Identification of Novel Natural Substrates of Fibroblast Activation Protein-alpha by Differential Degradomics and Proteomics.

Fibroblast activation protein-alpha (FAP) is a cell-surface transmembrane-anchored dimeric protease. This unique, constitutively active serine protease has both dipeptidyl aminopeptidase and endopeptidase activities and can hydrolyze the post-proline bond. FAP expression is very low in adult organs but is upregulated by activated fibroblasts in sites of tissue remodeling, including fibrosis, atherosclerosis, arthritis and tumors. To identify the endogenous substrates of FAP, we immortalized primary mouse embryonic fibroblasts (MEFs) from FAP gene knockout embryos and then stably transduced them to express either enzymatically active or inactive FAP. The MEF secretomes were then analyzed using degradomic and proteomic techniques. Terminal amine isotopic labeling of substrates (TAILS)-based degradomics identified cleavage sites in collagens, many other extracellular matrix (ECM) and associated proteins, and lysyl oxidase-like-1, CXCL-5, CSF-1, and C1qT6, that were confirmed in vitro In addition, differential metabolic labeling coupled with quantitative proteomic analysis also implicated FAP in ECM-cell interactions, as well as with coagulation, metabolism and wound healing associated proteins. Plasma from FAP-deficient mice exhibited slower than wild-type clotting times. This study provides a significant expansion of the substrate repertoire of FAP and provides insight into the physiological and potential pathological roles of this enigmatic protease.

The stromal cell-surface protease fibroblast activation protein-α localizes to lipid rafts and is recruited to invadopodia.

Fibroblast activation protein alpha (FAPα) is a cell surface protease expressed by cancer-associated fibroblasts in the microenvironment of most solid tumors. As there is increasing evidence for proteases having non-catalytic functions, we determined the FAPα interactome in cancer-associated fibroblasts using the quantitative immunoprecipitation combined with knockdown (QUICK) method. Complex formation with adenosin deaminase, erlin-2, stomatin, prohibitin, Thy-1 membrane glycoprotein, and caveolin-1 was further validated by immunoblotting. Co-immunoprecipitation (co-IP) of the known stoichiometric FAPα binding partner dipeptidyl-peptidase IV (DPPIV) corroborated the proteomic strategy. Reverse co-IPs validated the FAPα interaction with caveolin-1, erlin-2, and stomatin while co-IP upon RNA-interference mediated knock-down of DPPIV excluded adenosin deaminase as a direct FAPα interaction partner. Many newly identified FAPα interaction partners localize to lipid rafts, including caveolin-1, a widely-used marker for lipid raft localization. We hypothesized that this indicates a recruitment of FAPα to lipid raft structures. In density gradient centrifugation, FAPα co-fractionates with caveolin-1. Immunofluorescence optical sectioning microscopy of FAPα and lipid raft markers further corroborates recruitment of FAPα to lipid rafts and invadopodia. FAPα is therefore an integral component of stromal lipid rafts in solid tumors. In essence, we provide one of the first interactome analyses of a cell surface protease and translate these results into novel biological aspects of a marker protein for cancer-associated fibroblasts.

Expression of various insulin-like growth factor-1 mRNA isoforms in colorectal cancer.

AIM OF THE STUDY: Several epidemiological studies have attempted to demonstrate a relationship between increased serum level of insulin-like growth factor 1 (IGF-1) and an augmented risk of developing colorectal cancers (CRC). The human IGF-1 gene is composed of 6 exons and demonstrated expression of 6 different splice variants (isoforms) of mRNA (IA, IB, IC, IIA, IIB and IIC). The aim of the study was to evaluate the expression of different isoforms of IGF-1 mRNA in CRC and normal colon tissue. MATERIAL AND METHODS: 13 paired tissue specimens (colorectal tumor and non-tumor tissues) were analyzed using both quantitative polymerase chain reaction (PCR) and immunocytochemistry methods (IHC). The expression of classes I and II and variants A, B, C of IGF-1 mRNA were measured. RESULTS: In CRC higher amounts of IGF-1 class II mRNA than class I mRNA were detected. Among A, B, C isoforms, A variant of IGF-1 mRNA prevailed. The amounts of IGF-1 class I and class II mRNAs and of IGF-1 variant B mRNA were lowered in CRC as compared to the control. In CRC significant correlations were detected between reciprocal expression of class I and class II as well as between I and II isoforms and A, B and C. CONCLUSIONS: Expression of IGF-1 mRNA isoforms differs between normal and CRC tissues. Even if all isoforms of IGF-1 mRNA manifested correlations with each other in tissues of CRC, expression of all transcripts (except that of isoform A) was significantly decreased as compared to the control.

Identification of a novel anoikis signalling pathway using the fungal virulence factor gliotoxin.

Anoikis is a form of apoptosis induced by cell detachment. Integrin inactivation plays a major role in the process but the exact signalling pathway is ill-defined. Here we identify an anoikis pathway using gliotoxin (GT), a virulence factor of the fungus Aspergillus fumigatus, which causes invasive aspergillosis in humans. GT prevents integrin binding to RGD-containing extracellular matrix components by covalently modifying cysteines in the binding pocket. As a consequence, focal adhesion kinase (FAK) is inhibited resulting in dephosphorylation of p190RhoGAP, allowing activation of RhoA. Sequential activation of ROCK, MKK4/MKK7 and JNK then triggers pro-apoptotic phosphorylation of Bim. Cells in suspension or lacking integrin surface expression are insensitive to GT but are sensitised to ROCK-MKK4/MKK7-JNK-dependent anoikis upon attachment to fibronectin or integrin upregulation. The same signalling pathway is triggered by FAK inhibition or inhibiting integrin αV/ß3 with Cilengitide. Thus, GT can target integrins to induce anoikis on lung epithelial cells.

Differential effect of TGFß on the proteome of cancer associated fibroblasts and cancer epithelial cells in a co-culture approach - a short report.

BACKGROUND: Solid tumors contain various components that together form the tumor microenvironment. Cancer associated fibroblasts (CAFs) are capable of secreting and responding to signaling molecules and growth factors. Due to their role in tumor development, CAFs are considered as potential therapeutic targets. A prominent tumor-associated signaling molecule is transforming growth factor ß (TGFß), an inducer of epithelial-to-mesenchymal transition (EMT). The differential action of TGFß on CAFs and ETCs (epithelial tumor cells) has recently gained interest. Here, we aimed to investigate the effects of TGFß on CAFs and ETCs at the proteomic level. METHODS: We established a 2D co-culture system of differentially fluorescently labeled CAFs and ETCs and stimulated this co-culture system with TGFß. The respective cell types were separated using FACS and subjected to quantitative analyses of individual proteomes using mass spectrometry. RESULTS: We found that TGFß treatment had a strong impact on the proteome composition of CAFs, whereas ETCs responded only marginally to TGFß. Quantitative proteomic analyses of the different cell types revealed up-regulation of extracellular matrix (ECM) proteins in TGFß treated CAFs. In addition, we found that the TGFß treated CAFs exhibited increased N-cadherin levels. CONCLUSIONS: From our data we conclude that CAFs respond to TGFß treatment by changing their proteome composition, while ETCs appear to be rather resilient.

Tenascin-C Orchestrates Glioblastoma Angiogenesis by Modulation of Pro- and Anti-angiogenic Signaling.

High expression of the extracellular matrix component tenascin-C in the tumor microenvironment correlates with decreased patient survival. Tenascin-C promotes cancer progression and a disrupted tumor vasculature through an unclear mechanism. Here, we examine the angiomodulatory role of tenascin-C. We find that direct contact of endothelial cells with tenascin-C disrupts actin polymerization, resulting in cytoplasmic retention of the transcriptional coactivator YAP. Tenascin-C also downregulates YAP pro-angiogenic target genes, thus reducing endothelial cell survival, proliferation, and tubulogenesis. Glioblastoma cells exposed to tenascin-C secrete pro-angiogenic factors that promote endothelial cell survival and tubulogenesis. Proteomic analysis of their secretome reveals a signature, including ephrin-B2, that predicts decreased survival of glioma patients. We find that ephrin-B2 is an important pro-angiogenic tenascin-C effector. Thus, we demonstrate dual activities for tenascin-C in glioblastoma angiogenesis and uncover potential targeting and prediction opportunities.

Insulin-like growth factor-1 mRNA isoforms and insulin-like growth factor-1 receptor mRNA expression in chronic hepatitis C.

AIM: To evaluate the expression of different insulin-like growth factor (IGF)-1 mRNA isoforms and IGF-1 receptor (IGF-1R) mRNA in hepatitis C virus (HCV)-infected livers. METHODS: Thirty-four liver biopsy specimens from chronic hepatitis C (CH-C) patients were obtained before anti-viral therapy. Inflammatory activity (grading) and advancement of fibrosis (staging) were evaluated using a modified point scale of METAVIR. The samples were analyzed using quantitative real-time PCR technique. From fragments of liver biopsies and control liver that were divided and ground in liquid nitrogen, RNA was isolated using RNeasy Fibrous Tissue Mini Kit according to the manufacturer's instruction. Expression levels of IGF-1 mRNA isoforms (IGF-1A, IGF-1B, IGF-1C, P1, and P2) and IGF-1R mRNA were determined through normalization of copy numbers in samples as related to reference genes: glyceraldehyde-3-phosphate dehydrogenase and hydroxymethylbilane synthase. Results on liver expression of the IGF-1 mRNA isoforms and IGF-1R transcript were compared to histological alterations in liver biopsies and with selected clinical data in the patients. Statistical analysis was performed using Statistica PL v. 9 software. RESULTS: The study showed differences in quantitative expression of IGF-1 mRNA variants in HCV-infected livers, as compared to the control. Higher relative expression of total IGF-1 mRNA and of IGF-1 mRNAs isoforms (P1, A, and C) in HCV-infected livers as compared to the control were detected. Within both groups, expression of the IGF-1A mRNA isoform significantly prevailed over expressions of B and C isoforms. Expression of P1 mRNA was higher than that of P2 only in CH-C. Very high positive correlations were detected between reciprocal expressions of IGF-1 mRNA isoforms P1 and P2 (r = 0.876). Expression of P1 and P2 mRNA correlated with IGF-1A mRNA (r = 0.891; r = 0.821, respectively), with IGF-1B mRNA (r = 0.854; r = 0.813, respectively), and with IGF-1C mRNA (r = 0.839; r = 0.741, respectively). Expression of IGF-1A mRNA significantly correlated with isoform B and C mRNA (r = 0.956; r = 0.869, respectively), and B with C isoforms (r = 0.868) (P < 0.05 in all cases). Lower expression of IGF-1A and B transcripts was noted in the more advanced liver grading (G2) as compared to G1. Multiple negative correlations were detected between expression of various IGF-1 transcripts and clinical data (e.g., alpha fetoprotein, HCV RNA, steatosis, grading, and staging). Expression of IGF-1R mRNA manifested positive correlation with grading and HCV-RNA. CONCLUSION: Differences in quantitative expression of IGF-1 mRNA isoforms in HCV-infected livers, as compared to the control, suggest that HCV may induce alteration of IGF-1 splicing profile.

Differential expression of IGF-1 mRNA isoforms in colorectal carcinoma and normal colon tissue.

The insulin-like growth factor (IGF)-1 gene consists of 6 exons resulting in the expression of 6 variant forms of mRNA (IA, IB, IC, IIA, IIB and IIC) due to an alternative splicing. The mechanisms of IGF-1 gene splicing and the role of local expression manifested by IGF-1 mRNA variants in colorectal carcinoma (CRC) have not been extensively investigated. Therefore, the aim of our study was to analyse the expression of IGF-1 mRNA isoforms [A, B, C, P1 (class I) and P2 (class II)], as well as the protein expression in CRC and control samples isolated from 28 patients. The expression of Ki-67 was also analysed and clinical data were obtained. For this purpose, we used quantitative real-time PCR (qPCR) and immunocytochemistry. The expression of mRNAs coding for all splicing isoforms of IGF-1 was observed in every tissue sample studied, with a significantly lower expression noted in the CRC as compared to the control samples. The cytoplasmic expression of IGF-1 protein was found in 50% of the CRC and in ~40% of the non-tumor tissues; however, no significant quantitative inter-group differences were observed. The expression of the IGF-1 gene in the 2 groups of tissues was controlled by the P1 and P2 promoters in a similar manner. No significant differences were detected in the expression of the IGF-1 A and B isoforms; however, their expression was significantly higher compared to that of isoform C. No significant differences were observed between the expression of Ki-67 mRNA in the CRC and control tissue even though the expression of the Ki-67 protein was higher in the CRC compared to the control samples. Ki-67 protein expression was associated with the macroscopic and microscopic aspects of CRC. A significant positive correlation was found between the local production of total mRNA and isoform A and the expression of Ki-67 mRNA, although only in the non-tumor tissues. In CRC samples, the local expression of the total IGF-1 mRNA and all splicing isoforms of IGF-1 mRNA decreased as compared to the normal colon tissues, although however, with conservation of both gene promoter activities and with the continued principal splicing IGF-1 mRNA isoforms.

IGF1 mRNA isoform expression in the cervix of HPV-positive women with pre-cancerous and cancer lesions.

Human papillomavirus (HPV) plays a crucial role in cervical cancer etiology. However, not all HPV-infected women develop cancer, indicating that additional cellular factors facilitate carcinogenesis. The aim of this study was to analyze the expression profile of insulin-like growth factor 1 (IGF1) isoforms in the context of FOX2, SP1 and IGF1 receptor (IGF1R) expression during HPV-dependent cervical carcinogenesis. One hundred and nine epithelial tissue samples from women with pre-cancerous and cancer lesions of the cervix were analyzed. HPV DNA was identified by PCR, and real-time PCR was used to quantify the expression levels of the analyzed genes. All IGF1 mRNA splicing isoforms were up-regulated in pre-cancerous cells, and a shift in the balance towards mitogenic IGF1Eb was observed in the cancer samples. IGF1 expression was controlled mainly by the P1 promoter, and an increase in P2 usage was observed in the cancer. Correlations between IGF1 mRNA splicing isoforms and the FOX2 splicing factor, as well as P1/P2 activity and SP1 transcription factor expression levels were detected. No correlation was observed between the expression of IGF1 and its receptor IGF1R. Our results suggest that IGF1, in particular its splicing profile, may be an additional prognostic factor in cervical carcinogenesis.