Jessica Brandi

Secretome protein signature of human pancreatic cancer stem-like cells.

UNLABELLED Emerging research has demonstrated that pancreatic ductal adenocarcinoma (PDAC) contains a sub-population of cancer stem cells (CSCs) characterized by self-renewal, anchorage-independent-growth, long-term proliferation and chemoresistance. The secretome analysis of pancreatic CSCs has not yet been performed, although it may provide insight into tumour/microenvironment interactions and intracellular processes, as well as to identify potential biomarkers. To characterize the secreted proteins of pancreatic CSCs, we performed an iTRAQ-based proteomic analysis to compare the secretomes of Panc1 cancer stem-like cells (Panc1 CSCs) and parental cell line. A total of 72 proteins were found up-/down-regulated in the conditioned medium of Panc1 CSCs. The pathway analysis revealed modulation of vital physiological pathways including glycolysis, gluconeogenesis and pentose phosphate. Through ELISA immunoassays we analysed the presence of the three proteins most highly secreted by Panc1 CSCs (ceruloplasmin, galectin-3, and MARCKS) in sera of PDAC patient. ROC curve analysis suggests ceruloplasmin as promising marker for patients negative for CA19-9. Overall, our study provides a systemic secretome analysis of pancreatic CSCs revealing a number of secreted proteins which participate in pathological conditions including cancer differentiation, invasion and metastasis. They may serve as a valuable pool of proteins from which biomarkers and therapeutic targets can be identified. BIOLOGICAL SIGNIFICANCE The secretome of CSCs is a rich reservoir of biomarkers of cancer progression and molecular therapeutic targets, and thus is a topic of great interest for cancer research. The secretome analysis of pancreatic CSCs has not yet been performed. Recently, our group has demonstrated that Panc-1 CSCs isolated from parental cell line by using the CSC selective medium, represent a model of great importance to deepen the understanding of the biology of pancreatic adenocarcinoma. To our knowledge, this is the first proteomic study of pancreatic CSC secretome. We performed an iTRAQ-based analysis to compare the secretomes of Panc1 CSCs and Panc1 parental cell line and identified a total of 43 proteins secreted at higher level by pancreatic cancer stem cells. We found modulation of different vital physiological pathways (such as glycolysis and gluconeogenesis, pentose phosphate pathway) and the involvement of CSC secreted proteins (for example 72kDa type IV collagenase, galectin-3, alpha-actinin-4, and MARCKS) in pathological conditions including cancer differentiation, invasion and metastasis. By ELISA verification we found that MARCKS and ceruloplasmin discriminate between controls and PDAC patients; in addition ROC curve analyses indicate that MARCKS does not have diagnostic accuracy, while ceruloplasmin could be a promising marker only for patients negative for CA19-9. We think that the findings reported in our manuscript advance the understanding of the pathways implicated in tumourigenesis, metastasis and chemoresistance of pancreatic cancer, and also identify a pool of proteins from which novel candidate diagnostic and therapeutic biomarkers could be discovered.

Pancreatic ductal adenocarcinoma cell lines display a plastic ability to bi‑directionally convert into cancer stem cells

Pancreatic ductal adenocarcinoma (PDAC) is often diagnosed when metastatic events have occurred. Cancer stem cells (CSCs) play an important role in tumor initiation, metastasis, chemoresistance and relapse. A growing number of studies have suggested that CSCs exist in a dynamic equilibrium with more differentiated cancer cells via a bi‑directional regeneration that is dependent on the environmental stimuli. In this investigation, we obtain, by using a selective medium, PDAC CSCs from five out of nine PDAC cell lines, endowed with different tumorsphere‑forming ability. PDAC CSCs were generally more resistant to the action of five anticancer drugs than parental cell lines and were characterized by an increased expression of EpCAM and CD44v6, typical stem cell surface markers, and a decreased expression of E‑cadherin, the main marker of the epithelial state. PDAC CSCs were able to re‑differentiate into parental cells once cultured in parental growth condition, as demonstrated by re‑acquisition of the epithelial morphology, the decreased expression levels of EpCAM and CD44v6 and the increased sensitivity to anticancer drugs. Finally, PDAC CSCs injected into nude mice developed a larger subcutaneous tumor mass and showed a higher metastatic activity compared to parental cells. The present study demonstrates the ability to obtain CSCs from several PDAC cell lines and that these cells are differentially resistant to various anticancer agents. This variability renders them a model of great importance to deeply understand pancreatic adenocarcinoma biology, to discover new biomarkers and to screen new therapeutic compounds.

Proteomic analysis of pancreatic cancer stem cells: Functional role of fatty acid synthesis and mevalonate pathways

Recently, we have shown that the secretome of pancreatic cancer stem cells (CSCs) is characterized by proteins that participate in cancer differentiation, invasion, and metastasis. However, the differentially expressed intracellular proteins that lead to the specific characteristics of pancreatic CSCs have not yet been identified, and as a consequence the deranged metabolic pathways are yet to be elucidated. To identify the modulated proteins of pancreatic CSCs, iTRAQ-based proteomic analysis was performed to compare the proteome of Panc1 CSCs and Panc1 parental cells, identifying 230 modulated proteins. Pathway analysis revealed activation of glycolysis, the pentose phosphate pathway, the pyruvate-malate cycle, and lipid metabolism as well as downregulation of the Krebs cycle, the splicesome and non-homologous end joining. These findings were supported by metabolomics and immunoblotting analysis. It was also found that inhibition of fatty acid synthase by cerulenin and of mevalonate pathways by atorvastatin have a greater anti-proliferative effect on cancer stem cells than parental cells. Taken together, these results clarify some important aspects of the metabolic network signature of pancreatic cancer stem cells, shedding light on key and novel therapeutic targets and suggesting that fatty acid synthesis and mevalonate pathways play a key role in ensuring their viability. BIOLOGICAL SIGNIFICANCE To better understand the altered metabolic pathways of pancreatic cancer stem cells (CSCs), a comprehensive proteomic analysis and metabolite profiling investigation of Panc1 and Panc1 CSCs were carried out. The findings obtained indicate that Panc1 CSCs are characterized by upregulation of glycolysis, pentose phosphate pathway, pyruvate-malate cycle, and lipid metabolism and by downregulation of Krebs cycle, spliceosome and non-homologous end joining. Moreover, fatty acid synthesis and mevalonate pathways are shown to play a critical contribution to the survival of pancreatic cancer stem cells. This study is helpful for broadening the knowledge of pancreatic cancer stem cells and could accelerate the development of novel therapeutic strategies.

The antioxidant uncoupling protein 2 stimulates hnRNPA2/B1, GLUT1 and PKM2 expression and sensitizes pancreas cancer cells to glycolysis inhibition

Several evidence indicate that metabolic alterations play a pivotal role in cancer development. Here, we report that the mitochondrial uncoupling protein 2 (UCP2) sustains the metabolic shift from mitochondrial oxidative phosphorylation (mtOXPHOS) to glycolysis in pancreas cancer cells. Indeed, we show that UCP2 sensitizes pancreas cancer cells to the treatment with the glycolytic inhibitor 2-deoxy-D-glucose. Through a bidimensional electrophoresis analysis, we identify 19 protein species differentially expressed after treatment with the UCP2 inhibitor genipin and, by bioinformatic analyses, we show that these proteins are mainly involved in metabolic processes. In particular, we demonstrate that the antioxidant UCP2 induces the expression of hnRNPA2/B1, which is involved in the regulation of both GLUT1 and PKM2 mRNAs, and of lactate dehydrogenase (LDH) increasing the secretion of L-lactic acid. We further demonstrate that the radical scavenger N-acetyl-L-cysteine reverts hnRNPA2/B1 and PKM2 inhibition by genipin indicating a role for reactive oxygen species in the metabolic reprogramming of cancer cells mediated by UCP2. We also observe an UCP2-dependent decrease in mtOXPHOS complex I (NADH dehydrogenase), complex IV (cytochrome c oxidase), complex V (ATPase) and in mitochondrial oxygen consumption, suggesting a role for UCP2 in the counteraction of pancreatic cancer cellular respiration. All these results reveal novel mechanisms through which UCP2 promotes cancer cell proliferation with the concomitant metabolic shift from mtOXPHOS to the glycolytic pathway.

Comparative proteomic and phosphoproteomic profiling of pancreatic adenocarcinoma cells treated with CB1 or CB2 agonists

The pancreatic adenocarcinoma cell line Panc1 was treated with cannabinoid receptor ligands (arachidonylcyclopropylamide or GW405833) in order to elucidate the molecular mechanism of their anticancer effect. A proteomic approach was used to analyze the protein and phosphoprotein profiles. Western blot and functional data mining were also employed in order to validate results, classify proteins, and explore their potential relationships. We demonstrated that the two cannabinoids act through a widely common mechanism involving up‐ and down‐regulation of proteins related to energetic metabolism and cell growth regulation. Overall, the results reported might contribute to the development of a therapy based on cannabinoids for pancreatic adenocarcinoma.

Trichostatin A alters cytoskeleton and energy metabolism of pancreatic adenocarcinoma cells: an in depth proteomic study

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal of all human cancers with a high mortality rate. Resistance to conventional treatments and chemotherapeutics is a typical feature of PDAC. To investigate the causes of drug resistance it is essential to deeply investigate the mechanism of action of chemotherapeutics. In this study, we performed an in depth shotgun proteomic approach using the label‐free proteomic SWATH‐MS analysis to investigate novel insights of the mechanism of action of the histone deacetylase (HDAC) inhibitor trichostatin A (TSA) in PDAC cells. This proteomic analysis in PaCa44 cells and data elaboration of TSA‐regulated proteins by bioinformatics showed an overall up‐regulation of cytokeratins and other proteins related to the cytoskeleton organization, keratinization, and apoptotic cell death. On the contrary, a large amount of the down‐regulated proteins by TSA treatment belongs to the cellular energetic metabolism and to the machinery of protein synthesis, such as ribosomal proteins, determining synergistic cell growth inhibition by the combined treatment of TSA and the glycolytic inhibitor 2‐deoxy‐d‐glucose in a panel of PDAC cell lines. Data are available via ProteomeXchange with identifier PXD007801.

Tissue proteomics of splenic marginal zone lymphoma

Splenic marginal zone lymphoma (SMZL) is a rare chronic B lymphoproliferative disease, whose molecular pathogenesis has still not been well established. For the first time, a proteomic approach was undertaken to analyse the protein profiles of SMZL tissue. 1D and 2D Western blot, immunohistochemical analysis, and functional data mining were also performed in order to validate results, investigate protein species specific regulation, classify proteins, and explore their potential relationships. We demonstrated that SMZL is characterized by modulation of protein species related to energetic metabolism and apoptosis pathways. We also reported specific protein species (such as biliverdin reductase A, manganese superoxide dismutase, beta‐2 microglobulin, growth factor receptor‐bound protein 2, acidic leucine‐rich nuclear phosphoprotein 32 family member A, and Set nuclear oncogene) directly involved in NF‐kB and BCR pathways, as well as in chromatin remodelling and cytoskeleton. Our findings shed new light on SMZL pathogenesis and provide a basis for the future development of novel biomarkers. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium with the dataset identifier PXD001124.

Proteomic approaches to decipher cancer cell secretome

In this review, we give an overview of the actual proteomic approaches used in the study of cancer cells secretome. In particular, we describe the proteomic strategies to decipher cancer cell secretome initially focusing on the different aspects of sample preparation. We examine the issues related to the presence of low abundant proteins, the analysis of secreted proteins in the conditioned media with or without the removal of fetal bovine serum and strategies developed to reduce intracellular protein contamination. As regards the identification and quantification of secreted proteins, we described the different proteomic approaches used, i.e. gel-based, MS-based (label-based and label-free), and the antibody and array-based methods, together with some of the most recent applications in the field of cancer research. Moreover, we describe the bioinformatics tools developed for the in silico validation and characterization of cancer cells secretome. We also discuss the most important available tools for protein annotation and for prediction of classical and non-classical secreted proteins. In summary in this review advances, concerns and challenges in the field of cancer secretome analysis are discussed.

Myristic acid induces proteomic and secretomic changes associated with steatosis, cytoskeleton remodeling, endoplasmic reticulum stress, protein turnover and exosome release in HepG2 cells

Myristic acid, the 14-carbon saturated fatty acid (C14:0), is associated to an increased cardiovascular disease risk. Since it is found in low concentration in cells, its specific properties have not been fully analyzed. The aim of this study was to explore the cell response to this fatty acid to help explaining clinical findings on the relationship between C14:0 and cardiovascular disease. The human liver HepG2 cell line was used to investigate the hepatic response to C14:0 in a combined proteomic and secretomic approach. A total of 47 intracellular and 32 secreted proteins were deregulated after treatments with different concentrations of C14:0. Data are available via ProteomeXchange (PXD007902). In addition, C14:0 treatment of primary murine hepatocytes confirmed that C14:0 induces lipid droplet accumulation and elevates perilipin-2 levels. Functional enrichment analysis revealed that C14:0 modulates lipid droplet formation and cytoskeleton organization, induce ER stress, changes in exosome and extracellular miRNA sorting in HepG2cells. Our data provide for the first time a proteomic profiling of the effects of C14:0 in human hepatoma cells and contribute to the elucidation of molecular mechanisms through which this fatty acid may cause adverse health effects. BIOLOGICAL SIGNIFICANCE Myristic acid is correlated with an increase in plasma cholesterol and mortality due to cardiovascular diseases. This study is the first example of an integration of proteomic and secretomic analysis of HepG2 cells to investigate the specific properties and functional roles of myristic acid on hepatic cells. Our analyses will lead to a better understanding of the myristic acid induced effects and can elicit new diagnostic and treatment strategies based on altered proteins.

An integrated approach identifies new oncotargets in melanoma

Melanoma is an aggressive skin cancer; an early detection of the primary tumor may improve its prognosis. Despite many genes have been shown to be involved in melanoma, the full framework of melanoma transformation has not been completely explored. The characterization of pathways involved in tumor restraint in in vitro models may help to identify oncotarget genes. We therefore aimed to probe novel oncotargets through an integrated approach involving proteomic, gene expression and bioinformatic analysis We investigated molecular modulations in melanoma cells treated with ascorbic acid, which is known to inhibit cancer growth at high concentrations. For this purpose a proteomic approach was applied. A deeper insight into ascorbic acid anticancer activity was achieved; the discovery of deregulated processes suggested further biomarkers. In addition, we evaluated the expression of identified genes as well as the migration ability in several melanoma cell lines. Data obtained by a multidisciplinary approach demonstrated the involvement of Enolase 1 (ENO1), Parkinsonism-associated deglycase (PARK7), Prostaglansin E synthase 3 (PTGES3), Nucleophosmin (NPM1), Stathmin 1 (STMN1) genes in cell transformation and identified Single stranded DNA binding protein 1 (SSBP1) as a possible onco-suppressor in melanoma cancer.