Cancer-associated transcription factors in DNA damage response.

Transcription factors (TFs) constitute a wide and highly diverse group of proteins capable of controlling gene expression. Their roles in oncogenesis, tumor progression, and metastasis have been established, but recently their role in the DNA damage response pathway (DDR) has emerged. Many of them can affect elements of canonical DDR pathways, modulating their activity and deciding on the effectiveness of DNA repair. In this review, we focus on the latest reports on the effects of two TFs with dual roles in oncogenesis and metastasis (hypoxia-inducible factor-1 α (HIF1α), proto-oncogene MYC) and three epithelial-mesenchymal transition (EMT) TFs (twist-related protein 1 (TWIST), zinc-finger E-box binding homeobox 1 (ZEB1), and zinc finger protein 281 (ZNF281)) associated with control of canonical DDR pathways.

Do the Successive Waves of SARS-CoV-2, Vaccination Status and Place of Infection Influence the Clinical Picture and COVID-19 Severity among Patients with Persistent Clinical Symptoms? The Retrospective Study of Patients from the STOP-COVID Registry of the PoLoCOV-Study.

The severity of ailments caused by SARS-CoV-2 varies and the clinical picture has already evolved during the pandemic, complicating diagnostics. In Poland, no study has been performed to assess the clinical picture of patients across the successive pandemic waves. The aim of the study was to present the characteristics of patients who present to medical center because of persistent symptoms after COVID-19, and to study differences between hospitalized/non-hospitalized, vaccinated/non-vaccinated individuals and between different waves in Poland. This is a retrospective study evaluating the clinical presentation of COVID-19 patients from the STOP-COVID registry of the PoLoCOV-Study. This registry includes patients who present to the medical center because of persistent clinical symptoms after the isolation. The patients' data were obtained from individuals who suffered from COVID-19 between September 2020 and December 2021.The patients were divided into groups according to the infection rate increase pattern (II/III/IV pandemic wave), status of vaccination and place of isolation. Regardless of the pandemic wave, the patients' most commonly reported weaknesses were a cough and a headache. The arterial hypertension and hyperlipidemia were the most frequent concomitant chronic conditions. Hospitalized patients more often reported weakness or a cough while home-isolated patients were more likely to have rhinitis or a headache. Patients who completed the vaccination course showed a shorter duration of clinical symptoms and a lower mean number of symptoms. Additionally, vaccinated individuals reported less taste and/or olfactory dysfunction than unvaccinated individuals. To conclude, the persistence of the pandemic has resulted in significant changes observed in the clinical picture. Successive waves caused deterioration in the subjective assessment of the disease severity. A cough seemed to occur more frequently in the later pandemic waves.

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.

Discovering biomarkers for hormone-dependent tumors: in silico study on signaling pathways implicated in cell cycle and cytoskeleton regulation.

Malignancies dependent on hormone homeostasis include breast, ovary, cervical, prostate, testis and uterine tumors. Hormones are involved in signal transduction which orchestrate processes, such as apoptosis, proliferation, cell cycle or cytoskeleton organization. Currently, there is a need for novel biomarkers which would help to diagnose cancers efficiently. In this study, the genes implicated in signaling that is important in hormone-sensitive carcinogenesis were investigated regarding their prognostic significance. Data of seven cancer cohorts were collected from FireBrowse. 54 gene sets implicated in specific pathways were browsed through MSig database. Profiling was assessed via Monocle3, while gene ontology through PANTHER. For confirmation, correlation analysis was performed using WGCNA. Protein-protein networks were visualized via Cytoscape and impact of genes on survival, as well as cell cycle or cytoskeleton-related prognostic signatures, was tested. Several differences in expression profile were identified, some of them allowed to distinguish histology. Functional annotation revealed that various regulation of cell cycle, adhesion, migration, apoptosis and angiogenesis underlie these differences. Clinical traits, such as histological type or cancer staging, were found during evaluation of module-trait relationships. Of modules, the TopHubs (COL6A3, TNR, GTF2A1, NKX3-1) interacted directly with, e.g., PDGFB, ITGA10, SP1 or AKT3. Among TopHubs and interacting proteins, many showed an impact on hazard ratio and affected the cell cycle or cytoskeleton-related prognostic signatures, e.g., COL1A1 or PDGFB. In conclusion, this study laid the foundation for further hormone-sensitive carcinogenesis research through identification of genes which prove that crosstalk between cell cycle and cytoskeleton exists, opening avenues for future therapeutic strategies.

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.

Corrigendum: Fragile Gene WWOX Guides TFAP2A/TFAP2C-Dependent Actions Against Tumor Progression in Grade II Bladder Cancer.

[This corrects the article DOI: 10.3389/fonc.2021.621060.].

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.

WWOX Loses the Ability to Regulate Oncogenic AP-2γ and Synergizes with Tumor Suppressor AP-2α in High-Grade Bladder Cancer.

The cytogenic locus of the WWOX gene overlaps with the second most active fragile site, FRA16D, which is present at a higher frequency in bladder cancer (BLCA) patients with smoking habit, a known risk factor of this tumor. Recently, we demonstrated the relevance of the role of WWOX in grade 2 BLCA in collaboration with two AP-2 transcription factors whose molecular actions supported or opposed pro-cancerous events, suggesting a distinct character. As further research is needed on higher grades, the aim of the present study was to examine WWOX-AP-2 functionality in grade 3 and 4 BLCA using equivalent in vitro methodology with additional transcriptome profiling of cellular variants. WWOX and AP-2α demonstrated similar anti-cancer functionality in most biological processes with subtle differences in MMP-2/9 regulation; this contradicted that of AP-2γ, whose actions potentiated cancer progression. Simultaneous overexpression of WWOX and AP-2α/AP-2γ revealed that single discrepancies appear in WWOX-AP-2α collaboration but only at the highest BLCA grade; WWOX-AP-2α collaboration was considered anti-cancer. However, WWOX only appeared to have residual activity against oncogenic AP-2γ in grade 3 and 4: variants with either AP-2γ overexpression alone or combined WWOX and AP-2γ overexpression demonstrated similar pro-tumoral behavior. Transcriptome profiling with further gene ontology certified biological processes investigated in vitro and indicated groups of genes consisting of AP-2 targets and molecules worth investigation as biomarkers. In conclusion, tumor suppressor synergism between WWOX and AP-2α is unimpaired in high-grade BLCA compared to intermediate grade, yet the ability of WWOX to guide oncogenic AP-2γ is almost completely lost.

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.

Fragile Gene WWOX Guides TFAP2A/TFAP2C-Dependent Actions Against Tumor Progression in Grade II Bladder Cancer.

INTRODUCTION: The presence of common fragile sites is associated with no-accidental chromosomal instability which occurs prior to carcinogenesis. The WWOX gene spans the second most active fragile site: FRA16D. Chromosomal breakage at this site is more common in bladder cancer patients who are tobacco smokers which suggests the importance of WWOX gene loss regarding bladder carcinogenesis. Tryptophan domains of WWOX are known to recognize motifs of other proteins such as AP-2α and AP-2γ allowing protein-protein interactions. While the roles of both AP-2 transcription factors are important for bladder carcinogenesis, their nature is different. Based on the literature, AP-2γ appears to be oncogenic, whereas AP-2α mainly exhibits tumor suppressor character. Presumably, the interaction between WWOX and both transcription factors regulates thousands of genes, hence the aim of the present study was to determine WWOX, AP-2α, and AP-2γ function in modulating biological processes of bladder cancer. METHODS: RT-112 cell line (grade II bladder cancer) was subjected to two stable lentiviral transductions. Overall, this resulted in six variants to investigate distinct WWOX, AP-2α, or AP-2γ function as well as WWOX in collaboration with a particular transcription factor. Cellular models were examined with immunocytochemical staining and in terms of differences in biological processes using assays investigating cell viability, proliferation, apoptosis, adhesion, clonogenicity, migration, activity of metalloproteinases and 3D culture growth. RESULTS: WWOX overexpression increased apoptosis but decreased cell viability, migration and large spatial colonies. AP-2α overexpression decreased tumor cell viability, migratory potential, matrix metalloproteinase-2 activity and clonogenicity. AP-2γ overexpression decreased matrix metalloproteinase-2 activity but increased wound healing, adhesion, clonogenicity and spatial colony formation. WWOX and AP-2α overexpression induced apoptosis but decreased cell viability, adhesion, matrix metalloproteinase-2 activity, overall number of cultured colonies and migration rate. WWOX and AP-2γ overexpression decreased tumor cell viability, proliferation potential, adhesion, clonogenicity and the ability to create spatial structures, but also increased apoptosis or migration rate. CONCLUSION: Co-overexpression of WWOX with AP-2α or WWOX with AP-2γ resulted in a net anti-tumor effect. However, considering this research findings and the difference between AP-2α and AP-2γ, we suggest that this similarity is due to a divergent behavior of WWOX.

MicroRNAs: Their Role in Metastasis, Angiogenesis, and the Potential for Biomarker Utility in Bladder Carcinomas.

Angiogenesis is the process of generating new capillaries from pre-existing blood vessels with a vital role in tumor growth and metastasis. MicroRNAs (miRNAs) are noncoding RNAs that exert post-transcriptional control of protein regulation. They participate in the development and progression of several cancers including bladder cancer (BLCA). In cancer tissue, changes in microRNA expression exhibit tissue specificity with high levels of stability and detectability. miRNAs are less vulnerable to degradation, making them novel targets for therapeutic approaches. A suitable means of targeting aberrant activated signal transduction pathways in carcinogenesis of BLCA is possibly through altering the expression of key miRNAs that regulate them, exerting a strong effect on signal transduction. Precaution must be taken, as the complexity of miRNA regulation might result in targeting several downstream tumor suppressors or oncogenes, enhancing the effect further. Since exosomes contain both mRNA and miRNA, they could therefore possibly be more effective in targeting a recipient cell if they deliver a specific miRNA to modify the recipient cell protein production and gene expression. In this review, we discuss the molecules that have been shown to play a significant role in BLCA tumor development. We also discuss the roles of various miRNAs in BLCA angiogenesis and metastasis. Advances in the management of metastatic BLCA have been limited; miRNA mimics and molecules targeted at miRNAs (anti-miRs) as well as exosomes could serve as therapeutic modalities or as diagnostic biomarkers.

Functional genomics of AP-2α and AP-2γ in cancers: in silico study.

BACKGROUND: Among all causes of death, cancer is the most prevalent and is only outpaced by cardiovascular diseases. Molecular theory of carcinogenesis states that apoptosis and proliferation are regulated by groups of tumor suppressors or oncogenes. Transcription factors are example of proteins comprising representatives of both cancer-related groups. Exemplary family of transcription factors which exhibits dualism of function is Activating enhancer-binding Protein 2 (AP-2). Scientific reports concerning their function in carcinogenesis depend on particular family member and/or tumor type which proves the issue to be unsolved. Therefore, the present study examines role of the best-described AP-2 representatives, AP-2α and AP-2γ, through ontological analysis of their target genes and investigation what processes are differentially regulated in 21 cancers using samples deposited in Genomic Data Analysis Center (GDAC) Firehose. METHODS: Expression data with clinical annotation was collected from TCGA-dedicated repository GDAC Firehose. Transcription factor targets were obtained from Gene Transcription Regulation Database (GTRD), TRANScription FACtor database (TRANSFAC) and Transcriptional Regulatory Relationships Unraveled by Sentence-based Text mining (TRRUST). Monocle3 R package was used for global samples profiling while Protein ANalysis THrough Evolutionary Relationships (PANTHER) tool was used to perform gene ontology analysis. RESULTS: With RNA-seq data and Monocle3 or PANTHER tools we outlined differences in many processes and signaling pathways, separating tumor from normal tissues or tumors from each other. Unexpectedly, a number of alterations in basal-like breast cancer were identified that distinguished it from other subtypes, which could bring future clinical benefits. CONCLUSIONS: Our findings indicate that while the AP-2α/γ role remains ambiguous, their activity is based on processes that underlie the cancer hallmarks and their expression could have potential in diagnosis of selected tumors.

The biological characteristics of transcription factors AP-2α and AP-2γ and their importance in various types of cancers.

The Activator Protein 2 (AP-2) transcription factor (TF) family is vital for the regulation of gene expression during early development as well as carcinogenesis process. The review focusses on the AP-2α and AP-2γ proteins and their dualistic regulation of gene expression in the process of carcinogenesis. Both AP-2α and AP-2γ influence a wide range of physiological or pathological processes by regulating different pathways and interacting with diverse molecules, i.e. other proteins, long non-coding RNAs (lncRNA) or miRNAs. This review summarizes the newest information about the biology of two, AP-2α and AP-2γ, TFs in the carcinogenesis process. We emphasize that these two proteins could have either oncogenic or suppressive characteristics depending on the type of cancer tissue or their interaction with specific molecules. They have also been found to contribute to resistance and sensitivity to chemotherapy in oncological patients. A better understanding of molecular network of AP-2 factors and other molecules may clarify the atypical molecular mechanisms occurring during carcinogenesis, and may assist in the recognition of new diagnostic biomarkers.