Skip to main content

Advertisement

SpringerLink
Log in

Mutant GNAS limits tumor aggressiveness in established pancreatic cancer via antagonizing the KRAS-pathway

  • Original Article—Liver, Pancreas, and Biliary Tract
  • Published:
Journal of Gastroenterology Aims and scope Submit manuscript

Abstract

Background

Mutations in GNAS drive pancreatic tumorigenesis and frequently occur in intraductal papillary mucinous neoplasm (IPMN); however, their value as a therapeutic target is yet to be determined. This study aimed at evaluating the involvement of mutant GNAS in tumor aggressiveness in established pancreatic cancer.

Methods

CRISPR/Cas9-mediated GNAS R201H silencing was performed using human primary IPMN-associated pancreatic cancer cells. The role of oncogenic GNAS in tumor maintenance was evaluated by conducting cell culture and xenograft experiments, and western blotting and transcriptome analyses were performed to uncover GNAS-driven signatures.

Results

Xenografts of GNAS wild-type cells were characterized by a higher Ki-67 labeling index relative to GNAS-mutant cells. Phenotypic alterations in the GNAS wild-type tumors resulted in a significant reduction in mucin production accompanied by solid with massive stromal components. Transcriptional profiling suggested an apparent conflict of mutant GNAS with KRAS signaling. A significantly higher Notch intercellular domain (NICD) was observed in the nuclear fraction of GNAS wild-type cells. Meanwhile, inhibition of protein kinase A (PKA) induced NICD in GNAS-mutant IPMN cells, suggesting that NOTCH signaling is negatively regulated by the GNAS-PKA pathway. GNAS wild-type cells were characterized by a significant invasive property relative to GNAS-mutant cells, which was mediated through the NOTCH regulatory pathway.

Conclusions

Oncogenic GNAS induces mucin production, not only via MUC2 but also via MUC5AC/B, which may enlarge cystic lesions in the pancreas. The mutation may also limit tumor aggressiveness by attenuating NOTCH signaling; therefore, such tumor-suppressing effects must be considered when therapeutically inhibiting the GNAS pathway.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

References

  1. Bray F, Ferlay J, Soerjomataram I, et al. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. 2018;68:394–424.

    PubMed  Google Scholar 

  2. Siegel RL, Miller KD, Jemal A. Cancer statistics, 2020. CA Cancer J Clin. 2020;70:7–30.

    PubMed  Google Scholar 

  3. Basturk O, Hong SM, Wood LD, et al. A revised classification system and recommendations from the Baltimore consensus meeting for neoplastic precursor lesions in the pancreas. Am J Surg Pathol. 2015;39:1730–41.

    PubMed  PubMed Central  Google Scholar 

  4. Ren B, Liu X, Suriawinata AA. Pancreatic ductal adenocarcinoma and its precursor lesions: histopathology, cytopathology, and molecular pathology. Am J Pathol. 2019;189:9–21.

    PubMed  Google Scholar 

  5. Ryan DP, Hong TS, Bardeesy N. Pancreatic adenocarcinoma. N Engl J Med. 2014;371:1039–49.

    CAS  PubMed  Google Scholar 

  6. Patra KC, Bardeesy N, Mizukami Y. Diversity of precursor lesions for pancreatic cancer: the genetics and biology of intraductal papillary mucinous neoplasm. Clin Transl Gastroenterol. 2017;8:e86.

    PubMed  PubMed Central  Google Scholar 

  7. Watanabe K, Nakamura T, Onodera S, et al. A novel GNAS-mutated human induced pluripotent stem cell model for understanding GNAS-mutated tumors. Tumour Biol. 2020;42:1010428320962588.

    CAS  PubMed  Google Scholar 

  8. Drelon C, Berthon A, Sahut-Barnola I, et al. PKA inhibits WNT signalling in adrenal cortex zonation and prevents malignant tumour development. Nat Commun. 2016;7:12751.

    CAS  PubMed  PubMed Central  Google Scholar 

  9. Xing F, Luan Y, Cai J, et al. The anti-Warburg effect elicited by the cAMP-PGC1alpha pathway drives differentiation of glioblastoma cells into astrocytes. Cell Rep. 2017;18:468–81.

    CAS  PubMed  PubMed Central  Google Scholar 

  10. Furukawa T, Kuboki Y, Tanji E, et al. Whole-exome sequencing uncovers frequent GNAS mutations in intraductal papillary mucinous neoplasms of the pancreas. Sci Rep. 2011;1:161.

    PubMed  PubMed Central  Google Scholar 

  11. Wu J, Matthaei H, Maitra A, et al. Recurrent GNAS mutations define an unexpected pathway for pancreatic cyst development. Sci Transl Med. 2011;3:92ra66.

    CAS  PubMed  PubMed Central  Google Scholar 

  12. Omori Y, Ono Y, Kobayashi T, et al. How does intestinal-type intraductal papillary mucinous neoplasm emerge? CDX2 plays a critical role in the process of intestinal differentiation and progression. Virchows Arch. 2020;477:21–31.

    CAS  PubMed  Google Scholar 

  13. Yamada M, Sekine S, Ogawa R, et al. Frequent activating GNAS mutations in villous adenoma of the colorectum. J Pathol. 2012;228:113–8.

    CAS  PubMed  Google Scholar 

  14. Nault JC, Fabre M, Couchy G, et al. GNAS-activating mutations define a rare subgroup of inflammatory liver tumors characterized by STAT3 activation. J Hepatol. 2012;56:184–91.

    CAS  PubMed  Google Scholar 

  15. Ritterhouse LL, Vivero M, Mino-Kenudson M, et al. GNAS mutations in primary mucinous and non-mucinous lung adenocarcinomas. Mod Pathol. 2017;30:1720–7.

    CAS  PubMed  Google Scholar 

  16. Taki K, Ohmuraya M, Tanji E, et al. GNAS(R201H) and Kras(G12D) cooperate to promote murine pancreatic tumorigenesis recapitulating human intraductal papillary mucinous neoplasm. Oncogene. 2016;35:2407–12.

    CAS  PubMed  Google Scholar 

  17. Ideno N, Yamaguchi H, Ghosh B, et al. GNAS(R201C) induces pancreatic cystic neoplasms in mice that express activated kras by inhibiting YAP1 signaling. Gastroenterology. 2018;155:1593-1607 e12.

    CAS  PubMed  Google Scholar 

  18. Patra KC, Kato Y, Mizukami Y, et al. Mutant GNAS drives pancreatic tumourigenesis by inducing PKA-mediated SIK suppression and reprogramming lipid metabolism. Nat Cell Biol. 2018;20:811–22.

    CAS  PubMed  PubMed Central  Google Scholar 

  19. He X, Zhang L, Chen Y, et al. The G protein alpha subunit Galphas is a tumor suppressor in Sonic hedgehog-driven medulloblastoma. Nat Med. 2014;20:1035–42.

    CAS  PubMed  PubMed Central  Google Scholar 

  20. Iglesias-Bartolome R, Torres D, Marone R, et al. Inactivation of a Galpha(s)-PKA tumour suppressor pathway in skin stem cells initiates basal-cell carcinogenesis. Nat Cell Biol. 2015;17:793–803.

    CAS  PubMed  PubMed Central  Google Scholar 

  21. Zauber P, Marotta SP, Sabbath-Solitare M. GNAS gene mutation may be present only transiently during colorectal tumorigenesis. Int J Mol Epidemiol Genet. 2016;7:24–31.

    CAS  PubMed  PubMed Central  Google Scholar 

  22. Felsenstein M, Noe M, Masica DL, et al. IPMNs with co-occurring invasive cancers: neighbours but not always relatives. Gut. 2018;67:1652–62.

    CAS  PubMed  Google Scholar 

  23. Omori Y, Ono Y, Tanino M, et al. Pathways of progression from intraductal papillary mucinous neoplasm to pancreatic ductal adenocarcinoma based on molecular features. Gastroenterology. 2019;156:647-61 e2.

    CAS  PubMed  Google Scholar 

  24. Noe M, Niknafs N, Fischer CG, et al. Genomic characterization of malignant progression in neoplastic pancreatic cysts. Nat Commun. 2020;11:4085.

    CAS  PubMed  PubMed Central  Google Scholar 

  25. Kamiyama H, Kamiyama M, Hong SM, et al. In vivo and in vitro propagation of intraductal papillary mucinous neoplasms. Lab Invest. 2010;90:665–73.

    PubMed  PubMed Central  Google Scholar 

  26. Ran FA, Hsu PD, Wright J, et al. Genome engineering using the CRISPR-Cas9 system. Nat Protoc. 2013;8:2281–308.

    CAS  PubMed  PubMed Central  Google Scholar 

  27. Subramanian A, Tamayo P, Mootha VK, et al. Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc Natl Acad Sci U S A. 2005;102:15545–50.

    CAS  PubMed  PubMed Central  Google Scholar 

  28. Schneider CA, Rasband WS, Eliceiri KW. NIH Image to ImageJ: 25 years of image analysis. Nat Methods. 2012;9:671–5.

    CAS  PubMed  PubMed Central  Google Scholar 

  29. Liberzon A, Birger C, Thorvaldsdottir H, et al. The molecular signatures database (MSigDB) hallmark gene set collection. Cell Syst. 2015;1:417–25.

    CAS  PubMed  PubMed Central  Google Scholar 

  30. De La OJ, Emerson LL, Goodman JL, et al. Notch and Kras reprogram pancreatic acinar cells to ductal intraepithelial neoplasia. Proc Natl Acad Sci U S A. 2008;105:18907–12.

    Google Scholar 

  31. Thomas MM, Zhang Y, Mathew E, et al. Epithelial Notch signaling is a limiting step for pancreatic carcinogenesis. BMC Cancer. 2014;14:862.

    PubMed  PubMed Central  Google Scholar 

  32. Komatsu H, Tanji E, Sakata N, et al. A GNAS mutation found in pancreatic intraductal papillary mucinous neoplasms induces drastic alterations of gene expression profiles with upregulation of mucin genes. PLoS ONE. 2014;9:e87875.

    PubMed  PubMed Central  Google Scholar 

  33. Zhang H, Kong Q, Wang J, et al. Complex roles of cAMP-PKA-CREB signaling in cancer. Exp Hematol Oncol. 2020;9:32.

    CAS  PubMed  PubMed Central  Google Scholar 

  34. Pattabiraman DR, Bierie B, Kober KI, et al. Activation of PKA leads to mesenchymal-to-epithelial transition and loss of tumor-initiating ability. Science. 2016;351:aad3680.

    PubMed  PubMed Central  Google Scholar 

  35. Pan Y, Wang C, Wang B. Phosphorylation of Gli2 by protein kinase A is required for Gli2 processing and degradation and the Sonic hedgehog-regulated mouse development. Dev Biol. 2009;326:177–89.

    CAS  PubMed  Google Scholar 

  36. Djouder N, Tuerk RD, Suter M, et al. PKA phosphorylates and inactivates AMPKalpha to promote efficient lipolysis. EMBO J. 2010;29:469–81.

    CAS  PubMed  Google Scholar 

  37. McGill MA, McGlade CJ. Mammalian numb proteins promote Notch1 receptor ubiquitination and degradation of the Notch1 intracellular domain. J Biol Chem. 2003;278:23196–203.

    CAS  PubMed  Google Scholar 

  38. Lahiry M, Kumar S, Hari K, et al. AMPK-Fyn signaling promotes Notch1 stability to potentiate hypoxia-induced breast cancer stemness and drug resistance. BioRxiv. 2020. https://doi.org/10.2139/ssrn.3586992.

    Article  Google Scholar 

  39. Greer RL, Staley BK, Liou A, et al. Numb regulates acinar cell dedifferentiation and survival during pancreatic damage and acinar-to-ductal metaplasia. Gastroenterology. 2013;145:1088-97.e8.

    CAS  PubMed  Google Scholar 

  40. Inaguma S, Kasai K, Ikeda H. GLI1 facilitates the migration and invasion of pancreatic cancer cells through MUC5AC-mediated attenuation of E-cadherin. Oncogene. 2011;30:714–23.

    CAS  PubMed  Google Scholar 

  41. Valque H, Gouyer V, Gottrand F, et al. MUC5B leads to aggressive behavior of breast cancer MCF7 cells. PLoS ONE. 2012;7:e46699.

    CAS  PubMed  PubMed Central  Google Scholar 

  42. Leir SH, Harris A. MUC6 mucin expression inhibits tumor cell invasion. Exp Cell Res. 2011;317:2408–19.

    CAS  PubMed  Google Scholar 

  43. Yang KS, Ciprani D, O’Shea A, et al. Extracellular vesicle analysis allows for identification of invasive IPMN. Gastroenterology. 2021;160:1345–5811.

    CAS  PubMed  Google Scholar 

  44. Huang Y, Nahar S, Nakagawa A, et al. Regulation of GLI underlies a role for BET bromodomains in pancreatic cancer growth and the tumor microenvironment. Clin Cancer Res. 2016;22:4259–70.

    CAS  PubMed  PubMed Central  Google Scholar 

  45. Wilson CH, McIntyre RE, Arends MJ, et al. The activating mutation R201C in GNAS promotes intestinal tumourigenesis in Apc(Min/+) mice through activation of Wnt and ERK1/2 MAPK pathways. Oncogene. 2010;29:4567–75.

    CAS  PubMed  PubMed Central  Google Scholar 

  46. Nomura R, Saito T, Mitomi H, et al. GNAS mutation as an alternative mechanism of activation of the Wnt/beta-catenin signaling pathway in gastric adenocarcinoma of the fundic gland type. Hum Pathol. 2014;45:2488–96.

    CAS  PubMed  Google Scholar 

  47. O’Hayre M, Vazquez-Prado J, Kufareva I, et al. The emerging mutational landscape of G proteins and G-protein-coupled receptors in cancer. Nat Rev Cancer. 2013;13:412–24.

    CAS  PubMed  PubMed Central  Google Scholar 

  48. Coles GL, Cristea S, Webber JT, et al. Unbiased proteomic profiling uncovers a targetable GNAS/PKA/PP2A axis in small cell lung cancer stem cells. Cancer Cell. 2020;38:129-43.e7.

    CAS  PubMed  PubMed Central  Google Scholar 

  49. Zimmerman NP, Roy I, Hauser AD, et al. Cyclic AMP regulates the migration and invasion potential of human pancreatic cancer cells. Mol Carcinog. 2015;54:203–15.

    CAS  PubMed  Google Scholar 

  50. Wehbe N, Slika H, Mesmar J, et al. The role of Epac in cancer progression. Int J Mol Sci. 2020;21:6489.

    CAS  PubMed Central  Google Scholar 

  51. Almahariq M, Chao C, Mei FC, et al. Pharmacological inhibition and genetic knockdown of exchange protein directly activated by cAMP 1 reduce pancreatic cancer metastasis in vivo. Mol Pharmacol. 2015;87:142–9.

    PubMed  PubMed Central  Google Scholar 

  52. Avila JL, Kissil JL. Notch signaling in pancreatic cancer: oncogene or tumor suppressor? Trends Mol Med. 2013;19:320–7.

    CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We would like to thank Rika Kakisaka and Atsuko Nishikawa (Sapporo Higashi Tokushukai Hospital) for performing IHC quantification of the xenografts; Ayumu Sugitani (Sapporo Higashi Tokushukai Hospital) for supporting statistical analyses; and Eiko Aoyanagi (Sapporo Higashi Tokushukai Hospital) for tissue sample preparation. We also thank Krushna C. Patra (Department of Cancer Biology at University of Cincinnati) for critical reading of the manuscript.

Funding

This work was supported by Pancreas Research Foundation of Japan and Grants-in-Aid for Regional R&D Proposal-Based Program from Northern Advancement Center for Science & Technology of Hokkaido in Japan (to H. Kawabata) and by JSPS KAKENHI Grant Number 20K17009 (to H. Kawabata), 20K07671 (to Y.O.), 20K09070 (to H. Karasaki) and 20H03655 (to Y.M.).

Author information

Authors and Affiliations

  1. Department of Medicine, Asahikawa Medical University, 2-1 Midorigaoka Higashi, Asahikawa, Hokkaido, 078-8510, Japan

    Hidemasa Kawabata, Yusuke Ono, Nobue Tamamura, Hiroki Sato, Kenji Takahashi, Kenzui Taniue, Tetsuhiro Okada, Syugo Fujibayashi, Akihiro Hayashi, Takuma Goto, Katsuro Enomoto, Hiroaki Konishi, Mikihiro Fujiya, Yusuke Mizukami & Toshikatsu Okumura

  2. Department of Cardiovascular Surgery, Asahikawa Medical University, Asahikawa, Hokkaido, 078-8510, Japan

    Kyohei Oyama

  3. Department of Advanced Medical Science, Asahikawa Medical University, Asahikawa, Hokkaido, 078-8510, Japan

    Jun Ueda

  4. Department of Surgical Pathology, Asahikawa Medical University, Asahikawa, Hokkaido, 078-8510, Japan

    Keita Miyakawa & Mishie Tanino

  5. Division of Tumor Pathology, Department of Pathology, Asahikawa Medical University, Asahikawa, Hokkaido, 078-8510, Japan

    Yuji Nishikawa

  6. Department of Microscopic Anatomy and Cell Biology, Asahikawa Medical University, Asahikawa, Hokkaido, 078-8510, Japan

    Daisuke Koga & Tsuyoshi Watanabe

  7. Institute of Biomedical Research, Sapporo-Higashi Tokushukai Hospital, Sapporo, Hokkaido, 065-0033, Japan

    Yusuke Ono, Chiho Maeda, Hidenori Karasaki & Yusuke Mizukami

  8. Isotope Science Center, The University of Tokyo, Tokyo, 113-0032, Japan

    Kenzui Taniue

  9. Division of Gastrointestinal and Oncologic Surgery, Massachusetts General Hospital, Boston, MA, 02114, USA

    Hiroki Sato & Andrew S. Liss

Authors
  1. Hidemasa Kawabata
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Yusuke Ono
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Nobue Tamamura
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Kyohei Oyama
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Jun Ueda
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Hiroki Sato
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Kenji Takahashi
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Kenzui Taniue
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Tetsuhiro Okada
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Syugo Fujibayashi
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Akihiro Hayashi
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Takuma Goto
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Katsuro Enomoto
    View author publications

    You can also search for this author in PubMed Google Scholar

  14. Hiroaki Konishi
    View author publications

    You can also search for this author in PubMed Google Scholar

  15. Mikihiro Fujiya
    View author publications

    You can also search for this author in PubMed Google Scholar

  16. Keita Miyakawa
    View author publications

    You can also search for this author in PubMed Google Scholar

  17. Mishie Tanino
    View author publications

    You can also search for this author in PubMed Google Scholar

  18. Yuji Nishikawa
    View author publications

    You can also search for this author in PubMed Google Scholar

  19. Daisuke Koga
    View author publications

    You can also search for this author in PubMed Google Scholar

  20. Tsuyoshi Watanabe
    View author publications

    You can also search for this author in PubMed Google Scholar

  21. Chiho Maeda
    View author publications

    You can also search for this author in PubMed Google Scholar

  22. Hidenori Karasaki
    View author publications

    You can also search for this author in PubMed Google Scholar

  23. Andrew S. Liss
    View author publications

    You can also search for this author in PubMed Google Scholar

  24. Yusuke Mizukami
    View author publications

    You can also search for this author in PubMed Google Scholar

  25. Toshikatsu Okumura
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Yusuke Mizukami.

Ethics declarations

Conflict of interest

YO and YM receive funding from Hitachi High-Tech Corporation. The other authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Kawabata, H., Ono, Y., Tamamura, N. et al. Mutant GNAS limits tumor aggressiveness in established pancreatic cancer via antagonizing the KRAS-pathway. J Gastroenterol 57, 208–220 (2022). https://doi.org/10.1007/s00535-021-01846-4

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00535-021-01846-4

Keywords

Search

Navigation