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CCAAT/Enhancer-Binding Protein delta (C/EBPδ) is a transcription factor involved in differentiation and inflammation. While sparsely expressed in adult tissues, aberrant expression of C/EBPδ has been associated with different cancers. Initially, re-expression of C/EBPδ in cell cultures limited tumor cell proliferation, assigning it a tumor suppressor role. However, opposing observations were made in pre-clinical models and patients, suggesting that C/EBPδ not only mediates cell proliferation but dictates a broader spectrum of tumorigenesis-related effects. It is now widely accepted that C/EBPδ contributes to an inflammatory, tumor-promoting microenvironment, aids hypoxia adaption and contributes to the recruitment of blood vessels for improved nutrient supply to tumor cells and facilitated extravasation. This review summarizes the work published on this transcription factor in the field of cancer over the past decade. It points out areas in which a consensus on C/EBPδ’s role appears to emerge and seek to explain seemingly contradictory results.

Pancreatic ductal adenocarcinoma (PDAC) is a dismal disease with a poor clinical prognosis and unsatisfactory treatment options. We previously found that the transcription factor CCAAT/Enhancer-Binding Protein Delta (C/EBPδ) is lowly expressed in PDAC compared to healthy pancreas duct cells, and that patient survival and lymph node involvement in PDAC is correlated with the expression of C/EBPδ in primary tumor cells. C/EBPδ shares a homologous DNA-binding sequence with other C/EBP-proteins, leading to the presumption that other C/EBP-family members might act redundantly and compensate for the loss of C/EBPδ. This implies that patient stratification could be improved when expression levels of multiple C/EBP-family members are considered simultaneously. In this study, we assessed whether the quantification of C/EBPβ or C/EBPγ in addition to that of C/EBPδ might improve the prediction of patient survival and lymph node involvement using a cohort of 68 resectable PDAC patients. Using Kaplan–Meier analyses of patient groups with different C/EBP-expression levels, we found that both C/EBPβ and C/EBPγ can partially compensate for low C/EBPδ and improve patient survival. Further, we uncovered C/EBPβ as a novel predictor of a decreased likelihood of lymph node involvement in PDAC, and found that C/EBPβ and C/EBPδ can compensate for the lack of each other in order to reduce the risk of lymph node involvement. C/EBPγ, on the other hand, appears to promote lymph node involvement in the absence of C/EBPδ. Altogether, our results show that the redundancy of C/EBP-family members might have a profound influence on clinical prognoses and that the expression of both C/EPBβ and C/EBPγ should be taken into account when dichotomizing patients according to C/EBPδ expression.

CCAAT/enhancer-binding protein delta (C/EBPδ) is a member of the C/EBP family of transcription factors. According to the current paradigm, C/EBPδ potentiates cytokine production and modulates macrophage function thereby enhancing the inflammatory response. Remarkably, however, C/EBPδ deficiency does not consistently lead to a reduction in Lipopolysaccharide (LPS)-induced cytokine production by macrophages. Here, we address this apparent discrepancy and show that the effect of C/EBPδ on cytokine production and macrophage function depends on both the macrophage subtype and the LPS concentration used. Using CRISPR-Cas generated macrophages in which the transactivation domain of C/EBPδ was deleted from the endogenous locus (ΔTAD macrophages), we next show that the context-dependent role of C/EBPδ in macrophage biology relies on compensatory transcriptional activity in the absence of C/EBPδ. We extend these findings by revealing a large discrepancy between transcriptional programs in C/EBPδ knock-out and C/EBPδ transactivation dead (ΔTAD) macrophages implying that compensatory mechanisms do not specifically modify C/EBPδ-dependent inflammatory responses but affect overall macrophage biology. Overall, these data imply that knock-out approaches are not suited for identifying the genuine transcriptional program regulated by C/EBPδ, and we suggest that this phenomenon applies for transcription factor families in general.

Simple Summary The clinical efficacy of systemic chemotherapy is limited in pancreatic cancer (PDAC) due to toxicity-dependent dose-limitations often leading to premature cessation of therapy. Targeted delivery of chemotherapeutic drugs to cancer cells, without affecting healthy nontumor cells, will largely reduce collateral toxicity. Reductions in collateral toxicity will allow increased drug concentrations to be used, thereby increasing the efficacy of chemotherapy. In the current study, we designed and validated a PDAC-specific protease-dependent drug release system. More specifically, we generated capped mesoporous silica nanoparticles that only release their cargo after proteolytic removal of the cap by PDAC-expressed proteases. We demonstrated the feasibility of protease-mediated targeted drug delivery in PDAC through the release of paclitaxel, resulting in cytotoxicity in cultured PDAC cells. Abstract Pancreatic ductal adenocarcinoma (PDAC) has the worst survival rate of all cancers. This poor prognosis results from the lack of efficient systemic treatment regimens, demanding high-dose chemotherapy that causes severe side effects. To overcome dose-dependent toxicities, we explored the efficacy of targeted drug delivery using a protease-dependent drug-release system. To this end, we developed a PDAC-specific drug delivery system based on mesoporous silica nanoparticles (MSN) functionalized with an avidin–biotin gatekeeper system containing a protease linker that is specifically cleaved by tumor cells. Bioinformatic analysis identified ADAM9 as a PDAC-enriched protease, and PDAC cell-derived conditioned medium efficiently cleaved protease linkers containing ADAM9 substrates. Cleavage was PDAC specific as conditioned medium from leukocytes was unable to cleave the ADAM9 substrate. Protease linker-functionalized MSNs were efficiently capped with avidin, and cap removal was confirmed to occur in the presence of PDAC cell-derived ADAM9. Subsequent treatment of PDAC cells in vitro with paclitaxel-loaded MSNs indeed showed high cytotoxicity, whereas no cell death was observed in white blood cell-derived cell lines, confirming efficacy of the nanoparticle-mediated drug delivery system. Taken together, this research introduces a novel ADAM9-responsive, protease-dependent, drug delivery system for PDAC as a promising tool to reduce the cytotoxicity of systemic chemotherapy.

End‐stage renal disease, the final stage of all chronic kidney disorders, is associated with renal fibrosis and inevitably leads to renal failure and death. Transition of tubular epithelial cells (TECs) into mesenchymal fibroblasts constitutes a proposed mechanism underlying the progression of renal fibrosis and here we assessed whether protease‐activated receptor (PAR)‐1, which recently emerged as an inducer of epithelial‐to‐mesenchymal transition (EMT), aggravates renal fibrosis. We show that PAR‐1 activation on TECs reduces the expression of epithelial markers and simultaneously induces mesenchymal marker expression reminiscent of EMT. We next show that kidney damage was reduced in PAR‐1‐deficient mice during unilateral ureter obstruction (UUO) and that PAR‐1‐deficient mice develop a diminished fibrotic response. Importantly, however, we did hardly observe any signs of mesenchymal transition in both wild‐type and PAR‐1‐deficient mice suggesting that diminished fibrosis in PAR‐1‐deficient mice is not due to reduced EMT. Instead, the accumulation of macrophages and fibroblasts was significantly reduced in PAR‐1‐deficient animals which were accompanied by diminished production of MCP‐1 and TGF‐β. Overall, we thus show that PAR‐1 drives EMT of TECs in vitro and aggravates UUO‐induced renal fibrosis although this is likely due to PAR‐1‐dependent pro‐fibrotic cytokine production rather than EMT.

CCAAT/enhancer-binding protein delta (CEBPD) is associated with the regulation of apoptosis and cell proliferation and is a candidate tumor suppressor gene. Here, we investigated its role in hepatocellular carcinoma (HCC). We observe that CEBPD mRNA expression is significantly downregulated in HCC tumors as compared with adjacent tissues. Protein levels of CEBPD are also lower in tumors relative to adjacent tissues. Reduced expression of CEBPD in the tumor correlates with worse clinical outcome. In both Huh7 and HepG2 cells, shRNA-mediated CEBPD knockdown significantly reduces cell proliferation, single cell colony formation and arrests cells in the G0/G1 phase. Subcutaneous xenografting of Huh7 in nude mice show that CEBPD knockdown results in smaller tumors. Gene expression analysis shows that CEBPD modulates interleukin-1 signaling. We conclude that CEBPD expression uncouples cancer compartment expansion and clinical outcome in HCC, potentially by modulating interleukin-1 signaling. Thus, although our results support the notion that CEBPD acts as a tumor suppressor in HCC, its action does not involve impairing compartment expansion per se but more likely acts through improving anticancer immunity.

Vorapaxar-dependent protease-activated receptor (PAR)-1 inhibition diminishes diabetic nephropathy in experimental type 1 diabetes. As most patients with diabetic nephropathy suffer from type 2 diabetes, the aim of this study was to investigate whether PAR-1 inhibition also limits diabetic nephropathy in experimental type 2 diabetes. Consequently, leptin-deficient black and tan brachyuric (BTBRob/ob) mice were randomly assigned to vorapaxar (1.75 mg/kg; twice weekly via oral gavage) or vehicle treatment, whereas matched wild-type (WT) BTBR (BTBRWT) mice served as nondiabetic controls. Weight and (nonfasting) blood glucose levels were monitored for up to 18 wk, after which kidney function and histologic damage was evaluated postmortem. We show that blood glucose levels and body weight increased in diabetic BTBRob/ob mice compared with nondiabetic BTBRWT controls. Vorapaxar-dependent PAR-1 inhibition reduced but did not normalize blood glucose levels in BTBRob/ob mice, whereas it potentiated the increase in body weight. Vorapaxar did not, however, preserve kidney function, whereas it only minimally reduced histopathological signs of kidney injury. Overall, we thus show that PAR-1 inhibition reduces blood glucose levels during the progression of diabetic nephropathy in experimental type 2 diabetes but does not improve renal function. This is in contrast to the therapeutic potential of vorapaxar in type 1 diabetes-induced nephropathy, highlighting the importance of disease-dependent treatment modalities.-Waasdorp, M., Florquin, S., Duitman, J., Spek, C. A. Pharmacological PAR-1 inhibition reduces blood glucose levels but does not improve kidney function in experimental type 2 diabetic nephropathy.

Idiopathic pulmonary fibrosis (IPF) is a chronic lung disease of unknown etiology with minimal treatment options. Repetitive alveolar epithelial injury has been suggested as one of the causative mechanisms of this disease. Type 2 alveolar epithelial cells (AEC2) play a crucial role during fibrosis by functioning as stem cells able to repair epithelial damage. The DNA demethylase Tet methylcytosine dioxygenase 2 (TET2) regulates the stemness of multiple types of stem cells, but whether it also affects the stemness of AEC2 during fibrosis remains elusive. To study the role of TET2 in AEC2 during fibrosis, we first determined TET2 protein levels in the lungs of IPF patients and compared TET2 expression in AEC2 of IPF patients and controls using publicly available data sets. Subsequently, pulmonary fibrosis was induced by the intranasal administration of bleomycin to wild-type and AEC2-specific TET2 knockout mice to determine the role of TET2 in vivo. Fibrosis was assessed by hydroxyproline analysis and fibrotic gene expression. Additionally, macrophage recruitment and activation, and epithelial injury were analyzed. TET2 protein levels and gene expression were downregulated in IPF lungs and AEC2, respectively. Bleomycin inoculation induced a robust fibrotic response as indicated by increased hydroxyproline levels and increased expression of pro-fibrotic genes. Additionally, increased macrophage recruitment and both M1 and M2 activation were observed. None of these parameters were, however, affected by AEC2-specific TET2 deficiency. TET2 expression is reduced in IPF, but the absence of TET2 in AEC2 cells does not affect the development of bleomycin-induced pulmonary fibrosis., peer-reviewed

Pancreatic acinar cells have high plasticity and can transdifferentiate into ductal‐like cells. This acinar‐to‐ductal metaplasia (ADM) contributes to tissue maintenance but may also contribute to the premalignant transformation that can eventually progress to pancreatic ductal adenocarcinoma (PDAC). Macrophages are key players in ADM, and macrophage‐secreted matrix metalloproteinase (MMP)‐9 induces ADM through yet unknown mechanisms. As we previously identified MMP9 as a novel agonist of protease‐activated receptor 1 (PAR1), a receptor that is known to orchestrate the cross‐talk between macrophages and tumor cells in PDAC, we here assessed the contribution of PAR1 to pancreatic cell fates. We found that genetic deficiency for PAR1 increases acinar gene expression programs in the healthy pancreas and that PAR1 deficiency limits ductal transdifferentiation in experimental systems for ADM. Moreover, PAR1 silencing in PDAC cells increases acinar marker expression. Changes in PDAC cell lines were associated with a downregulation of known Myc‐target genes, and Myc inhibition mimics PAR1 deficiency in enhancing acinar programs in healthy organoids and PDAC cells. Overall, we identify the PAR1‐Myc axis as a driver of ductal cell fates in premalignant pancreas and PDAC. Moreover, we show that cellular plasticity is not unique to acinar cells and that ductal regeneration into acinar‐like cells is possible even in the context of oncogenic KRAS activation., Using healthy pancreatic tissue, premalignant transdifferentiation, and ductal carcinoma models, we demonstrated that protease‐activated receptor 1 (PAR1) drives ductal cell fate. PAR1 deficiency prevented ductal transdifferentiation in models for acinar‐to‐ductal metaplasia. We demonstrated that downstream of PAR1 activation, Myc activity drives ductal differentiation programs which goes in line with the known effector functions of Myc in pancreatic ductal adenocarcinoma.

Background Pancreatic ductal adenocarcinoma (PDAC) is a grim disease with high mortality rates. Increased macrophage influx in PDAC is a common hallmark and associated with poor prognosis. Macrophages have high cellular plasticity, which can differentiate into both anti- and pro-tumorigenic properties. Here, we investigated how naïve (M0) macrophages differ from other macrophages in their anti-tumorigenic activities. Methods In vitro BrdU proliferation and Annexin V cell death analyses were performed on PANC-1 and MIA PaCa-2 PDAC cell lines exposed to conditioned medium of different macrophage subsets. Macrophage secreted factors were measured by transcript analysis and ELISA. Therapeutic antibodies were used to functionally establish the impact of the identified cytokine on PDAC proliferation. Results Proliferation and cell death assays revealed that only M0 macrophages harbor anti-tumorigenic activities and that M1, M2, and TAMs do not. mRNA analysis and ELISA results suggested TNF-α as a potential candidate to mediate M0 macrophage induced cell death. To demonstrate the importance of TNF-α in M0 macrophage-induced cell death, PANC-1 and MIA PaCa-2 cell-lines were exposed to M0 macrophage conditioned medium in the presence of the TNF-α inhibitor Infliximab, which effectively diminished the anti-tumor activities of M0 macrophages. Conclusion Newly tumor-infiltrated naive M0 macrophages exert anti-tumorigenic activities via TNF-α secretion. Their subsequent differentiation into either M1, M2, or TAM subsets reduces TNF-α levels, thereby abolishing their cytotoxic activity on PDAC cells. These data suggest that reestablishing TNF-α secretion in differentiated macrophages might yield a therapeutic benefit.

CCAAT/enhancer-binding protein &delta, (C/EBP&delta, ) is a transcription factor involved in growth arrest and differentiation, which has consequently been suggested to harbor tumor suppressive activities. However, C/EBP&delta, over-expression correlates with poor prognosis in glioblastoma and promotes genomic instability in cervical cancer, hinting at an oncogenic role of C/EBP&delta, in these contexts. Here, we explore the role of C/EBP&delta, in pancreatic cancer. We determined C/EBP&delta, expression in biopsies from pancreatic cancer patients using public gene-expression datasets and in-house tissue microarrays. We found that C/EBP&delta, is highly expressed in healthy pancreatic ductal cells but lost in pancreatic ductal adenocarcinoma. Furthermore, loss of C/EBP&delta, correlated with increased lymph node involvement and shorter overall survival in pancreatic ductal adenocarcinoma patients. In accordance with this, in vitro experiments showed reduced clonogenic capacity and proliferation of pancreatic ductal adenocarcinoma cells following C/EBP&delta, re-expression, concurrent with decreased sphere formation capacity in soft agar assays. We thus report a previously unrecognized but important tumor suppressor role of C/EBP&delta, in pancreatic ductal adenocarcinoma. This is of particular interest since only few tumor suppressors have been identified in the context of pancreatic cancer. Moreover, our findings suggest that restoration of C/EBP&delta, activity could hold therapeutic value in pancreatic ductal adenocarcinoma, although the latter claim needs to be substantiated in future studies.

Purpose Targeting tumor-infiltrating macrophages limits progression and improves chemotherapeutic responses in pancreatic ductal adenocarcinoma (PDAC). Protease-activated receptor (PAR)1 drives monocyte/macrophage recruitment, and stromal ablation of PAR1 limits cancer growth and enhances gemcitabine sensitivity in experimental PDAC. However, the functional interplay between PAR1, macrophages and tumor cells remains unexplored. Here we address the PAR1-macrophage-tumor cell crosstalk and assess its contributions to tumor progression. Methods PAR1 expression and macrophage infiltration were correlated in primary PDAC biopsies using gene expression datasets and tissue microarrays. Medium transfer experiments were used to evaluate the functional consequences of macrophage-tumor cell crosstalk and to assess the contribution of PAR1 to the observed responses. PAR1 cleavage assays were used to identify a macrophage-secreted PAR1 agonist, and the effects of candidate proteases were assessed in medium transfer experiments with specific inhibitors and/or recombinant agonist. Results PAR1 expression correlates with macrophage infiltration in primary PDACs, and macrophages induce mesenchymal transition of PDAC cells through PAR1 activation. Protease profiling identified macrophage-secreted matrix metalloprotease 9 (MMP9) as the relevant PAR1 agonist in PDAC. PAR1 and/or MMP9 inhibition limited macrophage-driven mesenchymal transition. Likewise, preventing mesenchymal transition by silencing ZEB1 or by pharmacological inhibition of the MMP9/PAR1 axis significantly reduced the ability of tumor cells to survive the anti-tumor activities of macrophages. Conclusion Macrophages secrete MMP9, which acts upon PDAC cell PAR1 to induce mesenchymal transition. This macrophage-induced mesenchymal transition supports the tumor-promoting role of macrophage influx, explaining the dichotomous contributions of these immune cells to tumor growth.

Advocating the use of therapeutic treatment strategies in preclinical IPF research remains challenginghttps://bit.ly/39Ob77c

Additional file 1: Table S1. Primer sequences for qPCR analysis of mRNA expression.

Protease activated receptor-1 (PAR1) expression is associated with disease progression and overall survival in a variety of cancers. However, the importance of tumor cell PAR1 in pancreatic ductal adenocarcinomas (PDAC) remains unexplored. Utilizing orthotopic models with wild type and PAR1-targeted PDAC cells, we show that tumor cell PAR1 negatively affects PDAC growth, yet promotes metastasis. Mechanistically, we show that tumor cell-specific PAR1 expression correlates with mesenchymal signatures in PDAC and that PAR1 is linked to the maintenance of a partial mesenchymal cell state. Indeed, loss of PAR1 expression results in well-differentiated pancreatic tumors in vivo, with enhanced epithelial characteristics both in vitro and in vivo. Taken together, we have identified a novel growth inhibitory role of PAR1 in PDAC, which is linked to the induction, and maintenance of a mesenchymal-like phenotype. The recognition that PAR1 actively limits pancreatic cancer cell growth suggest that the contributions of PAR1 to tumor growth differ between cancers of epithelial origin and that its targeting should be applied with care.

// Maaike Waasdorp 1 , JanWillem Duitman 1, 2, 3 , Sandrine Florquin 4 and C. Arnold Spek 1 1 Center for Experimental and Molecular Medicine, Academic Medical Center, Amsterdam, The Netherlands 2 Inserm UMR1152, Physiopathologie et Epidemiologie des maladies respiratoires, Medical School Xavier Bichat, Paris, France 3 Departement Hospitalo-Universitaire FIRE (Fibrosis, Inflammation and Remodeling) and LabEx Inflamex, Universite Paris Diderot, Sorbonne Paris Cite, Paris, France 4 Department of Pathology, Academic Medical Center, Amsterdam, The Netherlands Correspondence to: Maaike Waasdorp, email: M.Waasdorp@amc.uva.nl Keywords: diabetic nephropathy; vorapaxar; protease-activated receptor-1; mesangial expansion; streptozotocin; Pathology Received: December 15, 2017 Accepted: March 22, 2018 Published: April 24, 2018 ABSTRACT Background: Twenty years after the onset of diabetes, up to 40% of patients develop diabetic nephropathy. Protease-activated receptor-1 (PAR-1) has recently been shown to aggravate the development of experimental diabetic nephropathy. PAR-1 deficient mice develop less albuminuria and glomerular lesions and PAR-1 stimulation induces proliferation and fibronectin production in mesangial cells in vitro . Vorapaxar is a clinically available PAR-1 inhibitor which is currently used for secondary prevention of ischemic events. Objectives: The aim of this study was to investigate in a preclinical setting whether vorapaxar treatment may be a novel strategy to reduce diabetes-induced kidney damage. Results: While control treated diabetic mice developed significant albuminuria, mesangial expansion and glomerular fibronectin deposition, diabetic mice on vorapaxar treatment did not show any signs of kidney damage despite having similar levels of hyperglycemia. Conclusions: These data show that PAR-1 inhibition by vorapaxar prevents the development of diabetic nephropathy in this preclinical animal model for type I diabetes and pinpoint PAR-1 as a novel therapeutic target to pursue in the setting of diabetic nephropathy. Materials and Methods: 22 C57Bl/6 mice were made diabetic using multiple low-dose streptozotocin injections (50 mg/kg) and 22 littermates served as non-diabetic controls. Four weeks after the induction of diabetes, 11 mice of each group were assigned to control or vorapaxar treatment. Mice were sacrificed after 20 weeks of treatment and kidney damage was evaluated.

Background Idiopathic pulmonary fibrosis is a devastating fibrotic diffuse parenchymal lung disorder that remains refractory to pharmacological therapies. Therefore, novel treatments are urgently required. CCAAT/enhancer binding protein delta (C/EBPδ) is a transcription factor that mediates critical cellular functions in pathophysiology and which was recently suggested to be a key regulatory component in IPF. The purpose of this study was to prove or refute the importance of C/EBPδ in pulmonary fibrosis. Methods Pulmonary fibrosis was induced by intranasal instillation of bleomycin into wild-type and C/EBPδ deficient mice. At different time intervals after bleomycin instillation, fibrosis was assessed by hydroxyproline analysis, histochemistry and q-PCR for fibrotic marker expression. Results C/EBPδ deficient mice developed pulmonary fibrosis to a similar degree as wildtype mice as evident from similar Ashcroft scores, hydroxyproline levels and expression levels of collagen, fibronectin and α-smooth muscle actin at both 14 and 21 days after bleomycin instillation. The resolution of fibrosis, assessed at 48 days after bleomycin instillation, was also similar in wildtype and C/EBPδ deficient mice. In line with the lack of effect of C/EBPδ on fibrosis progression/resolution, macrophage recruitment and/or differentiation were also not different in wildtype or C/EBPδ deficient mice. Conclusions Overall, C/EBPδ does not seem to affect bleomycin-induced experimental pulmonary fibrosis and we challenge the importance of C/EBPδ in pulmonary fibrosis. Relevance for patients This study shows that the transcription factor C/EBPδ does not play a major role in the development of pulmonary fibrosis. Pharmacological targeting of C/EBPδ is therefore not likely to have a beneficial effect for patients suffering from pulmonary fibrosis.

Hedgehog proteins are pivotal morphogens acting through a canonical pathway involving first activation of ligand binding to Patched followed by alleviation of Smoothened receptor inhibition, leading to activation of Gli transcription factors. Noncanonical Hedgehog signaling remains poorly characterized but is thought to be mainly dependent on Smoothened. However, Smoothened inhibitors have yielded only partial success in combating Hedgehog signal transduction-dependent cancer, suggesting that noncanonical Smoothened-independent pathways also are clinically relevant. Moreover, several Smoothened-dependent effects (e.g. neurite projection) do not require transcriptional activation, further suggesting biological importance of noncanonical Smoothened-dependent pathways. We comprehensively characterized the cellular kinome in Hedgehog-challenged murine WT and Smoothened(-/-) fibroblasts as well as Smoothened agonist-stimulated cells. A peptide assay-based kinome analysis (in which cell lysates are used to phosphorylate specific kinase substrates), along with endocytosis, Lucifer Yellow-based, and immunoblotting assays, identified an elaborate signaling network of both Smoothened-dependent and -independent pathways that mediates actin reorganization through Src-like kinases, activates various proinflammatory signaling cascades, and concomitantly stimulates Wnt and Notch signaling while suppressing bone morphogenetic protein (BMP) signaling. The contribution of noncanonical Smoothened-independent signaling to the overall effects of Hedgehog on cellular physiology appears to be much larger than previously envisioned and may explain the transcriptionally independent effects of Hedgehog signaling on cytoskeleton. The observation that Patched-dependent, Smoothened-independent, noncanonical Hedgehog signaling increases Wnt/Notch signaling provides a possible explanation for the failure of Smoothened antagonists in combating Hedgehog-dependent but Smoothened inhibitor-resistant cancer. Our findings suggest that inhibiting Hedgehog-Patched interaction could result in more effective therapies as compared with conventional Smoothened-directed therapies.

Pancreatic cancer is a dismal disorder that is histologically characterized by a dense fibrotic stroma around the tumor cells. As the extracellular matrix comprises the bulk of the stroma, matrix degrading proteases may play an important role in pancreatic cancer. It has been suggested that matrix metalloproteases are key drivers of both tumor growth and metastasis during pancreatic cancer progression. Based upon this notion, changes in matrix metalloprotease expression levels are often considered surrogate markers for pancreatic cancer progression and/or treatment response. Indeed, reduced matrix metalloprotease levels upon treatment (either pharmacological or due to genetic ablation) are considered as proof of the anti-tumorigenic potential of the mediator under study. In the current review, we aim to establish whether matrix metalloproteases indeed drive pancreatic cancer progression and whether decreased matrix metalloprotease levels in experimental settings are therefore indicative of treatment response. After a systematic review of the studies focusing on matrix metalloproteases in pancreatic cancer, we conclude that the available literature is not as convincing as expected and that, although individual matrix metalloproteases may contribute to pancreatic cancer growth and metastasis, this does not support the generalized notion that matrix metalloproteases drive pancreatic ductal adenocarcinoma progression.

Diabetic nephropathy is a major microvascular complication of diabetes mellitus, and the leading cause of end stage renal disease worldwide. The pathogenesis of diabetic nephropathy is complex, making the development of novel treatments that stop or reverse the progression of microalbuminuria into end stage renal disease a challenge. Protease activated receptor (PAR)-2 has recently been shown to aggravate disease progression in diabetic nephropathy based upon which it was suggested that PAR-2 would be a potential target for the treatment of diabetic nephropathy. To fully appreciate the translational potential of PAR-2 in diabetic nephropathy, we evaluated the effect of PAR-2 deficiency on the development of diabetic nephropathy in a streptozotocin-induced diabetes model characteristic of type 1 diabetes. Although diabetic PAR-2 deficient mice showed reduced albuminuria compared to diabetic wild type mice, an increase in mesangial expansion was evident in the PAR-2 deficient mice. No differences were observed in blood glucose levels, podocyte numbers or in glomerular vascular density. These results show that PAR-2 plays a dual role in the development of streptozotocin-induced diabetic nephropathy and may thus not be the eagerly awaited novel target to combat diabetic nephropathy. Targeting PAR-2 should consequently only be pursued with great care in a clinical setting.

Background Idiopathic pulmonary fibrosis is a devastating fibrotic diffuse parenchymal lung disorder that remains refractory to pharmacological therapies. Therefore, novel treatments are urgently required. CCAAT/enhancer binding protein delta (C/EBPδ) is a transcription factor that mediates critical cellular functions in pathophysiology and which was recently suggested to be a key regulatory component in IPF. The purpose of this study was to prove or refute the importance of C/EBPδ in pulmonary fibrosis. Methods Pulmonary fibrosis was induced by intranasal instillation of bleomycin into wild-type and C/EBPδ deficient mice. At different time intervals after bleomycin instillation, fibrosis was assessed by hydroxyproline analysis, histochemistry and q-PCR for fibrotic marker expression. Results C/EBPδ deficient mice developed pulmonary fibrosis to a similar degree as wildtype mice as evident from similar Ashcroft scores, hydroxyproline levels and expression levels of collagen, fibronectin and α-smooth muscle actin at both 14 and 21 days after bleomycin instillation. The resolution of fibrosis, assessed at 48 days after bleomycin instillation, was also similar in wildtype and C/EBPδ deficient mice. In line with the lack of effect of C/EBPδ on fibrosis progression/resolution, macrophage recruitment and/or differentiation were also not different in wildtype or C/EBPδ deficient mice. Conclusions Overall, C/EBPδ does not seem to affect bleomycin-induced experimental pulmonary fibrosis and we challenge the importance of C/EBPδ in pulmonary fibrosis. Relevance for patients This study shows that the transcription factor C/EBPδ does not play a major role in the development of pulmonary fibrosis. Pharmacological targeting of C/EBPδ is therefore not likely to have a beneficial effect for patients suffering from pulmonary fibrosis.

Idiopathic pulmonary fibrosis is the most devastating diffuse fibrosing lung disease of unknown aetiology. Compelling evidence suggests that both protease-activated receptor (PAR)-1 and PAR-2 participate in the development of pulmonary fibrosis. Previous studies have shown that bleomycin-induced lung fibrosis is diminished in both PAR-1 and PAR-2 deficient mice. We thus have been suggested that combined inactivation of PAR-1 and PAR-2 would be more effective in blocking pulmonary fibrosis. Human and murine fibroblasts were stimulated with PAR-1 and PAR-2 agonists in the absence or presence of specific PAR-1 or PAR-2 antagonists after which fibrotic markers like collagen and smooth muscle actin were analysed by Western blot. Pulmonary fibrosis was induced by intranasal instillation of bleomycin into wild-type and PAR-2 deficient mice with or without a specific PAR-1 antagonist (P1pal-12). Fibrosis was assessed by hydroxyproline quantification and (immuno)histochemical analysis. We show that specific PAR-1 and/or PAR-2 activating proteases induce fibroblast migration, differentiation and extracellular matrix production. Interestingly, however, combined activation of PAR-1 and PAR-2 did not show any additive effects on these pro-fibrotic responses. Strikingly, PAR-2 deficiency as well as pharmacological PAR-1 inhibition reduced bleomycin-induced pulmonary fibrosis to a similar extent. PAR-1 inhibition in PAR-2 deficient mice did not further diminish bleomycin-induced pulmonary fibrosis. Finally, we show that the PAR-1-dependent pro-fibrotic responses are inhibited by the PAR-2 specific antagonist. Targeting PAR-1 and PAR-2 simultaneously is not superior to targeting either receptor alone in bleomycin-induced pulmonary fibrosis. We postulate that the pro-fibrotic effects of PAR-1 require the presence of PAR-2.

Idiopathic pulmonary fibrosis (IPF) is a destructive disease in part resulting from premature or mature cellular aging. Protease-activated receptor-1 (PAR-1) recently emerged as a critical component in the context of fibrotic lung diseases. Therefore, we aimed to study the role of macrophages in PAR-1-mediated idiopathic pulmonary fibrosis. The number of macrophages were significantly reduced in lungs of PAR-1 antagonist (P1pal-12) treated animals upon bleomycin instillation. In line with these data, PAR-1 stimulation increased monocyte / macrophage recruitment in response to epithelium injury in in vitro trans-well assays. Moreover, macrophages induced fibroblasts migration, differentiation and secretion of collagen, which were inhibited in the presence of TGF-beta receptor inhibitors. Interestingly, these profibrotic effects were partially inhibited by treatment with the PAR-1 inhibitor P1pal-12. Using shRNA mediated PAR-1 knock down in fibroblasts, we demonstrate that fibroblast PAR-1 contributes to TGF-beta activation and production. Finally, we show that the macrophage-dependent induction of PAR-1 driven TGF-beta activation was mediated by FXa. Our data identify novel mechanisms by which PAR-1 stimulation on different cell types can contribute to IPF and identify macrophages as key players in PAR-1 dependent development of this devastating disease. IPF may result from cellular senescence mediated by macrophages in the lung.

Idiopathic pulmonary fibrosis (IPF) is a devastating lung disease, characterized by accumulation of extracellular matrix (ECM) proteins. Matrix metalloprotease 1 (MMP-1) is an endopeptidase capable of degrading ECM proteins like fibrillar collagens, although pleiotropic actions have been described. We investigated MMP-1 expression in a cohort of IPF patients and determined the effect of genetic ablation of MMP-1 in the development of lung fibrosis in mice. We confirmed that MMP-1 levels are significantly increased in IPF lungs as compared to control lungs at both the mRNA and protein level. Interestingly, MMP-1 protein levels negatively correlated with Forced Vital Capacity (Spearman r = -0.45, p = 0.009). Next, wildtype and MMP-1a null (MMP-1a-/-) mice were subjected to bleomycin-induced lung fibrosis. MMP-1a-/- mice were protected from bleomycin-induced lung fibrosis as assessed by lung hydroxyproline content at d14, compared to wildtype mice (196±16 μg vs 146±11 μg/lung). Inflammatory cell influx in the BALF was significantly lower in MMP-1a-/- mice at d14 compared to wildtype mice (1.18x10^6±2.7x10^5 vs 0.54x10^6±0.67x10^5 cells/mL), whereas there was no difference at d7. We conclude that MMP-1 plays a detrimental role in the pathogenesis of lung fibrosis in mice and human. These data support our efforts in targeting MMP-1 as a new option against lung fibrosis progression.

Streptococcus pneumoniae is the most common causative pathogen in community-acquired pneumonia. Protease-activated receptor 2 (PAR2) is expressed by different cell types in the lungs and can mediate inflammatory responses. We sought to determine the role of PAR2 during pneumococcal pneumonia. Pneumococcal pneumonia or sepsis was induced in wild-type and PAR2 knock-out (Par2−/−) mice by infection with viable S. pneumoniae. Par2−/− mice demonstrated improved host defense, a largely preserved lung barrier integrity, and reduced mortality during pneumococcal pneumonia. PAR2 deficiency did not influence bacterial growth after intravenous infection. Inhibition of the endogenous PAR2 activating proteases tissue factor/factor VIIa or tryptase did not impact on bacterial burdens during pneumonia. In a PAR2 reporter cell line it was demonstrated that S. pneumoniae-derived proteases are able to cleave PAR2. These results show that S. pneumoniae is able to cleave and exploit PAR2 to disseminate systemically from the airways.

Background Protease-activated receptor-1 (PAR-1) potentiates diabetic nephropathy (DN) as evident from reduced kidney injury in diabetic PAR-1 deficient mice. Although thrombin is the prototypical PAR-1 agonist, anticoagulant treatment does not limit DN in experimental animal models suggesting that thrombin is not the endogenous PAR-1 agonist driving DN. Objectives To identify the endogenous PAR-1 agonist potentiating diabetes-induced nephropathy. Methods Unbiased protease expression profiling in glomeruli from human kidneys with DN was performed using publically available microarray data. The identified prime candidate PAR-1 agonist was subsequently analysed for PAR-1-dependent induction of fibrosis in vitro. Results Of the 553 proteases expressed in the human genome, 247 qualified as potential PAR-1 agonists of which 71 were significantly expressed above background in diabetic glomeruli. The recently identified PAR-1 agonist plasmin(ogen), together with its physiological activator tissue plasminogen activator, were among the highest expressed proteases. Plasmin did however not induce mesangial proliferation and/or fibronectin deposition in vitro. In a PAR-1 independent manner, plasmin even reduced fibronectin deposition. Conclusion Expression profiling identified plasmin as potential endogenous PAR-1 agonist driving DN. Instead of inducing fibronectin expression, plasmin however reduced mesangial fibronectin deposition in vitro. Therefore we conclude that plasmin may not be the endogenous PAR-1 agonist potentiating DN., Highlights • Plasmin is highly expressed in kidneys of diabetic nephropathy patients. • Plasmin limits fibronectin deposition by mesangial cells. • Plasmin-dependent PAR-1 activation does not drive diabetic nephropathy.

Background Protease activated receptor (PAR)-1 expression is increased in a variety of tumor cells. In preclinical models, tumor cell PAR-1 appeared to be involved in the regulation of lung tumor growth and metastasis; however the role of PAR-1 in the lung tumor microenvironment, which is emerging as a key compartment in driving cancer progression, remained to be explored. Methods In the present study, PAR-1 gene expression was determined in lung tissue from patients with non-small-cell lung cancer (NSCLC) using a combination of publicly available RNA microarray datasets and in house-made tissue microarrays including tumor biopsies of 94 patients with NSCLC (40 cases of adenocarcinoma, 42 cases of squamous cell carcinoma and 12 cases of other type of NSCLC at different stages). Results PAR-1 gene expression strongly correlated with tumor stromal markers (i.e. macrophage, endothelial cells and (myo) fibroblast markers) but not with epithelial cell markers. Immunohistochemical analysis confirmed the presence of PAR-1 in the tumor stroma and showed that PAR-1 expression was significantly upregulated in malignant tissue compared with normal lung tissue. The overexpression of PAR-1 in tumor stroma of NSCLC appeared to be independent from tumor type, tumor stage, histopathological differentiation status, disease progression and patient survival. Conclusion Overall, our data provide evidence that PAR-1 in NSCLC is mainly expressed on cells that constitute the pulmonary tumor microenvironment, including vascular endothelial cells, macrophages and stromal fibroblasts. Electronic supplementary material The online version of this article (doi:10.1186/s12885-017-3081-3) contains supplementary material, which is available to authorized users.

Protease-activated receptor-2 (PAR-2) is a G protein-coupled receptor that functions as a cell-surface sensor for coagulation factors and other proteases associated with the tumour microenvironment. Pancreatic cancer cells express high levels of PAR-2 and activation of PAR-2 may induce their proliferation and migration. Interestingly, however, PAR-2 expression is increased in stroma-rich pancreatic cancer regions, suggesting a potential role of PAR-2 in the tumour microenvironment. Here, we assessed the importance of PAR-2 in the stromal compartment by utilizing an orthotopic pancreatic cancer model, in which tumour cells are PAR-2-positive, whereas stromal cells are PAR-2-negative. We assessed tumour weight and volume and analysed proliferation and (lymph)angiogenesis both in vivo and in vitro. We show that genetic ablation of PAR-2 from the stromal compartment inhibits primary tumour growth, which is accompanied by reduced vascularization in primary tumours and reduced in tube formation of vascular endothelial cells in vitro. In contrast to smaller primary tumours, the number of lymph node metastases was increased in PAR-2-deficient animals, which was accompanied by an increased number of lymphatic vessels. In vitro tube-formation assays show that PAR-2 does not inhibit the intrinsic tube-forming capacity of lymphatic endothelial cells, but that PAR-2 actually inhibits cancer cell-induced tube formation. Overall, stromal PAR-2 thus plays a dual role in pancreatic cancer development by potentiating primary tumour growth but limiting lymphangiogenesis and subsequent lymph node metastasis. Our data identify a novel role of PAR-2 in the tumour microenvironment and pinpoint PAR-2 as a negative regulator of lymphangiogenesis.

Protease activated receptor (PAR)-1 expression in tumor cells is associated with disease progression and overall survival in a variety of cancers of epithelial origin; however, the importance of PAR-1 in the tumor microenvironment remains unexplored. Utilizing an orthotopic pancreatic cancer model in which tumor cells are PAR-1 positive whereas stromal cells are PAR-1 negative, we show that PAR-1 expression in the microenvironment drives progression and induces chemoresistance of pancreatic cancer. PAR-1 enhances monocyte recruitment into the tumor microenvironment by regulating monocyte migration and fibroblast dependent chemokine production thereby inducing chemoresistance. Overall, our data identify a novel role of PAR-1 in the pancreatic tumor microenvironment and suggest that PAR-1 may be an attractive target to reduce drug resistance in pancreatic cancer.

Endogenously administered activated protein C ameliorates diabetic nephropathy (DN) in a protease-activated receptor-1 (PAR-1)-dependent manner, suggesting that PAR-1 activation limits the progression of DN. Activation of PAR-1 in fibroblast-like cells, however, induces proliferation and extracellular matrix production, thereby driving fibrotic disease. Considering the key role of mesangial proliferation and extracellular matrix production during DN, PAR-1 may in fact potentiate diabetes-induced kidney injury. To determine the net effect of PAR-1 in DN, streptozotocin-induced DN was studied in wild type and PAR-1 deficient mice. Subsequent mechanistic insight was obtained by assessing profibrotic responses of mesangial and tubular epithelial cells in vitro, following PAR-1 stimulation and inhibition. Despite having similar glucose levels, PAR-1 deficient mice developed less kidney damage after induction of diabetes, as evidenced by diminished proteinuria, plasma cystatin C levels, expansion of the mesangial area, and tubular atrophy. In vitro, PAR-1 signaling in mesangial cells led to increased proliferation and expression of matrix proteins fibronectin and collagen IV. Conversely, a reduction in both proliferation and fibronectin deposition was observed in diabetic PAR-1 deficient mice. Overall, we show that PAR-1 plays an important role in the development of DN and PAR-1 might therefore be an attractive therapeutic target to pursue in DN.

Background The most frequent pathogen that causes bacterial meningitis is the Gram-positive bacterium Streptococcus (S.) pneumoniae. CCAAT/enhancer binding protein δ is a transcription factor that has recently been hypothesized to play a detrimental role in outcome of meningitis caused by S. pneumoniae. Here, we studied the role of C/EBPδ prior to the development of pneumococcal meningitis. Methods Wild-type and C/EBPδ-deficient mice (C/EBPδ−/−) were intraveneously infected with S. pneumoniae and sacrificed after 24 or 48 h. cebpδ expression, bacterial loads, inflammatory response and pathology in the brain were assessed. Results S. pneumoniae induces cebpδ expression in the brain during blood-borne brain infection. In comparison to wild-type mice, C/EBPδ−/− animals showed decreased bacterial loads in blood and brain 48 h after inoculation. In the blood compartment, the host inflammatory response was significantly lower upon infection in C/EBPδ−/− mice as compared to wild-type mice. Conclusion C/EBPδ facilitates bacterial dissemination to the brain and enhances the immune response in the blood compartment. Our study suggests that C/EBPδ plays a detrimental role during the initial development of blood-borne brain infection.

Prolactin is best known as the polypeptide anterior pituitary hormone, which regulates the development of the mammary gland. However, it became clear over the last decade that prolactin contributes to a broad range of pathologies, including breast cancer. Prolactin is also involved in angiogenesis via the release of pro-angiogenic factors by leukocytes and epithelial cells. However, whether prolactin also influences endothelial cells, and whether there are functional consequences of prolactin-induced signalling in the perspective of angiogenesis, remains so far elusive. In the present study, we show that prolactin induces phosphorylation of ERK1/2 and STAT5 and induces tube formation of endothelial cells on Matrigel. These effects are blocked by a specific prolactin receptor antagonist, del1-9-G129R-hPRL. Moreover, in an in vivo model of the chorioallantoic membrane of the chicken embryo, prolactin enhances vessel density and the tortuosity of the vasculature and pillar formation, which are hallmarks of intussusceptive angiogenesis. Interestingly, while prolactin has only little effect on endothelial cell proliferation, it markedly stimulates endothelial cell migration. Again, migration was reverted by del1-9-G129R-hPRL, indicating a direct effect of prolactin on its receptor. Immunohistochemistry and spectral imaging revealed that the prolactin receptor is present in the microvasculature of human breast carcinoma tissue. Altogether, these results suggest that prolactin may directly stimulate angiogenesis, which could be one of the mechanisms by which prolactin contributes to breast cancer progression, thereby providing a potential tool for intervention.

CCAAT/enhancer-binding protein δ (C/EBPδ) recently emerged as an essential player in the inflammatory response to bacterial infections. C/EBPδ levels increase rapidly after a proinflammatory stimulus, and increasing C/EBPδ levels seem to be indispensable for amplification of the inflammatory response. Here we aimed to elucidate the role of C/EBPδ in host defense in community-acquired pneumococcal pneumonia. We show that C/EBPδ −/− mice are relatively resistant to pneumococcal pneumonia, as indicated by delayed and reduced mortality, diminished outgrowth of pneumococci in lungs, and reduced dissemination of the infection. Moreover, expression of platelet-activating factor receptor (PAFR), which is known to potentiate bacterial translocation of Gram-positive bacteria, was significantly reduced during infection in C/EBPδ −/− mice compared with WT controls. Importantly, cell stimulation experiments revealed that C/EBPδ potentiates PAFR expression induced by lipoteichoic acid and pneumococci. Thus, C/EBPδ exaggerates bacterial dissemination during Streptococcus pneumoniae -induced pulmonary infection, suggesting an important role for PAFR-dependent bacterial translocation.

Chronic inflammatory disorders constitute a heterogeneous group of complex and multifactorial diseases, which are often associated with increased cardiovascular morbidity and mortality, independent of the established cardiovascular risk factors. In keeping with this observation, hypercoagulability is frequently observed in patients suffering from atherosclerosis, chronic obstructive pulmonary disease and rheumatoid arthritis although the physiological significance of activated coagulation remained elusive. However, the identification of protease activated receptors (PAR) seem to provide a link between coagulation activation and disease progression as their activation by coagulation factors triggers a broad range of signaling pathways relevant for chronic inflammatory disorders. In experimental animal models, anticoagulation and/or genetic ablation of PAR signaling affords protection against the perpetuation of atherosclerosis, chronic obstructive pulmonary disease and rheumatoid arthritis. It is thus tempting to speculate that targeting the coagulation-PAR axis might have clinical relevance in the setting of chronic inflammatory disorders. In the current review, we discuss the current knowledge on coagulation activation in inflammatory disorders, we discuss the relationship between atherosclerosis, chronic obstructive pulmonary disease and rheumatoid arthritis and we review the current knowledge on PAR signaling in these disorders.