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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., (Published by Oxford University Press 2024.)

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., (Published 2024. This article is a U.S. Government work and is in the public domain in the USA. International Journal of Cancer published by John Wiley & Sons Ltd on behalf of UICC.)

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., (Published by Oxford University Press 2024.)

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., Competing Interests: Declaration of interests The authors declare no competing interest., (Copyright © 2023. Published by Elsevier Inc.)

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., (Published by Oxford University Press 2022.)

The metabolic dependencies of cancer cells have substantial potential to be exploited to improve the diagnosis and treatment of cancer. Creatine riboside (CR) is identified as a urinary metabolite associated with risk and prognosis in lung and liver cancer. However, the source of high CR levels in patients with cancer as well as their implications for the treatment of these aggressive cancers remain unclear. By integrating multiomics data on lung and liver cancer, we have shown that CR is a cancer cell-derived metabolite. Global metabolomics and gene expression analysis of human tumors and matched liquid biopsies, together with functional studies, revealed that dysregulation of the mitochondrial urea cycle and a nucleotide imbalance were associated with high CR levels and indicators of a poor prognosis. This metabolic phenotype was associated with reduced immune infiltration and supported rapid cancer cell proliferation that drove aggressive tumor growth. CRhi cancer cells were auxotrophic for arginine, revealing a metabolic vulnerability that may be exploited therapeutically. This highlights the potential of CR not only as a poor-prognosis biomarker but also as a companion biomarker to inform the administration of arginine-targeted therapies in precision medicine strategies to improve survival for patients with cancer.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by its highly reactive inflammatory desmoplastic stroma with evidence of an extensive tumor stromal interaction largely mediated by inflammatory factors. KRAS mutation and inflammatory signaling promote protumorigenic events, including metabolic reprogramming with several inter-regulatory crosstalks to fulfill the high demand of energy and regulate oxidative stress for tumor growth and progression. Notably, the more aggressive molecular subtype of PDAC enhances influx of glycolytic intermediates. This review focuses on the interactive role of inflammatory signaling and metabolic reprogramming with emerging evidence of crosstalk, which supports the development, progression, and therapeutic resistance of PDAC. Understanding the emerging crosstalk between inflammation and metabolic adaptations may identify potential targets and develop novel therapeutic approaches for PDAC., Competing Interests: Declaration of interests The authors have no interests to declare., (Published by Elsevier Inc.)

Long noncoding RNAs (lncRNAs) are often associated with polysomes, indicating coding potential. However, only a handful of endogenous proteins encoded by putative lncRNAs have been identified and assigned a function. Here, we report the discovery of a putative gastrointestinal-tract-specific lncRNA ( LINC00675 ) that is regulated by the pioneer transcription factor FOXA1 and encodes a conserved small protein of 79 amino acids which we termed FORCP ( FO XA1- R egulated C onserved Small P rotein). FORCP transcript is undetectable in most cell types but is abundant in well-differentiated colorectal cancer (CRC) cells where it functions to inhibit proliferation, clonogenicity, and tumorigenesis. The epitope-tagged and endogenous FORCP protein predominantly localizes to the endoplasmic reticulum (ER). In response to ER stress, FORCP depletion results in decreased apoptosis. Our findings on the initial characterization of FORCP demonstrate that FORCP is a novel, conserved small protein encoded by a mis-annotated lncRNA that regulates apoptosis and tumorigenicity in well-differentiated CRC cells., Competing Interests: XL, LP, WT, BM, MJ, SL, ED, CH, IG, QH, QS, AS, JM, BT, LJ, AR, RW, SA, RC, SS, MG, SH, CH, PM, KP, MA No competing interests declared, SD, TA is affiliated with Leidos Biomedical Research, Inc. AL Reviewing editor, eLife

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; • (nitric oxide)-mediated signaling pathway was elucidated. The level of kynurenine, a tryptophan metabolite, was associated with high NOS2 expression, and a higher level of kynurenine predicted poor survival in patients (n = 63, p = 0.01). Gene expression analysis in PDAC tumors (n = 63) showed a positive correlation between the expression of NOS2 and the tryptophan/kynurenine pathway genes, including indoleamine-2,3-dioxygenase 1 (IDO1) and several aryl hydrocarbon receptor (AHR)-target genes including NFE2L2 (NRF2), SERPINB2, IL1b, IL6 and IL8, which are implicated in pancreatic cancer. Consistently, treatment of pancreatic cancer cell lines with NO • donor induced IDO1, kynurenine production and the expression of AHR-target genes. Furthermore, kynurenine treatment enhanced spheroid growth and invasive potential of pancreatic cancer cell lines. Mechanistically, NO • -induced IDO1/Kynurenine/AHR signaling was mediated by RUNX3 transcription factor. Our findings identified a novel NO • /RUNX3/Kynurenine metabolic axis, which enhances disease aggressiveness in pancreatic cancer and may have potential translational significance in improving disease outcome., (© 2019 UICC.)

Background: Despite the near universal occurrence of activating codon 12 KRAS somatic variants in pancreatic cancer, there is considerable heterogeneity in the molecular make-up, MAPK/ERK pathway activation states, and clinical outcome in this disease. We analyzed the expression levels of CNKSR1, a scaffold that influences MAPK/ERK pathway activity, in clinical pancreas cancer specimens and their impact on survival of patients with pancreatic cancer., Methods: Immunohistochemical staining for CNKSR1 expression was performed on 120 specimens from three independent pancreatic cancer tissue registries, phospho-ERK levels were measured in 86 samples. Expression was divided into CNKSR1 low and CNKSR1 high and correlated with clinicopathological variables including overall survival using multivariate Cox proportional hazard ratio models., Results: CNKSR1 expression was increased in tumors compared to matched normal uninvolved resection specimens (p = 0.004). 28.3% (34/120) of patient specimens stained as CNKSR1 low compared to 71.7% (86/120) of specimens which stained as CNKSR1 high. High CNKSR1 expression was more prevalent in low grade tumors (p = 0.04). In multivariate analysis, low CNKSR1 expression status was independently correlated with decreased overall survival (HR = 2.146; 95% CI 1.34 to 3.43). When stratifying primary, non-metastatic tumor biopsies by CNKSR1 expression, resection was associated with improved survival in patients with high CNKSR1 expression (p < 0.0001) but not low CNKSR1 expression (p = 0.3666). Pancreatic tumors with nuclear in addition to cytoplasmic CNKSR1 staining (32/107) showed increased nuclear phospho-ERK expression compared to tumor with cytoplasmic CNKSR1 staining only (p = 0.017)., Conclusion: CNKSR1 expression is increased in pancreatic tissue specimens and was found to be an independent prognostic marker of overall survival. CNKSR1 may help to identify patient subgroups with unfavorable tumor biology in order to improve risk stratification and treatment selection. Cellular distribution of CNKSR1 was correlated with nuclear pERK expression.

Significance: Pancreatic tumors express high level of nitric oxide synthases (NOSs) in particular inducible (iNOS/NOS2) and endothelial (eNOS/NOS3) forms. However, the role of nitric oxide (NO • ) in the development and progression of pancreatic cancer is not clearly defined. Delineating the NO • -induced signaling in pancreatic cancer and its potential contribution in disease aggressiveness may provide therapeutic targets to improve survival in this lethal malignancy. Recent Advances: An increased expression of NOS2/iNOS in tumors is associated with poorer survival in early stage resected patients with pancreatic ductal adenocarcinoma (PDAC). Furthermore, genetic deletion of NOS2 enhanced survival in mice with autochthonous PDAC. Additionally, targeting NOS3/eNOS reduced the abundance of precursor lesions in mice, which trended toward improved survival., Critical Issues: The extremely poor prognosis in pancreatic cancer is due to the late diagnosis and lack of effective therapy in advanced disease. One of the most critical issues is to decipher the underlying mechanism of disease aggressiveness and therapeutic resistance for identifying potential therapeutic target and effective treatment. Given the evidence of a strong association between inflammation and pancreatic cancer and clinical evidence, which suggests an association between NOS2 and disease aggressiveness, it is critical to define the role of NO • signaling in this lethal malignancy., Future Directions: Recent preclinical and clinical evidences indicate a potential therapeutic significance of targeting NO • signaling in pancreatic cancer. With the emergence of new preclinical models, including the patient-derived organoids, further preclinical evaluation using clinically tested NOS inhibitors is needed for designing future clinical investigation. Antioxid. Redox Signal. 26, 1000-1008.

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., Competing Interests: None, (©2016 American Association for Cancer Research.)

Inflammatory maturation of M1 macrophages by proinflammatory stimuli such as toll like receptor ligands results in profound metabolic reprogramming resulting in commitment to aerobic glycolysis as evidenced by repression of mitochondrial oxidative phosphorylation (OXPHOS) and enhanced glucose utilization. In contrast, "alternatively activated" macrophages adopt a metabolic program dominated by fatty acid-fueled OXPHOS. Despite the known importance of these developmental stages on the qualitative aspects of an inflammatory response, relatively little is know regarding the regulation of these metabolic adjustments. Here we provide evidence that the immunosuppressive cytokine IL-10 defines a metabolic regulatory loop. Our data show for the first time that lipopolysaccharide (LPS)-induced glycolytic flux controls IL-10-production via regulation of mammalian target of rapamycin (mTOR) and that autocrine IL-10 in turn regulates macrophage nitric oxide (NO) production. Genetic and pharmacological manipulation of IL-10 and nitric oxide (NO) establish that metabolically regulated autocrine IL-10 controls glycolytic commitment by limiting NO-mediated suppression of OXPHOS. Together these data support a model where autocine IL-10 production is controlled by glycolytic flux in turn regulating glycolytic commitment by preserving OXPHOS via suppression of NO. We propose that this IL-10-driven metabolic rheostat maintains metabolic equilibrium during M1 macrophage differentiation and that perturbation of this regulatory loop, either directly by exogenous cellular sources of IL-10 or indirectly via limitations in glucose availability, skews the cellular metabolic program altering the balance between inflammatory and immunosuppressive phenotypes., (Copyright © 2016. Published by Elsevier B.V.)

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., Competing Interests: None.

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., (©2016 American Association for Cancer Research.)

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive neoplasm characterized by a marked fibro-inflammatory microenvironment, the presence of which can promote both cancer induction and growth. Therefore, selective manipulation of local cytokines is an attractive, although unrealized, therapeutic approach. T cells possess a unique mechanism of p38 mitogen-activated protein kinase (MAPK) activation downstream of T cell receptor (TCR) engagement through the phosphorylation of Tyr323 (pY323). This alternative p38 activation pathway is required for pro-inflammatory cytokine production. Here we show in human PDAC that a high percentage of infiltrating pY323(+) T cells was associated with large numbers of tumor necrosis factor (TNF)-α- and interleukin (IL)-17-producing CD4(+) tumor-infiltrating lymphocytes (TILs) and aggressive disease. The growth of mouse pancreatic tumors was inhibited by genetic ablation of the alternative p38 pathway, and transfer of wild-type CD4(+) T cells, but not those lacking the alternative pathway, enhanced tumor growth in T cell-deficient mice. Notably, a plasma membrane-permeable peptide derived from GADD45-α, the naturally occurring inhibitor of p38 pY323(+) (ref. 7), reduced CD4(+) TIL production of TNF-α, IL-17A, IL-10 and secondary cytokines, halted growth of implanted tumors and inhibited progression of spontaneous KRAS-driven adenocarcinoma in mice. Thus, TCR-mediated activation of CD4(+) TILs results in alternative p38 activation and production of protumorigenic factors and can be targeted for therapeutic benefit.

MicroRNAs (miRNAs) regulate the expression of hundreds of genes. However, identifying the critical targets within a miRNA-regulated gene network is challenging. One approach is to identify miRNAs that exert a context-dependent effect, followed by expression profiling to determine how specific targets contribute to this selective effect. In this study, we performed miRNA mimic screens in isogenic KRAS-Wild-type (WT) and KRAS-Mutant colorectal cancer (CRC) cell lines to identify miRNAs selectively targeting KRAS-Mutant cells. One of the miRNAs we identified as a selective inhibitor of the survival of multiple KRAS-Mutant CRC lines was miR-126. In KRAS-Mutant cells, miR-126 over-expression increased the G1 compartment, inhibited clonogenicity and tumorigenicity, while exerting no effect on KRAS-WT cells. Unexpectedly, the miR-126-regulated transcriptome of KRAS-WT and KRAS-Mutant cells showed no significant differences. However, by analyzing the overlap between miR-126 targets with the synthetic lethal genes identified by RNAi in KRAS-Mutant cells, we identified and validated a subset of miR-126-regulated genes selectively required for the survival and clonogenicity of KRAS-Mutant cells. Our strategy therefore identified critical target genes within the miR-126-regulated gene network. We propose that the selective effect of miR-126 on KRAS-Mutant cells could be utilized for the development of targeted therapy for KRAS mutant tumors.

Molecular profiling of primary tumors may facilitate the classification of patients with cancer into more homogenous biological groups to aid clinical management. Metabolomic profiling has been shown to be a powerful tool in characterizing the biological mechanisms underlying a disease but has not been evaluated for its ability to classify cancers by their tissue of origin. Thus, we assessed metabolomic profiling as a novel tool for multiclass cancer characterization. Global metabolic profiling was employed to identify metabolites in paired tumor and non-tumor liver (n=60), breast (n=130) and pancreatic (n=76) tissue specimens. Unsupervised principal component analysis showed that metabolites are principally unique to each tissue and cancer type. Such a difference can also be observed even among early stage cancers, suggesting a significant and unique alteration of global metabolic pathways associated with each cancer type. Our global high-throughput metabolomic profiling study shows that specific biochemical alterations distinguish liver, pancreatic and breast cancer and could be applied as cancer classification tools to differentiate tumors based on tissue of origin.

Inflammation is widely recognized as an inducer of cancer progression. The inflammation-associated enzyme, inducible nitric oxide synthase (NOS2), has emerged as a candidate oncogene in estrogen receptor (ER)-negative breast cancer, and its increased expression is associated with disease aggressiveness and poor survival. Although these observations implicate NOS2 as an attractive therapeutic target, the mechanisms of both NOS2 induction in tumors and nitric oxide (NO)-driven cancer progression are not fully understood. To enhance our mechanistic understanding of NOS2 induction in tumors and its role in tumor biology, we used stimulants of NOS2 expression in ER(-) and ER(+) breast cancer cells and examined downstream NO-dependent effects. Herein, we show that up-regulation of NOS2 occurs in response to hypoxia, serum withdrawal, IFN-γ, and exogenous NO, consistent with a feed-forward regulation of NO production by the tumor microenvironment in breast cancer biology. Moreover, we found that key indicators of an aggressive cancer phenotype including increased S100 calcium binding protein A8, IL-6, IL-8, and tissue inhibitor matrix metalloproteinase-1 are up-regulated by these NOS2 stimulants, whereas inhibition of NOS2 in MDA-MB-231 breast cancer cells suppressed these markers. Moreover, NO altered cellular migration and chemoresistance of MDA-MB-231 cells to Taxol. Most notably, MDA-MB-231 tumor xenographs and cell metastases from the fat pad to the brain were significantly suppressed by NOS2 inhibition in nude mice. In summary, these results link elevated NOS2 to signals from the tumor microenvironment that arise with cancer progression and show that NO production regulates chemoresistance and metastasis of breast cancer cells.

Purpose: To identify metabolic pathways that are perturbed in pancreatic ductal adenocarcinoma (PDAC), we investigated gene-metabolite networks with integration of metabolomics and transcriptomics., Experimental Design: We conducted global metabolite profiling analysis on two independent cohorts of resected PDAC cases to identify critical metabolites alteration that may contribute to the progression of pancreatic cancer. We then searched for gene surrogates that were significantly correlated with the key metabolites, by integrating metabolite and gene expression profiles., Results: Fifty-five metabolites were consistently altered in tumors as compared with adjacent nontumor tissues in a test cohort (N = 33) and an independent validation cohort (N = 31). Weighted network analysis revealed a unique set of free fatty acids (FFA) that were highly coregulated and decreased in PDAC. Pathway analysis of 157 differentially expressed gene surrogates revealed a significantly altered lipid metabolism network, including key lipolytic enzymes PNLIP, CLPS, PNLIPRP1, and PNLIPRP2. Gene expressions of these lipases were significantly decreased in pancreatic tumors as compared with nontumor tissues, leading to reduced FFAs. More importantly, a lower gene expression of PNLIP in tumors was associated with poorer survival in two independent cohorts. We further showed that two saturated FFAs, palmitate and stearate, significantly induced TRAIL expression, triggered apoptosis, and inhibited proliferation in pancreatic cancer cells., Conclusions: Our results suggest that impairment in a lipolytic pathway involving lipases, and a unique set of FFAs, may play an important role in the development and progression of pancreatic cancer and provide potential targets for therapeutic intervention., (©2013 AACR.)

The forkhead box L1 (FOXL1) transcription factor regulates epithelial proliferation and development of gastrointestinal tract and has been implicated in gastrointestinal tumorigenesis in mouse models. However, the role of FOXL1 in pancreatic cancer development and progression remains to be elucidated. Here, we report that higher expression of FOXL1 is significantly associated with better clinical outcome in human pancreatic ductal adenocarcinoma (PDAC). A lower FOXL1 expression is correlated with metastasis and advanced pathologic stage of pancreatic cancer. Mechanistic analyses showed that overexpression of FOXL1 induces apoptosis and inhibits proliferation and invasion in pancreatic cancer cells, whereas silencing of FOXL1 by siRNA inhibits apoptosis and enhances tumor cell growth and invasion. Furthermore, FOXL1 overexpression significantly suppressed the growth of tumor xenografts in nude mice. FOXL1 promoted apoptosis partly through the induction of TNF-related apoptosis-inducing ligand (TRAIL) in pancreatic cancer cells. In addition, FOXL1 suppressed the transcription of zinc finger E-box-binding homeobox 1 (ZEB1), an activator of epithelial-mesenchymal transition, and the negative regulation of ZEB1 contributed to the inhibitory effect of FOXL1 on tumor cell invasion. Taken together, our findings suggest that FOXL1 expression is a candidate predictor of clinical outcome in patients with resected PDAC and it plays an inhibitory role in pancreatic tumor progression.

MIF is a proinflammatory cytokine and is implicated in cancer. A higher MIF level is found in many human cancer and cancer-prone inflammatory diseases, including chronic pancreatitis and pancreatic cancer. We tested the hypothesis that MIF contributes to pancreatic cancer aggressiveness and predicts disease outcome in resected cases. Consistent with our hypothesis we found that an elevated MIF mRNA expression in tumors was significantly associated with poor outcome in resected cases. Multivariate Cox-regression analysis further showed that MIF is independently associated with patients' survival (HR = 2.26, 95% CI = 1.17-4.37, p = 0.015). Mechanistic analyses revealed that MIF overexpression decreased E-cadherin and increased vimentin mRNA and protein levels in pancreatic cancer cell lines, consistent with the features of epithelial-to-mesenchymal transition (EMT). Furthermore, MIF-overexpression significantly increased ZEB1/2 and decreased miR-200b expression, while shRNA-mediated inhibition of MIF increased E-cadherin and miR-200b expression, and reduced the expression of ZEB1/2 in Panc1 cells. Re-expression of miR-200b in MIF overexpressing cells restored the epithelial characteristics, as indicated by an increase in E-cadherin and decrease in ZEB1/2 and vimentin expression. A reduced sensitivity to the chemotherapeutic drug, gemcitabine, occurred in MIF-overexpressing cells. Indicative of an increased malignant potential, MIF over-expressing cells showed significant increase in their invasion ability in vitro, and tumor growth and metastasis in an orthotopic xenograft mouse model. These results support a role of MIF in disease aggressiveness, indicating its potential usefulness as a candidate target for designing improved treatment in pancreatic cancer., (Copyright © 2012 UICC.)

Mutant KRAS in lung cancers induces molecular pathways that regulate cellular proliferation, survival and inflammation, which enhance tumorigenesis. Inducible nitric oxide synthase (NOS2) upregulation and sustained nitric oxide generation are induced during the inflammatory response and correlate positively with lung tumorigenesis. To explore the mechanistic contribution of NOS2 to KRAS-induced lung tumorigenesis and inflammation, we used a genetic strategy of crossing NOS2 knockout (NOS2KO) C57BL6 inbred mice with a KRAS(G12D)-driven mouse lung cancer model. KRAS(G12D);NOS2KO mice exhibited delayed lung tumorigenesis and a longer overall survival time compared to that of KRAS(G12D);NOS2WT (wild-type) controls. Correspondingly, tumors in KRAS(G12D);NOS2KO mice had reduced tumor cell proliferation in adenomas and carcinomas. NOS2 deficiency also led to markedly suppressed inflammatory response by attenuation of macrophage recruitment into alveoli and within tumor foci. In contrast, FOXP3+ regulatory T cells were increased in tumors from KRAS(G12D) ;NOS2KO mice. We further analyzed the expression of microRNA-21 (miR-21), an oncogenic noncoding RNA involved in oncogenic Ras signaling, by quantitative reverse-transcription polymerase chain reaction and in situ hybridization. Lung carcinomas dissected from KRAS(G12D);NOS2KO mice showed a significantly reduced miR-21 expression along with decreased tumor cell proliferation, suggesting that NOS2 deficiency could attenuate RAS signaling pathways that transactivate miR-21 expression. Therefore, deletion of NOS2 decreases lung tumor growth as well as inflammatory responses initiated by oncogenic KRAS, suggesting that both KRAS and NOS2 cooperate in driving lung tumorigenesis and inflammation. Inhibition of NOS2 may have a therapeutic value in lung cancers with oncogenic KRAS mutations., (Copyright © 2012 UICC.)

microRNA (miRNA) are small non-coding RNA targeting mRNAs leading to their instability and diminished translation. Altered expression of miRNA is associated with cancer. Inflammation and nitric oxide modulates the development of lymphomas in p53 knockout mice and there exists a negative feedback loop between p53 and NOS2. Using a genetic strategy, we tested the hypothesis that inflammation-induced oxidative and nitrosative stress modulates miRNA expression in mouse model deficient in either p53 or NOS2. Mice treated with Corynebacterium parvum (C. parvum), to induce inflammation, clearly separated from controls by their miRNA profiles in wild-type, p53- and NOS2-knockout genetic backgrounds. C. parvum-induced inflammation significantly (p < 0.005) increased miR-21, miR-29b and miR-34a/b/c and decreased (p < 0.005) mir-29c and mir-181a/c expression in the spleen of C57BL mice. However, p53-knockout C57BL mice did not show a significant increase in the mir-34b/c or a decrease in mir-29c expression following C. parvum-induced inflammation. Expression of mir-21, mir-29b and mir-181a was independent of p53-status. NOS2-knockout C57BL mice showed a significant increase in miR-21 and miR-34a/b/c and decrease in miR-181a similar to the wild-type (WT) mice following C. parvum-induced inflammation. However, in contrast to the WT mice, miR-29b/c expression was not affected following C. parvum-induced inflammation in NOS2 knockout mice. N-acetyl cysteine, an anti-oxidant, reduced the expression of miR-21 and miR-29b in C. parvum-treated WT mice (p < 0.005) as compared with control C. parvum-treated mice. These data are consistent with the hypothesis that inflammation modulates miRNA expression in vivo and the alteration in specific miRNA under an inflammatory microenvironment, can be influenced by p53 (miR-34b/c) and NO(•) (29b/c)., (Copyright © 2011 UICC.)

Background: Cellular senescence can be a functional barrier to carcinogenesis. We hypothesized that inflammation modulates carcinogenesis through senescence and DNA damage response (DDR). We examined the association between senescence and DDR with macrophage levels in inflammatory bowel disease (IBD). In vitro experiments tested the ability of macrophages to induce senescence in primary cells. Inflammation modulating microRNAs were identified in senescence colon tissue for further investigation., Methodology/principal Findings: Quantitative immunohistochemistry identified protein expression by colon cell type. Increased cellular senescence (HP1γ; P = 0.01) or DDR (γH2A.X; P = 0.031, phospho-Chk2, P = 0.014) was associated with high macrophage infiltration in UC. Co-culture with macrophages (ANA-1) induced senescence in >80% of primary cells (fibroblasts MRC5, WI38), illustrating that macrophages induce senescence. Interestingly, macrophage-induced senescence was partly dependent on nitric oxide synthase, and clinically relevant NO• levels alone induced senescence. NO• induced DDR in vitro, as detected by immunofluorescence. In contrast to UC, we noted in Crohn's disease (CD) that senescence (HP1γ; P<0.001) and DDR (γH2A.X; P<0.05, phospho-Chk2; P<0.001) were higher, and macrophages were not associated with senescence. We hypothesize that nitric oxide may modulate senescence in CD; epithelial cells of CD had higher levels of NOS2 expression than in UC (P = 0.001). Microarrays and quantitative-PCR identified miR-21 expression associated with macrophage infiltration and NOS2 expression., Conclusions: Senescence was observed in IBD with senescence-associated β-galactosidase and HP1γ. Macrophages were associated with senescence and DDR in UC, and in vitro experiments with primary human cells showed that macrophages induce senescence, partly through NO•, and that NO• can induce DDR associated with senescence. Future experiments will investigate the role of NO• and miR-21 in senescence. This is the first study to implicate macrophages and nitrosative stress in a direct effect on senescence and DDR, which is relevant to many diseases of inflammation, cancer, and aging.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancers worldwide. To identify biologically relevant genes with prognostic and therapeutic significance in PDAC, we first performed the microarray gene-expression profiling in 45 matching pairs of tumor and adjacent non-tumor tissues from resected PDAC cases. We identified 36 genes that were associated with patient outcome and also differentially expressed in tumors as compared with adjacent non-tumor tissues in microarray analysis. Further evaluation in an independent validation cohort (N = 27) confirmed that DPEP1 (dipeptidase 1) expression was decreased (T:N ratio ∼0.1, P<0.01) in tumors as compared with non-tumor tissues. DPEP1 gene expression was negatively correlated with histological grade (Spearman correlation coefficient = -0.35, P = 0.004). Lower expression of DPEP1 in tumors was associated with poor survival (Kaplan Meier log rank) in both test cohort (P = 0.035) and validation cohort (P = 0.016). DPEP1 expression was independently associated with cancer-specific mortality when adjusted for tumor stage and resection margin status in both univariate (hazard ratio = 0.43, 95%CI = 0.24-0.76, P = 0.004) and multivariate analyses (hazard ratio = 0.51, 95%CI = 0.27-0.94, P = 0.032). We further demonstrated that overexpression of DPEP1 suppressed tumor cells invasiveness and increased sensitivity to chemotherapeutic agent Gemcitabine. Our data also showed that growth factor EGF treatment decreased DPEP1 expression and MEK1/2 inhibitor AZD6244 increased DPEP1 expression in vitro, indicating a potential mechanism for DPEP1 gene regulation. Therefore, we provide evidence that DPEP1 plays a role in pancreatic cancer aggressiveness and predicts outcome in patients with resected PDAC. In view of these findings, we propose that DPEP1 may be a candidate target in PDAC for designing improved treatments.

ING2 (inhibitor of growth family, member 2) is a member of the plant homeodomain (PHD)-containing ING family of putative tumor suppressors. As part of mSin3A-HDAC corepressor complexes, ING2 binds to tri-methylated lysine 4 of histone H3 (H3K4me3) to regulate chromatin modification and gene expression. ING2 also functionally interacts with the tumor suppressor protein p53 to regulate cellular senescence, apoptosis and DNA damage response in vitro, and is thus expected to modulate carcinogenesis and aging. Here we investigate the developmental and physiological functions of Ing2 through targeted germline disruption. Consistent with its abundant expression in mouse and human testes, male mice deficient for Ing2 showed abnormal spermatogenesis and were infertile. Numbers of mature sperm and sperm motility were significantly reduced in Ing2(-/-) mice (∼2% of wild type, P<0.0001 and ∼10% of wild type, P<0.0001, respectively). Their testes showed degeneration of seminiferous tubules, meiotic arrest before pachytene stage with incomplete meiotic recombination, induction of p53, and enhanced apoptosis. This phenotype was only partially abrogated by concomitant loss of p53 in the germline. The arrested spermatocytes in Ing2(-/-) testes were characterized by lack of specific HDAC1 accumulation and deregulated chromatin acetylation. The role of Ing2 in germ cell maturation may extend to human ING2 as well. Using publicly available gene expression datasets, low expression of ING2 was found in teratozoospermic sperm (>3-fold reduction) and in testes from patients with defective spermatogenesis (>7-fold reduction in Sertoli-cell only Syndrome). This study establishes ING2 as a novel regulator of spermatogenesis functioning through both p53- and chromatin-mediated mechanisms, suggests that an HDAC1/ING2/H3K4me3-regulated, stage-specific coordination of chromatin modifications is essential to normal spermatogenesis, and provides an animal model to study idiopathic and iatrogenic infertility in men. In addition, a bona fide tumor suppressive role of Ing2 is demonstrated by increased incidence of soft-tissue sarcomas in Ing2(-/-) mice.

Using an orthotopic model of renal cell carcinoma, we showed previously that IL-2/anti-CD40 immunotherapy resulted in synergistic anti-tumor responses, whereas IL-2 or α-CD40 alone mediated partial transient anti-tumor effects. We now show that treatment of tumor-bearing mice with IL-2/α-CD40, but not IL-2 or α-CD40, induced significant nitric oxide synthase (NOS) 2 expression in tumor-associated macrophages. In control-treated mice (low NO), NOS2 inhibition reduced tumor burden. However, during immunotherapy (high NO), NOS2 inhibition or macrophage depletion reversed the ability of IL-2/α-CD40 treatment to reduce lung metastases but had no effect on primary tumor burden. Furthermore, IL-2/α-CD40 induced the IFN-γ- and NO-dependent decrease in matrix metalloproteinase (MMP) expression and activity, concomitant with increases in tissue inhibitor of metalloproteinase (TIMP) 1 and E-cadherin expression within tumors. Finally, treatment of tumor-bearing mice with the NO donor JS-K significantly reduced metastases. These data differentiate the mechanism for primary anti-tumor effects of IL-2/α-CD40 immunotherapy, which are independent of NO, from the NO-dependent inhibition of metastases. Furthermore, reduced MMP9 activity implicates M1-polarized macrophages within the tumor microenvironment as critical components of therapeutic response. Our data demonstrate the mechanistic basis for IL-2/α-CD40-mediated control of metastases and suggest that the context-dependent application of NO donors may hold promise for prevention of metastatic disease.

Hepatocellular carcinoma (HCC) is a common cancer, and hepatitis B virus (HBV) is a major etiological agent. Convincing epidemiological and experimental evidence also links HCC to aflatoxin, a naturally occurring mycotoxin that produces a signature p53-249(ser) mutation. Recently, we have reported that tumor-derived HBx variants encoded by HBV exhibited attenuated transactivation and proapoptotic functions but retained their ability to block p53-mediated apoptosis. These results indicate that mutations in HBx may contribute to the development of HCC. In this study, we determined whether tumor-derived HBx mutants along, or in cooperation with p53-249(ser), could alter cell proliferation and chromosome stability of normal human hepatocytes. To test this hypothesis, we established a telomerase immortalized normal human hepatocycte line HHT4 that exhibited a near diploid karyotype and expressed many hepatocyte-specific genes. We found that overexpression one of the tumor-derived HBx mutants, CT, significantly increased colony forming efficiency (CFE) while its corresponding wild-type allele CNT significantly decreased CFE in HHT4 cells. p53-249(ser) rescued CNT-mediated inhibition of colony formation. Although HHT4 cells lacked an anchorage independent growth capability as they did not form any colonies in soft agar, the CT-expressing HHT4 cells could form colonies, which could be significantly enhanced by p53-249(ser). Induction of aneuploidy could be observed in HHT4 cells expressing CT, but additionally recurring chromosome abnormalities could only be detected in cells coexpressing CT and p53-249(ser). Our results are consistent with the hypothesis that certain mutations in HBx and p53 at codon 249 may cooperate in contributing to liver carcinogenesis.

Nitric oxide (NO(*)), an important signaling molecule and a component of inflammatory response, is involved in tumorigenesis. However, the quantity of NO(*) and the cellular microenvironment influences the role of NO(*) in tumor development. We used a genetic strategy to test the hypothesis that an inflammatory microenvironment with an enhanced level of NO(*) accelerates spontaneous tumor development. C. parvum-induced inflammation and increased NO(*) synthase-2 (NOS2) expression coincided with accelerated spontaneous tumor development, mostly lymphomas, in p53-/-NOS2+/+ C57BL6 mice when compared with the controls (P = 0.001). However, p53-/-NOS2-/- mice did not show any difference in tumor latency between C. parvum-treated and control groups. In C. parvum-treated p53-/-NOS2+/+ mice, tumor development was preceded by a higher expression of NOS2 and phosphorylated Akt-Ser(473) (pAkt-Ser473) in spleen, increased cell proliferation measured by Ki-67 IHC in spleen and thymus, and a lower apoptotic index and CD95-L expression in spleen and thymus. C. parvum-treated p53-/-NOS2+/+ mice showed an increase in the number of Foxp3(+) T-reg cells, dendritic cells (DC), as well as increased CD80(+), CD86(+), CD40(+), and CD83(+) on DC in the spleen. Regulatory T-cells (T-reg) and the maturation of DC may modulate tumorigenesis. An increase in the FoxP3(+)T-reg cells in C. parvum-treated p53-/-NOS2+/+ mice indicates a role of NO(*) in the regulation of T-reg cells that may contribute to a protumor shift of the immune environment favoring an accelerated tumor development. These data provide genetic and mechanistic evidence that an inflammatory microenvironment and an increased level of NO(*) can accelerate tumor development.

Infection and chronic inflammation contribute to about 1 in 4 of all cancer cases. Mediators of the inflammatory response, e.g., cytokines, free radicals, prostaglandins and growth factors, can induce genetic and epigenetic changes including point mutations in tumor suppressor genes, DNA methylation and post-translational modifications, causing alterations in critical pathways responsible for maintaining the normal cellular homeostasis and leading to the development and progression of cancer. Recent discovery of an interaction between microRNAs and innate immunity during inflammation has further strengthened the association between inflammation and cancer., ((c) 2007 Wiley-Liss, Inc.)

Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide and the major risk factors include chronic infections with the hepatitis B (HBV) or C (HCV) virus, and exposure to dietary aflatoxin B(1) (AFB(1)) or alcohol consumption. Multiple genetic and epigenetic changes are involved in the molecular pathogenesis of HCC, for example, somatic mutations in the p53 tumor suppressor gene (TP53) and the activation of the WNT signal transduction pathway. AFB(1) frequently induces G:C to T:A transversions at the third base in codon 249 of TP53 and cooperates with HBV in causing p53 mutations in HCC. The detection of TP53 mutant DNA in plasma is a biomarker of both AFB(1) exposure and HCC risk. Chronic infection with HBV and HCV viruses, and oxyradical disorders including hemochromatosis, also generate reactive oxygen/nitrogen species that can both damage DNA and mutate cancer-related genes such as TP53. Certain mutant p53 proteins may exhibit a 'gain of oncogenic function'. The p53 biological network is a key responder to this oxidative and nitrosative stress. Depending on the extent of the DNA damage, p53 regulates the transcription of protective antioxidant genes and with extensive DNA damage, transactivates pro-oxidant genes that contribute to apoptosis. The X gene of HBV (HBx) is the most common open reading frame integrated into the host genome in HCC and the integrated HBx is frequently mutated. Mutant HBx proteins still retain their ability to bind to p53, and attenuate DNA repair and p53-mediated apoptosis. In summary, both viruses and chemicals are implicated in the etiology of TP53 mutations during the molecular pathogenesis of HCC.

Independently, superoxide (O2-) and nitric oxide (NO) are biologically important signaling molecules. When co-generated, these radicals react rapidly to form powerful oxidizing and nitrating intermediates. Although this reaction was once thought to be solely cytotoxic, herein we demonstrate using MCF7, macrophage, and endothelial cells that when nanomolar levels of NO and O2- were produced concomitantly, the effective NO concentration was established by the relative fluxes of these two radicals. Differential regulation of sGC, pERK, HIF-1alpha, and p53 were used as biological dosimeters for NO concentration. Introduction of intracellular- or extracellular-generated O2- during NO generation resulted in a concomitant increase in oxidative intermediates with a decrease in steady-state NO concentrations and a proportional reduction in the levels of sGC, ERK, HIF-1alpha, and p53 regulation. NO responses were restored by addition of SOD. The intermediates formed from the reactions of NO with O2- were non-toxic, did not form 3-nitrotyrosine, nor did they elicit any signal transduction responses. H2O2 in bolus or generated from the dismutation of O2- by SOD, was cytotoxic at high concentrations and activated p53 independent of NO. This effect was completely inhibited by catalase, suppressed by NO, and exacerbated by intracellular catalase inhibition. We conclude that the reaction of O2- with NO is an important regulatory mechanism, which modulates signaling pathways by limiting steady-state levels of NO and preventing H2O2 formation from O2-.

In lung tumors, the p53 tumor suppressor gene is commonly mutated with a characteristic mutation spectrum. The amount of and alterations in plasma DNA, such as mutations in p53, were associated with several cancers. Few studies used quantitative methods of high sensitivity. Previously, we observed p53 mutations in the noncancerous tissue that differed from those in lung tumors using the highly sensitive p53 mutation load assay. Based on our observation of an increased p53 mutation load in nontumorous lung tissue in smokers, we hypothesized that plasma DNA may contain mutant p53 indicative of tobacco smoke exposure and will be an effective biomarker of lung cancer or smoking exposure. We modified the p53 mutation load assay to detect mutations at p53 codons 248 and 249, common mutations in lung cancer, in plasma DNA samples with a sensitivity of 1:5,000. The assay was applied to a set of lung cancer cases (n = 39), hospital controls (n = 21), and population controls (n = 20) from a larger study. Controls were selected to consist of equal numbers of both ever and never smokers. The p53 mutation load (mutated p53 copies per total number of p53 copies) was associated with smoking (P = 0.06), but not with lung cancer (P = 0.59). Most of the individuals with p53 mutations observed in plasma DNA were ever smokers and the p53 mutation load was higher in those who smoked for longer durations (P = 0.04). In summary, we were able to detect p53 mutations in plasma DNA from healthy individuals and our data suggest that p53 mutations in plasma DNA may be a marker of carcinogen exposure from tobacco smoke.

Nitric oxide (NO.), an important mediator of inflammation, and beta-catenin, a component of the Wnt-adenomatous polyposis coli signaling pathway, contribute to the development of cancer. We have identified two T-cell factor 4 (Tcf-4)-binding elements (TBE1 and TBE2) in the promoter of human inducible NO synthase 2 (NOS2). We tested the hypothesis that beta-catenin regulates human NOS2 gene. Mutation in either of the two TBE sites decreased the basal and cytokine-induced NOS2 promoter activity in different cell lines. The promoter activity was significantly reduced when both TBE1 and TBE2 sites were mutated (P < 0.01). Nuclear extract from HCT116, HepG2, or DLD1 cells bound to NOS2 TBE1 or TBE2 oligonucleotides in electrophoretic mobility shift assays and the specific protein-DNA complexes were supershifted with anti-beta-catenin or anti-Tcf-4 antibody. Overexpression of beta-catenin and Tcf-4 significantly increased both basal and cytokine-induced NOS2 promoter activity (P < 0.01), and the induction was dependent on intact TBE sites. Overexpression of beta-catenin or Tcf-4 increased NOS2 mRNA and protein expression in HCT116 cells. Lithium chloride (LiCl), an inhibitor of glycogen synthase kinase-3beta, increased cytosolic and nuclear beta-catenin level, NOS2 expression, and NO. production in primary human and rat hepatocytes and cancer cell lines. Treatment with Wnt-3A-conditioned medium increased beta-catenin and NOS2 expression in fetal human hepatocytes. When administered in vivo, LiCl increased hepatic beta-catenin level in a dose-dependent manner with simultaneous increase in NOS2 expression. These data are consistent with the hypothesis that beta-catenin up-regulates NOS2 and suggest a novel mechanism by which the Wnt/beta-catenin signaling pathway may contribute to cancer by increasing NO. production.

p53 as a key molecular node in the stress response pathway, including inflammation. p53 is involved in several critical pathways including cell cycle arrest, apoptosis, DNA repair, and cellular senescence, which are essential for normal cellular homeostasis and maintaining genome integrity. The alteration of the TP53 gene or posttranslational modification in the p53 protein can alter its response to cellular stress. The molecular archaeology of the TP53 mutation spectrum generates hypotheses concerning the etiology and molecular pathogenesis of human cancer. The spectrum of somatic mutations in the TP53 gene implicates environmental carcinogens, and both endogenous agents and processes in the etiology of human cancer.

Activation of the p53 network plays a central role in the inflammatory stress response associated with ulcerative colitis and may modulate cancer risk in patients afflicted with this chronic disease. Here, we describe the gene expression profiles associated with four microenvironmental components of the inflammatory response (NO*, H2O2, DNA replication arrest, and hypoxia) that result in p53 stabilization and activation. Isogenic HCT116 and HCT116 TP53-/- colon cancer cells were exposed to the NO* donor Sper/NO, H2O2, hypoxia, or hydroxyurea, and their mRNA was analyzed using oligonucleotide microarrays. Overall, 1,396 genes changed in a p53-dependent manner (P < 0.001), with the majority representing a "unique" profile for each condition. Only 14 genes were common to all four conditions. Included were eight known p53 target genes. Hierarchical sample clustering distinguished early (1 and 4 hours) from late responses (8, 12, and 24 hours), and each treatment was differentiated from the others. Overall, NO* and hypoxia stimulated similar transcriptional responses. Gene ontology analysis revealed cell cycle as a key feature of stress responses and confirmed the similarity between NO* and hypoxia. Cell cycle profiles analyzed by flow cytometry showed that NO* and hypoxia induced quiescent S-phase and G2-M arrest. Using a novel bioinformatic algorithm, we identified several putative p53-responsive elements among the genes induced in a p53-dependent manner, including four [KIAA0247, FLJ12484, p53CSV (HSPC132), and CNK (PLK3)] common to all exposures. In summary, the inflammatory stress response is a complex, integrated biological network in which p53 is a key molecular node regulating gene expression.

Chronic inflammation contributes to tumorigenesis. The retinoblastoma protein (pRb), in its hyperphosphorylated form, releases E2 promoter binding factor-1 (E2F1), which drives cell proliferation. Here, we show that pRb is hyperphosphorylated in both mouse and human colitis. In turn, pRb hyperphosphorylation is associated with release of E2F1 from pRb, resulting in the activation of E2F1 target molecules involved in proliferation and apoptosis. These observations provide insight into the in vivo mechanisms associated with chronic colon inflammation and increased colon cancer risk.

Inflammation influences the development of cancer. The nitric oxide synthase (NOS2) is induced by inflammatory cytokines, e.g., tumor necrosis factor alpha and interleukin 1beta, and produces nitric oxide (NO*), a critical mediator of the inflammatory response. Because p53 governs NO* production by transcriptionally transrepressing NOS2, we used a genetic strategy to determine whether NO* and p53 cooperatively regulate tumorigenesis. Lymphomas developed more rapidly in p53-/-NOS2-/- or p53-/-NOS2+/- mice than in p53-/-NOS2+/+ mice that were cross-bred into a >95% C57BL6 background and maintained in a pathogen-free condition. Likewise, sarcomas and lymphomas developed faster in p53+/-NOS2-/- or p53+/-NOS2+/- than in p53+/-NOS2+/+ mice. When compared with the double knockout mice, p53-/-NOS2+/+ mice showed a higher apoptotic index and a decreased proliferation index with an increased expression of death receptor ligands, CD95-L and tumor necrosis factor-related apoptosis-inducing ligand, and the cell cycle checkpoint protein, p21(waf1), in the spleen and thymus before tumor development. Furthermore, mice deficient in both p53 and NOS2 produced a high level of anti-inflammatory interleukin 10 when compared with p53-deficient mice. These studies provide genetic and mechanistic evidence that NO* can suppress tumorigenesis.

p53-mediated apoptosis may involve the induction of redox-controlling genes, resulting in the production of reactive oxygen species. Microarray expression analysis of doxorubicin exposed, related human lymphoblasts, p53 wild-type (WT) Tk6, and p53 mutant WTK1 identified the p53-dependent up-regulation of manganese superoxide dismutase (MnSOD) and glutathione peroxidase 1 (GPx). Consensus p53 binding sequences were identified in human MnSOD and GPx promoter regions. A 3-fold increase in the MnSOD promoter activity was observed after the induction of p53 in Li-Fraumeni syndrome (LFS) fibroblast, TR9-7, expressing p53 under the control of a tetracycline-regulated promoter. An increased protein expression of endogenous MnSOD and GPx also positively correlated with the level of p53 induction in TR9-7 cells. However, catalase (CAT) protein expression remained unaltered after p53 induction. We also examined the expression of MnSOD, GPx, and CAT in a panel of normal or LFS fibroblasts, containing either WT or mutant p53. We found increased MnSOD enzymatic activity, MnSOD mRNA expression, and MnSOD and GPx protein in LFS fibroblasts carrying a WT p53 allele when compared with homozygous mutant p53 isogenic cells. The CAT protein level was unchanged in these cells. We observed both the release of cytochrome C and Ca(2+) from the mitochondria into the cytoplasm and an increased frequency of apoptotic cells after p53 induction in the TR9-7 cells that coincided with an increased expression of MnSOD and GPx, and the level of reactive oxygen species. The increase in apoptosis was reduced by the antioxidant N-acetylcysteine. These results identify a novel mechanism of p53-dependent apoptosis in which p53-mediated up-regulation of MnSOD and GPx, but not CAT, produces an imbalance in antioxidant enzymes and oxidative stress.

Since its discovery 25 years ago, the p53 protein has emerged as a key tumor suppressor protein at the crossroads of cellular stress response pathways. Through these pathways, which can lead to cell-cycle arrest, DNA repair, cellular senescence, differentiation and apoptosis, p53 facilitates the repair and survival of damaged cells or eliminates severely damaged cells from the replicative pool to protect the organism. Because of these dynamic and multiple functions of p53, which are largely lost following mutations in the gene encoding p53, this molecule continues to be studied intensively in biomedical research, including the fields of toxicology and pharmacology. In this article, we briefly review the first 25 years of research on p53.

Among genetic alterations, the activation of proto-oncogenes and inactivation of tumour suppressor genes in affected cells are considered to be the core molecular events that provide a selective growth advantage and clonal expansion during the multistep process of carcinogenesis. The TP53 tumour suppressor gene is mutated in about half of all human cancer cases. The p53 protein modulates multiple cellular functions, such as gene transcription, DNA synthesis and repair, cell cycle arrest, senescence and apoptosis. Mutations in the TP53 gene can abrogate these functions, leading to genetic instability and progression to cancer. The molecular archaeology of the TP53 mutation spectrum generates hypotheses concerning the etiology and molecular pathogenesis of each type of cancer. The spectrum of somatic mutations in the TP53 gene, of which 75% are missense mutations, implicates environmental carcinogens and endogenous processes in the etiology of human cancer. The presence of a characteristic TP53 mutation can also manifest a molecular link between exposure to a particular carcinogen and a specific type of human cancer, e.g. exposure to aflatoxin B1 (AFB1) and codon 249 mutations in hepatocellular carcinoma; exposure to ultraviolet (UV) light and C:C-->T:T tandem mutations in skin cancer; and cigarette smoking and the prevalence of G-->T transversions in lung cancer. Although exogenous carcinogens have been shown to target p53 selectively, evidence supporting the endogenous insult of TP53 from oxyradicals and nitrogen-oxyradicals is also accumulating. TP53 mutations can be a biomarker of carcinogen effect. Determining the characteristic TP53 mutation load in non-tumorous tissue, using a highly sensitive mutation assay, can indicate exposure to a specific carcinogen and may also help in identifying individuals at an increased risk of cancer.