LMO3 is a suppressor of the basal-like/squamous subtype and reduces disease aggressiveness of pancreatic cancer through glycerol 3-phosphate metabolism.

Pancreatic ductal adenocarcinoma (PDAC) encompasses diverse molecular subtypes, including the classical/progenitor and basal-like/squamous subtypes, each exhibiting distinct characteristics, with the latter known for its aggressiveness. We employed an integrative approach combining transcriptome and metabolome analyses to pinpoint potential genes contributing to the basal-like/squamous subtype differentiation. Applying this approach to our NCI-UMD-German and a validation cohort, we identified LIM Domain Only 3 (LMO3), a transcription co-factor, as a candidate suppressor of the basal-like/squamous subtype. Reduced LMO3 expression was significantly associated with higher pathological grade, advanced disease stage, induction of the basal-like/squamous subtype and decreased survival among PDAC patients. In vitro experiments demonstrated that LMO3 transgene expression inhibited PDAC cell proliferation and migration/invasion, concurrently downregulating the basal-like/squamous gene signature. Metabolome analysis of patient tumors and PDAC cells revealed a metabolic program linked to elevated LMO3 and the classical/progenitor subtype, characterized by enhanced lipogenesis and suppressed amino acid metabolism. Notably, glycerol 3-phosphate (G3P) levels positively correlated with LMO3 expression and associated with improved patient survival. Furthermore, glycerol-3-phosphate dehydrogenase 1 (GPD1), a crucial enzyme in G3P synthesis, showed upregulation in LMO3-high and classical/progenitor PDAC, suggesting its potential role in mitigating disease aggressiveness. Collectively, our findings suggest that heightened LMO3 expression reduces transcriptome and metabolome characteristics indicative of basal-like/squamous tumors with decreased disease aggressiveness in PDAC patients. The observations describe LMO3 as a candidate for diagnostic and therapeutic targeting in PDAC.

MIF/NR3C2 axis regulates glucose metabolism reprogramming in pancreatic cancer through MAPK-ERK and AP-1 pathways.

Inflammation and aberrant cellular metabolism are widely recognized as hallmarks of cancer. In pancreatic ductal adenocarcinoma (PDAC), inflammatory signaling and metabolic reprogramming are tightly interwoven, playing pivotal roles in the pathogenesis and progression of the disease. However, the regulatory functions of inflammatory mediators in metabolic reprogramming in pancreatic cancer have not been fully explored. Earlier, we demonstrated that pro-inflammatory mediator macrophage migration inhibitory factor (MIF) enhances disease progression by inhibiting its downstream transcriptional factor nuclear receptor subfamily 3 group C member 2 (NR3C2). Here, we provide evidence that MIF and NR3C2 interactively regulate metabolic reprogramming, resulting in MIF-induced cancer growth and progression in PDAC. MIF positively correlates with the HK1 (hexokinase 1), HK2 (hexokinase 2) and LDHA (lactate dehydrogenase) expression and increased pyruvate and lactate production in PDAC patients. Additionally, MIF augments glucose uptake and lactate efflux by upregulating HK1, HK2 and LDHA expression in pancreatic cancer cells in vitro and in mouse models of PDAC. Conversely, a reduction in HK1, HK2 and LDHA expression is observed in tumors with high NR3C2 expression in PDAC patients. NR3C2 suppresses HK1, HK2 and LDHA expression, thereby inhibiting glucose uptake and lactate efflux in pancreatic cancer. Mechanistically, MIF-mediated regulation of glycolytic metabolism involves the activation of the mitogen-activated protein kinase-ERK signaling pathway, whereas NR3C2 interacts with the activator protein 1 to regulate glycolysis. Our findings reveal an interactive role of the MIF/NR3C2 axis in regulating glucose metabolism supporting tumor growth and progression and may be a potential target for designing novel approaches for improving disease outcome.

ADRA2A promotes the classical/progenitor subtype and reduces disease aggressiveness of pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) manifests diverse molecular subtypes, including the classical/progenitor and basal-like/squamous subtypes, with the latter known for its aggressiveness. We employed integrative transcriptome and metabolome analyses to identify potential genes contributing to the molecular subtype differentiation and its metabolic features. Our comprehensive analysis revealed that adrenoceptor alpha 2A (ADRA2A) was downregulated in the basal-like/squamous subtype, suggesting its potential role as a candidate suppressor of this subtype. Reduced ADRA2A expression was significantly associated with a high frequency of lymph node metastasis, higher pathological grade, advanced disease stage, and decreased survival among PDAC patients. In vitro experiments demonstrated that ADRA2A transgene expression and ADRA2A agonist inhibited PDAC cell invasion. Additionally, ADRA2A-high condition downregulated the basal-like/squamous gene expression signature, while upregulating the classical/progenitor gene expression signature in our PDAC patient cohort and PDAC cell lines. Metabolome analysis conducted on the PDAC cohort and cell lines revealed that elevated ADRA2A levels were associated with suppressed amino acid and carnitine/acylcarnitine metabolism, which are characteristic metabolic profiles of the classical/progenitor subtype. Collectively, our findings suggest that heightened ADRA2A expression induces transcriptome and metabolome characteristics indicative of classical/progenitor subtype with decreased disease aggressiveness in PDAC patients. These observations introduce ADRA2A as a candidate for diagnostic and therapeutic targeting in PDAC.

ELAPOR1 induces the classical/progenitor subtype and contributes to reduced disease aggressiveness through metabolic reprogramming in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is a heterogeneous disease with distinct molecular subtypes described as classical/progenitor and basal-like/squamous PDAC. We hypothesized that integrative transcriptome and metabolome approaches can identify candidate genes whose inactivation contributes to the development of the aggressive basal-like/squamous subtype. Using our integrated approach, we identified endosome-lysosome associated apoptosis and autophagy regulator 1 (ELAPOR1/KIAA1324) as a candidate tumor suppressor in both our NCI-UMD-German cohort and additional validation cohorts. Diminished ELAPOR1 expression was linked to high histological grade, advanced disease stage, the basal-like/squamous subtype, and reduced patient survival in PDAC. In vitro experiments demonstrated that ELAPOR1 transgene expression not only inhibited the migration and invasion of PDAC cells but also induced gene expression characteristics associated with the classical/progenitor subtype. Metabolome analysis of patient tumors and PDAC cells revealed a metabolic program associated with both upregulated ELAPOR1 and the classical/progenitor subtype, encompassing upregulated lipogenesis and downregulated amino acid metabolism. 1-Methylnicotinamide, a known oncometabolite derived from S-adenosylmethionine, was inversely associated with ELAPOR1 expression and promoted migration and invasion of PDAC cells in vitro. Taken together, our data suggest that enhanced ELAPOR1 expression promotes transcriptome and metabolome characteristics that are indicative of the classical/progenitor subtype, whereas its reduction associates with basal-like/squamous tumors with increased disease aggressiveness in PDAC patients. These findings position ELAPOR1 as a promising candidate for diagnostic and therapeutic targeting in PDAC.

Dysregulation of HNF1B/Clusterin axis enhances disease progression in a highly aggressive subset of pancreatic cancer patients.

Pancreatic ductal adenocarcinoma (PDAC) is a lethal malignancy and is largely refractory to available treatments. Identifying key pathways associated with disease aggressiveness and therapeutic resistance may characterize candidate targets to improve patient outcomes. We used a strategy of examining the tumors from a subset of PDAC patient cohorts with the worst survival to understand the underlying mechanisms of aggressive disease progression and to identify candidate molecular targets with potential therapeutic significance. Non-negative matrix factorization (NMF) clustering, using gene expression profile, revealed three patient subsets. A 142-gene signature specific to the subset with the worst patient survival, predicted prognosis and stratified patients with significantly different survival in the test and validation cohorts. Gene-network and pathway analysis of the 142-gene signature revealed dysregulation of Clusterin (CLU) in the most aggressive patient subset in our patient cohort. Hepatocyte nuclear factor 1 b (HNF1B) positively regulated CLU, and a lower expression of HNF1B and CLU was associated with poor patient survival. Mechanistic and functional analyses revealed that CLU inhibits proliferation, 3D spheroid growth, invasiveness and epithelial-to-mesenchymal transition (EMT) in pancreatic cancer cell lines. Mechanistically, CLU enhanced proteasomal degradation of EMT-regulator, ZEB1. In addition, orthotopic transplant of CLU-expressing pancreatic cancer cells reduced tumor growth in mice. Furthermore, CLU enhanced sensitivity of pancreatic cancer cells representing aggressive patient subset, to the chemotherapeutic drug gemcitabine. Taken together, HNF1B/CLU axis negatively regulates pancreatic cancer progression and may potentially be useful in designing novel strategies to attenuate disease progression in PDAC patients.

NO• /RUNX3/kynurenine metabolic signaling enhances disease aggressiveness in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is a lethal malignancy and is refractory to available treatments. Delineating the regulatory mechanisms of metabolic reprogramming, a key event in pancreatic cancer progression, may identify candidate targets with potential therapeutic significance. We hypothesized that inflammatory signaling pathways regulate metabolic adaptations in pancreatic cancer. Metabolic profiling of tumors from PDAC patients with a high- (>median, n = 31) and low-NOS2 (inducible nitric oxide synthase;

ADRA2A promotes the classical/progenitor subtype and reduces disease aggressiveness of pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) manifests diverse molecular subtypes, including the classical/progenitor and basal-like/squamous subtypes, with the latter known for its aggressiveness. We employed integrative transcriptome and metabolome analyses to identify potential genes contributing to the molecular subtype differentiation and its metabolic features. Transcriptome analysis in PDAC patient cohorts revealed downregulation of adrenoceptor alpha 2A (ADRA2A) in the basal-like/squamous subtype, suggesting its potential role as a candidate suppressor of this subtype. Reduced ADRA2A expression was significantly associated with a high frequency of lymph node metastasis, higher pathological grade, advanced disease stage, and decreased survival among PDAC patients. In vitro experiments demonstrated that ADRA2A transgene expression and ADRA2A agonist inhibited PDAC cell invasion. Additionally, ADRA2A-high condition downregulated the basal-like/squamous gene expression signature, while upregulating the classical/progenitor gene expression signature in our PDAC patient cohort and PDAC cell lines. Metabolome analysis conducted on the PDAC cohort and cell lines revealed that elevated ADRA2A levels were associated with suppressed amino acid and carnitine/acylcarnitine metabolism, which are characteristic metabolic profiles of the classical/progenitor subtype. Collectively, our findings suggest that heightened ADRA2A expression induces transcriptome and metabolome characteristics indicative of classical/progenitor subtype with decreased disease aggressiveness in PDAC patients. These observations introduce ADRA2A as a candidate for diagnostic and therapeutic targeting in PDAC.

Involvement of S100A14 protein in cell invasion by affecting expression and function of matrix metalloproteinase (MMP)-2 via p53-dependent transcriptional regulation.

S100 proteins have been implicated in tumorigenesis and metastasis. As a member of S100 proteins, the role of S100A14 in carcinogenesis has not been fully understood. Here, we showed that ectopic overexpression of S100A14 promotes motility and invasiveness of esophageal squamous cell carcinoma cells. We investigated the underlying mechanisms and found that the expression of matrix metalloproteinase (MMP)-2 is obviously increased after S100A14 gene overexpression. Inhibition of MMP2 by a specific MMP2 inhibitor at least partly reversed the invasive phenotype of cells overexpressing S100A14. By serendipity, we found that S100A14 could affect p53 transactivity and stability. Thus, we further investigated whether the effect of MMP2 by S100A14 is dependent on p53. A series of biochemical assays showed that S100A14 requires functional p53 to affect MMP2 transcription, and p53 potently transrepresses the expression of MMP2. Finally, RT-quantitative PCR analysis of human breast cancer specimens showed a significant correlation between S100A14 mRNA expression and MMP2 mRNA expression in cases with wild-type p53 but not in cases with mutant p53. Collectively, our data strongly suggest that S100A14 promotes cell motility and invasiveness by regulating the expression and function of MMP2 in a p53-dependent manner.

SERPINB3-MYC axis induces the basal-like/squamous subtype and enhances disease progression in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) exhibits distinct molecular subtypes: classical/progenitor and basal-like/squamous. Our study aimed to identify genes contributing to the development of the basal-like/squamous subtype, known for its aggressiveness. Transcriptome analyses revealed consistent upregulation of SERPINB3 in basal-like/squamous PDAC, correlating with reduced patient survival. SERPINB3 transgene expression in PDAC cells enhanced in vitro invasion and promoted lung metastasis in a mouse PDAC xenograft model. Metabolome analyses unveiled a metabolic signature linked to both SERPINB3 and the basal-like/squamous subtype, characterized by heightened carnitine/acylcarnitine and amino acid metabolism, associated with poor prognosis in patients with PDAC and elevated cellular invasiveness. Further analysis uncovered that SERPINB3 inhibited the cysteine protease calpain, a key enzyme in the MYC degradation pathway, and drove basal-like/squamous subtype and associated metabolic reprogramming through MYC activation. Our findings indicate that the SERPINB3-MYC axis induces the basal-like/squamous subtype, proposing SERPINB3 as a potential diagnostic and therapeutic target for this variant.

Carnitine palmitoyltransferase 1C regulates cancer cell senescence through mitochondria-associated metabolic reprograming.

Cellular senescence is a fundamental biological process that has profound implications in cancer development and therapeutics, but the underlying mechanisms remain elusive. Here we show that carnitine palmitoyltransferase 1C (CPT1C), an enzyme that catalyzes carnitinylation of fatty acids for transport into mitochondria for ß-oxidation, plays a major role in the regulation of cancer cell senescence through mitochondria-associated metabolic reprograming. Metabolomics analysis suggested alterations in mitochondria activity, as revealed by the marked decrease in acylcarnitines in senescent human pancreatic carcinoma PANC-1 cells, indicating low CPT1C activity. Direct analyses of mRNA and protein show that CPT1C is significantly reduced in senescent cells. Furthermore, abnormal mitochondrial function was observed in senescent PANC-1 cells, leading to lower cell survival under metabolic stress and suppressed tumorigenesis in a mouse xenograft model. Knock-down of CPT1C in PANC-1 cells induced mitochondrial dysfunction, caused senescence-like growth suppression and cellular senescence, suppressed cell survival under metabolic stress, and inhibited tumorigenesis in vivo. Further, CPT1C knock-down suppressed xenograft tumor growth in situ. Silencing of CPT1C in five other tumor cell lines also caused cellular senescence. On the contrary, gain-of-function of CPT1C reversed PANC-1 cell senescence and enhanced mitochondrial function. This study identifies CPT1C as a novel biomarker and key regulator of cancer cell senescence through mitochondria-associated metabolic reprograming, and suggests that inhibition of CPT1C may represent a new therapeutic strategy for cancer treatment through induction of tumor senescence.

Inducible nitric oxide synthase enhances disease aggressiveness in pancreatic cancer.

Pancreatic cancer is one of the most lethal malignancies and is refractory to the available treatments. Pancreatic ductal adenocarcinoma (PDAC) expresses high level of inducible nitric oxide synthase (NOS2), which causes sustained production of nitric oxide (NO). We tested the hypothesis that an aberrantly increased NO-release enhances the development and progression of PDAC. Enhanced NOS2 expression in tumors significantly associated with poor survival in PDAC patients (N = 107) with validation in independent cohorts. We then genetically targeted NOS2 in an autochthonous mouse model of PDAC to examine the effect of NOS2-deficiency on disease progression and survival. Genetic ablation of NOS2 significantly prolonged survival and reduced tumor severity in LSL-KrasG12D/+; LSL-Trp53R172H/+; Pdx-1-Cre (KPC) mice. Primary tumor cells isolated from NOS2-deficient KPC (NKPC) mice showed decreased proliferation and invasiveness as compared to those from KPC mice. Furthermore, NKPC tumors showed reduced expression of pERK, a diminished inactivation of Forkhead box transcription factor O (FOXO3), a tumor suppressor, and a decrease in the expression of oncomir-21, when compared with tumors in KPC mice. Taken together, these findings showed that NOS2 is a predictor of prognosis in early stage, resected PDAC patients, and provide proof-of-principle that targeting NOS2 may have potential therapeutic value in this lethal malignancy.

Endothelial Nitric Oxide Synthase Traffic Inducer (NOSTRIN) is a Negative Regulator of Disease Aggressiveness in Pancreatic Cancer.

PURPOSE: Pancreatic ductal adenocarcinoma (PDAC) is refractory to available treatments. Delineating critical pathways, responsible for disease aggressiveness and therapeutic resistance, may identify effective therapeutic targets. We aimed to identify key pathways contributing to disease aggressiveness by comparing gene expression profiles of tumors from early-stage PDAC cases with extremely poor survival (≤7 months) and those surviving 2 years or more following surgical resection. EXPERIMENTAL DESIGN: Gene expression profiling was performed in tumors in a test cohort of PDAC (N = 50), which included short (≤7 months, N = 11) and long surviving (≥2 years, N = 14) patients, using affymetrix GeneChip Human 1.0 ST array. Key genes associated with disease aggressiveness were identified, using Cox regression, Kaplan-Meier, and pathway analyses with validations in independent cohorts for mechanistic and functional analyses. RESULTS: Gene expression profiling identified 1,820 differentially expressed genes between short and long survival groups with inflammatory gene network ranking first. Lower expression of endothelial nitric oxide synthase traffic inducer (NOSTRIN) was associated with worst survival indicating its potential inhibitory role in disease progression. NOSTRIN overexpression suppressed migration and invasion of pancreatic cancer cells and enhanced sensitivity to chemotherapeutic drug gemcitabine. NOSTRIN inhibited production of nitric oxide (NO) by suppressing the activation of endothelial nitric oxide synthase (eNOS). Furthermore, miR-221, bound to the 3'UTR of NOSTRIN and suppressed its expression, and an increased miR-221 expression associated with poor survival in PDAC. CONCLUSIONS: Our findings showed that NOSTRIN is a potential negative regulator of disease aggressiveness, which may be targeted for designing improved treatment strategy in PDAC. Clin Cancer Res; 22(24); 5992-6001. ©2016 AACR.

A Novel MIF Signaling Pathway Drives the Malignant Character of Pancreatic Cancer by Targeting NR3C2.

Pancreatic cancers with aberrant expression of macrophage migration inhibitory factor (MIF) are particularly aggressive. To identify key signaling pathways that drive disease aggressiveness in tumors with high MIF expression, we analyzed the expression of coding and noncoding genes in high and low MIF-expressing tumors in multiple cohorts of pancreatic ductal adenocarcinoma (PDAC) patients. The key genes and pathways identified were linked to patient survival and were mechanistically, functionally, and clinically characterized using cell lines, a genetically engineered mouse model, and PDAC patient cohorts. Here, we report evidence of a novel MIF-driven signaling pathway that inhibits the orphan nuclear receptor NR3C2, a previously undescribed tumor suppressor that impacts aggressiveness and survival in PDAC. Mechanistically, MIF upregulated miR-301b that targeted NR3C2 and suppressed its expression. PDAC tumors expressing high levels of MIF displayed elevated levels of miR-301b and reduced levels of NR3C2. In addition, reduced levels of NR3C2 expression correlated with poorer survival in multiple independent cohorts of PDAC patients. Functional analysis showed that NR3C2 inhibited epithelial-to-mesenchymal transition and enhanced sensitivity to the gemcitabine, a chemotherapeutic drug used in PDAC standard of care. Furthermore, genetic deletion of MIF disrupted a MIF-mir-301b-NR3C2 signaling axis, reducing metastasis and prolonging survival in a genetically engineered mouse model of PDAC. Taken together, our results offer a preclinical proof of principle for candidate therapies to target a newly described MIF-miR-301b-NR3C2 signaling axis for PDAC management. Cancer Res; 76(13); 3838-50. ©2016 AACR.

Reversal effect of Tween-20 on multidrug resistance in tumor cells in vitro.

Multidrug resistance (MDR) is a major barrier for chemotherapy of many cancers. Non-ionic surfactants have great potential to reverse the MDR by preventing onset or delay progression of the carcinogenic process. However, the role of Tween-20 in the development of MDR remains unknown. The aim of this study was to explore the reversal effect and potential mechanism of Tween-20 on tumor cells in vitro. Alamar Blue assay was used to examine the reversal index of Tween-20 to vincristine (VCR), doxorubicin (DOX) and 5-fluorouracil (5-FU) in KBv200, HepG2/R and Bel-7402/5-FU, respectively. Morphological change was determined by Gimsa and Hoechst 33258 staining. The acumulation of DOX was confirmed by spectrofluorimetric assay. Cell cycle analysis was performed using flow cytometry. The mRNA and protein expression levels of MDR were assessed by semiquantitative RT-PCR and dot blot, respectively. The results showed that Tween-20 at concentrations of 0.0025%, 0.005%, 0.01% had little cytotoxicity. When combined with the cancer drugs, it significantly promoted the sensitivity of MDR cells. Fluorescence staining confirmed that the percentage of apoptotic cell increased when combined with Tween-20. This notion was further supported by the observation that Tween-20 treatment potentiated VIN-induced G2/M arrest of the cell cycle. Furthermore, Tween-20 treatment increased significantly intracellular accumulation of DOX. RT-PCR and dot blot revealed that Tween-20 could downregulate the expression of MDR and P-glycoprotein. Low concentrations of Tween-20 can efficiently reverse the multidrug resistance phenotype by enhancing accumulation of the anticancer drugs. The potential mechanism may be via inhibiting the multidrug-resistant gene expression.