Erratum: Structural, topological, and functional characterization of transmembrane proteins TMEM213, 207, 116, 72 and 30B provides a potential link to ccRCC etiology.

[This corrects the article on p. 1863 in vol. 13, PMID: 37293153.].

ISGF3 and STAT2/IRF9 Control Basal and IFN-Induced Transcription through Genome-Wide Binding of Phosphorylated and Unphosphorylated Complexes to Common ISRE-Containing ISGs.

In addition to the canonical ISGF3 and non-canonical STAT2/IRF9 complexes, evidence is emerging of the role of their unphosphorylated counterparts in IFN-dependent and -independent ISG transcription. To better understand the relation between ISGF3 and U-ISGF3 and STAT2/IRF9 and U-STAT2/IRF9 in IFN-I-stimulated transcriptional responses, we performed RNA-Seq and ChIP-Seq, in combination with phosphorylation inhibition and antiviral experiments. First, we identified a group of ISRE-containing ISGs that were commonly regulated in IFNα-treated WT and STAT1-KO cells. Thus, in 2fTGH and Huh7.5 WT cells, early and long-term IFNα-inducible transcription and antiviral activity relied on the DNA recruitment of the ISGF3 components STAT1, STAT2 and IRF9 in a phosphorylation- and time-dependent manner. Likewise, in ST2-U3C and Huh-STAT1KO cells lacking STAT1, delayed IFN responses correlated with DNA binding of phosphorylated STAT2/IRF9 but not U-STAT2/IRF9. In addition, comparative experiments in U3C (STAT1-KO) cells overexpressing all the ISGF3 components (ST1-ST2-IRF9-U3C) revealed U-ISGF3 (and possibly U-STAT2/IRF9) chromatin interactions to correlate with phosphorylation-independent ISG transcription and antiviral activity. Together, our data point to the dominant role of the canonical ISGF3 and non-canonical STAT2/IRF9, without a shift to U-ISGF3 or U-STAT2/IRF9, in the regulation of early and prolonged ISG expression and viral protection. At the same time, they suggest the threshold-dependent role of U-ISFG3, and potentially U-STAT2/IRF9, in the regulation of constitutive and possibly long-term IFNα-dependent responses.

Time-dependent recruitment of GAF, ISGF3 and IRF1 complexes shapes IFNα and IFNγ-activated transcriptional responses and explains mechanistic and functional overlap.

To understand in detail the transcriptional and functional overlap of IFN-I- and IFN-II-activated responses, we used an integrative RNAseq-ChIPseq approach in Huh7.5 cells and characterized the genome-wide role of pSTAT1, pSTAT2, IRF9 and IRF1 in time-dependent ISG expression. For the first time, our results provide detailed insight in the timely steps of IFNα- and IFNγ-induced transcription, in which pSTAT1- and pSTAT2-containing ISGF3 and GAF-like complexes and IRF1 are recruited to individual or combined ISRE and GAS composite sites in a phosphorylation- and time-dependent manner. Interestingly, composite genes displayed a more heterogeneous expression pattern, as compared to GAS (early) and ISRE genes (late), with the time- and phosphorylation-dependent recruitment of GAF, ISGF3 and IRF1 after IFNα stimulation and GAF and IRF1 after IFNγ. Moreover, functional composite genes shared features of GAS and ISRE genes through transcription factor co-binding to closely located sites, and were able to sustain IFN responsiveness in STAT1-, STAT2-, IRF9-, IRF1- and IRF9/IRF1-mutant Huh7.5 cells compared to Wt cells. Thus, the ISRE + GAS composite site acted as a molecular switch, depending on the timely available components and transcription factor complexes. Consequently, STAT1, STAT2 and IRF9 were identified as functional composite genes that are part of a positive feedback loop controlling long-term IFNα and IFNγ responses. More important, in the absence of any one of the components, the positive feedback regulation of the ISGF3 and GAF components appeared to be preserved. Together, these findings provide further insight in the existence of a novel ISRE + GAS composite-dependent intracellular amplifier circuit prolonging ISG expression and controlling cellular responsiveness to different types of IFNs and subsequent antiviral activity. It also offers an explanation for the existing molecular and functional overlap between IFN-I- and IFN-II-activated ISG expression.

Structural, topological, and functional characterization of transmembrane proteins TMEM213, 207, 116, 72 and 30B provides a potential link to ccRCC etiology.

Due to their involvement in the development of various cancers Transmembrane Proteins (TMEMs) are the focus of many recent studies. Previously we reported TMEM de-regulation in clear cell Renal Cell Carcinoma (ccRCC) with TMEM213, 207, 116, 72 and 30B being among the most downregulated on mRNA level. TMEM down-regulation was also more pronounced in advanced ccRCC tumors and was potentially linked to clinical parameters such as: metastasis (TMEM72 and 116), Fuhrman grade (TMEM30B) and overall survival (TMEM30B). To further investigate these findings, first, we set off to prove experimentally that selected TMEMs are indeed membrane-bound as predicted in silico, we verified the presence of signaling peptides on their N-termini, orientation of TMEMs within the membrane and validated their predicted cellular localization. To investigate the potential role of selected TMEMs in cellular processes overexpression studies in HEK293 and HK-2 cell lines were carried out. Additionally, we tested TMEM isoform expression in ccRCC tumors, identified mutations in TMEM genes and examined chromosomal aberrations in their loci. We confirmed the membrane-bound status of all selected TMEMs, assigned TMEM213, and 207 to early endosomes, TMEM72 to early endosomes and plasma membrane, TMEM116 and 30B to the endoplasmic reticulum. The N-terminus of TMEM213 was found to be exposed to the cytoplasm, the C-terminus of TMEM207, 116 and 72 were directed toward the cytoplasm, and both termini of TMEM30B faced the cytoplasm. Interestingly, TMEM mutations and chromosomal aberrations were infrequent in ccRCC tumors, yet we identified potentially damaging mutations in TMEM213 and TMEM30B and found deletions in the TMEM30B locus in nearly 30% of the tumors. Overexpression studies suggested selected TMEMs may take part in carcinogenesis processes such as cell adhesion, regulation of epithelial cell proliferation, and regulation of adaptive immune response, which could indicate a link to the development and progression of ccRCC.

Preoperative cell-free DNA concentration in plasma as a diagnostic and prognostic biomarker of clear cell renal cell carcinoma.

Introduction: Assessment of renal tumour masses is based on conventional imaging studies (computer tomography or magnetic resonance), which does not allow characterisation of the histopathological type. Moreover, the prediction of prognosis in localised and metastatic renal cell carcinoma requires improvement as well. Analysis of circulating free DNA (cfDNA) in blood is one of the variants of liquid biopsy that may improve diagnostics and prognosis issues of patients with renal tumour masses suspected to be renal cell carcinoma. The aim of the study was to assess the diagnostic and prognostic role of preoperative cfDNA concentration in the plasma samples of clear cell renal cell carcinoma (ccRCC) patients. Material and methods: The preoperative plasma cfDNA concentration was assessed in ccRCC patients (n = 46) and healthy individuals (control group) (n = 17). The circulating free DNA concentration was reflected by the 90 bp DNA fragments determined by real-time polymerase chain reaction. Results: The median cfDNA concentration was significantly higher in ccRCC patients (n = 46) compared to the control g roup (n = 17) (2588 ±2554 copies/ml vs. 960 ±490 copies/ml, p < 0.01). In multivariate analysis, the preoperative plasma cfDNA concentration was the significant factor increasing the probability of ccRCC detection (OR: 1.003; 95% CI: 1.001-1.005). The median cfDNA concentration depended on the stage of ccRCC; it was higher in metastatic ccRCC patients (n = 11) compared to non-metastatic ccRCC patients (n = 35) (3619 ±4059 copies/ml vs. 2473 ±1378 copies/ml, p < 0.03). Kaplan-Meier survival analysis demon-strated that patients with high cfDNA values (above 2913 copies/ml) had significantly worse cancer-specific survival (HR: 4.5; 95% CI: 1.3-16.9, log-rank Mantel-Cox test p = 0.015). Conclusions: Preoperative plasma cfDNA concentration has diagnostic and prognostic potential in ccRCC pa-tients.

Dysregulated Interferon Response and Immune Hyperactivation in Severe COVID-19: Targeting STATs as a Novel Therapeutic Strategy.

A disease outbreak in December 2019, caused by a novel coronavirus SARS-CoV-2, was named COVID-19. SARS-CoV-2 infects cells from the upper and lower respiratory tract system and is transmitted by inhalation or contact with infected droplets. Common clinical symptoms include fatigue, fever, and cough, but also shortness of breath and lung abnormalities. Still, some 5% of SARS-CoV-2 infections progress to severe pneumonia and acute respiratory distress syndrome (ARDS), with pulmonary edema, acute kidney injury, and/or multiple organ failure as important consequences, which can lead to death. The innate immune system recognizes viral RNAs and triggers the expression of interferons (IFN). IFNs activate anti-viral effectors and components of the adaptive immune system by activating members of the STAT and IRF families that induce the expression of IFN-stimulated genes (ISG)s. Among other coronaviruses, such as Middle East respiratory syndrome coronavirus (MERS-CoV) and SARS-CoV, common strategies have been identified to antagonize IFN signaling. This typically coincides with hyperactive inflammatory host responses known as the "cytokine storm" that mediate severe lung damage. Likewise, SARS-CoV-2 infection combines a dysregulated IFN response with excessive production of inflammatory cytokines in the lungs. This excessive inflammatory response in the lungs is associated with the local recruitment of immune cells that create a pathogenic inflammatory loop. Together, it causes severe lung pathology, including ARDS, as well as damage to other vulnerable organs, like the heart, spleen, lymph nodes, and kidney, as well as the brain. This can rapidly progress to multiple organ exhaustion and correlates with a poor prognosis in COVID-19 patients. In this review, we focus on the crucial role of different types of IFN that underlies the progression of SARS-CoV-2 infection and leads to immune cell hyper-activation in the lungs, exuberant systemic inflammation, and multiple organ damage. Consequently, to protect from systemic inflammation, it will be critical to interfere with signaling cascades activated by IFNs and other inflammatory cytokines. Targeting members of the STAT family could therefore be proposed as a novel therapeutic strategy in patients with severe COVID-19.

Identification of RCC Subtype-Specific microRNAs-Meta-Analysis of High-Throughput RCC Tumor microRNA Expression Data.

Renal cell carcinoma (RCC) is one of the most common cancers worldwide with a nearly non-symptomatic course until the advanced stages of the disease. RCC can be distinguished into three subtypes: papillary (pRCC), chromophobe (chRCC) and clear cell renal cell carcinoma (ccRCC) representing up to 75% of all RCC cases. Detection and RCC monitoring tools are limited to standard imaging techniques, in combination with non-RCC specific morphological and biochemical read-outs. RCC subtype identification relays mainly on results of pathological examination of tumor slides. Molecular, clinically applicable and ideally non-invasive tools aiding RCC management are still non-existent, although molecular characterization of RCC is relatively advanced. Hence, many research efforts concentrate on the identification of molecular markers that will assist with RCC sub-classification and monitoring. Due to stability and tissue-specificity miRNAs are promising candidates for such biomarkers. Here, we performed a meta-analysis study, utilized seven NGS and seven microarray RCC studies in order to identify subtype-specific expression of miRNAs. We concentrated on potentially oncocytoma-specific miRNAs (miRNA-424-5p, miRNA-146b-5p, miRNA-183-5p, miRNA-218-5p), pRCC-specific (miRNA-127-3p, miRNA-139-5p) and ccRCC-specific miRNAs (miRNA-200c-3p, miRNA-362-5p, miRNA-363-3p and miRNA-204-5p, 21-5p, miRNA-224-5p, miRNA-155-5p, miRNA-210-3p) and validated their expression in an independent sample set. Additionally, we found ccRCC-specific miRNAs to be differentially expressed in ccRCC tumor according to Fuhrman grades and identified alterations in their isoform composition in tumor tissue. Our results revealed that changes in the expression of selected miRNA might be potentially utilized as a tool aiding ccRCC subclass discrimination and we propose a miRNA panel aiding RCC subtype distinction.

Distinct cellular phenotype linked to defective DNA interstrand crosslink repair and homologous recombination.

Repair of DNA interstrand crosslinks (ICLs) predominantly involves the Fanconi anemia (FA) pathway and homologous recombination (HR). The HR repair system eliminates DNA double strand breaks (DSBs) that emerge during ICLs removal. The current study presents a novel cell line, CL­V8B, representing a new complementation group of Chinese hamster cell mutants hypersensitive to DNA crosslinking factors. CL­V8B exhibits increased sensitivity to various DNA­damaging agents, including compounds leading to DSBs formation (bleomycin and 6­thioguanine), and is extremely sensitive to poly (ADP-ribose) polymerase inhibitor (>400­fold), which is typical for HR­defective cells. In addition, this cell line exhibits a reduced number of spontaneous and induced sister chromatid exchanges, which suggests likely impairment of HR in CL­V8B cells. However, in contrast to other known HR mutants, CL­V8B cells do not show defects in Rad51 foci induction, but only slight alterations in the focus formation kinetics. CL­V8B is additionally characterized by a considerable chromosomal instability, as indicated by a high number of spontaneous and MMC­induced chromosomal aberrations, and a twice as large proportion of cells with abnormal centrosomes than that in the wild type cell line. The molecular defect present in CL­V8B does not affect the efficiency and stabilization of replication forks. However, stalling of the forks in response to replication stress is observed relatively rarely, which suggests an impairment of a signaling mechanism. Exposure of CL­V8B to crosslinking agents results in S­phase arrest (as in the wild type cells), but also in larger proportion of G2/M­phase cells and apoptotic cells. CL­V8B exhibits similarities to HR­ and/or FA­defective Chinese hamster mutants sensitive to DNA crosslinking agents. However, the unique phenotype of this new mutant implies that it carries a defect of a yet unidentified gene involved in the repair of ICLs.

Genetic characterization of Polish ccRCC patients: somatic mutation analysis of PBRM1, BAP1 and KDMC5, genomic SNP array analysis in tumor biopsy and preliminary results of chromosome aberrations analysis in plasma cell free DNA.

BACKGROUND: Mutation analysis and cytogenetic testing in clear cell renal cell carcinoma (ccRCC) is not yet implemented in a routine diagnostics of ccRCC. MATERIAL AND METHODS: We characterized the chromosomal alterations in 83 ccRCC tumors from Polish patients using whole genome SNP genotyping assay. Moreover, the utility of next generation sequencing of cell free DNA (cfDNA) in patients plasma as a potential tool for non-invasive cytogenetic analysis was tested. Additionally, tumor specific somatic mutations in PBRM1, BAP1 and KDM5C were determined. RESULTS: We confirmed a correlation between deletions at 9p and higher tumor size, and deletion of chromosome 20 and the survival time. In Fuhrman grade 1, only aberrations of 3p and 8p deletion, gain of 5q and 13q and gains of chromosome 7 and 16 were present. The number of aberrations increased with Fuhrman grade, all chromosomes displayed cytogenetic changes in G3 and G4. ccRCC specific chromosome aberrations were observed in cfDNA, although discrepancies were found between cfDNA and tumor samples. In total 12 common and 94 rare variants were detected in PBRM1, BAP1 and KDM5C, with four potentially pathogenic variants. We observed markedly lower mutation load in PBRM1. CONCLUSIONS: Cytogenetic analysis of cfDNA may allow more accurate diagnosis of tumor aberrations and therefore the correlation between the chromosome aberrations in cfDNA and clinical outcome should be studied in larger cohorts. The functional studies on in BAP1, KDM5C, PBRM1 mutations in large, independent sample set would be necessary for the assessment of their prognostic and diagnostic potential.

Potential Approaches and Recent Advances in Biomarker Discovery in Clear-Cell Renal Cell Carcinoma.

The early diagnosis and monitoring of clear-cell Renal Cell Carcinoma (ccRCC), which is the most common renal malignancy, remains challenging. The late diagnosis and lack of tools that can be used to assess the progression of the disease and metastasis significantly influence the chance of survival of ccRCC patients. Molecular biomarkers have been shown to aid the diagnosis and disease monitoring for other cancers, but such markers are not currently available for ccRCC. Recently, plasma and serum circulating nucleic acids, nucleic acids present in urine, and plasma and urine proteins gained interest in the field of cancer biomarker discovery. Here, we describe the applicability of plasma and urine nucleic acids as cancer biomarkers with a particular focus on DNA, small RNA, and protein markers for ccRCC.

The epigenetic landscape of clear-cell renal cell carcinoma.

Clear cell renal cell carcinoma (ccRCC) is the most common subtype of all kidney tumors. During the last few years, epigenetics has emerged as an important mechanism in ccRCC pathogenesis. Recent reports, involving large-scale methylation and sequencing analyses, have identified genes frequently inactivated by promoter methylation and recurrent mutations in genes encoding chromatin regulatory proteins. Interestingly, three of detected genes (PBRM1, SETD2 and BAP1) are located on chromosome 3p, near the VHL gene, inactivated in over 80% ccRCC cases. This suggests that 3p alterations are an essential part of ccRCC pathogenesis. Moreover, most of the proteins encoded by these genes cooperate in histone H3 modifications. The aim of this review is to summarize the latest discoveries shedding light on deregulation of chromatin machinery in ccRCC. Newly described ccRCC-specific epigenetic alterations could potentially serve as novel diagnostic and prognostic biomarkers and become an object of novel therapeutic strategies.

[The role of centrosomes in cells and their potential contribution to carcinogenesis]. / Rola centrosomów w komórkach i ich potencjalny udzial w kancerogenezie.

The centrosomes are subcellular organelles composed of two centrioles surrounded by a pericentriolar material. In animal cells they are responsible for the organization of the interphase microtubule cytoskeleton including microtubule nucleation and elongation, their attachment and release. The centrosomes are also involved in the construction of the mitotic spindle and chromosome segregation. More than a century ago it was suggested that these structures might be involved in human diseases, including cancer. Cancer cells show a high frequency of centrosome aberrations, especially amplification. Centrosome defects may increase the incidence of multipolar mitoses that lead to chromosomal segregation abnormalities and aneuploidy, which is the predominant type of genomic instability found in human solid tumors. The number of these organelles in cells is strictly controlled and is dependent on the proper process of centrosome duplication. Multiple genes that are frequently found mutated in cancers encode proteins which participate in the regulation of centrosome duplication and the numeral integrity of centrosomes. In recent years there has been growing interest in the potential participation of centrosomes in the process of carcinogenesis, especially because centrosome abnormalities are observed in premalignant stages of cancer development. The common presence of abnormal centrosomes in cancer cells and the role these organelles play in the cells suggest that the factors controlling the number of centrosomes may be potential targets for cancer therapy.

[The role of the Fanconi anemia pathway in DNA repair and maintenance of genome stability]. / Rola bialek szlaku niedokrwistosci Fanconiego w naprawie DNA i utrzymaniu stabilnosci genomu.

The Fanconi anemia (FA) pathway is one of the DNA repair systems involved in removal of DNA crosslinks. Proteins which belong to this pathway are crucial to the protection of genetic information, whereas disturbances in their function have serious implications for the whole organism. Biallelic mutations in FA genes are the cause of Fanconi anemia - a genetic disease which manifests itself through numerous congenital abnormalities, chromosomal instability and increased predisposition to cancer. The FA pathway is composed of fifteen proteins. Eight of them, in the presence of DNA interstrand crosslinks (ICLs), form a nuclear core complex responsible for monoubiquitination of FANCD2 and FANCI, which is a key step of ICL repair. FA proteins which are not involved in the monoubiquitination step participate in repair of DNA double strand breaks via homologous recombination. Some of the FA proteins, besides having a direct role in the repair of DNA damage, are engaged in replication, cell cycle control and mitosis. The unperturbed course of those processes determines the maintenance of genome stability.

[Poly(ADP-ribose) polymerase (PARP) inhibitors in BRCA1/2 cancer therapy]. / Inhibitory polimerazy poli(ADP-rybozy) (PARP) w terapii nowotworów z mutacjami BRCA1/2.

 A majority of currently used anticancer drugs belong to a group of chemical agents that damage DNA. The efficiency of the treatment is limited by effective DNA repair systems functioning in cancer cells. Many chemotherapeutic compounds cause strong systemic toxicity. Therefore, there is still a need for new anticancer agents which are less toxic for nontransformed cells and selectively kill cancer cells. One of the most promising molecular targets in cancer therapy is poly(ADP-ribose) polymerases (PARP). PARP play an essential role in repairing DNA strand breaks. Small molecule inhibitors of these enzymes have been developed and have proved to be extremely toxic for cancer cells that lack the functional BRCA1 and BRCA2 proteins that are involved in homologous recombination, a complex repair mechanism of DNA double strand breaks. Mutations in BRCA1/2 genes are associated with genetically inherited breast and ovarian cancers. Therefore PARP inhibitors may prove to be very effective and selective in the treatment of these cancer types. This review is focused on the function of BRCA1/2 proteins and poly(ADP-ribose) polymerases in DNA repair systems, especially in the homologous recombination process. A short history of the studies that led to synthesis of high specificity small molecule PARP inhibitors is also presented, as well as the results of clinical trials concerning the most effective PARP inhibitors in view of their potential application in oncological treatment, particularly breast cancers.

Deficiency in the repair of DNA damage by homologous recombination and sensitivity to poly(ADP-ribose) polymerase inhibition.

Deficiency in either of the breast cancer susceptibility proteins BRCA1 or BRCA2 induces profound cellular sensitivity to the inhibition of poly(ADP-ribose) polymerase (PARP) activity. We hypothesized that the critical role of BRCA1 and BRCA2 in the repair of double-strand breaks by homologous recombination (HR) was the underlying reason for this sensitivity. Here, we examine the effects of deficiency of several proteins involved in HR on sensitivity to PARP inhibition. We show that deficiency of RAD51, RAD54, DSS1, RPA1, NBS1, ATR, ATM, CHK1, CHK2, FANCD2, FANCA, or FANCC induces such sensitivity. This suggests that BRCA-deficient cells are, at least in part, sensitive to PARP inhibition because of HR deficiency. These results indicate that PARP inhibition might be a useful therapeutic strategy not only for the treatment of BRCA mutation-associated tumors but also for the treatment of a wider range of tumors bearing a variety of deficiencies in the HR pathway or displaying properties of 'BRCAness.'

[Complex role of the FA proteins in providing genome stability]. / Kompleksowa rola bialek niedokrwistosci Fanconiego w utrzymaniu stabilnosci genomu.

FA is a rare genetic disorder characterized by developmental abnormalities, bone marrow failure and cancer susceptibility. Cells that are derived from patients with FA display spontaneous chromosomal instability and hypersensitivity to DNA crosslinking agents that is used in FA clinical diagnostics. FA is genetically heterogeneous and caused by mutations in at least 11 distinct genes, FANCA, FANCA, B, C, D1, D2, E, F, G, I, J and L. FA proteins interact with various proteins involved in DNA damage response and cell cycle checkpoint regulation, such as: RAD51, BRCA1, BRCA2, ATM or NBS1. Moreover, BRCA2 that plays a crucial role in homologous recombination is one of FA proteins. Collectively, all these data indicate, that the FA pathway is involved in different molecular processes that prevent DNA and control genomic stability, although its precise role still remains undefined.