A plethora of circumstantial evidence implicates downregulation of the cyclin-dependent kinase (CDK) inhibitor p27 in prostate cancer. While greater than 85% of terminally differentiated secretory cells in normal human prostate display strong nuclear staining for p27, all cases of high-grade prostatic intraepithelial neoplasia, invasive carcinoma, and pelvic lymph node metastases studied by DeMarzo et al. showed down-regulation of p27 . In addition, low p27 expression correlates with higher mean Gleason scores, a number of prognostic morphological features, and decreased survival [2–4]. Thus, p27 may be a prostate tumor suppressor.
In support of this notion, the p27 protein has been identified as a target of viral oncoproteins [5, 6]. However, unlike traditional tumor suppressors, the p27 gene rarely shows homozygous inactivation in cancer cells [7–9], a finding that points towards alternative mechanisms of p27 inactivation.
p27 specifically inhibits CDKs, which mediate entry into S phase [10, 11]. The level of p27 is higher in quiescent than in proliferating cells, and this increase in p27 abundance is required for an effective cell cycle exit . The cell cycle-dependent variations in p27 levels are not reflected by similar changes in p27 mRNA . Many aggressive prostate cancers display decreased p27 protein levels in the presence of high p27 mRNA , suggesting that p27 depletion may result from ectopic proteolysis. In fact, p27 depletion in several cancers was shown to result from increased proteolysis via the ubiquitin/proteasome system [15–18].
This system employs a cascade of enzymatic reactions that covalently attach a ubiquitin chain to substrate proteins, thereby targeting them to the proteasome . The ubiquitin transfer reaction involves three enzymes: E1, which mediates the ATP-dependent activation of ubiquitin, and E2, or ubiquitin conjugating enzyme (UBC), which, together with an E3 ubiquitin ligase, transfers ubiquitin to the target protein.
Biochemical studies identified SCFSKP2, an E3 that mediates p27 ubiquitylation in vitro[20, 21]. This complex consists of at least four proteins: SKP1, CUL1, HRT1 (=RBX1/ROC1), and SKP2. SKP2 contains a so-called F-box, which mediates binding to SKP1, and C-terminal leucine-rich repeats that recognize p27. CUL1, in turn binds to SKP1, and together with HRT1, mediates the interaction with the ubiquitin-conjugating enzyme CDC34/UBC3. CKS1, a small protein that associates with CDKs and greatly stimulates p27 ubiquitylation, was recently identified as a forth SCFSKP2 component [22, 23].
Two rate-limiting steps for p27 ubiquitylation were defined: (1.) phosphorylation of p27 by CDK2 at threonine 187 [24–26], and (2.) binding of phosphorylated p27 to SKP2 [20, 21]. SKP2 is down-regulated in resting cells with stable p27, but strongly up-regulated in cells, which progress into S phase [27, 28]. In some tissue culture cells, overexpression of SKP2 is sufficient to induce p27 degradation and S phase entry [29–32], and can cooperate with ras in transformation [33, 34]. Significantly, overexpression of SKP2 has been observed in many cancer cell lines [28, 35] as well as in primary cancer specimens and many of these tumors also display down-regulation of p27 [33, 36–38].
Here, we used the androgen-sensitive human prostate cancer cell line LNCaP  as a model system to address the role of SKP2 in androgen-mediated cell cycle control. This cell line undergoes reversible G1 arrest in response to androgens [40–44]. The G1 arrest is accompanied by p27 upregulation [42, 43, 45], however the pathway leading to p27 upregulation is unknown. We show that androgen-induced p27 upregulation is paralleled by p27 stabilization and SKP2 downregulation. SKP2 overexpression is sufficient to overcome androgen-mediated p27 accumulation, indicating that it is a major mediator of androgen-mediated cell cycle control.