Contribution of galectin-1, a glycan-binding protein, to gastrointestinal tumor progression

Gastrointestinal cancer is a group of tumors that affect multiple sites of the digestive system, including the stomach, liver, colon and pancreas. These cancers are very aggressive and rapidly metastasize, thus identifying effective targets is crucial for treatment. Galectin-1 (Gal-1) belongs to a family of glycan-binding proteins, or lectins, with the ability to cross-link specific glycoconjugates. A variety of biological activities have been attributed to Gal-1 at different steps of tumor progression. Herein, we summarize the current literature regarding the roles of Gal-1 in gastrointestinal malignancies. Accumulating evidence shows that Gal-1 is drastically up-regulated in human gastric cancer, hepatocellular carcinoma, colorectal cancer and pancreatic ductal adenocarcinoma tissues, both in tumor epithelial and tumor-associated stromal cells. Moreover, Gal-1 makes a crucial contribution to the pathogenesis of gastrointestinal malignancies, favoring tumor development, aggressiveness, metastasis, immunosuppression and angiogenesis. We also highlight that alterations in Gal-1-specific glycoepitopes may be relevant for gastrointestinal cancer progression. Despite the findings obtained so far, further functional studies are still required. Elucidating the precise molecular mechanisms modulated by Gal-1 underlying gastrointestinal tumor progression, might lead to the development of novel Gal-1-based diagnostic methods and/or therapies.Fil: Bacigalupo, Maria Lorena. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas ; ArgentinaFil: Carabias, Pablo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas ; ArgentinaFil: Troncoso, María Fernanda. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas ; Argentin

Tumor-derived exosomes inhibit natural killer cell function in the pre-metastatic niche of pancreatic cancer

Introduction: Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal malignancies worldwide. More than 50% of patients are diagnosed with late-stage disease. Exosomes are a group of extracellular vesicles released by different types of cells, containing proteins, nucleic acids and lipids, mediating intercellular communication, and thus affecting physiological and pathological conditions. Tumor- derived exosomes have been shown to induce a pre-metastatic niche in the target organ to promote metastasis. Methods: We isolated exosomes from cell culture supernatants of a highly metastatic pancreatic cell line L3.6pl and a PDAC patient derived primary cell line TBO368 by ultracentrifugation. Exosomes were characterized by Western blotting, nanoparticle tracking analysis and transmission electron microscopy. The protein content of exosomes was analyzed by mass spectrometry. The potential effects of pancreatic cancer-derived exosomes on NK cells were investigated by immunofluorescence and flow cytometry. The exosomal TGF-β1 levels in serum of patients with PDAC were quantified by ELISA. Results: We found that adhesion receptors, especially integrins such as integrin αv and integrin β5, which are associated with liver-specific metastases, were enriched in pancreatic cancer-derived exosomes. These exosomes also displayed a variety of immune regulatory factors, such as TGF-β1, Nectin-2 and PVR. Then we co-cultured NK cells with exosomes derived from pancreatic cancer cells. After co-culture, the expression of NKG2D, CD107a, TNF-α and INF-γ in NK cells was significantly downregulated. NK cells also exhibited the decreased level of CD71 and CD98, as well as impaired glucose uptake ability. In addition, NK cell cytotoxicity against pancreatic cancer stem cells was attenuated. Moreover, pancreatic cancer-derived exosomes induced the phosphorylation of Smad2/3 in NK cells. Compared to healthy donors, serum exosomal TGF-β1 was significantly increased in patients with PDAC. Conclusion: In this study, we show that tumor-derived exosomes are responsible for pre-metastatic niche formation in the liver of PDAC. The inhibitory effects of pancreatic cancer-derived exosomes on NK cells represent a mechanism allowing metastatic tumor cells to escape from NK cell immune surveillance in the pre-metastatic niche. We also demonstrate that serum exosomal TGF-β1 was significantly increased in patients with PDAC. In conclusion, these findings emphasize the immunosuppressive role of pancreatic cancer-derived exosomes and provide new insights into our understanding of NK cell dysfunction in the pre-metastatic niche formation of PDAC

Lack of a 5.9 kDa peptide C-terminal fragment of fibrinogen α chain precedes fibrosis progression in patients with liver disease

Early detection of fibrosis progression is of major relevance for the diagnosis and management of patients with liver disease. This study was designed to find non-invasive biomarkers for fibrosis in a clinical context where this process occurs rapidly, HCV-positive patients who underwent liver transplantation (LT). We analyzed 93 LT patients with HCV recurrence, 41 non-LT patients with liver disease showing a fibrosis stage F≥1 and 9 patients without HCV recurrence who received antiviral treatment before LT, as control group. Blood obtained from 16 healthy subjects was also analyzed. Serum samples were fractionated by ion exchange chromatography and their proteomic profile was analyzed by SELDI-TOF-MS. Characterization of the peptide of interest was performed by ion chromatography and electrophoresis, followed by tandem mass spectrometry identification. Marked differences were observed between the serum proteome profile of LT patients with early fibrosis recurrence and non-recurrent LT patients. A robust peak intensity located at 5905 m/z was the distinguishing feature of non-recurrent LT patients. However, the same peak was barely detected in recurrent LT patients. Similar results were found when comparing samples of healthy subjects with those of non-LT fibrotic patients, indicating that our findings were not related to either LT or HCV infection. Using tandem mass-spectrometry, we identified the protein peak as a C-terminal fragment of the fibrinogen α chain. Cell culture experiments demonstrated that TGF-β reduces α-fibrinogen mRNA expression and 5905 m/z peak intensity in HepG2 cells, suggesting that TGF-β activity regulates the circulating levels of this protein fragment. In conclusion, we identified a 5.9 kDa C-terminal fragment of the fibrinogen α chain as an early serum biomarker of fibrogenic processes in patients with liver disease

Charakterisierung des sekretierten Proteoms im Adenokaryinom des Pankreas unter Einfluss verschiedenen Stimuli der Mikroumgebung des Tumors

Pancreatic cancer is categorized fourth in all cancer-related mortalities worldwide. Much of the dismal properties of this malignancy are attributable to the high heterogeneity of its microenvironment and the intense desmoplasia formation. A prominent feature of pancreatic desmoplasia is the infiltration of tumor mass with high numbers of activated pancreatic stellate cells (PSCs). In the first part of the study, a prominent observation is the elucidation of TNF-a as a key element in PSC activation.In addition, TNF along with CCL-4, contributes to changing the PSC proteome towards monitoring the endothelial system and angiogenesis. On the other hand, FGF-2 and IL-6 induction led to better cellular survival and decreased apoptotic activity of the stellate cells by the former and a decreasing apoptotic activity through modulation of ionized calcium. Both functional and network analysis have indicated a possible role the transcription factor proto-oncogene c-Fos in PSC activation. I followed up the functional and network analysis from Part I about the potential role of c-Fos as transcriptional regulator of PSCs activation via TNF-a. The treatment with TNF-a in stably transfected PSCs with c-Fos siRNA failed to propagate the cells to express fibrogenic factors. Interestingly, activated PSCs constitutively expressed intra- and extra-cellular TNF-a when grown in normal conditions. The knockdown of c-Fos diminished such capacity suggesting that there is a synergistic feedback loop between TNF-a and c-Fos used by PSCs to maintain proliferation and fibrotic activity independently to exogenous TNF-a from the pancreatic tumor microenvironment. The expression of eIF4E in pancreatic cancer cells were increased by active PSCs. Inhibition of eIF4E activation by siRNA blocked the effect of activated PSCs on pancreatic cancer cells and inhibited tumor growth in vitro. Our findings provides new evidence for the modulation of PSCs by tumor microenvironment and further insight to the role of stromal cells in pancreatic carcinogenesis and cancer progression. The fourth part Our findings indicate that inhibition PSC activation in combination with ribavirin can significantly inhibit the proliferation and migration, induce apoptosis, arrest the cell cycle, and decrease expression of translation initiation genes as modifying several other cancer-related processes in PDAC cells.Pankreaskrebs ist die vierthäufigste Krebstodesursache weltweit. Viele der malignen Eigenschaften dieser Tumorerkrankung sind auf die hohe Heterogenität der Mikroumgebung und die ausgeprägte Desmoplasie zurückzuführen. Ein wesentliches Merkmal der Desmoplasie ist die Infiltration der Tumormasse mit einer hohen Anzahl von aktivierten Sternzellen der Bauchspeicheldrüse (PSCs). Außerdem trägt TNF-a, zusammen mit CCL-4, zu einer Veränderung des PSC-Proteoms bei, welches das endotheliale System und die Angiogenese beeinflusst. Des weiteren konnte gezeigt werden, dass FGF-2 IL-6 ein vermehrtes zelluläres Überleben sowie, durch Modulation des Kalzium-Ionen-Spiegels, eine verringerte apoptotische Aktivität der Sternzellen bewirken. Sowohl funktionelle, als auch Netzwerk-Analysen haben auf eine mögliche Rolle des c-Fos in der PSC-Aktivierung hingewiesen. Im zweiten Teil untersuchten wir, basierend auf den funktionellen und Netzwerk-Analysen des ersten Teils, die potenzielle Rolle von c-Fos als Transkriptionsregulator in der TNF-a-induzierten PSC-Aktivierung. Interessanterweise exprimieren aktivierte PSCs konstitutiv intra- und extrazelluläres TNF-a, wenn sie unter normalen Bedingungen wachsen. Der Knockdown von c-Fos verringert jedoch diese Kapazität, was darauf hindeutet, dass eine synergistische Rückkopplungsschleife zwischen TNF-a und c-Fos existiert, welche von PSCs genutzt wird, um ihre Proliferation und ihre fibrotische Aktivität unabhängig von exogenem TNF-a aus der Pankreastumorumgebung aufrechtzuerhalten. Daneben haben die Ergebnisse aus Proteomanalyse und qRT-PCR gezeigt, dass die Zellüberstände von aktivierten PSCs die Apoptose von Pankreaskarzinom-Zellen reduzieren, sowie deren Proliferation und Migration fördern können. Außerdem wurde die Expression von elF4E in Pankreaskarzinom-Zellen durch aktivierte PSC erhöht. Unterdrückung der eIF4E-Aktivierung mit siRNA hat den Einfluss von aktivierten PSC auf Pankreaskarzinom-Zellen blockiert und das in-vitro Tumorwachstum inhibiert. Anschließend wurden die vielversprechendsten Wirkstoffe in einem Co-Kultur-Modell von Tumorzellen und PSCs sowie in einem Tier-Modell für Pankreasfibrose getestet. Unsere Ergebnisse zeigten, dass eine Hemmung der PSC-Aktivierung mit Ribavirin signifikant die Proliferation und Migration inhibiert, Apoptose induziert, den Zellzyklus anhält. Demzufolge könnte die Behandlung von PSC mit Ribavirin eine vielversprechende Therapie-Strategie bei Pankreaskrebs bieten

Tumour–stroma interactions in an organotypic culture model of pancreatic cancer

PhDPancreatic cancer is characterised by an intense fibrotic, or desmoplastic, stroma, which contributes to tumour progression. Three-dimensional in vitro culture models incorporating this non-tumour component may more closely recapitulate the complex in vivo situation. The aim of my project was to develop a physiologically relevant, three-dimensional organotypic culture model of pancreatic cancer to study the tumour-stroma interactions and its modulation by novel therapeutic agents. Cancer cells cultured on top of collagen/Matrigel gels, embedded with or without stromal cells (hTERT immortalised PS1 stellate cells or MRC5 fibroblasts), differentiated into luminal structures, exhibiting a central apoptotic core with a proliferating peripheral rim and apicobasal polarity. Stromal cells induced a reduction in total tumour cell number, which was associated with a decrease in E-cadherin expression, upregulated β-catenin expression and translocation of ezrin from the apical to the basal aspect of cancer cells, where it was associated with invasive activity. Subsequently, this organotypic model was raised to an air-liquid interface to study the direct and indirect effects of all-trans retinoic acid (ATRA), which rendered stellate cells back to their quiescent phenotype. Indirect effects of quiescent stellate cells on pancreatic cancer cells included changes in proliferation (decrease), apoptosis (increase), invasion (decrease), Wnt/β-catenin signalling (decrease) and an altered morphology. The Wnt/β-catenin signalling 6 perturbations were mediated by restoration of sFRP4 (secreted frizzled-related protein 4) secretion by quiescent stellate cells, resulting in reduced cancer cell invasion (reporter and invasion assays). All such observations could be validated in human pancreatic cancer tissue samples. Taken together, pancreatic organotypic culture offers a reproducible, in vitro three-dimensional culture model, which allows the study of tumour-stroma interactions in a physiologically relevant system. For treatment of pancreatic cancer, a tumour characterised by a poor response to conventional chemotherapeutic drugs, targeting the tumour-stroma cross-talk with agents such as ATRA offers an exciting novel therapeutic strategy.European Association of Cancer Researc

Proteomic analysis of immediate-early response plasma proteins after 70% and 90% partial hepatectomy

AIM: Partial hepatectomy (PH) induces robust hepatic regenerative and metabolic responses that are considered to be triggered by humoral factors. The aim of the study was to identify plasma protein factors that potentially trigger or reflect the body's immediate-early responses to liver mass reduction. METHODS: Male C57BL/6 mice were subjected to sham operation, 70% PH or 90% PH. Blood was collected from the inferior vena cava at 20, 60 and 180 min after surgery. RESULTS: Using a label-free quantitative mass spectrometry-based proteomics approach, we identified 399 proteins exhibiting significant changes in plasma expression between any two groups. Of the 399 proteins, 167 proteins had multiple unique sequences and high peptide ID confidence (>90%) and were defined as priority 1 proteins. A group of plasma proteins largely associated with metabolism is enriched after 70% PH. Among the plasma proteins that respond to 90% PH are a dominant group of proteins that are also associated with metabolism and one known cytokine (platelet factor 4). Ninety percent PH and 70% PH induces similar changes in plasma protein profile. CONCLUSION: Our findings enable us to gain insight into the immediate-early response of plasma proteins to liver mass loss. Our data support the notion that increased metabolic demands of the body after massive liver mass loss may function as a sensor that calibrates hepatic regenerative response

The role of Heparin-binding proteins in normal pancreas and acute pancreatitis

Acute pancreatitis (AP) is a leading cause for hospitalisation and has significant quality of life implications for the patient and cost implications for the National Health Service. Although most episodes of AP are mild and self-limiting, the severe form of the disease is associated with a high mortality. In the absence of definitive treatment, management is mainly supportive. There is an urgent need to develop more effective biomarkers and drugs to manage AP. Genome-wide studies have demonstrated that proteins that bind to heparin (HBPs) form highly interconnected networks which are functionally important in health and disease. It was hypothesized that this is true in the pancreas and in AP. Testing this hypothesis, using mRNA as a proxy for protein, it was shown that HBPs constitute an important extracellular sub-proteome within the normal pancreas and in major pancreatic diseases that is likely to provide a rich repository of potential biomarkers and drug targets. Building upon this work, a proteomic analysis of HBPs in normal pancreas (NP) and in caerulein-induced mouse AP was undertaken. This has more than doubled the number of HBPs to 883, with 460 new HBPs identified. These may represent the most interconnected set of extracellular proteins and therefore with the greatest regulatory potential. Non canonical HBPs such as NDUFS4, NDUFS6, NDUFS7, NDUFS8, NDUFA9, NDUFA10, NDUFA9 and NDUFA10 were identified and found to be underexpressed in AP as compared to NP. These may have potential moonlighting roles, not previously known. By virtue of being extracellular and binding to heparin, HBPs are accessible and are potential biomarkers and drug targets in AP. In addition to identifying existing biomarkers in AP such as pancreatic amylase, a number of HBPs with biomarkers potential such as HRG, CD14 and FN1 were identified and need further investigation. HBPs such as SERPINC1, VEGFA and PIP5K1C need further evaluation in drug development. These along with modified heparins, heparin mimetics and matrix therapy in AP provide exciting areas for future research

Pathophysiological implications of urinary peptides in hepatocellular carcinoma

SIMPLE SUMMARY: In this study, the application of capillary electrophoresis mass spectrometry enabled identification of 31 urinary peptides significantly associated with hepatocellular carcinoma diagnosis and prognosis. Further assessment of these peptides lead to prediction of cellular proteases involved in their development namely Meprin A subunit α and Kallikrein-6. Subsequent identification of the proteases was verified by immunohistochemistry in normal liver, cirrhosis and hepatocellular carcinoma. Histopathological assessment of the proteases revealed numerical gradient staining signifying their involvement in liver fibrosis and hepatocellular carcinoma formation. The discovered urinary peptides offered a potential noninvasive tool for diagnosis and prognosis of hepatocellular carcinoma. ABSTRACT: Hepatocellular carcinoma (HCC) is known to be associated with protein alterations and extracellular fibrous deposition. We investigated the urinary proteomic profiles of HCC patients in this prospective cross sectional multicentre study. 195 patients were recruited from the UK (Coventry) and Germany (Hannover) between 1 January 2013 and 30 June 2019. Out of these, 57 were HCC patients with a background of liver cirrhosis (LC) and 138 were non-HCC controls; 72 patients with LC, 57 with non-cirrhotic liver disease and 9 with normal liver function. Analysis of the urine samples was performed by capillary electrophoresis (CE) coupled to mass spectrometry (MS). Peptide sequences were obtained and 31 specific peptide markers for HCC were identified and further integrated into a multivariate classification model. The peptide model demonstrated 79.5% sensitivity and 85.1% specificity (95% CI: 0.81–0.93, p < 0.0001) for HCC and 4.1-fold increased risk of death (95% CI: 1.7–9.8, p = 0.0005). Proteases potentially involved in HCC progression were mapped to the N- and C-terminal sequence motifs of the CE-MS peptide markers. In silico protease prediction revealed that kallikrein-6 (KLK6) elicits increased activity, whilst Meprin A subunit α (MEP1A) has reduced activity in HCC compared to the controls. Tissue expression of KLK6 and MEP1A was subsequently verified by immunohistochemistry

Proteomic characterisation of prostate cancer intercellular communication reveals cell type-selective signalling and TMSB4X-dependent fibroblast reprogramming

Background: In prostate cancer, the tumour microenvironment (TME) represents an important regulator of disease progression and response to treatment. In the TME, cancer-associated fbroblasts (CAFs) play a key role in tumour progression, however the mechanisms underpinning fbroblast-cancer cell interactions are incompletely resolved. Here, we address this by applying cell type-specifc labelling with amino acid precursors (CTAP) and mass spectrometry (MS)-based (phospho) proteomics to prostate cancer for the frst time. Methods: Reciprocal interactions between PC3 prostate cancer cells co-cultured with WPMY-1 prostatic fbroblasts were characterised using CTAP-MS. Signalling network changes were determined using Metascape and Enrichr and visualised using Cytoscape. Thymosin β4 (TMSB4X) overexpression was achieved via retroviral transduction and assayed by ELISA. Cell motility was determined using Transwell and random cell migration assays and expression of CAF markers by indirect immunofuorescence. Results: WPMY-1 cells co-cultured with PC3s demonstrated a CAF-like phenotype, characterised by enhanced PDGFRB expression and alterations in signalling pathways regulating epithelial-mesenchymal transition, cytoskeletal organisation and cell polarisation. In contrast, co-cultured PC3 cells exhibited more modest network changes, with alterations in mTORC1 signalling and regulation of the actin cytoskeleton. The expression of the actin binding protein TMSB4X was signifcantly decreased in co-cultured WPMY-1 fbroblasts, and overexpression of TMSB4X in fbroblasts decreased migration of cocultured PC3 cells, reduced fbroblast motility, and protected the fbroblasts from being educated to a CAF-like phenotype by prostate cancer cells. Conclusions: This study highlights the potential of CTAP-MS to characterise intercellular communication within the prostate TME and identify regulators of cellular crosstalk such as TMSB4X.Yunjian Wu, Kimberley C. Clark, Elizabeth V. Nguyen, Birunthi Niranjan, Lisa G. Horvath, Renea A. Taylor, Roger J. Dal