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1.  HuR’s post-transcriptional regulation of death receptor 5 in pancreatic cancer cells 
Cancer Biology & Therapy  2012;13(10):946-955.
Apoptosis is one of the core signaling pathways disrupted in pancreatic ductal adenocarcinoma (PDA). Death receptor 5 (DR5) is a member of the tumor necrosis factor (TNF)-receptor superfamily that is expressed in cancer cells. Binding of TNF-related apoptosis-inducing ligand (TRAIL) to DR5 is a potent trigger of the extrinsic apoptotic pathway, and numerous clinical trials are based on DR5-targeted therapies for cancer, including PDA. Human antigen R (HuR), an RNA-binding protein, regulates a select number of transcripts under stress conditions. Here we report that HuR translocates from the nucleus to the cytoplasm of PDA cells upon treatment with a DR5 agonist. High doses of DR5 agonist induce cleavage of both HuR and caspase 8. HuR binds to DR5 mRNA at the 5′-untranslated region (UTR) in PDA cells in response to different cancer-associated stressors and subsequently represses DR5 protein expression; silencing HuR augments DR5 protein production by enabling its translation and thus enhances apoptosis. In PDA specimens (n = 53), negative HuR cytoplasmic expression correlated with elevated DR5 expression (odds ratio 16.1, p < 0.0001). Together, these data demonstrate a feedback mechanism elicited by HuR-mediated repression of the key apoptotic membrane protein DR5.
doi:10.4161/cbt.20952
PMCID: PMC3414415  PMID: 22785201
DR5; TRAIL; TRAIL-resistance; TRAILR2; apoptosis; pancreatic cancer; pancreatic ductal adenocarcinoma; post-transcriptional regulation
2.  Immunohistochemical and Genetic Evaluation of Deoxycytidine Kinase in Pancreatic Cancer: Relationship to Molecular Mechanisms of Gemcitabine Resistance and Survival 
Gemcitabine is considered the standard first-line therapy for patients with advanced pancreatic cancer. More recent strategies have focused on improving the efficacy of gemcitabine by either improving the method of delivery or by combining gemcitabine with other non-cross-resistant chemotherapy agents or with small-molecule drugs. However, the clinical benefits, response rates, and duration of responses have been modest. Deoxycytidine kinase (dCK) is the rate-limiting enzyme involved in the metabolism of gemcitabine. The expression of dCK has been postulated to be correlative of gemcitabine resistance. We determined the relationship of dCK immunohistochemical protein expression and/or genetic status of dCK in a panel of human pancreatic cancer tissues and pancreatic cancer cell lines and determined the relationship of these variables to the clinical outcome of patients treated with gemcitabine. We report that dCK protein expression is expressed in the majority of pancreatic cancers analyzed (40 of 44 cases, 91%) and showed a range of labeling intensities ranging from 1+ (labeling weaker in intensity than normal lymphocytes present in same section) to 3+ (labeling greater in intensity than normal lymphocytes present in same section). When labeling intensity was compared with survival, low dCK expression (1+ labeling) was correlated with both overall survival (P < 0.009) and progression-free survival following gemcitabine treatment (P < 0.04). Low dCK labeling intensity was also significantly correlated with patient age (70.3 ± 8.1 versus 59.8 ± 7.4 years; P < 0.0006), suggesting that age-related methylation of the dCK gene may account in part for the observed differences. Sequencing of the entire dCK coding sequence in 17 cell lines and 9 patients’ cancer tissues with disease progression while on gemcitabine did not identify any mutations, suggesting that genetic alterations of dCK are not a common mechanism of resistance to gemcitabine for this tumor type. Moreover, dCK labeling showed similar patterns and intensities of labeling among matched pretreatment and post-treatment tissues. In summary, pretreatment levels of dCK protein are most correlated with overall survival following gemcitabine treatment and are stable even after resistance to gemcitabine is clinically documented.
doi:10.1158/1078-0432.CCR-05-2655
PMCID: PMC3619210  PMID: 16638857
3.  The Novel Triad of Dorsal Agenesis of the Pancreas with Concurrent Pancreatic Ductal Adenocarcinoma and Nonalcoholic Chronic Calcific Pancreatitis: A Case Series and Review of the Literature 
Journal of Gastrointestinal Surgery  2011;15(9):1643-1649.
Introduction
Dorsal agenesis of the pancreas (DAP) is a rare congenital anomaly, with only 44 cases having been reported in the English literature since 1966.
Materials and Methods
A retrospective review of our IRB-approved pancreatic surgery database was performed from November 2005 to November 2010 searching for cases of DAP.
Discussion
Disorders in the retinoic acid (Raldh) and hedgehog (Hh) signaling pathways, which appear to play a role in the development of DAP, have been implicated in other diseases of the pancreas such as pancreatic ductal adenocarcinoma (PDA) and nonalcoholic chronic calcific pancreatitis (NCCP).
Conclusion
In this report, we describe three cases of DAP in the setting of PDA, two of which include the third component of NCCP. We provide a discussion of the clinical features of this novel triad and address the molecular pathways that relate to these respective diseases.
doi:10.1007/s11605-011-1542-6
PMCID: PMC3282461  PMID: 21512846
Dorsal agenesis of the pancreas; Pancreatic ductal adenocarcinoma; Nonalcoholic chronic calcific pancreatitis
4.  TUMOR-SPECIFIC EXPRESSION AND ALTERNATIVE SPLICING OF THE COL6A3 GENE IN PANCREATIC CANCER 
Surgery  2011;150(2):306-315.
Introduction
Pancreatic ductal adenocarcinoma (PDA) is a highly lethal disease in which a prominent desmoplastic reaction is a defining characteristic. Fibrillar collagens, such as collagen I and to a lesser extent, collagen III and V comprise the majority of this stromal fibrosis. Type VI collagen (COL6) forms a microfibrillar network associated with type I collagen fibrils. The expression of COL6 has been linked to inflammation and survival. Importantly, tumor-specific alternative splicing in COL6A3 has been identified in several cancers by genome exon arrays. We evaluated the expression and localization of COL6A3 in PDA and premalignant lesions and explored the presence of alternative splicing events.
Methods
We analyzed paired PDA-normal (n=18), IPMN (n=5), pancreatic cystadenoma (n=5), and eight PDA cell lines with RT-PCR, using unique primers that identify total COL6A3 gene and alternative splicing sites in several of its exons. Western blot analysis and immunohistochemistry were used to analyze the expression levels and localization of COL6A3 protein in the different lesions, and in two animal models of PDA.
Results
COL6A3 protein levels were significantly upregulated in 77% of the paired PDA-adjacent tissue examined. COL6A3 was mainly present in the desmoplastic stroma of PDA, with high deposition around the malignant ducts and in between the sites of stromal fatty infiltration. Analysis of the COL6A3 splice variants showed tumor-specific consistent inclusion of exons 3 and 6 in 17 of the 18 (94%) paired PDA-adjacent tissues. Inclusion of exon 4 was exclusively tumor-specific, with barely detectable expression in the adjacent tissues. IPMN and pancreatic cystadenomas showed no expression of any of the examined exons. Total COL6A3 mRNA and exon 6 were identified in six PDA cell lines, but only two cell lines (MIA PACA-2 and ASPC-1) expressed exons 3 and 4. In both the xenograft and transgenic models of PDA, COL6A3 immunoreactivity was present in the stroma and some PDA cells.
Conclusions
We describe, for the first time, a dynamic process of tumor-specific alternative splicing in several exons of stromal COL6A3. Alternatively spliced proteins may contribute to the etiology or progression of cancer and may serve as markers for cancer diagnosis. Identification of COL6A3 isoforms as PDA-specific provides the basis for future studies to explore the oncogenic and diagnostic potential of these alternative splicing events.
doi:10.1016/j.surg.2011.05.011
PMCID: PMC3163121  PMID: 21719059
pancreatic cancer; COL6A3; stroma; microenvironment
5.  Oncogene-induced Nrf2 transcription promotes ROS detoxification and tumorigenesis 
Nature  2011;475(7354):106-109.
Reactive oxygen species (ROS) are mutagenic and may thereby promote cancer1. Normally, ROS levels are tightly controlled by an inducible antioxidant program that responds to cellular stressors and is predominantly regulated by the transcription factor Nrf2 and its repressor protein Keap12-5. In contrast to the acute physiological regulation of Nrf2, in neoplasia there is evidence for increased basal activation of Nrf2. Indeed, somatic mutations that disrupt the Nrf2-Keap1 interaction to stabilize Nrf2 and increase the constitutive transcription of Nrf2 target genes were recently identified, suggesting that enhanced ROS detoxification and additional Nrf2 functions may in fact be pro-tumorigenic6. Here, we investigated ROS metabolism in primary murine cells following the expression of endogenous oncogenic alleles of K-Ras, B-Raf and Myc, and find that ROS are actively suppressed by these oncogenes. K-RasG12D, B-RafV619E and MycERT2 each increased the transcription of Nrf2 to stably elevate the basal Nrf2 antioxidant program and thereby lower intracellular ROS and confer a more reduced intracellular environment. Oncogene-directed increased expression of Nrf2 is a novel mechanism for the activation of the Nrf2 antioxidant program, and is evident in primary cells and tissues of mice expressing K-RasG12D and B-RafV619E, and in human pancreatic cancer. Furthermore, genetic targeting of the Nrf2 pathway impairs K-RasG12D-induced proliferation and tumorigenesis in vivo. Thus, the Nrf2 antioxidant and cellular detoxification program represents a previously unappreciated mediator of oncogenesis.
doi:10.1038/nature10189
PMCID: PMC3404470  PMID: 21734707
6.  Is there a role for the quantification of RRM1 and ERCC1 expression in pancreatic ductal adenocarcinoma? 
BMC Cancer  2012;12:104.
Background
RRM1 and ERCC1 overexpression has been extensively investigated as potential predictive markers of tumor sensitivity to conventional chemotherapy agents, most thoroughly in lung cancer. However, data in pancreatic cancer are scarce.
Methods
We investigated the mRNA and protein expression of ERCC1 and RRM1 by RT-PCR and immunohistochemistry (IHC) in formalin-fixed, paraffin-embedded pancreatic ductal carcinoma (PDA) tissues. The primary outcome investigated was the association between RRM1 and ERCC1 expression and overall survival (OS) or disease-free survival (DFS).
Results
A total of 94 patients with resected PDA were included in this study. Most of them (87%) received gemcitabine based chemotherapy. Data for OS analysis was available in all cases but only 68% had enough information to estimate DFS. IHC analysis revealed information for 99% (93/94) and 100% of the cases for RRM1 and ERCC1 expression respectively. However, PCR data interpretation was possible in only 49 (52%) and 79 (84%) cases respectively. There was no significant association between high or low expression of either RRM1 or ERCC1, detected by IHC and OS (14.4 vs. 19.9 months; P = 0.5 and 17.1 vs. 19.9; P = 0.83 respectively) or PCR and OS (48.0 vs. 24.1 months; P = 0.21 and 22.0 vs. 16.0 months; P = 0.39 respectively). Similar results were obtained for DFS.
Conclusions
RRM1 and ERCC1 expression does not seem to have a clear predictive or prognostic value in pancreatic cancer. Our data raise some questions regarding the real clinical and practical significance of analyzing these molecules as predictors of outcomes.
doi:10.1186/1471-2407-12-104
PMCID: PMC3364898  PMID: 22436573
7.  Phase I Evaluation of Intravenous Ascorbic Acid in Combination with Gemcitabine and Erlotinib in Patients with Metastatic Pancreatic Cancer 
PLoS ONE  2012;7(1):e29794.
Background
Preclinical data support further investigation of ascorbic acid in pancreatic cancer. There are currently insufficient safety data in human subjects, particularly when ascorbic acid is combined with chemotherapy.
Methods and Findings
14 subjects with metastatic stage IV pancreatic cancer were recruited to receive an eight week cycle of intravenous ascorbic acid (three infusions per week), using a dose escalation design, along with standard treatment of gemcitabine and erlotinib. Of 14 recruited subjects enrolled, nine completed the study (three in each dosage tier). There were fifteen non-serious adverse events and eight serious adverse events, all likely related to progression of disease or treatment with gemcitabine or erlotinib. Applying RECIST 1.0 criteria, seven of the nine subjects had stable disease while the other two had progressive disease.
Conclusions
These initial safety data do not reveal increased toxicity with the addition of ascorbic acid to gemcitabine and erlotinib in pancreatic cancer patients. This, combined with the observed response to treatment, suggests the need for a phase II study of longer duration.
Trial Registration
Clinicaltrials.gov NCT00954525
doi:10.1371/journal.pone.0029794
PMCID: PMC3260161  PMID: 22272248
8.  Nanoparticulate delivery of diphtheria toxin DNA effectively kills Mesothelin expressing pancreatic cancer cells 
Cancer biology & therapy  2008;7(10):1584-1590.
Pancreatic cancer is the fourth leading cause of cancer-related deaths in this country, and there is currently no effective targeted treatment for this deadly disease. A dire need exists to rapidly translate our molecular understanding of this devastating disease into effective, novel therapeutic options. Mesothelin is a candidate target protein shown by a number of laboratories to be specifically overexpressed in pancreatic cancers and not in the adjacent normal tissue. Translational investigations have shown promising results using this molecule as a therapeutic target (e.g., vaccine strategies). In addition, the mesothelin promoter has been cloned and dissected and can therefore be used as a vehicle for regulating expression of DNA sequences. Using a novel, proven, biodegradable nanoparticulate system, we sought to target mesothelin-expressing pancreatic cancer cells with a potent suicide gene, diphtheria toxin-A (DT-A). We first confirmed reports that a majority of pancreatic cancer cell lines and resected pancreatic ductal adenocarcinoma specimens overexpressed mesothelin at the mRNA and protein levels. High mesothelin-expressing pancreatic cancer cell lines produced more luciferase than cell lines with undetectable mesothelin expression when transfected with a luciferase sequence under the regulation of the mesothelin promoter. We achieved dramatic inhibition of protein translation (>95%) in mesothelin-expressing pancreatic cancer cell lines when DT-A DNA, driven by the mesothelin promoter, was delivered to pancreatic cancer cells. We show that this inhibition effectively targets the death of pancreatic cancer cells that overexpress mesothelin. The work presented here provides evidence that this strategy will work in pre-clinical mouse pancreatic cancer models, and suggests that such a strategy will work in the clinical setting against the majority of pancreatic tumors, most of which overexpress mesothelin.
PMCID: PMC3218426  PMID: 19039293
nanoparticles; mesothelin; pancreatic cancer; targeted DNA delivery
9.  CanScript, an 18-Base pair DNA sequence, boosts tumor cell-specific promoter activity 
Cancer Biology & Therapy  2010;10(9):878-884.
Gene therapy protocols for the treatment of cancer often employ gene promoter sequences that are known to be overexpressed in specific tumor cell types relative to normal cells. These promoters, while specific, are often weakly active. It would be desirable to increase the activity of such promoters, while at the same time retain specificity, so that the therapeutic gene is more robustly expressed. Using a luciferase reporter DNA construct in both in vitro cell transfection assays and in vivo mouse tumor models, we have determined that in the absence of any other DNA sequence, a previously identified 18-base pair enhancer sequence called CanScript, lying upstream of the MSLN gene, has ∼25% of the promoter activity of CAG, a very strong non-specific promoter/enhancer, in tumor cells in which MSLN is highly expressed. Furthermore, tandem repeat copies of CanScript enhance transcription in a dose-dependent manner and, when coupled with promoter sequences that are active in tumor cells, increase promoter activity. These findings suggest that the incorporation of CanScript into gene constructs may have application in enhancing activity of promoters used in cancer-targeting gene therapy strategies, thereby improving therapeutic efficacy.
doi:10.4161/cbt.10.9.13234
PMCID: PMC3040857  PMID: 20798601
targeted gene therapy; mesothelin; canscript; gene promoter
10.  Serum Monocyte Chemoattractant Protein-1 in Pancreatic Cancer 
Journal of Oncology  2011;2011:518394.
Background/Aims. Pancreatic ductal adenocarcinoma (PDA) has etiological association with chronic inflammation. Elevated circulating levels of inflammatory mediators, such as monocyte chemoattractant protein-1 (MCP-1), are found in obese individuals. We hypothesized that serum MCP-1 levels are elevated in obese PDA patients. Methods. ELISA was used to analyze MCP-1 serum levels in PDA (n = 62) and intraductal papillary mucinous neoplasms (IPMN) (n = 27). Recursive partitioning statistical analysis investigated the relationship between log MCP-1 and clinicopathological parameters. Results. Log MCP-1 values were significantly (P < 0.05) elevated in patients with BMI ≥ 37.5. In patients with BMI < 37.5, average log MCP-1 values were significantly elevated in PDA patients when compared to IPMN patients. Within the IPMN group, higher log MCP-1 levels correlated with increased age. Recursive partitioning analysis of IPMN versus PDA revealed a strategy of predicting characteristics of patients who are more likely to have cancer. This strategy utilizes log MCP-1 as the primary factor and also utilizes smoking status, gender, and age. Conclusion. MCP-1 is a promising biomarker in pancreatic cancer. The potential of using MCP-1 to distinguish PDA from IPMN patients must be studied in larger populations to validate and demonstrate its eventual clinical utility.
doi:10.1155/2011/518394
PMCID: PMC3184439  PMID: 21977031
12.  Genotyping and expression analysis of IDO2 in human pancreatic cancer: a novel, active target 
Background
We recently discovered that the enzyme indoleamine 2,3-dioxygenase (IDO) is overexpressed in primary pancreatic ductal adenocarcinomas (PDA) and in lymph node metastases (J Am Coll Surg. 5:849-54: 2008). IDO2 is a recently discovered relative of IDO that has unique signaling properties (Cancer Res. 67:7082-7087: 2007). Notably, the IDO2 gene has two functional polymorphisms commonly found in human populations that abolish its enzymatic activity (R235W and Y359STOP). Both IDO and IDO2 repress the immune system and we hypothesize that expression of these enzymes in PDA may help cancer cells evade immune detection.
Methods
Based on evidence that the IDO2 may be a preferential target of D-1-methyl-tryptophan (1-MT), a clinical lead inhibitor of IDO currently being evaluated in Phase I trials, we sequenced IDO2 in 36 resected PDAs and evaluated its expression in relation to the two known genetic polymorphisms.
Results
In our patient cohort, we found that 58% (21/36) of the cases were heterozygous for the R235W polymorphism; 28% (10/36) were homozygous wild-type; and only 14% (5/36) were homozygous for the functionally inactive polymorphism. Interestingly, IDO2 had a homozygous wild-type configuration in two pancreatic cancer cell lines whereas one cell line (MiaPaCa2 cells) was homozygous for the R235W polymorphism. As for the Y359STOP polymorphism (seen in the cell line Hs766T), we found that 27% (10/36) of the cases were heterozygous, 62% (22/36) were homozygous wild-type, and only 11% (4/36) were homozygous for this functionally inactive allele. Ruling out the possibility of compound polymorphic variants, we estimated 75% of our resected patient cohort had an active IDO2 enzyme with a conservative estimate that 58% of the patients had at least one functional allele. In immunohistochemical analyses, we found that IDO2 was equally overexpressed in pancreatic cancer tissue from each genetially polymorphic subgroup. We also detected IDO2 protein expression in the genetically distinct pancreatic cancer cell lines after exposure with IFN-γ, establishing that even functionally polymorphic IDO2 sequences can generate IDO2 protein.
Conclusions
These are the first data to report IDO2 expression in PDA and indicate that IDO2 genetic polymorphisms do not negate IFN-γ-inducible protein expression. IDO2 genotyping and expression analysis of our PDA patient tissue bank and cell lines show that IDO2 is active and expressed in a majority of PDA patients. Taken together, these data strongly suggest that the clinical lead compound D-1-MT acting through IDO2 might be useful in treatment of PDA, either alone or in combination with other anti-tumor modalities.
doi:10.1016/j.jamcollsurg.2008.12.018
PMCID: PMC3176891  PMID: 19476837
13.  INDUCTION OF MONOCYTE CHEMOATTRACTANT PROTEIN-1 BY NICOTINE IN PANCREATIC DUCTAL ADENOCARCINOMA CELLS: ROLE OF OSTEOPONTIN 
Surgery  2010;148(2):298-309.
Introduction
Cigarette smoke and nicotine are among the leading environmental risk factors for developing pancreatic ductal adenocarcinoma (PDA). We showed recently that nicotine induces osteopontin (OPN), a protein that plays critical roles in inflammation and tumor metastasis. We identified an OPN isoform, OPNc, that is selectively inducible by nicotine and highly expressed in PDA tissue from smokers. In this study, we explored the potential proinflammatory role of nicotine in PDA through studying its effect on the expression of monocyte chemoattractant protein- (MCP)-1 and evaluated the role of OPN in mediating these effects.
Methods
MCP-1 mRNA and protein in PDA cells treated with or without nicotine (3–300 nM) or OPN (0.15–15 nM) were analyzed by real time PCR and ELISA. Luciferase-labeled promoter studies evaluated the effects of nicotine and OPN on MCP-1 transcription. Intracellular and tissue colocalization of OPN and MCP-1 were examined by immunofluorescence and immunohistochemistry.
Results
Nicotine treatment significantly increased MCP-1 expression in PDA cells. Interestingly, blocking OPN with siRNA or OPN antibody abolished these effects. Transient transfection of the OPNc gene in PDA cells or their treatment with recombinant OPN protein significantly (P<0.05) increased MCP-1 mRNA and protein and induced its promoter activity. MCP-1 was found in 60% of invasive PDA lesions, of which 66% were smokers. MCP-1 colocalized with OPN in PDA cells and in the malignant ducts, and correlated well with higher expression levels of OPN in the tissue from patients with invasive PDA.
Conclusions
Our data suggest that cigarette smoking and nicotine may contribute to PDA inflammation through inducing MCP-1 and provide a novel insight into a unique role for OPN in mediating these effects.
doi:10.1016/j.surg.2010.05.002
PMCID: PMC2908036  PMID: 20579680
pancreatic cancer; nicotine; osteopontin; monocyte chemoattractant protein-1
14.  A Lethally Irradiated Allogeneic Granulocyte-Macrophage Colony Stimulating Factor-Secreting Tumor Vaccine for Pancreatic Adenocarcinoma: A Phase II Trial of Safety, Efficacy, and Immune Activation 
Annals of surgery  2011;253(2):328-335.
Purpose
Surgical resection provides the only possibility of cure for pancreas cancer. A standard adjuvant approach has not been established. We tested the safety and efficacy of a granulocyte-macrophage colony-stimulating factor (GM-CSF)-based immunotherapy administered in patients with resected pancreatic adenocarcinoma.
Patients and Methods
A single institution phase II study of 60 patients with resected pancreatic adenocarcinoma was performed. Each immunotherapy treatment consisted of a total of 5 × 108 GM-CSF-secreting cells distributed equally among 3 lymph node regions. The first immunotherapy treatment was administered 8 to 10 weeks after surgical resection. Subsequently, patients received 5-FU based chemoradiation. Patients who remained disease-free after completion of chemoradiotherapy received treatments 2 to 4, each 1 month apart. A fifth and final booster was administered 6 months after the fourth immunotherapy. The primary endpoint was disease free survival and secondary endpoints were overall survival and toxicity, and the induction of mesothelin-specific T cell responses.
Results
The median disease-free survival is 17.3 months (95% CI, 14.6–22.8) with median survival of 24.8 months (95% CI, 21.2–31.6). The administration of immunotherapy was well tolerated. In addition, the postimmunotherapy induction of mesothelin-specific CD8+ T cells in HLA-A1+ and HLA-A2+ patients correlates with disease-free survival.
Conclusions
An immunotherapy approach integrated with chemoradiation is safe and demonstrates an overall survival that compares favorably with published data for resected pancreas cancer. These data suggest additional boost immunotherapies given at regular intervals beyond 1 year postsurgery should be tested in future studies, and provide the rationale for conducting a multicenter phase II study.
doi:10.1097/SLA.0b013e3181fd271c
PMCID: PMC3085934  PMID: 21217520
15.  Evaluating the drug-target relationship between thymidylate synthase expression and tumor response to 5-fluorouracil 
Cancer biology & therapy  2008;7(7):986-994.
Thymidylate synthase is a target of 5-fluoruracil, a pyrimidine analog used to treat gastrointestinal and other cancers. The 5-fluorouracil metabolite, fluoro-deoxyuridine monophosphate, forms a ternary complex with thymidylate synthase and 5,10-methylene tetrahydrofolate. The purpose of this study was to evaluate the time-honored connection between thymidylate synthase and 5-fluorouracil. From our literature search spanning reports from 1995 to 2007 published in journals having an impact factor greater than two, we stratified the tumors within each article, according to low versus high thymidylate synthase expression. These groups were subdivided into responders, stable disease or disease progression. The relationship between thymidylate synthase expression and 5-fluorouracil response was analyzed for the overall group, as well as for subsets. Overall, the literature supported an approximately two-fold inverse relationship between thymidylate synthase expression and response to 5-fluoruracil. We found no change in the trend for a relationship between thymidylate synthase and 5-fluorouracil when the literature was stratified by date of publication, impact factor of the journal in which the report was published, or substrate (mRNA versus protein) for measuring thymidylate synthase expression. Of note, there is no significant change in the trend when comparing 5-fluorouracil treatment alone or in combination with leucovorin. We found a decline of this trend when certain chemotherapeutics were used in combination with 5-fluorouracil. In sum, the connection between thymidylate synthase expression and patient response to 5-fluorouracil does not satisfy expectations for an effective drug-target relationship; and thus, studies of the thymidylate synthase tandem repeat status might only be clinically valuable in regards to patient toxicity. Thus, we question the reliability of thymidylate synthase expression as a clinical predictor of 5-fluorouracil response. Future research could perhaps be directed towards alternate targets and metabolites of 5-fluorouracil, in an effort to find a clinically relevant biomarker panel for response and to optimize fluoropyrimidine-based therapy.
PMCID: PMC3081718  PMID: 18443433
thymidylate synthase; 5-fluorouracil; clinical response; pharmacogenomics; chemotherapeutic resistance; pharamocogenetic window; resistance; sensitivity
16.  EXPRESSION OF A PRO-METASTATIC SPLICE VARIANT OF OSTEOPONTIN, OPNC, IN HUMAN PANCREATIC DUCTAL ADENOCARCINOMA 
Surgery  2009;146(2):232-240.
Introduction
Osteopontin (OPN) is a secreted phosphoprotein that confers on cancer cells a migratory phenotype. We showed recently that nicotine, a major risk factor in pancreatic ductal adenocarcinoma (PDA), increases OPN expression in PDA cells. An OPN splice variant, OPNc, supports anchorage independence and maybe the most potent OPN isoform to convey metastatic behavior. In this study, we tested the effect of nicotine on OPNc expression, and analyzed the correlation between total OPN/OPNc levels and patients’ smoking history.
Methods
Real time PCR and UV-light-illumination of ethidium-bromide staining were used to examine the mRNA expression in tissue and in PDA cells treated with or without nicotine (3-300 nM). OPN and OPNc were localized by immunohisotchemistry, and ELISA was used to analyze OPN serum levels.
Results
Nicotine treatment of PDA cells selectively induced denovo expression of OPNc. OPNc was found in 87% of invasive PDA lesions, of which 73% were smokers. The levels of OPNc correlated well with higher expression levels of total OPN in the tissue and serum from patients with invasive PDA.
Conclusions
Our data suggest that smoking and nicotine may contribute to PDA metastatic potential through promoting OPNc expression. Although the direct role of OPNc in PDA progression is not defined, OPNc may have value as a diagnostic and prognostic marker, especially in invasive PDA.
doi:10.1016/j.surg.2009.03.036
PMCID: PMC2777713  PMID: 19628079
pancreatic cancer; nicotine; osteopontin
17.  Limits to TYMS and TP53 genes as predictive determinants for fluoropyrimidine sensitivity and further evidence for an RNA-based toxicity as a major influence 
Cancer research  2009;69(3):984-991.
The major determinants of 5-flurouracil response would appear, based on accumulated literature, to be thymidylate synthase (TYMS, TS) expression levels, TS gene modifications, and TP53 status. We tested 5-fluorouracil sensitivity in yeast and human cancer cell models in which TS or TP53 alleles and expression were varied. Polymorphic TS tandem repeat status, TS expression levels reported, TS intragenic mutations, and TP53 status in outbred and experimental cancer cell lines did not predict 5-FU sensitivity or resistance. Novel observations included a dose-resistant persistence of unbound TS protein in many cancers and, upon 5-FU treatment of the colon cancer cell line, HCT116, evidence of allelic switching favoring transcripts of the mutant TS allele. The reported alleles having an intragenic mutation could not be causally associated with major degrees of 5-FU sensitivity. In yeast, TS protein was altered upon treatment with fluoro-deoxyuridine monophosphate, but 5-FU toxicity appeared largely to be RNA-based, being rescued by uridine rather than by thymidine. Cancer cell lines were also rescued from 5-FU toxicity with uridine rather than thymidine. Additionally, a TS (CDC21) knockout yeast strain, obviating any potential role for TS protein as a target, was hypersensitive to 5-FU. When denatured proteins from cancer cells treated with radio-labeled 5-FU were, labeled species with alternative molecular weights other than TS were visualized, providing further evidence for alternative 5-FU protein targets. These data emphasize that TS and TP53 status do not consistently explain the variance in responses of fluoropyrimidine-treated cancer cells, in part due to RNA-based toxicity.
doi:10.1158/0008-5472.CAN-08-3610
PMCID: PMC2887094  PMID: 19155291
Thymidylate synthase expression; 5-fluorouracil; sensitivity; chemotherapeutic resistance; p53
18.  HuR’s role in gemcitabine efficacy in pancreatic cancer: HuR upregulates the expression of the gemcitabine metabolizing enzyme, deoxycytidine kinase 
Cancer research  2009;69(11):4567-4572.
RNA-binding protein HuR binds U- or AU-rich sequences in the 3′-untranslated regions (UTRs) of target mRNAs, stabilizing them and/or modulating their translation. Given HuR’s links with cancer, we studied the consequences of modulating HuR levels in pancreatic cancer cells. HuR-overexpressing cancer cells, in some instances, are up to 30-fold more sensitive to treatment with gemcitabine (GEM), the main chemotherapeutic component of treatment regimens for pancreatic ductal adenocarcinoma (PDA), compared to control cells. In pancreatic cancer cells, HuR associates with deoxycytidine kinase (dCK) mRNA, which encodes the enzyme that metabolizes and thereby activates GEM. GEM exposure to pancreatic cancer cells, enriches the association between HuR and dCK mRNA and increases cytoplasmic HuR levels. Accordingly, HuR overexpression elevates, while HuR silencing reduces, dCK protein expression in pancreatic cancer cells. In a clinical correlate study of GEM treatment, we found a 7-fold increase in risk of mortality in PDA patients with low cytoplasmic HuR levels compared to patients with high HuR levels, after adjusting for other treatments and demographic variables. These data support the notion that HuR is a key mediator of GEM efficacy in cancer cells, at least in part through its ability to regulate dCK levels post-transcriptionally. We propose that HuR levels in PDA modulate the therapeutic efficacy of GEM, thus serving as a marker of the clinical utility of this common chemotherapeutic agent and a potential target for intervention in pancreatic cancer.
doi:10.1158/0008-5472.CAN-09-0371
PMCID: PMC2744447  PMID: 19487279
pancreatic ductal adenocarcinoma; pancreatic cancer; HuR; gemcitabine; deoxycytidine kinase; mRNA binding protein; gemcitabine response; RNA binding proteins
19.  DPC4 Gene Status of the Primary Carcinoma Correlates With Patterns of Failure in Patients With Pancreatic Cancer 
Journal of Clinical Oncology  2009;27(11):1806-1813.
Purpose
Contrary to the extensive data accumulated regarding pancreatic carcinogenesis, the clinical and molecular features characteristic of advanced stage (stage III and IV) disease are unknown. A comprehensive study of pancreatic cancers from patients who have succumbed to their disease has the potential to greatly expand our understanding of the most lethal stage of this disease and identify novel areas for intervention.
Materials and Methods
Rapid autopsies were performed on 76 patients with documented pancreatic cancer. The histologic features of end stage disease were determined and correlated to the stage at initial diagnosis, patterns of failure (locally destructive v metastatic disease) and the status of the KRAS2, TP53, and DPC4 genes.
Results
At autopsy, 30% of patients died with locally destructive pancreatic cancer, and 70% died with widespread metastatic disease. These divergent patterns of failure found at autopsy (locally destructive v metastatic) were unrelated to clinical stage at initial presentation, treatment history, or histopathologic features. However, Dpc4 immunolabeling status of carcinoma tissues harvested at autopsy, a sensitive marker of DPC4 genetic status, was highly correlated with the presence of widespread metastasis but not with locally destructive tumors (P = .007).
Conclusion
Pancreatic cancers are represented by distinct genetic subtypes with significantly different patterns of failure. Determinations of DPC4 status at initial diagnosis may be of value in stratifying patients into treatment regimens related to local control versus systemic therapy.
doi:10.1200/JCO.2008.17.7188
PMCID: PMC2668706  PMID: 19273710
20.  Multifocal Neoplastic Precursor Lesions Associated With Lobular Atrophy of the Pancreas in Patients Having a Strong Family History of Pancreatic Cancer 
We screened 116 patients with a strong family history of pancreatic cancer using a combination of endoscopic ultrasound and computed tomography. Ten of these patients underwent surgical resection at our institution, providing an opportunity to define the morphology of pancreatic precursor lesions in patients with a strong family history of pancreatic cancer. Eight of the 10 pancreata were available and these were entirely submitted for histologic examination. The number of pancreatic intraepithelial neoplasia (PanIN) lesions and intraductal papillary mucinous neoplasms (IPMNs) were compared with age-matched controls. Parenchymal changes were defined. Selected precursor neoplasms from 6 pancreata were microdissected and analyzed for KRAS gene mutations. PanINs were significantly more common in the 8 cases (mean of 10.7% of the duct profiles, range 1.0% to 27.3%) than in the controls (mean 1.9%, range 0% to 9.2%, P<0.01). Different KRAS gene mutations were identified in separately microdissected precursor lesions in 2 of 6 cases. IPMNs were identified in 4 of the 8 cases, including 2 pancreata each having 2 distinct IPMNs. Both the IPMNs and the PanINs, even the low-grade PanIN-1 lesions, were associated with lobular parenchymal atrophy. Some individuals with a strong family history of pancreatic cancer develop multifocal, noninvasive epithelial precursor lesions of the pancreas. PanINs and IPMNs produce obstructive lobular atrophy, and this atrophy is likely the source of the chronic pancreatitis-like changes seen in these patients. The multifocal nature of familial pancreatic neoplasia suggests that surveillance of these patients is warranted after partial pancreatectomy.
PMCID: PMC2746409  PMID: 16931950
familial cancer; pancreatic cancer; pancreatic intraepithelial neoplasia; intraductal papillary mucinous neoplasm
21.  Leak after Whipple Resection: Preventable? 
HPB Surgery  1998;11(2):133-136.
doi:10.1155/1998/23464
PMCID: PMC2423961  PMID: 9893247
22.  Mini-Incision Versus Laparoscopic Cholecystectomy 
HPB Surgery  1997;10(5):338-340.
Background: We report a prospective randomised comparison between laparoscopic and small-incision cholecystectomy in 200 patients which was designed to eliminate bias for or against either technique.
Methods: Patients were randomised in the operating theatre and anaesthetic technique and pain-control methods were standardised. Four experienced surgeons did both types of procedure. Identical wound dressings were applied in both groups so that carers could be kept blind to the type of operation.
Findings: There was no significant difference between the groups for age, sex, body mass index, and American Society of Anaesthesiologists grade. Laparoscopic cholecystectomy took significantly longer than small-incision cholecystectomy (median 65 [range 27-140] min vs 40 [18-142] min, p<0.001). The operating time included operative cholangiography which was attempted in all patients. We found no significant difference between the groups for hospital stay (postoperative nights in hospital, median 3.0 [1-17] nights for laparoscopic vs 3.0 [1-14] nights for small-incision, p=0.74), time back to work for employed persons (median 5.0 weeks vs 4.0 weeks; p=0.39), and time to full activity (median 3.0 weeks vs 3.0 weeks; p=0.15).
Interpretation: Laparoscopic cholecystectomy takes longer to do than small-incision cholecystectomy and does not have any significant advantages in terms of hostital stay or 13 ostoperative recovery.
doi:10.1155/1997/27210
PMCID: PMC2423893  PMID: 9298392
23.  Intraductal Papillary Mucinous Neoplasm (IPMN) with Extra-Pancreatic Mucin: A Case Series and Review of the Literature 
Background
Intraductal papillary mucinous neoplasm (IPMN) is an increasingly recognized pancreatic neoplasm characterized by excessive mucin secretion by ductal epithelial cells resulting in a cystic dilation of the pancreatic duct.
Aim
The objective of this study was to review Thomas Jefferson University’s experience and the literature to determine the significance of extra-pancreatic mucin when associated with an IPMN.
Results
A retrospective analysis at our institution revealed only two cases of IPMN associated with extra-pancreatic mucin, which were classic IPMNs with rupture of the pancreatic duct and peritoneal mucin spillage. This specific finding is not previously described, although is assumed as five cases were reported in the literature with IPMN and mucin extension demonstrated by pseudomyxoma peritonei (PMP). We propose IPMN of the pancreas may be grossly compared to a mucocele of the appendix, as both are characterized by excessive secretion of mucin by ductal epithelial cells. A morbid complication of a mucocele is PMP. The presence of extra-pancreatic mucin with an IPMN could present a rare but important marker of the eventual seeding of tumor outside the primary IPMN. This has been documented with cases of iatrogenic spilling of pancreatic mucin, as well as multiple cases of IPMN associated with pseudomyxoma peritonei.
Conclusions
At this time, there is scant reporting and consensus for the treatment of IPMN with extra-pancreatic mucin.
doi:10.1007/s11605-012-1823-8
PMCID: PMC3309120  PMID: 22258877
Intraductal papillary mucinous neoplasm (IPMN); Extra-pancreatic mucin; Pancreatic cancer; Pseudomyxoma peritonei
24.  pp32 (ANP32A) Expression Inhibits Pancreatic Cancer Cell Growth and Induces Gemcitabine Resistance by Disrupting HuR Binding to mRNAs 
PLoS ONE  2010;5(11):e15455.
The expression of protein phosphatase 32 (PP32, ANP32A) is low in poorly differentiated pancreatic cancers and is linked to the levels of HuR (ELAV1), a predictive marker for gemcitabine response. In pancreatic cancer cells, exogenous overexpression of pp32 inhibited cell growth, supporting its long-recognized role as a tumor suppressor in pancreatic cancer. In chemotherapeutic sensitivity screening assays, cells overexpressing pp32 were selectively resistant to the nucleoside analogs gemcitabine and cytarabine (ARA-C), but were sensitized to 5-fluorouracil; conversely, silencing pp32 in pancreatic cancer cells enhanced gemcitabine sensitivity. The cytoplasmic levels of pp32 increased after cancer cells are treated with certain stressors, including gemcitabine. pp32 overexpression reduced the association of HuR with the mRNA encoding the gemcitabine-metabolizing enzyme deoxycytidine kinase (dCK), causing a significant reduction in dCK protein levels. Similarly, ectopic pp32 expression caused a reduction in HuR binding of mRNAs encoding tumor-promoting proteins (e.g., VEGF and HuR), while silencing pp32 dramatically enhanced the binding of these mRNA targets. Low pp32 nuclear expression correlated with high-grade tumors and the presence of lymph node metastasis, as compared to patients' tumors with high nuclear pp32 expression. Although pp32 expression levels did not enhance the predictive power of cytoplasmic HuR status, nuclear pp32 levels and cytoplasmic HuR levels associated significantly in patient samples. Thus, we provide novel evidence that the tumor suppressor function of pp32 can be attributed to its ability to disrupt HuR binding to target mRNAs encoding key proteins for cancer cell survival and drug efficacy.
doi:10.1371/journal.pone.0015455
PMCID: PMC2994932  PMID: 21152064

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