A rare germline CDKN2A variant (47T>G; p16-L16R) predisposes carriers to pancreatic cancer by reducing cell cycle inhibition

Isaac P. Horn, David L. Marks, Amanda N. Koenig, Tara L. Hogenson, Luciana L. Almada, Lauren E. Goldstein, Paola A. Romecin Duran, Renzo Vera, Anne M. Vrabel, Gaofeng Cui, Kari G. Rabe, William R. Bamlet, Georges Mer, Hugues Sicotte, Cheng Zhang, Hu Li, Gloria M. Petersen, Martin E. Fernandez-Zapico

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Germline mutations in CDKN2A, encoding the tumor suppressor p16, are responsible for a large proportion of familial melanoma cases and also increase risk of pancreatic cancer. We identified four families through pancreatic cancer probands that were affected by both cancers. These families bore a germline missense variant of CDKN2A (47T>G), encoding a p16-L16R mutant protein associated with high cancer occurrence. Here, we investigated the biological significance of this variant. When transfected into p16-null pancreatic cancer cells, p16-L16R was expressed at lower levels than wild-type (WT) p16. In addition, p16-L16R was unable to bind CDK4 or CDK6 compared with WT p16, as shown by coimmunoprecipitation assays and also was impaired in its ability to inhibit the cell cycle, as demonstrated by flow cytometry analyses. In silico molecular modeling predicted that the L16R mutation prevents normal protein folding, consistent with the observed reduction in expression/stability and diminished function of this mutant protein. We isolated normal dermal fibroblasts from members of the families expressing WT or L16R proteins to investigate the impact of endogenous p16-L16R mutant protein on cell growth. In culture, p16-L16R fibroblasts grew at a faster rate, and most survived until later passages than p16-WT fibroblasts. Further, western blotting demonstrated that p16 protein was detected at lower levels in p16-L16R than in p16-WT fibroblasts. Together, these results suggest that the presence of a CDKN2A (47T>G) mutant allele contributes to an increased risk of pancreatic cancer as a result of reduced p16 protein levels and diminished p16 tumor suppressor function.

Original languageEnglish (US)
Article number100634
JournalJournal of Biological Chemistry
StatePublished - Apr 3 2021

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology


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