Cancer-associated fibroblast-derived acetate promotes pancreatic cancer development by altering polyamine metabolism via the ACSS2-SP1-SAT1 axis.

The ability of tumour cells to thrive in harsh microenvironments depends on the utilization of nutrients available in the milieu. Here we show that pancreatic cancer-associated fibroblasts (CAFs) regulate tumour cell metabolism through the secretion of acetate, which can be blocked by silencing ATP citrate lyase (ACLY) in CAFs. We further show that acetyl-CoA synthetase short-chain family member 2 (ACSS2) channels the exogenous acetate to regulate the dynamic cancer epigenome and transcriptome, thereby facilitating cancer cell survival in an acidic microenvironment. Comparative H3K27ac ChIP-seq and RNA-seq analyses revealed alterations in polyamine homeostasis through regulation of SAT1 gene expression and enrichment of the SP1-responsive signature. We identified acetate/ACSS2-mediated acetylation of SP1 at the lysine 19 residue that increased SP1 protein stability and transcriptional activity. Genetic or pharmacologic inhibition of the ACSS2-SP1-SAT1 axis diminished the tumour burden in mouse models. These results reveal that the metabolic flexibility imparted by the stroma-derived acetate enabled cancer cell survival under acidosis via the ACSS2-SP1-SAT1 axis.

Vitamin B6 Competition in the Tumor Microenvironment Hampers Antitumor Functions of NK Cells.

Nutritional factors play crucial roles in immune responses. The tumor-caused nutritional deficiencies are known to affect antitumor immunity. Here, we demonstrate that pancreatic ductal adenocarcinoma (PDAC) cells can suppress NK-cell cytotoxicity by restricting the accessibility of vitamin B6 (VB6). PDAC cells actively consume VB6 to support one-carbon metabolism, and thus tumor cell growth, causing VB6 deprivation in the tumor microenvironment. In comparison, NK cells require VB6 for intracellular glycogen breakdown, which serves as a critical energy source for NK-cell activation. VB6 supplementation in combination with one-carbon metabolism blockage effectively diminishes tumor burden in vivo. Our results expand the understanding of the critical role of micronutrients in regulating cancer progression and antitumor immunity, and open new avenues for developing novel therapeutic strategies against PDAC. SIGNIFICANCE: The nutrient competition among the different tumor microenvironment components drives tumor growth, immune tolerance, and therapeutic resistance. PDAC cells demand a high amount of VB6, thus competitively causing NK-cell dysfunction. Supplying VB6 with blocking VB6-dependent one-carbon metabolism amplifies the NK-cell antitumor immunity and inhibits tumor growth in PDAC models. This article is featured in Selected Articles from This Issue, p. 5.

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.

The cholesterol pathway: impact on immunity and cancer.

Cholesterol is a multifaceted metabolite that is known to modulate processes in cancer, atherosclerosis, and autoimmunity. A common denominator between these diseases appears to be the immune system, in which many cholesterol-associated metabolites impact both adaptive and innate immunity. Many cancers display altered cholesterol metabolism, and recent studies demonstrate that manipulating systemic cholesterol metabolism may be useful in improving immunotherapy responses. However, cholesterol can have both proinflammatory and anti-inflammatory roles in mammals, acting via multiple immune cell types, and depending on context. Gaining mechanistic insights into various cholesterol-related metabolites can improve our understanding of their functions and extensive effects on the immune system, and ideally will inform the design of future therapeutic strategies against cancer and/or other pathologies.

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.

Metabolic and Immunological Subtypes of Esophageal Cancer Reveal Potential Therapeutic Opportunities.

BACKGROUND: Esophageal cancer has the sixth highest rate of cancer-associated deaths worldwide, with many patients displaying metastases and chemotherapy resistance. We sought to find subtypes to see if precision medicine could play a role in finding new potential targets and predicting responses to therapy. Since metabolism not only drives cancers but also serves as a readout, metabolism was examined as a key reporter for differences. METHODS: Unsupervised and supervised classification methods, including hierarchical clustering, partial least squares discriminant analysis, k-nearest neighbors, and machine learning techniques, were used to discover and display two major subgroups. Genes, pathways, gene ontologies, survival, and immune differences between the groups were further examined, along with biomarkers between the groups and against normal tissue. RESULTS: Esophageal cancer had two major unique metabolic profiles observed between the histological subtypes esophageal squamous cell carcinoma (ESCC) and esophageal adenocarcinoma (EAC). The metabolic differences suggest that ESCC depends on glycolysis, whereas EAC relies more on oxidative metabolism, catabolism of glycolipids, the tricarboxylic acid (TCA) cycle, and the electron transport chain. We also noted a robust prognostic risk associated with COQ3 expression. In addition to the metabolic alterations, we noted significant alterations in key pathways regulating immunity, including alterations in cytokines and predicted immune infiltration. ESCC appears to have increased signature associated with dendritic cells, Th17, and CD8 T cells, the latter of which correlate with survival in ESCC. We bioinformatically observed that ESCC may be more responsive to checkpoint inhibitor therapy than EAC and postulate targets to enhance therapy further. Lastly, we highlight correlations between differentially expressed enzymes and the potential immune status. CONCLUSION: Overall, these results highlight the extreme differences observed between the histological subtypes and may lead to novel biomarkers, therapeutic strategies, and differences in therapeutic response for targeting each esophageal cancer subtype.

JNK signaling contributes to skeletal muscle wasting and protein turnover in pancreatic cancer cachexia.

Cancer cachexia patients experience significant muscle wasting, which impairs the quality of life and treatment efficacy for patients. Skeletal muscle protein turnover is imparted by increased expression of ubiquitin-proteasome pathway components. Mitogen-activated protein kinases p38 and ERK have been shown to augment E3 ubiquitin ligase expression. Utilizing reverse-phase protein arrays, we identified pancreatic cancer cell-conditioned media-induced activation of JNK signaling in myotubes differentiated from C2C12 myoblasts. Inhibition of JNK signaling with SP600125 reduced cancer cell-conditioned media-induced myotube atrophy, myosin heavy chain protein turnover, and mRNA expression of cachexia-specific ubiquitin ligases Trim63 and Fbxo32. Furthermore, utilizing an orthotopic pancreatic cancer cachexia mouse model, we demonstrated that treatment of tumor-bearing mice with SP600125 improved longitudinal measurements of forelimb grip strength. Post-necropsy measurements demonstrated that SP600125 treatment rescued body weight, carcass weight, and gastrocnemius muscle weight loss without impacting tumor growth. JNK inhibitor treatment also rescued myofiber degeneration and reduced the muscle expression of Trim63 and Fbxo32. These data demonstrate that JNK signaling contributes to muscle wasting in cancer cachexia, and its inhibition has the potential to be utilized as an anti-cachectic therapy.

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.

Tumor-intrinsic CD47 signal regulates glycolysis and promotes colorectal cancer cell growth and metastasis.

Rationale: CD47 plays a vital role in the immune escape of tumor cells, but its role in regulating immune-unrelated biological processes such as proliferation and metastasis remains unclear. We seek to explore the immune-independent functions of CD47 in colorectal cancer (CRC). Methods: The expression of CD47 in CRC was determined by immunohistochemistry. The biological effect of CD47 signaling on tumor cell proliferation and metastasis was evaluated in vitro and in vivo. RNA sequencing analysis was performed to identify pivotal signaling pathways modulated by CD47. The interaction between CD47 and ENO1 was verified by co-immunoprecipitation (co-IP). The effect of CD47 on glycolytic metabolites was analyzed by seahorse XF and targeted metabolomics. Results: The expression of CD47 was upregulated and correlated to poor prognosis in CRC patients. Functional assays revealed that CD47 promoted CRC cell growth and metastasis in vitro and in vivo. Our mechanistic investigations demonstrated that CD47 interacted with ENO1 and protected it from ubiquitin-mediated degradation, subsequently promoting glycolytic activity and phosphorylation of ERK in CRC cells. Inhibition of ENO1 diminished CD47-mediated cell growth and migration. Clinically, the combined expression of CD47 and ENO1 provided reliable predictive biomarkers for the prognosis of CRC patients. Conclusions: CD47 is overexpressed in CRC, and its expression is associated with poor prognosis. Through stabilizing ENO1, CD47 enhances the aerobic glycolysis and ERK activity in CRC cells, thereby promoting the progression of CRC. Our studies reveal an unconventional role of CD47, suggesting that targeting the CD47-ENO1 axis may provide a novel therapeutic avenue for CRC.

MUC1 oncoprotein mitigates ER stress via CDA-mediated reprogramming of pyrimidine metabolism.

The Mucin 1 (MUC1) protein is overexpressed in various cancers and mediates chemotherapy resistance. However, the mechanism is not fully understood. Given that most chemotherapeutic drugs disrupt ER homeostasis as part of their toxicity, and MUC1 expression is regulated by proteins involved in ER homeostasis, we investigated the link between MUC1 and ER homeostasis. MUC1 knockdown in pancreatic cancer cells enhanced unfolded protein response (UPR) signaling and cell death upon ER stress induction. Transcriptomic analysis revealed alterations in the pyrimidine metabolic pathway and cytidine deaminase (CDA). ChIP and CDA activity assays showed that MUC1 occupied CDA gene promoter upon ER stress induction correlating with increased CDA expression and activity in MUC1-expressing cells as compared with MUC1 knockdown cells. Inhibition of either the CDA or pyrimidine metabolic pathway diminished survival in MUC1-expressing cancer cells upon ER stress induction. Metabolomic analysis demonstrated that MUC1-mediated CDA activity corresponded to deoxycytidine to deoxyuridine metabolic reprogramming upon ER stress induction. The resulting increase in deoxyuridine mitigated ER stress-induced cytotoxicity. In addition, given (1) the established roles of MUC1 in protecting cells against reactive oxygen species (ROS) insults, (2) ER stress-generated ROS further promote ER stress and (3) the emerging anti-oxidant property of deoxyuridine, we further investigated if MUC1 regulated ER stress by a deoxyuridine-mediated modulation of ROS levels. We observed that deoxyuridine could abrogate ROS-induced ER stress to promote cancer cell survival. Taken together, our findings demonstrate a novel MUC1-CDA axis of the adaptive UPR that provides survival advantage upon ER stress induction.

Evaluating the Metabolic Alterations in Pancreatic Cancer.

Metabolic reprograming is an established hallmark of cancer cells. Pancreatic cancer cells, by virtue of the underlying oncogenic drivers, demonstrate metabolic reprograming to sustain growth, invasiveness, and therapy resistance. The increased demands of the growing tumor cells alter the metabolic and signaling pathways to meet the growing nutrient requirements. Investigating the metabolic vulnerabilities of tumor cells can help in developing effective therapeutics to target pancreatic cancer. In this chapter, we explain in detail the methods to evaluate the metabolic changes occurring in the tumor. This includes the glucose/glutamine uptake assays and the measurement of reactive oxygen species, extracellular acidification rate, and oxygen consumption rate in the tumor cells. All these physiological assays help in understanding the metabolic nature of the tumor.

Transcriptional Profiling Using RNA-Seq to Study Hypoxia-Mediated Gene Regulation.

Exposing cells to a hypoxic environment leads to significant physiological and molecular alterations. Most of the hypoxic responses are regulated by the transcription factors known as hypoxia-inducible factors (HIFs). HIF1, a heterodimer of hypoxia-stabilized subunit HIF-1alpha and a constitutively expressed subunit HIF-1beta, serves as a key transcription factor that regulates gene expressions which are involved in cell growth, metabolism, and proliferation. The global expression patterns can be analyzed by utilizing RNA-Seq to understand the cellular alterations in hypoxia. This technique enables us to understand the comprehensive regulation of gene expression by specific factors or environmental stimuli. Here, we describe the complete process of studying hypoxia-mediated gene expression by using RNA-Seq, including the hypoxic treatment of cells, RNA isolation, RNA quality check, cDNA library preparation, and library quality check.

Genomic alterations in mucins across cancers.

The significance of mucins in cancers has led to the development of novel biomarkers and therapeutic agents against cancers. Despite significant advances in the understanding of mucins, systemic investigations into the role of mucins in cancer biology focusing particularly on the histological subtypes and stages, along with other variables, are yet to be carried out to discover potential novel functions and cancer-specific roles. Here, we investigated 11 mucin expressing cancers for DNA mutations, mRNA expression, copy number, methylation, and the impacts these genomic features may have on patient survival by utilizing The Cancer Genome Atlas dataset. We demonstrate that mucin DNA mutations have a significant rate, pattern, and impact on cancer patient survival depending on the tissue of origin. This includes a frequent T112P mutation in MUC1 that is seen in half of the pancreatic MUC1 mutations, as well as being present in other cancers. We also observed a very frequent MUC4 mutation at H4205, which correlated with survival outcomes in patients. Furthermore, we observed significant alterations in mucin mRNA expression in multiple tumor types. Our results demonstrate de novo expression of certain mucins in cancer tissues, including MUC21 in colorectal cancers. We observed a general decrease in promoter methylation for mucins, which correlated with decreased expression of many genes, such as MUC15 in kidney cancers. Lastly, several mucin gene loci demonstrated copy number increase in multiple histological subtypes. Thus, our study presents a comprehensive analysis of genomic alterations in mucins and their corresponding roles in cancer progression.

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.