Deregulation of p27 by oncogenic signaling and its prognostic significance in breast cancer

Corresponding author: Joyce Slingerland (e-mail: ) 2 0 Braman Breast Cancer Institute, University of Miami School of Medicine , Miami, FL , USA 1 Sunnybrook and Women's Health Sciences Centre, University of Toronto , Toronto, Ontario , Canada 2 Joyce Slingerland, Braman Breast Cancer Institute, Sylvester Compre- hensive Cancer Center, University of Miami School of Medicine , 1475 NW 12th Avenue (D8-4), Miami, FL 33136 USA. Tel: p27 is a key regulator of progression from G1 to S phase. Although the gene encoding p27 is rarely mutated in human cancers, p27 is functionally inactivated in a majority of human cancers through accelerated p27 proteolysis, through sequestration by cyclin D-cyclin-dependent kinase complexes and by cytoplasmic mislocalization. Here we review mechanisms whereby oncogenic activation of receptor tyrosine kinase and Ras pathways lead to accelerated p27 proteolysis and p27 mislocalization in cancer cells. The prognostic significance of p27 in human breast cancer is also reviewed. - Introduction G1 progression is governed by cyclin-dependent kinases (Cdks) [13]. The Cdks regulate biochemical pathways, or checkpoints, that integrate mitogenic and growth inhibitory signals and coordinate cell cycle transitions [4,5]. The Cdks are regulated by both activating and inhibitory phosphorylation, by cyclin binding and by two different families of Cdk inhibitors. In early G1 phase, mitogens increase D-type cyclins, which bind and activate Cdk4 and Cdk6 [6]. The subsequent activation of cyclin E and cyclin ACdk2 complexes regulate S phase entry and progression. Two families of Cdk inhibitors regulate the cyclinCdk complexes [2,7,8]. The inhibitor of Cdk4 (INK4) family members, which include p15INK4B, p16INK4A, p18INK4C, and p19INK4D, specifically bind Cdk4 and Cdk6 and inhibit cyclin D association. Members of the kinase inhibitor protein (KIP) family, p21CIP1, p27Kip1, and p57Kip2, bind and inhibit cyclin E-bound and cyclin A-bound Cdk2. Although p21 and p27 are major inhibitors of Cdk2, they also promote G1 progression by facilitating the assembly of cyclin DCdk4 and cyclin DCdk6 complexes [9,10]. In early G1, p27 assembles cyclin D1Cdks in the cyto Regulation of the cell cycle inhibitor p27KIP1 p27 was discovered in cells arrested by transforming growth factor- (TGF-), by contact inhibition, and by lovastatin [1114]. p27 acts in G0 and early G1 to inhibit cyclin ECdk2. Assembly of cyclin DCdk complexes by p27 is activated in early G1 and involves changes in p27 phosphorylation [15]. Mitogenic growth factor signaling mediates a decrease in p27 protein levels. Whereas p27 mRNA concentrations are constant throughout the cell cycle, p27 concentrations are the highest in quiescent cells, decrease during G1 phase and are minimal in S phase [16]. p27 translation is maximal in quiescence and falls rapidly after exit from G0 [16,17]. p27 is also importantly regulated by proteolysis, with the p27 t1/2 decreasing fivefold to eightfold with passage from G0 to S phase [18,19]. Transcriptional regulation Although p27 regulation occurs predominantly at the levels of translation and protein stability, transcriptional AFX = acute lymphocytic leukaemia-1 fused gene from chromosome X; Cdk = cyclin-dependent kinase; INK = inhibitor of Cdk; KIP = kinase inhibitor protein; MAPK = mitogen-activated protein kinase; MEK = MAPK kinase; PI3K = phosphoinositide 3-kinase; PKB = protein kinase B; PTEN = phosphatase and tensin homolog deleted on chromosome 10; SCF = Skp1, Cul1, F-box protein; TGF- = transforming growth factor-; TSC2 = tumor suppressor tuberin-2. regulation of p27 has been demonstrated. Normal quiescent T cells express high concentrations of p27 mRNA and protein, both of which decline rapidly after T cell activation [20]. Regulation of p27 mRNA concentrations also occurs after androgen depletion in breast cancer cells [21], in normal prostate tissue, and benign prostatic hyperplasia [22]. In melanoma cells, interleukin-6 signaling activates signal transduction and activators of transcription-3 (STAT3) and increases p27 mRNA [23]. The p27 promoter contains binding sites for several transcription factors including Sp1, cAMP-response element, Myb, NFB, and acute lymphocytic leukaemia-1 fused gene from chromosome X (AFX). AFX is a forkhead transcription factor recently shown to activate p27 transcription [24]. Phosphorylation of AFX by protein kinase B (PKB) inactivates this transcription factor and might thereby decrease p27 transcription. The relevance of transcriptional regulation of p27 to human cancers is unclear because most reduction of p27 in human cancers is thought to occur through proteolysis (see below and [7]). Regulation of p27 localization p27 localization is also cell cycle regulated: p27 is nuclear in G0 and early G1 and appears transiently in the cytoplasm at the G1/S transition [25]. The nuclear import of p27 depends on a bipartite nuclear localization signal in the carboxy-terminal region of the protein [26]. Interaction of p27 with the nuclear pore protein NPAP60 [27,28] is important in p27 nuclear import and might also regulate p27 export [27]. In response to mitogenic stimulation, at least part of the nuclear p27 pool undergoes nuclear export dependent on phosphorylation at serine 10 [25,29,30]. Human kinase interacting stathmin (hKis) can phosphorylate p27 at serine 10 [29]. p27 is bound to the exportin CRM1 in early G1, and binding of CRM1 to p27 increases with G1 progression [25]. p27 contains a nuclear export signal (NES) whose mutation decreases p27CRM1 binding, nuclear export, and p27 degradation [25]. Active CRM1RanGTP-mediated nuclear export of p27 is linked to cytoplasmic proteolysis of p27 in early G1. Proteolytic degradation of p27 p27 proteolysis is regulated by at least two distinct mechanisms. In early G1, mitogens seem to activate an exportlinked degradation mechanism that is followed in late G1 and S phases by a cyclin ECdk2-dependent degradation of p27. The late G1 and early S phase of p27 proteolysis is regulated by its phosphorylation at threonine 187 (T187) by cyclin ECdk2 [18,3133]. Phosphorylation of p27 at T187 promotes the interaction of p27 with Skp2, the F box component of the SCFSkp2 (Skp1, Cul1, F-box protein) ubiquitin ligase. Once p27 phosphorylated on T187 is recognized by its SCF-type E3 ligase, composed of Skp1, Cul1, the F-box protein, Skp2 and Roc1, and the Cks1 cofactor [3439], this complex then mediates the subsequent degradation of p27 by the 26S proteasome. Recent data from T187A knock-in and Skp2/ mice also suggest that p27 proteolysis in early G1 is independent of T187 phosphorylation [19,40]. In early G1, growth factors stimulate p27 proteolysis in a manner independent of T187 phosphorylation and possibly also of Skp2 [19,40]. This initial mitogen-stimulated p27 degradation in early G1 might be linked to p27 export [25] and would allow an incremental activation of cyclin ECdk2 that is then followed by (...truncated)