Search

Supplementary Movie S2 from Development and Characterization of a Novel In vivo Model of Carcinoid Syndrome

PDF file - 1066KB, MEK inhibition alone and combination PI3K + MEK inhibition decreases serotonin secretion, while BEZ235 decreases CgA expression. (A) BON and (B) QGP-1 cells were plated in 24 well plates at a density of 1x105 cells/cm2. After 24h, cells were washed and maintained with serum-free medium. Cells were subsequently treated with either (upper panels) control, BKM120 (5 �M), PD0325901 (100 nM), or combination BKM120 + PD0325901 (5 �M/100 nM), or (lower panels) control, BEZ235 (1000 nM), PD0325901 (100 nM), or combination BEZ235 + PD0325901 (1000 nM/100 nM) for 1h. Media was collected and serotonin measured by serotonin ELISA (* p < 0.05 vs. control). (C) Whole cell lysates were collected following 24h of drug treatment with either control, BKM120 (5 �M), PD0325901 (100 nM), or combination BKM120 + PD0325901 (5 �M/100 nM), or control, BEZ235 (1000 nM), PD0325901 (100 nM), or combination BEZ235 + PD0325901 (1000 nM/100 nM) and analyzed for CgA expression. β-actin was used as a loading control.

PDF file - 649KB, Combination PI3K and MEK inhibitor treatment results in a more pronounced decrease in NCI-H727 and QGP-1 cell proliferation. (A) NCI-H727 and (B) QGP-1 cells were plated in 24 well plates at a density of 3x104 cells/well. Treatments were initiated the following morning with either control, BKM120 (5 μM), PD0325901 (100 nM), or combination BKM120 + PD0325901 (5 μM/100 nM) (upper panels), or control, BEZ235 (1000 nM), PD0325901 (100 nM), or combination BEZ235 + PD0325901 (1000 nM/100 nM) (lower panels). Media was exchanged for fresh drug media at 36h. Proliferation was assessed directly by cell counting at 72h (* p < 0.05 vs. control; � p < 0.05 vs. monotherapy).

PDF file - 812 KB, PI3K inhibition decreases cell proliferation in NCI-H727 and QGP-1 cell lines. (A) NCI-H727 and (B) QGP-1 cells were plated in 24 well plates at a density of 3x104 cells/well. Treatments were initiated the following morning with increasing doses of either BKM120 (0-5 μM) (upper panels) or BEZ235 (0-1000 nM) (lower panels). Media was exchanged for fresh drug media again at 36h. Proliferation was assessed directly by cell counting at 72h (* p < 0.05 vs. control). (C) BON cells transfected with shRNA directed against the alpha subunit of PI3K were plated in 24 well plates at a density of 3x104 cells/well and proliferation assessed at 24h intervals up to 96h.

Purpose: Carcinoid syndrome, characterized by flushing, diarrhea, and valvular heart disease, can occur following carcinoid tumor metastasis to the liver and systemic release of bioactive hormones into the systemic circulation. Treatment of this devastating disease is hampered by the lack of an in vivo model that recapitulates the clinical syndrome.Experimental Design: Here, we have injected BON cells, a novel human carcinoid cell line established in our laboratory, into the spleens of athymic nude mice to establish liver metastases.Results: The majority of mice injected intrasplenically with BON cells developed significant increases in plasma serotonin and urine 5-hydroxyindoleacetic acid, and several mice exhibited mesenteric fibrosis, diarrhea, and fibrotic cardiac valvular disease reminiscent of carcinoid syndrome by both echocardiographic and histopathologic evaluation. Mice pretreated with octreotide, a long-acting somatostatin analogue, or bevacizumab, a vascular endothelial growth factor inhibitor, developed fewer liver metastases and manifestations of carcinoid syndrome, including valvular heart disease.Conclusion: We have provided an important in vivo model to further delineate novel treatment modalities for carcinoid syndrome that will also be useful to elucidate the factors contributing to the sequelae of carcinoid disease (e.g., mesenteric fibrosis and valvular heart disease).

PDF file - 741KB, PI3K inhibition enhances apoptosis in NCI-H727 and QGP-1 cell lines. (A) NCI-H727 and (B) QGP-1 cells were plated in 24 well plates at a density of 5x104 cells/cm2. Treatments were initiated the following morning with increasing doses of either BKM120 (0-5 μM) (upper panels) or BEZ235 (0-1000 nM) (lower panels) and continued for 24h. Apoptosis was measured by DNA fragmentation using the Cell Death Detection ELISAplus (* p < 0.05 vs. control).

Background: The precise involvement of the PI3K/mTOR and RAS/MEK pathways in carcinoid tumors is not well defined. Therefore, the purpose of our study was to evaluate the role these pathways play in carcinoid cell proliferation, apoptosis, and secretion and to determine the effects of combined treatment on carcinoid tumor inhibition.Methods: The human neuroendocrine cell lines BON (pancreatic carcinoid), NCI-H727 (lung carcinoid), and QGP-1 (somatostatinoma) were treated with either the pan-PI3K inhibitor, BKM120, or the dual PI3K–mTOR inhibitor, BEZ235, alone or in combination with the MEK inhibitor, PD0325901; proliferation, apoptosis, and protein expression were assessed. Peptide secretion was evaluated in BON and QGP-1 cells. The antiproliferative effect of BEZ235, alone or combined with PD0325901, was then tested in vivo.Results: Both BKM120 and BEZ235 decreased proliferation and increased apoptosis; combination with PD0325901 significantly enhanced the antineoplastic effects of either treatment alone. In contrast, neurotensin peptide secretion was markedly stimulated with BKM120 treatment, but not BEZ235. The combination of BEZ235 + PD0325901 significantly inhibited the growth of BON xenografts without systemic toxicity.Conclusions: Both BKM120 and BEZ235 effectively inhibited neuroendocrine tumor (NET) cell proliferation and stimulated apoptosis. However, inhibition of the PI3K pathway alone with BKM120 significantly stimulated neurotensin peptide secretion; this did not occur with the dual inhibition of both PI3K and mTOR using BEZ235 suggesting that this would be a more effective treatment regimen for NETs. Moreover, the combination of BEZ235 and the MEK inhibitor PD0325901 was a safe and more effective therapy in vivo compared with single agents alone. Clin Cancer Res; 20(5); 1212–22. ©2014 AACR.

PDF file - 1080KB, Combination PI3K + MEK inhibition enhances apoptosis compared to monotherapy. (A) NCI-H727 and (B) QGP-1 cells were plated in 24 well plates at a density of 5x104 cells/cm2. Treatments were initiated the following morning with either control, BKM120 (5 μM), PD0325901 (100 nM), or combination BKM120 + PD0325901 (5 μM/100 nM) (upper panels), or control, BEZ235 (1000 nM), PD0325901 (100 nM), or combination BEZ235 + PD0325901 (1000 nM/100 nM) (lower panels) and continued for 24 hours. Apoptosis was measured by DNA fragmentation using the Cell Death Detection ELISAplus (* p < 0.05 vs. control; � p < 0.05 vs. monotherapy). (C) BON cells were plated in 6 well plates at 9 x 105 cells/well. After 24h of treatment, cells were collected, stained with annexin V Alexa Fluor 488, and analyzed by flow cytometry (* p < 0.05 vs. control; � p < 0.05 vs. monotherapy).

PDF file - 1864KB, mTOR inhibition decreases proliferation but does not increase apoptosis or enhance effect in combination with a MEK inhibitor. (A) BON, (B) NCI-H727 and (C) QGP-1 cells were plated in 24 well plates at a density of 3x104 cells/well. Treatments were initiated the following morning with either control, rapamycin (20 nM), PIK-75 (1 μM), PD98059 (10 μM), rapaymycin + PD98059 (20 nM/10 μM), or PIK-75 + PD98059 (1 μM /10 μM). Proliferation (upper panels) was assessed directly by cell counting at 72h (* p < 0.05 vs. control; � p < 0.05 vs. monotherapy). Apoptosis (middle panels) was measured by DNA fragmentation using the Cell Death Detection ELISAplus (* p < 0.05 vs. control; � p < 0.05 vs. monotherapy). For Western blot analysis (lower panels), cells were plated in 6 well plates at a density of 5 x104 cells/cm2. Whole cell lysates were collected following 24h of drug treatment. β-actin was used as a loading control.

Background: Peptide receptor radionuclide therapy (PRRT) is a novel radiotherapy approved for gastroenteropancreatic neuroendocrine tumors (GEP-NETs); however, PRRT has a relatively low treatment response rate of 20-30%. We hypothesize that addition of 3-aminopyridine-2-carboxaldehyde thiosemicarbazone (3-AP), a ribonucleotide reductase (RnR) inhibitor that inhibits the rate-limiting enzyme of DNA synthesis and repair, can improve radiotherapy outcomes. The purpose of this study is to determine the therapeutic rationale of RnR-targeted therapy and evaluate 3-AP as a radiosensitizer in treating GEP-NET in vitro and in vivo. Methods: We utilized two human GEP-NET cell lines: BON, a pancreatic carcinoid tumor, and QGP-1, a somatostatinoma. (i) Cells were treated with 3-AP for 96h and cell proliferation assays were performed using a sulforhodamine B (SRB) kit. Next, cells were treated with 3-AP for 24, 48, 72, 96h and cell cycle analyses were performed with Beckman Coulter cell cycle kit and quantified by flow cytometry. Expression of cell cycle arrest markers (i.e., pCdk-1, cyclin D1) was assessed by immunoblotting. (ii) To determine radiosensitivity in vitro, cells were treated with 3-AP for 16h, followed by X-ray radiotherapy (XRT), and incubation in drug-free medium. Cell survival was assessed by colony formation assay and quantified by SRB kit at 14d post-XRT. Apoptosis was measured at 72 and 96h post-XRT, and expression of apoptotic proteins (i.e., cleaved PARP and cleaved CASPASE 3) was examined by immunoblotting. (iii) To determine radiosensitivity in vivo, we randomized 20 nude mice bearing QGP-1 xenografts into four groups: control; 3-AP (10mg/Kg); XRT (2 Gy); and combination (XRT immediately followed by 3-AP); each mouse received 5 consecutive treatments. Results: (i) A dose of 2500nM 3-AP arrested cells before entering G2/M phase, significantly reducing percentage of G2/M cells from 8.4% to 0.5% at 72h. Concurrently, we observed increased expression of checkpoint proteins (pRb, pCdk1, pCdk2, pCdc2) and decreased expression of cell cycle inhibitory proteins (cyclin D1, p21, p27 Kip). (ii) Exposure of QGP-1 and BON cells to 3-AP prior to XRT resulted in a synergistic decrease (i.e., 85% and 90%, respectively) in clonogenicity. Furthermore, the combination of 3-AP and XRT increased apoptosis 5-fold and cleaved PARP expression in BON cells. (iii) A significant decrease in tumor size was noted in mice with XRT alone compared to control (146mm2 vs. 280 mm2, respectively); combination treatment (3-AP + XRT) demonstrated a further 48% decrease in tumor size compared to XRT alone. There was no significant decrease in tumor size with 3-AP treatment alone. Conclusion: 3-AP treatment prior to XRT therapy resulted in significant increase in apoptosis in vitro and decrease in tumor size in vivo, suggesting synergistic cytotoxicity and radiosensitivity. Selective RNR inhibition by 3-AP provides a potent therapeutic strategy as a radiosensitizer and offers a novel approach in treating advanced GEP-NETs. Citation Format: Zeta Chow, Jeremy Johnson, Aman Chauhan, Tadahide Isumi, Lowell Anthony, Courtney M. Townsend, B. Mark Evers, Piotr Rychahou. 3-Aminopyridine-2-carboxaldehyde thiosemicarbazone (3-AP): A promising radiochemotherapy for gastroenteropancreatic neuroendocrine tumors [abstract]. In: Proceedings of the AACR Virtual Special Conference on Radiation Science and Medicine; 2021 Mar 2-3. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(8_Suppl):Abstract nr PO-014.

Background: Neuroendocrine tumors (NETs) are a heterogeneous group of neoplasms arising from multiple organs. Peptide Receptor Radionuclide Therapy (PRRT) is a novel therapy approved for treating NETs with a treatment response rate of 20-30%. To improve therapeutic response of PRRT, 3-AP (Triapine), which is a ribonucleotide reductase (RR) inhibitor with antineoplastic and radiation sensitizing properties, can be used in combination with PRRT. The purpose of our study is to examine effects of Triapine treatment on cellular proliferation, apoptosis and activation of DNA repair pathway in NET cells in vitro. Methods: For these studies, we utilized two human NET cell lines: BON, a pancreatic carcinoid tumor, and QGP-1, a somatostatinoma. (i) To determine effective dose range of Triapine (500 - 2500nM), proliferation was measured at 48, 72, and 96 h with sulforhodamine B (SRB) assay. (ii) To evaluate the effects of radiosensitivity, cultures were treated with increasing doses (250 - 5,000nM), received 2 Gy radiation (XRT) 1h later, exposed to Triapine for 24 hours, rinsed, and fed Triapine-free media; colonies were counted and quantified with SRB assays 14 days later and survival curves were generated. (iii) To further characterize cellular responses, cells were treated with Triapine (250 - 5,000 nM), irradiated 1h later, and harvested 24 h later to examine expression levels of Cyclin D1, cleaved PARP, p-DNA-PK (S2056), p-ATM (S1981) and p-ATR (S428) by Western blot. Results: (i) SRB proliferation assay demonstrated the effective dose range, determined by half maximal inhibitory concentration (IC-50), was 1,3 µM in BON cells and 1.73 µM in QGP-1 cells at 96 h. (ii) Exposure to Triapine before irradiation resulted in a significant increase in the radiosensitivity of each of the tumor cell lines with dose enhancement factors at a surviving fraction of 0.10 of 1.8 and 1.6 for BON and QGP-1, respectively. (iii) Triapine treatment alone activated DNA repair pathway in a dose-dependent fashion as determined by pDNA-PK (S2056) expression. Increasing p-ATM (S1981) expression was observed in a dose-dependent fashion in QGP-1 cells, but not in BON cells. In addition, p-ATR (S428) expression was not significantly different in either cell line. (iv) Triapine treatment alone or in combination with 2 and 4 Gy XRT resulted in dose-dependent decrease in cyclin D1 expression in both cell lines, however, no significant increase in cleaved PARP expression at 24 and 48h. Conclusion: Our results demonstrated rapid and dose dependent decrease in cyclin D1 expression after Triapine treatment, dose-dependent activation of DNA-PK, the key component of the non-homologous end joining pathway and significant increase in the radiosensitivity of each of the tumor cell lines. In conclusion, our findings provide a rationale for inclusion of Triapine as a radiosensitizer in combination with PRRT in treating NETs. Citation Format: Zeta Chow, Aman Chauhan, Lowell Anthony, Courtney Townsend, B. Mark Evers, Piotr Rychahou. Effect of 3AP on neuroendocrine tumor cell proliferation, apoptosis and activation of DNA repair pathway [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6269.

Introduction: Neuroendocrine tumors (NETs) are a heterogeneous group of neoplasms arising from multiple organs. The mammalian Target of Rapamycin (mTOR) and phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway has a well-established role in NETs that was supported by the clinical efficacy, shown by the rapamycin analog, Everolimus. However, treatment with everolimus results in activation of Akt as an escape mechanism for cancer cells. Novel dual mTOR/PI3K inhibitors, PF-04691502 and PKI-402, were shown to have superior antineoplastic effect, however, not have been tested in NETs. The purpose of our current study was to test the effects of PF-04691502 and PKI-402 on proliferation, apoptosis and Akt activation of NET cells in vitro. Methods: For these studies, we utilized two human NET cell lines: BON, a pancreatic carcinoid tumor, and QGP-1, a somatostatinoma. (i) BON and QGP-1 cells were treated with increasing concentrations of PF-04691502 (100nM – 10,000nM) and PKI-402 (50nM – 1,000nM) at 72, 96 and 120 h to determine half maximal inhibitory concentration (IC-50) by sulforhodamine B (SRB) assay. (ii) BON and QGP-1 cells were then treated with and harvested 24 h later to further examine expression of cyclin D1 and PARP by Western blot. In addition, phospho-Akt (S473), phospho-S6 (S235/236), and phospho-4E-BP1(Thr37/46) expression was evaluated by Western blot to assess the effect of PI3K/mTOR inhibition on downstream signaling proteins in NET cells. Results: (i) SRB assays demonstrated that IC-50 was 74nM for PF-04691502 in BON cells, 196nM for PF-04691502 in QGP-1 cells, 40nM for PKI-402 in BON cells, and 45nM for PKI-402 in QGP-1 cells at 96 h. (ii) Cyclin D1 expression was decreased in a dose-dependent fashion in both BON and QGP-1 cells. PARP expression was increased in QGP-1 cells treated with PF-04691502 (500nM). In contrast, we did not observe a significant increase in PARP expression in BON cells following PF-04691502 treatment or with PKI-402 treatment of either cell line. (iii) A dose-dependent decrease in p-AKT (S473) and p-6S (S235/236) expression was noted in cells treated with PF-04691502 at 24 h and with PKI-402 at 4 h. We did not observe significant difference in p4E-BP1(Thr37/46) expression in QGP-1 cells after treatment with PF-04691502 at 24h and PKI-402 at 4h. Conclusion: Our results demonstrate that both PF-04691502 and PKI-402 have a cytostatic inhibitory effect on proliferation in both BON and QGP-1 cells. In addition, treatment with PF-04691502 causes cytotoxic induction of apoptosis in QGP-1 cells, but not in BON cells. Our results suggest that PI3K/mTOR inhibition with PF-04691502 could be a novel therapeutic approach to the treatment of NETs. Citation Format: Zeta Chow, Courtney Townsend, B. Mark Evers, Piotr Rychahou. Effect of novel mTOR-PI3K dual inhibitors on neuroendocrine tumor cell proliferation and apoptosis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 676.

Neurotensin is a peptide hormone released from enteroendocrine cells in the small intestine in response to fat ingestion. Although the mechanisms regulating neurotensin secretion are still incompletely understood, our recent findings implicate a role for extracellular signal-regulated kinase 1 and 2 as positive regulators of free fatty acid-stimulated neurotensin secretion. Previous studies have shown that kinase suppressor of Ras 1 acts as a molecular scaffold of the Raf/MEK/extracellular signal-regulated kinase 1 and 2 kinase cascade and regulates intensity and duration of extracellular signal-regulated kinase 1 and 2 signaling. Here, we demonstrate that inhibition of kinase suppressor of Ras 1 attenuates neurotensin secretion and extracellular signal-regulated kinase 1 and 2 signaling in human endocrine cells. Conversely, we show that overexpression of kinase suppressor of Ras 1 enhances neurotensin secretion and extracellular signal-regulated kinase 1 and 2 signaling. We also show that inhibition of extracellular signal-regulated kinase 2 and exocyst complex component 70, a substrate of extracellular signal-regulated kinase 2 and mediator of secretory vesicle exocytosis, potently inhibits basal and docosahexaenoic acid-stimulated neurotensin secretion, whereas overexpression of exocyst complex component 70 enhances basal and docosahexaenoic acid-stimulated neurotensin secretion. Together, our findings demonstrate a role for kinase suppressor of Ras 1 as a positive regulator of neurotensin secretion from human endocrine cells and indicate that this effect is mediated by the extracellular signal-regulated kinase 1 and 2 signaling pathway. Moreover, we reveal a novel role for exocyst complex component 70 in regulation of neurotensin vesicle exocytosis through its interaction with the extracellular signal-regulated kinase 1 and 2 signaling pathway.

Neurotensin (NT), a 13 amino-acid peptide, is predominantly released from enteroendocrine cells of the small bowel in response to fat ingestion. Free fatty acid receptors (FFARs) FFAR1 and FFAR4 regulate secretion of gut hormones and insulin. Here, we show that docosahexaenoic acid, a long-chain fatty acid, has the most dramatic effect on NT release. FFAR1 agonists slightly stimulate and FFAR4 agonists dramatically stimulate and amplify NT secretion. Double knockdown of FFAR1 and FFAR4 decreases NT release, whereas overexpression of FFAR4, but not FFAR1, increases NT release. Administration of cpdA, an FFAR4 agonist, but not TAK-875, a selective FFAR1 agonist, increases plasma NT levels and further increases olive oil-stimulated plasma NT levels. Inhibition of MAPK kinase (MEK)/ERK1/2 decreased fatty acid-stimulated NT release but increased AMP-activated protein kinase (AMPK) phosphorylation. In contrast, inhibition of AMPK further increased NT secretion and ERK1/2 phosphorylation mediated by FFAR1 or FFAR4. Our results indicate that FFAR4 plays a more critical role than FFAR1 in mediation of fat-regulated NT release and in inhibitory crosstalk between MEK/ERK1/2 and AMPK in the control of NT release downstream of FFAR1 and FFAR4.

// Ji Tae Kim 1 , Jing Li 1, 2 , Jun Song 1, 2 , Eun Y. Lee 1, 3 , Heidi L. Weiss 1 , Courtney M. Townsend, Jr. 4 , B. Mark Evers 1, 2 1 Markey Cancer Center, University of Kentucky, Lexington, KY, USA 2 Department of Surgery, University of Kentucky, Lexington, KY, USA 3 Department of Pathology and Laboratory Medicine, University of Kentucky, Lexington, KY, USA 4 Department of Surgery, University of Texas Medical Branch, Galveston, TX, USA Correspondence to: B. Mark Evers, e-mail: mark.evers@uky.edu Keywords: neuroendocrine tumor, neurotensin receptor 1, promoter methylation, cell growth, cell migration Received: April 20, 2015 Accepted: July 15, 2015 Published: July 27, 2015 ABSTRACT Neurotensin (NTS), localized predominantly to the small bowel, stimulates the growth of a variety of cancers, including neuroendocrine tumors (NETs), mainly through its interaction with the high-affinity NTS receptor 1 (NTSR1). Here, we observed increased expression of NTSR1 in almost all tested clinical NET samples, but not in normal tissues. Through RT-PCR analysis, we found that the expression of NTSR1 and NTSR2 was either variable (NTSR1) or absent (NTSR2) in human NET cell lines. In contrast, NTSR3 and NTS were expressed in all NET cells. Treatment with 5-aza-2′-deoxycytidine, a demethylating agent, increased levels of NTSR1 and NTSR2 suggesting that DNA methylation contributes to NTSR1/2 expression patterns, which was confirmed by methylation analyses. In addition, we found that knockdown of NTSR1 decreased proliferation, expression levels of growth-related proteins, and anchorage-independent growth of BON human carcinoid cells. Moreover, stable silencing of NTSR1 suppressed BON cell growth, adhesion, migration and invasion. Our results show that high expression of NTSR1 is found in clinical NETs and that promoter methylation is an important mechanism controlling the differential expression of NTSR1 and silencing of NTSR2 in NET cells. Furthermore, knockdown of NTSR1 in BON cells suppressed oncogenic functions suggesting that NTSR1 contributes to NET tumorigenesis.

We recently developed and validated a prognostic model that accurately predicts the 2-year risk of emergent gallstone-related hospitalization in older patients presenting with symptomatic gallstones.We used 100% Texas Medicare data (2000 to 2011) to identify patients aged 66 years and older with an initial episode of symptomatic gallstones not requiring emergency hospitalization. At presentation, we calculated each patient's risk of 2-year gallstone-related emergent hospitalization using the previously validated model. Patients were placed into the following risk groups based on model estimates:30%, 30% to60%, and ≥ 60%. Within each risk group, we calculated the percent of elective cholecystectomies (≤ 2.5 months from initial episode) performed.In all, 161,568 patients had an episode of symptomatic gallstones. Mean age was 76.5 ± 7.3 years and 59.9% were female. The 2-year risk of gallstone-related hospitalizations increased from 15.9% to 41.5% to 65.2% across risk groups. For the overall cohort, 22.3% in the low-risk group, 20.9% in the moderate-risk group, and 23.2% in the high-risk group underwent elective cholecystectomy in the 2.5 months after the initial symptomatic episode. In patients with no comorbidities, elective cholecystectomy rates decreased from 34.2% in the low-risk group to 26.7% in the high-risk group. Of patients who did not undergo cholecystectomy, only 9.5% were seen by a surgeon in the 2.5 months after the initial episode.The risk of recurrent acute biliary symptoms requiring hospitalization has no influence, or even a paradoxical negative influence, on the decision to perform elective cholecystectomy after an initial symptomatic episode. Translation of the risk prediction model into clinical practice can better align treatment with risk and improve outcomes in older patients with symptomatic gallstones.

Neurotensin (NTS), a 13–amino acid peptide which is distributed predominantly along gastrointestinal tract, has multiple physiologic and pathologic functions, and its effects are mediated by three distinct NTS receptors (NTSRs). Overexpression and activation of NTS signaling components, especially NTS and/or NTSR1, are closely linked with cancer progression and metastasis in various types of cancers including neuroendocrine tumors (NETs). Although deregulation of NTSR3/sortilin has been implicated in a variety of human diseases, the expression and role of NTSR3/sortilin in NETs have not been elucidated. In this study, we investigated the expression and oncogenic effect of NTSR3/sortilin in NETs. Increased protein levels of NTSR3/sortilin were noted in the majority of human clinical NETs (n = 21) by immunohistochemical analyses compared with normal tissues (n = 12). Expression of NTS and NTSR3/sortilin was also noted in all tested NET cell lines. In addition, small interfering RNA–mediated knockdown of NTSR3/sortilin decreased cell number without alteration of cell cycle progression and apoptosis induction in NET cell lines BON and QGP-1. Moreover, silencing of NTSR3/sortilin significantly suppressed cell adhesion and cell migration with inhibition of focal adhesion kinase and Src phosphorylation in the NET cells. Our results demonstrate increased expression of NTSR3/sortilin in NET patient tissues and a critical role of NTSR3/sortilin on NET cell adhesion and migration suggesting that NTSR3/sortilin contributes to NET tumorigenesis.

Wnt/β-catenin signaling plays a pivotal role in regulating cell growth and differentiation by activation of the β-catenin/T-cell factor (TCF) complex and subsequent regulation of a set of target genes that have one or more TCF-binding elements (TBEs). Hyperactivation of this pathway has been implicated in numerous malignancies including human neuroendocrine tumors (NETs). Neurotensin (NT), an intestinal hormone, induces proliferation of several gastrointestinal (GI) cancers including cancers of the pancreas and colon. Here, we analyzed the human NT promoter in silico and found at least four consensus TBEs within the proximal promoter region. Using a combination of ChIP and luciferase reporter assays, we identified one TBE (located ∼900 bp proximal from the transcription start site) that was immunoprecipitated efficiently by TCF4-targeting antibody; mutation of this site attenuated the responsiveness to β-catenin. We also confirmed that the promoter activity and the mRNA and protein expression levels of NT were increased by various Wnt pathway activators and decreased by Wnt inhibitors in NET cell lines BON and QGP-1, which express and secrete NT. Similarly, the intracellular content and secretion of NT were induced by Wnt3a in these cells. Finally, inhibition of NT signaling suppressed cell proliferation and anchorage-independent growth and decreased expression levels of growth-related proteins in NET cells. Our results indicate that NT is a direct target of the Wnt/β-catenin pathway and may be a mediator for NET cell growth.

Objectives Patients with advanced pancreatic neuroendocrine tumors have limited therapeutic options. Everolimus (RAD001), an inhibitor of the mammalian target of rapamycin (mTOR) pathway, has been shown to increase progression-free survival, but not overall survival, indicating a need to identify additional therapeutic targets. Inhibition of mTOR complex 1 by RAD001 may induce upstream AKT upregulation. We hypothesized that dual inhibition of AKT along with mTOR will overcome the limited activity of RAD001 alone. Methods The BON cell line has been used as a model to study pancreatic neuroendocrine tumor cell biology. Western blots and cell growth assays were performed with mTOR inhibitor RAD001 (50 nM), mitogen-activated protein kinase inhibitor PD0325901 (50 nM), PI3K (phosphatidylinositol 3-kinase) inhibitor LY294002 (25 μM), or vehicle control. Nude mice were treated daily for 6 weeks with RAD001 (oral gavage) and with LY29400 (subcutaneous) 1 week after intrasplenic injection of BON cells. Results Cellular proliferation was most attenuated with the combination therapy of LY29400 and RAD001. Similarly, the volume of liver metastasis was lowest in the group treated with both LY29400 (100 mg/kg per week, subcutaneous) and RAD001 (2.5 mg/kg per day) compared with that in the vehicle group (P = 0.04). Conclusion The combination therapy of LY29400 and RAD001 decreased the cell growth in vitro and progression of liver metastasis in vivo compared with vehicle or with single-drug therapy.

Current guidelines recommend minimally invasive breast biopsy (MIBB) as the gold standard for the diagnosis of breast lesions. The purpose of this study was to describe geographic patterns and time trends in the use of MIBB in Texas.We used 100% Texas Medicare claims data (2000-2008) to identify women older than 66 years of age who underwent breast biopsy. Biopsies were classified as open or MIBB. Time trends, racial/ethnic variation, and geographic variation in the use of biopsy techniques were examined.A total of 87,165 breast biopsies were performed on 75,518 breast masses in 67,582 women; 65.8% of the initial biopsies were MIBB. Radiologists performed 70.3% and surgeons performed 26.2% of MIBB. Surgeons performed 94.2% of open biopsies. Hispanic women were less likely to undergo MIBB (55.9%) compared with white (66.6%) and black (68.9%) women (p0.0001). Women undergoing MIBB were also more likely to live in metropolitan areas and have higher income and educational levels (p0.0001). The rate of MIBB increased from 44.4% in 2001 to 79.1% in 2008 (p0.0001). There are clear geographic patterns in MIBB use, with highest use near major cities. Although rates are increasing overall, rates of improvement in the use of MIBB vary considerably across geographic regions and remain persistently low in more rural areas.Despite an increase in the use of MIBB over time, MIBB use was consistently lower than recommended. We must identify specific barriers in rural areas to effectively change practice and achieve the statewide goal of 90% MIBB.

Importance Mentorship is considered a key element for career satisfaction and retention in academic surgery. Stakeholders of an effective mentorship program should include the mentor, the mentee, the department, and the institution. Objective The objective of this study was to characterize the status of mentorship programs in departments of surgery in the United States, including the roles of all 4 key stakeholders, because to our knowledge, this has never been done. Design, Setting, and Participants A survey was sent to 155 chairs of departments of surgery in the United States in July 2014 regarding the presence and structure of the mentorship program in their department. The analysis of the data was performed in November 2014 and December 2014. Main Outcomes and Measures Presence and structure of a mentorship program and involvement of the 4 key stakeholders. Results Seventy-six of 155 chairs responded to the survey, resulting in a 49% response rate. Forty-one of 76 of department chairs (54%) self-reported having an established mentorship program. Twenty-five of 76 departments (33%) described no formal or informal pairing of mentors with mentees. In 62 (82%) and 59 (78%) departments, no formal training existed for mentors or mentees, respectively. In 42 departments (55%), there was no formal requirement for the frequency of scheduled meetings between the mentor and mentee. In most departments, mentors and mentees were not required to fill out evaluation forms, but when they did, 28 of 31 were reviewed by the chair (90%). In 70 departments (92%), no exit strategy existed for failed mentor-mentee relationships. In more than two-thirds of departments, faculty mentoring efforts were not recognized formally by either the department or the institution, and only 2 departments (3%) received economic support for the mentoring program from the institution. Conclusions and Relevance These data show that only half of departments of surgery in the United States have established mentorship programs, and most are informal, unstructured, and do not involve all of the key stakeholders. Given the importance of mentorship to career satisfaction and retention, development of formal mentorship programs should be considered for all academic departments of surgery.

The goals of this focused meeting were to verify and clarify the causes and extent of the general surgery (GS) workforce shortfalls. We also sought to define workable solutions within the existing framework of medical accreditation and certification.Numerous peer-reviewed and lay reports describe a current and worsening availability of GS services, affecting rural areas as well as large cities, academia, and the military.Primary recommendations were broadly agreed upon by attendee surgeons who were selected from numerous different professional scenarios and included 2 nonmedical observers.(1) enhance the number of GS trainees and the breadth of training, (2) incorporate more flexibility and breadth in residency, (3) minimally invasive surgery should largely return to GS, (4) broader use of community hospitals in these efforts, (5) publicize loan forgiveness and improved visa status for international medical graduates going into GS, and (6) select candidates with a bias toward a general surgical career.These methods are promising approaches to this serious deficiency but will require regular reporting and publicity for the recording of actual increases in GS output.

Human colorectal cancer (CRC) cells are resistant to the anti-proliferative effect of transforming growth factor-β (TGF-β), suggesting that disruption of TGF-β signaling plays an important role in colorectal carcinogenesis. Ecotropic virus integration site-1 (Evi-1) oncoprotein represses TGF-β signaling by interacting with Smads, but its role in CRC has not been established. The purpose of this study is to determine whether Evi-1 plays role(s) in CRCs and to characterize Evi-1 transcript(s) in CRCs. Evi-1 was overexpressed in 53% of human CRC samples, 100% of colon adenoma samples, and 100% of human colon cancer cell lines tested. Using 5' RACE, we cloned a novel Evi-1 transcript (Evi-1e) from a human CRC tissue and found that this novel transcript was expressed at a higher level in CRC tissues than in normal tissues and was the major Evi-1 transcript in CRCs. Transient Evi-1 transfection inhibited TGF-β-induced transcriptional activity and reversed the growth inhibitory effect of TGF-β in MC-26 mouse colon cancer cells. In conclusion, we have identified overexpression of Evi-1 oncoprotein as a novel mechanism by which a subset of human CRCs may escape TGF-β regulation. We have also identified a novel Evi-1 transcript, Evi-1e, as the major Evi-1 transcript expressed in human CRCs.

The recommended therapy for mild gallstone pancreatitis is cholecystectomy on initial hospitalization.Using a 5% national Medicare sample (1996-2005), we evaluated adherence to current recommendations for gallstone pancreatitis (cholecystectomy rates on initial hospitalization and the use of endoscopic retrograde cholangiopancreatography [ERCP]/sphincterotomy). We evaluated predictors of cholecystectomy, gallstone-related readmissions, and 2-year mortality.Adherence to current guidelines was low. Only 57% of 8,452 Medicare beneficiaries presenting to an acute care hospital with a first episode of mild gallstone pancreatitis underwent cholecystectomy on initial hospitalization. Of the patients who did not undergo cholecystectomy, 55% were never evaluated by a surgeon. Likewise, only 28% of patients who did not undergo cholecystectomy had a sphincterotomy. The 2-year readmission rates were higher among patients who did not undergo cholecystectomy (44% vs 4%; P.0001), and 33% of these patients required cholecystectomy after discharge. In the no cholecystectomy group, ERCP prevented readmissions (hazard ratio, 0.53; 95% confidence interval, 0.47-0.61) and when readmissions occurred they were less likely to be for gallstone pancreatitis in patients who had an ERCP (27.8% vs 53.2%; P.0001). On multivariate analysis, patients who were older, black, admitted to a nonsurgical service, lived in certain US regions, and had specific comorbidities were less likely to undergo cholecystectomy.Adherence to current recommendations for the management of mild gallstone pancreatitis is low in older patients. Our data suggest that40% of patients who did not undergo cholecystectomy would have benefited from early definitive therapy. Implementation of policies to increase adherence to guidelines would prevent gallstone-related morbidity and mortality in older patients.

The mammalian target of rapamycin (mTOR) signaling exists in two complexes: mTORC1 and mTORC2. Neurotensin (NT), an intestinal hormone secreted by enteroendocrine (N) cells in the small bowel, has important physiological effects in the gastrointestinal tract. The human endocrine cell line BON abundantly expresses the NT gene and synthesizes and secretes NT in a manner analogous to that of N cells. Here, we demonstrate that the inhibition of mTORC1 by rapamycin (mTORC1 inhibitor), torin1 (both mTORC1 and mTORC2 inhibitor) or short hairpin RNA-mediated knockdown of mTOR, regulatory associated protein of mTOR (RAPTOR), and p70 S6 kinase (p70S6K) increased basal NT release via upregulating NT gene expression in BON cells. c-Jun activity was increased by rapamycin or torin1 or p70S6K knockdown. c-Jun overexpression dramatically increased NT promoter activity, which was blocked by PD98059, an mitogen-activated protein kinase kinase (MEK) inhibitor. Furthermore, overexpression of MEK1 or extracellular signal-regulated kinase 1 (ERK1) increased c-Jun expression and NT promoter activity. More importantly, PD98059 blocked rapamycin- or torin1-enhanced NT secretion. Consistently, rapamycin and torin1 also increased NT gene expression in Hep3B cells, a human hepatoma cell line that, similar to BON, expresses high levels of NT. Phosphorylation of c-Jun and ERK1/2 was also increased by rapamycin and torin1 in Hep3B cells. Finally, we showed activation of mTOR in BON cells treated with amino acids, high glucose, or serum and, concurrently, the attenuation of ERK1/2 and c-Jun phosphorylation and NT secretion. Together, mTORC1, as a nutrient sensor, negatively regulates NT secretion via the MEK/ERK/c-Jun signaling pathway. Our results identify a physiological link between mTORC1 and MEK/ERK signaling in controlling intestinal hormone gene expression and secretion.

Dietary fiber intake links to decreased risk of colorectal cancers. The underlying mechanisms remain unclear. Recently, we found that butyrate, a short-chain fatty acid produced in gut by bacterial fermentation of dietary fiber, enhances TGF-β signaling in rat intestinal epithelial cells (RIE-1). Furthermore, TGF-β represses inhibitors of differentiation (Ids), leading to apoptosis. We hypothesized that dietary fiber enhances TGF-β's growth inhibitory effects on gut epithelium via inhibition of Id2. In this study, Balb/c and DBA/2N mice were fed with a regular rodent chow or supplemented with a dietary fiber (20% pectin) and Smad3 level in gut epithelium was measured. In vitro, RIE-1 cells were treated with butyrate and TGF-β1, and cell functions were evaluated. Furthermore, the role of Ids in butyrate- and TGF-β-induced growth inhibition was investigated. We found that pectin feeding increased Smad3 protein levels in the jejunum (1.47 ± 0.26-fold, P = 0.045, in Balb/c mice; 1.49 ± 0.19-fold, P = 0.016, in DBA/2N mice), and phospho-Smad3 levels (1.92 ± 0.27-fold, P = 0.009, in Balb/c mice; 1.83 ± 0.28-fold, P = 0.022, in DBA/2N mice). Butyrate or TGF-β alone inhibited cell growth and induced cell cycle arrest. The combined treatment of butyrate and TGF-β synergistically induced cell cycle arrest and apoptosis in RIE-1 cells and repressed Id2 and Id3 levels. Furthermore, knockdown of Id2 gene expression by use of small interfering RNA caused cell cycle arrest and apoptosis. We conclude that dietary fiber pectin enhanced Smad3 expression and activation in the gut. Butyrate and TGF-β induced cell cycle arrest and apoptosis, which may be mediated by repression of Id2. Our results implicate a novel mechanism of dietary fiber in reducing the risk of colorectal cancer development.

Evidence-based guidelines recommend cholecystectomy during initial hospitalization for complicated gallstone disease. Previous studies and quality initiative data from our institution demonstrated that only 40% to 75% of patients underwent cholecystectomy on index admission.In January 2009, we implemented a critical pathway to improve cholecystectomy rates for all patients emergently admitted for acute cholecystitis, mild gallstone pancreatitis, or common bile duct stones. We compared cholecystectomy rates during initial hospitalization, time to cholecystectomy, length of initial stay, and readmission rates in prepathway (January 2005 to February 2008) and postpathway patients (January 2009 to May 2010).Demographic and clinical characteristics were similar between prepathway (n = 455) and postpathway patients (n = 112). Cholecystectomy rates during initial hospitalization increased from 48% to 78% after pathway implementation (p0.0001). There were no differences in operative mortality or operative complications between the 2 groups. For patients undergoing cholecystectomy on initial hospitalization, the mean length of stay decreased after pathway implementation (7.1 days to 4.5 days; p0.0001), primarily due to a decrease in the time from admission to cholecystectomy (4.1 days to 2.1 days; p0.0001). Thirty-three percent of prepathway and 10% of postpathway patients required readmission for gallstone-related problems or operative complications (p0.0001), and each readmission generated an average of $19,000 in additional charges.Implementation of a multidisciplinary critical pathway improved cholecystectomy rates on initial hospitalization and lowered costs by shortening length of stay and markedly decreasing readmission rates for gallstone-related problems. Broader implementation of similar pathways offers the potential to translate evidence-based guidelines into clinical practice and minimize the cost of medical care.

OBJECTIVES: To evaluate time trends in surgical resection rates and operative mortality in older adults diagnosed with locoregional pancreatic cancer and to determine the effect of age on surgical resection rates and 2-year survival after surgical resection. DESIGN: Retrospective cohort study using data from the Surveillance, Epidemiology, and End Results (SEER) and linked Medicare claims database (1992�2005). SETTING: Secondary data analysis of population-based tumor registry and linked claims data. PARTICIPANTS: Medicare beneficiaries aged 66 and older diagnosed with locoregional pancreatic cancer (N=9,553), followed from date of diagnosis to time of death or censorship. MEASUREMENTS: Percentage of participants undergoing surgical resection, 30-day operative mortality after resection, and 2-year survival according to age group. RESULTS: Surgical resection rates increased significantly, from 20% in 1992 to 29% in 2005, whereas 30-day operative mortality rates decreased from 9% to 5%. After controlling for multiple factors, participants were less likely to be resected with older age. Resection was associated with lower hazard of death, regardless of age, with hazard ratios of 0.46, 0.51, 0.47, 0.43, and 0.35 for resected participants younger than 70, 70 to 74, 75 to 79, 80 to 84, and 85 and older respectively compared with unresected participants younger than 70 (P

Expression of the gene encoding neurotensin/neuromedin N (NT/N) is mostly limited to the brain and specialized enteroendocrine N cells in the distal small intestine. We have identified key regulatory elements in the promoter region that are involved in human NT/N (hNT/N) gene expression in the novel human endocrine cell line, BON, which resembles intestinal N cells in several important aspects including NT/N precursor protein processing, ratios of different NT/N mRNA isoforms, and high levels of constitutive expression of the NT/N gene. In this study, we demonstrated multiple cis-regulatory elements including a proximal region containing a cAMP-responsive element (CRE)/AP-1-like element that binds both the AP-1 and CRE-binding protein (CREB)/ATF proteins (c-Jun, ATF-1, ATF-2, JunD, and CREB). Similar to the rat NT/N gene, this region is critical for constitutive hNT/N gene expression. Moreover, we identified a novel region that binds the orphan hormone receptor, NR2F2. We have demonstrated that the C terminus of NR2F2 strongly represses hNT/N transcription, whereas an N-terminal domain antagonizes this repressive effect. Regulation of NT/N expression by NR2F2 may have important consequences for lipid metabolism. We speculate that a complex interplay between the proximal CRE/AP-1-like motif and NR2F2 binding region exists to regulate hNT/N expression, which is critical for the high level of constitutive expression of NT/N in enteroendocrine cells. Finally, the BON cell line provides a unique model to characterize the factors regulating expression of the hNT/N gene and to better understand the mechanisms responsible for terminal differentiation of the N cell lineage in the gut.

Carcinoid tumors are slow growing and highly vascular neuroendocrine neoplasms that are increasing in incidence. Previously, we showed that carcinoid tumors express vascular endothelial growth factor receptor 2 (VEGFR-2) in the epithelial compartment of carcinoid tumor sections; yet, its role is not completely understood. The purpose of our study was to: (i) assess the expression of VEGFR-2 in the novel human carcinoid cell line BON, (ii) to determine the role of PI3K/Akt signaling on VEGFR-2 expression and (iii) to assess the effect of VEGFR-2 on BON cell invasion, migration and proliferation. We found that, although VEGFR-2 is expressed in BON cells, reduction in VEGFR-2 expression actually enhanced proliferation, invasion, and migration of the BON cell line. Also, expression of VEGFR-2 was inversely related to PI3K signaling. Carcinoid liver metastases in mice demonstrated decreased VEGFR-2 expression. Furthermore, the expression of a truncated, soluble form of VEGFR-2 (sVEGFR-2), a protein demonstrated to inhibit cell growth, was detected in BON cells. The presence of VEGFR-2 in the epithelial component of carcinoid tumors and in the BON cell line suggests an alternate role for VEGFR-2, in addition to its well-defined role in angiogenesis. The expression of sVEGFR-2 may explain the inverse relationship between VEGFR-2 expression and PI3K/Akt signaling and the inhibitory effect VEGFR-2 has on BON cell proliferation, migration and invasion.

Cholecystectomy during initial hospitalization is the current recommended therapy for acute cholecystitis. The rate of cholecystectomy and subsequent health care trajectory in elderly patients with acute cholecystitis has not been evaluated.We used 5% national Medicare sample claims data from 1996 to 2005 to identify a cohort of patients aged 66 years and older, requiring urgent or emergent admission for acute cholecystitis. We evaluated cholecystectomy rates on initial hospitalization, factors independently predicting receipt of cholecystectomy, factors predicting further gallstone-related complications, and 2-year survival in the cholecystectomy and no cholecystectomy groups in univariate and multivariate models.There were 29,818 Medicare beneficiaries who were urgently or emergently admitted for acute cholecystitis from 1996 to 2005. Mean age was 77.7 +/- 7.3 years, 89% of patients were white, and 58% were female. Twenty-five percent of patients did not undergo cholecystectomy during the index admission. Lack of definitive therapy was associated with a 27% subsequent cholecystectomy rate and a 38% gallstone-related readmission rate in the 2 years after discharge; the readmission rate was only 4% in patients undergoing cholecystectomy (p0.0001). No cholecystectomy on initial hospitalization was associated with worse 2-year survival (hazard ratio 1.56, 95% CI 1.47 to 1.65) even after controlling for patient demographics and comorbidities. Readmissions led to an additional $7,000 in Medicare payments per readmission.Our study demonstrated that 25% of cholecystectomies on Medicare beneficiaries were not performed on initial hospitalization, leading to readmissions in 38% of surviving patients. For patients requiring readmission, the percentage of open procedures was increased, and the additional Medicare payment was $7,000 per re-admission. Cholecystectomy for acute cholecystitis in elderly patients should be performed during initial hospitalization to prevent recurrent episodes of cholecystitis, multiple readmissions, higher readmission rates, and increased costs.

Purpose: Carcinoid syndrome, characterized by flushing, diarrhea, and valvular heart disease, can occur following carcinoid tumor metastasis to the liver and systemic release of bioactive hormones into the systemic circulation. Treatment of this devastating disease is hampered by the lack of an in vivo model that recapitulates the clinical syndrome. Experimental Design: Here, we have injected BON cells, a novel human carcinoid cell line established in our laboratory, into the spleens of athymic nude mice to establish liver metastases. Results: The majority of mice injected intrasplenically with BON cells developed significant increases in plasma serotonin and urine 5-hydroxyindoleacetic acid, and several mice exhibited mesenteric fibrosis, diarrhea, and fibrotic cardiac valvular disease reminiscent of carcinoid syndrome by both echocardiographic and histopathologic evaluation. Mice pretreated with octreotide, a long-acting somatostatin analogue, or bevacizumab, a vascular endothelial growth factor inhibitor, developed fewer liver metastases and manifestations of carcinoid syndrome, including valvular heart disease. Conclusion: We have provided an important in vivo model to further delineate novel treatment modalities for carcinoid syndrome that will also be useful to elucidate the factors contributing to the sequelae of carcinoid disease (e.g., mesenteric fibrosis and valvular heart disease).

Transforming growth factor-beta (TGF-beta) regulates epithelial tissue homeostasis by activating processes that control cell cycle arrest, differentiation and apoptosis. Disruption of the TGF-beta signaling pathway often occurs in colorectal cancers. Earlier, we have shown that TGF-beta induces apoptosis through the transcription factor Smad3. Affymetrix oligonucleotide microarrays were used to identify TGF-beta/Smad3 target genes that regulate apoptosis in rat intestinal epithelial cells (RIE-1). We found that TGF-beta repressed the expression of the inhibitor of differentiation (Id) gene family. Knockdown of Id1 and Id2 gene expression induced apoptosis in RIE-1 cells, whereas overexpression of Id2 attenuated TGF-beta-induced apoptosis. TranSignal Protein/DNA arrays were used to identify the hypoxia-inducing factor-1 (HIF-1) as a downstream target of TGF-beta. HIF-1 is a basic helix-loop-helix protein, and overexpression of Id2 blocked HIF-1 activation by TGF-beta. Furthermore, knockdown of HIF-1 blocked TGF-beta-induced apoptosis. Thus, we have identified HIF-1 as a novel mediator downstream of Id2 in the pathway of TGF-beta-induced apoptosis.

Background A strong volume-outcome relationship has been demonstrated for pancreatic resection, and regionalization of care to high-volume centers (>11 resections/year) has been recommended. However, it is unclear if volume alone should be the sole criteria for regionalization. The objective of this study is to evaluate variability in outcomes among high-volume hospitals (>11 resections/year). Methods We used the Texas Hospital Inpatient Discharge Database from 1999 through 2005 to evaluate variability in outcomes after pancreatic resection among high-volume hospitals in Texas. The outcome variables of interest were mortality, length of stay, discharge to a skilled nursing facility, operation within 24 hours of hospital admission, and total hospital charges. Unadjusted and adjusted models were performed. Results A total of 12 high-volume hospitals were in Texas. The number of resections at each hospital ranged from 78–608 cases for the 7-year time period studied. In unadjusted models, there was significant variability in mortality (range, 0.7%–7.7%, P < .0001), duration of stay (range of medians, 9–21 days, P < .0001), the need for ongoing nursing care at discharge (range, 0.7%–41.4%, P < .0001), operation within 24 hours of admission (range, 41%–96%, P < .0001), and total hospital charges (median range, $38,318–$110,860, P < .0001). There were significant differences in the demographics, risks of mortality, and illness severity among the 12 high-volume hospitals. Therefore, multivariate models were used to control for age group, sex, race/ethnicity, risk of mortality, illness severity, admission status, diagnosis, procedure, and insurance status. In the multivariate models, the particular hospital at which the pancreatic surgery was performed was a significant independent predictor of every outcome variable except mortality. Conclusions For pancreatic resection, there is significant variability in outcomes even among high-volume providers. Individual hospitals likely account for much of the variability not explained by hospital volume. Although the structure measure of hospital volume is easy to measure, these data suggest that it is not a reliable single measure of quality or outcomes after pancreatic surgery.

Overexpression of cyclooxygenase-2 (COX-2), an inducible enzyme regulating prostaglandin release, is mechanistically linked to the development, growth, and spread of gastrointestinal (GI) cancers. GI peptide bombesin (BBS) was reported to stimulate COX-2 gene expression. Here we show that TGF-beta1 dramatically enhances the BBS-induced expression of COX-2 mRNA and protein, and the release of PGE2 in the model rat intestinal epithelial cell (RIE-1) line. The synergistic increase in COX-2 levels results from a combination of enhanced COX-2 transcription and reduced mRNA degradation. BBS, but not TGF-beta1, stimulated COX-2 promoter activity, and TGF-beta1 enhanced COX-2 mRNA stability through a p38(MAPK)-dependent pathway. The synergistic regulation of COX-2 expression by TGF-beta1 and BBS may contribute to the upregulation of COX-2 in GI cancers.

The current recommendation is that pancreatic resections be performed at hospitals doing10 pancreatic resections annually.To evaluate the extent of regionalization of pancreatic resection and the factors predicting resection at high-volume centers (10 cases/year) in Texas.Using the Texas Hospital Inpatient Discharge Public Use Data File, we evaluated trends in the percentage of patients undergoing pancreatic resection at high-volume centers (10 cases/year) from 1999 to 2004 and determined the factors that independently predicted resection at high-volume centers.A total of 3,189 pancreatic resections were performed in the state of Texas. The unadjusted in-hospital mortality was higher at low-volume centers (7.4%) compared to high-volume centers (3.0%). Patients resected at high-volume centers increased from 54.5% in 1999 to 63.3% in 2004 (P = 0.0004). This was the result of a decrease in resections performed at centers doing less than five resections/year (35.5% to 26.0%). In a multivariate analysis, patients who were75 (OR = 0.51), female (OR = 0.86), Hispanic (OR = 0.58), having emergent surgery (OR = 0.39), diagnosed with periampullary cancer (OR = 0.68), and living75 mi from a high-volume center (OR = 0.93 per 10-mi increase in distance, P0.05 for all OR) were less likely to be resected at high-volume centers. The odds of being resected at a high-volume center increased 6% per year.Whereas regionalization of pancreatic resection at high-volume centers in the state of Texas has improved slightly over time, 37% of patients continue to undergo pancreatic resection at low-volume centers, with more than 25% occurring at centers doing less than five per year. There are obvious demographic disparities in the regionalization of care, but additional unmeasured barriers need to be identified.

Transforming growth factor (TGF)-beta-dependent apoptosis is important in the elimination of damaged or abnormal cells from normal tissues in vivo. Previously, we have shown that TGF-beta inhibits the growth of rat intestinal epithelial (RIE)-1 cells. However, RIE-1 cells are relatively resistant to TGF-beta-induced apoptosis due to a low endogenous Smad3-to-Akt ratio. Overexpression of Smad3 sensitizes RIE-1 cells (RIE-1/Smad3) to TGF-beta-induced apoptosis by altering the Smad3-to-Akt ratio in favor of apoptosis. In this study, we utilized a genomic approach to identify potential downstream target genes that are regulated by TGF-beta/Smad3. Total RNA samples were analyzed using Affymetrix oligonucleotide microarrays. We found that TGF-beta regulated 518 probe sets corresponding to its target genes. Interestingly, among the known apoptotic genes included in the microarray analyses, only caspase-3 was induced, which was confirmed by real-time RT-PCR. Furthermore, TGF-beta activated caspase-3 through protein cleavage. Upstream of caspase-3, TGF-beta induced mitochondrial depolarization, cytochrome c release, and cleavage of caspase-9, which suggests that the intrinsic apoptotic pathway mediates TGF-beta-induced apoptosis in RIE-1/Smad3 cells.

AMP-activated protein kinase (AMPK), a critical fuel-sensing enzyme, regulates the metabolic effects of various hormones. Neurotensin (NT) is a 13-amino acid peptide predominantly localized in enteroendocrine cells of the small bowel and released by fat ingestion. Increased fasting plasma levels of pro-NT (a stable NT precursor fragment produced in equimolar amounts relative to NT) are associated with an increased risk of diabetes, cardiovascular disease, and mortality; however, the mechanisms regulating NT release are not fully defined. We previously reported that inhibition of the mammalian target of rapamycin (mTOR) complex 1 (mTORC1) increases NT secretion and gene expression through activation of the MEK/ERK pathway. Here, we show that activation of AMPK increases NT secretion from endocrine cell lines (BON and QGP-1) and isolated mouse crypt cells enriched for NT-positive cells. In addition, plasma levels of NT increase in mice treated with 5-aminoimidazole-4-carboxamide riboside, a pharmacologic AMPK activator. Small interfering RNA-mediated knockdown of AMPKα decrease, whereas overexpression of the subunit significantly enhances, NT secretion from BON cells treated with AMPK activators or oleic acid. Similarly, small interfering RNA knockdown of the upstream AMPK kinases, liver kinase B1 and Ca2+ calmodulin-dependent protein kinase kinase 2, also attenuate NT release and AMPK phosphorylation. Moreover, AMPK activation increases NT secretion through inhibition of mTORC1 signaling. Together, our findings show that AMPK activation enhances NT release through inhibition of mTORC1 signaling, thus demonstrating an important cross talk regulation for NT secretion.

It is unknown whether the improved survival seen at high-volume centers has been translated to all patients with pancreatic cancer.To use the Surveillance, Epidemiology, and End Results (SEER) database to evaluate population-based trends in surgical resection and survival.All patients diagnosed with pancreatic cancer from 1988-1999 were identified. The survival and proportion of patients undergoing surgical resection were compared for each of three equal time periods.There were 24,016 patients with pancreatic cancer. 19,533 had stage data available. 9% had localized, 29% had regional, and 62% had distant disease. Resection rates increased for patients with localized and regional disease over the three time periods. Survival increased for patients with regional and distant disease. For regional pancreatic cancer patients, 2-year survival increased from 9.5% to 13.5% (p0.0001) and from 21.5% to 28.9% following surgical resection (p = 0.002). For resected local/regional pancreatic cancer, the year of diagnosis was and independent predictor of improved survival (p = 0.0001).SEER patients with regional and distant pancreatic cancer have improved survival over the past decade in both unadjusted and adjusted models. The improvement is most striking for patients with regional disease and reflects increased resection rates and improved resection techniques over time.

Carcinoid tumors are rare neuroendocrine tumors with a predilection for the gastrointestinal tract. Protein kinase D (PKD), a novel serine/threonine protein kinase, has been implicated in the regulation of transport processes in certain cell types. We have reported an important role for PKD in stimulated peptide secretion from a human (BON) carcinoid cell line; however, the role of PKD isoforms, including PKD2, in the proliferation and invasion of carcinoid tumors remains unclear. In the present study, we found that overexpression of PKD2 by stable transfection of BON cells with PKD2-wild type (PKD2WT) significantly increased proliferation and invasion compared to cells transfected with PKD2-kinase dead (PKD2KD) or pcDNA3 (control). Similarly, inhibition of PKD2 activity with small interfering RNA (siRNA) significantly decreased proliferation and invasion compared to cells transfected with non-targeting control (NTC) siRNA. These data support an important role for PKD2 in carcinoid tumor progression. Targeted inhibition of the PKD family may prove to be a novel treatment option for patients with carcinoid tumors.

Background During the early stages of colorectal carcinogenesis, the phosphatidylinositol 3-kinase (PI3K)/Akt pathway is activated, enabling the transformed cells to survive and grow in the absence of anchorage to extracellular matrix. Transforming growth factor β (TGF-β) is an important tumor suppressor in the colon, and it is inactivated during later stages of colorectal carcinogenesis. The purpose of this study was to determine whether TGF-β inhibits Akt-induced anchorage-independent growth and resistance to anoikis in gut epithelial cells. Methods Rat intestinal epithelial cells (RIE-1) were infected with a retrovirus containing pLXSN-mAkt, and three independent clones were selected. Anchorage-independent growth was examined by colony formation in soft agar and cell counting in ultralow attachment plates. Anoikis was analyzed with the use of Annexin V staining. Results All three clones of RIE-1/mAkt formed colonies in soft agar, which were decreased by TGF-β. TGF-β induced anoikis and treatment with a general caspase inhibitor, zVAD-fluoromethyl ketone, blocked TGF-β–mediated decrease in colony formation. Conclusions TGF-β attenuated Akt-induced anchorage-independent growth in RIE-1 cells in part by enhancing anoikis. Our data demonstrate a novel tumor-suppressor activity of TGF-β and provide the molecular justification for the required activation of the PI3K/Akt pathway and the subsequent inactivation of TGF-β signaling during colorectal carcinogenesis.