The Diagnostic Value of ACSL1, ACSL4, and ACSL5 and the Clinical Potential of an ACSL Inhibitor in Non-Small-Cell Lung Cancer.

Abnormal expression of ACSL members 1, 3, 4, 5, and 6 is frequently seen in human cancer; however, their clinical relevance is unclear. In this study, we analyzed the expression of ACSLs and investigated the effects of the ACSL inhibitor Triacsin C (TC) in lung cancer. We found that, compared to normal human bronchial epithelial (NHBE) cells, ACSL1, ACSL4, and ACSL6 were highly expressed, while ACSL3 and ACSL5 were lost in the majority of lung cancer cell lines. ACSL activity was associated with the expression levels of the ACSLs. In primary lung tumors, a higher expression of ACSL1, ACSL4, and ACSL5 was significantly correlated with adenocarcinoma (ADC). Moreover, ACSL5 was significantly reversely related to the proliferation marker Ki67 in low-grade tumors, while ACSL3 was positively associated with Ki67 in high-grade tumors. Combination therapy with TC and Gemcitabine enhanced the growth-inhibitory effect in EGFR wild-type cells, while TC combined with EGFR-TKIs sensitized the EGFR-mutant cells to EGFR-TKI treatment. Taken together, the data suggest that ACSL1 may be a biomarker for lung ADC, and ACSL1, ACSL4, and ACSL5 may be involved in lung cancer differentiation, and TC, in combination with chemotherapy or EGFR-TKIs, may help patients overcome drug resistance.

Targeted Disruption of the MORG1 Gene in Mice Causes Embryonic Resorption in Early Phase of Development.

The mitogen-activated protein kinase organizer 1 (MORG1) is a scaffold molecule for the ERK signaling pathway, but also binds to prolyl-hydroxylase 3 and modulates HIFα expression. To obtain further insight into the role of MORG1, knockout-mice were generated by homologous recombination. While Morg1+/- mice developed normally without any apparent phenotype, there were no live-born Morg1-/- knockout offspring, indicating embryonic lethality. The intrauterine death of Morg1-/- embryos is caused by a severe failure to develop brain and other neuronal structures such as the spinal cord and a failure of chorioallantoic fusion. On E8.5, Morg1-/- embryos showed severe underdevelopment and proliferative arrest as indicated by absence of Ki67 expression, impaired placental vascularization and altered phenotype of trophoblast giant cells. On E9.5, the malformed Morg1-/- embryos showed defective turning into the final fetal position and widespread apoptosis in many structures. In the subsequent days, apoptosis and decomposition of embryonic tissue progressed, accompanied by a massive infiltration of inflammatory cells. Developmental aberrancies were accompanied by altered expression of HIF-1/2α and VEGF-A and caspase-3 activation in embryos and extraembryonic tissues. In conclusion, the results suggest a multifactorial process that causes embryonic death in homozygous Morg1 mutant mice, described here, to the best of our knowledge, for the first time.

Targeted Interleukin-9 delivery in pulmonary hypertension: Comparison of immunocytokine formats and effector cell study.

AIMS: Pulmonary hypertension (PH) is accompanied by pulmonary vascular remodelling. By targeted delivery of Interleukin-9 (IL9) via the immunocytokine F8IL9, beneficial effects could be demonstrated in a mouse model of PH. This study aimed to compare two immunocytokine formats (single-chain Fv and full IgG) and to identify potential target cells of IL9. METHODS: The Monocrotaline mouse model of PH (PH, n = 12) was chosen to evaluate the treatment effects of F8IL9F8 (n = 12) and F8IgGIL9 (n = 6) compared with sham-induced animals (control, n = 10), the dual endothelin receptor antagonist Macitentan (MAC, n = 12) or IL9-based immunocytokines with irrelevant antigen specificity (KSFIL9KSF, n = 12; KSFIgGIL9 n = 6). Besides comparative validation of treatment effects, the study was focused on the detection and quantification of mast cells (MCs) and regulatory T cells (Tregs). RESULTS: There was a significantly elevated systolic right ventricular pressure (104 ± 36 vs. 45 ± 17 mmHg) and an impairment of right ventricular echocardiographic parameters (RVbasal: 2.52 ± 0.25 vs. 1.94 ± 0.13 mm) in untreated PH compared with controls (p < 0.05). Only the groups treated with F8IL9, irrespective of the format, showed consistent beneficial effects (p < 0.05). Moreover, F8IL9F8 but not F8IgGIL9 treatment significantly reduced lung tissue damage compared with untreated PH mice (p < 0.05). There was a significant increase in Tregs in F8IL9-treated compared with control animals, the untreated PH and the MAC group (p < 0.05). CONCLUSIONS: Beneficial treatment effects of targeted IL9 delivery in a preclinical model of PH could be convincingly validated. IL9-mediated recruitment of Tregs into lung tissue might play a crucial role in the induction of anti-inflammatory and anti-proliferative mechanisms potentially contributing to a novel disease-modifying concept.

Invasion-Associated Reorganization of Laminin 332 in Oral Squamous Cell Carcinomas: The Role of the Laminin γ2 Chain in Tumor Biology, Diagnosis, and Therapy.

Invasion of the connective tissue by carcinoma cells is accompanied by disintegration and reorganization of the hemidesmosomes, which connect the basement membrane to the basal epithelial cells. In terms of mediating the basement membrane, i.e., basal cell interactions, the heterotrimeric laminin 332 is the most important bridging molecule. Due to this distinct function, laminin 332, especially its gamma 2 chain, came into the focus of cancer research. Specific de novo synthesis and deposition patterns of laminin 332 are evident upon development and progression of oral squamous cell carcinomas (OSCCs). Loss from the basement membrane, cytoplasmic accumulation, and extracellular deposition are associated with crucial processes such as stromal activation and immune response, epithelial to mesenchymal transition, and tumor cell budding. In networks with components of the tumor microenvironment, altered expression of laminin 332 chains, proteolytic processing, and interaction with integrin receptors seem to promote cancer cell migration. Indeed, reorganization patterns are shown to have a high diagnostic and prognostic value. Here, we summarize the current knowledge on laminin 332 reorganization in OSCCs with special focus on its gamma 2 chain and provide, based on the current literature, evidence on its promising role as a grading and monitoring parameter and as a potential therapeutic target.

The natural compound atraric acid suppresses androgen-regulated neo-angiogenesis of castration-resistant prostate cancer through angiopoietin 2.

Castration-resistant prostate cancer (CRPC) is an aggressive lethal form of prostate cancer (PCa). Atraric acid (AA) not only inhibits the wild-type androgen receptor (AR) but also those AR mutants that confer therapy resistance to other clinically used AR antagonists, indicating a different mode of AR antagonism. AA induces cellular senescence and inhibits CRPC tumour growth in in vivo xenograft mouse model associated with reduced neo-angiogenesis suggesting the repression of intratumoural neo-angiogenesis by AA. In line with this, the secretome of CRPC cells mediates neo-angiogenesis in an androgen-dependent manner, which is counteracted by AA. This was confirmed by two in vitro models using primary human endothelial cells. Transcriptome sequencing revealed upregulated angiogenic pathways by androgen, being however VEGF-independent, and pointing to the pro-angiogenic factor angiopoietin 2 (ANGPT2) as a key driver of neo-angiogenesis induced by androgens and repressed by AA. In agreement with this, AA treatment of native patient-derived PCa tumour samples ex vivo inhibits ANGPT2 expression. Mechanistically, in addition to AA, immune-depletion of ANGPT2 from secretome or blocking ANGPT2-receptors inhibits androgen-induced angiogenesis. Taken together, we reveal a VEGF-independent ANGPT2-mediated angiogenic pathway that is inhibited by AA leading to repression of androgen-regulated neo-angiogenesis.

Tumor vascular remodeling by thrombospondin-1 enhances drug delivery and antineoplastic activity.

The disorganized and inefficient tumor vasculature is a major obstacle to the delivery and efficacy of antineoplastic treatments. Antiangiogenic agents can normalize the tumor vessels, improving vessel function and boosting the distribution and activity of chemotherapy. The type III repeats (T3R) domain of thrombospondin-1 contains different potential antiangiogenic sequences. We therefore hypothesized that it might affect the tumor vasculature. Ectopic expression of the T3R domain by the tumor cells or by the host, or administration of recombinant T3R, delayed the in vivo growth of experimental tumors. Tumors presented marked reorganization of the vasculature, with abundant but smaller vessels, associated with substantially less necrosis. Mechanistically, the use of truncated forms of the domain, containing different active sequences, pointed to the FGF2/FGFR/ERK axis as a target for T3R activity. Along with reduced necrosis, the expression of T3R promoted tumor distribution of chemotherapy (paclitaxel), with a higher drug concentration and more homogeneous distribution, as assessed by HPLC and MALDI imaging mass spectrometry. T3R-expressing tumors were more responsive to paclitaxel and cisplatin. This study shows that together with its known role as a canonical inhibitor of angiogenesis, thrombospondin-1 can also remodel tumor blood vessels, affecting the morphological and functional properties of the tumor vasculature. The ability of T3R to reduce tumor growth and improve the response to chemotherapy opens new perspectives for therapeutic strategies based on T3R to be used in combination therapies.

Serum Liberation of Fetal Fibronectin Variants in Patients with Pulmonary Hypertension: ED-A+ Fn as Promising Novel Biomarker of Pulmonary Vascular and Right Ventricular Myocardial Remodeling.

BACKGROUND AND AIMS: Pulmonary Hypertension (PH) represents an aetiologically and clinically heterogeneous disorder accompanied by a severely impaired prognosis. Key steps of PH pathogenesis are vascular and right ventricular myocardial remodelling entailing the re-occurrence of fetal variants of the cell adhesion modulating protein fibronectin (Fn) being virtually absent in healthy adult tissues. These variants are liberated into circulation and are therefore qualified as excellent novel serum biomarkers. Moreover, these molecules might serve as promising therapeutic targets. The current study was aimed at quantifying the serum levels of two functionally important fetal Fn variants (ED-A+ and ED-B+ Fn) in patients suffering from PH due to different aetiologies compared to healthy controls. METHODS: Serum levels of ED-A+ and ED-B+ Fn were quantified using novel ELISA protocols established and validated in our group in 80 PH patients and 40 controls. Results were analysed with respect to clinical, laboratory, echocardiographic and functional parameters. RESULTS: Serum levels of ED-A+ Fn (p = 0.001) but not ED-B+ Fn (p = 0.722) were significantly increased in PH patients compared to healthy controls. Thus, the following analyses were performed only for ED-A+ Fn. When dividing PH patients into different aetiological groups according to current ESC guidelines, the increase in ED-A+ Fn in PH patients compared to controls remained significant for group 1 (p = 0.032), 2 (p = 0.007) and 3 (p = 0.001) but not for group 4 (p = 0.156). Correlation analysis revealed a significant relation between ED-A+ Fn and brain natriuretic peptide (BNP) (r = 0.310; p = 0.002), six minutes' walk test (r = -0.275; p = 0.02) and systolic pulmonary artery pressure (PAPsys) (r = 0.364; p < 0.001). By logistic regression analysis (backward elimination WALD) including a variety of potentially relevant patients' characteristics, only chronic kidney disease (CKD) (OR: 8.866; CI: 1.779-44.187; p = 0.008), C reactive protein (CRP) (OR: 1.194; CI: 1.011-1.410; p = 0.037) and ED-A+ Fn (OR: 1.045; CI: 1.011-1.080; p = 0.009) could be identified as independent predictors of the presence of PH. CONCLUSIONS: Against the background of our results, ED-A+ Fn could serve as a promising novel biomarker of PH with potential value for initial diagnosis and aetiological differentiation. Moreover, it might contribute to more precise risk stratification of PH patients. Beyond that, the future role of ED-A+ Fn as a therapeutic target has to be evaluated in further studies.

CCN-Based Therapeutic Peptides Modify Pancreatic Ductal Adenocarcinoma Microenvironment and Decrease Tumor Growth in Combination with Chemotherapy.

The prominent desmoplastic stroma of pancreatic ductal adenocarcinoma (PDAC) is a determinant factor in tumor progression and a major barrier to the access of chemotherapy. The PDAC microenvironment therefore appears to be a promising therapeutic target. CCN2/CTGF is a profibrotic matricellular protein, highly present in the PDAC microenvironment and associated with disease progression. Here we have investigated the therapeutic value of the CCN2-targeting BLR100 and BLR200, two modified synthetic peptides derived from active regions of CCN3, an endogenous inhibitor of CCN2. In a murine orthotopic PDAC model, the two peptides, administered as monotherapy at low doses (approximating physiological levels of CCN3), had tumor inhibitory activity that increased with the dose. The peptides affected the tumor microenvironment, inhibiting fibrosis and vessel formation and reducing necrosis. Both peptides were active in preventing ascites formation. An increased activity was obtained in combination regimens, administering BLR100 or BLR200 with the chemotherapeutic drug gemcitabine. Pharmacokinetic analysis indicated that the improved activity of the combination was not mainly determined by the substantial increase in gemcitabine delivery to tumors, suggesting other effects on the tumor microenvironment. The beneficial remodeling of the tumor stroma supports the potential value of these CCN3-derived peptides for targeting pathways regulated by CCN2 in PDAC.

Hyperthermia affects collagen fiber architecture and induces apoptosis in pancreatic and fibroblast tumor hetero-spheroids in vitro.

Desmoplasia, an aberrant production of extracellular matrix (ECM), is considered as one predictive marker of malignancy of pancreatic cancer. In this paper, we study the effect of mild hyperthermia on fibrillary collagen architecture in murine Achilles tendons and in a pancreatic cancer model, in vitro, i.e. 3D hetero-type tumor spheroids, consisting of pancreatic cancer (Panc-1) cells and fibroblasts (WI-38), producing collagen fibers. We clearly demonstrate that i) mild hyperthermia (40 °C, 42 °C) damages the collagen architecture in murine Achilles tendons. ii) Mild extrinsic (hot air) and iron oxide nanoparticle based magnetic hyperthermia reduce the level of collagen fiber architecture in the generated hetero-type tumor spheroids. iii) Mild magnetic hyperthermia reduces cell vitality mainly through apoptotic and necrotic processes in the generated tumor spheroids. In conclusion, hetero-type 3D tumor spheroids are suitable for studying the effect of hyperthermia on collagen fibers, in vitro.

Cellular inflammation in pulmonary hypertension: Detailed analysis of lung and right ventricular tissue, circulating immune cells and effects of a dual endothelin receptor antagonist.

Growing evidence suggests that inflammation is crucially involved in the pathogenesis of pulmonary hypertension (PH) and consecutive right heart failure. The present study analyzed the inflammatory response in lung and right ventricle in a rat model of PH and evaluated the effects of the dual endothelin receptor antagonist (ERA) Macitentan. PH was induced by monocrotalin (60 mg/kg body weight s.c.) in Sprague-Dawley rats (PH, n = 10) and compared to healthy controls (CON, n = 10) as well as monocrotalin-induced, macitentan-treated rats (THER, n = 10). Detection of Dendritic cells (DCs), regulatory T cells (Tregs) and others as well as RT-PCR based inflammatory gene expression analysis were performed. Circulating DCs and Tregs were quantified by flow cytometry in the rat model and in PH patients (n = 70) compared to controls (n = 52). Inflammatory cells were increased in lung and right ventricular tissue, whereas DCs and Tregs were decreased in blood. Expression of 17 genes in the lung and 20 genes in the right ventricle were relevantly (>2.0 fold) regulated in the PH group. These effects were, at least in part, attenuated in response to Macitentan treatment. In humans as well as rats, immune cells showed significant correlations to clinical, echocardiographic, and haemodynamic parameters. PH is accompanied by a distinct inflammatory response in lung and right but not left ventricular tissue attenuated by Macitentan. Correlations of circulating DCs as well as tissue resident immune cells with parameters reflecting right ventricular function raise the idea of both, promising biomarkers and novel treatment strategies.

Targeting Wnt Signaling via Notch in Intestinal Carcinogenesis.

Proliferation and differentiation of intestinal epithelial cells is assisted by highly specialized and well-regulated signaling cascades. The Wnt pathway, which is one of the fundamental pathways in the intestine, contributes to the organization of proliferative intestinal crypts by positioning and cycling of intestinal stem cells and their derivatives. The Wnt pathway promotes differentiation of intestinal secretory cell types along the crypt-plateau and crypt-villus axis. In contrast to the Wnt pathway, the intestinal Notch cascade participates in cellular differentiation and directs progenitor cells towards an absorptive fate with diminished numbers of Paneth and goblet cells. Opposing activities of Notch and Wnt signaling in the regulation of intestinal stem cells and the enterocytic cell fate have been elucidated recently. In fact, targeting Notch was able to overcome tumorigenesis of intestinal adenomas, prevented carcinogenesis, and counteracted Paneth cell death in the absence of caspase 8. At present, pharmacological Notch inhibition is considered as an interesting tool targeting the intrinsic Wnt pathway activities in intestinal non-neoplastic disease and carcinogenesis.

Identification of Proteomic Markers in Head and Neck Cancer Using MALDI-MS Imaging, LC-MS/MS, and Immunohistochemistry.

PURPOSE: The heterogeneity of squamous cell carcinoma tissue greatly complicates diagnosis and individualized therapy. Therefore, characterizing the heterogeneity of tissue spatially and identifying appropriate biomarkers is crucial. MALDI-MS imaging (MSI) is capable of analyzing spatially resolved tissue biopsies on a molecular level. EXPERIMENTAL DESIGN: MALDI-MSI is used on snap frozen and formalin-fixed and paraffin-embedded (FFPE) tissue samples from patients with head and neck cancer (HNC) to analyze m/z values localized in tumor and nontumor regions. Peptide identification is performed using LC-MS/MS and immunohistochemistry (IHC). RESULTS: In both FFPE and frozen tissue specimens, eight characteristic masses of the tumor's epithelial region are found. Using LC-MS/MS, the peaks are identified as vimentin, keratin type II, nucleolin, heat shock protein 90, prelamin-A/C, junction plakoglobin, and PGAM1. Lastly, vimentin, nucleolin, and PGAM1 are verified with IHC. CONCLUSIONS AND CLINICAL RELEVANCE: The combination of MALDI-MSI, LC-MS/MS, and subsequent IHC furnishes a tool suitable for characterizing the molecular heterogeneity of tissue. It is also suited for use in identifying new representative biomarkers to enable a more individualized therapy.

MiR-192, miR-200c and miR-17 are fibroblast-mediated inhibitors of colorectal cancer invasion.

Colorectal cancer remains a leading cause of cancer-related death worldwide. A previous transcriptomics based study characterized molecular subgroups of which the stromal subgroup was associated with the worst clinical outcome. Micro-RNAs (miRNAs) are well-known regulators of gene expression and can follow a non-linear repression mechanism. We set up a model combining piecewise linear and linear regression and applied this combined regression model to a comprehensive colon adenocarcinoma dataset. We identified miRNAs involved in regulating characteristic gene sets, particularly extracellular matrix remodeling in the stromal subgroup. Comparison of expression data from separated (epithelial) cancer cells and stroma cells or fibroblasts associate these regulatory interactions with infiltrating stromal or tumor-associated fibroblasts. MiR-200c, miR-17 and miR-192 were identified as the most promising candidates regulating genes crucial for extracellular matrix remodeling. We validated our computational findings by in vitro assays. Enforced expression of either miR-200c, miR-17 or miR-192 in untransformed human colon fibroblasts down-regulated 85% of all predicted target genes. Expressing these miRNAs singly or in combination in human colon fibroblasts co-cultured with colon cancer cells considerably reduced cancer cell invasion validating these miRNAs as cancer cell infiltration suppressors in tumor associated fibroblasts.

Cystatin A suppresses tumor cell growth through inhibiting epithelial to mesenchymal transition in human lung cancer.

Cystatin A (CSTA), belonging to type 1 cystatin super-family, is expressed primarily in epithelial and lymphoid tissues for protecting cells from proteolysis of cytoplasmic and cytoskeletal proteins by cathepsins B, H and L. CSTA acts as a tumor suppressor in esophageal cancer, however, its role in lung cancer has not yet been elucidated. Here we found that CSTA was down-regulated in all lung cancer cell lines compared to normal lung epithelial cells. CSTA was restored in most lung cancer cell lines after treatment with demethylation agent 5-aza-2-deoxycytidine and deacetylation agent Trichostatin. Bisulfite sequencing revealed that CSTA was partially methylated in the promoter and exon 1. In primary lung tumors, squamous cell carcinoma (SCC) significantly expressed more CSTA compared to adenocarcinoma (p<0.00001), and higher expression of CSTA was significantly associated with lower tumor grade (p<0.01). CSTA stable transfection reduced the activity of cathepsin B and inhibited the ability of colony formation, migration and invasion, and enhanced gemcitabine-induced apoptosis. CSTA overexpression resulted in reduced activity of ERK, p-38, and AKT. Additionally, CSTA overexpression led to a mesenchymal to epithelial transition (MET) and prevented the TGF-ß1-induced epithelial to mesenchymal transition (EMT) through inhibiting the ERK/MAPK pathway. In conclusion, our date indicate 1) epigenetic regulation is associated with CSTA gene silencing; 2) CSTA exerts tumor suppressive function through inhibiting MAPK and AKT pathways; 3) Overexpression of CSTA leads to MET and prevents TGF-ß1-induced EMT by modulating the MAPK pathway; 4) CSTA may be a potential biomarker for lung SCC and tumor differentiation.

Increased Serum Levels of Fetal Tenascin-C Variants in Patients with Pulmonary Hypertension: Novel Biomarkers Reflecting Vascular Remodeling and Right Ventricular Dysfunction?

Pulmonary vascular remodeling is a pathophysiological feature that common to all classes of pulmonary hypertension (PH) and right ventricular dysfunction, which is the major prognosis-limiting factor. Vascular, as well as cardiac tissue remodeling are associated with a re-expression of fetal variants of cellular adhesion proteins, including tenascin-C (Tn-C). We analyzed circulating levels of the fetal Tn-C splicing variants B⁺ and C⁺ Tn-C in serum of PH patients to evaluate their potential as novel biomarkers reflecting vascular remodeling and right ventricular dysfunction. Serum concentrations of B⁺ and C⁺ Tn-C were determined in 80 PH patients and were compared to 40 healthy controls by enzyme-linked immunosorbent assay. Clinical, laboratory, echocardiographic, and functional data were correlated with Tn-C levels. Serum concentrations of both Tn-C variants were significantly elevated in patients with PH (p < 0.05). Significant correlations could be observed between Tn-C and echocardiographic parameters, including systolic pulmonary artery pressure (B⁺ Tn-C: r = 0.31, p < 0.001, C⁺ Tn-C: r = 0.26, p = 0.006) and right atrial area (B⁺ Tn-C: r = 0.46, p < 0.001, C⁺ Tn-C: r = 0.49, p < 0.001), and laboratory values like BNP (B⁺ Tn-C: r = 0.45, p < 0.001, C⁺ Tn-C: r = 0.42, p < 0.001). An inverse correlation was observed between Tn-C variants and 6-minute walk distance as a functional parameter (B⁺ Tn-C: r = -0.54, p < 0.001, C⁺ Tn-C: r = -0.43, p < 0.001). In a multivariate analysis, B⁺ Tn-C, but not C⁺ Tn-C, was found to be an independent predictor of pulmonary hypertension. Both fetal Tn-C variants may represent novel biomarkers that are capable of estimating both pulmonary vascular remodeling and right ventricular load. The potential beneficial impact of Tn-C variants for risk stratification in patients with PH needs further investigation.

Arginase Inhibition Reverses Monocrotaline-Induced Pulmonary Hypertension.

Pulmonary hypertension (PH) is a heterogeneous disorder associated with a poor prognosis. Thus, the development of novel treatment strategies is of great interest. The enzyme arginase (Arg) is emerging as important player in PH development. The aim of the current study was to determine the expression of ArgI and ArgII as well as the effects of Arg inhibition in a rat model of PH. PH was induced in 35 Sprague-Dawley rats by monocrotaline (MCT, 60 mg/kg as single-dose). There were three experimental groups: sham-treated controls (control group, n = 11), MCT-induced PH (MCT group, n = 11) and MCT-induced PH treated with the Arg inhibitor Nω-hydroxy-nor-l-arginine (nor-NOHA; MCT/NorNoha group, n = 13). ArgI and ArgII expression was determined by immunohistochemistry and Western blot. Right ventricular systolic pressure (RVPsys) was measured and lung tissue remodeling was determined. Induction of PH resulted in an increase in RVPsys (81 ± 16 mmHg) compared to the control group (41 ± 15 mmHg, p = 0.002) accompanied by a significant elevation of histological sum-score (8.2 ± 2.4 in the MCT compared to 1.6 ± 1.6 in the control group, p < 0.001). Both, ArgI and ArgII were relevantly expressed in lung tissue and there was a significant increase in the MCT compared to the control group (p < 0.01). Arg inhibition resulted in a significant reduction of RVPsys to 52 ± 19 mmHg (p = 0.006) and histological sum-score to 5.8 ± 1.4 compared to the MCT group (p = 0.022). PH leads to increased expression of Arg. Arg inhibition leads to reduction of RVPsys and diminished lung tissue remodeling and therefore represents a potential treatment strategy in PH.

Myofibroblasts have an impact on expression, dimerization and signaling of different ErbB receptors in OSCC cells.

INTRODUCTION: Receptors of the ErbB family belong to the key players in cancer development and are targets of several therapeutic approaches. Their functional dependency on the tumor microenvironment, especially on CAFs is albeit still poorly understood. Our objective was to investigate the impact of CAF secretome on ErbB receptor expression and signaling behavior in OSCC. METHODS: Stimulation of PE/CA-PJ15 OSCC cells with conditioned media of TGF-ß1-activated fibroblasts was used as model system for CAF to cancer cell communication. Thereby costimulation with inhibitors against matrix metalloproteinases (MMPs), epidermal growth factor receptor (EGFR), MAPK/ERK kinase (MEK), phosphoinositide-3 kinase (PI3-K), signal transducer and activator of transcription 3 (Stat3) or knockdown of Her3 by siRNA was utilized for detailed investigation of the expression, dimerization and signaling pattern of ErbB in western blot and coimmunoprecipitation. RESULTS: Our results show that soluble factors in activated fibroblast secretome stimulate metalloproteinase activity in the membrane of cancer cells. Thereby ligands are released that activate EGFR and subsequently upregulates EGFR expression via the STAT3 pathway. Simultaneously, the expression of PKCɛ was enhanced via a PI3-kinase/Akt-mediated pathway and a negative feedback regulation loop on EGFR downstream signaling generated. Furthermore, the activated fibroblasts secretome stimulated the highly oncogenic hetero-dimerization between HER3 and p95HER2. That protein association is inversely dependent on the expression level of HER3. CONCLUSIONS: Our results demonstrate that the activated fibroblasts secretome can induce a counterbalanced regulation of protein expression, downstream signaling and the dimerization patterns of different ErbB receptor subtypes in the cancer cell. Thus, the combinatorial targeting of CAFs and selective ErbB receptor subtype inhibitors may provide a useful approach in cancer therapy.

Lung tissue remodelling in MCT-induced pulmonary hypertension: a proposal for a novel scoring system and changes in extracellular matrix and fibrosis associated gene expression.

Pulmonary hypertension (PH) is associated with vasoconstriction and remodelling. We studied lung tissue remodelling in a rat model of PH with special focus on histology and extracellular matrix (ECM) remodelling. After induction of PH by monocrotaline, lung tissue was analysed histologically, by gene expression analysis and immunofluorescence labelling of ED-A domain containing fibronectin (ED-A+ Fn), B domain containing tenascin-C (B+ Tn-C) as well as alpha-smooth muscle actin (α-SMA). Serum concentrations of ED-A+ Fn were determined by ELISA. Systolic right ventricular pressure (RVPsys) values were significantly elevated in PH (n = 18; 75 ± 26.4 mmHg) compared to controls (n = 10; 29 ± 19.3 mmHg; p = 0.015). The histological sum-score was significantly increased in PH (8.0 ± 2.2) compared to controls (2.5 ± 1.6; p < 0.001). Gene expression analysis revealed relevant induction of several key genes of extracellular matrix remodelling. Increased protein deposition of ED-A+ Fn but not of B+ Tn-C and α-SMA in lung tissue was found in PH (2.88 ± 3.19 area%) compared to controls (1.32 ± 0.16 area%; p = 0.030). Serum levels of ED-A+ Fn were significantly higher in PH (p = 0.007) positively correlating with RVPsys (r = 0.618, p = 0.019). We here present a novel histological scoring system to assess lung tissue remodelling in PH. Gene expression analysis revealed induction of candidate genes involved in collagen matrix turnover, fibrosis and vascular remodelling. The stable increased tissue deposition of ED-A+ Fn in PH as well as its dynamics in serum suggests a role as a promising novel biomarker and potential therapeutic target.

Detection of Soluble ED-A(+) Fibronectin and Evaluation as Novel Serum Biomarker for Cardiac Tissue Remodeling.

Background and Aims. Fibronectin containing the extra domain A (ED-A(+) Fn) was proven to serve as a valuable biomarker for cardiac remodeling. The study was aimed at establishing an ELISA to determine ED-A(+) Fn in serum of heart failure patients. Methods. ED-A(+) Fn was quantified in serum samples from 114 heart failure patients due to ischemic (ICM, n = 44) and dilated (DCM, n = 39) cardiomyopathy as well as hypertensive heart disease (HHD, n = 31) compared to healthy controls (n = 12). Results. In comparison to healthy volunteers, heart failure patients showed significantly increased levels of ED-A(+) Fn (p < 0.001). In particular in ICM patients there were significant associations between ED-A(+) Fn serum levels and clinical parameters, for example, increased levels with rising NYHA class (p = 0.013), a negative correlation with left ventricular ejection fraction (p = 0.026, r: -0.353), a positive correlation with left atrial diameter (p = 0.008, r: 0.431), and a strong positive correlation with systolic pulmonary artery pressure (p = 0.002, r: 0.485). In multivariate analysis, ED-A(+) Fn was identified as an independent predictor of an ischemic heart failure etiology. Conclusions. The current study could clearly show that ED-A(+) Fn is a promising biomarker in cardiovascular diseases, especially in heart failure patients due to an ICM. We presented a valid ELISA method, which could be applied for further studies investigating the value of ED-A(+) Fn.

Parameters of Stromal Activation and Epithelial to Mesenchymal Transition as Predictive Biomarkers for Induction Chemotherapy in Patients With Locally Advanced Oral Cavity and Oropharyngeal Squamous Cell Cancer.

OBJECTIVES: Induction chemotherapy (IC) is likely to be effective for biologically distinct subgroups of oral cancer and biomarker development may lead to identification of those patients. METHODS: We evaluated immune cell infiltration, stroma formation and structure of the invasive front as well as the immunohistochemical expression of alpha smooth muscle actin (ASMA), CD163, E-cadherin, N-cadherin, and the laminin gamma 2 chain in pretreatment biopsy specimens and surgical resections after IC in 20 patients with locally advanced oral cancer who were treated in a prospective, ongoing, phase II trial on IC using docetaxel, cisplatin, and 5-fluorouracil (TPF). RESULTS: Significant negative prognostic factors for incomplete pathological tumor response to IC were alcohol abuse (P=0.032), cN+ (P=0.042), and <30% tumor reduction after first cycle of IC (P=0.034). Of the investigated histological parameters and biomarkers only a low membrane-bound expression of E-cadherin showed a trend to be associated with incomplete response to IC (P=0.061). Low expression of ASMA in stromal vessels and a strong tumor invasion front were significantly associated to tumor recurrence (P=0.024 and P=0.004, respectively). The median follow-up of all patients was 35 months. Alcohol abuse (P<0.001), <30% tumor reduction after first cycle of IC (P=0.005), and a strong tumor invasion front (P=0.019) were negative prognostic factors for overall survival. CONCLUSION: A strong predictive biomarker among the investigated parameters for benefitting from TPF IC could not be found. The extent of the tumor invasion front was a negative prognostic marker for recurrence and survival in oral cancer treated by TPF IC followed by surgery and postoperative radiochemotherapy.