GNASR201C Induces Pancreatic Cystic Neoplasms in Mice That Express Activated KRAS by Inhibiting YAP1 Signaling

Noboru Ideno, Hiroshi Yamaguchi, Bidyut Ghosh, Sonal Gupta, Takashi Okumura, Dana J. Steffen, Catherine G. Fisher, Laura D. Wood, Aatur D. Singhi, Masafumi Nakamura, J. Silvio Gutkind, Anirban Maitra

Research output: Contribution to journalArticle

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Abstract

Background & Aims: Mutations at hotspots in GNAS, which encodes stimulatory G-protein, α subunits, are detected in approximately 60% of intraductal papillary mucinous neoplasms (IPMNs) of the pancreas. We generated mice with KRAS-induced IPMNs that also express a constitutively active form of GNAS in pancreas and studied tumor development. Methods: We generated p48-Cre; LSL-KrasG12D; Rosa26R-LSL-rtTA-TetO-GnasR201C mice (Kras;Gnas mice); pancreatic tissues of these mice express activated KRAS and also express a mutant form of GNAS (GNASR201C) upon doxycycline administration. Mice that were not given doxycycline were used as controls, and survival times were compared by Kaplan-Meier analysis. Pancreata were collected at different time points after doxycycline administration and analyzed by histology. Pancreatic ductal adenocarcinomas (PDACs) were isolated from mice and used to generate cell lines, which were analyzed by reverse transcription polymerase chain reaction, immunoblotting, immunohistochemistry, and colony formation and invasion assays. Full-length and mutant forms of yes-associated protein (YAP) were expressed in PDAC cells. IPMN specimens were obtained from 13 patients with IPMN undergoing surgery and analyzed by immunohistochemistry. Results: All Kras;Gnas mice developed pancreatic cystic lesions that resemble human IPMNs; the grade of epithelial dysplasia increased with time. None of the control mice developed cystic lesions. Approximately one third of Kras;Gnas mice developed PDACs at a median of 30 weeks after doxycycline administration, whereas 33% of control mice developed PDACs. Expression of GNASR201C did not accelerate the development of PDACs compared with control mice. However, the neoplasms observed in Kras;Gnas mice were more differentiated, and expressed more genes associated with ductal phenotypes, than in control mice. PDACs isolated from Kras;Gnas mice had activation of the Hippo pathway; in cells from these tumors, phosphorylated YAP1 was sequestered in the cytoplasm, and this was also observed in human IPMNs with GNAS mutations. Sequestration of YAP1 was not observed in PDAC cells from control mice. Conclusions: In mice that express activated KRAS in the pancreas, we found expression of GNASR201C to cause development of more differentiated tumors, with gene expression pattern associated with the ductal phenotype. Expression of mutant GNAS caused phosphorylated YAP1 to be sequestered in the cytoplasm, altering tumor progression.

Original languageEnglish
Pages (from-to)1593-1607.e12
JournalGastroenterology
Volume155
Issue number5
DOIs
Publication statusPublished - Nov 1 2018

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Pancreatic Neoplasms
Adenocarcinoma
Doxycycline
Neoplasms
Pancreas
Cytoplasm
Immunohistochemistry
Phenotype
Mutation
Protein Subunits
Kaplan-Meier Estimate
GTP-Binding Proteins
Immunoblotting
Reverse Transcription
Histology

All Science Journal Classification (ASJC) codes

  • Hepatology
  • Gastroenterology

Cite this

Ideno, N., Yamaguchi, H., Ghosh, B., Gupta, S., Okumura, T., Steffen, D. J., ... Maitra, A. (2018). GNASR201C Induces Pancreatic Cystic Neoplasms in Mice That Express Activated KRAS by Inhibiting YAP1 Signaling. Gastroenterology, 155(5), 1593-1607.e12. https://doi.org/10.1053/j.gastro.2018.08.006

GNASR201C Induces Pancreatic Cystic Neoplasms in Mice That Express Activated KRAS by Inhibiting YAP1 Signaling. / Ideno, Noboru; Yamaguchi, Hiroshi; Ghosh, Bidyut; Gupta, Sonal; Okumura, Takashi; Steffen, Dana J.; Fisher, Catherine G.; Wood, Laura D.; Singhi, Aatur D.; Nakamura, Masafumi; Gutkind, J. Silvio; Maitra, Anirban.

In: Gastroenterology, Vol. 155, No. 5, 01.11.2018, p. 1593-1607.e12.

Research output: Contribution to journalArticle

Ideno, N, Yamaguchi, H, Ghosh, B, Gupta, S, Okumura, T, Steffen, DJ, Fisher, CG, Wood, LD, Singhi, AD, Nakamura, M, Gutkind, JS & Maitra, A 2018, 'GNASR201C Induces Pancreatic Cystic Neoplasms in Mice That Express Activated KRAS by Inhibiting YAP1 Signaling', Gastroenterology, vol. 155, no. 5, pp. 1593-1607.e12. https://doi.org/10.1053/j.gastro.2018.08.006
Ideno, Noboru ; Yamaguchi, Hiroshi ; Ghosh, Bidyut ; Gupta, Sonal ; Okumura, Takashi ; Steffen, Dana J. ; Fisher, Catherine G. ; Wood, Laura D. ; Singhi, Aatur D. ; Nakamura, Masafumi ; Gutkind, J. Silvio ; Maitra, Anirban. / GNASR201C Induces Pancreatic Cystic Neoplasms in Mice That Express Activated KRAS by Inhibiting YAP1 Signaling. In: Gastroenterology. 2018 ; Vol. 155, No. 5. pp. 1593-1607.e12.
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abstract = "Background & Aims: Mutations at hotspots in GNAS, which encodes stimulatory G-protein, α subunits, are detected in approximately 60{\%} of intraductal papillary mucinous neoplasms (IPMNs) of the pancreas. We generated mice with KRAS-induced IPMNs that also express a constitutively active form of GNAS in pancreas and studied tumor development. Methods: We generated p48-Cre; LSL-KrasG12D; Rosa26R-LSL-rtTA-TetO-GnasR201C mice (Kras;Gnas mice); pancreatic tissues of these mice express activated KRAS and also express a mutant form of GNAS (GNASR201C) upon doxycycline administration. Mice that were not given doxycycline were used as controls, and survival times were compared by Kaplan-Meier analysis. Pancreata were collected at different time points after doxycycline administration and analyzed by histology. Pancreatic ductal adenocarcinomas (PDACs) were isolated from mice and used to generate cell lines, which were analyzed by reverse transcription polymerase chain reaction, immunoblotting, immunohistochemistry, and colony formation and invasion assays. Full-length and mutant forms of yes-associated protein (YAP) were expressed in PDAC cells. IPMN specimens were obtained from 13 patients with IPMN undergoing surgery and analyzed by immunohistochemistry. Results: All Kras;Gnas mice developed pancreatic cystic lesions that resemble human IPMNs; the grade of epithelial dysplasia increased with time. None of the control mice developed cystic lesions. Approximately one third of Kras;Gnas mice developed PDACs at a median of 30 weeks after doxycycline administration, whereas 33{\%} of control mice developed PDACs. Expression of GNASR201C did not accelerate the development of PDACs compared with control mice. However, the neoplasms observed in Kras;Gnas mice were more differentiated, and expressed more genes associated with ductal phenotypes, than in control mice. PDACs isolated from Kras;Gnas mice had activation of the Hippo pathway; in cells from these tumors, phosphorylated YAP1 was sequestered in the cytoplasm, and this was also observed in human IPMNs with GNAS mutations. Sequestration of YAP1 was not observed in PDAC cells from control mice. Conclusions: In mice that express activated KRAS in the pancreas, we found expression of GNASR201C to cause development of more differentiated tumors, with gene expression pattern associated with the ductal phenotype. Expression of mutant GNAS caused phosphorylated YAP1 to be sequestered in the cytoplasm, altering tumor progression.",
author = "Noboru Ideno and Hiroshi Yamaguchi and Bidyut Ghosh and Sonal Gupta and Takashi Okumura and Steffen, {Dana J.} and Fisher, {Catherine G.} and Wood, {Laura D.} and Singhi, {Aatur D.} and Masafumi Nakamura and Gutkind, {J. Silvio} and Anirban Maitra",
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T1 - GNASR201C Induces Pancreatic Cystic Neoplasms in Mice That Express Activated KRAS by Inhibiting YAP1 Signaling

AU - Ideno, Noboru

AU - Yamaguchi, Hiroshi

AU - Ghosh, Bidyut

AU - Gupta, Sonal

AU - Okumura, Takashi

AU - Steffen, Dana J.

AU - Fisher, Catherine G.

AU - Wood, Laura D.

AU - Singhi, Aatur D.

AU - Nakamura, Masafumi

AU - Gutkind, J. Silvio

AU - Maitra, Anirban

PY - 2018/11/1

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N2 - Background & Aims: Mutations at hotspots in GNAS, which encodes stimulatory G-protein, α subunits, are detected in approximately 60% of intraductal papillary mucinous neoplasms (IPMNs) of the pancreas. We generated mice with KRAS-induced IPMNs that also express a constitutively active form of GNAS in pancreas and studied tumor development. Methods: We generated p48-Cre; LSL-KrasG12D; Rosa26R-LSL-rtTA-TetO-GnasR201C mice (Kras;Gnas mice); pancreatic tissues of these mice express activated KRAS and also express a mutant form of GNAS (GNASR201C) upon doxycycline administration. Mice that were not given doxycycline were used as controls, and survival times were compared by Kaplan-Meier analysis. Pancreata were collected at different time points after doxycycline administration and analyzed by histology. Pancreatic ductal adenocarcinomas (PDACs) were isolated from mice and used to generate cell lines, which were analyzed by reverse transcription polymerase chain reaction, immunoblotting, immunohistochemistry, and colony formation and invasion assays. Full-length and mutant forms of yes-associated protein (YAP) were expressed in PDAC cells. IPMN specimens were obtained from 13 patients with IPMN undergoing surgery and analyzed by immunohistochemistry. Results: All Kras;Gnas mice developed pancreatic cystic lesions that resemble human IPMNs; the grade of epithelial dysplasia increased with time. None of the control mice developed cystic lesions. Approximately one third of Kras;Gnas mice developed PDACs at a median of 30 weeks after doxycycline administration, whereas 33% of control mice developed PDACs. Expression of GNASR201C did not accelerate the development of PDACs compared with control mice. However, the neoplasms observed in Kras;Gnas mice were more differentiated, and expressed more genes associated with ductal phenotypes, than in control mice. PDACs isolated from Kras;Gnas mice had activation of the Hippo pathway; in cells from these tumors, phosphorylated YAP1 was sequestered in the cytoplasm, and this was also observed in human IPMNs with GNAS mutations. Sequestration of YAP1 was not observed in PDAC cells from control mice. Conclusions: In mice that express activated KRAS in the pancreas, we found expression of GNASR201C to cause development of more differentiated tumors, with gene expression pattern associated with the ductal phenotype. Expression of mutant GNAS caused phosphorylated YAP1 to be sequestered in the cytoplasm, altering tumor progression.

AB - Background & Aims: Mutations at hotspots in GNAS, which encodes stimulatory G-protein, α subunits, are detected in approximately 60% of intraductal papillary mucinous neoplasms (IPMNs) of the pancreas. We generated mice with KRAS-induced IPMNs that also express a constitutively active form of GNAS in pancreas and studied tumor development. Methods: We generated p48-Cre; LSL-KrasG12D; Rosa26R-LSL-rtTA-TetO-GnasR201C mice (Kras;Gnas mice); pancreatic tissues of these mice express activated KRAS and also express a mutant form of GNAS (GNASR201C) upon doxycycline administration. Mice that were not given doxycycline were used as controls, and survival times were compared by Kaplan-Meier analysis. Pancreata were collected at different time points after doxycycline administration and analyzed by histology. Pancreatic ductal adenocarcinomas (PDACs) were isolated from mice and used to generate cell lines, which were analyzed by reverse transcription polymerase chain reaction, immunoblotting, immunohistochemistry, and colony formation and invasion assays. Full-length and mutant forms of yes-associated protein (YAP) were expressed in PDAC cells. IPMN specimens were obtained from 13 patients with IPMN undergoing surgery and analyzed by immunohistochemistry. Results: All Kras;Gnas mice developed pancreatic cystic lesions that resemble human IPMNs; the grade of epithelial dysplasia increased with time. None of the control mice developed cystic lesions. Approximately one third of Kras;Gnas mice developed PDACs at a median of 30 weeks after doxycycline administration, whereas 33% of control mice developed PDACs. Expression of GNASR201C did not accelerate the development of PDACs compared with control mice. However, the neoplasms observed in Kras;Gnas mice were more differentiated, and expressed more genes associated with ductal phenotypes, than in control mice. PDACs isolated from Kras;Gnas mice had activation of the Hippo pathway; in cells from these tumors, phosphorylated YAP1 was sequestered in the cytoplasm, and this was also observed in human IPMNs with GNAS mutations. Sequestration of YAP1 was not observed in PDAC cells from control mice. Conclusions: In mice that express activated KRAS in the pancreas, we found expression of GNASR201C to cause development of more differentiated tumors, with gene expression pattern associated with the ductal phenotype. Expression of mutant GNAS caused phosphorylated YAP1 to be sequestered in the cytoplasm, altering tumor progression.

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