Intrinsic Cooperation between p16INK4a and p21Waf1/Cip1 in the Onset of Cellular Senescence and Tumor Suppression In vivo

Shinji Takeuchi,Saburo Sone,Akiko Takahashi,Tomoko Tajima,Atsushi Hirao,Yuichi Ishikawa,Shigeru Yanagi,Noriko Motoi,Shin Yoshimoto,Kiyoko Fukami,Kimi Yamakoshi,Eiji Hara,Naoko Ohtani

doi:10.1158/0008-5472.can-10-0801

Abstract

Although the p16(INK4a) and p21Waf1/Cip1 cyclin-dependent kinase (CDK) inhibitors are known to play key roles in cellular senescence in vitro, their roles in senescence remain rather poorly understood in vivo. This situation is partly due to the possibility of compensatory effect(s) between p16INK4a and p21Waf1/Cip1 or to the upregulation of functionally related CDK inhibitors. To directly address the cooperative roles of p16INK4a and p21Waf1/Cip1 in senescence in vivo, we generated a mouse line simply lacking both p16INK4a and p21Waf1/Cip1 genes [double-knockout (DKO)]. Mouse embryonic fibroblasts (MEF) derived from DKO mice displayed no evidence of cellular senescence when cultured serially in vitro. Moreover, DKO MEFs readily escaped Ras-induced senescence and overrode contact inhibition in culture. This was not the case in MEFs lacking either p16INK4a or p21Waf1/Cip1, indicating that p16(INK4a) and p21Waf1/Cip1 play cooperative roles in cellular senescence and contact inhibition in vitro. Notably, we found the DKO mice to be extremely susceptible to 7,12-dimethylbenz(a)anthracene/12-O-tetradecanoylphorbol-13-acetate-induced skin carcinogenesis that involves oncogenic mutation of the H-ras gene. Mechanistic investigations suggested that the high incidence of cancer in DKO mice likely reflected a cooperative effect of increased benign skin tumor formation caused by p21Waf1/Cip1 loss, with increased malignant conversion of benign skin tumors caused by p16(INK4a) loss. Our findings establish an intrinsic cooperation between p16INK4a and p21Waf1/Cip1 in the onset of cellular senescence and tumor suppression in vivo.

Highlights

The p16INK4a and p21Waf1/Cip1 cyclin-dependent kinase (CDK) inhibitors are known to play key roles in the onset of cellular senescence, a state of permanent cell cycle arrest in culture [1,2,3,4,5,6]
We have generated for the first time the p16INK4a;p21Waf1/Cip1 double mutant mice (DKO mice) on a C57BL/6 background, a carcinogenesis-resistant strain, and analyzed the effects of p16INK4a and p21Waf1/Cip1 deficiency on the susceptibility to cancer in cooperation with oncogenic Ras expression
Our analyses clearly show a significant increase of cancer incidence in the DKO background because p21Waf1/Cip1 deletion promotes benign skin tumor development and p16INK4a deletion promotes the incidence of malignant conversion of benign skin tumors (Table 1)

Summary

Introduction

The p16INK4a and p21Waf1/Cip cyclin-dependent kinase (CDK) inhibitors are known to play key roles in the onset of cellular senescence, a state of permanent cell cycle arrest in culture [1,2,3,4,5,6]. The simultaneous induction of p21Waf1/Cip1and p16Ink4a expression cooperatively blocks the activation of both cyclin D kinase (CDK4/6) and cyclin E kinase (CDK2), allowing the accumulation of the dephosphorylated form of the retinoblastoma tumor suppressor protein (pRb) and thereby causing permanent cell cycle arrest [7,8,9,10]. Are resistant to the onset and the maintenance of cellular senescence in culture These results indicate that p16INK4a plays a critical role in cooperating with p21Waf1/Cip in tumor suppression in vivo

Materials and Methods

Results

Findings

Discussion