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Background: HuR is an RNA-binding protein that translocates from the nucleus to the cytoplasm with its mRNA cargo in response to stress, and regulates mRNA stability and translation of these transcripts. HuR has been implicated as an important regulator of pro-survival pathways in cancer, and increased cytoplasmic expression of HuR has been observed in numerous cancer types. The regulatory mechanisms of metabolic pathways under metabolic stress are poorly understood. We investigated HuR's role in regulating pancreatic cancer metabolism. Methods: We measured HuR cytoplasmic expression in pancreatic cancer cells in response to glucose deprivation. Ribonucleoprotein precipitation (RIP) of HuR was performed to survey candidate metabolic mRNA targets. HuR levels were depleted with siRNA oligos under metabolic stress, and effects on cell survival, biochemicals in the media, and metabolic enzyme targets were determined. Experiments were performed in MiaPaca2, BXPC3, Panc1 cell lines. Results: Cytoplasmic protein expression of HuR was increased by immunoblot and immunofluorescence after 6 hours of glucose deprivation in all 3 cell lines (vs. 25 mM glucose). In addition, HuR depletion with siRNA impaired survival in of all 3 cell lines in low glucose media ( Conclusions: HuR cytoplasmic expression is critical for pancreatic cancer cell survival under glucose deprivation. HuR likely supports a ‘Warburg’ phenotype characterized by increased lactate production. Surprisingly, HuR activation led to decreased glucose uptake. Novel metabolic targets of HuR were identified, and coordination of these targets may be responsible for the observed findings. Improved understanding of how HuR acutely regulates metabolism in response to stress should uncover novel therapeutic targets against pancreatic cancer. Citation Format: Jordan M. Winter, Richard A. Burkhart, Danielle M. Pineda, Saswati Chand, Joseph Cozzitorto, Charles J. Yeo, Jonathan R. Brody. HuR affects pancreatic cancer metabolism through post-transcriptional control of core metabolic enzymes. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5394. doi:10.1158/1538-7445.AM2013-5394

The poor outcome of pancreatic cancer is hypothesized to be due to a subset of cancer cells that have elevated propensity to metastasize and are resistant to current chemotherapeutics. In vitro, the process of epithelial to mesenchymal transition (EMT) has been shown to grant cells with the aforementioned properties. Hence, it has been hypothesized that the process of EMT might have answers to controlling the metastatic cascade. Using gene expression microarrays, MRC2 expression was found to be upregulated when cells undergo EMT. MRC2 is a transmembrane protein known for its role in collagen degradation. Its role in the progression of metastatic pancreatic cancer and in vivo expression was assayed for validation as a drug target. MRC2 upregulation by TGF-β1 did not promote increased transition to a mesenchymal phenotype or migratory ability in the absence of a chemotactic gradient. However, using boyden chamber and matrigel invasion assays, it was observed that MRC2 affects chemotactic and invasive ability. Immunohistochemical analysis of over 250 pancreatic tumors revealed that MRC2 is upregulated in the cancer samples especially in the stromal compartment. MRC2 staining patterns were consistent with involvement in invasion, including elevation in individual, invading cancer cells and stromal elevations in peri-tumoral and poorly differentiated regions. Blinded immunohistochemical studies disclosed a correlation between elevated stromal MRC2 levels and poor prognosis. Flow cytometry is being used on primary tumors to confirm the dual stain results hinting towards the elevation of MRC2 on a subset of pancreatic cancer cells in vivo. Dual stains on tissue microarrays are also being used to identify the stromal cell population showing MRC2 elevation. Based on the role of MRC2 in chemotactic invasion and extracellular matrix remodeling, loss of cells expressing MRC2 might be a deterrent for metastasis. Since MRC2 is part of an endocytic pathway,upon confirmation of MRC2 as a marker of the mesenchymal cancer cells, it has the potential for use as an adjuvant targeted therapy for the treatment of pancreatic cancer. Citation Format: Arkadeep Sinha, Aprill Watanabe, Jordan Winter, Peter Allen, Galen Hostetter, Brian B. Haab. Analysis of the endocytic receptor MRC2 as a target for mesenchymal pancreatic cancer cells. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr B60.

Pancreatic Fistula; UTI, Urinary Tract Infection; DGE, Delayed Gastric Emptying; C. diff. colitis, Clostridium difficile colitis; DVT/PE, Deep Vein Thrombosis/Pulmonary Embolism. All values represent medians. *Tobacco Use data not available for 2 patients, EBL data not available for 4 patients. Total numbers for these variables exclude these patients **Number of patients with one or more complications. ***Only includes patients with date of surgery from 10/2005-06/2011.

Metabolic transformation towards a glycolytic phenotype is well described in pancreatic ductal adenocarcinoma (PDAs) and other cancers, yet the underlying molecular mechanisms remain largely unknown. To date, literature has focused principally on genetic changes in cancers such as somatic mutations. However, it is more likely that post-transcriptional regulatory elements play a significant role in metabolic adaptive responses given the rapid and dynamic nature of conditions in vivo. One element, the RNA-binding protein HuR, has been shown to direct the dynamic cellular response to various cancer-associated stressors by translocating from the nucleus to the cytoplasm and augmenting target expression. Recently discovered HuR-metabolic targets include HIF-1α and the glucose transporter GLUT-1. We hypothesize therefore that HuR is an important post-transcriptional regulator of cellular metabolism in pancreatic cancer. We began by exposing pancreatic cancer cell lines (MiaPaca-2, PL5, PL45, BxPC-3, ASPC-1, and Panc1) to the known glycolytic inhibitors citrate and hypoglycemia. When citrate was added to culture media at doses between 0.1 to 10 mM (for 2-12 hrs), we identified robust HuR cytoplasmic translocation by both western blot and immunofluorescence. When these cells are exposed to a known α-ketoglutarate agonist and hypoxia mimetic, Dimethyloxalylglycine, HuR is also activated to the cytoplasm at doses between 0.1 and 1 mM. Similarly, reducing the glucose in standard media (25mM) to physiologic levels (5mM) and below (down to 0.5 mM) induces HuR cytoplasmic translocation. Interestingly, with continued metabolic stress cytoplasmic HuR protein returned to pre-stress levels after 24 hours of treatment. We then studied the affects of these metabolic stressors (4–6 hours) on validated HuR mRNA targets (dCK, HIF-1α, and SUMO1), and observed upregulation using quantitative PCR (qPCR) analysis of total RNA. Finally, we performed ribonucleoprotein-immunoprecipitation (RNP-IP) of HuR bound mRNA cargo in PDA cells and detected that the HuR target mRNAs dCK and HuR were upregulated under conditions of hypoglycemia as compared to normoglycemia. These data are the first to demonstrate that HuR is activated to the cytoplasm in pancreatic cancer cells as a result of metabolic stress. Additionally, we demonstrate that HuR activation during changes in the metabolic mileau affects mRNA levels of HuR targets. Based on these preliminary findings, HuR is a likely regulator of an acute metabolic transformation necessary for pancreatic cancer cells. Ongoing studies will elucidate HuR's role in adapting and maintaining the metabolic phenotype that is characteristic of malignancy. This may additionally lead to the discovery of novel metabolic targets regulated acutely by these post-transcriptional mechanisms. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5144. doi:1538-7445.AM2012-5144

Background The role of adjuvant chemoradiation therapy for ampullary carcinoma is unknown. Previous literature suggests that certain populations with high risk factors for recurrence may benefit from adjuvant chemoradiation. We combined the experience of two institutions to better delineate which patients may benefit from adjuvant chemoradiation. Methods Patients who underwent curative surgery for ampullary carcinoma at the Johns Hopkins Hospital (n = 290; 1992-2007) and at the Mayo Clinic (n = 130; 1977-2005) were reviewed. Patients with Results Median overall-survival was 39.9 months with 2- and 5-year survival rates of 62.4% and 39.1%. On univariate analysis, adverse prognostic factors for overall survival included T3/T4 stage disease (RR = 1.86, p = 0.002), node positive status (RR = 3.18, p < 0.001), and poor histological grade (RR = 1.69, p = 0.011). Patients who received adjuvant chemoradiation (n = 66) vs. surgery alone (n = 120) showed a higher rate of T3/T4 stage disease (57.6% vs. 30.8%, P < 0.001), lymph node involvement (72.7% vs. 30.0%, P < 0.001), and close or positive margins (4.6% vs. 0.0%, P = 0.019). Five year survival rates among node negative and node positive patients were 58.7% and 18.4% respectively. When compared with surgery alone, use of adjuvant chemoradiation improved survival among node positive patients (mOS 32.1 vs. 15.7 mos, 5 yr OS: 27.5% vs. 5.9%; RR = 0.47, P = 0.004). After adjusting for adverse prognostic factors on multivariate analysis, patients treated with adjuvant chemoradiation demonstrated a significant survival benefit (RR = 0.40, P < 0.001). Disease relapse occurred in 37.1% of all patients, most commonly metastatic disease in the liver or peritoneum. Conclusions Node-positive patients with resected ampullary adenocarcinoma may benefit from 5-FU based adjuvant chemoradiation. Since a significant proportion of patients develop metastatic disease, there is a need for more effective systemic treatment.

4020 Background: Thousands of transcripts and proteins have been reported as overexpressed in pancreatic cancer, although their prognostic significance remains uncertain. Methods: Representative se...

333 Background: Primary pancreatic small cell carcinoma (SCC) is rare, with just over 30 cases reported in the literature. Only 7 of these patients underwent surgical resection with a median survival of 6 months. Prognosis of SCC is therefore considered to be poor, and the role of adjuvant therapy is uncertain. Here we report two institutions' experience with resectable pancreatic SCC. Methods: Six patients with pancreatic SCC at the Johns Hopkins Hospital (4 patients) and the Mayo Clinic (2 patients) were identified from prospectively collected pancreatic cancer databases and re-reviewed by pathology. All six patients underwent a pancreaticoduodenectomy. Clinicopathologic data was analyzed, and the literature on pancreatic SCC was reviewed. Results: Median age at diagnosis was 50 years (range 27-60). Half of the patients were male, and half were known smokers. All six masses were limited to the pancreatic head. Median tumor size was 3 cm, and all cases had positive lymph nodes except for one patient who only had five nodes sampled. There was no perioperative mortality, although three patients had postoperative complications. All six patients received adjuvant chemotherapy therapy, five of whom were given cisplatin and etoposide. Of these five patients, three were known to have received radiation, while the remaining two had a plan for radiation at an outside facility. Median survival was 20 months with a range of 9-173 months. The patient who lived for 9 months received chemotherapy only, while the patient who lived for 173 months was given chemoradiation with cisplatin and etoposide and represents the longest reported survival time from pancreatic SCC to date. Conclusions: Pancreatic SCC is an extremely rare form of cancer with a poor prognosis. Patients in this surgical series showed improved survival rates when compared to prior experiences with both resected and unresectable cases. Cisplatin and etoposide appears to be the preferred chemotherapy regimen, although its efficacy remains uncertain, as does the role of combined modality treatment with radiation. No significant financial relationships to disclose.

15044 Background: The objective of the current study was to examine the impact of a family history of pancreatic adenocarcinoma (PCA) on the outcome of patients receiving adjuvant chemoradiation therapy (CRT) following pancreaticoduodenectomy (PD). Patients and Methods: Between August 1993 and February 2005, 902 patients underwent PD for pancreatic adenocarcinoma. Following PD, 405 patients received no adjuvant CRT, while 346 patients received CRT. Another 151 patients were excluded because they received protocol treatment, neoadjuvant CRT, or were lost to follow-up. Patients who received adjuvant CRT were treated with 5-FU (97.4%) based CRT (median dose 50 Gy) and maintenance 5-FU or gemcitabine. Survival was estimated using the Kaplan-Meier method and differences in survival were examined using the log-rank test. Cox regression analysis was used to control for family history. Results: Of the 751 patients included in the study, 158 (21%) patients had a known family history of pancreatic adenocarcinoma (only one family member n=119; >=2 either first or second degree relatives, n=39). Clinicopathologic characteristics of patients with a family history of PCA were similar to those of patients who did not have a family history (age, race, positive lymph node status, primary tumor size, and proportion receiving adjuvant CRT; all P>0.05). In an analysis of the entire patient cohort, adjuvant CRT was associated with an improvement in median overall survival compared with no adjuvant CRT (21.0 months vs. 14.6 months, respectively; P= 0.001). Family history of PCA (>=1 family member) was not associated with overall survival (positive family history, 20.0 months vs. negative family history, 17.3 months; P = 0.12). Family history of PCA also did not modify the effect of CRT on overall survival. Specifically, on multivariate analysis, after stratifying on family history (>=1 family member), CRT remained significantly associated with an improved survival (Hazard ratio=0.71; P=0.001). Conclusion: Adjuvant 5-FU based CRT improves the median survival of patients with resected pancreatic adenocarcinoma. The improvement in median survival associated with adjuvant CRT was independent of a familial history of pancreatic adenocarcinoma. No significant financial relationships to disclose.