The concept that CDK2 is essential for controlled entry into S phase and the onset of DNA replication was challenged a few years ago when two independent groups reported the generation of CDK2−/−
). These mice are viable, develop normally and only show meiotic defects. These results were surprising, as CDK2 was thought to be required for promoting G1/S transition. In addition, it was also demonstrated that cancer cells can proliferate in absence of CDK2 (15
). Thus, the data accumulated in the last few years has brought the concept that CDK2 is not an adequate target for therapeutic intervention. In this report we prove that CDK2 inhibition can still be considerate as an adequate target for therapeutic intervention depending on the particular oncogenic pathway involved.
We have previously shown that forced expression of CDK4 in mouse skin induces keratinocyte proliferation and malignant progression in a ras-dependent skin tumorigenesis model (7
). Here, we have examined the skin of CDK2−/−
mice which is structural and functional similar to that of wild type siblings. It has been reported that CDK1 activation compensates for the absence of CDK2 in thymocytes, although this mechanism depends on the presence of p27Kip1
). However, biochemical analysis of CDK2−/−
epidermis shows reduction rather than activation of CDK1. We also observed increase activity of CDK4 in the epidermis of CDK2−/−
mice (). Since p27Kip1
stabilize CDK4/D-type cyclin complexes (37
), it was possible that in absence of CDK2, redistribution of p27Kip1
increase CDK4/D-type cyclin/p27Kip1
complex formation. Nevertheless, biochemical analysis did not show increased formation of those complexes (data no shown). Whether the increase in CDK4 activity compensates for the absence of CDK2 in the epidermis merits further investigation. The importance of the CDK4-CDK2 link in keratinocyte proliferation was demonstrated by the activation of CDK2, through titration of p27/p21, by forced expression of CDK4 (7
) () and the reduction of the total number of proliferative cells in K5CDK4/CDK2−/−
epidermis compared to K5CDK4 mice. Therefore, activation of both, CDK4 and CDK2 kinases are necessary for epidermal hyperproliferation observed in K5CDK4 mice ().
We also studied whether CDK2 is required for CDK4-induced malignant progression to SCCs (20
). In our experimental setting overexpression of CDK4 reduces papilloma multiplicity, although the number of mice with papilloma (incidence) was similar between K5CDK4 and wild type mice. Lack of CDK2 leads to a significant reduction of papilloma incidence and multiplicity in K5CDK4/CDK2−/−
mice. More importantly, lack of CDK2 expression in K5CDK4 mice (K5CDK4/CDK2−/−
mice) results in a severe reduction of CDK4-induced malignant progression to SCCs. Therefore, lack of CDK2 affects papilloma development and more importantly CDK2 expression is required for CDK4-induced malignant progression. Histopathological analysis of tumors from the four genotypes confirms the role of CDK2 in malignant progression. Tumors from K5CDK4 mice were classified as regular papillomas and SCC I, II, and III; whereas most of the K5CDK4/CDK2−/−
tumors were regular papillomas and no SCCs II or III were observed. In addition, K5CDK4 carcinomas showed areas of strong positive immunofluorescence staining for keratin 13 (K13), a malignancy marker in squamous epithelia (31
), whereas SCC from K5CDK4/CDK2−/−
mice were K13-negative.
We have previously demonstrated that cyclin D1 and CDK4 expression plays an important role in papilloma development (38
). We determined that cyclin D1 expression is not affected in CDK2−/−
papillomas (data no shown). The latter confirms that reduced papilloma formation and progression in CDK2−/−
compound mice is not due to altered D-type cyclin levels. We also observed a mild reduction of the expression of transgenic CDK4 in tumors from K5CDK4/CDK2−/−
mice compared with K5CDK4 siblings (). However, the CDK4 protein levels were still higher than wild type mice. Thus, the reduction of transgenic-CDK4 is not responsible for the reduction in the number of papillomas and SCC in K5CDK4/CDK2−/−
mice. Supporting these results we previously reported, that two independent K5CDK4 transgenic lines exhibiting different levels of CDK4 protein expression develop SCCs at a similar rate (20
). Similar to CDK1 expression in epidermis, CDK1 protein level was variable among tumors from the same genotype, but there are no relevant differences in CDK1 levels among the four genotypes. Interesting, p15Ink4b
expression was reduced in K5CDK4 mice independently of the CDK2 status. p15Ink4b
transcription can be down-regulated via phosphorylation and inactivation of Smad3 by CDK4 (32
), but decrease p15Ink4b
was observed in both K5CDK4 and K5CDK4/CDK2−/−
, suggesting that its downregulation is not responsible for the CDK4-induced malignant progression.
The present study allows us to define an important role of CDK2 in malignant progression. Interestingly, Duensing et al. determined that CDK2 is dispensable for normal centrosome duplication, but required for oncogene-induced centrosome overduplication (42
). Thus, lack of CDK2 could also abrogate the centrosome overduplication in epithelial tumor cells protecting the cells against genetic instability, a mechanism involved in malignant progression. The present study also shows a reduction in the total number of chemically induced tumors in both CDK2−/−
mice suggesting that CDK2 may play an important role in cell survival. To this effect Huang et al have recently shown that CDK2 influences survival of cells under genotoxic stress by inhibiting the forkhead transcription factor, FOXO1. Importantly, CDK2 phosphorylate and inhibits the pro-apoptotic function of FOXO1 (43
). Thus, CDK2 ablation could restore or increase FOXO1 activity leading to reduce tumorigenesis.
We have previously demonstrated that CDK4 ablation, but not cyclin D2, inhibits myc-mediated oral tumorigenesis (23
). Here we show that deletion of CDK2 in a K5Myc background does not affect tumor development as 93% of K5Myc/CDK2−/−
mice developed oral tumors. These tumors were classified as SCCs showing a variable degree of squamous differentiation. No difference in tumor incidences or the degree of differentiation was observed between K5Myc and K5Myc/CDK2−/−
mice as opposed to K5Myc/CDK4−/−
mice which did not develop tumors (23
). Immunostaining analysis of myc-induced oral tumors revealed equal levels of proliferation (BrdU-positive cells) in the epithelial compartment of the oral tumors from K5Myc and K5Myc/CDK2−/−
mice (data no shown). Thus, it is clear that lack of CDK2 does not affect the proliferation mediated by myc. To determine the effect of myc in other tissues, we also analyzed mouse epidermis from K5Myc and K5Myc/Cdk2−/−
mice. In contrast to the reduced proliferation mediated by lack of CDK4, ablation of CDK2 did not result in suppression of the epidermal phenotype observed in K5Myc mice. As we previously reported, overexpression of CDK4 was observed in K5Myc epidermis and it was not affected by CDK2 ablation (23
). Consequently, lack of CDK2 cannot suppress the epidermal phenotype mediated by myc suggesting that CDK4 plays a unique role in myc-induced keratinocyte proliferation (45
). Thus, our results suggest that CDK2 is expendable for myc-induce proliferation not only in the gingival epithelium, but as a general characteristic for myc-induced proliferation in epithelial tissues. In addition to inducing proliferation, myc can also drive apoptosis which serves as a barrier to unchecked cellular proliferation (47
). In this sense, it was recently described that the removal of CDK2 suppressed the ability of myc to induce apoptosis (49
). Thus, it is possible that loss of CDK2 reduces the myc-mediated apoptosis supporting tumor development.
In summary, our data suggest that the efficacy of targeting CDK2 in tumor development and malignant progression is dependent on the specific oncogenic pathway involved. Similarly, other groups have shown that different oncogenic pathways respond differently to the inhibition of cell cycle regulators. For instance, cyclin D1-deficient mice are resistant to breast cancers induced by ras, but remains fully sensitive to other oncogenic pathways such as those driven by myc or Wnt-1 (50
). Also, CDK1 inhibition induces apoptosis in tumor cell lines transformed with myc, but not Ras or a variety of other activated oncogenes (51
Whether loss of CDK2 inhibits tumorigenesis mediated by other pathways such us neu or wnt remains to be determined. Overall we have established that ras/CDK4-induced tumors are more sensitive to CDK2 inhibition than myc-induced tumors. Therefore, the suitability of CDK2 as a target for therapeutic intervention must be considered in the context of the particular pathway affected.