequently detected, and Blast-confirmed, viruses included human endogenous retrovirus K113, human herpesviruses 7 and 6B, Megavirus chilensis, cytomegalovirus (CMV) and Epstein-Barr virus (EBV), with one patient showing EBV enrichment in primary tumour and metastases. EBV was PCR-validated in 80 pairs of CRC primary tumour and their corresponding normal tissues; in 21 of these pairs (26.3%), it was detectable in primary tumours only. The number of viral species was increased and bacterial species decreased in CRCs compared with normal tissues, and we could discriminate primary CRCs from metastases and normal tissues by applying the Hutcheson t-test on the Shannon indices based on viral and bacterial species. Taken together, our results descriptively support hypotheses on microorganisms as potential (co)risk factors of CRC and extend putative suggestions on critical microbiome species in CRC metastasis.

Dietary Modulation of Bacteriophages as an Additional Player in Inflammation and Cancer.

Natural compounds such as essential oils and tea have been used successfully in naturopathy and folk medicine for hundreds of years. Current research is unveiling the molecular role of their antibacterial, anti-inflammatory, and anticancer properties. Nevertheless, the effect of these compounds on bacteriophages is still poorly understood. The application of bacteriophages against bacteria has gained a particular interest in recent years due to, e.g., the constant rise of antimicrobial resistance to antibiotics, or an increasing awareness of different types of microbiota and their potential contribution to gastrointestinal diseases, including inflammatory and malignant conditions. Thus, a better knowledge of how dietary products can affect bacteriophages and, in turn, the whole gut microbiome can help maintain healthy homeostasis, reducing the risk of developing diseases such as diverse types of gastroenteritis, inflammatory bowel disease, or even cancer. The present review summarizes the effect of dietary compounds on the physiology of bacteriophages. In a majority of works, the substance class of polyphenols showed a particular activity against bacteriophages, and the primary mechanism of action involved structural damage of the capsid, inhibiting bacteriophage activity and infectivity. Some further dietary compounds such as caffeine, salt or oregano have been shown to induce or suppress prophages, whereas others, such as the natural sweeter stevia, promoted species-specific phage responses. A better understanding of how dietary compounds could selectively, and specifically, modulate the activity of individual phages opens the possibility to reorganize the microbial network as an additional strategy to support in the combat, or in prevention, of gastrointestinal diseases, including inflammation and cancer.

The Chicken Egg Chorioallantoic Membrane (CAM) Model as an In Vivo Method for the Investigation of the Invasion and Metastasis Cascade of Malignant Tumor Cells.

The CAM model enables an in vivo analysis of the individual sub-steps of the metastatic cascade like local invasion, intravasation, or the establishment of metastasis in particular organs. Incubated fertilized chicken eggs are inoculated with human tumor cells and further processed for up to 9-10 days. The invasion and metastasis of these cells is then detected quantitatively with high specificity and sensitivity by means of a PCR for human ALU sequences, using the genomic DNA isolated from distant portions of the CAM, as well as from diverse internal organs of the developing embryo.

MicroRNAs in the Tumor Microenvironment.

The tumor microenvironment (TME) is decisive for the eradication or survival of any tumor mass. Moreover, it plays a pivotal role for metastasis and for providing the metastatic niche. The TME offers special physiological conditions and is composed of, for example, surrounding blood vessels, the extracellular matrix (ECM), diverse signaling molecules, exosomes and several cell types including, but not being limited to, infiltrated immune cells, cancer-associated endothelial cells (CAEs), and cancer-associated fibroblasts (CAFs). These cells can additionally and significantly contribute to tumor and metastasis progression, especially also by acting via their own deregulated micro (mi) RNA expression or activity. Thus, miRNAs are essential players in the crosstalk between cancer cells and the TME. MiRNAs are small non-coding (nc) RNAs that typically inhibit translation and stability of messenger (m) RNAs, thus being able to regulate several cell functions including proliferation, migration, differentiation, survival, invasion, and several steps of the metastatic cascade. The dynamic interplay between miRNAs in different cell types or organelles such as exosomes, ECM macromolecules, and the TME plays critical roles in many aspects of cancer development. This chapter aims to give an overview on the multiple contributions of miRNAs as players within the TME, to summarize the role of miRNAs in the crosstalk between different cell populations found within the TME, and to illustrate how they act on tumorigenesis and the behavior of cells in the TME context. Lastly, the potential clinical utility of miRNAs for cancer therapy is discussed.

NF1-RAC1 axis regulates migration of the melanocytic lineage.

Metastases's spreading is the main cause of mortality for advanced stage cancer patients, including melanoma. The formation of metastases is favored by enhanced migratory and invasive capacities of tumor cells. Tumor suppressor gene NF1 is a negative regulator of RAS and its deregulation plays an important role in several aspects of melanoma transformation and progression. However, very little is described about the role of NF1 in cellular migration and invasion. In this study, our results show on the one hand, that the loss of NF1 expression delays migration of human melanoblasts via a RAC1-dependent mechanism. On the other hand, our data indicate that NF1 loss in melanoma cells is enhancing migration, intravasation and metastases formation in vivo. Moreover, not only this phenotype is associated with an upregulation of PREX1 but also patient-derived melanoma samples with low NF1 expression present increased levels of PREX1. In sum, our study brings new elements on the mechanism controlling cellular migration in the context of NF1 loss. These data are of prime interest to improve treatment strategies against all NF1-mutated tumors, including this subtype of melanoma.

The lncRNA lincNMR regulates nucleotide metabolism via a YBX1 - RRM2 axis in cancer.

Long intergenic non-coding RNA-Nucleotide Metabolism Regulator (lincNMR) is a long non-coding RNA (lncRNA) which is induced in hepatocellular carcinoma. Its depletion invokes a proliferation defect, triggers senescence and inhibits colony formation in liver, but also breast and lung cancer cells. Triple-label SILAC proteomics profiles reveal a deregulation of key cell cycle regulators in lincNMR-depleted cells like the key dNTP synthesizing enzymes RRM2, TYMS and TK1, implicating lincNMR in regulating nucleotide metabolism. LincNMR silencing decreases dNTP levels, while exogenous dNTPs rescues the proliferation defect induced by lincNMR depletion. In vivo RNA Antisense Purification (RAP-MS) identifies YBX1 as a direct interaction partner of lincNMR which regulates RRM2, TYMS and TK1 expression and binds to their promoter regions. In a Chick Chorioallantoic Membrane (CAM) in vivo model, lincNMR-depleted tumors are significantly smaller. In summary, we discover a lincRNA, lincNMR, which regulates tumor cell proliferation through a YBX1-RRM2-TYMS-TK1 axis governing nucleotide metabolism.

maxRatio improves the detection of samples with abnormal amplification profiles on QIAgen's artus HIV-1 qPCR assay.

Background: Accurate viral load (VL) determination is paramount to determine the efficacy of anti-HIV-1 therapy. The conventional method used, fit-point (FP), assumes an equal efficiency in the polymerase chain reaction (PCR) among samples that might not hold for low-input templates. An alternative approach, maxRatio, was introduced to compensate for inhibition in PCR. Methods: Herein, we assessed whether maxRatio could improve VL quantification using 2,544 QIAgen artus HI virus-1 RT-PCR reactions. The assay's standard dilutions were used to build external standard curves with either FP or maxRatio that re-calculated the VLs. Results: FP and maxRatio were highly comparable (Pearson's ρ=0.994, Cohen's  κ=0.885), and the combination of the two methods identified samples (n=41) with aberrant amplification profiles. Conclusions: The combination of maxRatio and FP could improve the predictive value of the assay.

Defining the "Metastasome": Perspectives from the genome and molecular landscape in colorectal cancer for metastasis evolution and clinical consequences.

Metastasis still poses the highest challenge for personalized therapy in cancer, partly due to a still incomplete understanding of its molecular evolution. We recently presented the most comprehensive whole-genome study of colorectal metastasis vs. matched primary tumors and suggested novel components of disease progression and metastasis evolution, some of them potentially relevant for targeted therapy. In this review, we try to put these findings into perspective with latest discoveries of colleagues and recent literature, and propose a systematic international team effort to collectively define the "metastasome", a term we introduce to summarize all genomic, epigenomic, transcriptomic, further -omic, molecular and functional characteristics rendering metastases different from primary tumors. Based on recent discoveries, we propose a revised metastasis model for colorectal cancer which is based on a common ancestor clone, early dissemination but flexible early or late stage clonal separation paralleling stromal interactions. Furthermore, we discuss hypotheses on site-specific metastasis, colorectal cancer progression, metastasis-targeted diagnosis and therapy, and metastasis prevention based on latest metastasome data.

Filtering maxRatio results with machine learning models increases quantitative PCR accuracy over the fit point method.

With qPCR reaching thousands of reactions per run, assay validation needs automation. We applied support vector machine to qPCR analysis and we could identify reactions with 100% accuracy, dispensing them from further validation. We achieved a greatly reduced workload that could improve high-throughput qPCR analysis.

Methylglyoxal down-regulates the expression of cell cycle associated genes and activates the p53 pathway in human umbilical vein endothelial cells.

Although methylglyoxal (MGO) has emerged as key mediator of diabetic microvascular complications, the influence of MGO on the vascular transcriptome has not thoroughly been assessed. Since diabetes is associated with low grade inflammation causing sustained nuclear factor-kappa B (NF-κB) activation, the current study addressed 1) to what extent MGO changes the transcriptome of human umbilical vein endothelial cells (HUVECs) exposed to an inflammatory milieu, 2) what are the dominant pathways by which these changes occur and 3) to what extent is this affected by carnosine, a putative scavenger of MGO. Microarray analysis revealed that exposure of HUVECs to high MGO concentrations significantly changes gene expression, characterized by prominent down-regulation of cell cycle associated genes and up-regulation of heme oxygenase-1 (HO-1). KEGG-based pathway analysis identified six significantly enriched pathways of which the p53 pathway was the most affected. No significant enrichment of inflammatory pathways was found, yet, MGO did inhibit VCAM-1 expression in Western blot analysis. Carnosine significantly counteracted MGO-mediated changes in a subset of differentially expressed genes. Collectively, our results suggest that MGO initiates distinct transcriptional changes in cell cycle/apoptosis genes, which may explain MGO toxicity at high concentrations. MGO did not augment TNF-α induced inflammation.

Mingle: A Command Line Utility for Merging Multi-fasta Files.

Massively parallel sequencing (MPS) has become a standard technique in molecular biology whose application has spread from the analysis of the human genome to that of virtually all other organisms. MPS requires reference genomes to be performed and, in some cases, multiple genomes need to be handled as a single unit to carry out genetic analysis. Nucleic acid sequences are typically stored in "fasta" files, which can contain multiple genomes ("multi-fasta"). Although it is possible to convert a multi-fasta file into a single sequence using specific computer commands, the resulting file will not keep track of the boundaries of the original sequences, making it difficult to determine to what genome read obtained from MPS belong to. In this study we introduce mingle, a shell script that can be used to create custom reference genome by merging multi-fasta files while providing a list of boundaries of the individual genomes that can be used for downstream analysis.