PFKFB4 interacts with FBXO28 to promote HIF-1α signaling in glioblastoma.

Glioblastoma is a highly aggressive brain tumor for which there is no cure. The metabolic enzyme 6-Phosphofructo-2-Kinase/Fructose-2,6-Biphosphatase 4 (PFKFB4) is essential for glioblastoma stem-like cell (GSC) survival but its mode of action is unclear. Understanding the role of PFKFB4 in tumor cell survival could allow it to be leveraged in a cancer therapy. Here, we show the importance of PFKFB4 for glioblastoma growth in vivo in an orthotopic patient derived mouse model. In an evaluation of patient tumor samples of different cancer entities, PFKFB4 protein was found to be overexpressed in prostate, lung, colon, mammary and squamous cell carcinoma, with expression level correlating with tumor grade. Gene expression profiling in PFKFB4-silenced GSCs revealed a downregulation of hypoxia related genes and Western blot analysis confirmed a dramatic reduction of HIF (hypoxia inducible factor) protein levels. Through mass spectrometric analysis of immunoprecipitated PFKFB4, we identified the ubiquitin E3 ligase, F-box only protein 28 (FBXO28), as a new interaction partner of PFKFB4. We show that PFKFB4 regulates the ubiquitylation and subsequent proteasomal degradation of HIF-1α, which is mediated by the ubiquitin ligase activity of FBXO28. This newly discovered function of PFKFB4, coupled with its cancer specificity, provides a new strategy for inhibiting HIF-1α in cancer cells.

AAMP is a binding partner of costimulatory human B7-H3.

Background: Targeted immunotherapies are of growing interest in the treatment of various cancers. B7 homolog 3 protein (B7-H3), a member of the co-stimulatory/-inhibitory B7-family, exerts immunosuppressive and pro-tumorigenic functions in various cancer types and is under evaluation in ongoing clinical trials. Unfortunately, interaction partner(s) remain unknown which restricts the druggability. Methods: Aiming to identify potential binding partner(s) of B7-H3, a yeast two-hybrid and a mass spectrometry screen were performed. Potential candidates were evaluated by bimolecular fluorescence complementation (BiFC) assay, co-immunoprecipitation (co-IP), and functionally in a 3H-thymidine proliferation assay of Jurkat cells, a T-cell lineage cell line. Prognostic value of angio-associated migratory cell protein (AAMP) and B7-H3 expression was evaluated in isocitrate dehydrogenase 1 wildtype (IDH1wt) glioblastoma (GBM) patients from The Cancer Genome Atlas (TCGA)-GBM cohort. Results: Of the screening candidates, CD164, AAMP, PTPRA, and SLAMF7 could be substantiated via BiFC. AAMP binding could be further confirmed via co-IP and on a functional level. AAMP was ubiquitously expressed in glioma cells, immune cells, and glioma tissue, but did not correlate with glioma grade. Finally, an interaction between AAMP and B7-H3 could be observed on expression level, hinting toward a combined synergistic effect. Conclusions: AAMP was identified as a novel interaction partner of B7-H3, opening new possibilities to create a targeted therapy against the pro-tumorigenic costimulatory protein B7-H3.

The Wnt-specific astacin proteinase HAS-7 restricts head organizer formation in Hydra.

BACKGROUND: The Hydra head organizer acts as a signaling center that initiates and maintains the primary body axis in steady state polyps and during budding or regeneration. Wnt/beta-Catenin signaling functions as a primary cue controlling this process, but how Wnt ligand activity is locally restricted at the protein level is poorly understood. Here we report a proteomic analysis of Hydra head tissue leading to the identification of an astacin family proteinase as a Wnt processing factor. RESULTS: Hydra astacin-7 (HAS-7) is expressed from gland cells as an apical-distal gradient in the body column, peaking close beneath the tentacle zone. HAS-7 siRNA knockdown abrogates HyWnt3 proteolysis in the head tissue and induces a robust double axis phenotype, which is rescued by simultaneous HyWnt3 knockdown. Accordingly, double axes are also observed in conditions of increased Wnt activity as in transgenic actin::HyWnt3 and HyDkk1/2/4 siRNA treated animals. HyWnt3-induced double axes in Xenopus embryos could be rescued by coinjection of HAS-7 mRNA. Mathematical modelling combined with experimental promotor analysis indicate an indirect regulation of HAS-7 by beta-Catenin, expanding the classical Turing-type activator-inhibitor model. CONCLUSIONS: We show the astacin family protease HAS-7 maintains a single head organizer through proteolysis of HyWnt3. Our data suggest a negative regulatory function of Wnt processing astacin proteinases in the global patterning of the oral-aboral axis in Hydra.

Chicken lens development: complete signature of expression of galectins during embryogenesis and evidence for their complex formation with α-, ß-, δ-, and τ-crystallins, N-CAM, and N-cadherin obtained by affinity chromatography.

The emerging multifunctionality of galectins by specific protein-glycan/protein interactions explains the interest to determine their expression during embryogenesis. Complete network analysis of all seven chicken galectins (CGs) is presented in the course of differentiation of eye lens that originates from a single type of progenitor cell. It answers the questions on levels of expression and individual patterns of distribution. A qualitative difference occurs in the CG-1A/B paralogue pair, underscoring conspicuous divergence. Considering different cell phenotypes, lens fiber and also epithelial cells can both express the same CG, with developmental upregulation for CG-3 and CG-8. Except for expression of the lens-specific CG (C-GRIFIN), no other CG appeared to be controlled by the transcription factors L-Maf and Pax6. Studying presence and nature of binding partners for CGs, we tested labeled galectins in histochemistry and in ligand blotting. Mass spectrometric (glyco)protein identification after affinity chromatography prominently yielded four types of crystallins, N-CAM, and, in the cases of CG-3 and CG-8, N-cadherin. Should such pairing be functional in situ, it may be involved in tightly packing intracellular lens proteins and forming membrane contact as well as in gaining plasticity and stability of adhesion processes. The expression of CGs throughout embryogenesis is postulated to give meaning to spatiotemporal alterations in the local glycome.

Distinct Origin of Claudin7 in Early Tumor Endosomes Affects Exosome Assembly.

Microvesicles are the body's most powerful intercellular communication system and cancer-initiating cell microvesicles (CIC-TEX) reprogram Non-CIC towards fortified malignancy. Claudin7, a CIC-biomarker in gastrointestinal tumors, is recovered in TEX. Recent evidence suggesting individual cells delivering distinct microvesicles became of particular interest for claudin7, which is part of tight junctions (TJ) and glycolipid-enriched membrane domains (GEM), GEM-located claudin7 is palmitoylated. This offered the unique possibility of exploring the contribution of a CIC marker and its origin from distinct membrane domains on CIC-TEX biogenesis and activities. Proteome and miRNA analysis of wild-type, claudin7-knockdown and a rescue with claudin7 harboring a mutated palmitoylation site (mP) of a rat pancreatic and a human colon cancer line uncovered significant, only partly overlapping contributions of palmitoylated and non-palmitoylated claudin7 to TEX composition. Palmitoylated claudin7 facilitates GEM-integrated plasma membrane and associated signaling molecule recruitment; non-palmitoylated claudin7 supports recruitment of trafficking components, proteins engaged in fatty acid metabolism and TJ proteins into TEX. Claudin7mP also assists TEX recovery of selected miRNA. Thus, distinctly located claudin7 affects CIC-TEX composition and TJ-derived cld7 might play a unique role in equipping CIC-TEX with transporters and lipid metabolism-regulating molecules, awareness of distinct TEX populations being crucial facing therapeutic translation.

The Pancreatic Cancer-Initiating Cell Marker CD44v6 Affects Transcription, Translation, and Signaling: Consequences for Exosome Composition and Delivery.

Pancreatic cancer-initiating cells (PaCIC) express CD44v6 and Tspan8. A knockdown (kd) of these markers hinders the metastatic capacity, which can be rescued, if the cells are exposed to CIC-exosomes (TEX). Additional evidence that CD44v6 regulates Tspan8 expression prompted us to explore the impact of these PaCIC markers on nonmetastatic PaCa and PaCIC-TEX. We performed proteome, miRNA, and mRNA deep sequencing analyses on wild-type, CD44v6kd, and Tspan8kd human PaCIC and TEX. Database comparative analyses were controlled by qRT-PCR, Western blot, flow cytometry, and confocal microscopy. Transcriptome analysis of CD44 versus CD44v6 coimmunoprecipitating proteins in cells and TEX revealed that Tspan8, several signal-transducing molecules including RTK, EMT-related transcription factors, and proteins engaged in mRNA processing selectively associate with CD44v6 and that the membrane-attached CD44 intracytoplasmic tail supports Tspan8 and NOTCH transcription. Deep sequencing uncovered a CD44v6 contribution to miRNA processing. Due to the association of CD44v6 with Tspan8 in internalization prone tetraspanin-enriched membrane domains (TEM) and the engagement of Tspan8 in exosome biogenesis, most CD44v6-dependent changes were transferred into TEX such that the input of CD44v6 to TEX activities becomes largely waved in both a CD44v6kd and a Tspan8kd. Few differences between CD44v6kd- and Tspan8kd-TEX rely on CD44v6 being also recovered in non-TEM derived TEX, highlighting distinct TEX delivery from individual cells that jointly account for TEX-promoted target modulation. This leads us to propose a model in which CD44v6 strongly supports tumor progression by cooperating with signaling molecules, altering transcription of key molecules, and through its association with the mRNA processing machinery. The association of CD44v6 with Tspan8, which plays a crucial role in vesicle biogenesis, promotes metastases by transferring CD44v6 activities into TEM and TEM-independently derived TEX. Further investigations of the lead position of CD44v6 in shifting metastasis-promoting activities into CIC-TEX may offer a means of targeting TEX-CD44v6 in therapeutic applications.

SILAC-Based Quantification of TGFBR2-Regulated Protein Expression in Extracellular Vesicles of Microsatellite Unstable Colorectal Cancers.

Microsatellite unstable (MSI) colorectal cancers (CRCs) are characterized by mutational inactivation of Transforming Growth Factor Beta Receptor Type 2 (TGFBR2). TGFBR2-deficient CRCs present altered target gene and protein expression. Such cellular alterations modulate the content of CRC-derived extracellular vesicles (EVs). EVs function as couriers of proteins, nucleic acids, and lipids in intercellular communication. At a qualitative level, we have previously shown that TGFBR2 deficiency causes overall alterations in the EV protein content. To deepen the basic understanding of altered protein dynamics, this work aimed to determine TGFBR2-dependent EV protein signatures in a quantitative manner. Using a stable isotope labeling with amino acids in cell culture (SILAC) approach for mass spectrometry-based quantification, 48 TGFBR2-regulated proteins were identified in MSI CRC-derived EVs. Overall, TGFBR2 deficiency caused upregulation of several EV proteins related to the extracellular matrix and nucleosome as well as downregulation of proteasome-associated proteins. The present study emphasizes the general overlap of proteins between EVs and their parental CRC cells but also highlights the impact of TGFBR2 deficiency on EV protein composition. From a clinical perspective, TGFBR2-regulated quantitative differences of protein expression in EVs might nominate novel biomarkers for liquid biopsy-based MSI typing in the future.

Deciphering the galectin-12 protein interactome reveals a major impact of galectin-12 on glutamine anaplerosis in colon cancer cells.

Galectins are ß-galactoside binding proteins which possess a variety of functions including modulation of apoptosis, growth and differentiation. Hence, alterations in the expression profile have been associated with loss of cellular homeostasis contributing to tumor growth and progression. Though galectin-12 is significantly downregulated in several tumor entities, including colon cancer, its impact on cellular homeostasis as well as galectin-12 specific binding partners have not been identified so far. We therefore established an experimental strategy which is based on reversible cross-link immunoprecipitation to capture the galectin-12 protein interactome in colon cancer cells. By applying this approach, we identified 10 novel candidates of galectin-12 interacting proteins including the neutral amino acid exchanger SLC1A5. Remarkably, we uncovered that binding of galectin-12 to SLC1A5 significantly reduced glutamine uptake in our model cell line. Consequently, utilization of glutamine carbon for biomass synthesis was profoundly affected, suggesting galectin-12 as a novel inhibitor of glutamine anaplerosis in colon cancer cells. More detailed analysis revealed that colon cancer cells can counteract galectin-12 mediated glutamine deprivation by induction of compensatory mechanisms which facilitate adaption to low-glutamine conditions and thus survival.

Claudin7-dependent exosome-promoted reprogramming of nonmetastasizing tumor cells.

Claudin7 (cld7) is a cancer-initiating cell (CIC) marker in gastrointestinal tumors, a cld7-knockdown (kd) being accompanied by loss of tumor progression. Tumor exosomes (TEX) restoring CIC activities, we explored the contribution of cld7. This became particularly interesting, as tight junction (TJ)- and glycolipid-enriched membrane domain (GEM)-derived cld7 is recruited into distinct TEX. TEXs were derived from CIC or cld7kd cells of a rat pancreatic and a human colon cancer line. TEX derived from pancreatic cancer cld7kd cells rescued with palmitoylation site-deficient cld7 (cld7mP) allowed selectively evaluating the contribution of GEM-derived TEX, only palmitoylated cld7 being integrated into GEM. Cld7 CIC-TEX promoted tumor cell dissemination and metastatic growth without a major impact on proliferation, apoptosis resistance and epithelial-mesenchymal transition. Instead, migration, invasion and (lymph)angiogenesis were strongly supported, only migration being selectively fostered by GEM-derived cld7 TEX. CIC-TEX coculture of cld7kd cells uncovered significant changes in the cld7kd cell protein and miRNA profiles. However, changes did not correspond to the CIC-TEX profile, CIC-TEX rather initiating integrin, protease and RTK, particularly lymphangiogenic receptor activation. CIC-TEX preferentially rescuing cld7kd-associated defects in signal transduction was backed up by an RTK inhibitor neutralizing the impact of CIC-TEX on tumor progression. In conclusion, cld7 contributes to selective steps of the metastatic cascade. Defects of cld7kd and cld7mP cells in migration, invasion and (lymph)angiogenesis are effaced by CIC-TEX that act by signaling cascade activation. Accordingly, RTK inhibitors are an efficient therapeutic defeating CIC-TEX.

Copying Life: Synthesis of an Enzymatically Active Mirror-Image DNA-Ligase Made of D-Amino Acids.

Our objective is the creation of a mirror-image synthetic biology: that is, to mimic, entirely independent of Nature, a biological system and to re-create it from artificial component parts. Utilizing enantiomeric L-nucleotides and D-amino acids rather than the natural components, we use chemical synthesis toward a basic, self-replicating mirror-image biological system. Here, we report the synthesis of a functional DNA-ligase in the D-enantiomeric conformation, which is an exact mirror-image of the natural enzyme, exhibiting DNA ligation activity on chirally inverted nucleic acids in L-conformation, but not acting on natural substrates and with natural co-factors. Starting from the known structure of the Paramecium bursaria chlorella virus 1 DNA-ligase and the homologous but shorter DNA-ligase of Haemophilus influenza, we designed and synthesized chemically peptides, which could then be assembled into a full-length molecule yielding a functional protein. The structure and the activity of the mirror-image ligase were characterized, documenting its enantiospecific functionality.

Modeling and multiscale characterization of the quantitative imaging based fibrosis index reveals pathophysiological, transcriptome and proteomic correlates of lung fibrosis induced by fractionated irradiation.

Pulmonary fibrosis represents a leading cause of morbidity and mortality worldwide. Therapy induced lung fibrosis constitutes a pivotal dose-limiting side effect of radiotherapy and other anticancer agents. We aimed to develop objective criteria for assessment of fibrosis and discover pathophysiological and molecular correlates of lung fibrosis as a function of fractionated whole thoracic irradiation. Dose-response series of fractionated irradiation was utilized to develop a non-invasive and quantitative measure for the degree of fibrosis - the fibrosis index (FI). The correlation of FI with histopathology, blood-gas, transcriptome and proteome responses of the lung tissue was analyzed. Macrophages infiltration and polarization was assessed by immunohistochemistry. Fibrosis development followed a slow kinetic with maximum lung fibrosis levels detected at 24-week post radiation insult. FI favorably correlated with radiation dose and surrogates of lung fibrosis i.e., enhanced pro-inflammatory response, tissue remodeling and extracellular matrix deposition. The loss of lung architecture correlated with decreased epithelial marker, loss of microvascular integrity with decreased endothelial and elevated mesenchymal markers. Lung fibrosis was further attributed to a switch of the inflammatory state toward a macrophage/T-helper cell type 2-like (M2/Th2) polarized phenotype. Together, the multiscale characterization of FI in radiation-induced lung fibrosis (RILF) model identified pathophysiological, transcriptional and proteomic correlates of fibrosis. Pathological immune response and endothelial/epithelial to mesenchymal transition were discovered as critical events governing lung tissue remodeling. FI will be instrumental for deciphering the molecular mechanisms governing lung fibrosis and discovery of novel targets for treatment of this devastating disease with an unmet medical need.

Detection of malignancy-associated phosphoproteome changes in human colorectal cancer induced by cell surface binding of growth-inhibitory galectin-4.

Emerging evidence on efficient tumor growth regulation by endogenous lectins directs interest to determine on a proof-of-principle level the range of information on alterations provided by full-scale analysis using phosphoproteomics. In our pilot study, we tested galectin-4 (gal-4) that is a growth inhibitor for colon cancer cells (CRC), here working with the LS 180 line. In order to cover monitoring of short- and long-term effects stable isotope labeling by amino acids in cell culture-based quantitative phosphoproteomic analyses were conducted on LS 180 cell preparations collected 1 and 72 h after adding gal-4 to the culture medium. After short-term treatment, 981 phosphosites, all of them S/T based, were detected by phosphoproteomics. Changes higher than 1.5-fold were seen for eight sites in seven proteins. Most affected were the BET1 homolog (BET1), whose level of phosphorylation at S50 was about threefold reduced, and centromere protein F (CENPF), extent of phosphorylation at S3119 doubling in gal-4-treated cells. Phosphoproteome analysis after 72 h of treatment revealed marked changes at 33 S/T-based phosphosites from 29 proteins. Prominent increase of phosphorylation was observed for cofilin-1 at position S3. Extent of phosphorylation of the glutamine transporter SLC1A5 at position S503 was decreased by a factor of 3. Altered phosphorylation of BET1, CENPF, and cofilin-1 as well as a significant effect of gal-4 treatment on glutamine uptake by cells were substantiated by independent methods in the Vaco 432, Colo 205, CX 1, and HCT 116 cell lines. With the example of gal-4 which functions as a tumor suppressor in CRC cells, we were able to prove that cell surface binding of the lectin not only markedly influences the cell proteome, but also has a bearing on malignancy-associated intracellular protein phosphorylation. These results underscore the potential of this approach to give further work on elucidating the details of signaling underlying galectin-triggered growth inhibition a clear direction. © 2018 IUBMB Life, 71(3):364-375, 2019.

Chicken GRIFIN: binding partners, developmental course of localization and activation of its lens-specific gene expression by L-Maf/Pax6.

Tissue lectins appear to be involved in a broad range of physiological processes, as reflected for the members of the family of galectins by referring to them as adhesion/growth-regulatory effectors. In order to clarify the significance of galectin presence, key challenges are to define their binding partners and the profile of localization. Having identified the chicken galectin-related interfiber protein (C-GRIFIN) as lens-specific protein present in the main body of adult lens, we here report its interaction with lens proteins in ligand blotting. The assumption for pairing with α-, ß- and δ-crystallins was ascertained by mass spectrometric detection of their presence in eluted fractions obtained by affinity chromatography. Biochemical and immunohistochemical monitoring revealed protein presence from about 3-day-old embryos onwards, mostly in the cytoplasm of elongated posterior cells, later in secondary lens fiber cells. On the level of gene expression, its promoter was activated by transcription factor L-Maf alone and together with Pax6 like a crystallin gene, substantiating C-GRIFIN's status as lens-specific galectin. Using this combined strategy for counterreceptor and expression profiling by bio- and histochemical methods including light, electron and fluorescence microscopy, respective monitoring in lens development can now be taken to the level of the complete galectin family.

Widespread expression of perilipin 5 in normal human tissues and in diseases is restricted to distinct lipid droplet subpopulations.

Diseases associated with the accumulation of lipid droplets are increasing in western countries. Lipid droplet biogenesis, structure and degradation are regulated by proteins of the perilipin family. Perilipin 5 has been shown to regulate basal lipolysis in oxidative tissues. We examine perilipin 5 in normal human tissues and in diseases using protein biochemical and microscopic techniques. Perilipin 5 was constitutively located at small lipid droplets in skeletal myocytes, cardiomyocytes and brown adipocytes. In addition, perilipin 5 was detected in the epithelia of the gastrointestinal and urogenital tract, especially in hepatocytes, the mitochondria-rich parietal cells of the stomach, tubular kidney cells and ductal cells of the salivary gland and pancreas. Granular cytoplasmic expression, without a lipid droplet-bound localization was detected elsewhere. In cardiomyopathies, in skeletal muscle diseases and during hepatocyte steatogenesis, perilipin 5 was upregulated and localized to larger and more numerous lipid droplets. In steatotic human hepatocytes, perilipin 5 was moderately increased and colocalized with perilipins 1 and 2 but not with perilipin 3 at lipid droplets. In liver diseases implicated in alterations of mitochondria, such as mitochondriopathies, alcoholic liver disease, Wilson's disease and acute liver injury, perilipin 5 was frequently localized to small lipid droplets and less in the cytoplasm. In tumorigenesis, perilipin 5 was especially upregulated in lipo-, leio- and rhabdomyosarcoma and hepatocellular and renal cell carcinoma. In summary, our study provides evidence that perilipin 5 is not restricted to certain cell types but localizes to distinct lipid droplet subpopulations reflecting a possible function in oxidative energy supply in normal tissues and in diseases.

Identification of CRKII, CFL1, CNTN1, NME2, and TKT as Novel and Frequent T-Cell Targets in Human IDH-Mutant Glioma.

Purpose: Successful immunotherapies for IDHmut gliomas require better knowledge of T-cell target antigens. Here, we elucidated their antigen repertoire recognized by spontaneous T-cell responses using an unbiased proteomic approach.Experimental Design: Protein fractionations of tissue lysates from IDHmut gliomas (n = 4) were performed. Fractions were tested by IFNγ ELISpot assay for recognition through patients' T cells. Proteins of immunogenic fractions were identified by mass spectrometry and validated by in silico-predicted synthetic long peptides in patients of origin, additional IDHmut glioma patients (n = 16), and healthy donors (n = 13). mRNA and protein expression of immunogenic antigens was analyzed in tumor tissues and IDHmut glioma stem-like cells (GSC). HLA-A*02-restricted T-cell epitopes were functionally determined by short peptides and numbers of antigen-specific T cells by HLA-peptide tetramer analysis.Results: A total of 2,897 proteins were identified in immunogenic tumor fractions. Based on a thorough filter process, 79 proteins were selected as potential T-cell antigens. Twenty-six of these were recognized by the patients' T cells, and five of them (CRKII, CFL1, CNTN1, NME2, and TKT) in up to 56% unrelated IDHmut glioma patients. Most immunogenic tumor-associated antigens (TAA) were expressed in IDHmut gliomas and GSCs, while being almost absent in normal brain tissues. Finally, we identified HLA-A*02-restricted epitopes for CRKII, NME2, and TKT that were recognized by up to 2.82% of antigen-specific peripheral cytotoxic T cells in IDHmut glioma patients.Conclusions: By analyzing the repertoire of T-cell target antigens in IDHmut glioma patients, we identified five novel immunogenic TAAs and confirmed their expression on IDHmut tumors and GSCs. Clin Cancer Res; 24(12); 2951-62. ©2018 AACR.

Two-Step Coimmunoprecipitation (TIP) Enables Efficient and Highly Selective Isolation of Native Protein Complexes.

Coimmunoprecipitation (co-IP) is one of the most frequently used techniques to study protein-protein (PPIs) or protein-nucleic acid interactions (PNIs). However, the presence of coprecipitated contaminants is a well-recognized issue associated with single-step co-IPs. To overcome this limitation, we developed the two-step co-IP (TIP) strategy that enables sequential coimmunoprecipitations of endogenous protein complexes. TIP can be performed with a broad range of mono- and polyclonal antibodies targeting a single protein or different components of a given complex. TIP results in a highly selective enrichment of protein complexes and thus outperforms single-step co-IPs for downstream applications such as mass spectrometry for the identification of PPIs and quantitative PCR for the analysis of PNIs. We benchmarked TIP for the identification of CD95/FAS-interacting proteins in primary human CD4+ T cells, which recapitulated all major known interactors, but also enabled the proteomics discovery of PPM1G and IPO7 as new interaction partners. For its feasibility and high performance, we propose TIP as an advanced tool for the isolation of highly purified protein-protein and protein-nucleic acid complexes under native expression conditions.

Integrin α7 Is a Functional Marker and Potential Therapeutic Target in Glioblastoma.

Functionally relevant markers of glioblastoma stem-like cells (GSCs) have potential for therapeutic targeting to treat this aggressive disease. Here we used generation and screening of thousands of monoclonal antibodies to search for receptors and signaling pathways preferentially enriched in GSCs. We identified integrin α7 (ITGA7) as a major laminin receptor in GSCs and in primary high-grade glioma specimens. Analyses of mRNA profiles in comprehensive datasets revealed that high ITGA7 expression negatively correlated with survival of patients with both low- and high-grade glioma. In vitro and in vivo analyses showed that ITGA7 plays a key functional role in growth and invasiveness of GSCs. We also found that targeting of ITGA7 by RNAi or blocking mAbs impaired laminin-induced signaling, and it led to a significant delay in tumor engraftment plus a strong reduction in tumor size and invasion. Our data, therefore, highlight ITGA7 as a glioblastoma biomarker and candidate therapeutic target.

Database-augmented Mass Spectrometry Analysis of Exosomes Identifies Claudin 3 as a Putative Prostate Cancer Biomarker.

In prostate cancer and other malignancies sensitive and robust biomarkers are lacking or have relevant limitations. Prostate specific antigen (PSA), the only biomarker widely used in prostate cancer, is suffering from low specificity. Exosomes offer new perspectives in the discovery of blood-based biomarkers. Here we present a proof-of principle study for a proteomics-based identification pipeline, implementing existing data sources, to exemplarily identify exosome-based biomarker candidates in prostate cancer.Exosomes from malignant PC3 and benign PNT1A cells and from FBS-containing medium were isolated using sequential ultracentrifugation. Exosome and control samples were analyzed on an LTQ-Orbitrap XL mass spectrometer. Proteomic data is available via ProteomeXchange with identifier PXD003651. We developed a scoring scheme to rank 64 proteins exclusively found in PC3 exosomes, integrating data from four public databases and published mass spectrometry data sets. Among the top candidates, we focused on the tight junction protein claudin 3. Retests under serum-free conditions using immunoblotting and immunogold labeling confirmed the presence of claudin 3 on PC3 exosomes. Claudin 3 levels were determined in the blood plasma of patients with localized (n = 58; 42 with Gleason score 6-7, 16 with Gleason score ≥8) and metastatic prostate cancer (n = 11) compared with patients with benign prostatic hyperplasia (n = 15) and healthy individuals (n = 15) using ELISA, without prior laborious exosome isolation. ANOVA showed different CLDN3 plasma levels in these groups (p = 0.004). CLDN3 levels were higher in patients with Gleason ≥8 tumors compared with patients with benign prostatic hyperplasia (p = 0.012) and Gleason 6-7 tumors (p = 0.029). In patients with localized tumors CLDN3 levels predicted a Gleason score ≥ 8 (AUC = 0.705; p = 0.016) and did not correlate with serum PSA.By using the described workflow claudin 3 was identified and validated as a potential blood-based biomarker in prostate cancer. Furthermore this workflow could serve as a template to be used in other cancer entities.

TGFBR2-dependent alterations of exosomal cargo and functions in DNA mismatch repair-deficient HCT116 colorectal cancer cells.

BACKGROUND: Colorectal cancers (CRCs) that lack DNA mismatch repair function exhibit the microsatellite unstable (MSI) phenotype and are characterized by the accumulation of frameshift mutations at short repetitive DNA sequences (microsatellites). These tumors recurrently show inactivating frameshift mutations in the tumor suppressor Transforming Growth Factor Beta Receptor Type 2 (TGFBR2) thereby abrogating downstream signaling. How altered TGFBR2 signaling affects exosome-mediated communication between MSI tumor cells and their environment has not been resolved. Here, we report on molecular alterations of exosomes shed by MSI cells and the biological response evoked in recipient cells. METHODS: Exosomes were isolated and characterized by electron microscopy, nanoparticle tracking, and western blot analysis. TGFBR2-dependent effects on the cargo and functions of exosomes were studied in a MSI CRC model cell line enabling reconstituted and inducible TGFBR2 expression and signaling. Microsatellite frameshift mutations in exosomal and cellular DNA were examined by PCR-based DNA fragment analysis and exosomal protein profiles were identified by mass spectrometry. Uptake of fluorescent-labeled exosomes by hepatoma recipient cells was monitored by confocal microscopy. TGFBR2-dependent exosomal effects on secreted cytokine levels of recipient cells were analyzed by Luminex technology and ELISA. RESULTS: Frameshift mutation patterns in microsatellite stretches of TGFBR2 and other MSI target genes were found to be reflected in the cargo of MSI CRC-derived exosomes. At the proteome level, reconstituted TGFBR2 expression and signaling uncovered two protein subsets exclusively occurring in exosomes derived from TGFBR2-deficient (14 proteins) or TGFBR2-proficient (five proteins) MSI donor cells. Uptake of these exosomes by recipient cells caused increased secretion (2-6 fold) of specific cytokines (Interleukin-4, Stem Cell Factor, Platelet-derived Growth Factor-B), depending on the TGFBR2 expression status of the tumor cell. CONCLUSION: Our results indicate that the coding MSI phenotype of DNA mismatch repair-deficient CRC cells is maintained in their exosomal DNA. Moreover, we uncovered that a recurrent MSI tumor driver mutation like TGFBR2 can reprogram the protein content of MSI cell-derived exosomes and in turn modulate the cytokine secretion profile of recipient cells. Apart from its diagnostic potential, these TGFBR2-dependent exosomal molecular and proteomic signatures might help to understand the signaling routes used by MSI tumors. Fricke et al. uncovered coding microsatellite instability-associated mutations of colorectal tumor driver genes like TGFBR2 in MSI tumor cellderived exosomes. Depending on the TGFBR2 expression status of their donor cells, shed exosomes show distinct proteomic signatures and promote altered cytokine secretion profiles in recipient cells.

Deciphering the Acute Cellular Phosphoproteome Response to Irradiation with X-rays, Protons and Carbon Ions.

Radiotherapy is a cornerstone of cancer therapy. The recently established particle therapy with raster-scanning protons and carbon ions landmarks a new era in the field of high-precision cancer medicine. However, molecular mechanisms governing radiation induced intracellular signaling remain elusive. Here, we present the first comprehensive proteomic and phosphoproteomic study applying stable isotope labeling by amino acids in cell culture (SILAC) in combination with high-resolution mass spectrometry to decipher cellular response to irradiation with X-rays, protons and carbon ions. At protein expression level limited alterations were observed 2 h post irradiation of human lung adenocarcinoma cells. In contrast, 181 phosphorylation sites were found to be differentially regulated out of which 151 sites were not hitherto attributed to radiation response as revealed by crosscheck with the PhosphoSitePlus database.Radiation-induced phosphorylation of the p(S/T)Q motif was the prevailing regulation pattern affecting proteins involved in DNA damage response signaling. Because radiation doses were selected to produce same level of cell kill and DNA double-strand breakage for each radiation quality, DNA damage responsive phosphorylation sites were regulated to same extent. However, differential phosphorylation between radiation qualities was observed for 55 phosphorylation sites indicating the existence of distinct signaling circuitries induced by X-ray versus particle (proton/carbon) irradiation beyond the canonical DNA damage response. This unexpected finding was confirmed in targeted spike-in experiments using synthetic isotope labeled phosphopeptides. Herewith, we successfully validated uniform DNA damage response signaling coexisting with altered signaling involved in apoptosis and metabolic processes induced by X-ray and particle based treatments.In summary, the comprehensive insight into the radiation-induced phosphoproteome landscape is instructive for the design of functional studies aiming to decipher cellular signaling processes in response to radiotherapy, space radiation or ionizing radiation per se Further, our data will have a significant impact on the ongoing debate about patient treatment modalities.