P16 methylation increases the sensitivity of cancer cells to the CDK4/6 inhibitor palbociclib

Autoři: Paiyun Li aff001;  Xuehong Zhang aff001;  Liankun Gu aff001;  Jing Zhou aff001;  Dajun Deng aff001
Působiště autorů: Key Laboratory of Carcinogenesis and Translational Research (MOE/Beijing), Division of Etiology, Peking University Cancer Hospital and Institute, Beijing, China aff001
Vyšlo v časopise: PLoS ONE 14(10)
Kategorie: Research Article
doi: https://doi.org/10.1371/journal.pone.0223084


The P16 (CDKN2Aink4a) gene is an endogenous CDK4/6 inhibitor. Palbociclib (PD0332991) is an anti-CDK4/6 chemical for cancer treatment. P16 is most frequently inactivated by copy number deletion and DNA methylation in cancers. It is well known that cancer cells with P16 deletion are more sensitive to palbociclib than those without. However, whether P16 methylation is related to palbociclib sensitivity is not known. By analyzing public pharmacogenomic datasets, we found that the IC50 of palbociclib in cancer cell lines (n = 522) was positively correlated with both the P16 expression level and P16 gene copy number. Our experimental results further showed that cancer cell lines with P16 methylation were more sensitive to palbociclib than those without. To determine whether P16 methylation directly increased the sensitivity of cancer cells to palbociclib, we induced P16 methylation in the lung cancer cell lines H661 and HCC827 and the gastric cancer cell line BGC823 via an engineered P16-specific DNA methyltransferase (P16-Dnmt) and found that the sensitivity of these cells to palbociclib was significantly increased. The survival rate of P16-Dnmt cells was significantly lower than that of vector control cells 48 hrs post treatment with palbociclib (10 μM). Notably, palbociclib treatment also selectively inhibited the proliferation of the P16-methylated subpopulation of P16-Dnmt cells, further indicating that P16 methylation can increase the sensitivity of cells to this CDK4/6 inhibitor. These results were confirmed in an animal experiment. In conclusion, inactivation of the P16 gene by DNA methylation can increase the sensitivity of cancer cells to palbociclib.

Klíčová slova:

Breast cancer – Cancer treatment – Cell cycle and cell division – DNA methylation – Lung and intrathoracic tumors – Methylation – Polymerase chain reaction – SW480 cells


1. Hanahan D, Weinberg RA. Hallmarks of Cancer: The Next Generation. Cell. 2011;144(5):646–74. doi: 10.1016/j.cell.2011.02.013 PubMed PMID: WOS:000288007100007. 21376230

2. Nurse P. Cyclin dependent kinases and cell cycle control (Nobel lecture). Chembiochem. 2002;3(7):596–+. doi: 10.1002/1439-7633(20020703)3:7<596::AID-CBIC596>3.0.CO;2-U PubMed PMID: WOS:000176625200001. 12324993

3. Lim SH, Kaldis P. Cdks, cyclins and CKIs: roles beyond cell cycle regulation. Development. 2013;140(15):3079–93. doi: 10.1242/dev.091744 PubMed PMID: WOS:000321864900002. 23861057

4. Malumbres M, Barbacid M. To cycle or not to cycle: A critical decision in cancer. Nature Reviews Cancer. 2001;1(3):222–31. doi: 10.1038/35106065 PubMed PMID: WOS:000180397200014. 11902577

5. Macaluso M, Montanari M, Giordano A. Rb family proteins as modulators of gene expression and new aspects regarding the interaction with chromatin remodeling enzymes. Oncogene. 2006;25(38):5263–7. doi: 10.1038/sj.onc.1209680 PubMed PMID: WOS:000240064100011. 16936746

6. Harbour JW, Luo RX, Santi AD, Postigo AA, Dean DC. Cdk phosphorylation triggers sequential intramolecular interactions that progressively block Rb functions as cells move through G1. Cell. 1999;98(6):859–69. doi: 10.1016/s0092-8674(00)81519-6 PubMed PMID: WOS:000082679200012. 10499802

7. Shapiro GI. Cyclin-dependent kinase pathways as targets for cancer treatment. J Clin Oncol. 2006;24(11):1770–83. doi: 10.1200/JCO.2005.03.7689 PubMed PMID: WOS:000236783900017. 16603719

8. Asghar U, Witkiewicz AK, Turner NC, Knudsen ES. The history and future of targeting cyclin-dependent kinases in cancer therapy. Nature Reviews Drug Discovery. 2015;14(2):130–46. doi: 10.1038/nrd4504 PubMed PMID: WOS:000348968100017. 25633797

9. Fry DW, Harvey PJ, Keller PR, Elliott WL, Meade MA, Trachet E, et al. Specific inhibition of cyclin-dependent kinase 4/6 by PD 0332991 and associated antitumor activity in human tumor xenografts. Molecular Cancer Therapeutics. 2004;3(11):1427–37. PubMed PMID: WOS:000225070800010. 15542782

10. DeMichele A, Clark AS, Tan KS, Heitjan DF, Gramlich K, Gallagher M, et al. CDK 4/6 Inhibitor Palbociclib (PD0332991) in Rb+ Advanced Breast Cancer: Phase II Activity, Safety, and Predictive Biomarker Assessment. Clinical Cancer Research. 2015;21(5):995–1001. doi: 10.1158/1078-0432.CCR-14-2258 PubMed PMID: WOS:000351982800011. 25501126

11. Hortobagyi GN, Stemmer SM, Burris HA, Yap YS, Sonke GS, Paluch-Shimon S, et al. Ribociclib as First-Line Therapy for HR-Positive, Advanced Breast Cancer. New Engl J Med. 2016;375(18):1738–48. doi: 10.1056/NEJMoa1609709 PubMed PMID: WOS:000387007300007. 27717303

12. Infante JR, Cassier PA, Gerecitano JF, Witteveen PO, Chugh R, Ribrag V, et al. A Phase I Study of the Cyclin-Dependent Kinase 4/6 Inhibitor Ribociclib (LEE011) in Patients with Advanced Solid Tumors and Lymphomas. Clinical Cancer Research. 2016;22(23):5696–705. doi: 10.1158/1078-0432.CCR-16-1248 PubMed PMID: WOS:000389438100011. 27542767

13. Patnaik A, Rosen LS, Tolaney SM, Tolcher AW, Goldman JW, Gandhi L, et al. Efficacy and Safety of Abemaciclib, an Inhibitor of CDK4 and CDK6, for Patients with Breast Cancer, Non-Small Cell Lung Cancer, and Other Solid Tumors. Cancer discovery. 2016;6(7):740–53. doi: 10.1158/2159-8290.CD-16-0095 PubMed PMID: WOS:000383354500025. 27217383

14. Ingham M, Schwartz GK. Cell-Cycle Therapeutics Come of Age. J Clin Oncol. 2017;35(25):2949–+. doi: 10.1200/JCO.2016.69.0032 PubMed PMID: WOS:000408568300015. 28580868

15. O'Leary B, Finn RS, Turner NC. Treating cancer with selective CDK4/6 inhibitors. Nature reviews Clinical oncology. 2016;13(7):417–30. Epub 2016/04/01. doi: 10.1038/nrclinonc.2016.26 27030077.

16. Sherr CJ, Beach D, Shapiro GI. Targeting CDK4 and CDK6: From Discovery to Therapy. Cancer discovery. 2016;6(4):353–67. doi: 10.1158/2159-8290.CD-15-0894 PubMed PMID: WOS:000375843800020. 26658964

17. Roskoski R. Cyclin-dependent protein serine/threonine kinase inhibitors as anticancer drugs. Pharmacological Research. 2019;139:471–88. doi: 10.1016/j.phrs.2018.11.035 PubMed PMID: WOS:000458709000047. 30508677

18. Beaver JA, Amiri-Kordestani L, Charlab R, Chen W, Palmby T, Tilley A, et al. FDA Approval: Palbociclib for the Treatment of Postmenopausal Patients with Estrogen Receptor-Positive, HER2-Negative Metastatic Breast Cancer. Clinical Cancer Research. 2015;21(21):4760–6. doi: 10.1158/1078-0432.CCR-15-1185 PubMed PMID: WOS:000364488100005. 26324739

19. Shah A, Bloomquist E, Tang SH, Fu WT, Bi YW, Liu Q, et al. FDA Approval: Ribociclib for the Treatment of Postmenopausal Women with Hormone Receptor-Positive, HER2-Negative Advanced or Metastatic Breast Cancer. Clinical Cancer Research. 2018;24(13):2999–3004. doi: 10.1158/1078-0432.CCR-17-2369 PubMed PMID: WOS:000437270800005. 29437768

20. Corona SP, Generali D. Abemaciclib: a CDK4/6 inhibitor for the treatment of HR+/HER2-advanced breast cancer. Drug Des Dev Ther. 2018;12:321–30. doi: 10.2147/Dddt.S137783 PubMed PMID: WOS:000425328800001. 29497278

21. Schettini F, De Santo I, Rea CG, De Placido P, Formisano L, Giuliano M, et al. CDK 4/6 Inhibitors as Single Agent in Advanced Solid Tumors. Front Oncol. 2018;8. doi: 10.3389/fonc.2018.00608 PubMed PMID: WOS:000453070700002. 30631751

22. Teh JLF, Aplin AE. Arrested Developments: CDK4/6 Inhibitor Resistance and Alterations in the Tumor Immune Microenvironment. Clinical Cancer Research. 2019;25(3):921–7. doi: 10.1158/1078-0432.CCR-18-1967 PubMed PMID: WOS:000457395900007. 30287548

23. Zhao R, Choi BY, Lee MH, Bode AM, Dong ZG. Implications of Genetic and Epigenetic Alterations of CDKN2A (p16(INK4a)) in Cancer. EBioMedicine. 2016;8:30–9. doi: 10.1016/j.ebiom.2016.04.017 PubMed PMID: WOS:000378622200017. 27428416

24. Kong Y, Sheng X, Wu X, Yan J, Ma M, Yu J, et al. Frequent Genetic Aberrations in the CDK4 Pathway in Acral Melanoma Indicate the Potential for CDK4/6 Inhibitors in Targeted Therapy. Clin Cancer Res. 2017. Epub 2017/08/24. doi: 10.1158/1078-0432.CCR-17-0070 28830923.

25. Konecny GE, Winterhoff B, Kolarova T, Qi JW, Manivong K, Dering J, et al. Expression of p16 and Retinoblastoma Determines Response to CDK4/6 Inhibition in Ovarian Cancer. Clinical Cancer Research. 2011;17(6):1591–602. doi: 10.1158/1078-0432.CCR-10-2307 PubMed PMID: WOS:000288435300039. 21278246

26. Cen L, Carlson BL, Schroeder MA, Ostrem JL, Kitange GJ, Mladek AC, et al. p16-Cdk4-Rb axis controls sensitivity to a cyclin-dependent kinase inhibitor PD0332991 in glioblastoma xenograft cells. Neuro Oncol. 2012;14(7):870–81. Epub 2012/06/20. doi: 10.1093/neuonc/nos114 22711607; PubMed Central PMCID: PMC3379801.

27. Young RJ, Waldeck K, Martin C, Foo JH, Cameron DP, Kirby L, et al. Loss of CDKN2A expression is a frequent event in primary invasive melanoma and correlates with sensitivity to the CDK4/6 inhibitor PD0332991 in melanoma cell lines. Pigment Cell Melanoma Res. 2014;27(4):590–600. Epub 2014/02/06. doi: 10.1111/pcmr.12228 24495407.

28. Wiedemeyer WR, Dunn IF, Quayle SN, Zhang JH, Chheda MG, Dunn GP, et al. Pattern of retinoblastoma pathway inactivation dictates response to CDK4/6 inhibition in GBM. P Natl Acad Sci USA. 2010;107(25):11501–6. doi: 10.1073/pnas.1001613107 PubMed PMID: WOS:000279058000063. 20534551

29. Cui C, Gan Y, Gu L, Wilson J, Liu Z, Zhang B, et al. P16-specific DNA methylation by engineered zinc finger methyltransferase inactivates gene transcription and promotes cancer metastasis. Genome biology. 2015;16:252. doi: 10.1186/s13059-015-0819-6 26592237; PubMed Central PMCID: PMC4656189.

30. Barretina J, Caponigro G, Stransky N, Venkatesan K, Margolin AA, Kim S, et al. The Cancer Cell Line Encyclopedia enables predictive modelling of anticancer drug sensitivity. Nature. 2012;483(7391):603–7. doi: 10.1038/nature11003 PubMed PMID: WOS:000302006100040. 22460905

31. Garnett MJ, Edelman EJ, Heidorn SJ, Greenman CD, Dastur A, Lau KW, et al. Systematic identification of genomic markers of drug sensitivity in cancer cells. Nature. 2012;483(7391):570–U87. doi: 10.1038/nature11005 PubMed PMID: WOS:000302006100033. 22460902

32. Herman JG, Graff JR, Myohanen S, Nelkin BD, Baylin SB. Methylation-specific PCR: A novel PCR assay for methylation status of CpG islands. P Natl Acad Sci USA. 1996;93(18):9821–6. doi: 10.1073/pnas.93.18.9821 PubMed PMID: WOS:A1996VF61400091. 8790415

33. Zhou J, Cao J, Lu ZM, Liu HW, Deng DJ. A 115-bp MethyLight assay for detection of p16 (CDKN2A) methylation as a diagnostic biomarker in human tissues. Bmc Med Genet. 2011;12. doi: 10.1186/1471-2350-12-67 PubMed PMID: WOS:000291947000001. 21569495

34. Zhang BZ, Xiang SY, Zhong QM, Yin YR, Gu LK, Deng DJ. The p16-Specific Reactivation and Inhibition of Cell Migration Through Demethylation of CpG Islands by Engineered Transcription Factors. Hum Gene Ther. 2012;23(10):1071–81. doi: 10.1089/hum.2012.070 PubMed PMID: WOS:000310364400483. 22738793

35. Gan Y, Ma WR, Wang XH, Qiao JL, Zhang BZ, Cui CH, et al. Coordinated transcription of ANRIL and P16 genes is silenced by P16 DNA methylation. Chinese J Cancer Res. 2018;30(1):93–+. doi: 10.21147/j.issn.1000-9604.2018.01.10 PubMed PMID: WOS:000426439800010. 29545723

36. Tian W, Du YT, Ma YW, Gu LK, Zhou J, Deng DJ. MALAT1-miR663a negative feedback loop in colon cancer cell functions through direct miRNA-lncRNA binding. Cell Death & Disease. 2018;9. doi: 10.1038/s41419-018-0925-y PubMed PMID: WOS:000443087500014. 30154407

37. Green JL, Okerberg ES, Sejd J, Palafox M, Monserrat L, Alemayehu S, et al. Direct CDKN2 Modulation of CDK4 Alters Target Engagement of CDK4 Inhibitor Drugs. Mol Cancer Ther. 2019;18(4):771–9. Epub 2019/03/07. doi: 10.1158/1535-7163.MCT-18-0755 30837298.

38. Finn RS, Crown JP, Lang I, Boer K, Bondarenko IM, Kulyk SO, et al. The cyclin-dependent kinase 4/6 inhibitor palbociclib in combination with letrozole versus letrozole alone as first-line treatment of oestrogen receptor-positive, HER2-negative, advanced breast cancer (PALOMA-1/TRIO-18): a randomised phase 2 study. Lancet Oncol. 2015;16(1):25–35. doi: 10.1016/S1470-2045(14)71159-3 PubMed PMID: WOS:000346912800042. 25524798

39. Finn R, Jiang Y, Rugo H, Moulder SL, Im SA, Gelmon KA, et al. Biomarker analyses from the phase 3 PALOMA-2 trial of palbociclib (P) with letrozole (L) compared with placebo (PLB) plus L in postmenopausal women with ER +/HER2-advanced breast cancer (ABC). Annals of Oncology. 2016;27. doi: 10.1093/annonc/mdw435.5 PubMed PMID: WOS:000393913000694.

40. Finn RS, Liu Y, Martin M, Rugo HS, Dieras V, Im SA, et al. Comprehensive gene expression biomarker analysis of CDK 4/6 and endocrine pathways from the PALOMA-2 study. Cancer Research. 2018;78(4). PubMed PMID: WOS:000425489400257.

41. Liu Z, Lin H, Gan Y, Cui C, Zhang B, Gu L, et al. P16 Methylation Leads to Paclitaxel Resistance of Advanced Non-Small Cell Lung Cancer. J Cancer. 2019;10(7):1726–33. Epub 2019/06/18. doi: 10.7150/jca.26482 31205528; PubMed Central PMCID: PMC6547999.

Článek vyšel v časopise


2019 Číslo 10
Nejčtenější tento týden