SLIT-ROBO families of proteins mediate axon pathfinding and their expression is not solely confined to nervous system. Aberrant expression of SLIT-ROBO genes was repeatedly shown in a wide variety of cancers, yet data about their collective behavior in hepatocellular carcinoma (HCC) is missing. Hence, we quantified SLIT-ROBO transcripts in HCC cell lines, and in normal and tumor tissues from liver.

Accuracy in the diagnosis of breast cancer and classification of cancer subtypes has improved over the years with the development of well-established immunohistopathological criteria. More recently, diagnostic gene-sets at the mRNA expression level have been tested as better predictors of disease state. However, breast cancer is heterogeneous in nature; thus extraction of differentially expressed gene-sets that stably distinguish normal tissue from various pathologies poses challenges. Meta-analysis of high-throughput expression data using a collection of statistical methodologies leads to the identification of robust tumor gene expression signatures.

CpG hypermethylation in gene promoters is a frequent mechanism of tumor suppressor gene silencing in various types of cancers. It usually occurs at early steps of cancer progression and can be detected easily, giving rise to development of promising biomarkers for both detection and progression of cancer, including breast cancer. 5-aza-2'-deoxycytidine (AZA) is a DNA demethylating and anti-cancer agent resulting in induction of genes suppressed via DNA hypermethylation.

WDR81 (WD repeat-containing protein 81) is associated with cerebellar ataxia, mental retardation and disequilibrium syndrome (CAMRQ2, [MIM 610185]). Human and mouse studies suggest that it might be a gene of importance during neurodevelopment. This study aimed at fully characterizing the structure of the wdr81 transcript, detecting the possible transcript variants and revealing its expression profile in zebrafish, a powerful model organism for studying development and disease.

Background: Prognostic biomarkers for cancer have the power to change the course of disease if they add value beyond known prognostic factors, if they can help shape treatment protocols, and if they are reliable. The aim of this study was to identify such biomarkers for colon cancer and to understand the molecular mechanisms leading to prognostic stratifications based on these biomarkers. Methods and Findings: We used an in house R based script (SSAT) for the in silico discovery of stage-independent prognostic biomarkers using two cohorts, GSE17536 and GSE17537, that include 177 and 55 colon cancer patients, respectively. This identified 2 genes, ULBP2 and SEMA5A, which when used jointly, could distinguish patients with distinct prognosis. We validated our findings using a third cohort of 48 patients ex vivo. We find that in all cohorts, a combined ULBP2/SEMA5A classification (SU-GIB) can stratify distinct prognostic sub-groups with hazard ratios that range from 2.4 to 4.5 (p≤0.01) when overall- or cancer-specific survival is used as an end-measure, independent of confounding prognostic parameters. In addition, our preliminary analyses suggest SU-GIB is comparable to Oncotype DX colon(®) in predicting recurrence in two different cohorts (HR: 1.5-2; p≤0.02). SU-GIB has potential as a companion diagnostic for several drugs including the PI3K/mTOR inhibitor BEZ235, which are suitable for the treatment of patients within the bad prognosis group. We show that tumors from patients with worse prognosis have low EGFR autophosphorylation rates, but high caspase 7 activity, and show upregulation of pro-inflammatory cytokines that relate to a relatively mesenchymal phenotype. Conclusions: We describe two novel genes that can be used to prognosticate colon cancer and suggest approaches by which such tumors can be treated. We also describe molecular characteristics of tumors stratified by the SU-GIB signature.

The steroid receptor RNA activator 1 (SRA1) gene is involved in the progression of various cancers via different molecular mechanisms mediated by long non-coding RNA SRA (lncRNA SRA). This study aimed to evaluate the lncRNA SRA effect on the tumor progression of colorectal cancer (CRC).

Pancreatic cancer (PCA) is one of the most lethal malignancies worldwide with a 5-year survival rate of 9%. Despite the advances in the field, the need for an earlier detection and effective therapies is paramount. PCA high heterogeneity suggests that epigenetic alterations play a key role in tumour development. However, only few epigenetic biomarkers or therapeutic targets have been identified so far. Here we explored the potential of distinct DNA methylation signatures as biomarkers for early detection and prognosis of PCA. PI3K/AKT-related genes differentially expressed in PCA were identified using the Pancreatic Expression Database (n = 153). Methylation data from PCA patients was obtained from The Cancer Genome Atlas (n = 183), crossed with clinical data to evaluate the biomarker potential of the epigenetic signatures identified and validated in independent cohorts. The majority of selected genes presented higher expression and hypomethylation in tumour tissue. The methylation signatures of specific genes in the PI3K/AKT pathway could distinguish normal from malignant tissue at initial disease stages with AUC > 0.8, revealing their potential as PCA diagnostic tools. ITGA4, SFN, ITGA2, and PIK3R1 methylation levels could be independent prognostic indicators of patients' survival. Methylation status of SFN and PIK3R1 were also associated with disease recurrence. Our study reveals that the methylation levels of PIK3/AKT genes involved in PCA could be used to diagnose and predict patients' clinical outcome with high sensitivity and specificity. These results provide new evidence of the potential of epigenetic alterations as biomarkers for disease screening and management and highlight possible therapeutic targets.

Transcriptome experiments are performed to assess protein abundance through mRNA expression analysis. Expression levels of genes vary depending on the experimental conditions and the cell response. Transcriptome data must be diverse and yet comparable in reference to stably expressed genes, even if they are generated from different experiments on the same biological context from various laboratories. In this study, expression patterns of 9090 microarray samples grouped into 381 NCBI-GEO datasets were investigated to identify novel candidate reference genes using randomizations and Receiver Operating Characteristic (ROC) curves. The analysis demonstrated that cell type specific reference gene sets display less variability than a united set for all tissues. Therefore, constitutively and stably expressed, origin specific novel reference gene sets were identified based on their coefficient of variation and percentage of occurrence in all GEO datasets, which were classified using Medical Subject Headings (MeSH). A large number of MeSH grouped reference gene lists are presented as novel tissue specific reference gene lists. The most commonly observed 17 genes in these sets were compared for their expression in 8 hepatocellular, 5 breast and 3 colon carcinoma cells by RT-qPCR to verify tissue specificity. Indeed, commonly used housekeeping genes GAPDH, Actin and EEF2 had tissue specific variations, whereas several ribosomal genes were among the most stably expressed genes in vitro. Our results confirm that two or more reference genes should be used in combination for differential expression analysis of large-scale data obtained from microarray or next generation sequencing studies. Therefore context dependent reference gene sets, as presented in this study, are required for normalization of expression data from diverse technological backgrounds.

Cancer-testis (CT) genes are expressed in various cancers but not in normal tissues other than in cells of the germline. Although DNA demethylation of promoter-proximal CpGs of CT genes is linked to their expression in cancer, the mechanisms leading to demethylation are unknown. To elucidate such mechanisms we chose to study the Caco-2 colorectal cancer cell line during the course of its spontaneous differentiation in vitro, as we found CT genes, in particular PAGE2, -2B and SPANX-B, to be up-regulated during this process. Differentiation of these cells resulted in a mesenchymal-to-epithelial transition (MET) as evidenced by the gain of epithelial markers CDX2, Claudin-4 and E-cadherin, and a concomitant loss of mesenchymal markers Vimentin, Fibronectin-1 and Transgelin. PAGE2 and SPAN-X up-regulation was accompanied by an increase in Ten-eleven translocation-2 (TET2) expression and cytosine 5-hydroxymethylation as well as the disassociation of heterochromatin protein 1 and the polycomb repressive complex 2 protein EZH2 from promoter-proximal regions of these genes. Reversal of differentiation resulted in down-regulation of PAGE2, -2B and SPANX-B, and induction of epithelial-to-mesenchymal transition (EMT) markers, demonstrating the dynamic nature of CT gene regulation in this model.

The zebrafish has become a popular model for studying normal brain aging due to its large fecundity, conserved genome, and available genetic tools; but little data exists about neurobiological age-related alterations. The current study tested the hypothesis of an association between brain aging and synaptic protein loss across males and females. Western blot analysis of synaptophysin (SYP), a presynaptic vesicle protein, and postsynaptic density-95 (PSD-95) and gephyrin (GEP), excitatory and inhibitory postsynaptic receptor-clustering proteins, respectively, was performed in young, middle-aged, and old male and female zebrafish (Danio rerio) brains. Univariate and multivariate analyses demonstrated that PSD-95 significantly increased in aged females and SYP significantly decreased in males, but GEP was stable. Thus, these key synaptic proteins vary across age in a sexually dimorphic manner, which has been observed in other species, and these consequences may represent selective vulnerabilities for aged males and females. These data expand our knowledge of normal aging in zebrafish, as well as further establish this model as an appropriate one for examining human brain aging.

microRNA expression and sequence analysis database (http://konulab.fen.bilkent.edu.tr/mirna/) (mESAdb) is a regularly updated database for the multivariate analysis of sequences and expression of microRNAs from multiple taxa. mESAdb is modular and has a user interface implemented in PHP and JavaScript and coupled with statistical analysis and visualization packages written for the R language. The database primarily comprises mature microRNA sequences and their target data, along with selected human, mouse and zebrafish expression data sets. mESAdb analysis modules allow (i) mining of microRNA expression data sets for subsets of microRNAs selected manually or by motif; (ii) pair-wise multivariate analysis of expression data sets within and between taxa; and (iii) association of microRNA subsets with annotation databases, HUGE Navigator, KEGG and GO. The use of existing and customized R packages facilitates future addition of data sets and analysis tools. Furthermore, the ability to upload and analyze user-specified data sets makes mESAdb an interactive and expandable analysis tool for microRNA sequence and expression data.

As mutations in SARS-CoV-2 virus accumulate rapidly, novel primers that amplify this virus sensitively and specifically are in demand. We have developed a webserver named CoVrimer by which users can search for and align existing or newly designed conserved/degenerate primer pair sequences against the viral genome and assess the mutation load of both primers and amplicons. CoVrimer uses mutation data obtained from an online platform established by NGDC-CNCB (12 May 2021) to identify genomic regions, either conserved or with low levels of mutations, from which potential primer pairs are designed and provided to the user for filtering based on generalized and SARS-CoV-2 specific parameters. Alignments of primers and probes can be visualized with respect to the reference genome, indicating variant details and the level of conservation. Consequently, CoVrimer is likely to help researchers with the challenges posed by viral evolution and is freely available at http://konulabapps.bilkent.edu.tr:3838/CoVrimer/.

Numerous natural products exhibit antiproliferative activity against cancer cells by modulating various biological pathways. In this study, we investigated the potential use of eight natural compounds (apigenin, curcumin, epigallocatechin gallate, fisetin, forskolin, procyanidin B2, resveratrol, urolithin A) and two repurposed agents (fulvestrant and metformin) as chemotherapy enhancers and mesenchymal-to-epithelial (MET) inducers of cancer cells. Screening of these compounds in various colon, breast, and pancreatic cancer cell lines revealed anti-cancer activity for all compounds, with curcumin being the most effective among these in all cell lines. Although some of the natural products were able to induce MET in some cancer cell lines, the MET induction was not related to increased synergy with either 5-FU, irinotecan, gemcitabine, or gefitinib. When synergy was observed, for example with curcumin and irinotecan, this was unrelated to MET induction, as assessed by changes in E-cadherin and vimentin expression. Our results show that MET induction is compound and cell line specific, and that MET is not necessarily related to enhanced chemosensitivity.

Although glioblastomas are common, there remains a need to elucidate the underlying mechanisms behind their initiation and progression and identify molecular pathways for improving treatment. In this study, sixteen fresh-frozen glioblastoma samples and seven samples of healthy brain tissues were analyzed with miRNA and whole transcriptome microarray chips. Candidate miRNAs and mRNAs were selected to validate expression in fifty patient samples in total with the criteria of abundance, relevance and prediction scores. miRNA and target mRNA relationships were assessed by inhibiting selected miRNAs in glioblastoma cells. Functional tests have been conducted in order to see the effects of miRNAs on invasion, migration and apoptosis of GBM cells. Analyses were carried out to determine correlations between selected molecules and clinicopathological features. 1332 genes and 319 miRNAs were found to be dysregulated by the microarrays. The results were combined and analyzed with Transcriptome Analysis Console 3 software and the DAVID online database. Primary differential pathways included Ras, HIF-1, MAPK signaling and cell adhesion. OncomiR candidates 21-5p, 92b-3p, 182-5p and 339-5p for glioblastoma negatively correlated with notable mRNA targets both in tissues and in in vitro experiments. miR-21-5p and miR-339-5p significantly affected migration, invasion and apoptosis of GBM cells in vitro. Significant correlations with overall survival, tumor volume, recurrence and age at diagnosis were discovered. In this article we present valuable integrated microarray analysis of glioblastoma samples regarding miRNA and gene-expression levels. Notable biomarkers and miRNA-mRNA interactions have been identified, some of which correlated with clinicopathological features in our cohort.

Background. There is not yet an agreed adjuvant treatment for melanoma patients with American Joint Committee on Cancer stages III B and C. We report administration of an autologous melanoma vaccine to prevent disease recurrence. Patients and Methods. 126 patients received eight doses of irradiated autologous melanoma cells conjugated to dinitrophenyl and mixed with BCG. Delayed type hypersensitivity (DTH) response to unmodified melanoma cells was determined on the vaccine days 5 and 8. Gene expression analysis was performed on 35 tumors from patients with good or poor survival. Results. Median overall survival was 88 months with a 5-year survival of 54%. Patients attaining a strong DTH response had a significantly better (p = 0.0001) 5-year overall survival of 75% compared with 44% in patients without a strong response. Gene expression array linked a 50-gene signature to prognosis, including a cluster of four cancer testis antigens: CTAG2 (NY-ESO-2), MAGEA1, SSX1, and SSX4. Thirty-five patients, who received an autologous vaccine, followed by ipilimumab for progressive disease, had a significantly improved 3-year survival of 46% compared with 19% in nonvaccinated patients treated with ipilimumab alone (p = 0.007). Conclusion. Improved survival in patients attaining a strong DTH and increased response rate with subsequent ipilimumab suggests that the autologous vaccine confers protective immunity.

Cholinergic Receptor Nicotinic Alpha 5 (CHRNA5) is an important susceptibility locus for nicotine addiction and lung cancer. Depletion of CHRNA5 has been associated with reduced cell viability, increased apoptosis and alterations in cellular motility in different cancers yet not in breast cancer. Herein we first showed the expression of CHRNA5 was variable and positively correlated with the fraction of total genomic alterations in breast cancer cell lines and tumors indicating its potential role in DNA damage response (DDR). Next, we demonstrated that silencing of CHRNA5 expression in MCF7 breast cancer cell line by RNAi affected expression of genes involved in cytoskeleton, TP53 signaling, DNA synthesis and repair, cell cycle, and apoptosis. The transcription profile of CHRNA5 depleted MCF7 cells showed a significant positive correlation with that of A549 lung cancer cell line while exhibiting a negative association with the CHRNA5 co-expression profile obtained from Cancer Cell Line Encylopedia (CCLE). Moreover, it exhibited high similarities with published MCF7 expression profiles obtained from exposure to TP53 inducer nutlin-3a and topoisomerase inhibitors. We then demonstrated that CHRNA5 siRNA treatment reduced cell viability and DNA synthesis indicating G1 arrest while it significantly increased apoptotic sub-G1 cell population. Accordingly, we observed lower levels of phosphorylated RB (Ser807/811) and an increased BAX/BCL2 ratio in RNAi treated MCF7 cells. We also showed that CHRNA5 RNAi transcriptome correlated negatively with DDR relevant gene expression profile in breast cancer gene expression datasets while the coexposure to topoisomerase inhibitors in the presence of CHRNA5 RNAi enhanced chemosensitivity potentially due to reduced DDR. CHRNA5 RNAi consistently lowered total CHEK1 mRNA and protein levels as well as phosphorylated CHEK1 (Ser345) in MCF7 cells. We also detected a significant positive correlation between the expression levels of CHRNA5 and CHEK1 in CCLE, TCGA and METABRIC breast cancer datasets. Our study suggests CHRNA5 RNAi is associated with cell cycle inhibition, apoptosis as well as reduced DDR and increased drug sensitivity in breast cancer yet future studies are warranted since dose- and cell line-specific differences exist in response to CHRNA5 depletion. Gene expression microarray data can be accessed from GEO database under the accession number GSE89333.

Acetylcholinesterase (AChE), an enzyme responsible for degradation of acetylcholine, has been identified as a prognostic marker in liver cancer. Although in vivo Ache tumorigenicity assays in mouse are present, no established liver cancer xenograft model in zebrafish using an ache mutant background exists. Herein, we developed an embryonic zebrafish xenograft model using epithelial (Hep3B) and mesenchymal (SKHep1) liver cancer cell lines in wild-type and ache sb55 sibling mutant larvae after characterization of cholinesterase expression and activity in cell lines and zebrafish larvae. The comparison of fluorescent signal reflecting tumor size at 3-days post-injection (dpi) revealed an enhanced tumorigenic potential and a reduced migration capacity in cancer cells injected into homozygous ache sb55 mutants when compared with the wild-type. Increased tumor load was confirmed using an ALU based tumor DNA quantification method modified for use in genotyped xenotransplanted zebrafish embryos. Confocal microscopy using the Huh7 cells stably expressing GFP helped identify the distribution of tumor cells in larvae. Our results imply that acetylcholine accumulation in the microenvironment directly or indirectly supports tumor growth in liver cancer. Use of this model system for drug screening studies holds potential in discovering new cholinergic targets for treatment of liver cancers.

Despite the availability of various treatment protocols, response to therapy in patients with Acute Myeloid Leukemia (AML) remains largely unpredictable. Transcriptomic profiling studies have thus far revealed the presence of molecular subtypes of AML that are not accounted for by standard clinical parameters or by routinely used biomarkers. Such molecular subtypes of AML are predicted to vary in response to chemotherapy or targeted therapy. The Renin-Angiotensin System (RAS) is an important group of proteins that play a critical role in regulating blood pressure, vascular resistance and fluid/electrolyte balance. RAS pathway genes are also known to be present locally in tissues such as the bone marrow, where they play an important role in leukemic hematopoiesis. In this study, we asked if the RAS genes could be utilized to predict drug responses in patients with AML. We show that the combined in silico analysis of up to five RAS genes can reliably predict sensitivity to Doxorubicin as well as Etoposide in AML. The same genes could also predict sensitivity to Doxorubicin when tested in vitro. Additionally, gene set enrichment analysis revealed enrichment of TNF-alpha and type-I IFN response genes among sensitive, and TGF-beta and fibronectin related genes in resistant cancer cells. However, this does not seem to reflect an epithelial to mesenchymal transition per se. We also identified that RAS genes can stratify patients with AML into subtypes with distinct prognosis. Together, our results demonstrate that genes present in RAS are biomarkers for drug sensitivity and the prognostication of AML.

Senescence is a permanent proliferation arrest in response to cell stress such as DNA damage. It contributes strongly to tissue aging and serves as a major barrier against tumor development. Most tumor cells are believed to bypass the senescence barrier (become "immortal") by inactivating growth control genes such as TP53 and CDKN2A. They also reactivate telomerase reverse transcriptase. Senescence-to-immortality transition is accompanied by major phenotypic and biochemical changes mediated by genome-wide transcriptional modifications. This appears to happen during hepatocellular carcinoma (HCC) development in patients with liver cirrhosis, however, the accompanying transcriptional changes are virtually unknown. We investigated genome-wide transcriptional changes related to the senescence-to-immortality switch during hepatocellular carcinogenesis. Initially, we performed transcriptome analysis of senescent and immortal clones of Huh7 HCC cell line, and identified genes with significant differential expression to establish a senescence-related gene list. Through the analysis of senescence-related gene expression in different liver tissues we showed that cirrhosis and HCC display expression patterns compatible with senescent and immortal phenotypes, respectively; dysplasia being a transitional state. Gene set enrichment analysis revealed that cirrhosis/senescence-associated genes were preferentially expressed in non-tumor tissues, less malignant tumors, and differentiated or senescent cells. In contrast, HCC/immortality genes were up-regulated in tumor tissues, or more malignant tumors and progenitor cells. In HCC tumors and immortal cells genes involved in DNA repair, cell cycle, telomere extension and branched chain amino acid metabolism were up-regulated, whereas genes involved in cell signaling, as well as in drug, lipid, retinoid and glycolytic metabolism were down-regulated. Based on these distinctive gene expression features we developed a 15-gene hepatocellular immortality signature test that discriminated HCC from cirrhosis with high accuracy. Our findings demonstrate that senescence bypass plays a central role in hepatocellular carcinogenesis engendering systematic changes in the transcription of genes regulating DNA repair, proliferation, differentiation and metabolism.

Long non-coding RNAs (lncRNAs) have been shown to have functional roles in cancer biology and are dys-regulated in many tumors. Colon Cancer Associated Transcript -1 (CCAT1) is a lncRNA, previously shown to be significantly up-regulated in colon cancer. The aim of this study is to determine expression levels of CCAT1 in gastric carcinoma (GC).