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

Abstract

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. 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. Germline mutations in CDKN2A affecting the encoded p16INK4A protein (hereafter referred to as p16) are a major risk factor for familial melanoma, with a high proportion (40–60%) of families in which multiple individuals develop melanoma-carrying germline mutations in CDKN2A (1Goldstein A.M. Chan M. Harland M. Hayward N.K. Demenais F. Timothy Bishop D. Azizi E. Bergman W. Bianchi-Scarra G. Bruno W. Calista D. Cannon Albright L.A. Chaudru V. Chompret A. Cuellar F. et al.Features associated with germline CDKN2A mutations: A GenoMEL study of melanoma-prone families from three continents.J. Med. Genet. 2007; 44: 99-106Crossref PubMed Scopus (279) Google Scholar). In addition, germline CDKN2A mutations are associated with higher risk of pancreatic adenocarcinoma (PDAC), with some evidence for increased cancers at other sites (2McWilliams R.R. Wieben E.D. Rabe K.G. Pedersen K.S. Wu Y. Sicotte H. Petersen G.M. 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Sander C.A. p16INK4a expression is frequently decreased and associated with 9p21 loss of heterozygosity in sporadic melanoma.J. Cutan. Pathol. 1998; 25: 291-296Crossref PubMed Scopus (76) Google Scholar). Thus, p16 inactivation plays an important role in the development of both pancreatic cancer and melanoma. p16 is a known tumor suppressor that functions by binding to and inhibiting cyclin-dependent and the of inhibiting cell CDK4 for cancer Cancer Res. PubMed Scopus Google Scholar). is that mutations in p16 to decreased cell of this protein. A of p16 mutations demonstrated to to melanoma to reduced in assays (1Goldstein A.M. Chan M. Harland M. Hayward N.K. Demenais F. Timothy Bishop D. Azizi E. Bergman W. Bianchi-Scarra G. Bruno W. Calista D. Cannon Albright L.A. Chaudru V. Chompret A. Cuellar F. et al.Features associated with germline CDKN2A mutations: A GenoMEL study of melanoma-prone families from three continents.J. Med. Genet. 2007; 44: 99-106Crossref PubMed Scopus (279) Google Scholar, A.M. Familial melanoma, pancreatic cancer and germline CDKN2A PubMed Scopus Google Scholar, C. S. D. J. M. Avril M.F. Chompret A. G.M. A. G. Bressac-de Paillerets B. and genetic of CDKN2A mutations identified in melanoma-prone families or PubMed Scopus Google Scholar, C. M. H. significance of mutations in 2010; PubMed Scopus Google Scholar). p16 of significance identified in multiple melanoma identified four familial cancer a germline missense CDKN2A (47T>G), that the variant protein CDKN2A tumor suppressor proteins by p16, and the the of p16. The of p16 is and A.M. Chan M. Harland M. Gillanders E.M. Hayward N.K. Avril M.F. Azizi E. Bianchi-Scarra G. Bishop D.T. Bressac-de Paillerets B. Bruno W. Calista D. Cannon Albright L.A. Demenais F. Elder D.E. et al.High-risk melanoma susceptibility genes and pancreatic cancer, neural system tumors, and uveal melanoma across GenoMEL.Cancer Res. 2006; 66: 9818-9828Crossref PubMed Scopus (290) Google Scholar). in silico that the p16-L16R variant K.G. R.R. J. Petersen G.M. Prevalence of mutations in cancer genes pancreatic cancer patients with a Med. 2018; PubMed Scopus (54) Google its We investigated the significance of this variant a of and cell biological to the by which expression of p16-L16R to increased expression in p16-null pancreatic cancer demonstrated that p16-L16R lower protein levels than wild-type (WT) p16 and that p16-L16R almost cell function. that compared normal fibroblasts from p16 WT with L16R individuals from these cancer that the p16-L16R fibroblasts increased compared with WT p16-L16R from with multiple cancers. These suggest that of the variant to a in cell these individuals to higher risk of cancer. of four through pancreatic cancer probands were by genetic at an of individuals were individuals pancreatic cancer, with melanoma, and three with both cancers. in these the germline CDKN2A by a genetic K.G. R.R. J. Petersen G.M. Prevalence of mutations in cancer genes pancreatic cancer patients with a Med. 2018; PubMed Scopus (54) Google Scholar, C. R. H. J. J. R. K.G. J. Wu Z. R. et germline mutations in cancer genes and risk of pancreatic 2018; PubMed Scopus Google Scholar). germline was on of and were members of three in which a with pancreatic cancer the CDKN2A (47T>G) were individuals were to the mutation these were cancers cancer, cancer, cancer, cancer, and pancreatic multiple cancers. The cancer In members were mutation cancers were pancreatic cancer pancreatic cancer and melanoma, and pancreatic cancer and cancer. melanoma and three other cancers these with cancer, four additional cancers. The of the mutation in these is consistent with of the cancer These a higher of cancer in than these a with the p16-L16R mutation was in which with melanoma A.M. risk of cancer in CDKN2A germline mutation Med. Genet. PubMed Google Scholar). the of the L16R mutation on the of p16, we molecular of WT and L16R mutant the of p16 as a Li J. K. A. P. suppressor of and of its with cyclin-dependent 1998; PubMed Scopus Google Scholar). the WT system in the of the was a in early in the for the p16-L16R mutant A and is as is in of by a to the protein we observed of p16 that of in most the of p16 with its binding We that contributes to in the of p16 in with CDK4 L. J.O. for of the cyclin-dependent by the suppressor 1998; PubMed Scopus Google Scholar). is important for the of the p16 of the L16R mutation is the of p16. the of the L16R p16-null and pancreatic cancer B. S. C. T. C. G. H. H. M. A. of pancreatic carcinoma cell Analysis of p16 and PubMed Scopus Google and an human pancreatic cell that and an p16 Z. H. J. Z. Li Z. H. D. P. of and in human pancreatic PubMed Scopus Google to were transfected with p16-WT or the of the p16 on the cell were by flow with p16-WT decreased cell in all three cell as shown by a in the of in the of and compared with with the A and In with p16-L16R a in the proportion of in the of and compared with and the in was than that observed with p16-WT A and The for p16 in was shown to for WT and for L16R p16 as by that in WT L16R p16 by blotting an of with p16-WT or p16-L16R demonstrated lower levels of p16-L16R compared with p16-WT on all three cell and These that the p16-L16R variant reduced protein levels and is in compared with the p16-WT the expression was WT and mutant p16 in at and that decreased p16-L16R protein levels by reduced p16-L16R of WT and L16R p16 in and a p16 also lower levels of L16R than WT p16 that reduced expression of L16R is by the presence of the an impact on protein and protein for Genet. PubMed Scopus Google Scholar, G. J. and in and 2018; 19: PubMed Scopus Google we the that the of of p16 from in p16-WT to in the L16R variant the of of the p16 protein. Thus, L16R were with and at this expressed by and for protein of these in decreased protein levels compared with p16-WT p16-L16R with and levels that were lower than that of WT p16 p16 lower levels than p16-WT and p16, that a the lower Together, these suggest that the decreased protein observed for p16-L16R is by a The of the L16R mutant protein in cell was by p16-L16R of p16-WT and flow cytometry The L16R mutant cell than the WT transfected at of or compared with p16-L16R and was in cell to the WT expressed of the compared with p16-L16R western blotting of from the that p16-L16R protein expression was higher than that of the p16-WT These results that the L16R mutant is than the WT p16 protein levels are The L16R mutant was by its ability to endogenous CDK4 and were transfected with p16-L16R or with a reduced of p16-WT to a of protein. cell were an and by western blotting for and The L16R mutant almost with the p16-WT levels of CDK4 and CDK6 that the p16-L16R a reduced for These results are consistent with the diminished ability of p16-L16R to inhibit cell and into the the increased of WT L16R p16, p16-WT protein expression was the of CDK4 or CDK6 to is affected by loss of binding to were for with or a were transfected with p16-WT for and by western CDK4 and CDK6 protein levels were decreased by was in expression with of CDK4 or CDK6 Thus, the binding of CDK4 or CDK6 to p16 impact on p16 or for and that p16-L16R in to its to bind CDK4 or We investigated a role in the levels of WT L16R p16. In cells, we that and p16-L16R protein levels were increased by with the p16-WT protein levels were increased to levels with expressed in and melanoma cells, a cell for p16-L16R and These results suggest that expressed p16-L16R protein is by the that the WT protein and is by In we identified a large of CDKN2A of significance in to p16-L16R that were associated with an increased risk of PDAC and melanoma (2McWilliams R.R. Wieben E.D. Rabe K.G. Pedersen K.S. Wu Y. Sicotte H. Petersen G.M. Prevalence of CDKN2A mutations in pancreatic cancer patients: Implications for genetic counseling.Eur. J. Hum. Genet. 2011; 19: 472-478Crossref PubMed Scopus (82) Google Scholar, R.R. Wieben E.D. K.G. L. Li D. G. J.E. H. J. W. et germline and risk of pancreatic cancer in 2018; PubMed Scopus Google Scholar). We compared a of these to WT and L16R p16 for protein expression and cell the of these lower protein expression compared with p16-WT the L16R mutant and variant a reduced cell compared with the WT protein Thus, we were unable to loss of function for most of these which with cancer is that in function of these were assays on p16 or that these other functions of p16 of which we A. p16 protein expression through of Cancer Res. 2018; PubMed Scopus Google Scholar, R. the Oncol. 2019; PubMed Scopus (4) Google Scholar, J. Liu T. M. D. Familial mutations in p16 its PubMed Scopus Google Scholar). The that p16-L16R reduced protein expression and diminished ability to cell compared with p16-WT expressed in p16-null We to loss of p16-L16R function detected this protein is expressed as in human Thus, normal human fibroblasts were isolated from from p16-L16R members with L16R and WT p16 and as a for that at risk of as a result of p16-L16R that all from identified L16R individuals were for CDKN2A and from identified as were the impact of p16-L16R expression on fibroblasts from were at for a and as was until from passages to were higher for p16-L16R than p16-WT cells, increase in cell A and of fibroblasts from to are shown in L16R fibroblasts from patients with or cancers and the When the four from individuals or cancers higher than WT of p16-L16R from with cancer were higher than WT at some fibroblasts to a higher for p16-L16R p16-WT fibroblasts at almost all passages We also cell of WT and L16R by WT a of in and than L16R fibroblasts When L16R were higher in and than WT of faster cell in p16 L16R WT as by cell A and p16-WT to p16-L16R The was from p16-L16R fibroblasts and for p16 WT Together, these results that on fibroblasts and to to later passages than p16-WT are the suggest that p16-L16R expression a on of fibroblasts were with age with multiple cancers and the were consistent with a and age in B. M. C. P. C. A. of fibroblasts from patients with J. Cancer. PubMed Scopus Google Scholar, Y. The in and in human Natl. S. A. PubMed Scopus Google Scholar). the three from patients with multiple cancers were in this age and was observed We also investigated the impact of the p16-L16R allele on p16 protein expression in endogenous p16 protein was by western blotting in WT or L16R fibroblasts p16 was detected in some of both In for in normal p16 protein levels were decreased in L16R were with is to increase p16 protein expression in fibroblasts M.J. A. A. S. E. C. G. fibroblasts in and an 2019; PubMed Scopus Google Scholar, C. of PubMed Scopus Google Scholar). Thus, fibroblasts were for with to this p16-L16R lower p16 protein expression than p16-WT fibroblasts and Thus, the of p16-L16R in to an in endogenous p16 protein consistent with p16 WT and L16R in multiple cell we observed that the p16-L16R from and we expressing WT or L16R p16 with and the by western impact on endogenous protein levels of WT or L16R p16 in and also impact on p16 protein levels of p16-WT and or p16-L16R Thus, the decreased levels of endogenous p16 detected in p16-L16R to to We identified the variant in four of patients that members with familial pancreatic cancer and melanoma. Here, we demonstrated that expressed in p16-null pancreatic cancer cells, p16-L16R ability to the cell and decreased binding to CDK4 and CDK6 compared with These evidence that p16-L16R is a consistent with the of this variant with increased of melanoma and pancreatic cancer. In addition, we that p16-L16R lower protein levels compared with the WT as demonstrated by expression in with p16-L16R also higher and lower levels of p16 consistent with the in pancreatic cancer Thus, through its to the cell and decreased protein the presence of p16-L16R at higher risk of melanoma and pancreatic cancer. We that p16-L16R protein was detected at a lower by western blotting compared with expressed in pancreatic cancer and a melanoma cell levels of WT and L16R p16 were were transfected at that decreased protein expression of p16-L16R is at the In addition, p16-L16R fibroblasts also expressed lower levels of p16 than p16-WT were in silico molecular modeling that p16-L16R is than p16-L16R protein was from by the in cell on expressed p16 WT or Thus, the lower levels of endogenous p16-L16R in to We also demonstrated that p16-WT is from by binding to the that p16-L16R is is to and we that other p16 of significance identified as associated with pancreatic cancer (2McWilliams R.R. Wieben E.D. Rabe K.G. Pedersen K.S. Wu Y. Sicotte H. Petersen G.M. Prevalence of CDKN2A mutations in pancreatic cancer patients: Implications for genetic counseling.Eur. J. Hum. Genet. 2011; 19: 472-478Crossref PubMed Scopus (82) Google Scholar, R.R. Wieben E.D. K.G. L. Li D. G. J.E. H. J. W. et germline and risk of pancreatic cancer in 2018; PubMed Scopus Google also lower protein compared with p16-WT The that the decreased protein expression of p16-L16R and other and the of lower expression for of these are the to investigate the impact of endogenous germline p16 on cell fibroblasts from normal of individuals in familial cancer of fibroblasts from cancer patients of germline mutation and of mutant shown that from cancer patients compared with normal fibroblasts M.J. is in fibroblasts from families J. Cancer. 79: PubMed Scopus Google Scholar, B. C. P. M. H. A. fibroblasts from cancer patients: and in and role of in Res. PubMed Scopus Google Scholar). study investigated alterations in p16 mutant fibroblasts compared with WT M. Lynch H.T. P. S. L. of normal fibroblasts for CDKN2A in familial to early PubMed Scopus Google Scholar). of fibroblasts from p16-L16R and p16-WT members of p16-L16R a to the impact of p16-L16R as expressed on cell and p16 protein assays demonstrated that p16-L16R on grew faster than WT western blotting that p16-L16R fibroblasts on expressed lower p16 protein than WT These suggest that decreased function and lower protein expression of p16-L16R to a than in WT We that the p16-L16R with the were from individuals a history of multiple cancers. of we the that from individuals with cancers were affected by to cancer or for the p16-L16R patients cancer a in higher than WT cells, the that p16-L16R in cancer. are to p16-L16R on and to cancers. is to individuals p16-L16R or other germline CDKN2A mutations develop melanoma, pancreatic cancer, or other and of these mutations is than (2McWilliams R.R. Wieben E.D. Rabe K.G. Pedersen K.S. Wu Y. Sicotte H. Petersen G.M. Prevalence of CDKN2A mutations in pancreatic cancer patients: Implications for genetic counseling.Eur. J. Hum. Genet. 2011; 19: 472-478Crossref PubMed Scopus (82) Google Scholar, D.T. Demenais F. A.M. Bergman W. Bishop Bressac-de Paillerets B. Chompret A. P. J. Harland M. Hayward M. et variation in the of CDKN2A mutations for melanoma.J. Natl. Cancer Inst. 2002; 94: PubMed Scopus Google Scholar, S. S. Brand R. pancreatic cancer J. PubMed Scopus Google Scholar, E. K. H. on and of familial atypical multiple mole melanoma 2016; PubMed Scopus (82) Google Scholar). in in and genetic as other mutations or gene as to or fibroblasts from p16 WT and mutant members of p16 mutant familial families a for or that are p16 WT mutant to genetic variation In we expression of p16 WT and L16R in pancreatic cancer cells, and normal fibroblasts from individuals in families that p16-L16R to molecular evidence that p16-L16R is a mutation with ability to with or cell mutation is to as a the loss of p16 function that frequently in pancreatic cancer and melanoma. is also that expression of p16-L16R to to faster or other in p16 tumor suppressor function. of the study human were and by the was from all a which was for the of the p16-L16R variant by and by genetic K.G. R.R. J. Petersen G.M. Prevalence of mutations in cancer genes pancreatic cancer patients with a Med. 2018; PubMed Scopus (54) Google Scholar, C. R. H. J. J. R. K.G. J. Wu Z. R. et germline mutations in cancer genes and risk of pancreatic 2018; PubMed Scopus Google Scholar). a and risk factor The were and to history of cancer and age of were from from the with WT or L16R p16 and with or a history of cancer. were on a of the and a in the a to by with to of were in a with with were and with and at for to until and were by the were into to and of were to a cell and at in a for in were in and in was until all were fibroblasts is these to of were for or later were for cell and molecular the p16 as WT L16R for genomic was isolated from the was the the were on and a were the were to and was the and pancreatic adenocarcinoma and melanoma were from human pancreatic transfected with mutant and p16 Z. H. J. Z. Li Z. H. D. P. of and in human pancreatic PubMed Scopus Google were a from Cancer cell were from were in at were in with in and in in normal in were transfected a of of CDK4 and CDK6 was to with and and WT p16 was from and into the and and and into the and L16R was the from The were and p16 were were in with and all are from to were three with were were in transfected cells, were three with and in were by three through a at and the of were the protein were in with and on or to or were to in with The were in in with for were at for with in with The were and from and were for with in with on were detected with and were on and to by or a were transfected for with WT or L16R p16 were in with to a of were by for with of at or was to of in for at by in of and were and at with were three in on were by of and at for These were to and western as and CDK4 and CDK6 on in were transfected for with WT or L16R p16 in the were in for and in at for were at were for in in for in in three in and for with at in these were three in and were on in with was a system an with a and an were the were in and the for were and were and A of were were expressed as of and were transfected as with or or at a of were and at in and cells, were with and at for were by in of with for on The were with and at for by in of with and for at in the and were with and at for were by of and of The were at The were with and at for by in of with and was and or were Analysis of cell was the cell to the of in the and from the cell fibroblasts from expressing WT or p16-L16R were a and in with were in at and for were and as at until increase in cell was by the of of the was from The was to of A of the was by were with and the were to and the genes was the with p16 levels were the the levels for and in the the we the of human p16 Li J. K. A. P. suppressor of and of its with cyclin-dependent 1998; PubMed Scopus Google as The and of the protein were from the was to the p16 L16R mutant P. K. for molecular PubMed Scopus Google Scholar). The were and with D. R. A molecular Scopus Google the D. M. M.J. S. J. H. S. D. L. K. C. et for molecular modeling and of B. 1998; PubMed Scopus Google Scholar). The was with in with a of were with and with to were in the WT system and for the of The were to with a of The and of system were by of and at with a was by for to a the A. with to an Scopus Google Scholar). The were on protein The with a of The for was were the T. D. Pedersen L. for in large Scopus Google with a of The B. H. A for molecular Scopus Google was to the and a was for all the were and were with multiple were expressed as the are this The that with the of this We to and for I. P. D. L. A. T. L. L. L. L. E. P. A. R. R. A. M. G. and C. Z. and K. G. W. R. H. I. P. D. L. and M. E. I. P. D. L. G. H. G. M. and M. E. and D. L. M. and M. E. H. G. M. and M. E. Cancer The of this is the of the and the of the of

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