Divergent Impact of Enzyme Replacement Therapy on Human Cardiomyocytes and Enterocytes Affected by Fabry Disease: Correlation with Mannose-6-phosphate Receptor Expression.

Background: The impact of enzyme replacement therapy (ERT) on cardiomyocytes and intestinal cells, affected by Fabry disease (FD), is still unclear. Methods: Six patients with FD, including five family members with GLA mutation c.666delC and one with GLA mutation c.658C > T, manifesting cardiomyopathy and intestinal symptoms (abdominal pain, diarrhea and malabsorption) were included in the study. Clinical outcome, cardiac magnetic resonance (CMR), endomyocardial and gastro-intestinal biopsies were evaluated before and after 2 years of treatment with agalsidase-α (0.2 mg/kg every other week). Immunohistochemistry and Western blot assessments of mannose-6-phosphate receptors (IGF-II-R) on intestinal and myocardial frozen tissue were obtained at diagnosis and after 2 years of ERT. Results: After ERT left ventricular maximal wall thickness, ranging from pre (<10.5 mm) to mild (<15 mm) and moderate hypertrophy (16 mm), was not associated with significant changes at CMR. Degree of dyspnea, mean cardiomyocyte diameter and % vacuolated areas of cardiomyocytes, representing intracellular GL3, remained unmodified. In contrast, intestinal symptoms improved with disappearance of diarrhea, recovery of anemia and weight gain, correlating with near complete clearance of the enterocytes from GL3 inclusions. IGF-II-R expression was remarkably higher even at histochemistry in intestinal tissue compared with myocardium (p < 0.001) either at baseline and after ERT, thus justifying intestinal recovery. Conclusions: Human cells affected by FD may respond differently to ERT: while cardiomyocytes retain their GL3 content after 2 years of treatment, gastro-intestinal cells show GL3 removal with recovery of function. This divergent response may be related to differences in cellular turnover, as well as tissue IGF-II-R expression.

CD73 induces GM-CSF/MDSC-mediated suppression of T cells to accelerate pancreatic cancer pathogenesis.

Metabolic alterations regulate cancer aggressiveness and immune responses. Given the poor response of pancreatic ductal adenocarcinoma (PDAC) to conventional immunotherapies, we investigated the link between metabolic alterations and immunosuppression. Our metabolic enzyme screen indicated that elevated expression of CD73, an ecto-5'-nucleotidase that generates adenosine, correlates with increased aggressiveness. Correspondingly, we observed increased interstitial adenosine levels in tumors from spontaneous PDAC mouse models. Diminishing CD73 by genetic manipulations ablated in vivo tumor growth, and decreased myeloid-derived suppressor cells (MDSC) in orthotopic mouse models of PDAC. A high-throughput cytokine profiling demonstrated decreased GM-CSF in mice implanted with CD73 knockdowns. Furthermore, we noted increased IFN-γ expression by intratumoral CD4+ and CD8+ T cells in pancreatic tumors with CD73 knockdowns. Depletion of CD4+ T cells, but not CD8+ T cells abrogated the beneficial effects of decreased CD73. We also observed that splenic MDSCs from Nt5e knockdown tumor-bearing mice were incompetent in suppressing T cell activation in the ex vivo assays. Replenishing GM-CSF restored tumor growth in Nt5e knockout tumors, which was reverted by MDSC depletion. Finally, anti-CD73 antibody treatment significantly improved gemcitabine efficacy in orthotopic models. Thus, targeting the adenosine axis presents a novel therapeutic opportunity for improving the anti-tumoral immune response against PDAC.

Metabolic Rewiring by Loss of Sirt5 Promotes Kras-Induced Pancreatic Cancer Progression.

BACKGROUND & AIMS: SIRT5 plays pleiotropic roles via post-translational modifications, serving as a tumor suppressor, or an oncogene, in different tumors. However, the role SIRT5 plays in the initiation and progression of pancreatic ductal adenocarcinoma (PDAC) remains unknown. METHODS: Published datasets and tissue arrays with SIRT5 staining were used to investigate the clinical relevance of SIRT5 in PDAC. Furthermore, to define the role of SIRT5 in the carcinogenesis of PDAC, we generated autochthonous mouse models with conditional Sirt5 knockout. Moreover, to examine the mechanistic role of SIRT5 in PDAC carcinogenesis, SIRT5 was knocked down in PDAC cell lines and organoids, followed by metabolomics and proteomics studies. A novel SIRT5 activator was used for therapeutic studies in organoids and patient-derived xenografts. RESULTS: SIRT5 expression negatively regulated tumor cell proliferation and correlated with a favorable prognosis in patients with PDAC. Genetic ablation of Sirt5 in PDAC mouse models promoted acinar-to-ductal metaplasia, precursor lesions, and pancreatic tumorigenesis, resulting in poor survival. Mechanistically, SIRT5 loss enhanced glutamine and glutathione metabolism via acetylation-mediated activation of GOT1. A selective SIRT5 activator, MC3138, phenocopied the effects of SIRT5 overexpression and exhibited antitumor effects on human PDAC cells. MC3138 also diminished nucleotide pools, sensitizing human PDAC cell lines, organoids, and patient-derived xenografts to gemcitabine. CONCLUSIONS: Collectively, we identify SIRT5 as a key tumor suppressor in PDAC, whose loss promotes tumorigenesis through increased noncanonic use of glutamine via GOT1, and that SIRT5 activation is a novel therapeutic strategy to target PDAC.

SIRT5 Inhibition Induces Brown Fat-Like Phenotype in 3T3-L1 Preadipocytes.

Brown adipose tissue (BAT) activity plays a key role in regulating systemic energy. The activation of BAT results in increased energy expenditure, making this tissue an attractive pharmacological target for therapies against obesity and type 2 diabetes. Sirtuin 5 (SIRT5) affects BAT function by regulating adipogenic transcription factor expression and mitochondrial respiration. We analyzed the expression of SIRT5 in the different adipose depots of mice. We treated 3T3-L1 preadipocytes and mouse primary preadipocyte cultures with the SIRT5 inhibitor MC3482 and investigated the effects of this compound on adipose differentiation and function. The administration of MC3482 during the early stages of differentiation promoted the expression of brown adipocyte and mitochondrial biogenesis markers. Upon treatment with MC3482, 3T3-L1 adipocytes showed an increased activation of the AMP-activated protein kinase (AMPK), which is known to stimulate brown adipocyte differentiation. This effect was paralleled by an increase in autophagic/mitophagic flux and a reduction in lipid droplet size, mediated by a higher lipolytic rate. Of note, MC3482 increased the expression and the activity of adipose triglyceride lipase, without modulating hormone-sensitive lipase. Our findings reveal that SIRT5 inhibition stimulates brown adipogenesis in vitro, supporting this approach as a strategy to stimulate BAT and counteract obesity.

miR-200c-3p Regulates Epitelial-to-Mesenchymal Transition in Epicardial Mesothelial Cells by Targeting Epicardial Follistatin-Related Protein 1.

Recent findings suggest that epithelial to mesenchymal transition (EMT), a key step during heart development, is involved in cardiac tissue repair following myocardial infarction (MI). MicroRNAs (miRNAs) act as key regulators in EMT processes; however, the mechanisms by which miRNAs target epicardial EMT remain largely unknown. Here, by using an in vitro model of epicardial EMT, we investigated the role of miRNAs as regulators of this process and their potential targets. EMT was induced in murine epicardial-mesothelial cells (EMCs) through TGF ß1 treatment for 48, 72, and 96 h as indicated by the expression of EMT-related genes by qRT-PCR, WB, and immunofluorescence. Further, enhanced expression of stemness genes was also detected. Among several EMT-related miRNAs, miR-200c-3p expression resulted as the most strongly suppressed. Interestingly, we also found a significant upregulation of Follistatin-related protein 1 (FSTL1), a miR-200c predicted target already identified as a potent cardiogenic factor produced by epicardial cells that promotes regeneration following MI. Dual-luciferase reporter assay demonstrated that miR-200c-3p directly targeted the 3'-untranslated region of FSTL1 in EMCs. Consistently, WB analysis showed that knockdown of miR-200c-3p significantly increased FSTL1 expression, whereas overexpression of miR-200c-3p counteracted TGF ß1-mediated FSTL1 upregulation. Importantly, FSTL1 silencing maintained epithelial features in EMCs, despite EMT induction by TGF ß1, and attenuated EMT-associated traits, including migration and stemness. In conclusion, epicardial FSTL1, an important cardiogenic factor in its secreted form, induces EMT, stemness, and migration of EMCs in a miR-200c-3p dependent pathway.

Sirtuins' control of autophagy and mitophagy in cancer.

Mammalian cells use a specialized and complex machinery for the removal of altered proteins or dysfunctional organelles. Such machinery is part of a mechanism called autophagy. Moreover, when autophagy is specifically employed for the removal of dysfunctional mitochondria, it is called mitophagy. Autophagy and mitophagy have important physiological implications and roles associated with cellular differentiation, resistance to stresses such as starvation, metabolic control and adaptation to the changing microenvironment. Unfortunately, transformed cancer cells often exploit autophagy and mitophagy for sustaining their metabolic reprogramming and growth to a point that autophagy and mitophagy are recognized as promising targets for ongoing and future antitumoral therapies. Sirtuins are NAD+ dependent deacylases with a fundamental role in sensing and modulating cellular response to external stresses such as nutrients availability and therefore involved in aging, oxidative stress control, inflammation, differentiation and cancer. It is clear, therefore, that autophagy, mitophagy and sirtuins share many common aspects to a point that, recently, sirtuins have been linked to the control of autophagy and mitophagy. In the context of cancer, such a control is obtained by modulating transcription of autophagy and mitophagy genes, by post translational modification of proteins belonging to the autophagy and mitophagy machinery, by controlling ROS production or major metabolic pathways such as Krebs cycle or glutamine metabolism. The present review details current knowledge on the role of sirtuins, autophagy and mitophagy in cancer to then proceed to discuss how sirtuins can control autophagy and mitophagy in cancer cells. Finally, we discuss sirtuins role in the context of tumor progression and metastasis indicating glutamine metabolism as an example of how a concerted activation and/or inhibition of sirtuins in cancer cells can control autophagy and mitophagy by impinging on the metabolism of this fundamental amino acid.

SIRT1-NOX4 signaling axis regulates cancer cachexia.

Approximately one third of cancer patients die due to complexities related to cachexia. However, the mechanisms of cachexia and the potential therapeutic interventions remain poorly studied. We observed a significant positive correlation between SIRT1 expression and muscle fiber cross-sectional area in pancreatic cancer patients. Rescuing Sirt1 expression by exogenous expression or pharmacological agents reverted cancer cell-induced myotube wasting in culture conditions and mouse models. RNA-seq and follow-up analyses showed cancer cell-mediated SIRT1 loss induced NF-κB signaling in cachectic muscles that enhanced the expression of FOXO transcription factors and NADPH oxidase 4 (Nox4), a key regulator of reactive oxygen species production. Additionally, we observed a negative correlation between NOX4 expression and skeletal muscle fiber cross-sectional area in pancreatic cancer patients. Knocking out Nox4 in skeletal muscles or pharmacological blockade of Nox4 activity abrogated tumor-induced cachexia in mice. Thus, we conclude that targeting the Sirt1-Nox4 axis in muscles is an effective therapeutic intervention for mitigating pancreatic cancer-induced cachexia.

Macrophages potentiate STAT3 signaling in skeletal muscles and regulate pancreatic cancer cachexia.

Incidence of cachexia is highly prevalent in pancreatic ductal adenocarcinoma (PDAC); advanced disease stage directly correlates with decreased muscle and fat mass in PDAC patients. The pancreatic tumor microenvironment is central to the release of systemic factors that govern lipolysis, proteolysis, and muscle and fat degeneration leading to the cachectic phenotype in cancer patients. The current study explores the role of macrophages, a key immunosuppressive player in the pancreatic tumor microenvironment, in regulating cancer cachexia. We observed a negative correlation between CD163-positive macrophage infiltration and muscle-fiber cross sectional area in human PDAC patients. To investigate the role of macrophages in myodegeneration, we utilized conditioned media transplant assays and orthotopic models of PDAC-induced cachexia in immune-competent mice with and without macrophage depletion. We observed that macrophage-derived conditioned medium, in combination with tumor cell-conditioned medium, promoted muscle atrophy through STAT3 signaling. Furthermore, macrophage depletion attenuated systemic inflammation and muscle wasting in pancreatic tumor-bearing mice. Targeting macrophage-mediated STAT3 activation or macrophage-derived interleukin-1 alpha or interleukin-6 diminished myofiber atrophy. Taken together, the current study identified the critical association between macrophages and cachexia phenotype in pancreatic cancer.

Metabolic Alterations in Pancreatic Cancer Progression.

Pancreatic cancer is the third leading cause of cancer-related deaths in the USA. Pancreatic tumors are characterized by enhanced glycolytic metabolism promoted by a hypoxic tumor microenvironment and a resultant acidic milieu. The metabolic reprogramming allows cancer cells to survive hostile microenvironments. Through the analysis of the principal metabolic pathways, we identified the specific metabolites that are altered during pancreatic cancer progression in the spontaneous progression (KPC) mouse model. Genetically engineered mice exhibited metabolic alterations during PanINs formation, even before the tumor development. To account for other cells in the tumor microenvironment and to focus on metabolic adaptations concerning tumorigenic cells only, we compared the metabolic profile of KPC and orthotopic tumors with those obtained from KPC-tumor derived cell lines. We observed significant upregulation of glycolysis and the pentose phosphate pathway metabolites even at the early stages of pathogenesis. Other biosynthetic pathways also demonstrated a few common perturbations. While some of the metabolic changes in tumor cells are not detectable in orthotopic and spontaneous tumors, a significant number of tumor cell-intrinsic metabolic alterations are readily detectable in the animal models. Overall, we identified that metabolic alterations in precancerous lesions are maintained during cancer development and are largely mirrored by cancer cells in culture conditions.

Hypoxia and Inflammation as a Consequence of ß-Fibril Accumulation: A Perspective View for New Potential Therapeutic Targets.

Amyloidoses are heterogeneous diseases that result from the deposition of toxic insoluble ß-sheet fibrillar protein aggregates in different tissues. The cascade of molecular events leading to amyloidoses and to the related clinical manifestations is not completely understood. Nevertheless, it is known that tissue damage associated to this disease involves alteration of tissue architecture, interaction with cell surface receptors, inflammation elicited by the amyloid protein deposition, oxidative stress, and apoptosis. However, another important aspect to consider is that systemic protein massive deposition not only subverts tissue architecture but also determines a progressive cellular hypertrophy and dilation of the extracellular space enlarging the volume of the organ. Such an alteration increases the distance between cells and vessels with a drop in pO2 that, in turn, causes both necrotic cell death and activation of the hypoxia transcription factor HIF-1α. Herewith, we propose the hypothesis that both cell death and hypoxia represent two important events for the pathogenesis of damage and progression of amyloidoses. In fact, molecules released by necrotic cells activate inflammatory cells from one side while binding to HIF-1α-dependent membrane receptors expressed on hypoxic parenchymal cells on the other side. This latter event generates a signaling cascade triggering NFκB activation and chronic inflammation. Finally, we also suggest that this scenario, once proved and detailed, might suggest important targets for new therapeutic interventions.

Mitophagy and Oxidative Stress in Cancer and Aging: Focus on Sirtuins and Nanomaterials.

Mitochondria are the cellular center of energy production and of several important metabolic processes. Mitochondrion health is maintained with a substantial intervention of mitophagy, a process of macroautophagy that degrades selectively dysfunctional and irreversibly damaged organelles. Because of its crucial duty, alteration in mitophagy can cause functional and structural adjustment in the mitochondria, changes in energy production, loss of cellular adaptation, and cell death. In this review, we discuss the dual role that mitophagy plays in cancer and age-related pathologies, as a consequence of oxidative stress, evidencing the triggering stimuli and mechanisms and suggesting the molecular targets for its therapeutic control. Finally, a section has been dedicated to the interplay between mitophagy and therapies using nanoparticles that are the new frontier for a direct and less invasive strategy.

Detecting Autologous Blood Transfusion in Doping Control: Biomarkers of Blood Aging and Storage Measured by Flow Cytofluorimetry.

BACKGROUND: Blood transfusions are banned by the World Anti-Doping Agency as a form of "blood doping". A method of detection of homologous blood transfusion (HBT) has been implemented by the accredited anti-doping laboratories worldwide; however, no internationally recognized method has been finalized so far for the direct detection of autologous blood transfusions, which can at present be revealed only by targeted longitudinal profiling of key blood parameters. METHODS: The present article reports the results of an investigation aimed to pre-select potential biomarkers of blood aging and storage that can be measured to identify the presence in the sample of reinfused blood. Microparticles from platelets and erythrocytes, erythrocytes size and density, annexin V (as a marker of phosphatidylserine externalization), and the membrane surface antigens CD 55 and CD 59, were specifically considered as potential biomarkers and measured by flow cytofluorimetric techniques. RESULTS AND CONCLUSION: Our results indicate that the parameters more strongly affected by the ex vivo storage of whole blood are erythrocytes size and density, annexin V and microparticles. Although the real diagnostic value of the proposed biomarkers shall obviously be confirmed by further studies carried out on blood samples collected after an actual autologous blood transfusion, these results appear very encouraging towards the development of a direct method for detecting autologous blood transfusion in sport doping.

Evaluating the Metabolic Impact of Hypoxia on Pancreatic Cancer Cells.

Hypoxia is frequently observed in human cancers and induces global metabolic reprogramming that includes an increase in glucose uptake and glycolysis, alterations in NAD(P)H/NAD(P)+ and intracellular ATP levels, and increased utilization of glutamine as the major precursor for fatty acid synthesis. In this chapter, we describe in detail various physiological assays that have been adopted to study the metabolic shift propagated by exposure to hypoxic conditions in pancreatic cell culture model that includes glucose uptake, glutamine uptake, and lactate release by pancreatic cancer cell lines. We have also elaborated the assays to evaluate the ratio of NAD(P)H/NAD(P)+ and intracellular ATP estimation using the commercially available kit to assess the metabolic state of cancer cells.

De Novo Lipid Synthesis Facilitates Gemcitabine Resistance through Endoplasmic Reticulum Stress in Pancreatic Cancer.

Pancreatic adenocarcinoma is moderately responsive to gemcitabine-based chemotherapy, the most widely used single-agent therapy for pancreatic cancer. Although the prognosis in pancreatic cancer remains grim in part due to poor response to therapy, previous attempts at identifying and targeting the resistance mechanisms have not been very successful. By leveraging The Cancer Genome Atlas dataset, we identified lipid metabolism as the metabolic pathway that most significantly correlated with poor gemcitabine response in pancreatic cancer patients. Furthermore, we investigated the relationship between alterations in lipogenesis pathway and gemcitabine resistance by utilizing tissues from the genetically engineered mouse model and human pancreatic cancer patients. We observed a significant increase in fatty acid synthase (FASN) expression with increasing disease progression in spontaneous pancreatic cancer mouse model, and a correlation of high FASN expression with poor survival in patients and poor gemcitabine responsiveness in cell lines. We observed a synergistic effect of FASN inhibitors with gemcitabine in pancreatic cancer cells in culture and orthotopic implantation models. Combination of gemcitabine and the FASN inhibitor orlistat significantly diminished stemness, in part due to induction of endoplasmic reticulum (ER) stress that resulted in apoptosis. Moreover, direct induction of ER stress with thapsigargin caused a similar decrease in stemness and showed synergistic activity with gemcitabine. Our in vivo studies with orthotopic implantation models demonstrated a robust increase in gemcitabine responsiveness upon inhibition of fatty acid biosynthesis with orlistat. Altogether, we demonstrate that fatty acid biosynthesis pathway manipulation can help overcome the gemcitabine resistance in pancreatic cancer by regulating ER stress and stemness. Cancer Res; 77(20); 5503-17. ©2017 AACR.

MUC1 and HIF-1alpha Signaling Crosstalk Induces Anabolic Glucose Metabolism to Impart Gemcitabine Resistance to Pancreatic Cancer.

Poor response to cancer therapy due to resistance remains a clinical challenge. The present study establishes a widely prevalent mechanism of resistance to gemcitabine in pancreatic cancer, whereby increased glycolytic flux leads to glucose addiction in cancer cells and a corresponding increase in pyrimidine biosynthesis to enhance the intrinsic levels of deoxycytidine triphosphate (dCTP). Increased levels of dCTP diminish the effective levels of gemcitabine through molecular competition. We also demonstrate that MUC1-regulated stabilization of hypoxia inducible factor-1α (HIF-1α) mediates such metabolic reprogramming. Targeting HIF-1α or de novo pyrimidine biosynthesis, in combination with gemcitabine, strongly diminishes tumor burden. Finally, reduced expression of TKT and CTPS, which regulate flux into pyrimidine biosynthesis, correlates with better prognosis in pancreatic cancer patients on fluoropyrimidine analogs.

MUC1-Mediated Metabolic Alterations Regulate Response to Radiotherapy in Pancreatic Cancer.

Purpose:MUC1, an oncogene overexpressed in multiple solid tumors, including pancreatic cancer, reduces overall survival and imparts resistance to radiation and chemotherapies. We previously identified that MUC1 facilitates growth-promoting metabolic alterations in pancreatic cancer cells. The present study investigates the role of MUC1-mediated metabolism in radiation resistance of pancreatic cancer by utilizing cell lines and in vivo models.Experimental Design: We used MUC1-knockdown and -overexpressed cell line models for evaluating the role of MUC1-mediated metabolism in radiation resistance through in vitro cytotoxicity, clonogenicity, DNA damage response, and metabolomic evaluations. We also investigated whether inhibition of glycolysis could revert MUC1-mediated metabolic alterations and radiation resistance by using in vitro and in vivo models.Results: MUC1 expression diminished radiation-induced cytotoxicity and DNA damage in pancreatic cancer cells by enhancing glycolysis, pentose phosphate pathway, and nucleotide biosynthesis. Such metabolic reprogramming resulted in high nucleotide pools and radiation resistance in in vitro models. Pretreatment with the glycolysis inhibitor 3-bromopyruvate abrogated MUC1-mediated radiation resistance both in vitro and in vivo, by reducing glucose flux into nucleotide biosynthetic pathways and enhancing DNA damage, which could again be reversed by pretreatment with nucleoside pools.Conclusions: MUC1-mediated nucleotide metabolism plays a key role in facilitating radiation resistance in pancreatic cancer and targeted effectively through glycolytic inhibition. Clin Cancer Res; 23(19); 5881-91. ©2017 AACR.

GOT1-mediated anaplerotic glutamine metabolism regulates chronic acidosis stress in pancreatic cancer cells.

The increased rate of glycolysis and reduced oxidative metabolism are the principal biochemical phenotypes observed in pancreatic ductal adenocarcinoma (PDAC) that lead to the development of an acidic tumor microenvironment. The pH of most epithelial cell-derived tumors is reported to be lower than that of plasma. However, little is known regarding the physiology and metabolism of cancer cells enduring chronic acidosis. Here, we cultured PDAC cells in chronic acidosis (pH 6.9-7.0) and observed that cells cultured in low pH had reduced clonogenic capacity. However, our physiological and metabolomics analysis showed that cells in low pH deviate from glycolytic metabolism and rely more on oxidative metabolism. The increased expression of the transaminase enzyme GOT1 fuels oxidative metabolism of cells cultured in low pH by enhancing the non-canonical glutamine metabolic pathway. Survival in low pH is reduced upon depletion of GOT1 due to increased intracellular ROS levels. Thus, GOT1 plays an important role in energy metabolism and ROS balance in chronic acidosis stress. Our studies suggest that targeting anaplerotic glutamine metabolism may serve as an important therapeutic target in PDAC.

SIRT1-SIRT3 Axis Regulates Cellular Response to Oxidative Stress and Etoposide.

Sirtuins are conserved NAD+ -dependent deacylases. SIRT1 is a nuclear and cytoplasmic sirtuin involved in the control of histones a transcription factors function. SIRT3 is a mitochondrial protein, which regulates mitochondrial function. Although, both SIRT1 and SIRT3 have been implicated in resistance to cellular stress, the link between these two sirtuins has not been studied so far. Here we aimed to unravel: i) the role of SIRT1-SIRT3 axis for cellular response to oxidative stress and DNA damage; ii) how mammalian cells modulate such SIRT1-SIRT3 axis and which mechanisms are involved. Therefore, we analyzed the response to different stress stimuli in WT or SIRT1-silenced cell lines. Our results demonstrate that SIRT1-silenced cells are more resistant to H2 O2 and etoposide treatment showing decreased ROS accumulation, γ-H2AX phosphorylation, caspase-3 activation and PARP cleavage. Interestingly, we observed that SIRT1-silenced cells show an increased SIRT3 expression. To explore such a connection, we carried out luciferase assays on SIRT3 promoter demonstrating that SIRT1-silencing increases SIRT3 promoter activity and that such an effect depends on the presence of SP1 and ZF5 recognition sequences on SIRT3 promoter. Afterwards, we performed co-immunoprecipitation assays demonstrating that SIRT1 binds and deacetylates the transcription inhibitor ZF5 and that there is a decreased interaction between SP1 and ZF5 in SIRT1-silenced cells. Therefore, we speculate that acetylated ZF5 cannot bind and sequester SP1 that is free, then, to increase SIRT3 transcription. In conclusion, we demonstrate that cells with low SIRT1 levels can maintain their resistance and survival by increasing SIRT3 expression. J. Cell. Physiol. 232: 1835-1844, 2017. © 2016 Wiley Periodicals, Inc.

Silibinin-mediated metabolic reprogramming attenuates pancreatic cancer-induced cachexia and tumor growth.

Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer-related deaths in the US. Cancer-associated cachexia is present in up to 80% of PDAC patients and is associated with aggressive disease and poor prognosis. In the present studies we evaluated an anti-cancer natural product silibinin for its effectiveness in targeting pancreatic cancer aggressiveness and the cachectic properties of pancreatic cancer cells and tumors. Our results demonstrate that silibinin inhibits pancreatic cancer cell growth in a dose-dependent manner and reduces glycolytic activity of cancer cells. Our LC-MS/MS based metabolomics data demonstrates that silibinin treatment induces global metabolic reprogramming in pancreatic cancer cells. Silibinin treatment diminishes c-MYC expression, a key regulator of cancer metabolism. Furthermore, we observed reduced STAT3 signaling in silibinin-treated cancer cells. Overexpression of constitutively active STAT3 was sufficient to substantially revert the silibinin-induced downregulation of c-MYC and the metabolic phenotype. Our in vivo investigations demonstrate that silibinin reduces tumor growth and proliferation in an orthotopic mouse model of pancreatic cancer and prevents the loss of body weight and muscle. It also improves physical activity including grip strength and latency to fall in tumor-bearing mice. In conclusion, silibinin-induced metabolic reprogramming diminishes cell growth and cachectic properties of pancreatic cancer cells and animal models.