Identification of novel interferon responsive protein partners of human leukocyte antigen A (HLA-A) using cross-linking mass spectrometry (CLMS) approach.

The interferon signalling system elicits a robust cytokine response against a wide range of environmental pathogenic and internal pathological signals, leading to induction of a subset of interferon-induced proteins. We applied DSS (disuccinimidyl suberate) mediated cross-linking mass spectrometry (CLMS) to capture novel protein-protein interactions within the realm of interferon induced proteins. In addition to the expected interferon-induced proteins, we identified novel inter- and intra-molecular cross-linked adducts for the canonical interferon induced proteins, such as MX1, USP18, OAS3, and STAT1. We focused on orthogonal validation of a cohort of novel interferon-induced protein networks formed by the HLA-A protein (H2BFS-HLA-A-HMGA1) using co-immunoprecipitation assay, and further investigated them by molecular dynamics simulation. Conformational dynamics of the simulated protein complexes revealed several interaction sites that mirrored the interactions identified in the CLMS findings. Together, we showcase a proof-of-principle CLMS study to identify novel interferon-induced signaling complexes and anticipate broader use of CLMS to identify novel protein interaction dynamics within the tumour microenvironment.

Identification of a Stable, Non-Canonically Regulated Nrf2 Form in Lung Cancer Cells.

Nrf2 (nuclear factor erythroid 2 (NF-E2)-related factor 2) transcription factor is recognized for its pro-survival and cell protective role upon exposure to oxidative, chemical, or metabolic stresses. Nrf2 controls a number of cellular processes such as proliferation, differentiation, apoptosis, autophagy, lipid synthesis, and metabolism and glucose metabolism and is a target of activation in chronic diseases like diabetes, neurodegenerative, and inflammatory diseases. The dark side of Nrf2 is revealed when its regulation is imbalanced (e.g., via oncogene activation or mutations) and under such conditions constitutively active Nrf2 promotes cancerogenesis, metastasis, and radio- and chemoresistance. When there is no stress, Nrf2 is instantly degraded via Keap1-Cullin 3 (Cul3) pathway but despite this, cells exhibit a basal activation of Nrf2 target genes. It is yet not clear how Nrf2 maintains the expression of its targets under homeostatic conditions. Here, we found a stable 105 kDa Nrf2 form that is resistant to Keap1-Cul3-mediated degradation and translocates to the nucleus of lung cancer cells. RNA-Seq analysis indicate that it might originate from the exon 2 or exon 3-truncated transcripts. This stable 105 kDa Nrf2 form might help explain the constitutive activity of Nrf2 under normal cellular conditions.

Functional Interfaces, Biological Pathways, and Regulations of Interferon-Related DNA Damage Resistance Signature (IRDS) Genes.

Interferon (IFN)-related DNA damage resistant signature (IRDS) genes are a subgroup of interferon-stimulated genes (ISGs) found upregulated in different cancer types, which promotes resistance to DNA damaging chemotherapy and radiotherapy. Along with briefly discussing IFNs and signalling in this review, we highlighted how different IRDS genes are affected by viruses. On the contrary, different strategies adopted to suppress a set of IRDS genes (STAT1, IRF7, OAS family, and BST2) to induce (chemo- and radiotherapy) sensitivity were deliberated. Significant biological pathways that comprise these genes were classified, along with their frequently associated genes (IFIT1/3, IFITM1, IRF7, ISG15, MX1/2 and OAS1/3/L). Major upstream regulators from the IRDS genes were identified, and different IFN types regulating these genes were outlined. Functional interfaces of IRDS proteins with DNA/RNA/ATP/GTP/NADP biomolecules featured a well-defined pharmacophore model for STAT1/IRF7-dsDNA and OAS1/OAS3/IFIH1-dsRNA complexes, as well as for the genes binding to GDP or NADP+. The Lys amino acid was found commonly interacting with the ATP phosphate group from OAS1/EIF2AK2/IFIH1 genes. Considering the premise that targeting IRDS genes mediated resistance offers an efficient strategy to resensitize tumour cells and enhances the outcome of anti-cancer treatment, this review can add some novel insights to the field.

Viruses, cancer and non-self recognition.

Virus-host interactions form an essential part of every aspect of life, and this review is aimed at looking at the balance between the host and persistent viruses with a focus on the immune system. The virus-host interaction is like a cat-and-mouse game and viruses have developed ingenious mechanisms to manipulate cellular pathways, most notably the major histocompatibility (MHC) class I pathway, to reside within infected cell while evading detection and destruction by the immune system. However, some of the signals sensing and responding to viral infection are derived from viruses and the fact that certain viruses can prevent the infection of others, highlights a more complex coexistence between the host and the viral microbiota. Viral immune evasion strategies also illustrate that processes whereby cells detect and present non-self genetic material to the immune system are interlinked with other cellular pathways. Immune evasion is a target also for cancer cells and a more detailed look at the interfaces between viral factors and components of the MHC class I peptide-loading complex indicates that these interfaces are also targets for cancer mutations. In terms of the immune checkpoint, however, viral and cancer strategies appear different.

Liver Cirrhosis in Chronic Hepatitis B Patients Is Associated with Genetic Variations in DNA Repair Pathway Genes.

Liver cirrhosis (LC), contributing to more than 1 million of deaths annually, is a major healthcare concern worldwide. Hepatitis B virus (HBV) is a major LC etiological factor, and 15% of patients with chronic HBV infection (CHB) develop LC within 5 years. Recently, novel host genetic determinants were shown to influence HBV lifecycle and CHB course. DNA repair enzymes can affect dynamics of liver damage and are involved in HBV covalently closed circular DNA (cccDNA) formation, an essential step for viral replication. This study aimed to evaluate the possible role of genes representing key DNA-repair pathways in HBV-induced liver damage. MALDI-TOF MS genotyping platform was applied to evaluate variations within XRCC1, XRCC4, ERCC2, ERCC5, RAD52, Mre11, and NBN genes. Apart from older age (p < 0.001), female sex (p = 0.021), portal hypertension (p < 0.001), thrombocytopenia (p < 0.001), high HBV DNA (p = 0.001), and high aspartate aminotransferase (AST) (p < 0.001), we found that G allele at rs238406 (ERCC2, p = 0.025), T allele at rs25487 (XRCC1, p = 0.012), rs13181 GG genotype (ERCC2, p = 0.034), and C allele at rs2735383 (NBN, p = 0.042) were also LC risk factors. The multivariate logistic regression model showed that rs25487 CC (p = 0.005) and rs238406 TT (p = 0.027) were independently associated with lower risk of LC. This study provides evidence for the impact of functional and potentially functional variations in key DNA-repair genes XRCC1 and ERCC2 in HBV-induced liver damage in a Caucasian population.

Genetic variation in IL-10 influences the progression of hepatitis B infection.

OBJECTIVES: The outcomes of hepatitis B virus (HBV) infection vary substantially among affected individuals, providing evidence of the role of host genetic background in the susceptibility to HBV persistence and the dynamics of liver injury progression to cirrhosis and hepatocellular carcinoma (HCC). METHODS: Six single-nucleotide polymorphisms within the interleukin 10 gene (IL10) were genotyped by MALDI-TOF mass spectrometry in 857 patients with chronic HBV infection (CHB), 48 patients with resolved HBV infection, and 100 healthy volunteers. Associations of the selected polymorphisms with susceptibility to chronic HBV infection, liver injury progression, and outcomes were investigated. RESULTS: IL10 -819T (rs1800871), -592A (rs1800872), and +504T (rs3024490) alleles were associated with treatment-induced hepatitis B surface antigen (HBsAg) seroclearance. Additionally, IL10 ATAC haplotype increased the chance of HBsAg loss and was significantly more frequent in patients with less liver injury. Moreover rs1800871TT, rs1518110TT, rs1800872AA, and rs3024490TT genotypes were identified as predictors of a lower FIB-4 score (<0.5). CONCLUSIONS: This study indicates that polymorphisms within the promoter region and intronic sequences of IL10 are associated with chronicity of hepatitis B and with HBV-induced liver damage.

MHC Class I Regulation: The Origin Perspective.

Viral-derived elements and non-coding RNAs that build up "junk DNA" allow for flexible and context-dependent gene expression. They are extremely dense in the MHC region, accounting for flexible expression of the MHC I, II, and III genes and adjusting the level of immune response to the environmental stimuli. This review brings forward the viral-mediated aspects of the origin and evolution of adaptive immunity and aims to link this perspective with the MHC class I regulation. The complex regulatory network behind MHC expression is largely controlled by virus-derived elements, both as binding sites for immune transcription factors and as sources of regulatory non-coding RNAs. These regulatory RNAs are imbalanced in cancer and associate with different tumor types, making them promising targets for diagnostic and therapeutic interventions.

Host genetic background affects the course of infection and treatment response in patients with chronic hepatitis B.

BACKGROUND: Hepatitis B virus (HBV) utilizes proteins encoded by the host to infect hepatocytes and replicate. Recently, several novel host factors have been identified and described as important to the HBV lifecycle. The influence of host genetic background on chronic hepatitis B (CHB) pathogenesis is still poorly understood. OBJECTIVES: Here, we aimed to investigate the association of NTCP, FXRα, HNF1α, HNF4α, and TDP2 genetic polymorphisms with the natural course of CHB and antiviral treatment response. STUDY DESIGN: We genotyped 18 single-nucleotide polymorphisms using MALDI-TOF mass spectrometry in 136 patients with CHB and 100 healthy individuals. We investigated associations of the selected polymorphisms with biochemical, serological and hepatic markers of disease progression and treatment response. RESULTS: No significant differences in genotypic or allelic distribution between CHB and control groups were observed. Within TDP2, rs3087943 variations were associated with treatment response, and rs1047782 modified the risk of advanced liver inflammation. Rs7154439 within NTCP was associated with HBeAg seroconversion after 48 weeks of nucleos(t)ide analogue treatment. HNF1α genotypes were associated with treatment response, liver damage and baseline HBeAg presence. HNF4α rs1800961 predicted PEG-IFNα treatment-induced HBsAg clearance in long-term follow up. CONCLUSIONS: This study indicates host genetic background relevance in the course of CHB and confirms the role of recently described genes for HBV infection. The obtained results might serve as a starting point for validation studies on the clinical application of selected genetic variants to predict individual risks of CHB-induced liver failure and treatment response.

Correction to: Reactivation of TAp73 tumor suppressor by protoporphyrin IX, a metabolite of aminolevulinic acid, induces apoptosis in TP53-deficient cancer cells.

[This corrects the article DOI: 10.1186/s13008-018-0043-3.].

TNF-α polymorphisms affect persistence and progression of HBV infection.

BACKGROUND: Hepatitis B virus (HBV) infections are a major threat worldwide. Disease progression and outcome is diverse and depends on host genetic background. Recently, a high rate of HBV reactivation in individuals receiving tumor necrosis factor-α (TNF-α) antagonists showed the importance of this cytokine in HBV infection control. Here, we investigated the influence of TNF-α promoter polymorphisms on susceptibility to chronic HBV infection (CHB), liver injury progression and outcomes. METHODS: A total of 231 patients with CHB constituted the study group and 100 healthy volunteers-the local control group. TNF-α -1031T/C, -863C/A, -857C/T, -308G/A, and -238G/A were genotyped using MALDI-TOF mass spectrometry. RESULTS: TNF-α -1031C and -863A alleles were observed more frequently in CHB group than in healthy controls. Carriers of TNF-α -1031C and -863A variant alleles had lower baseline levels of serum HBV DNA and lower liver necroinflammatory activity than dominant homozygotes. A -857CT genotype predisposed to higher necroinflammatory activity. No associations between TNF-α variants and liver fibrosis were found. CONCLUSION: This study indicates that TNF-α -863A and -1031C alleles are associated with increased susceptibility to CHB in individuals from northern Poland. The same variants determine the course of CHB, lowering viremia and reducing necroinflammatory activity of the liver.

Reactivation of TAp73 tumor suppressor by protoporphyrin IX, a metabolite of aminolevulinic acid, induces apoptosis in TP53-deficient cancer cells.

BACKGROUND: The p73 protein is a tumor suppressor that shares structural and functional similarity with p53. p73 is expressed in two major isoforms; the TA isoform that interacts with p53 pathway, thus acting as tumor suppressor and the N-terminal truncated ΔN isoform that inhibits TAp73 and p53 and thus, acts as an oncogene. RESULTS: By employing a drug repurposing approach, we found that protoporphyrin IX (PpIX), a metabolite of aminolevulinic acid applied in photodynamic therapy of cancer, stabilizes TAp73 and activates TAp73-dependent apoptosis in cancer cells lacking p53. The mechanism of TAp73 activation is via disruption of TAp73/MDM2 and TAp73/MDMX interactions and inhibition of TAp73 degradation by ubiquitin ligase Itch. Finally, PpIX showed potent antitumor effect and inhibited the growth of xenograft human tumors in mice. CONCLUSION: Our findings may in future contribute to the successful repurposing of PpIX into clinical practice.

Inhibition of cancer antioxidant defense by natural compounds.

All classic, non-surgical anticancer approaches like chemotherapy, radiotherapy or photodynamic therapy kill cancer cells by inducing severe oxidative stress. Even tough chemo- and radiotherapy are still a gold standard in cancer treatment, the identification of non-toxic compounds that enhance their selectivity, would allow for lowering their doses, reduce side effects and risk of second cancers. Many natural products have the ability to sensitize cancer cells to oxidative stress induced by chemo- and radiotherapy by limiting antioxidant capacity of cancer cells. Blocking antioxidant defense in tumors decreases their ability to balance oxidative insult and results in cell death. Though one should bear in mind that the same natural compound often exerts both anti-oxidant and pro-oxidant properties, depending on concentration used, cell type, exposure time and environmental conditions. Here we present a comprehensive overview of natural products that inhibit major antioxidant defense mechanisms in cancer cells and discuss their potential in clinical application.

Targeting of p53 and its homolog p73 by protoporphyrin IX.

The p53 tumor suppressor is recognized as a promising target for anti-cancer therapies. We previously reported that protoporphyrin IX (PpIX) disrupts the p53/murine double minute 2 (MDM2) complex and leads to p53 accumulation and activation of apoptosis in HCT 116 cells. Here we show the direct binding of PpIX to the N-terminal domain of p53. Furthermore, we addressed the induction of apoptosis in HCT 116 p53-null cells by PpIX and revealed interactions between PpIX and p73. We propose that PpIX disrupts the p53/MDM2 or MDMX and p73/MDM2 complexes and thereby activates the p53- or p73-dependent cancer cell death.

[Pharmacological activation of tumor suppressor, wild-type p53 as a promising strategy to fight cancer]. / Farmakologiczna aktywacja supresora nowotworu, natywnego bialka p53 jako obiecujaca strategia zwalczania nowotworów.

A powerful tumor suppressor--p53 protein is a transcription factor which plays a critical role in eliciting cellular responses to a variety of stress signals, including DNA damage, hypoxia and aberrant proliferative signals, such as oncogene activation. Since its discovery thirty one years ago, p53 has been connected to tumorigenesis as it accumulates in the transformed tumor cells. Cellular stress induces stabilization of p53 and promotes, depending on the stress level, cell cycle arrest or apoptosis in the irreversibly damaged cells. The p53 protein is found inactive in more than 50% of human tumors either by enhanced proteasomal degradation or due to the inactivating point mutations in its gene. Numerous data indicate that low molecular weight compounds, identified by molecular modeling or in the functional, cell-based assays, efficiently activate non-mutated p53 in cancer cells which in consequence leads to their elimination due to p53-dependent apoptosis. In this work we describe the structure and cellular function of p53 as well as the latest discoveries on the compounds with high anti-tumor activities aiming at reactivation of the tumor suppressor function of p53.

[The structure and cellular regulation of p73: their implication in anticancer therapy]. / Struktura i regulacja bialka p73 w komórce--implikacja w terapii przeciwnowotworowej.

p73 protein belongs, together with p63, to the p53 family. It is a relatively poorly studied structural and functional homolog of the well-described tumor suppressor protein p53, also known as the guardian of the genome. p73 protein, like p53, becomes activated by, for example, DNA damaging agents and it targets the same promoter sequences as p53. Both proteins participate in pathways of signal transduction whose activation leads to apoptosis induction or cell-cycle arrest. Studies concerning anticancer treatment focusing on the activation of p53 have been carried out extensively for about 10 years. It appears that a similar therapeutic strategy can be successfully applied in p73 activation as well. Unlike the TP53 gene, the gene encoding p73 protein is rarely mutated in tumors although the protein is found to be inactive. This can become very useful when designing molecules which will selectively activate p73 and consequently induce cancer-cell death. The aim of the present study was to describe in detail the structure, function, as well as cellular regulation of p73 in light of its therapeutic potential.

p73 tumor suppressor protein: a close relative of p53 not only in structure but also in anti-cancer approach?

The discovery of the p53 tumor suppressor protein in 1979 shed new light on cancer cell biology and introduced a trend in cancer research focusing on p53-like proteins. This in turn led to the discovery of two homologous proteins of p53-p63 in 1998 and p73 in 1997. The p53 family members are mainly involved in apoptosis induction under cellular stress, but also in early embryonic developmental processes. The p63 and p73 proteins activate the transcription of a number of p53 target genes. The precise role of p63 in cancer cells is not fully revealed yet, unlike that of p53 and p73. The p53 tumor suppressor protein is found inactive in approximately 50% of human cancers. However, p73 is not as often inactivated in tumors. Of importance, transcriptionally active forms of p73 induce apoptosis in cancer cells independent of p53 status. Moreover, the regulatory mechanisms governing p73 stability in cells are well described. These features promoted the research concerning p73-targeted anti-cancer treatment. The p73 protein is subject to sophisticated activatory and inhibitory regulatory mechanisms. The up-to-date anti-cancer compounds targeting p73 protein in vitro inhibit its negative regulators, which leads to the activation of p73 pro-apoptotic function in cancer cells. In the current review we present the recent scientific findings on p73 regulation in cells and the newest anti-cancer strategies concerning its tumor suppressor function.