- Email: [email protected]
RIN1 Axis in Pancreatic Cancer Cells
Tu1912
Background: Pancreatic cancer has an extremely poor prognosis. At the time of diagnosis, over 80% of patients have locally advanced or invasive disease. The carcinogenesis of pancreatic ductal adenocarcinoma (PDAC) involves stepwise progression from pancreatic intraepithelial neoplasia (PanIN) to invasive carcinoma. PanINs measure less than 5 mm and are not detectable on current imaging modalities. Precursor lesions, such as high-grade PanIN (PanIN-3) represent a target for early intervention. Low grade PanIN-1 and -2 are widely prevalent but their significance is unclear. Aims: To evaluate prevalence, significance and implications of PanIN-3 in PDAC. Methods: A retrospective review of the pathology database (1/2000 to 7/2013) at a tertiary care center. Demographic information, patient history, imaging studies, EUS findings and surgical pathology were reviewed. Results: A total of 607 pancreatic resections for cystic and solid lesions were reviewed. Neoadjuvant chemotherapy was given to 48 patients. In the remaining 559 patients, 198 (35.4%) had primary surgical resection for PDAC. Among these patients, 74.5% (of 185) showed the presence of any subtype of PanIN. High grade or PanIN-3 was present in 36.2% (of 185) resections for PDAC. Further, PanIN-3 was mostly found in PDAC (90.5%, p-value <0.001) compared to all other lesion types. Among patients with PDACs, who did not receive neoadjuvant therapy, univariate analysis (tables 1 and 2), demonstrated that Caucasian race and the presence of perineural invasion were significantly associated with PanIN-3. Pathological evidence of chronic pancreatitis and higher number of positive lymph nodes trended towards significant. Using these variables in a binomial logistic regression model demonstrated that Caucasian race (OR 10.9, p-value 0.02, 95% confidence interval 1.4, 86.6) and the presence of perineural invasion (OR 6.4, p-value 0.02, 95% confidence interval 1.4, 29.2) were significantly associated with PanIN-3. Interestingly, a comparison of PanIN3 among PDACs who underwent primary surgical resection versus post-neoadjuvant chemotherapy resection revealed that there was an absence of significant reduction in PanIN-3 among post-neoadjuvant resected PDACs (66 of 168 (39.3%) vs. 12 of 36 (33.3%), p = 0.57). Conclusion: High grade PanIN lesions are particularly prevalent in PDACs. In patients undergoing primary surgical resection for PDACs, high grade PanIN-3 lesions were notably associated with Caucasian race and perineural invasion. Supporting prior literature that PanIN-3 lesions may harbor cells with invasive features, the finding of PanIN-3 association with perineural invasion needs to be validated by larger studies. Table 1. Univariate analysis of clinicopathologic categorical variables of PanIN-1 and -2 vs. PanIN-3
AGA Abstracts
Determining Novel Clinicopathologic Correlations of High Grade Pancreatic Intraepithelial Neoplasia (PanIN) in Pancreatic Ductal Adenocarcinoma (PDAC) Jean R. Park, Feng Li, Michael Wellner, Jordan Thomas, Brett C. Sklaw, Brett C. Sklaw, Kevin M. Cronley, Veeral Oza, Jasleen Grewal, Jeffery R. Groce, Benjamin Swanson, Tanios Bekaii-Saab, Darwin Conwell, Somashekar G. Krishna
*Significant or trending towards significant variables Tu1913 CTNNB1 Mutational Analysis of Solid-Pseudopapillary Neoplasms of the Pancreas Using Endoscopic Ultrasound-Guided Fine-Needle Aspiration and Next-Generation Deep Sequencing Yoshimasa Kubota, Hiroshi Kawakami, Mitsuteru Natsuizaka, Kazumichi Kawakubo, Katsuji Marukawa, Taiki Kudo, Yoko Abe, Kimitoshi Kubo, Masaki Kuwatani, Yutaka Hatanaka, Tomoko Mitsuhashi, Yoshihiro Matsuno, Naoya Sakamoto Background: Solid-pseudopapillary neoplasm (SPN) of the pancreas is a rare neoplasm that is seen mostly in young female patients. Mutational analyses by surgically resected specimen and direct sequencing have revealed mutations in exon3 of the cadherin-associated protein beta 1 (CTNNB1) gene. Objectives: This study was intended to determine the feasibility of mutational analyses using samples obtained by endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) and next-generation sequencing (NGS). Methods: Thirty-four samples were tested: SPNs (n = 7) and other pancreatic lesions (n = 27; pancreatic ductal adenocarcinomas (PDAC), 16; pancreatic neuroendocrine tumors (PNET), 7; acinar cell carcinoma, 1; autoimmune pancreatitis, 1; and focal pancreatitis, 2). Samples were obtained by either EUS-FNA (n = 32) or surgery (n = 2) at Hokkaido University Hospital, Sapporo, Japan, between December 2008 and October 2013. EUS-FNA was performed using a curvilinear echoendoscope and 22-gauge needles. Sequencing was performed on Ion PGMTM (Personal Genome Machine) Sequencer by using an Ion 314TM chip (Life Technologies) with an IonPGMTM Sequencing 200 kit (Life Technologies). Obtained sequences were mapped onto the human reference genome hg19, and variants were detected using Ion Torrent Suite v2.2 software (Life Technologies). Results: A single-base pair missense mutations in exon3 of CTNNB1 was observed in all 7 SPNs. The mutations were found in codon 32 (n=3), codon 37 (n=2), and codon 41 (n=2). A N390 C/T (Thr41Ile) mutation was detected in 1 of 7 PNET samples. However, mutations were not observed in the tissue samples of any of the 15 PDAC or other 5 pancreatic disease cases. The variant frequency of CTNNB1 ranged from 5.39% to 48.77%, and the average base coverage depth of the samples with mutations ranged from 4,490 to 84,609. Conclusions: Mutational analysis of CTNNB1 by NGS is feasible and achieved using SPN samples obtained by EUS-FNA with high sensitivity and accuracy. Further exploration of CTNNB1 mutations in other neuroendocrine tumors will help identifying the genetic alterations that occur in PNET. Tu1914 Identification of a Novel Protein Kinase D1 (PKD1)/ RIN1 Axis in Pancreatic Cancer Cells Sushovan Guha, James Sinnett-Smith, Enrique Rozengurt Background: Pancreatic ductal adenocarcinoma (PDAC) is a devastating disease with 5-year survival rate of only 3-5%. As the current therapies offer limited survival benefits, novel therapeutic targets and strategies are needed to treat this aggressive disease. Protein kinase D1 (PKD1) is emerging as a critical node in the control of the ERK pathway leading to PDAC proliferation and thus, as a potential novel target in PDAC therapy. In order to elucidate the mechanism(s) by which PKD leads to ERK cascade activation, we examined the PKD/RIN1 pathway in PDAC cells. RIN1 (Ras and Rab interactor 1) is a multidomain protein that binds GTP-bound RAS with high affinity and inhibits the activating interaction between Ras and Raf. Consequently, RIN1 is a potent inhibitor of Ras-induced ERK activation. We demonstrated that PKD1 directly phosphorylates RIN1 at Ser351, a site that promotes binding of 14-3-3 proteins. Given the importance of Ras signaling in promoting PDAC carcinogenesis, we determined whether the PKD1/RIN1 axis operates in pancreatic cancer cells. Methods and Results: Initially, we found that RIN1 protein is expressed by PDAC cells, including PANC-1, MIA PaCa-2, AsPC-1, BxPC-3, and Panc-28. To test whether PKD1 phosphorylates RIN1 in these cells, we determined whether stimulation of PANC-1 cells with G protein-coupled receptor (GPCR) agonists that stimulate PKD1 activation, including the GI peptide neurotensin (NT), induces RIN1 phosphorylation. We used an antibody that recognizes the phosphorylated state of the consensus motif targeted by PKD. PANC-1 cells were treated with or without the preferential PKD family inhibitor CRT0066101 and subsequently stimulated with NT for 5 min or 30 min. Lysates of these cells were immunoprecipitated with a RIN1 antibody. Analysis of the immunoprecipitates with the PKD motif phospho-antibody demonstrated striking increase in the phosphorylation of RIN1 in a CRT0066101-sensitive manner, implying that NT induced PKD1-mediated RIN1 phosphorylation. We also examined whether GPCR/PKD1 activation led to complex formation between RIN1 and 14-3-3 proteins. We found that stimulation of PANC-1 cells with NT for 5 min or 30 min induced co-immunoprecipitation of RIN1 with 14-3-3 proteins. According with our hypothesis, RIN1 acts as an inhibitor of Ras/Raf interaction and consequently should reduce the proliferation of PDAC cells. In agreement with this hypothesis, we found that forced expression of RIN1 in PANC-1 or Mia PaCa-2 reduced the proliferation of these cells. Conclusions: Collectively, our results support the hypothesis that PKD1-mediated RIN1 phosphorylation leads to complex formation with 14-3-3 proteins, uncouples RIN1 from activated K-Ras thereby allowing K-Ras/Raf interaction and stimulation of the proliferation of PDAC cells. These findings increase the rationale for targeting PKD1 for PDAC therapy.
*Significant or trending towards significant variables Table 2. Univariate analysis of clinicopathologic continuous variables of PanIN-1 and -2 vs. PanIN-3
S-871
AGA Abstracts
Background: Pancreatic cancer has an extremely poor prognosis. At the time of diagnosis, over 80% of patients have locally advanced or invasive disease. The carcinogenesis of pancreatic ductal adenocarcinoma (PDAC) involves stepwise progression from pancreatic intraepithelial neoplasia (PanIN) to invasive carcinoma. PanINs measure less than 5 mm and are not detectable on current imaging modalities. Precursor lesions, such as high-grade PanIN (PanIN-3) represent a target for early intervention. Low grade PanIN-1 and -2 are widely prevalent but their significance is unclear. Aims: To evaluate prevalence, significance and implications of PanIN-3 in PDAC. Methods: A retrospective review of the pathology database (1/2000 to 7/2013) at a tertiary care center. Demographic information, patient history, imaging studies, EUS findings and surgical pathology were reviewed. Results: A total of 607 pancreatic resections for cystic and solid lesions were reviewed. Neoadjuvant chemotherapy was given to 48 patients. In the remaining 559 patients, 198 (35.4%) had primary surgical resection for PDAC. Among these patients, 74.5% (of 185) showed the presence of any subtype of PanIN. High grade or PanIN-3 was present in 36.2% (of 185) resections for PDAC. Further, PanIN-3 was mostly found in PDAC (90.5%, p-value <0.001) compared to all other lesion types. Among patients with PDACs, who did not receive neoadjuvant therapy, univariate analysis (tables 1 and 2), demonstrated that Caucasian race and the presence of perineural invasion were significantly associated with PanIN-3. Pathological evidence of chronic pancreatitis and higher number of positive lymph nodes trended towards significant. Using these variables in a binomial logistic regression model demonstrated that Caucasian race (OR 10.9, p-value 0.02, 95% confidence interval 1.4, 86.6) and the presence of perineural invasion (OR 6.4, p-value 0.02, 95% confidence interval 1.4, 29.2) were significantly associated with PanIN-3. Interestingly, a comparison of PanIN3 among PDACs who underwent primary surgical resection versus post-neoadjuvant chemotherapy resection revealed that there was an absence of significant reduction in PanIN-3 among post-neoadjuvant resected PDACs (66 of 168 (39.3%) vs. 12 of 36 (33.3%), p = 0.57). Conclusion: High grade PanIN lesions are particularly prevalent in PDACs. In patients undergoing primary surgical resection for PDACs, high grade PanIN-3 lesions were notably associated with Caucasian race and perineural invasion. Supporting prior literature that PanIN-3 lesions may harbor cells with invasive features, the finding of PanIN-3 association with perineural invasion needs to be validated by larger studies. Table 1. Univariate analysis of clinicopathologic categorical variables of PanIN-1 and -2 vs. PanIN-3
AGA Abstracts
Determining Novel Clinicopathologic Correlations of High Grade Pancreatic Intraepithelial Neoplasia (PanIN) in Pancreatic Ductal Adenocarcinoma (PDAC) Jean R. Park, Feng Li, Michael Wellner, Jordan Thomas, Brett C. Sklaw, Brett C. Sklaw, Kevin M. Cronley, Veeral Oza, Jasleen Grewal, Jeffery R. Groce, Benjamin Swanson, Tanios Bekaii-Saab, Darwin Conwell, Somashekar G. Krishna
*Significant or trending towards significant variables Tu1913 CTNNB1 Mutational Analysis of Solid-Pseudopapillary Neoplasms of the Pancreas Using Endoscopic Ultrasound-Guided Fine-Needle Aspiration and Next-Generation Deep Sequencing Yoshimasa Kubota, Hiroshi Kawakami, Mitsuteru Natsuizaka, Kazumichi Kawakubo, Katsuji Marukawa, Taiki Kudo, Yoko Abe, Kimitoshi Kubo, Masaki Kuwatani, Yutaka Hatanaka, Tomoko Mitsuhashi, Yoshihiro Matsuno, Naoya Sakamoto Background: Solid-pseudopapillary neoplasm (SPN) of the pancreas is a rare neoplasm that is seen mostly in young female patients. Mutational analyses by surgically resected specimen and direct sequencing have revealed mutations in exon3 of the cadherin-associated protein beta 1 (CTNNB1) gene. Objectives: This study was intended to determine the feasibility of mutational analyses using samples obtained by endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) and next-generation sequencing (NGS). Methods: Thirty-four samples were tested: SPNs (n = 7) and other pancreatic lesions (n = 27; pancreatic ductal adenocarcinomas (PDAC), 16; pancreatic neuroendocrine tumors (PNET), 7; acinar cell carcinoma, 1; autoimmune pancreatitis, 1; and focal pancreatitis, 2). Samples were obtained by either EUS-FNA (n = 32) or surgery (n = 2) at Hokkaido University Hospital, Sapporo, Japan, between December 2008 and October 2013. EUS-FNA was performed using a curvilinear echoendoscope and 22-gauge needles. Sequencing was performed on Ion PGMTM (Personal Genome Machine) Sequencer by using an Ion 314TM chip (Life Technologies) with an IonPGMTM Sequencing 200 kit (Life Technologies). Obtained sequences were mapped onto the human reference genome hg19, and variants were detected using Ion Torrent Suite v2.2 software (Life Technologies). Results: A single-base pair missense mutations in exon3 of CTNNB1 was observed in all 7 SPNs. The mutations were found in codon 32 (n=3), codon 37 (n=2), and codon 41 (n=2). A N390 C/T (Thr41Ile) mutation was detected in 1 of 7 PNET samples. However, mutations were not observed in the tissue samples of any of the 15 PDAC or other 5 pancreatic disease cases. The variant frequency of CTNNB1 ranged from 5.39% to 48.77%, and the average base coverage depth of the samples with mutations ranged from 4,490 to 84,609. Conclusions: Mutational analysis of CTNNB1 by NGS is feasible and achieved using SPN samples obtained by EUS-FNA with high sensitivity and accuracy. Further exploration of CTNNB1 mutations in other neuroendocrine tumors will help identifying the genetic alterations that occur in PNET. Tu1914 Identification of a Novel Protein Kinase D1 (PKD1)/ RIN1 Axis in Pancreatic Cancer Cells Sushovan Guha, James Sinnett-Smith, Enrique Rozengurt Background: Pancreatic ductal adenocarcinoma (PDAC) is a devastating disease with 5-year survival rate of only 3-5%. As the current therapies offer limited survival benefits, novel therapeutic targets and strategies are needed to treat this aggressive disease. Protein kinase D1 (PKD1) is emerging as a critical node in the control of the ERK pathway leading to PDAC proliferation and thus, as a potential novel target in PDAC therapy. In order to elucidate the mechanism(s) by which PKD leads to ERK cascade activation, we examined the PKD/RIN1 pathway in PDAC cells. RIN1 (Ras and Rab interactor 1) is a multidomain protein that binds GTP-bound RAS with high affinity and inhibits the activating interaction between Ras and Raf. Consequently, RIN1 is a potent inhibitor of Ras-induced ERK activation. We demonstrated that PKD1 directly phosphorylates RIN1 at Ser351, a site that promotes binding of 14-3-3 proteins. Given the importance of Ras signaling in promoting PDAC carcinogenesis, we determined whether the PKD1/RIN1 axis operates in pancreatic cancer cells. Methods and Results: Initially, we found that RIN1 protein is expressed by PDAC cells, including PANC-1, MIA PaCa-2, AsPC-1, BxPC-3, and Panc-28. To test whether PKD1 phosphorylates RIN1 in these cells, we determined whether stimulation of PANC-1 cells with G protein-coupled receptor (GPCR) agonists that stimulate PKD1 activation, including the GI peptide neurotensin (NT), induces RIN1 phosphorylation. We used an antibody that recognizes the phosphorylated state of the consensus motif targeted by PKD. PANC-1 cells were treated with or without the preferential PKD family inhibitor CRT0066101 and subsequently stimulated with NT for 5 min or 30 min. Lysates of these cells were immunoprecipitated with a RIN1 antibody. Analysis of the immunoprecipitates with the PKD motif phospho-antibody demonstrated striking increase in the phosphorylation of RIN1 in a CRT0066101-sensitive manner, implying that NT induced PKD1-mediated RIN1 phosphorylation. We also examined whether GPCR/PKD1 activation led to complex formation between RIN1 and 14-3-3 proteins. We found that stimulation of PANC-1 cells with NT for 5 min or 30 min induced co-immunoprecipitation of RIN1 with 14-3-3 proteins. According with our hypothesis, RIN1 acts as an inhibitor of Ras/Raf interaction and consequently should reduce the proliferation of PDAC cells. In agreement with this hypothesis, we found that forced expression of RIN1 in PANC-1 or Mia PaCa-2 reduced the proliferation of these cells. Conclusions: Collectively, our results support the hypothesis that PKD1-mediated RIN1 phosphorylation leads to complex formation with 14-3-3 proteins, uncouples RIN1 from activated K-Ras thereby allowing K-Ras/Raf interaction and stimulation of the proliferation of PDAC cells. These findings increase the rationale for targeting PKD1 for PDAC therapy.
*Significant or trending towards significant variables Table 2. Univariate analysis of clinicopathologic continuous variables of PanIN-1 and -2 vs. PanIN-3
S-871
AGA Abstracts