P53-Mediated Growth Suppression in Response to Nutlin-3 in Cyclin D1–Transformed Cells Occurs Independently of p21

Charlene E Kan,George R Stark,John T Patton,Mark W Jackson

doi:10.1158/0008-5472.can-07-0259

Abstract

Interaction of cyclin D1 with cyclin-dependent kinases (CDK) results in the hyperphosphorylation of the RB family of proteins, thereby inactivating the tumor-suppressive function of RB. Our previous findings suggest that constitutive cyclin D1/CDK activity inhibits p53-mediated gene repression by preventing the appropriate regulation of CDK activity by the CDK inhibitor p21, a transcriptional target of p53. To study the role of cyclin D1 in driving human mammary cell transformation, we expressed a constitutively active cyclin D1-CDK fusion protein (D1/CDK) in immortalized human mammary epithelial cells. D1/CDK-expressing human mammary epithelial cells grew anchorage-independently in the presence of wild-type p53, consistent with the idea that D1/CDK disrupts downstream p53 signaling. Using this transformation model, we examined the sensitivity of the D1/CDK-expressing cells to Nutlin-3, an HDM2 antagonist that activates p53. Surprisingly, treatment of D1/CDK-transformed cells with Nutlin-3 prevented their anchorage-independent growth. The Nutlin-3-induced growth arrest was enforced in D1/CDK-expressing cells despite the presence of hyperphosphorylated RB implicating a p53-dependent, RB-independent mechanism for growth suppression. Further analysis identified that CDC2 and cyclin B1, key cell cycle regulators, were stably down-regulated following p53 stabilization by Nutlin-3, consistent with direct interaction between p53 and the CDC2 and cyclin B1 promoters, leading to the repression of transcription by methylation. In contrast to D1/CDK expression, direct inactivation of p53 resulted in no repression of CDC2 and no cell cycle arrest. We conclude that induction of p53 by Nutlin-3 is a viable therapeutic strategy in cancers with constitutive CDK signaling due to the direct repression of specific p53 target genes.

Highlights

Cyclin D1 complexes with cyclin-dependent kinase (CDK) to promote the G1-S transition by phosphorylating RB family members, releasing E2F from inhibitory interactions with RB/ p130/p107, and de-repressing genes critical for cell cycle progression [1, 2]
To avoid the use of viral proteins and to develop a model to study cyclin D1–mediated transformation, we evaluated HME1 cells expressing a cyclin D1–CDK fusion protein (D1/CDK) fusion protein to inhibit RB, a short hairpin RNA (shRNA) to inhibit p53 and a constitutively active PI3K
We describe here the transformation of immortalized HME1 cells by a cyclin D1–CDK fusion protein that models the cyclin D1 overexpression commonly observed in cancer

Summary

Introduction

Cyclin D1 complexes with cyclin-dependent kinase (CDK) to promote the G1-S transition by phosphorylating RB family members, releasing E2F from inhibitory interactions with RB/ p130/p107, and de-repressing genes critical for cell cycle progression [1, 2]. Doi:10.1158/0008-5472.CAN-07-0259 complexes to the inhibit kinase activity that is necessary for cell cycle progression [3]. We reported previously that the cyclin D1–CDK2 fusion protein could inhibit the repression of a number of genes normally repressed in response to p53 activation. Inactivation of p53 by overexpression of HDM2 or use of short hairpin RNA (shRNA) to p53, overexpression of cyclin D1 or cyclin E, or inactivation of RB by the adenoviral E7 protein abrogated the repression of genes required for cell cycle progression, including PLK1 [7]

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