p21 Prevents the Exhaustion of CD4+ T Cells Within the Antitumor Immune Response Against Colorectal Cancer.

BACKGROUND & AIMS: T cells are crucial for the antitumor response against colorectal cancer (CRC). T-cell reactivity to CRC is nevertheless limited by T-cell exhaustion. However, molecular mechanisms regulating T-cell exhaustion are only poorly understood. METHODS: We investigated the functional role of cyclin-dependent kinase 1a (Cdkn1a or p21) in cluster of differentiation (CD) 4+ T cells using murine CRC models. Furthermore, we evaluated the expression of p21 in patients with stage I to IV CRC. In vitro coculture models were used to understand the effector function of p21-deficient CD4+ T cells. RESULTS: We observed that the activation of cell cycle regulator p21 is crucial for CD4+ T-cell cytotoxic function and that p21 deficiency in type 1 helper T cells (Th1) leads to increased tumor growth in murine CRC. Similarly, low p21 expression in CD4+ T cells infiltrated into tumors of CRC patients is associated with reduced cancer-related survival. In mouse models of CRC, p21-deficient Th1 cells show signs of exhaustion, where an accumulation of effector/effector memory T cells and CD27/CD28 loss are predominant. Immune reconstitution of tumor-bearing Rag1-/- mice using ex vivo-treated p21-deficient T cells with palbociclib, an inhibitor of cyclin-dependent kinase 4/6, restored cytotoxic function and prevented exhaustion of p21-deficient CD4+ T cells as a possible concept for future immunotherapy of human disease. CONCLUSIONS: Our data reveal the importance of p21 in controlling the cell cycle and preventing exhaustion of Th1 cells. Furthermore, we unveil the therapeutic potential of cyclin-dependent kinase inhibitors such as palbociclib to reduce T-cell exhaustion for future treatment of patients with colorectal cancer.

PU.1 controls fibroblast polarization and tissue fibrosis.

Fibroblasts are polymorphic cells with pleiotropic roles in organ morphogenesis, tissue homeostasis and immune responses. In fibrotic diseases, fibroblasts synthesize abundant amounts of extracellular matrix, which induces scarring and organ failure. By contrast, a hallmark feature of fibroblasts in arthritis is degradation of the extracellular matrix because of the release of metalloproteinases and degrading enzymes, and subsequent tissue destruction. The mechanisms that drive these functionally opposing pro-fibrotic and pro-inflammatory phenotypes of fibroblasts remain unknown. Here we identify the transcription factor PU.1 as an essential regulator of the pro-fibrotic gene expression program. The interplay between transcriptional and post-transcriptional mechanisms that normally control the expression of PU.1 expression is perturbed in various fibrotic diseases, resulting in the upregulation of PU.1, induction of fibrosis-associated gene sets and a phenotypic switch in extracellular matrix-producing pro-fibrotic fibroblasts. By contrast, pharmacological and genetic inactivation of PU.1 disrupts the fibrotic network and enables reprogramming of fibrotic fibroblasts into resting fibroblasts, leading to regression of fibrosis in several organs.

N-glycosylation Regulates Intrinsic IFN-γ Resistance in Colorectal Cancer: Implications for Immunotherapy.

BACKGROUND & AIMS: Advanced colorectal carcinoma (CRC) is characterized by a high frequency of primary immune evasion and refractoriness to immunotherapy. Given the importance of interferon (IFN)-γ in CRC immunosurveillance, we investigated whether and how acquired IFN-γ resistance in tumor cells would promote tumor growth, and whether IFN-γ sensitivity could be restored. METHODS: Spontaneous and colitis-associated CRC development was induced in mice with a specific IFN-γ pathway inhibition in intestinal epithelial cells. The influence of IFN-γ pathway gene status and expression on survival was assessed in patients with CRC. The mechanisms underlying IFN-γ resistance were investigated in CRC cell lines. RESULTS: The conditional knockout of the IFN-γ receptor in intestinal epithelial cells enhanced spontaneous and colitis-associated colon tumorigenesis in mice, and the loss of IFN-γ receptor α (IFNγRα) expression by tumor cells predicted poor prognosis in patients with CRC. IFNγRα expression was repressed in human CRC cells through changes in N-glycosylation, which decreased protein stability via proteasome-dependent degradation, inhibiting IFNγR-signaling. Downregulation of the bisecting N-acetylglucosaminyltransferase III (MGAT3) expression was associated with IFN-γ resistance in all IFN-γ-resistant cells, and highly correlated with low IFNγRα expression in CRC tissues. Both ectopic and pharmacological reconstitution of MGAT3 expression with all-trans retinoic acid increased bisecting N-glycosylation, as well as IFNγRα protein stability and signaling. CONCLUSIONS: Together, our results demonstrated that tumor-associated changes in N-glycosylation destabilize IFNγRα, causing IFN-γ resistance in CRC. IFN-γ sensitivity could be reestablished through the increase in MGAT3 expression, notably via all-trans retinoic acid treatment, providing new prospects for the treatment of immune-resistant CRC.

Epithelial presenilin-1 drives colorectal tumour growth by controlling EGFR-COX2 signalling.

OBJECTIVE: Psen1 was previously characterised as a crucial factor in the pathogenesis of neurodegeneration in patients with Alzheimer's disease. Little, if any, is known about its function in the gut. Here, we uncovered an unexpected functional role of Psen1 in gut epithelial cells during intestinal tumourigenesis. DESIGN: Human colorectal cancer (CRC) and control samples were investigated for PSEN1 and proteins of theγ-secretase complex. Tumour formation was analysed in the AOM-DSS and Apc min/+ mouse models using newly generated epithelial-specific Psen1 deficient mice. Psen1 deficient human CRC cells were studied in a xenograft tumour model. Tumour-derived organoids were analysed for growth and RNA-Seq was performed to identify Psen1-regulated pathways. Tumouroids were generated to study EGFR activation and evaluation of the influence of prostanoids. RESULTS: PSEN1 is expressed in the intestinal epithelium and its level is increased in human CRC. Psen1-deficient mice developed only small tumours and human cancer cell lines deficient in Psen1 had a reduced tumourigenicity. Tumouroids derived from Psen1-deficient Apc min/+ mice exhibited stunted growth and reduced cell proliferation. On a molecular level, PSEN1 potentiated tumour cell proliferation via enhanced EGFR signalling and COX-2 production. Exogenous administration of PGE2 reversed the slow growth of PSEN1 deficient tumour cells via PGE2 receptor 4 (EP4) receptor signalling. CONCLUSIONS: Psen1 drives tumour development by increasing EGFR signalling via NOTCH1 processing, and by activating the COX-2-PGE2 pathway. PSEN1 inhibition could be a useful strategy in treatment of CRC.

Neutrophil extracellular traps drive epithelial-mesenchymal transition of human colon cancer.

Neutrophil extracellular traps (NETs) are extracellular structures, composed of nuclear DNA and various proteins released from neutrophils. Evidence is growing that NETs exert manifold functions in infection, immunity and cancer. Recently, NETs have been detected in colorectal cancer (CRC) tissues, but their association with disease progression and putative functional impact on tumourigenesis remained elusive. Using high-resolution stimulated emission depletion (STED) microscopy, we showed that citrullinated histone H3 (H3cit) is sufficient to specifically detect citrullinated NETs in colon cancer tissues. Among other evidence, this was supported by the close association of H3cit with de-condensed extracellular DNA, the hallmark of NETs. Extracellular DNA was reliably differentiated from nuclear condensed DNA by staining with an anti-DNA antibody, providing a novel and valuable tool to detect NETs in formalin-fixed paraffin-embedded tissues. Using these markers, the clinical association of NETs was investigated in a cohort of 85 patients with colon cancer. NETs were frequently detected (37/85, 44%) in colon cancer tissue sections and preferentially localised either only in the tumour centre or both in the tumour centre and the invasive front. Of note, citrullinated NETs were significantly associated with high histopathological tumour grades and lymph node metastasis. In vitro, purified NETs induced filopodia formation and cell motility in CRC cell lines. This was associated with increased expression of mesenchymal marker mRNAs (vimentin [VIM], fibronectin [FN1]) and epithelial-mesenchymal transition promoting transcription factors (ZEB1, Slug [SNAI2]), as well as decreased expression of the epithelial markers E-cadherin (CDH1) and epithelial cell adhesion molecule (EPCAM). These findings indicated that NETs activate an epithelial-mesenchymal transition-like process in CRC cells and may contribute to the metastatic progression of CRC. © 2021 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Contribution of Blood Vessel Activation, Remodeling and Barrier Function to Inflammatory Bowel Diseases.

Inflammatory bowel diseases (IBDs) consist of a group of chronic inflammatory disorders with a complex etiology, which represent a clinical challenge due to their often therapy-refractory nature. In IBD, inflammation of the intestinal mucosa is characterized by strong and sustained leukocyte infiltration, resulting in the loss of epithelial barrier function and subsequent tissue destruction. This is accompanied by the activation and the massive remodeling of mucosal micro-vessels. The role of the gut vasculature in the induction and perpetuation of mucosal inflammation is receiving increasing recognition. While the vascular barrier is considered to offer protection against bacterial translocation and sepsis after the breakdown of the epithelial barrier, endothelium activation and angiogenesis are thought to promote inflammation. The present review examines the respective pathological contributions of the different phenotypical changes observed in the microvascular endothelium during IBD, and provides an overview of potential vessel-specific targeted therapy options for the treatment of IBD.

Anti-DNA-IgM Favors the Detection of NET-Associated Extracellular DNA.

During inflammatory responses, neutrophils enter the sites of attack where they execute various defense mechanisms. They (I) phagocytose microorganisms, (II) degranulate to release cytokines, (III) recruit various immune cells by cell-type specific chemokines, (IV) secrete anti-microbials including lactoferrin, lysozyme, defensins and reactive oxygen species, and (V) release DNA as neutrophil extracellular traps (NETs). The latter originates from mitochondria as well as from decondensed nuclei. This is easily detected in cultured cells by staining of DNA with specific dyes. However, in tissues sections the very high fluorescence signals emitted from the condensed nuclear DNA hamper the detection of the widespread, extranuclear DNA of the NETs. In contrast, when we employ anti-DNA-IgM antibodies, they are unable to penetrate deep into the tightly packed DNA of the nucleus, and we observe a robust signal for the extended DNA patches of the NETs. To validate anti-DNA-IgM, we additionally stained the sections for the NET-markers histone H2B, myeloperoxidase, citrullinated histone H3, and neutrophil elastase. Altogether, we have described a fast one-step procedure for the detection of NETs in tissue sections, which provides new perspectives to characterize neutrophil-associated immune reactions in disease.

Nucleotide-dependent farnesyl switch orchestrates polymerization and membrane binding of human guanylate-binding protein 1.

Dynamin-like proteins (DLPs) mediate various membrane fusion and fission processes within the cell, which often require the polymerization of DLPs. An IFN-inducible family of DLPs, the guanylate-binding proteins (GBPs), is involved in antimicrobial and antiviral responses within the cell. Human guanylate-binding protein 1 (hGBP1), the founding member of GBPs, is also engaged in the regulation of cell adhesion and migration. Here, we show how the GTPase cycle of farnesylated hGBP1 (hGBP1F) regulates its self-assembly and membrane interaction. Using vesicles of various sizes as a lipid bilayer model, we show GTP-dependent membrane binding of hGBP1F In addition, we demonstrate nucleotide-dependent tethering ability of hGBP1F Furthermore, we report nucleotide-dependent polymerization of hGBP1F, which competes with membrane binding of the protein. Our results show that hGBP1F acts as a nucleotide-controlled molecular switch by modulating the accessibility of its farnesyl moiety, which does not require any supportive proteins.

ß6 -integrin serves as a novel serum tumor marker for colorectal carcinoma.

Colorectal cancer (CRC) is one of the leading causes of cancer-related deaths worldwide and the need for novel biomarkers and therapeutic strategies to improve diagnosis and surveillance is obvious. This study aims to identify ß6 -integrin (ITGB6) as a novel serum tumor marker for diagnosis, prognosis, and surveillance of CRC. ITGB6 serum levels were validated in retro- and prospective CRC patient cohorts. ITGB6 serum levels were analyzed by ELISA. Using an initial cohort of 60 CRC patients, we found that ITGB6 is present in the serum of CRC, but not in non-CRC control patients. A cut-off of ≥2 ng/mL ITGB6 reveals 100% specificity for the presence of metastatic CRC. In an enlarged study cohort of 269 CRC patients, ITGB6 predicted the onset of metastatic disease and was associated with poor prognosis. Those data were confirmed in an independent, prospective cohort consisting of 40 CRC patients. To investigate whether ITGB6 can also be used for tumor surveillance, serum ITGB6-levels were assessed in 26 CRC patients, pre- and post-surgery, as well as during follow-up visits. After complete tumor resection, ITGB6 serum levels declined completely. During follow-up, a new rise in ITGB6 serum levels indicated tumor recurrence or the onset of new metastasis as confirmed by CT scan. ITGB6 was more accurate for prognosis of advanced CRC and for tumor surveillance as the established marker carcinoembryonic antigen (CEA). Our findings identify ITGB6 as a novel serum marker for diagnosis, prognosis, and surveillance of advanced CRC. This might essentially contribute to an optimized patient care.

Mbd2 enables tumourigenesis within the intestine while preventing tumour-promoting inflammation.

Epigenetic regulation plays a key role in the link between inflammation and cancer. Here we examine Mbd2, which mediates epigenetic transcriptional silencing by binding to methylated DNA. In separate studies the Mbd2-/- mouse has been shown (1) to be resistant to intestinal tumourigenesis and (2) to have an enhanced inflammatory/immune response, observations that are inconsistent with the links between inflammation and cancer. To clarify its role in tumourigenesis and inflammation, we used constitutive and conditional models of Mbd2 deletion to explore its epithelial and non-epithelial roles in the intestine. Using a conditional model, we found that suppression of intestinal tumourigenesis is due primarily to the absence of Mbd2 within the epithelia. Next, we demonstrated, using the DSS colitis model, that non-epithelial roles of Mbd2 are key in preventing the transition from acute to tumour-promoting chronic inflammation. Combining models revealed that prior to inflammation the altered Mbd2-/- immune response plays a role in intestinal tumour suppression. However, following inflammation the intestine converts from tumour suppressive to tumour promoting. To summarise, in the intestine the normal function of Mbd2 is exploited by cancer cells to enable tumourigenesis, while in the immune system it plays a key role in preventing tumour-enabling inflammation. Which role is dominant depends on the inflammation status of the intestine. As environmental interactions within the intestine can alter DNA methylation patterns, we propose that Mbd2 plays a key role in determining whether these interactions are anti- or pro-tumourigenic and this makes it a useful new epigenetic model for inflammation-associated carcinogenesis. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Soluble intercellular adhesion molecule-1 is a prognostic marker in colorectal carcinoma.

PURPOSE: Serological tumor markers are routinely used to monitor tumor onset and progression. In colorectal carcinoma (CRC), the carcinoembryonic antigen (CEA) is roughly elevated in 50% of patients at initial diagnosis. Soluble ICAM-1 (sICAM-1) is elevated in different cancers. The aim of this study was to evaluate the prognostic relevance of sICAM-1 combined with CEA in patients with CRC. METHODS: In blood samples of 297 CRC patients, sICAM-1 was determined by ELISA and CEA by microparticle enzyme immunoassay the day before oncologic resection. Separation in patients with sICAM-1high and sICAM-1low was performed by minimum p value approach; separation in CEA normal and elevated was performed according to the established diagnostic cutoff. Clinical data were obtained from the prospective collected data from the Erlangen Registry for Colorectal Carcinomas. RESULTS: Cancer-related 5-year survival rate of patients with sICAM-1low (< 290 ng/ml, n = 208) was significantly increased (83.4%) as compared to that of patients with sICAM-1high (≥ 290 ng/ml, n = 89) (66.2%; p < 0.001). Patients with normal CEA concentrations (n = 199; 90.8%) showed a significantly (p < 0.001) improved cancer-related 5-year survival rate compared to patients with elevated CEA concentrations (n = 98; 52.1%). Moreover, high sICAM-1 was an independent risk factor (hazard ratio 1.6) in multivariate analysis. Of note, increased sICAM-1 levels, either within normal or within elevated CEA, allowed to identify high-risk subgroups, both for overall (p < 0.001) and cancer-related survival (p < 0.001). CONCLUSION: Application of a novel risk score combining CEA/sICAM-1 serum concentrations allows the identification of high-risk groups for poor survival in CRC patients.

IFN-γ-response mediator GBP-1 represses human cell proliferation by inhibiting the Hippo signaling transcription factor TEAD.

Interferon-gamma (IFN-γ) is a pleiotropic cytokine that exerts important functions in inflammation, infectious diseases, and cancer. The large GTPase human guanylate-binding protein 1 (GBP-1) is among the most strongly IFN-γ-induced cellular proteins. Previously, it has been shown that GBP-1 mediates manifold cellular responses to IFN-γ including the inhibition of proliferation, spreading, migration, and invasion and through this exerts anti-tumorigenic activity. However, the mechanisms of GBP-1 anti-tumorigenic activities remain poorly understood. Here, we elucidated the molecular mechanism of the human GBP-1-mediated suppression of proliferation by demonstrating for the first time a cross-talk between the anti-tumorigenic IFN-γ and Hippo pathways. The α9-helix of GBP-1 was found to be sufficient to inhibit proliferation. Protein-binding and molecular modeling studies revealed that the α9-helix binds to the DNA-binding domain of the Hippo signaling transcription factor TEA domain protein (TEAD) mediated by the 376VDHLFQK382 sequence at the N-terminus of the GBP-1-α9-helix. Mutation of this sequence resulted in abrogation of both TEAD interaction and suppression of proliferation. Further on, the interaction caused inhibition of TEAD transcriptional activity associated with the down-regulation of TEAD-target genes. In agreement with these results, IFN-γ treatment of the cells also impaired TEAD activity, and this effect was abrogated by siRNA-mediated inhibition of GBP-1 expression. Altogether, this demonstrated that the α9-helix is the proliferation inhibitory domain of GBP-1, which acts independent of the GTPase activity through the inhibition of the Hippo transcription factor TEAD in mediating the anti-proliferative cell response to IFN-γ.

Azidothymidine Sensitizes Primary Effusion Lymphoma Cells to Kaposi Sarcoma-Associated Herpesvirus-Specific CD4+ T Cell Control and Inhibits vIRF3 Function.

Kaposi sarcoma-associated herpesvirus (KSHV) is linked with the development of Kaposi sarcoma and the B lymphocyte disorders primary effusion lymphoma (PEL) and multi-centric Castleman disease. T cell immunity limits KSHV infection and disease, however the virus employs multiple mechanisms to inhibit efficient control by these effectors. Thus KSHV-specific CD4+ T cells poorly recognize most PEL cells and even where they can, they are unable to kill them. To make KSHV-infected cells more sensitive to T cell control we treated PEL cells with the thymidine analogue azidothymidine (AZT), which sensitizes PEL lines to Fas-ligand and TRAIL challenge; effector mechanisms which T cells use. PELs co-cultured with KSHV-specific CD4+ T cells in the absence of AZT showed no control of PEL outgrowth. However in the presence of AZT PEL outgrowth was controlled in an MHC-restricted manner. To investigate how AZT sensitizes PELs to immune control we first examined BJAB cells transduced with individual KSHV-latent genes for their ability to resist apoptosis mediated by stimuli delivered through Fas and TRAIL receptors. This showed that in addition to the previously described vFLIP protein, expression of vIRF3 also inhibited apoptosis delivered by these stimuli. Importantly vIRF3 mediated protection from these apoptotic stimuli was inhibited in the presence of AZT as was a second vIRF3 associated phenotype, the downregulation of surface MHC class II. Although both vFLIP and vIRF3 are expressed in PELs, we propose that inhibiting vIRF3 function with AZT may be sufficient to restore T cell control of these tumor cells.

Processing and secretion of guanylate binding protein-1 depend on inflammatory caspase activity.

Human guanylate binding protein-1 (GBP-1) belongs to the family of large GTPases. The expression of GBP-1 is inducible by inflammatory cytokines, and the protein is involved in inflammatory processes and host defence against cellular pathogens. GBP-1 is the first GTPase which was described to be secreted by eukaryotic cells. Here, we report that precipitation of GBP-1 with GMP-agarose from cell culture supernatants co-purified a 47-kD fragment of GBP-1 (p47-GBP-1) in addition to the 67-kD full-length form. MALDI-TOF sequencing revealed that p47-GBP-1 corresponds to the C-terminal helical part of GBP-1 and lacks most of the globular GTPase domain. In silico analyses of protease target sites, together with cleavage experiments in vitro and in vivo, showed that p67-GBP-1 is cleaved by the inflammatory caspases 1 and 5, leading to the formation of p47-GBP-1. Furthermore, the secretion of p47-GBP-1 was found to occur via a non-classical secretion pathway and to be dependent on caspase-1 activity but independent of inflammasome activation. Finally, we showed that p47-GBP-1 represents the predominant form of secreted GBP-1, both in cell culture supernatants and, in vivo, in the cerebrospinal fluid of patients with bacterial meningitis, indicating that it may represent the biologically active form of extracellular GBP-1. These findings confirm the involvement of caspase-1 in non-classical secretion mechanisms and open novel perspectives for the extracellular function of secreted GBP-1.

Cocultivation of Mesenchymal Stem Cells and Endothelial Progenitor Cells Reveals Antiapoptotic and Proangiogenic Effects.

Integrating bioartificial tissues into the host vasculature is a prerequisite for tissue engineering applications. Endothelial progenitor cells (EPCs) display a high angiogenic potential and a low donor-site morbidity, making them ideal for tissue engineering applications. In our study we used a murine EPC cell line (T17b) and rat mesenchymal stem cells (MSCs) for cocultivation experiments. MSCs were cocultured with increasing T17b EPC amounts. Furthermore, MSCs in monoculture were treated with conditioned medium (CM) from T17b EPCs and T17b EPCs were treated with CM from MSCs. Proliferation and apoptosis were quantified with a bromodeoxyuridine ELISA and a DNA fragmentation ELISA, respectively. Osteogenic differentiation was detected with an alkaline phosphatase assay and bone morphogenetic protein-2 ELISA. The production of proangiogenic molecules was measured with a matrix metalloproteinase-3 and vascular endothelial growth factor ELISA as well as nitric oxide assay. We could show that T17b EPCs stimulated MSC proliferation but not vice versa. On the other hand, MSCs promoted the cell survival of EPCs. The growth-inducing and antiapoptotic effects were dependent on heterotypic cell contacts and paracrine mediators. Moreover, proangiogenic growth factors were found in the coculture. Collectively, our results indicate that the coapplication of MSCs and T17b EPCs provides new perspectives for tissue engineering applications.

VEGFR2 Signaling Prevents Colorectal Cancer Cell Senescence to Promote Tumorigenesis in Mice With Colitis.

BACKGROUND & AIMS: Senescence prevents cellular transformation. We investigated whether vascular endothelial growth factor (VEGF) signaling via its receptor, VEGFR2, regulates senescence and proliferation of tumor cells in mice with colitis-associated cancer (CAC). METHODS: CAC was induced in VEGFR2(ΔIEC) mice, which do not express VEGFR2 in the intestinal epithelium, and VEGFR2(fl/fl) mice (controls) by administration of azoxymethane followed by dextran sodium sulfate. Tumor development and inflammation were determined by endoscopy. Colorectal tissues were collected for immunoblot, immunohistochemical, and quantitative polymerase chain reaction analyses. Findings from mouse tissues were confirmed in human HCT116 colorectal cancer cells. We analyzed colorectal tumor samples from patients before and after treatment with bevacizumab. RESULTS: After colitis induction, VEGFR2(ΔIEC) mice developed significantly fewer tumors than control mice. A greater number of intestinal tumor cells from VEGFR2(ΔIEC) mice were in senescence than tumor cells from control mice. We found VEGFR2 to activate phosphatidylinositol-4,5-bisphosphate-3-kinase and AKT, resulting in inactivation of p21 in HCT116 cells. Inhibitors of VEGFR2 and AKT induced senescence in HCT116 cells. Tumor cell senescence promoted an anti-tumor immune response by CD8(+) T cells in mice. Patients whose tumor samples showed an increase in the proportion of senescent cells after treatment with bevacizumab had longer progression-free survival than patients in which the proportion of senescent tumor cells did not change before and after treatment. CONCLUSIONS: Inhibition of VEGFR2 signaling leads to senescence of human and mouse colorectal cancer cells. VEGFR2 interacts with phosphatidylinositol-4,5-bisphosphate-3-kinase and AKT to inactivate p21. Colorectal tumor senescence and p21 level correlate with patient survival during treatment with bevacizumab.

Kaposi's sarcoma associated herpesvirus tegument protein ORF75 is essential for viral lytic replication and plays a critical role in the antagonization of ND10-instituted intrinsic immunity.

Nuclear domain 10 (ND10) components are restriction factors that inhibit herpesviral replication. Effector proteins of different herpesviruses can antagonize this restriction by a variety of strategies, including degradation or relocalization of ND10 proteins. We investigated the interplay of Kaposi's Sarcoma-Associated Herpesvirus (KSHV) infection and cellular defense by nuclear domain 10 (ND10) components. Knock-down experiments in primary human cells show that KSHV-infection is restricted by the ND10 components PML and Sp100, but not by ATRX. After KSHV infection, ATRX is efficiently depleted and Daxx is dispersed from ND10, indicating that these two ND10 components can be antagonized by KSHV. We then identified the ORF75 tegument protein of KSHV as the viral factor that induces the disappearance of ATRX and relocalization of Daxx. ORF75 belongs to a viral protein family (viral FGARATs) that has homologous proteins in all gamma-herpesviruses. Isolated expression of ORF75 in primary cells induces a relocalization of PML and dispersal of Sp100, indicating that this viral effector protein is able to influence multiple ND10 components. Moreover, by constructing a KSHV mutant harboring a stop codon at the beginning of ORF75, we could demonstrate that ORF75 is absolutely essential for viral replication and the initiation of viral immediate-early gene expression. Using recombinant viruses either carrying Flag- or YFP-tagged variants of ORF75, we could further corroborate the role of ORF75 in the antagonization of ND10-mediated intrinsic immunity, and show that it is independent of the PML antagonist vIRF3. Members of the viral FGARAT family target different ND10 components, suggesting that the ND10 targets of viral FGARAT proteins have diversified during evolution. We assume that overcoming ND10 intrinsic defense constitutes a critical event in the replication of all herpesviruses; on the other hand, restriction of herpesviral replication by ND10 components may also promote latency as the default outcome of infection.

Guanylate binding protein 1-mediated interaction of T cell antigen receptor signaling with the cytoskeleton.

GTPases act as important switches in many signaling events in cells. Although small and heterotrimeric G proteins are subjects of intensive studies, little is known about the large IFN-inducible GTPases. In this article, we show that the IFN-γ-inducible guanylate binding protein 1 (GBP-1) is a regulator of T cell activation. Silencing of GBP-1 leads to enhanced activation of early T cell Ag receptor/CD3 signaling molecules, including Lck, that is translated to higher IL-2 production. Mass spectrometry analyses showed that regulatory cytoskeletal proteins, like plastin-2 that bundles actin fibers and spectrin ß-chain, brain 1 that links the plasma membrane to the actin cytoskeleton, are binding partners of GBP-1. The spectrin cytoskeleton influences cell spreading and surface expression of TCR/CD3 and the leukocyte phosphatase CD45. We found higher cell spreading and enhanced surface expression of TCR/CD3 and CD45 in GBP-1 silenced T cells that explain their enhanced TCR/CD3 signaling. We conclude that GBP-1 is a downstream processor of IFN-γ via which T cells regulate cytoskeleton-dependent cell functions.

Identification of Predictive Markers for Response to Neoadjuvant Chemoradiation in Rectal Carcinomas by Proteomic Isotope Coded Protein Label (ICPL) Analysis.

Neoadjuvant chemoradiation (nCRT) is an established procedure in stage union internationale contre le cancer (UICC) II/III rectal carcinomas. Around 53% of the tumours present with good tumor regression after nCRT, and 8%-15% are complete responders. Reliable selection markers would allow the identification of poor or non-responders prior to therapy. Tumor biopsies were harvested from 20 patients with rectal carcinomas, and stored in liquid nitrogen prior to therapy after obtaining patients' informed consent (Erlangen-No.3784). Patients received standardized nCRT with 5-Fluoruracil (nCRT I) or 5-Fluoruracil ± Oxaliplatin (nCRT II) according to the CAO/ARO/AIO-04 protocol. After surgery, regression grading (Dworak) of the tumors was performed during histopathological examination of the specimens. Tumors were classified as poor (Dworak 1 + 2) or good (Dworak 3 + 4) responders. Laser capture microdissection (LCM) for tumor enrichment was performed on preoperative biopsies. Differences in expressed proteins between poor and good responders to nCRT I and II were identified by proteomic analysis (Isotope Coded Protein Label, ICPL™) and selected markers were validated by immunohistochemistry. Tumors of 10 patients were classified as histopathologically poor (Dworak 1 or 2) and the other 10 tumor samples as histopathologically good (Dworak 3 or 4) responders to nCRT after surgery. Sufficient material in good quality was harvested for ICPL analysis by LCM from all biopsies. We identified 140 differentially regulated proteins regarding the selection criteria and the response to nCRT. Fourteen of these proteins were synchronously up-regulated at least 1.5-fold after nCRT I or nCRT II (e.g., FLNB, TKT, PKM2, SERINB1, IGHG2). Thirty-five proteins showed a complete reciprocal regulation (up or down) after nCRT I or nCRT II and the rest was regulated either according to nCRT I or II. The protein expression of regulated proteins such as PLEC1, TKT, HADHA and TAGLN was validated successfully by immunohistochemistry. ICPL is a valid method to identify differentially expressed proteins in rectal carcinoma tissue between poor vs. good responders to nCRT. The identified protein markers may act as selection criteria for nCRT in the future, but our preliminary findings must be reproduced and validated in a prospective cohort.

Absolute quantification of DcR3 and GDF15 from human serum by LC-ESI MS.

Biomarkers are widely used in clinical diagnosis, prognosis and therapy monitoring. Here, we developed a protocol for the efficient and selective enrichment of small and low concentrated biomarkers from human serum, involving a 95% effective depletion of high-abundant serum proteins by partial denaturation and enrichment of low-abundant biomarkers by size exclusion chromatography. The recovery of low-abundance biomarkers was above 97%. Using this protocol, we quantified the tumour markers DcR3 and growth/differentiation factor (GDF)15 from 100 µl human serum by isotope dilution mass spectrometry, using (15) N metabolically labelled and concatamerized fingerprint peptides for the both proteins. Analysis of three different fingerprint peptides for each protein by liquid chromatography electrospray ionization mass spectrometry resulted in comparable concentrations in three healthy human serum samples (DcR3: 27.23 ± 2.49 fmol/ml; GDF15: 98.11 ± 0.49 fmol/ml). In contrast, serum levels were significantly elevated in tumour patients for DcR3 (116.94 ± 57.37 fmol/ml) and GDF15 (164.44 ± 79.31 fmol/ml). Obtained data were in good agreement with ELISA and qPCR measurements, as well as with literature data. In summary, our protocol allows the reliable quantification of biomarkers, shows a higher resolution at low biomarker concentrations than antibody-based strategies, and offers the possibility of multiplexing. Our proof-of-principle studies in patient sera encourage the future analysis of the prognostic value of DcR3 and GDF15 for colon cancer patients in larger patient cohorts.