Downregulation of cyclin D1 sensitizes cancer cells to MDM2 antagonist Nutlin-3

// Peipei Yang 1, * , Weicai Chen 1, * , Xuhui Li 2 , Grant Eilers 3 , Quan He 1 , Lili Liu 1 , Yeqing Wu 1 , Yuehong Wu 1 , Wei Yu 1 , Jonathan A. Fletcher 3 , Wen-Bin Ou 1, 2, 3 1 Zhejiang Provincial Key Laboratory of Silkworm Bioreactor and Biomedicine, College of Life Sciences, Zhejiang Sci-Tech University, Hangzhou, China 2 Zhejiang Provincial Key Laboratory of Applied Enzymology, Yangtze Delta Region Institute of Tsinghua University, Jiaxing, Zhejiang, China 3 Department of Pathology, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA, USA * These authors contributed equally to this work Correspondence to: Wen-Bin Ou, email: ouwenbin@tsinghua.org.cn Keywords: cyclin D1, MDM2, p53, upregulation, Nutlin-3 Received: January 10, 2016 Accepted: March 31, 2016 Published: April 26, 2016 ABSTRACT The MDM2-p53 pathway has a prominent oncogenic function in the pathogenesis of various cancers. Nutlin-3, a small-molecule antagonist of MDM2-p53 interaction, inhibits proliferation in cancer cells with wild-type p53. Herein, we evaluate the expression of MDM2, both the full length and a splicing variant MDM2-A, and the sensitivity of Nutlin-3 in different cancer cell lines. Included are seven cell lines with wild-type p53 (four mesothelioma, one breast cancer, one chondrosarcoma, and one leiomyosarcoma), two liposarcoma cell lines harboring MDM2 amplification and wild-type p53, and one mesothelioma cell line harboring a p53 point mutation. Nutlin-3 treatment increased expression of cyclin D1, MDM2, and p53 in cell lines with wild-type p53. Additive effects were observed in cells containing wild-type p53 through coordinated attack on MDM2-p53 binding and cyclin D1 by lentivirual shRNA knockdown or small molecule inhibition, as demonstrated by immunoblots and cell viability analyses. Further results demonstrate that MDM2 binds to cyclin D1, and that an increase in cyclin D1 expression after Nutlin-3 treatment is correlated with expression and ubiquitin E3-ligase activity of MDM2. MDM2 and p53 knockdown experiments demonstrated inhibition of cyclin D1 by MDM2 but not p53. These results indicate that combination inhibition of cyclin D1 and MDM2-p53 binding warrants clinical evaluation as a novel therapeutic strategy in cancer cells harboring wild-type p53.