Regulation of succinate dehydrogenase and role of succinate in cancer.

Succinate dehydrogenase (SDH) has been classically considered a mitochondrial enzyme with the unique property to participate in both the citric acid cycle and the electron transport chain. However, in recent years, several studies have highlighted the role of the SDH substrate, i.e. succinate, in biological processes other than metabolism, tumorigenesis being the most remarkable. For this reason, SDH has now been defined a tumor suppressor and succinate an oncometabolite. In this review, we discuss recent findings regarding alterations in SDH activity leading to succinate accumulation, which include SDH mutations, regulation of mRNA expression, post-translational modifications and endogenous SDH inhibitors. Further, we report an extensive examination of the role of succinate in cancer development through the induction of epigenetic and metabolic alterations and the effects on epithelial to mesenchymal transition, cell migration and invasion, and angiogenesis. Finally, we have focused on succinate and SDH as diagnostic markers for cancers having altered SDH expression/activity.

Secreted molecules inducing epithelial-to-mesenchymal transition in cancer development.

The epithelial-mesenchymal transition (EMT) is a biologic process that allows a polarized epithelial cell to undergo multiple biochemical changes that enable it to assume a mesenchymal cell phenotype. EMT is involved in embryo development, wound healing, tissue regeneration, organ fibrosis and has also been proposed as the critical mechanism for the acquisition of malignant phenotypes by epithelial cancer cells. These cells have been shown to acquire a mesenchymal phenotype when localized at the invasive front of primary tumours increasing aggressiveness, invasiveness, metastatic potential and resistance to chemotherapy. There is now increasing evidence demonstrating that a crucial role in the development of this process is played by factors secreted by cells of the tumour microenvironment or by the tumour cells themselves. This review summarises the current knowledge of EMT induction in cancer by paracrine or autocrine mechanisms, by exosomes or free proteins and miRNAs.

Mutant p53 induces SIRT3/MnSOD axis to moderate ROS production in melanoma cells.

The TP53 tumor suppressor gene is the most frequently altered gene in tumors and mutant p53 isoforms can acquire oncogenic properties referred to as gain-of-function (GOF). In this study, we used wild-type (A375) and mutant p53 (MeWo) melanoma cell lines to assess the regulation of the mitochondrial antioxidant manganese superoxide dismutase (MnSOD) by mutant p53. The effects of mutant p53 were evaluated by qPCR, immunoblotting, enzyme activity assay, cell proliferation assay, reactive oxygen species (ROS) assay after cellular transfection. We demonstrate that mutant p53 induces MnSOD expression, which is recovered by the ROS scavenger N-acetyl-l-cysteine. This suggests MnSOD induction as a defense mechanism of melanoma cells to counterbalance the pro-oxidant conditions induced by mutant p53. We also demonstrate that mutant p53 induces the expression of Sirtuin3 (SIRT3), a major mitochondrial NAD+-dependent deacetylase, stimulating MnSOD deacetylation and enzymatic activity. Indeed, the restoration of SIRT3 reverses MnSOD activity decrease by mutant p53 knock-down. Finally, MnSOD knock-down further enhances mutant p53-mediated ROS increase, counteracting mutp53-dependent cell hyperproliferation. This indicates that SIRT3 and MnSOD act to maintain ROS levels controlled to promote cell proliferation and survival, providing new therapeutic opportunities to be further considered for clinical studies in cancer patients bearing mutant TP53 gene.

Extracellular Matrix Composition Modulates the Responsiveness of Differentiated and Stem Pancreatic Cancer Cells to Lipophilic Derivate of Gemcitabine.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal disease. Gemcitabine (GEM) is used as the gold standard drug in PDAC treatment. However, due to its poor efficacy, it remains urgent to identify novel strategies to overcome resistance issues. In this context, an intense stroma reaction and the presence of cancer stem cells (CSCs) have been shown to influence PDAC aggressiveness, metastatic potential, and chemoresistance. METHODS: We used three-dimensional (3D) organotypic cultures grown on an extracellular matrix composed of Matrigel or collagen I to test the effect of the new potential therapeutic prodrug 4-(N)-stearoyl-GEM, called C18GEM. We analyzed C18GEM cytotoxic activity, intracellular uptake, apoptosis, necrosis, and autophagy induction in both Panc1 cell line (P) and their derived CSCs. RESULTS: PDAC CSCs show higher sensitivity to C18GEM treatment when cultured in both two-dimensional (2D) and 3D conditions, especially on collagen I, in comparison to GEM. The intracellular uptake mechanisms of C18GEM are mainly due to membrane nucleoside transporters' expression and fatty acid translocase CD36 in Panc1 P cells and to clathrin-mediated endocytosis and CD36 in Panc1 CSCs. Furthermore, C18GEM induces an increase in cell death compared to GEM in both cell lines grown on 2D and 3D cultures. Finally, C18GEM stimulated protective autophagy in Panc1 P and CSCs cultured on 3D conditions. CONCLUSION: We propose C18GEM together with autophagy inhibitors as a valid alternative therapeutic approach in PDAC treatment.

Regulation of Autophagy by Nuclear GAPDH and Its Aggregates in Cancer and Neurodegenerative Disorders.

Several studies indicate that the cytosolic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has pleiotropic functions independent of its canonical role in glycolysis. The GAPDH functional diversity is mainly due to post-translational modifications in different amino acid residues or due to protein-protein interactions altering its localization from cytosol to nucleus, mitochondria or extracellular microenvironment. Non-glycolytic functions of GAPDH include the regulation of cell death, autophagy, DNA repair and RNA export, and they are observed in physiological and pathological conditions as cancer and neurodegenerative disorders. In disease, the knowledge of the mechanisms regarding GAPDH-mediated cell death is becoming fundamental for the identification of novel therapies. Here, we elucidate the correlation between autophagy and GAPDH in cancer, describing the molecular mechanisms involved and its impact in cancer development. Since autophagy is a degradative pathway associated with the regulation of cell death, we discuss recent evidence supporting GAPDH as a therapeutic target for autophagy regulation in cancer therapy. Furthermore, we summarize the molecular mechanisms and the cellular effects of GAPDH aggregates, which are correlated with mitochondrial malfunctions and can be considered a potential therapeutic target for various diseases, including cancer and neurodegenerative disorders.

The metabolic landscape of cancer stem cells.

Cancer stem cells (CSCs) are a sub-population of quiescent cells endowed with self-renewal properties that can sustain the malignant behavior of the tumor mass giving rise to more differentiated cancer cells. For this reason, the specific killing of CSCs represents one of the most important challenges of the modern molecular oncology. However, their particular resistance to traditional chemotherapy and radiotherapy imposes a thorough understanding of their biological and biochemical features. The metabolic peculiarities of CSCs may be a therapeutic and diagnostic opportunity in cancer research. In this review, we summarize the most significant discoveries on the metabolism of CSCs describing and critically analyzing the studies supporting either glycolysis or mitochondrial oxidative phosphorylation as a primary source of energy for CSCs.

UCP2, a mitochondrial protein regulated at multiple levels.

An ever-increasing number of studies highlight the role of uncoupling protein 2 (UCP2) in a broad range of physiological and pathological processes. The knowledge of the molecular mechanisms of UCP2 regulation is becoming fundamental in both the comprehension of UCP2-related physiological events and the identification of novel therapeutic strategies based on UCP2 modulation. The study of UCP2 regulation is a fast-moving field. Recently, several research groups have made a great effort to thoroughly understand the various molecular mechanisms at the basis of UCP2 regulation. In this review, we describe novel findings concerning events that can occur in a concerted manner at various levels: Ucp2 gene mutation (single nucleotide polymorphisms), UCP2 mRNA and protein expression (transcriptional, translational, and protein turn-over regulation), UCP2 proton conductance (ligands and post-transcriptional modifications), and nutritional and pharmacological regulation of UCP2.

UCP2 inhibition triggers ROS-dependent nuclear translocation of GAPDH and autophagic cell death in pancreatic adenocarcinoma cells.

Mitochondrial uncoupling protein 2 (UCP2) can moderate oxidative stress by favoring the influx of protons into the mitochondrial matrix, thus reducing electron leakage from respiratory chain and mitochondrial superoxide production. Here, we demonstrate that UCP2 inhibition by genipin or UCP2 siRNA strongly increases reactive oxygen species (ROS) production inhibiting pancreatic adenocarcinoma cell growth. We also show that UCP2 inhibition triggers ROS-dependent nuclear translocation of the glycolytic enzyme glyceraldehyde 3-phosphate dehydrogenase (GAPDH), formation of autophagosomes, and the expression of the autophagy marker LC3-II. Consistently, UCP2 over-expression significantly reduces basal autophagy confirming the anti-autophagic role of UCP2. Furthermore, we demonstrate that autophagy induced by UCP2 inhibition determines a ROS-dependent cell death, as indicated by the apoptosis decrease in the presence of the autophagy inhibitors chloroquine (CQ) or 3-methyladenine (3-MA), or the radical scavenger NAC. Intriguingly, the autophagy induced by genipin is able to potentiate the autophagic cell death triggered by gemcitabine, the standard chemotherapeutic drug for pancreatic adenocarcinoma, supporting the development of an anti-cancer therapy based on UCP2 inhibition associated to standard chemotherapy. Our results demonstrate for the first time that UCP2 plays a role in autophagy regulation bringing new insights into mitochondrial uncoupling protein field.

Targeting gemcitabine containing liposomes to CD44 expressing pancreatic adenocarcinoma cells causes an increase in the antitumoral activity.

Pancreatic adenocarcinoma is often diagnosed when metastatic events have occurred. The early spread of circulating cancer cells expressing the CD44 receptor may play a crucial role in this process. In this study, we have investigated the cellular delivery ability and both in vitro and in vivo anti-tumoral activity of liposomes conjugated with two different low molecular weight hyaluronic acids (HA 4.8kDa and HA 12kDa), the primary ligand of CD44, and containing a lipophilic gemcitabine (GEM) pro-drug. By confocal microscopy and flow cytometry analyses, we demonstrate that the cellular uptake into a highly CD44-expressing pancreatic adenocarcinoma cell line is higher with HA-conjugated (12kDa>4.8kDa) than non-conjugated liposomes. Consistently, in vitro cytotoxic assays display an increased sensitivity towards GEM containing HA-liposomes, compared to non-conjugated liposomes. Conversely, CD44 non-expressing normal cells show a similar uptake and in vitro cytotoxicity with both HA-conjugated and non-conjugated liposomes. Furthermore, we demonstrate that the HA-liposomes are taken up into the cells via lipid raft-mediated endocytosis. All the liposome formulations containing GEM show a higher antitumoral activity than free GEM in a mouse xenograft tumor model of human pancreatic adenocarcinoma. The 12kDa HA-liposomes have the strongest efficiency, while non-conjugated liposomes and the 4.8kDa HA-liposomes are similarly active. Taken together, our results provide a strong rationale for further development of HA-conjugated liposomes to treat pancreatic adenocarcinoma.

Role of mitochondrial uncoupling protein 2 in cancer cell resistance to gemcitabine.

Cancer cells exhibit an endogenous constitutive oxidative stress higher than that of normal cells, which renders tumours vulnerable to further reactive oxygen species (ROS) production. Mitochondrial uncoupling protein 2 (UCP2) can mitigate oxidative stress by increasing the influx of protons into the mitochondrial matrix and reducing electron leakage and mitochondrial superoxide generation. Here, we demonstrate that chemical uncouplers or UCP2 over-expression strongly decrease mitochondrial superoxide induction by the anticancer drug gemcitabine (GEM) and protect cancer cells from GEM-induced apoptosis. Moreover, we show that GEM IC(50) values well correlate with the endogenous level of UCP2 mRNA, suggesting a critical role for mitochondrial uncoupling in GEM resistance. Interestingly, GEM treatment stimulates UCP2 mRNA expression suggesting that mitochondrial uncoupling could have a role also in the acquired resistance to GEM. Conversely, UCP2 inhibition by genipin or UCP2 mRNA silencing strongly enhances GEM-induced mitochondrial superoxide generation and apoptosis, synergistically inhibiting cancer cell proliferation. These events are significantly reduced by the addition of the radical scavenger N-acetyl-l-cysteine or MnSOD over-expression, demonstrating a critical role of the oxidative stress. Normal primary fibroblasts are much less sensitive to GEM/genipin combination. Our results demonstrate for the first time that UCP2 has a role in cancer cell resistance to GEM supporting the development of an anti-cancer therapy based on UCP2 inhibition associated to GEM treatment.

Autophagy induced by p53-reactivating molecules protects pancreatic cancer cells from apoptosis.

TP53 mutations compromising p53 transcriptional function occur in more than 50 % of human cancers, including pancreatic adenocarcinoma, and render cancer cells more resistant to conventional therapy. In the last few years, many efforts have been addressed to identify p53-reactivating molecules able to restore the wild-type transcriptionally competent conformation of the mutated proteins. Here, we show that two of these compounds, CP-31398 and RITA, induce cell growth inhibition, apoptosis, and autophagy by activating p53/DNA binding and p53 phosphorylation (Ser15), without affecting the total p53 amount. These effects occur in both wild-type and mutant p53 pancreatic adenocarcinoma cell lines, whereas they are much less pronounced in normal human primary fibroblasts. Furthermore, CP-31398 and RITA regulate the axis SESN1-2/AMPK/mTOR by inducing AMPK phosphorylation on Thr172, which has a crucial role in the autophagic response. The protective role of autophagy in cell growth inhibition by CP-31398 and RITA is supported by the finding that the AMPK inhibitor compound C or the autophagy inhibitors chloroquine or 3-methyladenine sensitize both pancreatic adenocarcinoma cell lines to the apoptotic response induced by p53-reactivating molecules. Our results demonstrate for the first time a survival role for autophagy induced by p53-reactivating molecules, supporting the development of an anti-cancer therapy based on autophagy inhibition associated to p53 activation.

Expression of the antiapoptotic protein BAG3 is a feature of pancreatic adenocarcinoma and its overexpression is associated with poorer survival.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most deadly cancers, being the fourth leading cause of cancer-related deaths. Long-term survival reaching 15% is achieved in less than 5% of patients who undergo surgery, and median survival is only 6 months in those with inoperable lesions. A deeper understanding of PDAC biologic characteristics as well as novel prognostic markers are therefore required to improve outcomes. Herein we report that BAG3, a protein with recognized anti-apoptotic activity, was expressed in 346 PDACs analyzed, but was not expressed in the surrounding nonneoplastic tissue. In a cohort of 66 patients who underwent radical resection (R0), survival was significantly shorter in patients with high BAG3 expression (median, 12 months) than in those with low BAG3 expression (median, 23 months) (P = 0.001). Furthermore, we report that BAG3 expression in PDAC-derived cell lines protects from apoptosis and confers resistance to gemcitabine, offering a partial explanation for the survival data. Our results indicate that BAG3 has a relevant role in PDAC biology, and suggest that BAG3 expression level might be a potential marker for prediction of patient outcome.

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.

Mitochondrial Elongation and OPA1 Play Crucial Roles during the Stemness Acquisition Process in Pancreatic Ductal Adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is the most common type of pancreatic cancer with an overall 5-year survival rate of less than 9%. The high aggressiveness of PDAC is linked to the presence of a subpopulation of cancer cells with a greater tumorigenic capacity, generically called cancer stem cells (CSCs). CSCs present a heterogeneous metabolic profile that might be supported by an adaptation of mitochondrial function; however, the role of this organelle in the development and maintenance of CSCs remains controversial. To determine the role of mitochondria in CSCs over longer periods, which may reflect more accurately their quiescent state, we studied the mitochondrial physiology in CSCs at short-, medium-, and long-term culture periods. We found that CSCs show a significant increase in mitochondrial mass, more mitochondrial fusion, and higher mRNA expression of genes involved in mitochondrial biogenesis than parental cells. These changes are accompanied by a regulation of the activities of OXPHOS complexes II and IV. Furthermore, the protein OPA1, which is involved in mitochondrial dynamics, is overexpressed in CSCs and modulates the tumorsphere formation. Our findings indicate that CSCs undergo mitochondrial remodeling during the stemness acquisition process, which could be exploited as a promising therapeutic target against pancreatic CSCs.

Proteomics in pancreatic cancer research.

In this review, we give an overview of the actual role of proteomic technologies in the study of pancreatic cancers (PCs). We describe PC proteomics on the basis of sample origins, i.e. tissues, body fluids, and PC cell lines. As regards PC tissues, we report the identification of a number of candidate biomarkers of precursor lesions that may allow early diagnosis of this neoplasia. Moreover, we describe cytoskeletal and hypoxia-regulated proteins that confirm the involvement of cytoskeleton modifications and metabolism adaptations in carcinogenesis. We also discuss the most important biomarkers identified by proteomic analysis involved in local invasion and distant metastasis, and in the cross-talk between pancreatic tumor and the surrounding stroma. Furthermore, we report novel candidate biomarkers identified in serum, plasma, and pancreatic juice of cancer patients compared with cancer-free controls. Proteomic alterations in PC cell line models as compared to normal controls and studies on cell lines treated with drugs or new agents to understand their mechanism of pharmacological action or the onset of drug resistance are also presented. Finally, we discuss the recent improvements obtained in classical 2-DE and high-throughput proteomic strategies able to allow the overcoming of relevant proteomic drawbacks.

MeCP2/H3meK9 are involved in IL-6 gene silencing in pancreatic adenocarcinoma cell lines.

The aim of the present study was to analyse the molecular mechanisms involved in the Interleukin-6 (IL-6) silencing in pancreatic adenocarcinoma cell lines. Our results demonstrate that TNF-alpha, a major IL-6 inducer, is able to induce IL-6 only in three out of six cell lines examined. 5-aza-2'-deoxycytidine (DAC), but not trichostatin A (TSA), activates the expression of IL-6 in all cell lines, indicating that DNA methylation, but not histone deacetylation, plays an essential role in IL-6 silencing. Indeed, the IL-6 upstream region shows a methylation status that correlates with IL-6 expression and binds MeCP2 and H3meK9 only in the non-expressing cell lines. Our results suggest that critical methylations located from positions -666 to -426 relative to the transcription start site of IL-6 may act as binding sites for MeCP2.

Integrated lipidomics and proteomics reveal cardiolipin alterations, upregulation of HADHA and long chain fatty acids in pancreatic cancer stem cells.

Pancreatic cancer stem cells (PCSCs) play a key role in the aggressiveness of pancreatic ductal adenocarcinomas (PDAC); however, little is known about their signaling and metabolic pathways. Here we show that PCSCs have specific and common proteome and lipidome modulations. PCSCs displayed downregulation of lactate dehydrogenase A chain, and upregulation of trifunctional enzyme subunit alpha. The upregulated proteins of PCSCs are mainly involved in fatty acid (FA) elongation and biosynthesis of unsaturated FAs. Accordingly, lipidomics reveals an increase in long and very long-chain unsaturated FAs, which are products of fatty acid elongase-5 predicted as a key gene. Moreover, lipidomics showed the induction in PCSCs of molecular species of cardiolipin with mixed incorporation of 16:0, 18:1, and 18:2 acyl chains. Our data indicate a crucial role of FA elongation and alteration in cardiolipin acyl chain composition in PCSCs, representing attractive therapeutic targets in PDAC.

Progressively De-Differentiated Pancreatic Cancer Cells Shift from Glycolysis to Oxidative Metabolism and Gain a Quiescent Stem State.

Pancreatic ductal adenocarcinoma (PDAC) is typically characterized by high chemoresistance and metastatic spread, features mainly attributable to cancer stem cells (CSCs). It is of central interest the characterization of CSCs and, in particular, the study of their metabolic features in order to selectively identify their peculiarities for an efficient therapeutic approach. In this study, CSCs have been obtained by culturing different PDAC cell lines with a specific growth medium. Cells were characterized for the typical stem/mesenchymal properties at short-, medium-, and long-term culture. Metabolomics, proteomics, analysis of oxygen consumption rate in live cells, and the effect of the inhibition of lactate transporter on cell proliferation have been performed to delineate the metabolism of CSCs. We show that gradually de-differentiated pancreatic cancer cells progressively increase the expression of both stem and epithelial-to-mesenchymal transition markers, shift their metabolism from a glycolytic to an oxidative one, and lastly gain a quiescent state. These quiescent stem cells are characterized by high chemo-resistance, clonogenic ability, and metastatic potential. Re-differentiation reverts these features, re-activating their proliferative capacity and glycolytic metabolism, which generally correlates with high aggressiveness. These observations add an important piece of knowledge to the comprehension of the biology of CSCs, whose metabolic plasticity could be exploited for the generation of promising and selective therapeutic approaches for PDAC patients.

Synergistic effect of trichostatin A and 5-aza-2'-deoxycytidine on growth inhibition of pancreatic endocrine tumour cell lines: a proteomic study.

Our research group recently reported that pancreatic endocrine cancer cell lines are sensitive to the HDAC inhibitor trichostatin A (TSA). In the present paper, we show that the combined treatment of pancreatic endocrine tumour cell lines with TSA and the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine (DAC) determines a strong synergistic inhibition of proliferation mainly due to apoptotic cell death. Proteomic analysis demonstrates that the modulation of specific proteins correlates with the antiproliferative effect of the drugs. A schematic network clarifies the most important targets or pathways involved in pancreatic endocrine cancer growth inhibition by single or combined drug treatments, which include proteasome, mitochondrial apoptotic pathway and caspase related proteins, p53 and Ras related proteins. A comparison between the patterns of proteins regulated by TSA or DAC in endocrine and ductal pancreatic cancer cell lines is also presented.