Nathan G. Richards

HuR status is a powerful marker for prognosis and response to gemcitabine-based chemotherapy for resected pancreatic ductal adenocarcinoma patients.

Background:Pancreatic ductal adenocarcinoma (PDA) is a devastating disease that killed nearly 38,000 people in the United States this past year. Objective:Treatment of PDA typically includes surgery and/or chemotherapy with gemcitabine. No reliable biomarker exists for prognosis or response to chemotherapy. Two previously proposed prognostic markers, cyclooxygenase-2 (COX-2) and vascular endothelial growth factor (VEGF), are regulated by Hu protein antigen R (HuR), an mRNA binding protein that we have previously demonstrated to be a promising predictive marker of gemcitabine response. This study was designed to evaluate the clinical utility of HuR, COX-2, and VEGF as potential prognostic and predictive biomarkers for PDA. Methods:A tissue microarray of 53 PDA specimens from patients who underwent potentially curative pancreatic resection was analyzed. HuR, COX-2, and VEGF status were correlated with clinicopathologic and survival data. We also performed ribonucleoprotein immunoprecipitation assays using an HuR antibody to assess VEGF and COX-2 mRNA binding to HuR in pancreatic cancer cells. Results:Roughly 50% (27/53) of patients had high cytoplasmic HuR expression. These patients had worse pathologic features as assessed by T staging (P = 0.005). Only cytoplasmic HuR status correlated with tumor T staging, whereas VEGF (P = 1.0) and COX-2 (P = 0.39) expression did not correlate with T staging. Additionally, HuR status was an unprecedented positive predictive marker for overall survival in patients treated with gemcitabine, pushing median survival over 45 months in the high cytoplasmic HuR expressing patient population compared with less than 23 months in the low cytoplasmic HuR expressing patient group (P = 0.033 for log-rank test and P = 0.04 in a Cox regression model) for the low versus high cytoplasmic HuR expressing group. We also validated that mRNA transcripts for both VEGF and the gemcitabine metabolizing enzyme, deoxycytidine kinase, are specifically bound by HuR in pancreatic cancer cells. Conclusions:HuR is a useful prognostic biomarker for PDA patients as indicated by its association with higher tumor T stage. Additionally, HuR status is a robust predictor of outcome for patients with resected PDA in the setting of adjuvant gemcitabine therapy. Finally, HuR binds to VEGF mRNA implying that HuR, in part, regulates VEGF expression in PDA. This study supports the notion that HuR status should be used by clinicians for the individualized treatment of PDA in the future.

The Economic Costs of Obesity and the Impact of Bariatric Surgery

The obesity epidemic has far-reaching implications for the economic and health care future in the United States. Treatments that show reduction in health care costs over time should be approved and made available to as many patients as possible. It is our opinion that bariatric surgery meets this criterion. However, bariatric surgery cannot provide the impact necessary for reduction in health care and economic costs on a national scale. The obesity epidemic must be addressed by policy efforts at the local, state, and national levels. As experts on obesity, bariatric surgeons must be prepared to guide and inform these efforts.

Identifying pancreatic cancer patients for targeted treatment: the challenges and limitations of the current selection process and vision for the future.

Recent preclinical data have demonstrated that pancreatic adenocarcinoma (PDA) cells with defects in the Fanconi anemia/BRCA2 pathway are hypersensitive to interstrand crosslinking agents. The challenge is to efficiently identify patients who will benefit from these therapies. Patients were chosen for this study by evaluating personal history, ethnic background and family history of pancreatic malignancy. Molecular assays were performed on tissue samples. Patient A developed PDA in the context of a known BRCA2 frameshift mutation (2157delG), suspected because of her personal and multigenerational family history of breast cancer. She was treated with surgical resection, and targeted chemotherapy. Patient A continues to be disease free 32 months after her diagnosis and treatment. Patient B developed PDA in the context of a strong family history of PDA and Ashkenazi Jewish heritage. Genetic analysis on critical DNA repair genes revealed no alterations. This patient did not receive a tailored treatment regimen. This study highlights the challenge of treating PDA patients and selecting those eligible for targeted therapy. The current targeted treatment options for PDA are reviewed. A new multidisciplinary approach for stratifying PDA patients for promising targeted adjuvant therapy and familial risk counseling is proposed.

CanScript, an 18-Base pair DNA sequence, boosts tumor cell-specific promoter activity: implications for targeted gene therapy.

Gene therapy protocols for the treatment of cancer often employ gene promoter sequences that are known to be over-expressed 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.

Medical Management of Acute Rhinosinusitis in Children and Adults

Acute rhinosinusitis is a commonly seen disease that is frequently diagnosed based on clinical symptomatology without any objective evidence. The most common form of treatment is antibiotics, even though many cases may not have a bacterial etiology. The most common bacterial organisms in childhood sinusitis belong to the group that cause many childhood upper respiratory and lower respiratory infections and include Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis, and Staphylococcus aureus. Besides antibiotics, other treatments are aimed at assisting with drainage, and this can be accomplished by reducing the inflammation and edema that prevents venting of the sinus structures. These medications can include antihistamines, decongestants, intranasal steroids, nasal saline irrigation, and, in some countries, mucolytics. Complementary and alternative medicine (CAM) techniques including Chinese herbal medications are often used, but there is little scientific evidence to support their use. The use of immunotherapy has not been found to be effective in the management of acute rhinosinusitis in children or adults.

Caveolin-1 and Pancreatic Ductal Adenocarcinoma

Pancreatic ductal adenocarcinoma (PDA) is a devastating disease because the annual incidence nearly equals the annual mortality. Landmark studies have identified the key genetic alterations that play a role in PDA tumorigenesis. However, recent work has underscored the importance of the pancreatic tumor microenvironment and other proteins that may be alternatively regulated in PDA cells. Caveolin (Cav-1), an integral membrane protein that plays a role in endo and exocytosis and intracellular signal transduction, is an example of a protein that appears to play a central role in PDA tumorigenesis. Cav-1 has been shown to have both oncogenic and tumor suppressor properties in lung and prostate cancers depending on the experimental setting. PDA models have demonstrated similar roles for Cav-1. For example, Cav-1 contributes to anchorage independent cell survival by its interaction with a cell surface protein, carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6), which leads to PDA tumor growth and aggressiveness through the regulation of matrix metalloproteinases (MMPs). Conversely, Cav-1 can inhibit PDA tumor growth and development by regulating the EGFR-mitogen-activated protein kinase (MAPK) signaling pathway. Moreover, Cav-1 suppresses progression of PDA by inhibiting Rho GTPases which are small monomeric molecules that are typically involved in actin cytoskeleton rearrangement during cellular migration. Clinically, Cav-1 expression, as a biomarker, correlates with patient survival, pathologic features, and fatty acid synthase (FASN) expression. Ongoing studies will elucidate the intricate way in which Cav-1 facilitates the pancreatic tumorigenesis process (including its role in the reverse Warburg effect in the PDA environment) as well as treatment interventions (radioresistance) for this devastating disease. These studies will aid us in better understanding the meaning of Cav-1 expression levels in pancreatic tumor cells and its microenvironment.

Abstract 4100: HuR regulates death receptor-5 (DR5, TRAIL-R2)-targeted treatment of pancreatic cancer cells

Apoptosis has been identified as a core signaling pathway disrupted in pancreatic ductal adenocarcinoma (PDA) tumorigenesis. Death Receptor 5 (DR5, TRAIL-R2) is a membrane bound protein that initiates the extrinsic apoptotic pathway upon ligand exposure and is currently being explored as a ‘druggable’ target in multiple cancers including PDA. Identifying a mechanism that regulates DR5 in the tumor microenvironment (e.g. hypoxia, chemotherapeutic exposure) is critical for optimizing DR5 based-therapies. Human antigen R (HuR), an RNA binding protein, controls post-transcriptional gene expression by binding to specific regions of 3’and 5’ UTRs of mRNA target genes. Previously, HuR, a pro-survival molecule, has been shown to play an important role in the intrinsic apoptotic pathway. We identified DR5 mRNA as a HuR target in PDA cells and explored the significance of HuR9s role in functionally regulating the extrinsic apoptotic pathway in PDA cells. We also explored HuR as a modulator of DR5-targeted therapy for the treatment of PDA. Ribonucleoprotein immunoprecipitation (RNP-IP) assays were performed on PDA cells using HuR antibody (Ab) compared to a control (IgG Ab) under stress conditions, 3 hours with 1μM of the standard of care drug for PDA, gemcitabine; and 75 μM of a PARP inhibitor (PARPi). mRNA was converted to cDNA using RT-PCR, and then analyzed by qPCR. DR5 mRNA was validated as a HuR target with a 6-fold greater binding to HuR compared to the control. Strikingly, this binding increases 12- and 24-fold upon treatment with gemcitabine and the PARPi respectively. Silencing HuR expression, through siRNA transfections, leads to an increase of DR5 protein expression at 24 and 48 hours in multiple PDA cell lines. Additionally, silencing of HuR significantly enhances the action of a DR5-specific monoclonal Ab (0.8 mg/mL) against PDA cells within 36 hours (a 20% detected increase in cell death compared to control cells), most likely due to an enhanced availability of the DR5 receptor. Finally, in a training set of PDA clinical specimens, we found a significant inverse correlation between high/low HuR cytoplasmic expression and low/high DR5 levels (p value=0.03). In over 80% (26 of 31) of the specimens HuR cytoplasmic levels inversely correlated with DR5 expression levels, providing further evidence that elevated cytoplasmic HuR is repressing DR5 protein levels in patient tumor cells. In sum, we have shown that ‘activated HuR’ represses DR5 protein expression in PDA cells. Therefore, we conclude that low cytoplasmic HuR levels allow for greater availability of the target DR5, and will thus accordingly enhance the efficacy of DR5-targeted therapy. Thus, manipulating and/or utilizing HuR expression levels may serve as a clinically informative tool for optimizing DR5-targeted therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4100. doi:10.1158/1538-7445.AM2011-4100

Abstract 3643: Manipulation of HuR expression alters the efficacy of PARP-inhibitors in pancreatic and ovarian cancer cells

PARP-inhibitors (PARPi) are a novel class of agents that target cancer cells deficient in DNA repair. Early phase trials show promising success with PARP-inhibitors, yet drug resistance mechanisms and off-target pathways affected by drug exposure have not been explored. HuR (ELAV1) is a protein that binds and stabilizes mRNA transcripts upon certain stress stimuli. These HuR mRNA targets result in an increase in protein expression; and thus, protein function. Recently, we demonstrated that HuR binds and regulates deoxycytidine kinase, the gemcitabine metabolizing enzyme. Thus, HuR expression enhances gemcitabine efficacy against pancreatic cancer cells. We subsequently screened other chemotherapeutics that may stimulate the HuR stress response. In isogenic pancreatic cancer cell lines, stable HuR overexpression rendered cells up to 2-fold more sensitive to PARPi compared to control cells. Accordingly, siRNA knock down of HuR expression in both pancreatic and ovarian cancer cell lines caused PARPi resistance compared to a scramble-sequence control siRNA. In a pancreatic cancer cell line, MiaPaCa2, a 2.5-fold siRNA knock down of HuR mRNA expression detected by qPCR rendered cells approximately 4-fold more resistant to PARPi. Similarly, a modest siRNA knock down of HuR in an ovarian cancer cell line, A2780, rendered cells more resistant to PARPi. Immunofluorescence studies indicate that a 5 hour treatment with 75 µM PARPi directly caused HuR to transport from the nucleus to the cytoplasm, presumably moving ARE-rich mRNA cargoes after drug exposure. Focused gene expression analysis on HuR bound, immunoprecipitated RNA defined functional HuR downstream targets (including deoxycytidine kinase). Current studies will reveal and validate the importance of these downstream mRNA targets in PARP-inhibitor metabolism and efficacy. We hypothesize that HuR may be central to PARPi effectiveness and may be useful in understanding PARPi de novo drug resistance mechanisms. Future work will reveal whether HuR status can be utilized as a predictive marker for PARP-inhibitor-based therapy and if all clinically available PARP-inhibitors engage HuR9s regulation of unique mRNA cargoes in cancer cells upon treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3643.