To the editor:
We read with great interest the work published in Blood by Chen et al concerning the therapeutic restoration of p27KIP1 protein levels in cancer after applying the specific Skp2 inhibitor CpdA.1 Skp2 is an E3-ubiquitin ligase that mediates degradation of several cell-cycle regulators.2 Among its targets are negative cell-cycle regulators, including the kinase inhibitor protein (KIP) family member p27KIP1, and positive ones, such as cyclin E.2 In addition, p27KIP1 also inhibits cyclin E.2 Thus, Skp2, p27KIP1, and cyclin E form a tight network controlling S-phase entry.2
In various tumors Skp2 is frequently overexpressed and represents a major cause of p27KIP1 protein down-regulation.2 However, p27KIP1 gene alterations and transcriptional silencing, due to microRNA-dependent repression, promoter methylation, and transcriptional suppressors, is not a rare event, as the authors mention,1 but a significant source of p27KIP1 inactivation in several malignancies (Table S1, available on the Blood website; see the Supplemental Materials link at the top of the article). Consequently, we cannot exclude the possibility that Skp2 overexpression coexists with transcriptionally silenced and/or mutant p27KIP1. We have observed such a condition with the other KIP member and Skp2 target, p57KIP2.3
Based on the above, Skp2 inhibition, in cases with transcriptionally silenced and/or mutant p27KIP1, could result in increased expression of cyclin E with potential deleterious effects. Cyclin E provokes genomic instability, when overexpressed, by producing either DNA damage and/or centrosome amplification.4,5 Up-regulation of cyclin E is frequently observed in cancer, and is associated in various malignancies with poor survival.6
To test this hypothesis we mimicked the above scenario by silencing Skp2 alone or in combination with p27KIP1 in A549 cancer cells, which express high Skp2 levels.3 The experiment showed that sole Skp2 silencing resulted in elevation of p27KIP1, reduction of cyclin E expression (Figure 1A,B), and a decrease in growth,3 while Skp2/p27KIP1 double knockout, recapitulating the proposed scenario, led to increased levels of cyclin E (Figure 1A,B), centrosome amplification (Figure 1B), abnormal mitoses, and pronounced nuclear atypia characterized by micronuclei, lobulated nuclei, and nucleoplasmic bridges, features that are indicative of chromosomal instability (Figure 1C).7 In addition, marked p53 Ser-15 phosphorylation (Figure 1A), indicating a prominent DNA damage response, provides a mechanistic explanation for the observed genomic instability.8,9 Furthermore, the elevated levels of cyclin E comprised not only the full-length but also the low-molecular-weight isoforms.5,6 These isoforms are present in both cytoplasm and nucleus, and have increased affinity for cdk2.5,6 They have been associated with genomic instability, resistance to CIP/KIP inhibition, and poor outcome of patients with various malignancies.6
Similarly, is exclusive Skp2 targeting efficient in patients with p27KIP1 haploinsufficiency?10 In such cases, SKP2 blocking is effective when the levels of the remaining p27KIP1 allele are up-regulated above a threshold, able to exert its negative effect on cell-cycle progression. Below this threshold the CIP/KIP molecules will be sequestered by the cyclin D/cdk complexes, further promoting cyclin E/cdk2 activity.11
In conclusion, although the findings of Chen et al are significant and important, our results present an additional point of view, which stresses the impact of defining the transcriptional and/or mutational status of p27KIP1 before applying a therapeutic approach based exclusively on Skp2 inhibition.
The online version of this letter contains a data supplement.
This work was financially supported by the European Commission Seventh Framework Health Programme Genomic instability in cancer and precancer (FP7-GENICA) grant.
Conflict-of-interest disclosure: The authors declare no competing financial interests.