- Email: [email protected]
M1978 Activation of Calcium Sensing Receptor Suppresses Proliferation and Motility in Human Pancreatic Cancer Cells
TNF-R1 concentrations were significantly higher in both patient groups than in healthy subjects (IPMT, P=0.004; pancreatic adenocarcinoma, P=0.001). On the other hand, serum CA 19-9 concentrations were significantly higher in patients with pancreatic adenocarcinoma than in those with IPMTs (P=0.044) and healthy subjects (P=0.003). Conclusions. Serum TNF-R1 is a potentially good marker for selecting patients with pancreatic adenocarcinoma having distant metastases and those with main duct IPMTs.
Regulation of CA2+ Signaling in Pancreatic Cancer Jimmy Y. Chow, Ki-Nam Shim, Tiffany A. Ornelas, Flang Nguyen, John M. Carethers, Hui Dong Background: We recently reported that TGF-beta induces an increase in cytosolic Ca2+ ([Ca2+]cyt) in pancreatic cancer cells, but the mechanisms by which TGF-beta mediates [Ca2+]cyt homeostasis in these cells is currently unknown. Transient receptor potential (TRP) channels and Na+/Ca2+ exchangers (NCX) are membrane proteins that play prominent roles in controlling [Ca2+]cyt homeostasis in normal mammalian cells, but little is known regarding the regulation of [Ca2+]cyt in pancreatic cancer. We assessed the expression and function of NCX1 and TRPC1 proteins in pancreatic cancer cells. Methods: BxPc3 and CAPAN-1 cells were used. Real-time PCR was conducted to determine if TGF-beta treatment (10 ng/mL) could induce expression of NCX1 in BxPc3 pancreatic cancer cells. Cells were also assessed to determine functional expressions of NCX and TRPC channels analysed by a calcium imaging system. Cells were also pretreated with inhibitors for TRPC or an inhibitor for the Ca2+ entry mode of NCX to determine the roles of TRPC channels or NCX1 on TGF-beta-induced phosphorylation of PKC-alpha and [Ca2+]cyt. Changes of [Ca2+]cyt before and after treatment by various reagents were investigated by a calcium imaging system. Cell motility was assessed by Boyden chamber migration assay. Results: These cells exhibited functional expression of both NCX and TRPC channels. TGF-beta upregulated NCX1 mRNA expression and induced both intracellular Ca2+ release and extracellular Ca2+ entry in cells; however, inhibition of the TRPC channels or the Ca2+ entry mode of NCX markedly inhibited TGF-beta-induced increase in [Ca2+]cyt. Inhibition of the TRPC channels or NCX was also able to dose-dependently suppress PKC-alpha phosphorylation induced by TGF-beta. Inhibition of NCX1 also inhibited PKC-alpha phosphorylation induced by TGF-beta. Inhibition of NCX or TRPC channels reversed TGF-beta-induced pancreatic cancer cell motility. Conclusion: TGF-beta upregulates NCX1 expression and induces Ca2+ entry via TRPC1 and NCX1 to raise [Ca2+]cyt in pancreatic cancer cells, which is essential for PKC-alpha activation and subsequent tumor cell motility. Our data suggest that Ca2+ signaling via TRPC1 and NCX1 could be potential therapeutic targets for pancreatic cancer.
M1978 Activation of Calcium Sensing Receptor Suppresses Proliferation and Motility in Human Pancreatic Cancer Cells Ki-Nam Shim, Shanglei Liu, Christine L. Estrema, Tiffany A. Ornelas, Flang Nguyen, Mei Huang, John M. Carethers, Jimmy Y. Chow, Hui Dong Backgrounds & Aims: Calcium sensing receptor (CaSR), a member of the G-protein-coupled receptor, has been identified in a variety of mammalian tissues and suggested to have various regulatory functions, including cell proliferation and differentiation. Activation of CaSR increases metastasis in breast and prostate cancers, but decreases cell proliferation in parathyroid and colon cancer. Since the involvement of CaSR in pancreatic cancer is unknown, we sought to investigate the role of CaSR in proliferation and motility regulation in human pancreatic cancer cells. Methods: Expression of the CaSR in BxPC3 and MiaPaCa2 cells was detected by western blot analysis. ERK1/2 activity induced by CaSR agonists (Ca2+ or spermine) was studied using kinase assay kit. Cell proliferation and motility were determined using MTT assay, transwell migration assay, and scratch test. Results : Either Ca2+ (4 mM) and spermine (1mM) induced ERK1/2 phosphorylation in BxPC3. Inhibition of NCX1 by SN-6 (30 μM) inhibited CaSR-mediated ERK1/2 phosphorylation but potentiated CaRinduced down-regulation of cell proliferation and motility by SN-6 (5 μM) in BxPC3. Spermine (1mM) inhibited significantly the proliferation of MiaPaCa2 (P = 0.000) and Ca2+ (4 mM) inhibited cell motility of BxPC3 (P < 0.01). Pretreatment of calmodulin inhibitor, W-7 (40 μM) partially but significantly reversed CaSR-induced inhibition of cell proliferation of MiaPaCa2. Conclusion: Our results suggest that CaSR is functionally expressed in human pancreatic cancer cells and that activation of this receptor reduces cell proliferation and motility through a Ca2+/calmodulin pathway.
M1976 M1979
Identification of Palladin Gene Mutations Through Colorimetric Analysis in Patients With Pancreatic Malignancy and Unknown Family History Robert Ollar, Niket Sonpal, Sushil Duddempudi, Franklin Kasmin, Michael G. Wayne, Avram Cooperman
Adipocytes in the Pancreatic Tumor Microenvironment Promote Dissemination of Human Pancreatic Cancer Patrick B. White, Jey-Hsin Chen, Nicholas J. Zyromski, Abhishek Mathur, Keith D. Lillemoe, Henry A. Pitt
Cancer of the Pancreas (CaP) is the 4th leading cause of cancer death in the U.S and each year in the about 42,470 individuals are diagnosed with this condition and 35,240 die from the disease. The causes are multi-factorial, but genetic predisposition has a large role to play and several genes have been implicated in the pathogenesis of CaP. In fact, 10% of cases have been shown to have a familial clustering. One such gene is the Palladin loci (PALLD / 4q32-34), whose precise biological role is poorly understood, but it has been shown to play a role in cytoskeletal organization and embryonic development. It has been shown that Palladin RNA is overexpressed and mutated in pancreatic neoplasia. However, the Palladin mutation identified in familial pancreatic cancer may be unique to a single North American family, as this same mutation has not been found in any other European or North American populations. Our study aimed to examine a commercially available colorimetric based assay for the detection of germline Palladin gene mutations in lymphocytes of peripheral blood. This approach was targeted in patients with pancreatic malignancies with unknown previous family history as a way of delineating whether their CaP could be associated to their inheritance. Genomic DNA extracts were obtained from peripheral blood in 32 (14 males; 18 females) samples from patients (average age 60 years old) with known pancreatic disorders. The purified extracts were stored at -20 degrees Celcius and processed via a Polymerase Chain Reaction to generate amplicons containing the Palladin gene. The Trimgen Mutector Colorimetric Kit Assay was utilized to detect germline point mutations. In this assay, mutations were expressed as a numeric value called a Point Mutation Ratio. A ratio value of greater than or equal to 2 indicated a mutation. Our results found no paladin mutations in the 32 samples obtained. Our approach initially was aimed at identifying the inheritance background in those with CaP who lacked information about their ancestry. Our investigation based on a colorimetric approach, though inherently different than previous investigations using sequencer analysis, the findings echoed the two major studies in this realm. As cited by previous investigators in Europe and the United States, we also did not identify the mutation in any individuals; neither as a heterozygote or a homozygote. We will continue this study of the paladin mutations; however at this time we cannot support the assumption that this marker is useful in detecting a familial clustering in CaP. We believe that this marker should be studied in families with known familial pancreatic malignancy.
Background: Epidemiologic studies have established an association between obesity and several common malignancies. Human studies also have demonstrated that obesity accelerates human pancreatic cancer development and progression. We have recently reported that increased pancreatic neck fat is correlated with reduced survival and increased dissemination in human pancreatic cancer (JACS 2009, 208: 989-94). However, the fat content of the pancreatic tumors themselves was not analyzed. Therefore, we hypothesized that adipocytes in the pancreatic tumor microenvironment would be associated with increased dissemination and reduced survival in patients with resected pancreatic cancer. Methods: A case-control analysis was conducted in patients who had undergone resection for pancreatic adenocarcinoma. Twenty lymph node positive patients and twenty node negative patients were matched for age (59 vs. 63 years), gender (70% male vs. 60% male), BMI (24.5 vs. 25.6 kg/m2), medical comorbidities (hypertension, diabetes, hyperlipidemia), tumor size (2.8 vs. 2.6 cm), resection status (Ro-80% vs. 85%), and neural invasion (100% vs. 85%). Pancreatic tumor sections were reviewed for percentage fat, tumor, and fibrosis (including vessels and nerves) in a blinded fashion by two trained investigators. Fat cells, tumor cells, and fibrosis (percentage of field/ 10 high-powered fields) were recorded. In addition, the percentage of patients with adipocytes in the tumor microenvironment was determined. Discrepancies of more than 10% variation between observers were resolved by second observation and concurrence. Data were analyzed by Wilcoxian rank-sum and Fisher's exact test as appropriate. Results: Percentages of fat, tumor, and fibrosis in the two groups are presented in the table. More patients with fat in the tumor microenvironment were observed in the node positive than in the node negative group (79% vs. 37%, p<0.03). Conclusion: These data suggest that adipocytes within the tumor microenvironment promote the dissemination and lethality of pancreatic cancer. This analysis confirms and extends our previous observation that pancreatic steatosis enhances tumor spread and contributes to increased mortality in patients with pancreatic adenocarcinoma.
M1977 The Role of Inflammation in Patients With Intraductal Mucinous Tumors of the Pancreas and in Those With Pancreatic Adenocarcinoma Raffaele Pezzilli
*P<0.05 vs. Node Negative
Background. There are very few data regarding inflammation processes in patients with intraductal papillary mucinous tumors (IPMTs) of the pancreas. Aim. To evaluate the circulating concentrations of placental growth factor (PlGF), transforming growth factor-alpha (TGFα), transforming growth factor-beta1 (TGF-β1), tumor necrosis factor receptor 1 (TNF-R1) and matrix metalloproteinases-2 (MMP-2) in patients with IPMTs and in those with pancreatic adenocarcinoma and to also evaluate the concentrations of these molecules in comparison with CA 19-9. Patients. Sixty-nine patients (39 males, 30 females, mean age 69.8±10.4 years) were enrolled: 23 (33.3%) had IPMTs and 46 (66.7%) had histologically confirmed pancreatic adenocarcinoma. Thirteen healthy subjects were also studied. Methods. PlGF, TGF-α, TGF-β1, TNF-R1, MMP-2 were determined using commercially available kits. CA 19-9 was also assayed. Results. The only two substances showing significant differences among the three groups were TNF-R1 (P=0.003) and CA 19-9 (P=0.007). In particular,
M1980 Exogenously Added Trypsin is Not Required for High In Vitro Cytotoxicity of Newcastle Disease Virus Strain Lasota in Pancreatic Tumor Cells Bashar M. Attar, Robert J. Walter, Megan Delimata, Lakshminarayan Sooraj T K, Asad Rafiq Background: New, more effective treatment modalities are needed for pancreatic cancer. We showed previously that Newcastle disease virus (NDV) 73-T, a highly infectious bird pathogen, is highly cytotoxic to a variety of human tumor cells both In Vitro and In Vivo, but little is known regarding NDV's effects on pancreatic tumors. The 73-T strain and other mesogenic or velogenic strains are also problematic for clinical use due to possible inadvertent release into the environment. As a result, avirulent lentogenic strains may be better candidates
S-451
AGA Abstracts
AGA Abstracts
M1974
Regulation of CA2+ Signaling in Pancreatic Cancer Jimmy Y. Chow, Ki-Nam Shim, Tiffany A. Ornelas, Flang Nguyen, John M. Carethers, Hui Dong Background: We recently reported that TGF-beta induces an increase in cytosolic Ca2+ ([Ca2+]cyt) in pancreatic cancer cells, but the mechanisms by which TGF-beta mediates [Ca2+]cyt homeostasis in these cells is currently unknown. Transient receptor potential (TRP) channels and Na+/Ca2+ exchangers (NCX) are membrane proteins that play prominent roles in controlling [Ca2+]cyt homeostasis in normal mammalian cells, but little is known regarding the regulation of [Ca2+]cyt in pancreatic cancer. We assessed the expression and function of NCX1 and TRPC1 proteins in pancreatic cancer cells. Methods: BxPc3 and CAPAN-1 cells were used. Real-time PCR was conducted to determine if TGF-beta treatment (10 ng/mL) could induce expression of NCX1 in BxPc3 pancreatic cancer cells. Cells were also assessed to determine functional expressions of NCX and TRPC channels analysed by a calcium imaging system. Cells were also pretreated with inhibitors for TRPC or an inhibitor for the Ca2+ entry mode of NCX to determine the roles of TRPC channels or NCX1 on TGF-beta-induced phosphorylation of PKC-alpha and [Ca2+]cyt. Changes of [Ca2+]cyt before and after treatment by various reagents were investigated by a calcium imaging system. Cell motility was assessed by Boyden chamber migration assay. Results: These cells exhibited functional expression of both NCX and TRPC channels. TGF-beta upregulated NCX1 mRNA expression and induced both intracellular Ca2+ release and extracellular Ca2+ entry in cells; however, inhibition of the TRPC channels or the Ca2+ entry mode of NCX markedly inhibited TGF-beta-induced increase in [Ca2+]cyt. Inhibition of the TRPC channels or NCX was also able to dose-dependently suppress PKC-alpha phosphorylation induced by TGF-beta. Inhibition of NCX1 also inhibited PKC-alpha phosphorylation induced by TGF-beta. Inhibition of NCX or TRPC channels reversed TGF-beta-induced pancreatic cancer cell motility. Conclusion: TGF-beta upregulates NCX1 expression and induces Ca2+ entry via TRPC1 and NCX1 to raise [Ca2+]cyt in pancreatic cancer cells, which is essential for PKC-alpha activation and subsequent tumor cell motility. Our data suggest that Ca2+ signaling via TRPC1 and NCX1 could be potential therapeutic targets for pancreatic cancer.
M1978 Activation of Calcium Sensing Receptor Suppresses Proliferation and Motility in Human Pancreatic Cancer Cells Ki-Nam Shim, Shanglei Liu, Christine L. Estrema, Tiffany A. Ornelas, Flang Nguyen, Mei Huang, John M. Carethers, Jimmy Y. Chow, Hui Dong Backgrounds & Aims: Calcium sensing receptor (CaSR), a member of the G-protein-coupled receptor, has been identified in a variety of mammalian tissues and suggested to have various regulatory functions, including cell proliferation and differentiation. Activation of CaSR increases metastasis in breast and prostate cancers, but decreases cell proliferation in parathyroid and colon cancer. Since the involvement of CaSR in pancreatic cancer is unknown, we sought to investigate the role of CaSR in proliferation and motility regulation in human pancreatic cancer cells. Methods: Expression of the CaSR in BxPC3 and MiaPaCa2 cells was detected by western blot analysis. ERK1/2 activity induced by CaSR agonists (Ca2+ or spermine) was studied using kinase assay kit. Cell proliferation and motility were determined using MTT assay, transwell migration assay, and scratch test. Results : Either Ca2+ (4 mM) and spermine (1mM) induced ERK1/2 phosphorylation in BxPC3. Inhibition of NCX1 by SN-6 (30 μM) inhibited CaSR-mediated ERK1/2 phosphorylation but potentiated CaRinduced down-regulation of cell proliferation and motility by SN-6 (5 μM) in BxPC3. Spermine (1mM) inhibited significantly the proliferation of MiaPaCa2 (P = 0.000) and Ca2+ (4 mM) inhibited cell motility of BxPC3 (P < 0.01). Pretreatment of calmodulin inhibitor, W-7 (40 μM) partially but significantly reversed CaSR-induced inhibition of cell proliferation of MiaPaCa2. Conclusion: Our results suggest that CaSR is functionally expressed in human pancreatic cancer cells and that activation of this receptor reduces cell proliferation and motility through a Ca2+/calmodulin pathway.
M1976 M1979
Identification of Palladin Gene Mutations Through Colorimetric Analysis in Patients With Pancreatic Malignancy and Unknown Family History Robert Ollar, Niket Sonpal, Sushil Duddempudi, Franklin Kasmin, Michael G. Wayne, Avram Cooperman
Adipocytes in the Pancreatic Tumor Microenvironment Promote Dissemination of Human Pancreatic Cancer Patrick B. White, Jey-Hsin Chen, Nicholas J. Zyromski, Abhishek Mathur, Keith D. Lillemoe, Henry A. Pitt
Cancer of the Pancreas (CaP) is the 4th leading cause of cancer death in the U.S and each year in the about 42,470 individuals are diagnosed with this condition and 35,240 die from the disease. The causes are multi-factorial, but genetic predisposition has a large role to play and several genes have been implicated in the pathogenesis of CaP. In fact, 10% of cases have been shown to have a familial clustering. One such gene is the Palladin loci (PALLD / 4q32-34), whose precise biological role is poorly understood, but it has been shown to play a role in cytoskeletal organization and embryonic development. It has been shown that Palladin RNA is overexpressed and mutated in pancreatic neoplasia. However, the Palladin mutation identified in familial pancreatic cancer may be unique to a single North American family, as this same mutation has not been found in any other European or North American populations. Our study aimed to examine a commercially available colorimetric based assay for the detection of germline Palladin gene mutations in lymphocytes of peripheral blood. This approach was targeted in patients with pancreatic malignancies with unknown previous family history as a way of delineating whether their CaP could be associated to their inheritance. Genomic DNA extracts were obtained from peripheral blood in 32 (14 males; 18 females) samples from patients (average age 60 years old) with known pancreatic disorders. The purified extracts were stored at -20 degrees Celcius and processed via a Polymerase Chain Reaction to generate amplicons containing the Palladin gene. The Trimgen Mutector Colorimetric Kit Assay was utilized to detect germline point mutations. In this assay, mutations were expressed as a numeric value called a Point Mutation Ratio. A ratio value of greater than or equal to 2 indicated a mutation. Our results found no paladin mutations in the 32 samples obtained. Our approach initially was aimed at identifying the inheritance background in those with CaP who lacked information about their ancestry. Our investigation based on a colorimetric approach, though inherently different than previous investigations using sequencer analysis, the findings echoed the two major studies in this realm. As cited by previous investigators in Europe and the United States, we also did not identify the mutation in any individuals; neither as a heterozygote or a homozygote. We will continue this study of the paladin mutations; however at this time we cannot support the assumption that this marker is useful in detecting a familial clustering in CaP. We believe that this marker should be studied in families with known familial pancreatic malignancy.
Background: Epidemiologic studies have established an association between obesity and several common malignancies. Human studies also have demonstrated that obesity accelerates human pancreatic cancer development and progression. We have recently reported that increased pancreatic neck fat is correlated with reduced survival and increased dissemination in human pancreatic cancer (JACS 2009, 208: 989-94). However, the fat content of the pancreatic tumors themselves was not analyzed. Therefore, we hypothesized that adipocytes in the pancreatic tumor microenvironment would be associated with increased dissemination and reduced survival in patients with resected pancreatic cancer. Methods: A case-control analysis was conducted in patients who had undergone resection for pancreatic adenocarcinoma. Twenty lymph node positive patients and twenty node negative patients were matched for age (59 vs. 63 years), gender (70% male vs. 60% male), BMI (24.5 vs. 25.6 kg/m2), medical comorbidities (hypertension, diabetes, hyperlipidemia), tumor size (2.8 vs. 2.6 cm), resection status (Ro-80% vs. 85%), and neural invasion (100% vs. 85%). Pancreatic tumor sections were reviewed for percentage fat, tumor, and fibrosis (including vessels and nerves) in a blinded fashion by two trained investigators. Fat cells, tumor cells, and fibrosis (percentage of field/ 10 high-powered fields) were recorded. In addition, the percentage of patients with adipocytes in the tumor microenvironment was determined. Discrepancies of more than 10% variation between observers were resolved by second observation and concurrence. Data were analyzed by Wilcoxian rank-sum and Fisher's exact test as appropriate. Results: Percentages of fat, tumor, and fibrosis in the two groups are presented in the table. More patients with fat in the tumor microenvironment were observed in the node positive than in the node negative group (79% vs. 37%, p<0.03). Conclusion: These data suggest that adipocytes within the tumor microenvironment promote the dissemination and lethality of pancreatic cancer. This analysis confirms and extends our previous observation that pancreatic steatosis enhances tumor spread and contributes to increased mortality in patients with pancreatic adenocarcinoma.
M1977 The Role of Inflammation in Patients With Intraductal Mucinous Tumors of the Pancreas and in Those With Pancreatic Adenocarcinoma Raffaele Pezzilli
*P<0.05 vs. Node Negative
Background. There are very few data regarding inflammation processes in patients with intraductal papillary mucinous tumors (IPMTs) of the pancreas. Aim. To evaluate the circulating concentrations of placental growth factor (PlGF), transforming growth factor-alpha (TGFα), transforming growth factor-beta1 (TGF-β1), tumor necrosis factor receptor 1 (TNF-R1) and matrix metalloproteinases-2 (MMP-2) in patients with IPMTs and in those with pancreatic adenocarcinoma and to also evaluate the concentrations of these molecules in comparison with CA 19-9. Patients. Sixty-nine patients (39 males, 30 females, mean age 69.8±10.4 years) were enrolled: 23 (33.3%) had IPMTs and 46 (66.7%) had histologically confirmed pancreatic adenocarcinoma. Thirteen healthy subjects were also studied. Methods. PlGF, TGF-α, TGF-β1, TNF-R1, MMP-2 were determined using commercially available kits. CA 19-9 was also assayed. Results. The only two substances showing significant differences among the three groups were TNF-R1 (P=0.003) and CA 19-9 (P=0.007). In particular,
M1980 Exogenously Added Trypsin is Not Required for High In Vitro Cytotoxicity of Newcastle Disease Virus Strain Lasota in Pancreatic Tumor Cells Bashar M. Attar, Robert J. Walter, Megan Delimata, Lakshminarayan Sooraj T K, Asad Rafiq Background: New, more effective treatment modalities are needed for pancreatic cancer. We showed previously that Newcastle disease virus (NDV) 73-T, a highly infectious bird pathogen, is highly cytotoxic to a variety of human tumor cells both In Vitro and In Vivo, but little is known regarding NDV's effects on pancreatic tumors. The 73-T strain and other mesogenic or velogenic strains are also problematic for clinical use due to possible inadvertent release into the environment. As a result, avirulent lentogenic strains may be better candidates
S-451
AGA Abstracts
AGA Abstracts
M1974