PDF《Oncogene (2009) 28, 243–256》

Sowa et al., 1997;

Archer et al., 1998;

Kim et al., 1999;

Saito et al., 1999;

Sambucetti et al., 1999;

Richon et al., 2000;

Han et al., 2001;

Rocchi et al., 2005). p21WAF1/Cip1 arrests cell growth by inhibiting cyclin/Cdk complexes (Xiong et al., 1993) and can act as tumor suppressor gene by negatively regulating the cell cycle. In p21WAF1/Cip1 - de?cient mice, loss of p21WAF1/Cip1 accelerated the formation of spontaneous tumors (Martin-Caballero et al., 2001) and increased susceptibility to tumorigenesis in response to chemical carcinogens (Wouters et al., 1997;

Philipp et al., 1999;

Topley et al., 1999;

Poole et al., 2004). Thus, p21WAF1/Cip1 has many features of a bona ?de tumor suppressor gene. p21WAF1/Cip1 expression is commonly lost in a variety of human cancers and may therefore contribute to tumor progression. As mutations in the coding region of p21WAF1/Cip1 are rare events in cancer, epigenetic silencing Received

10 April 2008;

revised

19 August 2008;

accepted

26 August 2008;

published online

13 October

2008 Correspondence: Dr V Castronovo, Metastasis Research Laboratory, GIGA-Cancer (Centre for Experimental Cancer Research), University of Lie ` ge, Pathology Tour, B23, B-4000 Lie ` ge, Belgium. E-mail: [email protected] or Dr E Verdin, Gladstone Institute of Virology and Immunology,

1650 Owens Street, San Francisco, CA 94158, USA. E-mail: [email protected] Oncogene (2009) 28, 243C256 &

2009 Macmillan Publishers Limited All rights reserved 0950-9232/09 $32.00 www.nature.com/onc seems to be the main mechanism by which the p21WAF1/Cip1 gene is downregulated in tumors (Fang and Lu, 2002). Epigenetic suppression of p21WAF1/Cip1 is directly linked to HDAC activity, as inhibition of HDAC activities in cancer cells induces transcriptional reactivation of p21WAF1/Cip1 through hyperacetylation of histones H3 and H4 in the proximal promoter region (Sowa et al., 1997;

Richon et al., 2000;

Sawa et al., 2004). In other studies, p21Waf1/Cip1 induction by HDAC inhibitors was mediated through Sp1 sites through Sp1 family transcription factors, Sp1 and/or Sp3 (Nakano et al., 1997;

Sowa et al., 1997, 1999;

Han et al., 2001;

Wilson et al., 2006). To date, the most common model for p21WAF1/Cip1 repression in cancer cells is the recruit- ment of HDACs by Sp1/-Sp3 transcription factors in the proximal promoter region encompassing the Sp1/Sp3 binding sites, which in turn repress p21WAF1/Cip1 trans- cription by deacetylating histones H3 and H4. However, a role for p53 in HDAC-associated p21WAF1/Cip1 expres- sion has also been reported in several human cancer cell lines (Lagger et al., 2003;

Luo et al., 2004;

Roy et al., 2005;

Zhao et al., 2006). Regardless of their mechanism of action, HDAC inhibitors induce p21WAF1/Cip1 in various cancer cell lines. However, most HDAC inhibitors are global and non- speci?c for the various HDAC isoforms. Class I HDACs regulate p21WAF1/Cip1 gene expression (Huang et al., 2005, 2006;

Wilson et al., 2006;

Senese et al., 2007;

Spurling et al., 2008), but it is not known whether other HDACs are involved. To identify which HDACs regulate p21WAF1/Cip1 gene expression in cancer cells, we used small interfering RNAs (siRNAs) to target seven class I and class II HDACs (HDACs1C7). We found that silencing of HDAC4 consistently induces expression of p21WAF1/Cip1 in various human cancer cell lines. We also demon- strated that induction of p21WAF1/Cip1 is an important component of inhibition of cancer cell proliferation in vitro and arrest of tumor growth in vivo mediated by silencing of HDAC4, arguing that HDAC4 is a potential target for anticancer therapies. Results HDAC4 silencing induces expression of p21WAF1/Cip1 mRNA and protein HDAC inhibitors induce p21WAF1/Cip1 in many cancer cell lines (Nakano et al., 1997;