TRAF4-mediated nonproteolytic ubiquitination of androgen receptor promotes castration-resistant prostate cancer.

Castration-resistant prostate cancer (CRPC) poses a major clinical challenge with the androgen receptor (AR) remaining to be a critical oncogenic player. Several lines of evidence indicate that AR induces a distinct transcriptional program after androgen deprivation in CRPCs. However, the mechanism triggering AR binding to a distinct set of genomic loci in CRPC and how it promotes CRPC development remain unclear. We demonstrate here that atypical ubiquitination of AR mediated by an E3 ubiquitin ligase TRAF4 plays an important role in this process. TRAF4 is highly expressed in CRPCs and promotes CRPC development. It mediates K27-linked ubiquitination at the C-terminal tail of AR and increases its association with the pioneer factor FOXA1. Consequently, AR binds to a distinct set of genomic loci enriched with FOXA1- and HOXB13-binding motifs to drive different transcriptional programs including an olfactory transduction pathway. Through the surprising upregulation of olfactory receptor gene transcription, TRAF4 increases intracellular cAMP levels and boosts E2F transcription factor activity to promote cell proliferation under androgen deprivation conditions. Altogether, these findings reveal a posttranslational mechanism driving AR-regulated transcriptional reprogramming to provide survival advantages for prostate cancer cells under castration conditions.

Metabolic enzyme PFKFB4 activates transcriptional coactivator SRC-3 to drive breast cancer.

Alterations in both cell metabolism and transcriptional programs are hallmarks of cancer that sustain rapid proliferation and metastasis 1 . However, the mechanisms that control the interaction between metabolic reprogramming and transcriptional regulation remain unclear. Here we show that the metabolic enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 4 (PFKFB4) regulates transcriptional reprogramming by activating the oncogenic steroid receptor coactivator-3 (SRC-3). We used a kinome-wide RNA interference-based screening method to identify potential kinases that modulate the intrinsic SRC-3 transcriptional response. PFKFB4, a regulatory enzyme that synthesizes a potent stimulator of glycolysis 2 , is found to be a robust stimulator of SRC-3 that coregulates oestrogen receptor. PFKFB4 phosphorylates SRC-3 at serine 857 and enhances its transcriptional activity, whereas either suppression of PFKFB4 or ectopic expression of a phosphorylation-deficient Ser857Ala mutant SRC-3 abolishes the SRC-3-mediated transcriptional output. Functionally, PFKFB4-driven SRC-3 activation drives glucose flux towards the pentose phosphate pathway and enables purine synthesis by transcriptionally upregulating the expression of the enzyme transketolase. In addition, the two enzymes adenosine monophosphate deaminase-1 (AMPD1) and xanthine dehydrogenase (XDH), which are involved in purine metabolism, were identified as SRC-3 targets that may or may not be directly involved in purine synthesis. Mechanistically, phosphorylation of SRC-3 at Ser857 increases its interaction with the transcription factor ATF4 by stabilizing the recruitment of SRC-3 and ATF4 to target gene promoters. Ablation of SRC-3 or PFKFB4 suppresses breast tumour growth in mice and prevents metastasis to the lung from an orthotopic setting, as does Ser857Ala-mutant SRC-3. PFKFB4 and phosphorylated SRC-3 levels are increased and correlate in oestrogen receptor-positive tumours, whereas, in patients with the basal subtype, PFKFB4 and SRC-3 drive a common protein signature that correlates with the poor survival of patients with breast cancer. These findings suggest that the Warburg pathway enzyme PFKFB4 acts as a molecular fulcrum that couples sugar metabolism to transcriptional activation by stimulating SRC-3 to promote aggressive metastatic tumours.

Coactivator-Dependent Oscillation of Chromatin Accessibility Dictates Circadian Gene Amplitude via REV-ERB Loading.

A central mechanism for controlling circadian gene amplitude remains elusive. We present evidence for a "facilitated repression (FR)" model that functions as an amplitude rheostat for circadian gene oscillation. We demonstrate that ROR and/or BMAL1 promote global chromatin decondensation during the activation phase of the circadian cycle to actively facilitate REV-ERB loading for repression of circadian gene expression. Mechanistically, we found that SRC-2 dictates global circadian chromatin remodeling through spatial and temporal recruitment of PBAF members of the SWI/SNF complex to facilitate loading of REV-ERB in the hepatic genome. Mathematical modeling highlights how the FR model sustains proper circadian rhythm despite fluctuations of REV-ERB levels. Our study not only reveals a mechanism for active communication between the positive and negative limbs of the circadian transcriptional loop but also establishes the concept that clock transcription factor binding dynamics is perhaps a central tenet for fine-tuning circadian rhythm.

High MYC mRNA Expression Is More Clinically Relevant than MYC DNA Amplification in Triple-Negative Breast Cancer.

DNA abnormalities are used in inclusion criteria of clinical trials for treatments with specific targeted molecules. MYC is one of the most powerful oncogenes and is known to be associated with triple-negative breast cancer (TNBC). Its DNA amplification is often part of the targeted DNA-sequencing panels under the assumption of reflecting upregulated signaling. However, it remains unclear if MYC DNA amplification is a surrogate of its upregulated signaling. Thus, we investigated the difference between MYC DNA amplification and mRNA high expression in TNBCs utilizing publicly available cohorts. MYC DNA amplified tumors were found to have various mRNA expression levels, suggesting that MYC DNA amplification does not always result in elevated MYC mRNA expression. Compared to other subtypes, both MYC DNA amplification and mRNA high expression were more frequent in the TNBCs. MYC mRNA high expression, but not DNA amplification, was significantly associated with worse overall survival in the TNBCs. The TNBCs with MYC mRNA high expression enriched MYC target genes, cell cycle related genes, and WNT/ß-catenin gene sets, whereas none of them were enriched in MYC DNA amplified TNBCs. In conclusion, MYC mRNA high expression, but not DNA amplification, reflects not only its upregulated signaling pathway, but also clinical significance in TNBCs.

SRC-2 orchestrates polygenic inputs for fine-tuning glucose homeostasis.

Despite extensive efforts to understand the monogenic contributions to perturbed glucose homeostasis, the complexity of genetic events that fractionally contribute to the spectrum of this pathology remain poorly understood. Proper maintenance of glucose homeostasis is the central feature of a constellation of comorbidities that define the metabolic syndrome. The ability of the liver to balance carbohydrate uptake and release during the feeding-to-fasting transition is essential to the regulation of peripheral glucose availability. The liver coordinates the expression of gene programs that control glucose absorption, storage, and secretion. Herein, we demonstrate that Steroid Receptor Coactivator 2 (SRC-2) orchestrates a hierarchy of nutritionally responsive transcriptional complexes to precisely modulate plasma glucose availability. Using DNA pull-down technology coupled with mass spectrometry, we have identified SRC-2 as an indispensable integrator of transcriptional complexes that control the rate-limiting steps of hepatic glucose release and accretion. Collectively, these findings position SRC-2 as a major regulator of polygenic inputs to metabolic gene regulation and perhaps identify a previously unappreciated model that helps to explain the clinical spectrum of glucose dysregulation.

Nuclear receptor coactivators: master regulators of human health and disease.

Transcriptional coregulators (coactivators and corepressors) have emerged as the principal modulators of the functions of nuclear receptors and other transcription factors. During the decade since the discovery of steroid receptor coactivator-1 (SRC-1), the first authentic coregulator, more than 400 coregulators have been identified and characterized, and deciphering their function has contributed significantly to our understanding of their role in human physiology. Deregulated expression of coregulators has been implicated in diverse disease states and related pathologies. The advancement of molecular technologies has enabled us to better characterize the molecular associations of the SRC family of coactivators with other protein complexes in the context of gene regulation. These continuing discoveries not only expand our knowledge of the roles of coactivators in various human diseases but allow us to discover novel coactivator-targeting strategies for therapeutic intervention in these diseases.

MIEN1, a novel interactor of Annexin A2, promotes tumor cell migration by enhancing AnxA2 cell surface expression.

BACKGROUND: Migration and invasion enhancer 1 (MIEN1) is a novel gene found to be abundantly expressed in breast tumor tissues and functions as a critical regulator of tumor cell migration and invasion to promote systemic metastases. Previous studies have identified post-translational modifications by isoprenylation at the C-terminal tail of MIEN1 to favor its translocation to the inner leaflet of plasma membrane and its function as a membrane-bound adapter molecule. However, the exact molecular events at the membrane interface activating the MIEN1-driven tumor cell motility are vaguely understood. METHODS: MIEN1 was first studied using in-silico analysis on available RNA sequencing data of human breast tissues and its expression was ascertained in breast cells. We performed several assays including co-immunoprecipitation, wound healing, western blotting and immunofluorescence to decipher the molecular events involved in MIEN1-mediated tumor cell migration. RESULTS: Clinically, MIEN1 is predominantly overexpressed in Her-2 and luminal B subtypes of breast tumors, and its increased expression correlates with poor disease free survival. Molecular studies identified a phosphorylation-dependent activation signal in the immunoreceptor tyrosine based activation motif (ITAM) of MIEN1 and the phosphorylation-deficient MIEN1-mutants (Y39F/50 F) to regulate filopodia generation, migration and invasion. We found that ITAM-phosphorylation of MIEN1 is significantly impaired in isoprenylation-deficient MIEN1 mutants indicating that prenylation of MIEN1 and membrane association is required for cross-phosphorylation of tyrosine residues. Furthermore, we identified MIEN1 as a novel interactor of Annexin A2 (AnxA2), a Ca(2+) -dependent phospholipid binding protein, which serves as an extracellular proteolytic center regulating plasmin generation. Fluorescence resonance energy transfer (FRET) confirmed that MIEN1 physically interacts with AnxA2 and functional studies revealed that they mutually cooperate to accentuate tumor cell motility. Interestingly, our study identified that ectopic overexpression of MIEN1 significantly enhances Tyr23-phosphorylation on AnxA2, thereby stimulating cell surface translocation of AnxA2 and catalyzing the activation of its proteolytic activity. CONCLUSION: Our data show that the presence and interaction of both MIEN1 and AnxA2 in breast tumors are crucial drivers of cell motility. Our study has now deciphered a novel regulatory network governing the vicious process of breast tumor cell invasion-metastasis, and findings suggest MIEN1-AnxA2 as prospective targets to counter the deadly disease.

MicroRNA-940 suppresses prostate cancer migration and invasion by regulating MIEN1.

BACKGROUND: MicroRNAs (miRNAs) are crucial molecules that regulate gene expression and hence pathways that are key to prostate cancer progression. These non-coding RNAs are highly deregulated in prostate cancer thus facilitating progression of the disease. Among the many genes that have gained importance in this disease, Migration and invasion enhancer 1 (MIEN1), a novel gene located next to HER2/neu in the 17q12 amplicon of the human chromosome, has been shown to enhance prostate cancer cell migration and invasion, two key processes in cancer progression. MIEN1 is differentially expressed between normal and cancer cells and tissues. Understanding the regulation of MIEN1 by microRNA may enable development of better targeting strategies. METHODS: The miRNAs that could target MIEN1 were predicted by in silico algorithms and microarray analysis. The validation for miRNA expression was performed by qPCR and northern blotting in cells and by in situ hybridization in tissues. MIEN1 and levels of other molecules upon miRNA regulation was determined by Western blotting, qPCR, and immunofluorescence. The functional effects of miRNA on cells were determined by wound healing cell migration, Boyden chamber cell invasion, clonal and colony formation assays. For knockdown or overexpression of the miRNA or overexpression of MIEN1 3'UTR, cells were transfected with the oligomiRs and plasmids, respectively. RESULTS: A novel miRNA, hsa-miR-940 (miR-940), identified and validated to be highly expressed in immortalized normal cells compared to cancer cells, is a regulator of MIEN1. Analysis of human prostate tumors and their matched normal tissues confirmed that miR-940 is highly expressed in the normal tissues compared to its low to negligible expression in the tumors. While MIEN1 is a direct target of miR-940, miR-940 alters MIEN1 RNA, in a quantity as well as cell dependent context, along with altering its downstream effectors. The miR-940 inhibited migratory and invasive potential of cells, attenuated their anchorage-independent growth ability, and increased E-cadherin expression, implicating its role in mesenchymal-to-epithelial transition (MET). CONCLUSIONS: These results, for the first time, implicate miR-940, a regulator of MIEN1, as a promising novel diagnostic and prognostic tool for prostate cancer.

Protocol to detect and quantify tumor hypoxia in mice using photoacoustic imaging.

Photoacoustic imaging (PAI) with co-registered ultrasound (US) is a hybrid non-invasive imaging modality that enables visualization and quantification of tumor hypoxia in live animals. Here, using a breast tumor xenograft model as an example, we present a stepwise protocol describing animal preparation, positioning, instrument setup, and US-PAI image acquisition procedures. This protocol also guides through detailed data analysis, explains functional readouts obtained from PAI, and discusses the potential application of the technology to study the hypoxic tumor microenvironment. For complete details on the use and execution of this protocol, please refer to Dai et al.1.

Regulation of tumor metabolism by post translational modifications on metabolic enzymes.

Metabolic reprogramming is a hallmark of cancer development, progression, and metastasis. Several metabolic pathways such as glycolysis, tricarboxylic acid (TCA) cycle, lipid metabolism, and glutamine catabolism are frequently altered to support cancer growth. Importantly, the activity of the rate-limiting metabolic enzymes in these pathways are specifically modulated in cancer cells. This is achieved by transcriptional, translational, and post translational regulations that enhance the expression, activity, stability, and substrate sensitivity of the rate-limiting enzymes. These mechanisms allow the enzymes to retain increased activity supporting the metabolic needs of rapidly growing tumors, sustain their survival in the hostile tumor microenvironments and in the metastatic lesions. In this review, we primarily focused on the post translational modifications of the rate-limiting enzymes in the glucose and glutamine metabolism, TCA cycle, and fatty acid metabolism promoting tumor progression and metastasis.

Prenylated c17orf37 induces filopodia formation to promote cell migration and metastasis.

Post-translational modification by covalent attachment of isoprenoid lipids (prenylation) regulates the functions and biological activities of several proteins implicated in the oncogenic transformation and metastatic progression of cancer. The largest group of prenylated proteins contains a CAAX motif at the C-terminal that serves as a substrate for a series of post-translational modifications that convert these otherwise hydrophilic proteins to lipidated proteins, thus facilitating membrane association. C17orf37 (chromosome 17 open reading frame 37), also known as C35/Rdx12/MGC14832, located in the 17q12 amplicon, is overexpressed in human cancer, and its expression correlates with the migratory and invasive phenotype of cancer cells. Here we show that C17orf37 contains a functional CAAX motif and is post-translationally modified by protein geranylgeranyltransferase-I (GGTase-I). Geranylgeranylation of C17orf37 at the CAAX motif facilitates association of the protein to the inner leaflet of plasma membrane, enhances migratory phenotype of cells by inducing increased filopodia formation, and potentiates directional migration. A prenylation-deficient mutant of C17orf37 is functionally inactive and fails to trigger dissemination of tail vein-injected cells in a mouse model of metastasis. These findings demonstrate that prenylation is required for the function of the C17orf37 protein in cancer cells and imply that the post-translational modification may functionally regulate metastatic progression of disease.

Oncogenic activation in prostate cancer progression and metastasis: Molecular insights and future challenges.

Prostate cancer is a leading cause of death among men in the United States, and currently early diagnosis and appropriate treatment remain key approaches for patient care. Molecularly prostate cancer cells carry multiple perturbations that generate malignant phenotype capable of uncontrolled growth, survival, and invasion-metastasis to other organs. These alterations are acquired both by genetic and epigenetic changes in tumor cells resulting in the activation of growth factor receptors, signaling proteins, kinases, transcription factors and coregulators, and multiple proteases required for the progression of the disease. Recent advances provide novel insights into the molecular functions of these oncogenic activators, implicating potential therapeutic targeting opportunities for the treatment of prostate cancer.

Optimization and Characterization of a Bone Culture Model to Study Prostate Cancer Bone Metastasis.

Nearly 90% of patients with advanced prostate cancer manifest bone metastases. Distinct from the osteolytic metastasis mostly observed in other cancer types, prostate cancer bone metastasis is typically more osteoblastic, which is relatively understudied due to the lack of reliable and efficient models to resemble the indolent cellular growth and complexity of metastatic progression. In our previous studies, we developed bone-in-culture array (BICA) to primarily model the osteoblast-involved, pre-osteolytic stage of breast cancer bone metastasis. Given that the progression of prostate cancer bone metastasis is largely osteoblastic, it is reasonable to speculate that the original BICA model can be adjusted to investigate prostate cancer bone metastases. In this study, we refined BICA by reducing the surgical labor and improving its reproducibility and capacity. The optimized BICA can successfully recapitulate important features of prostate cancer bone metastasis such as the osteoblastic phenotype, indolent growth, cancer-niche interactions, and response to hormones. Our efforts address the long-standing need for reliable and efficient models to study prostate cancer bone metastasis.

Hypoxic activation of PFKFB4 in breast tumor microenvironment shapes metabolic and cellular plasticity to accentuate metastatic competence.

Cancer cells encounter a hostile tumor microenvironment (TME), and their adaptations to metabolic stresses determine metastatic competence. Here, we show that the metabolic enzyme 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase-4 (PFKFB4) is induced in hypoxic tumors acquiring metabolic plasticity and invasive phenotype. In mouse models of breast cancer, genetic ablation of PFKFB4 significantly delays distant organ metastasis, reducing local lymph node invasion by suppressing expression of invasive gene signature including integrin ß3. Photoacoustic imaging followed by metabolomics analyses of hypoxic tumors show that PFKFB4 drives metabolic flexibility, enabling rapid detoxification of reactive oxygen species favoring survival under selective pressure. Mechanistically, hypoxic induction triggers nuclear translocation of PFKFB4 accentuating non-canonical transcriptional activation of HIF-1α, and breast cancer patients with increased nuclear PFKFB4 in their tumors are found to be significantly associated with poor prognosis. Our findings imply that PFKFB4 induction is crucial for tumor cell adaptation in the hypoxic TME that determines metastatic competence.

Low expression of miR-195 is associated with cell proliferation, glycolysis and poor survival in estrogen receptor (ER)-positive but not in triple negative breast cancer.

MiR-195 is a tumor suppressive microRNA in breast cancer. Its clinical relevance remains debatable as it has only been studied via in vitro experiments or small cohort studies. We analyzed a total of 2,038 patients in the TCGA and METABRIC cohorts to assess whether low miR-195 expressing tumors are associated with aggressive cancer characteristics and poor prognostic outcomes. The median cutoff of miR-195 expression was used to split the groups into miR-195 high and low groups. Low miR-19 expressing tumors demonstrated high cell proliferating features by enriching the gene sets associated with cell proliferation, MKI67 expression and pathological grade. One-third of the top target miR-195 genes were related to cell proliferation. Low miR-195 expressing tumors were associated with both pro-cancerous and anti-cancerous immune cells. Low miR-195 expressing tumors were associated with enhanced glycolysis and poor survival in ER-positive tumors, but not other subtypes of breast cancer. In conclusion, low expression of miR-195 in ER-positive breast cancer was associated with enhanced cancer cell proliferation, glycolysis, and worse overall survival.

Steroid receptor coactivator 3 (SRC-3/AIB1) is enriched and functional in mouse and human Tregs.

A subset of CD4 + lymphocytes, regulatory T cells (Tregs), are necessary for central tolerance and function as suppressors of autoimmunity against self-antigens. The SRC-3 coactivator is an oncogene in multiple cancers and is capable of potentiating numerous transcription factors in a wide variety of cell types. Src-3 knockout mice display broad lymphoproliferation and hypersensitivity to systemic inflammation. Using publicly available bioinformatics data and directed cellular approaches, we show that SRC-3 also is highly enriched in Tregs in mice and humans. Human Tregs lose phenotypic characteristics when SRC-3 is depleted or pharmacologically inhibited, including failure of induction from resting T cells and loss of the ability to suppress proliferation of stimulated T cells. These data support a model for SRC-3 as a coactivator that actively participates in protection from autoimmunity and may support immune evasion of cancers by contributing to the biology of Tregs.

Transcriptional Repression of SIRT3 Potentiates Mitochondrial Aconitase Activation to Drive Aggressive Prostate Cancer to the Bone.

Metabolic dysregulation is a known hallmark of cancer progression, yet the oncogenic signals that promote metabolic adaptations to drive metastatic cancer remain unclear. Here, we show that transcriptional repression of mitochondrial deacetylase sirtuin 3 (SIRT3) by androgen receptor (AR) and its coregulator steroid receptor coactivator-2 (SRC-2) enhances mitochondrial aconitase (ACO2) activity to favor aggressive prostate cancer. ACO2 promoted mitochondrial citrate synthesis to facilitate de novo lipogenesis, and genetic ablation of ACO2 reduced total lipid content and severely repressed in vivo prostate cancer progression. A single acetylation mark lysine258 on ACO2 functioned as a regulatory motif, and the acetylation-deficient Lys258Arg mutant was enzymatically inactive and failed to rescue growth of ACO2-deficient cells. Acetylation of ACO2 was reversibly regulated by SIRT3, which was predominantly repressed in many tumors including prostate cancer. Mechanistically, SRC-2-bound AR formed a repressive complex by recruiting histone deacetylase 2 to the SIRT3 promoter, and depletion of SRC-2 enhanced SIRT3 expression and simultaneously reduced acetylated ACO2. In human prostate tumors, ACO2 activity was significantly elevated, and increased expression of SRC-2 with concomitant reduction of SIRT3 was found to be a genetic hallmark enriched in prostate cancer metastatic lesions. In a mouse model of spontaneous bone metastasis, suppression of SRC-2 reactivated SIRT3 expression and was sufficient to abolish prostate cancer colonization in the bone microenvironment, implying this nuclear-mitochondrial regulatory axis is a determining factor for metastatic competence. SIGNIFICANCE: This study highlights the importance of mitochondrial aconitase activity in the development of advanced metastatic prostate cancer and suggests that blocking SRC-2 to enhance SIRT3 expression may be therapeutically valuable. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/81/1/50/F1.large.jpg.

Signal transducer and activator of transcription 6 (STAT6) is a novel interactor of annexin A2 in prostate cancer cells.

Annexin A2 (AnxA2) is a multifunctional Ca(2+)-dependent phospholipid-binding protein, and its overexpression is implicated in malignant transformation of several cancers. In prostate cancer, however, the expression of AnxA2 is lost in prostate intraepithelial neoplasia and reappears in the high-grade tumors, suggesting a complex regulation of AnxA2 in the prostate microenvironment. Since a majority of the biological functions of AnxA2 are mediated by its interaction with other proteins, we performed a yeast two-hybrid assay to search for novel interactors of AnxA2. Our studies revealed that signal transducer and activator of transcription 6 (STAT6), a member of the STAT family of transcription factors, is a binding partner of AnxA2. We confirmed AnxA2-STAT6 interaction by in vitro co-immunoprecipitation and fluorescence resonance energy transfer (FRET) studies and demonstrated that AnxA2 interacts with phosphorylated STAT6. Furthermore, chromatin immunoprecipitation (ChIP) assay revealed that AnxA2 is associated with the STAT6 DNA-binding complex, and luciferase reporter assays demonstrated that AnxA2 upregulates the activity of STAT6. Upon interleukin-4 treatment, AnxA2 stabilizes the cytosolic levels of phosphorylated STAT6 and promotes its nuclear entry. These findings suggest that AnxA2-STAT6 interactions could have potential implications in prostate cancer progression. This report is the first to demonstrate the interaction of AnxA2 with STAT6 and suggests a possible mechanism by which AnxA2 contributes to the metastatic processes of prostate cancer.

Metabolic Dysregulation Controls Endocrine Therapy-Resistant Cancer Recurrence and Metastasis.

Cancer recurrence and metastasis involves many biological interactions, such as genetic, transcription, environmental, endocrine signaling, and metabolism. These interactions add a complex understanding of cancer recurrence and metastatic progression, delaying the advancement in therapeutic opportunities. We highlight the recent advances on the molecular complexities of endocrine-related cancers, focusing on breast and prostate cancer, and briefly review how endocrine signaling and metabolic programs can influence transcriptional complexes for metastasis competence. Nuclear receptors and transcriptional coregulators function as molecular nodes for the crosstalk between endocrine signaling and metabolism that alter downstream gene expression important for tumor progression and metastasis. This exciting regulatory axis may provide insights to the development of cancer therapeutics important for these desensitized endocrine-dependent cancers.

High expression of Annexin A2 is associated with DNA repair, metabolic alteration, and worse survival in pancreatic ductal adenocarcinoma.

BACKGROUND: Annexin A2 (ANXA2) is a known driver of cancer progression. We investigated what mechanism associates with ANXA2 high expression and its survival impact using a bioinformatic approach in pancreatic ductal adenocarcinoma. METHODS: Primary pancreatic tumor (n = 185) cohort in The Cancer Genome Atlas and Gene set enrichment analysis were used. RESULTS: There were no significant associations between ANXA2 expression and clinicopathologic features of the patients investigated. The ANXA2 high tumors enriched some of the known downstream signaling, such as NF-κB (P = .028) and tumor necrosis factor (P = .044) pathways, whereas others, such as angiogenesis or epithelial-mesenchymal transition, were not associated. ANXA2 high expression tumors enriched DNA repair-related gene sets (DNA repair; P = .011, p53 pathway; P = .036) and cell proliferation-related gene sets (MYC targets; P = .041). In addition, new association with metabolism related gene sets, such as glycolysis (P = .016), nucleic acid metabolism (P = .001), and pyrimidine metabolism (P = .004) were identified in the ANXA2 high group. Patients with high ANXA2 expression demonstrated significantly worse disease-free survival (P = .001) and overall survival (P = .014), with high ANXA2 being an independent risk factor. CONCLUSION: High ANXA2 expression was associated with NF-κB and tumor necrosis factor signaling, DNA repair, cell proliferation, and metabolic alteration and worse prognosis in pancreatic ductal adenocarcinoma.