Loss of p27(Kip1) but not p21(Cip1) decreases survival and synergizes with MYC in murine lymphomagenesis

Abstract

The cyclin-dependent kinase (CDK) inhibitors p21(Cip1) and p27(Kip1) are induced in response to anti-proliferative stimuli and block G(1)/S-phase progression through the inhibition of CDK2. Although the cyclin E-CDK2 pathway is often deregulated in tumors the relative contribution of p21(Cip1) and p27(Kip1) to tumorigenesis is still unclear. The MYC transcription factor is an important regulator of the G(1)/S transition and its expression is frequently altered in tumors. Previous reports suggested that p27(Kip1) is a crucial G(1) target of MYC. Our study shows that in mice, deficiency for p27(Kip1) but not p21(Cip1) results in decreased survival to retrovirally-induced lymphomagenesis. Importantly, in such p27(Kip1) deficient lymphomas an increased frequency of Myc activation is observed. p27(Kip1) deficiency was also shown to collaborate with MYC overexpression in transgenic lymphoma models. Thus, in vivo, the capacity of MYC to promote tumor growth is fully retained and even enhanced upon p27(Kip1) loss. We show that in lymphocytes, MYC overexpression and p27(Kip1) deficiency independently stimulate CDK2 activity and augment the fraction of cells in S phase, in support of their distinct roles in tumorigenesis.

Fig. 1. Decreased survival of p27–/– mice with MuLV-induced lymphomagenesis. The percentage of surviving animals is plotted against the days after infection. (A) Incidence of MuLV-induced tumors in WT and CKI KO mice. (B) Tumor survival of p27+/– mice in the presence or absence of p21.

Fig. 2. p27 deficiency accelerates spontaneous lymphomagenesis in Myc-transgenic mice. The percentage of surviving animals is plotted against their age. (A) Survival to spontaneous lymphomas in H2K-Myc mice in the presence or absence of p27. Lymphoma incidence in p21–/–; p27–/– animals is also depicted. (B) Tumor latency in Eµ-Myc mice in the presence or absence of p27.

Fig. 3. Pre-neoplastic phenotype of Eµ-Myc p27–/– spleens. Representative illustration of data collected from multiple groups of 2-week-old mice from all the genotypes indicated at the top. (A) Haematoxylin–eosin-stained sections from spleens. The scale specified is maintained in all panels. (B) Flow cytometric analysis of the DNA content of freshly isolated splenocytes stained with PI. PI uptake and relative cell number are shown by the horizontal and vertical axes, respectively. For each panel, the percentage of cells in the distinct phases of the cell cycle is shown in the upper right corner. The horizontal bar represents the percentage of proliferating cells (S+ G₂/M) for each genotype. (C and D) Flow cytometric analysis of splenocytes after staining for B220/TCRαβ (C) or IgM/B220 (D). The percentage of cells in each quadrant is indicated above the panels. In (D), the horizontal axis mean value for the B220⁺/IgM⁺ population (top right) is 360, 300, 470 and 900, from left to right.

Fig. 4. p27 deficiency enhances CDK2 activity in Eµ-Myc pre-neoplastic splenocytes. Representative data from multiple groups of 2-week-old mice is depicted. (A) Splenocytes from mice of the indicated genotypes were analyzed by western blotting (WB) using antibodies against cyclin E (cycE), cyclin A (cycA), CDK2, p27 and actin (loading control). (B) Upper panel: CDK2 immunoprecipitation (IP) of protein lysates from splenocytes was followed by kinase assay (KA) for histone H1 (HH1)-associated kinase activity. The genotypes analyzed are depicted at the top. Lower panel: expression of actin in the same extracts. (C) CoIP analysis of CDK2-bound p27 from Eµ-Myc and WT splenocytes (Spl) or WT thymocytes (Thy). The amount of p27 and CDK2 present in the same blot is shown. (D) Protein lysates from the three left Eµ-Myc samples depicted in (C) were pooled and CDK2–p27 CoIP analysis was carried out either on total (Total) or positively selected B220⁺ splenocytes (B220⁺). WT thymic lysates were used as control.

Fig. 5. Differential expression and activity of p27 in Eµ-Myc and WT MuLV-induced tumors. (A) Western blot (WB) analysis of the expression of p27, CDK2 and actin (loading control) in Eµ-Myc pre-neoplastic splenocytes (Spl) and tumors. WT splenocytes and thymocytes (Thy) were also analyzed. (B) CDK2–p27 co-immunoprecipitation (IP) analysis of the first six Eµ-Myc tumor samples shown in (A). The amount of p27 and CDK2 present in the same blot is shown. (C) CD4+/CD8+ thymic lymphomas from WT MuLV-infected mice were analyzed for p27 expression by WB. Protein lysates from WT and p27–/– (KO) thymus were used as control. Lymphomas with MuLV proviral integrations in the Myc loci are indicated by asterisks.