A Translational Investigation of IFN-α and STAT1 Signaling in Endothelial Cells during Septic Shock Provides Therapeutic Perspectives.

Septic shock and disseminated intravascular coagulation (DIC) are known to be characterized by an endothelial cell dysfunction. The molecular mechanisms underlying this relationship are, however, poorly understood. In this work, we aimed to investigate human circulating IFN-α in patients with septic shock-induced DIC and tested the potential role of endothelial Stat1 (signal transducer and activator of transcription 1) as a therapeutic target in a mouse model of sepsis. For this, circulating type I, type II, and type III IFNs and procoagulant microvesicles were quantified in a prospective cohort of patients with septic shock. Next, we used a septic shock model induced by cecal ligation and puncture in wild-type mice, in Ifnar1 (type I IFN receptor subunit 1)-knockout mice, and in Stat1 conditional knockout mice. In human samples, we observed higher concentrations of circulating IFN-α and IFN-α1 in patients with DIC compared with patients without DIC, whereas concentrations of IFN-ß, IFN-γ, IFN-λ1, IFN-λ2, and IFN-λ3 were not different. IFN-α concentration was positively correlated with CD105 microvesicle concentrations, reflecting endothelial injury. In Ifnar1-/- mice, cecal ligation and puncture did not induce septic shock and was characterized by lesser endothelial cell injury, with lower aortic inflammatory cytokine expression, endothelial inflammatory-related gene expression, and fibrinolysis. In mice in which Stat1 was specifically ablated in endothelial cells, a marked protection against sepsis was also observed, suggesting the relevance of an endothelium-targeted strategy. Our work highlights the key roles of type I IFNs as pathogenic players in septic shock-induced DIC and the potential pertinence of endothelial STAT1 as a therapeutic target.

The sVEGFR1-i13 splice variant regulates a ß1 integrin/VEGFR autocrine loop involved in the progression and the response to anti-angiogenic therapies of squamous cell lung carcinoma.

BACKGROUND: While lung adenocarcinoma patients can somewhat benefit from anti-angiogenic therapies, patients with squamous cell lung carcinoma (SQLC) cannot. The reasons for this discrepancy remain largely unknown. Soluble VEGF receptor-1, namely sVEGFR1-i13, is a truncated splice variant of the cell membrane-spanning VEGFR1 that has no transmembrane or tyrosine kinase domain. sVEGFR1-i13 is mainly viewed as an anti-angiogenic factor which counteracts VEGF-A/VEGFR signalling in endothelial cells. However, its role in tumour cells is poorly known. METHODS: mRNA and protein status were analysed by Real-Time qPCR, western blotting, ELISA assay, proximity ligation assay or immunohistochemistry in human tumour cell lines, murine tumourgrafts and non small cell lung carcinoma patients samples. RESULTS: We show that anti-angiogenic therapies specifically increase the levels of sVEGFR1-i13 in SQLC cell lines and chemically induced SQLC murine tumourgrafts. At the molecular level, we characterise a sVEGFR1-i13/ß1 integrin/VEGFR autocrine loop which determines whether SQLC cells proliferate or go into apoptosis, in response to anti-angiogenic therapies. Furthermore, we show that high levels of both sVEGFR1-i13 and ß1 integrin mRNAs and proteins are associated with advanced stages in SQLC patients and with a poor clinical outcome in patients with early stage SQLC. CONCLUSIONS: Overall, these results reveal an unexpected pro-tumoural function of sVEGFR1-i13 in SQLC tumour cells, which contributes to their progression and escape from anti-angiogenic therapies. These data might help to understand why some SQLC patients do not respond to anti-angiogenic therapies.

Splice Variants of the RTK Family: Their Role in Tumour Progression and Response to Targeted Therapy.

Receptor tyrosine kinases (RTKs) belong to a family of transmembrane receptors that display tyrosine kinase activity and trigger the activation of downstream signalling pathways mainly involved in cell proliferation and survival. RTK amplification or somatic mutations leading to their constitutive activation and oncogenic properties have been reported in various tumour types. Numerous RTK-targeted therapies have been developed to counteract this hyperactivation. Alternative splicing of pre-mRNA has recently emerged as an important contributor to cancer development and tumour maintenance. Interestingly, RTKs are alternatively spliced. However, the biological functions of RTK splice variants, as well as the upstream signals that control their expression in tumours, remain to be understood. More importantly, it remains to be determined whether, and how, these splicing events may affect the response of tumour cells to RTK-targeted therapies, and inversely, whether these therapies may impact these splicing events. In this review, we will discuss the role of alternative splicing of RTKs in tumour progression and response to therapies, with a special focus on two major RTKs that control proliferation, survival, and angiogenesis, namely, epidermal growth factor receptor (EGFR) and vascular endothelial growth factor receptor-1 (VEGFR1).

RNA splicing, cell signaling, and response to therapies.

PURPOSE OF REVIEW: PremRNA alternative splicing is more a rule than an exception as it affects more than 90% of multiexons genes and plays a key role in proteome diversity. Here, we discuss some recent studies published in the extensively growing field linking RNA splicing and cancer. RECENT FINDINGS: These last years, the development of high-throughput studies together with appropriate bioinformatic tools have led to the identification of new cancer-specific splicing patterns that allow to distinguish various cancer types, and provide new prognosis biomarkers. In addition, the functional consequences of hot spot mutations affecting various components of the spliceosome machinery in cancers have been described. As an example, missplicing of the enhancer of zeste homolog 2 histone methyltransferase premRNA in response to hot spot mutation of the splicing factor SRSF2 was found to participate to the pathogenesis of myelodysplastic syndrome. Moreover, proofs of principle that targeting the RNA splicing machinery can be used to correct aberrant missplicing, kill oncogene-driven cancer cells, or reverse resistance of tumor cells to targeted therapies have been done. As another example, the core spliceosomal function was recently found to be critical for the survival of Myc-driven breast cancer cells, rendering them hypersensitive to spliceosome inhibitors. SUMMARY: Dysregulation of premRNA alternative splicing appears to be one of the hallmarks of cancer. The characterization of novel splicing signatures in cancer as well as the identification of original signaling networks involving RNA splicing regulators should allow to decipher novel oncogenic mechanisms and to develop new therapeutic strategies.

Modulating tenascin-C functions by targeting the MAtrix REgulating MOtif, "MAREMO".

The extracellular matrix molecule Tenascin-C (TNC) promotes cancer and chronic inflammation by multiple mechanisms. Recently, TNC was shown to promote an immune suppressive tumor microenvironment (TME) through binding soluble chemoattracting factors, thus retaining leukocytes in the stroma. TNC also binds to fibronectin (FN) and other molecules, raising the question of a potential common TNC binding mechanism. By sequence comparison of two TNC-interacting domains in FN, the fifth (FN5) and thirteenth (FN13) fibronectin type III domains we identified a MAtrix REgulating MOtif "MAREMO" or M-motif that is highly conserved amongst vertebrates. By sequence analysis, structural modeling and functional analysis we found also putative M-motifs in TNC itself. We showed by negative staining electron microscopic imaging that the M-motif in FN mediates interactions with FN as well as with TNC. We generated two M-motif mimetic peptides P5 and P13 resembling the M-motif in FN5 and FN13, respectively. By using structural information we modelled binding of these M-motif mimetics revealing a putative MAREMO binding site MBS in FN5 and TN3, respectively overlapping with the M-motif. We further demonstrated that the M-motif mimetic peptides blocked several functions of TNC, such as binding of TNC to FN, cell rounding on a mixed FN/TNC substratum, FN matrix expression and subsequent assembly, TNC-induced signaling and gene expression, TNC chemokine binding and dendritic cell retention, thus providing novel opportunities to inhibit TNC actions. Our results suggest that targeting the MAREMO/MBS interaction could be exploited for reducing inflammation and matrix functions in cancer and fibrosis.

Novel Human Tenascin-C Function-Blocking Camel Single Domain Nanobodies.

The extracellular matrix (ECM) molecule Tenascin-C (TNC) is well-known to promote tumor progression by multiple mechanisms. However, reliable TNC detection in tissues of tumor banks remains limited. Therefore, we generated dromedary single-domain nanobodies Nb3 and Nb4 highly specific for human TNC (hTNC) and characterized the interaction with TNC by several approaches including ELISA, western blot, isothermal fluorescence titration and negative electron microscopic imaging. Our results revealed binding of both nanobodies to distinct sequences within fibronectin type III repeats of hTNC. By immunofluroescence and immunohistochemical imaging we observed that both nanobodies detected TNC expression in PFA and paraffin embedded human tissue from ulcerative colitis, solid tumors and liver metastasis. As TNC impairs cell adhesion to fibronectin we determined whether the nanobodies abolished this TNC function. Indeed, Nb3 and Nb4 restored adhesion of tumor and mesangial cells on a fibronectin/TNC substratum. We recently showed that TNC orchestrates the immune-suppressive tumor microenvironment involving chemoretention, causing tethering of CD11c+ myeloid/dendritic cells in the stroma. Here, we document that immobilization of DC2.4 dendritic cells by a CCL21 adsorbed TNC substratum was blocked by both nanobodies. Altogether, our novel TNC specific nanobodies could offer valuable tools for detection of TNC in the clinical practice and may be useful to inhibit the immune-suppressive and other functions of TNC in cancer and other diseases.

Tenascin-C immobilizes infiltrating T lymphocytes through CXCL12 promoting breast cancer progression.

Immune checkpoint therapy, where CD8 tumor infiltrating T lymphocytes (TIL) are reactivated, is a promising anti-cancer treatment approach, yet with low response rates. The extracellular matrix, in particular tenascin-C, may generate barriers for TIL. To investigate this possibility, we used a MMTV-NeuNT and syngeneic mammary gland grafting model derived thereof with engineered tenascin-C levels and observed accumulation of CD8 TIL in tenascin-C-rich stroma. Inhibition studies revealed that tenascin-C induced CXCL12 through TLR4. By binding CXCL12, tenascin-C retained CD8 TIL in the stroma. Blockade of CXCR4, the receptor of CXCL12, enhanced macrophage and CD8 TIL infiltration and reduced tumor growth and subsequent metastasis. Retention of CD8 TIL by tenascin-C/CXCL12 was also observed in human breast cancer by tissue staining. Moreover, whereas high CD8 TIL numbers correlated with longer metastasis-free survival, this was not the case when also tenascin-C and CXCL12 levels were high. Altogether, these results may be useful for improving tumor immunity as diagnostic tool and to formulate a future "TIL-matrix-release-and-reactivate" strategy.

Tenascin C Has a Modest Protective Effect on Acute Lung Pathology during Methicillin-Resistant Staphylococcus aureus-Induced Pneumonia in Mice.

Tenascin C (TNC) is an extracellular matrix protein with immunomodulatory properties that plays a major role during tissue injury and repair. TNC levels are increased in patients with pneumonia and pneumosepsis, and they are associated with worse outcomes. Methicillin-resistant Staphylococcus aureus (MRSA) is a Gram-positive bacterium that is a major causative pathogen in nosocomial pneumonia and a rising cause of community-acquired pneumonia. To study the role of TNC during MRSA-induced pneumonia, TNC sufficient (TNC+/+) and TNC-deficient (TNC-/-) mice were infected with MRSA via the airways and euthanized after 6, 24, and 48 h for analysis. Pulmonary transcription of TNC peaked at 6 h, while immunohistochemistry revealed higher protein levels at later time points. Although TNC deficiency was not associated with changes in bacterial clearance, TNC-/- mice showed increased levels of TNF-α and IL-6 in bronchoalveolar lavage fluid during the acute phase of infection when compared with TNC+/+ mice. In addition, TNC-/- mice showed more severe pulmonary pathology at 6, but not at 24 or 48 h, after infection. Together, these data suggest that TNC plays a moderate protective role against tissue pathology during the acute inflammatory phase, but not during the bacterial clearance phase, of MRSA-induced pneumonia. These results argue against an important role of TNC on disease outcome during MRSA-induced pneumonia. IMPORTANCE Recently, the immunomodulatory properties of TNC have drawn substantial interest. However, to date most studies made use of sterile models of inflammation. In this study, we examine the pathobiology of MRSA-induced pneumonia in a model of TNC-sufficient and TNC-deficient mice. We have studied the immune response and tissue pathology both during the initial insult and also during the resolution phase. We demonstrate that MRSA-induced pneumonia upregulates pulmonary TNC expression at the mRNA and protein levels. However, the immunomodulatory role of TNC during bacterial pneumonia is distinct from models of sterile inflammation, indicating that the function of TNC is context dependent. Contrary to previous descriptions of TNC as a proinflammatory mediator, TNC-deficient mice seem to suffer from enhanced tissue pathology during the acute phase of infection. Nonetheless, besides its role during the acute phase response, TNC does not seem to play a major role in disease outcome during MRSA-induced pneumonia.

Tenascin-C Deficiency Is Associated With Reduced Bacterial Outgrowth During Klebsiella pneumoniae-Evoked Pneumosepsis in Mice.

Tenascin C (TNC) is an extracellular matrix glycoprotein that recently emerged as an immunomodulator. TNC-deficient (TNC-/-) mice were reported to have a reduced inflammatory response upon systemic administration of lipopolysaccharide, the toxic component of gram-negative bacteria. Here, we investigated the role of TNC during gram-negative pneumonia derived sepsis. TNC+/+ and TNC-/- mice were infected with Klebsiella pneumoniae via the airways and sacrificed 24 and 42 h thereafter for further analysis. Pulmonary TNC protein levels were elevated 42 h after infection in TNC+/+ mice and remained undetectable in TNC-/- mice. TNC-/- mice showed modestly lower bacterial loads in lungs and blood, and a somewhat reduced local-but not systemic-inflammatory response. Moreover, TNC-/- and TNC+/+ mice did not differ with regard to neutrophil recruitment, lung pathology or plasma markers of distal organ injury. These results suggest that while TNC shapes the immune response during lipopolysaccharide-induced inflammation, this role may be superseded during pneumosepsis caused by a common gram-negative pathogen.

Impact of Tenascin-C on Radiotherapy in a Novel Syngeneic Oral Squamous Cell Carcinoma Model With Spontaneous Dissemination to the Lymph Nodes.

Radiotherapy, the most frequent treatment of oral squamous cell carcinomas (OSCC) besides surgery is employed to kill tumor cells but, radiotherapy may also promote tumor relapse where the immune-suppressive tumor microenvironment (TME) could be instrumental. We established a novel syngeneic grafting model from a carcinogen-induced tongue tumor, OSCC13, to address the impact of radiotherapy on OSCC. This model revealed similarities with human OSCC, recapitulating carcinogen-induced mutations found in smoking associated human tongue tumors, abundant tumor infiltrating leukocytes (TIL) and, spontaneous tumor cell dissemination to the local lymph nodes. Cultured OSCC13 cells and OSCC13-derived tongue tumors were sensitive to irradiation. At the chosen dose of 2 Gy mimicking treatment of human OSCC patients not all tumor cells were killed allowing to investigate effects on the TME. By investigating expression of the extracellular matrix molecule tenascin-C (TNC), an indicator of an immune suppressive TME, we observed high local TNC expression and TIL infiltration in the irradiated tumors. In a TNC knockout host the TME appeared less immune suppressive with a tendency towards more tumor regression than in WT conditions. Altogether, our novel syngeneic tongue OSCC grafting model, sharing important features with the human OSCC disease could be relevant for future anti-cancer targeting of OSCC by radiotherapy and other therapeutic approaches.

A mouse model of MSU-induced acute inflammation in vivo suggests imiquimod-dependent targeting of Il-1ß as relevant therapy for gout patients.

Rationale: The role of Monosodium Urate (MSU) crystals in gout pathophysiology is well described, as is the major impact of IL-1ß in the inflammatory reaction that constitutes the hallmark of the disease. However, despite the discovery of the NLRP3 inflammasome and its role as a Pattern Recognition Receptor linking the detection of a danger signal (MSU) to IL-1ß secretion in vitro, the precise mechanisms leading to joint inflammation in gout patients are still poorly understood. Methods: Acute urate crystal inflammation was obtained by subcutaneous injections of MSU crystals in mice. Symptoms were followed by scoring, cytokine quantification by ELISA and western blot, gene expression by RT-qPCR and RNAseq; Magnetic Resonance Imaging was also used to assess inflammation. Results: We provide an extensive clinical, biological and molecular characterization of an acute uratic inflammation mouse model which accurately mimics human gout. We report the efficacy of topical imiquimod treatment and its impact on Interferon-dependent down modulation of Il-1ß gene expression in this experimental model. Conclusion: Our work reveals several key features of MSU-dependent inflammation and identifies novel therapeutic opportunities for gout patients.

A VEGF-A/SOX2/SRSF2 network controls VEGFR1 pre-mRNA alternative splicing in lung carcinoma cells.

The splice variant sVEGFR1-i13 is a truncated version of the cell membrane-spanning VEGFR1 receptor that is devoid of its transmembrane and tyrosine kinase domains. We recently showed the contribution of sVEGFR1-i13 to the progression and the response of squamous lung carcinoma to anti-angiogenic therapies. In this study, we identify VEGF165, a splice variant of VEGF-A, as a regulator of sVEGFR1-i13 expression in these tumors, and further show that VEGF165 cooperates with the transcription factor SOX2 and the splicing factor SRSF2 to control sVEGFR1-i13 expression. We also demonstrate that anti-angiogenic therapies up-regulate sVEGFR1-i13 protein level in squamous lung carcinoma cells by a mechanism involving the VEGF165/SOX2/SRSF2 network. Collectively, our results identify for the first time a signaling network that controls VEGFR1 pre-mRNA alternative splicing in cancer cells.

Tenascin-C increases lung metastasis by impacting blood vessel invasions.

Metastasis is a major cause of death in cancer patients. The extracellular matrix molecule tenascin-C is a known promoter of metastasis, however the underlying mechanisms are not well understood. To further analyze the impact of tenascin-C on cancer progression we generated MMTV-NeuNT mice that develop spontaneous mammary tumors, on a tenascin-C knockout background. We also developed a syngeneic orthotopic model in which tumor cells derived from a MMTV-NeuNT tumor. Tumor cells were transfected with control shRNA or with shRNA to knockdown tenascin-C expression and, were grafted into the mammary gland of immune competent, wildtype or tenascin-C knockout mice. We show that stromal-derived tenascin-C increases metastasis by reducing apoptosis and inducing the cellular plasticity of cancer cells located in pulmonary blood vessels invasions (BVI), before extravasation. We characterized BVI as organized structures of tightly packed aggregates of proliferating tumor cells with epithelial characteristics, surrounded by Fsp1+ cells, internally located platelets and, a luminal monolayer of endothelial cells. We found extracellular matrix, in particular, tenascin-C, between the stromal cells and the tumor cell cluster. In mice lacking stromal-derived tenascin-C, the organization of pulmonary BVI was significantly affected, revealing novel functions of host-derived tenascin-C in supporting the integrity of the endothelial cell coat, increasing platelet abundance, tumor cell survival, epithelial plasticity, thereby promoting overall lung metastasis. Many effects of tenascin-C observed in BVI including enhancement of cellular plasticity, survival and migration, could be explained by activation of TGF-ß signaling. Finally, in several human cancers, we also observed BVI to be surrounded by an endothelial monolayer and to express tenascin-C. Expression of tenascin-C is specific to BVI and is not observed in lymphatic vascular invasions frequent in breast cancer, which lack an endothelial lining. Given that BVI have prognostic significance for many tumor types, such as shorter cancer patient survival, increased metastasis, vessel occlusion, and organ failure, our data revealing a novel mechanism by which stromal tenascin-C promotes metastasis in human cancer, may have potential for diagnosis and therapy.

VEGF165b, a splice variant of VEGF-A, promotes lung tumor progression and escape from anti-angiogenic therapies through a ß1 integrin/VEGFR autocrine loop.

Vascular endothelial growth factor-A (VEGF-A) is highly subjected to alternative pre-mRNA splicing that generates several splice variants. The VEGFxxx and VEGFxxxb families encode splice variants of VEGF-A that differ only at the level of six amino acids in their C-terminal part. The expression level of VEGFxxx splice variants and their function as pro-angiogenic factors during tumor neo-angiogenesis have been well-described. The role of VEGFxxxb isoforms is less well known, but they have been shown to inhibit VEGFxxx-mediated angiogenesis, while being partial or weak activators of VEGFR receptors in endothelial cells. On the opposite, their role on tumor cells expressing VEGFRs at their surface remains largely unknown. In this study, we find elevated levels of VEGF165b, the main VEGFxxxb isoform, in 36% of non-small cell lung carcinoma (NSCLC), mainly lung adenocarcinoma (46%), and show that a high VEGF165b/VEGF165 ratio correlates with the presence of lymph node metastases. At the molecular level, we demonstrate that VEGF165b stimulates proliferation and invasiveness of two lung tumor cell lines through a VEGFR/ß1 integrin loop. We further provide evidence that the isoform-specific knockdown of VEGF165b reduces tumor growth, demonstrating a tumor-promoting autocrine role for VEGF165b in lung cancer cells. Importantly, we show that bevacizumab, an anti-angiogenic compound used for the treatment of lung adenocarcinoma patients, increases the expression of VEGF165b and activates the invasive VEGFR/ß1 integrin loop. Overall, these data highlight an unexpected role of the VEGF165b splice variant in the progression of lung tumors and their response to anti-angiogenic therapies.