The Transcriptomic Profiles of ESR1 and MMP3 Stratify the Risk of Biochemical Recurrence in Primary Prostate Cancer beyond Clinical Features.

The molecular determinants of the heterogenic course of prostate cancer (PC) remain elusive. We aimed to determine the drivers predisposing to unfavorable PC outcomes anticipated by BCR events among patients of similar preoperative characteristics. The TCGA transcriptomic and clinical data of 497 PC individuals were used, stratified according to the risk of BCR by EAU-EANM-ESTRO-ESUR-SIOG. The relevance of the functional markers regarding BCR-free survival was examined by the cutp algorithm. Through UpSetR, subgroups of PC patients bearing an unfavorable signature were identified, followed by the hierarchical clustering of the major markers of the epithelial-to-mesenchymal transition (EMT). BCR-free survival was estimated with the Cox proportional hazards regression model. ESR1 significantly differentiated BCR-free survival, whereas AR did not. An elevation in KLK3 correlated with better prognosis, although PGR, KLK3, CDH1, and MMP3 predicted BCR better than the preoperative PSA level. Patients sharing an unfavorable profile of ESR1 and MMP3 together with lymph node status, Gleason score, T, and EAU risk groups were at a higher risk of BCR originating from mesenchymal features of PC cells. To conclude, we revealed an ESR1-driven unfavorable profile of EMT underpinning a worse PC trajectory. ESR1 may have a major role in PC progression; therefore, it could become a major focus for further investigations.

A Functional Genomics Review of Non-Small-Cell Lung Cancer in Never Smokers.

There is currently a dearth of information regarding lung cancer in never smokers (LCINS). Additionally, there is a difference in somatic mutations, tumour mutational burden, and chromosomal aberrations between smokers and never smokers (NS), insinuating a different disease entity in LCINS. A better understanding of actionable driver alterations prevalent in LCINS and the genomic landscape will contribute to identifying new molecular targets of relevance for NS that will drastically improve outcomes. Differences in treatment outcomes between NS and smokers, as well as sexes, with NSCLC suggest unique tumour characteristics. Epidermal growth factor receptor (EGFR) tyrosine kinase mutations and echinoderm microtubule-associated protein-like 4 anaplastic lymphoma kinase (EML4-ALK) gene rearrangements are more common in NS and have been associated with chemotherapy resistance. Moreover, NS are less likely to benefit from immune mediators including PD-L1. Unravelling the genomic and epigenomic underpinnings of LCINS will aid in the development of not only novel targeted therapies but also more refined approaches. This review encompasses driver genes and pathways involved in the pathogenesis of LCINS and a deeper exploration of the genomic landscape and tumour microenvironment. We highlight the dire need to define the genetic and environmental aspects entailing the development of lung cancer in NS.

Age- and Stage-Dependent Prostate Cancer Aggressiveness Associated with Differential Notch Signaling.

Prostate cancer (PC) remains a worldwide challenge, as does the question of how to distinguish its indolent from its aggressive form to reconcile proper management of the disease with age-related life expectations. This study aimed to differentiate the Notch-driven course of PC regarding patients' ages and stage of their disease. We analyzed 397 PC samples split into age subgroups of ≦55, 60−70, and >70 years old, as well as early vs. late stage. The clinical association of Notch signaling was evaluated by DFS and UpSet analyses. The clustering of downstream effectors was performed with ExpressCluster. Finally, for the most relevant findings, functional networks were constructed with MCODE and stringApp. The results have been validated with an independent cohort. We identified specific patterns of Notch expression associated with unfavorable outcomes, which were reflected by entering into a hybrid epithelial/mesenchymal state and thus reaching tumor plasticity with its all consequences. We characterized the molecular determinants of the age-related clinical behavior of prostate tumors that stem from different invasive properties depending on the route of the EMT program. Of the utmost relevance is the discovery of age- and stage-specific combinations of the Notch molecules predicting unfavorable outcomes and constituting a new prognostic and therapeutic approach for PCs.

The WWOX/HIF1A Axis Downregulation Alters Glucose Metabolism and Predispose to Metabolic Disorders.

Recent reports indicate that the hypoxia-induced factor (HIF1α) and the Warburg effect play an initiating role in glucotoxicity, which underlies disorders in metabolic diseases. WWOX has been identified as a HIF1α regulator. WWOX downregulation leads to an increased expression of HIF1α target genes encoding glucose transporters and glycolysis' enzymes. It has been proven in the normoglycemic mice cells and in gestational diabetes patients. The aim of the study was to determine WWOX's role in glucose metabolism regulation in hyperglycemia and hypoxia to confirm its importance in the development of metabolic disorders. For this purpose, the WWOX gene was silenced in human normal fibroblasts, and then cells were cultured under different sugar and oxygen levels. Thereafter, it was investigated how WWOX silencing alters the genes and proteins expression profile of glucose transporters and glycolysis pathway enzymes, and their activity. In normoxia normoglycemia, higher glycolysis genes expression, their activity, and the lactate concentration were observed in WWOX KO fibroblasts in comparison to control cells. In normoxia hyperglycemia, it was observed a decrease of insulin-dependent glucose uptake and a further increase of lactate. It likely intensifies hyperglycemia condition, which deepen the glucose toxic effect. Then, in hypoxia hyperglycemia, WWOX KO caused weaker glucose uptake and elevated lactate production. In conclusion, the WWOX/HIF1A axis downregulation alters glucose metabolism and probably predispose to metabolic disorders.

In vitro and in silico assessment of the effect of WWOX expression on invasiveness pathways associated with AP-2 transcription factors in bladder cancer.

BACKGROUND: WW Domain Containing Oxidoreductase (WWOX) belongs to the unusual tumor suppressors, whose molecular function is not fully understood in bladder cancer, especially regarding interaction with Activator Protein 2 (AP-2) α/γ transcription factors. Thus, using lentiviral systems we created an in vitro model overexpressing or downregulating WWOX in CAL-29 cell line to assess invasiveness pathways. Surprisingly, while WWOX overexpression was accompanied with increased expression of both AP-2 factors, its downregulation only affected AP-2α level but not AP-2γ which remained high. METHODS: Using cellular models and unpaired t-test or Wilcoxon test, we investigated significant changes in biological processes: clonogenicity, extracellular matrix adhesion, metalloproteinases activity, 3D culture growth, proliferation, mitochondrial redox potential and invasiveness. Relative gene expression acquired through Real-Time qPCR has been analyzed by Welch's t-test. Additionally, using oncoprint analysis we distinguished groups for bioinformatics analyzes in order to perform a follow-up of in vitro experiments. RESULTS: Downregulation of WWOX in bladder cancer cell line intensified ability of single cell to grow into colony, mitochondrial redox potential and proliferation rate. Moreover, these cells shown elevated pro-MMP-2/9 activity but reduced adhesion to collagen I or laminin I, as well as distinct 3D culture growth. Through global in silico profiling we determined that WWOX alters disease-free survival of bladder cancer patients and modulates vital processes through AP-2 downstream effectors. CONCLUSIONS: Our research indicates that WWOX possesses tumor suppressor properties in bladder cancer but consecutive examination is required to entirely understand the contribution of AP-2γ or AP-2α.

[Proteins in cancer multidrug resistance]. / Bialka w opornosci wielolekowej nowotworów.

Multidrug Resistance (MDR) is defined as insensitivity to administered medicines that are structurally unrelated and have different molecular targets. Cancers possess numerous mechanisms of drug resistance, involving various aspects of cell biology. A pivotal role in this phenomenon is played by proteins--enzymatic or structural parts of the cell. Membrane transporters, including the main members of ABC protein family--P-gp, MRP1 and BCRP, as well as LRP, which builds structure of vaults, determine the multidrug-resistant phenotype by decreasing drug concentration within the cell or modifying its distribution to intracellular compartments. The π isoform of protein enzyme--glutathione S-transferase (GSTP-1), is responsible for excessive intensity of detoxification of cytostatics. A common example of altered drug target site that does not respond to chemotherapy is topoisomerase II α (TopoIIa). Alterations of programmed cell death result from expression of metallothionein (MT)--inhibitor of the process, and cytokeratin 18 (CK18), which, if in high concentration, also prevents apoptosis of cells. Several methods of decreasing activity of these proteins have been developed, aiming to overcome MDR in cancer cells. However, for a variety of reasons, their clinical suitability is still very low, leading to continuous increase in death rate among patients. This paper presents current state of knowledge on the most important examples of proteins responsible for MDR of cancer cells and molecular mechanisms of their action.

Correlation between VEGFR-2 receptor kinase domain-containing receptor (KDR) mRNA and angiotensin II receptor type 1 (AT1-R) mRNA in endometrial cancer.

PURPOSE: Angiogenesis, a multistep process that results in new blood vessel formation from preexisting vasculature is essential for both the growth of solid tumour and for metastasis. Stimulation of vascular endothelial growth factor receptor (VEGFR), a transmembrane glycoprotein, results in mitogenesis. Within this family of receptors, VEGFR 2/kinase-insert-domain containing receptor appears to be principally upregulated during tumorigenesis. The aim of this study was to determine the expression of VEGFR-2/kinase-insert-domain containing receptor (KDR) and its correlation with angiotensin receptor type 1 (AT1-R) and clinical factors in endometrial carcinoma. METHODS: The expression of KDR and AT1-R was studied in endometrial carcinoma and normal endometrium by Real-time RT-PCR and Western blot analysis in 136 samples. The expression profile was correlated with the clinicopathological characteristics of endometrial adenocarcinoma. RESULTS: We noted a significant correlation between the expression of KDR and AT1-R in tumour grade G1, G2 and G3 (R(s)=0.50; p=0.002, R(s)=0.69; p=0.0001, R(s)=0.52; p=0.005, respectively). In stage I and stage II carcinoma, a significant correlation was also found between the expression of KDR and AT1-R (R(s)=0.70, p=0.0001, R(s)=0.67; p=0.001, respectively). Moreover significant correlation was observed between both KDR and AT1-R in tissue with different myometrial invasion (R(s)=0.54, p=0.0001, R(s)=0.68; p=0.0001; respectively for tumours with invasion into the inner half and invasion into the outer half). CONCLUSIONS: Basing on received correlation between AT1-R and KDR expression and previous results we speculate that angiotensin through AT1-R modulates KDR expression and thus have influence on local VEGF level. However, further studies are required to clarify the biological interaction between KDR, AT1-R and other hormonal regulators in endometrial carcinoma.

X or Y Cancer: An Extensive Analysis of Sex Differences in Lung Adenocarcinoma.

Background: Cellular metabolism is a tightly controlled process during which cell growth and survival are maintained. Lung cancer is a disease with clear sex differences, where female patients have better survival rates than males. Evidence of sex differences is demonstrated in cancer risk, prognosis and response to different therapies, yet a sex-specific approach to cancer studies is not widely considered. These different tumour characteristics attributed to sex that impact disease outcome, including constitutional genetic and somatic molecular differences, make it essential to assess viral and hormonal influences. Methods: In silico analysis of lung adenocarcinoma (LUAD) TCGA data, including K-means clustering algorithm, dimensional reduction with principal component analysis and differential expression analysis using EdgeR (p < 0.05), were used to explore some robust sex differences in LUAD that exist in core signalling pathways and metabolic processes between males and females. The correlation of differentially expressed genes (DEGs) expression with immune abundance in the LUAD cohort was analysed on TIMER2.0 and adjusted by tumour purity utilising Cox proportional hazard. Multiple factorial analysis heatmap visualisation was used to examine endogenous steroid hormonal effects on LUAD patients with different smoking status and age groups. Results: We found 161 DEGs showing key differences in regulation of immune system and cellular homeostasis, key elements of divergent cancer progression, between the two sexes. We also found male and female LUAD patients to favour different metabolic intermediates for energy production to support tumourigenesis. Additionally, high levels of Tregs accompanied by DEGs correlated with better LUAD prognosis, and circulating hormonal transcriptional targets affect proliferation and progression in males and females differently. Finally, we examined the role of oestrogen protection in men and pre-/postmenopausal women. Conclusions: Further studies should focus on sex-specific changes and investigate sex-specific gene regulatory networks of these DEGs. Several lifestyle factors, including tobacco smoking and diet, differ between males and females. These factors might affect metabolic pathways and can influence the activity of epigenetic regulators, resulting in significant global epigenetic changes.

Antineoplastic Nature of WWOX in Glioblastoma Is Mainly a Consequence of Reduced Cell Viability and Invasion.

Following the discovery of WWOX, research has moved in many directions, including the role of this putative tumor suppressor in the central nervous system and related diseases. The task of determining the nature of WWOX in glioblastoma (GBM) is still considered to be at the initial stage; however, the influence of this gene on the GBM malignant phenotype has already been reported. Because most of the available in vitro research does not consider several cellular GBM models or a wide range of investigated biological assays, the present study aimed to determine the main processes by which WWOX exhibits anticancer properties in GBM, while taking into account the phenotypic heterogeneity between cell lines. Ectopic WWOX overexpression was studied in T98G, DBTRG-05MG, U251MG, and U87MG cell lines that were compared with the use of assays investigating cell viability, proliferation, apoptosis, adhesion, clonogenicity, three-dimensional and anchorage-independent growth, and invasiveness. Observations presenting the antineoplastic properties of WWOX were consistent for T98G, U251MG, and U87MG. Increased proliferation and tumor growth were noted in WWOX-overexpressing DBTRG-05MG cells. A possible explanation for this, arrived at via bioinformatics tools, was linked to the TARDBP transcription factor and expression differences of USP25 and CPNE2 that regulate EGFR surface abundance. Collectively, and despite various cell line-specific circumstances, WWOX exhibits its anticancer nature mainly via a reduction of cell viability and invasiveness of glioblastoma.

Molecular landscapes of glioblastoma cell lines revealed a group of patients that do not benefit from WWOX tumor suppressor expression.

Introduction: Glioblastoma (GBM) is notorious for its clinical and molecular heterogeneity, contributing to therapeutic failure and a grim prognosis. WWOX is one of the tumor suppressor genes important in nervous tissue or related pathologies, which was scarcely investigated in GBM for reliable associations with prognosis or disease progression despite known alterations. Recently, we observed a phenotypic heterogeneity between GBM cell lines (U87MG, T98G, U251MG, DBTRG-05MG), among which the anti-GBM activity of WWOX was generally corresponding, but colony growth and formation were inconsistent in DBTRG-05MG. This prompted us to investigate the molecular landscapes of these cell lines, intending to translate them into the clinical context. Methods: U87MG/T98G/U251MG/DBTRG-05MG were subjected to high-throughput sequencing, and obtained data were explored via weighted gene co-expression network analysis, differential expression analysis, functional annotation, and network building. Following the identification of the most relevant DBTRG-distinguishing driver genes, data from GBM patients were employed for, e.g., differential expression analysis, survival analysis, and principal component analysis. Results: Although most driver genes were unique for each cell line, some were inversely regulated in DBTRG-05MG. Alongside driver genes, the differentially-expressed genes were used to build a WWOX-related network depicting protein-protein interactions in U87MG/T98G/U251MG/DBTRG-05MG. This network revealed processes distinctly regulated in DBTRG-05MG, e.g., microglia proliferation or neurofibrillary tangle assembly. POLE4 and HSF2BP were selected as DBTRG-discriminating driver genes based on the gene significance, module membership, and fold-change. Alongside WWOX, POLE4 and HSF2BP expression was used to stratify patients into cell lines-resembling groups that differed in, e.g., prognosis and treatment response. Some differences from a WWOX-related network were certified in patients, revealing genes that clarify clinical outcomes. Presumably, WWOX overexpression in DBTRG-05MG resulted in expression profile change resembling that of patients with inferior prognosis and drug response. Among these patients, WWOX may be inaccessible for its partners and does not manifest its anti-cancer activity, which was proposed in the literature but not regarding glioblastoma or concerning POLE4 and HSF2BP. Conclusion: Cell lines data enabled the identification of patients among which, despite high expression of WWOX tumor suppressor, no advantageous outcomes were noted due to the cancer-promoting profile ensured by other genes.

Delineating the glioblastoma stemness by genes involved in cytoskeletal rearrangements and metabolic alterations.

Literature data on glioblastoma ongoingly underline the link between metabolism and cancer stemness, the latter is one responsible for potentiating the resistance to treatment, inter alia due to increased invasiveness. In recent years, glioblastoma stemness research has bashfully introduced a key aspect of cytoskeletal rearrangements, whereas the impact of the cytoskeleton on invasiveness is well known. Although non-stem glioblastoma cells are less invasive than glioblastoma stem cells (GSCs), these cells also acquire stemness with greater ease if characterized as invasive cells and not tumor core cells. This suggests that glioblastoma stemness should be further investigated for any phenomena related to the cytoskeleton and metabolism, as they may provide new invasion-related insights. Previously, we proved that interplay between metabolism and cytoskeleton existed in glioblastoma. Despite searching for cytoskeleton-related processes in which the investigated genes might have been involved, not only did we stumble across the relation to metabolism but also reported genes that were found to be implicated in stemness. Thus, dedicated research on these genes in GSCs seems justifiable and might reveal novel directions and/or biomarkers that could be utilized in the future. Herein, we review the previously identified cytoskeleton/metabolism-related genes through the prism of glioblastoma stemness.

LINC01137/miR-186-5p/WWOX: a novel axis identified from WWOX-related RNA interactome in bladder cancer.

Introduction: The discovery of non-coding RNA (ncRNA) dates back to the pre-genomics era, but the progress in this field is still dynamic and leverages current post-genomics solutions. WWOX is a global gene expression modulator that is scarcely investigated for its role in regulating cancer-related ncRNAs. In bladder cancer (BLCA), the link between WWOX and ncRNA remains unexplored. The description of AP-2α and AP-2γ transcription factors, known as WWOX-interacting proteins, is more commonplace regarding ncRNA but still merits investigation. Therefore, this in vitro and in silico study aimed to construct an ncRNA-containing network with WWOX/AP-2 and to investigate the most relevant observation in the context of BLCA cell lines and patients. Methods: RT-112, HT-1376, and CAL-29 cell lines were subjected to two stable lentiviral transductions. High-throughput sequencing of cellular variants (deposited in the Gene Expression Omnibus database under the GSE193659 record) enabled the investigation of WWOX/AP-2-dependent differences using various bioinformatics tools (e.g., limma-voom, FactoMineR, multiple Support Vector Machine Recursive Feature Elimination (mSVM-RFE), miRDB, Arena-Idb, ncFANs, RNAhybrid, TargetScan, Protein Annotation Through Evolutionary Relationships (PANTHER), Gene Transcription Regulation Database (GTRD), or Evaluate Cutpoints) and repositories such as The Cancer Genome Atlas (TCGA) and Cancer Cell Line Encyclopedia. The most relevant observations from cap analysis gene expression sequencing (CAGE-seq) were confirmed using real-time PCR, whereas TCGA data were validated using the GSE31684 cohort. Results: The first stage of the whole study justified focusing solely on WWOX rather than on WWOX combined with AP-2α/γ. The most relevant observation of the developed ncRNA-containing network was LINC01137, i.e., long non-coding RNAs (lncRNAs) that unraveled the core network containing UPF1, ZC3H12A, LINC01137, WWOX, and miR-186-5p, the last three being a novel lncRNA/miRNA/mRNA axis. Patients' data confirmed the LINC01137/miR-186-5p/WWOX relationship and provided a set of dependent genes (i.e., KRT18, HES1, VCP, FTH1, IFITM3, RAB34, and CLU). Together with the core network, the gene set was subjected to survival analysis for both TCGA-BLCA and GSE31684 patients, which indicated that the increased expression of WWOX or LINC01137 is favorable, similar to their combination with each other (WWOX↑ and LINC01137↑) or with MIR186 (WWOX↑/LINC01137↑ but MIR186↓). Conclusion: WWOX is implicated in the positive feedback loop with LINC01137 that sponges WWOX-targeting miR-186-5p. This novel WWOX-containing lncRNA/miRNA/mRNA axis should be further investigated to depict its relationships in a broader context, which could contribute to BLCA research and treatment.

Determination of WWOX Function in Modulating Cellular Pathways Activated by AP-2α and AP-2γ Transcription Factors in Bladder Cancer.

Following the invention of high-throughput sequencing, cancer research focused on investigating disease-related alterations, often inadvertently omitting tumor heterogeneity. This research was intended to limit the impact of heterogeneity on conclusions related to WWOX/AP-2α/AP-2γ in bladder cancer which differently influenced carcinogenesis. The study examined the signaling pathways regulated by WWOX-dependent AP-2 targets in cell lines as biological replicates using high-throughput sequencing. RT-112, HT-1376 and CAL-29 cell lines were subjected to two stable lentiviral transductions. Following CAGE-seq and differential expression analysis, the most important genes were identified and functionally annotated. Western blot was performed to validate the selected observations. The role of genes in biological processes was assessed and networks were visualized. Ultimately, principal component analysis was performed. The studied genes were found to be implicated in MAPK, Wnt, Ras, PI3K-Akt or Rap1 signaling. Data from pathways were collected, explaining the differences/similarities between phenotypes. FGFR3, STAT6, EFNA1, GSK3B, PIK3CB and SOS1 were successfully validated at the protein level. Afterwards, a definitive network was built using 173 genes. Principal component analysis revealed that the various expression of these genes explains the phenotypes. In conclusion, the current study certified that the signaling pathways regulated by WWOX and AP-2α have more in common than that regulated by AP-2γ. This is because WWOX acts as an EMT inhibitor, AP-2γ as an EMT enhancer while AP-2α as a MET inducer. Therefore, the relevance of AP-2γ in targeted therapy is now more evident. Some of the differently regulated genes can find application in bladder cancer treatment.

WWOX and metabolic regulation in normal and pathological conditions.

WW domain-containing oxidoreductase (WWOX) spans the common fragile site FRA16D. There is evidence that translocations and deletions affecting WWOX accompanied by loss of expression are frequent in many cancers and often correlate with a worse prognosis. Additionally, WWOX germline mutations were also found to be the cause of pathologies of brain development. Because WWOX binds to some transcription factors, it is a modulator of many cellular processes, including metabolic processes. Recently, studies have linked WWOX to familial dyslipidemias, osteopenia, metabolic syndrome, and gestational diabetes, confirming its role as a regulator of steroid, cholesterol, glucose, and normal bone metabolism. The WW domain of WWOX is directly engaged in the control of the activity of transcription factors such as HIF1α and RUNX2; therefore, WWOX gene alterations are associated with some metabolic abnormalities. Presently, most interest is devoted to the associations between WWOX and glucose and basic energy metabolism disturbances. In particular, its involvement in the initiation of the Warburg effect in cancer or gestational diabetes and type II diabetes is of interest. This review is aimed at systematically and comprehensively presenting the current state of knowledge about the participation of WWOX in the metabolism of healthy and diseased organisms.

Prognostic significance of AP-2α/γ targets as cancer therapeutics.

Identifying genes with prognostic importance could improve cancer treatment. An increasing number of reports suggest the existence of successful strategies based on seemingly "untargetable" transcription factors. In addition to embryogenesis, AP-2 transcription factors are known to play crucial roles in cancer development. Members of this family can be used as prognostic factors in oncological patients, and AP-2α/γ transcription factors were previously investigated in our pan-cancer comparative study using their target genes. The present study investigates tumors that were previously found similar with an emphasis on the possible role of AP-2 factors in specific cancer types. The RData workspace was loaded back to R environment and 3D trajectories were built via Monocle3. The genes that met the requirement of specificity were listed using top_markers(), separately for mutual and unique targets. Furthermore, the candidate genes had to meet the following requirements: correlation with AP-2 factor (through Correlation AnalyzeR) and validated prognostic importance (using GEPIA2 and subsequently KM-plotter or LOGpc). Eventually, the ROC analysis was applied to confirm their predictive value; co-dependence of expression was visualized via BoxPlotR. Some similar tumors were differentiated by AP-2α/γ targets with prognostic value. Requirements were met by only fifteen genes (EMX2, COL7A1, GRIA1, KRT1, KRT14, SLC12A5, SEZ6L, PTPRN, SCG5, DPP6, NTSR1, ARX, COL4A3, PPEF1 and TMEM59L); of these, the last four were excluded based on ROC curves. All the above genes were confronted with the literature, with an emphasis on the possible role played by AP-2 factors in specific cancers. Following ROC analysis, the genes were verified using immunohistochemistry data and progression-related signatures. Staining differences were observed, as well as co-dependence on the expression of e.g. CTNNB1, ERBB2, KRAS, SMAD4, EGFR or MKI67. In conclusion, prognostic value of targets suggested AP-2α/γ as candidates for novel cancer treatment. It was also revealed that AP-2 targets are related to tumor progression and that some mutual target genes could be inversely regulated.

TGFα-EGFR pathway in breast carcinogenesis, association with WWOX expression and estrogen activation.

WWOX is a tumor-suppressive steroid dehydrogenase, which relationship with hormone receptors was shown both in animal models and breast cancer patients. Herein, through nAnT-iCAGE high-throughput gene expression profiling, we studied the interplay of estrogen receptors and the WWOX in breast cancer cell lines (MCF7, T47D, MDA-MB-231, BT20) under estrogen stimulation and either introduction of the WWOX gene by retroviral transfection (MDA-MB-231, T47D) or silenced with shRNA (MCF7, BT20). Additionally, we evaluated the consequent biological characteristics by proliferation, apoptosis, invasion, and adhesion assays. TGFα-EGFR signaling was found to be significantly affected in all examined breast cancer cell lines in response to estrogen and strongly associated with the level of WWOX expression, especially in ER-positive MCF7 cells. Under the influence of 17ß-estradiol presence, biological characteristics of the cell lines were also delineated. The study revealed modulation of adhesion, invasion, and apoptosis. The obtained results point at a complex role of the WWOX gene in the carcinogenesis of the breast tissue, which seems to be closely related to the presence of estrogen α and/or ß receptors.

Molecular analysis of WWOX expression correlation with proliferation and apoptosis in glioblastoma multiforme.

Glioblastoma multiforme is the most common type of primary brain tumor in adults. WWOX is a tumor suppressor gene involved in carcinogenesis and cancer progression in many different neoplasms. Reduced WWOX expression is associated with more aggressive phenotype and poor patient outcome in several cancers. We investigated alternations of WWOX expression and its correlation with proliferation, apoptosis and signal trafficking in 67 glioblastoma multiforme specimens. Moreover, we examined the level of WWOX LOH and methylation status in WWOX promoter region. Our results suggest that loss of heterozygosity (relatively frequent in glioblastoma multiforme) along with promoter methylation may decrease the expression of this tumor suppressor gene. Our experiment revealed positive correlations between WWOX and Bcl2 and between WWOX and Ki67. We also confirmed that WWOX is positively correlated with ErbB4 signaling pathway in glioblastoma multiforme.

WWOX oxidoreductase--substrate and enzymatic characterization.

WWOX is a tumour suppressor gene that spans the common fragile site FRA16D. Analysis of the WWOX expression pattern in normal human tissues showed the highest expression in testis, prostate, and ovary. Its altered expression has been demonstrated in different tissues and tumour types. The WWOX gene encodes a 414-amino acids protein, which is the first discovered protein with a short-chain dehydrogenase/reductase (SDR) central domain and two WW domains at the NH2 terminus. Due to its potential role in sex-steroid metabolism, using two bacterial expression systems, we have cloned WWOX fusion proteins showing oxidoreductase activity in a crude extract, defined a course of enzymatic reactions for selected steroid substrates, and determined related Km values. Our results show that the SDR domain of the WWOX protein has dehydrogenase activity and is reactive both in the presence of NAD+ and NADP+ for all examined steroid substrates. On the other hand, with the same substrates and reduced cofactors (NADH and NADPH) reduction activity was not observed.

Notch Signaling Pathway in Cancer-Review with Bioinformatic Analysis.

Notch signaling is an evolutionarily conserved pathway regulating normal embryonic development and homeostasis in a wide variety of tissues. It is also critically involved in carcinogenesis, as well as cancer progression. Activation of the Notch pathway members can be either oncogenic or suppressive, depending on tissue context. The present study is a comprehensive overview, extended with a bioinformatics analysis of TCGA cohorts, including breast, bladder, cervical, colon, kidney, lung, ovary, prostate and rectum carcinomas. We performed global expression profiling of the Notch pathway core components and downstream targets. For this purpose, we implemented the Uniform Manifold Approximation and Projection algorithm to reduce the dimensions. Furthermore, we determined the optimal cutpoint using Evaluate Cutpoint software to established disease-free and overall survival with respect to particular Notch members. Our results demonstrated separation between tumors and their corresponding normal tissue, as well as between tumors in general. The differentiation of the Notch pathway, at its various stages, in terms of expression and survival resulted in distinct profiles of biological processes such as proliferation, adhesion, apoptosis and epithelial to mesenchymal transition. In conclusion, whether oncogenic or suppressive, Notch signaling is proven to be associated with various types of malignancies, and thus may be of interest as a potential therapeutic target.

PLEK2, RRM2, GCSH: A Novel WWOX-Dependent Biomarker Triad of Glioblastoma at the Crossroads of Cytoskeleton Reorganization and Metabolism Alterations.

Glioblastoma is one of the deadliest human cancers. Its malignancy depends on cytoskeleton reorganization, which is related to, e.g., epithelial-to-mesenchymal transition and metastasis. The malignant phenotype of glioblastoma is also affected by the WWOX gene, which is lost in nearly a quarter of gliomas. Although the role of WWOX in the cytoskeleton rearrangement has been found in neural progenitor cells, its function as a modulator of cytoskeleton in gliomas was not investigated. Therefore, this study aimed to investigate the role of WWOX and its collaborators in cytoskeleton dynamics of glioblastoma. Methodology on RNA-seq data integrated the use of databases, bioinformatics tools, web-based platforms, and machine learning algorithm, and the obtained results were validated through microarray data. PLEK2, RRM2, and GCSH were the most relevant WWOX-dependent genes that could serve as novel biomarkers. Other genes important in the context of cytoskeleton (BMP4, CCL11, CUX2, DUSP7, FAM92B, GRIN2B, HOXA1, HOXA10, KIF20A, NF2, SPOCK1, TTR, UHRF1, and WT1), metabolism (MTHFD2), or correlation with WWOX (COL3A1, KIF20A, RNF141, and RXRG) were also discovered. For the first time, we propose that changes in WWOX expression dictate a myriad of alterations that affect both glioblastoma cytoskeleton and metabolism, rendering new therapeutic possibilities.