Cancer Letters

Cancer Letters

Volume 333, Issue 1, 1 June 2013, Pages 103-112
Cancer Letters

A novel CyclinE/CyclinA-CDK Inhibitor targets p27Kip1 degradation, cell cycle progression and cell survival: Implications in cancer therapy

https://doi.org/10.1016/j.canlet.2013.01.025Get rights and content

Abstract

p27Kip1 (p27) binds and inhibits the cyclin E- or cyclin A-associated cyclin-dependent kinases (CDKs)2 and other CDKs, and negatively regulates G1–G2 cell cycle progression. To develop specific CDK inhibitors, we have modeled the interaction between p27 and cyclin A-CDK2, and designed a novel compound that mimics p27 binding to cyclin A-CDK2. The chemically synthesized inhibitor exhibited high potency and selective inhibition towards cyclin E/cyclin A-CDK2 kinase in vitro but not other kinases. To facilitate permeability of the inhibitor, a cell penetrating peptide (CPP) was conjugated to the inhibitor to examine its effect in several cancer cell lines. The CPP-conjugated inhibitor significantly inhibited the proliferation of cancer cells. The treatment of the inhibitor resulted in the increased accumulation of p27 and p21Cip1/Waf1 (p21) and hypo-phosphorylation of retinoblastoma protein (Rb). The degradation of p27, mediated through the phosphorylation of threonine-187 in p27, was also inhibited. Consequently, exposure of cells to the inhibitor caused cell cycle arrest and apoptosis. We conclude that specific cyclinE/cyclin A-CDK2 inhibitors can be developed based on the interaction between p27 and cyclin/CDK to block cell cycle progression to prevent tumor growth and survival.

Highlights

► Novel small molecules have been designed and synthesized to inhibit Cyclin E/Cyclin A-CDK kinase. ► The inhibitor targets p27Kip1 degradation. ► The inhibitor is not ATP analogues. ► The inhibitor only inhibits Cyclin E/Cyclin A-CDK but not other kinases. ► The inhibitor inhibits cancer cell proliferation and induces cell cycle arrest.

Introduction

Cyclin-dependent kinases (CDKs) are a family of serine/threonine kinases that play key roles in controlling the entry into and passage through various phases of the cell cycle [1], [2], [3], [4]. In the cell cycle, distinct cyclin/CDK complexes are activated to regulate cell cycle progression. For example, while cyclin D/CDK4 or CDK6 regulates the progression of G1 phase, cyclin E/CDK2 is required for the G1/S transition. Another cyclin/CDK complex, cyclin A/CDK2, plays a critical role in the control of S phase and DNA replication. It is also essential for G2 progression. Cyclin A- and cyclin B-associated CDK1 (CDC2) regulates the G2/M phases. Altered activities of cyclin/CDKs, caused by over-expression, translocation, gene amplification, or other aberrant activation of cyclin D, cyclin E, or cyclin A, are associated with various malignant human cancers [1], [5]. The cell cycle is also negatively regulated by the presence of CDK inhibitors such as p27Kip1(p27) and p21Cip1/Waf1(p21). These inhibitory proteins interact with cyclin E or cyclin A/CDK2 or other cyclin/CDK binary complexes to inhibit their kinase activities [6], [7], [8], [9]. p21 is transcriptionally controlled by tumor suppressor protein p53 and loss or mutation of p53 in many cancers leads to the down-regulation of p21. Malignant cancers are also associated with the low or absent expression of p27 protein, which is typically associated with poor prognosis [10]. In the cell cycle, the protein level of p27 is primarily regulated by ubiquitin-dependent proteolysis [11], [12]. While the p27 protein level is high in early- and mid-G1 to prevent untimed activation of cyclin E/cyclin A-CDK2 to progress into the S phase, the p27 protein is targeted for degradation at the late G1 or in S phase through its phosphorylation at threonine 187 (T187) by kinases such as cyclin E/CDK2 or cyclin A/CDK2 [13], [14], [15], [16]. Evidence indicates the phosphorylation of T187 is a result of phosphorylation at tyrosine 88 in p27 by Src family kinases [17]. When p27 is phosphorylated on the conserved threonine residue 187, the F-box protein Skp2 and its associated CKS1 bind to the phosphorylated p27 and promotes p27 ubiquitin-dependent degradation by the SCFSKP2 ubiquitin E3 ligase [14], [18], [19]. Low p27 protein levels caused by excessive SCFSKP2-mediated proteolysis of p27 are associated with many types of aggressive tumors [10], [13]. Inhibiting cyclin E/cyclin A-CDK activity and prevention of p27 proteolysis should provide an excellent strategy to block the proliferation of malignant human cancers.

Because the elevated activities of CDKs are hallmarks of human cancer, CDKs represent an important therapeutic target for various types of human cancers. Considerable effort has been focused on development of small molecule inhibitors of CDK2 or other CDKs for potential anti-cancer purposes [20], [21], [22], [23], [24]. Most of these inhibitors have been developed against the ATP binding domain for these kinases. Although more than 50 CDK inhibitors have been reported [21], the chemical structures that act as CDK inhibitors are quite limited, since most of them are derived from relatively nonspecific protein kinase inhibitor scaffolds that inhibit the binding of ATP to CDKs and other kinases, such as staurosporins, flavonoids, indigoids, paulones, and purines. Structural information indicates that the core of the CDK catalytic center, consisting about 300 amino acid residues, shares significant homology with many other kinases [25]. The high degree of similarity between the kinase domain of CDK family members and other kinases in the ATP binding domain makes the selective inhibition of CDKs difficult.

The identification of CDK inhibitors p27 and p21 provide a new strategy to develop chemical inhibitors for CDKs. In this report, we have designed new CDK inhibitors based on the inhibitory binding of p27 to cyclin A/CDK2. Our data indicate that the inhibitor we have synthesized can selectively inhibit cyclinE- or cyclin A/CDK kinase activities both in vitro and in vivo. Our analysis indicates that the inhibitor can cause the cell cycle arrest and apoptosis of cancer cells.

Section snippets

Cell lines

The cancer cell lines HeLa, RKO, MCF-7 and PC3 were obtained from American Type Culture Collection (ATCC, Rockville, MD). All cell lines were cultured in DMEM medium containing L-glutamine supplemented with 10% fetal bovine serum (FBS). The immortalized human hepatocyte cell line MIHA was cultured as described before [26].

Expression and purification of GST-CyclinA/CDK2, GST-CyclinE/CDK2 kinase complexes

Human cDNAs for cyclin A, cyclin E and Cdk2 were cloned into the baculovirus expression vector pVL1392 (Pharmingen, San Diego, CA, USA) which is fused in frame at the carboxy

The interaction between the conserved p27 motif and the substrate recruitment site of the CyclinE/CyclinA-CDK2 complex

The CDK inhibitor p27 interacts with the binary cyclin E/CDK2 or cyclin A/CDK2 kinase complex as a substrate for phosphorylation. This process is mediated through the initial recognition of the conserved RNLFGP motif of p27 by the substrate recruitment site on cyclin E or cyclin A (Fig. 1A) [27]. The crystal structure of p27 bound to cyclinA/CDK2 provides a framework for such a specific interaction between p27 and the cyclin/CDK complex [28] (Fig. 1B and C). Association is driven by hydrophobic

Discussion

In a normal cell, there is a delicate balance between the activity of cyclin E- and cyclin A-associated CDK kinase complexes and the levels of p27 protein. Uncontrolled CDK activity leads to the aberrant cell cycle regulation and consequently causes various human cancers. Many lines of evidences, both from clinical studies and animal models, support the tumor-suppressor function of p27. The p27−/− mice have been shown to spontaneously develop adenomas of the intermediate lobe of the pituitary

Acknowledgements

We acknowledge financial support from the Hong Kong Research Grants Council (Projects: PolyU 5407/06M; PolyU 5638/07M; PolyU 5636/08M; PolyU 5634/09M; PolyU 5040/10P; PolyU 5037/11P; and PolyU 5020/12P) and The Hong Kong Polytechnic University (PolyU 5636/08M; and PolyU 5634/09M); Fong Shu Fook Tong Foundation and Joyce M. Kuok Foundation; The National Science Foundation of China (NSFC30971616, 21072007, 21133002 and 21272011); The Shenzhen Bureau of Science, Technology, Information

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