Skp2 directs Myc-mediated suppression of p27Kip1 yet has modest effects on Myc-driven lymphomagenesis

Abstract

The universal cyclin-dependent kinase inhibitor p27(Kip1) functions as a tumor suppressor, and reduced levels of p27(Kip1) connote poor prognosis in several human malignancies. p27(Kip1) levels are predominately regulated by ubiquitin-mediated turnover of the protein, which is marked for destruction by the E3 ubiquitin ligase SCF(Skp2) complex following its phosphorylation by the cyclin E-cyclin-dependent kinase 2 complex. Binding of phospho-p27(Kip1) is directed by the Skp2 F-box protein, and this is greatly augmented by its allosteric regulator Cks1. We have established that programmed expression of c-Myc in the B cells of Emu-Myc transgenic mice triggers p27(Kip1) destruction by inducing Cks1, that this response controls Myc-driven proliferation, and that loss of Cks1 markedly delays Myc-induced lymphomagenesis and cancels the dissemination of these tumors. Here, we report that elevated levels of Skp2 are a characteristic of Emu-Myc lymphomas and of human Burkitt lymphoma that bear MYC/Immunoglobulin chromosomal translocations. As expected, Myc-mediated suppression of p27(Kip1) was abolished in Skp2-null Emu-Myc B cells. However, the effect of Skp2 loss on Myc-driven proliferation and lymphomagenesis was surprisingly modest compared with the effects of Cks1 loss. Collectively, these findings suggest that Cks1 targets, in addition to p27(Kip1), are critical for Myc-driven proliferation and tumorigenesis.

FIGURE 1

Skp2 expression is elevated in pre-cancerous Eμ-Myc B cells and in Myc-induced lymphomas. A. SYBR-green real-time PCR analysis of levels of c-myc, Skp2 and p27 transcripts in bone marrow (BM) and splenic (spleen) B220⁺ B cells from 4 week-old wild type (wt, gray bars) or Eμ-Myc (black bars) littermate mice. Levels of mRNA were standardized to the expression of Ubiquitin (Ub), which is not regulated by Myc. B. Immunoblot analyses p27 and Skp2 expression in B220⁺ B cells from 4 week-old wild type or Eμ-Myc-transgenic littermate mice. Levels of c-Myc protein are also shown and Actin served as a loading control. C. Skp2 protein expression is elevated in Eμ-Myc lymphoma. Eμ-Myc lymphoma samples were analyzed by immunoblotting for Skp2 expression. B220⁺ B cells were used as a control. Actin immunoblotting served as a loading control. D. SKP2 expression is elevated in Burkitt lymphoma. Real-time PCR analysis of fourteen Burkitt lymphoma samples (black bars) compared to normal human CD19⁺ B cells (gray bar). Levels of mRNAs were standardized to the expression of Ubiquitin (Ub). E. Skp2 protein levels are elevated in Burkitt lymphoma. Thirteen human Burkitt lymphoma samples were analyzed by immunoblotting for Skp2 expression. CD19⁺ peripheral B cells were used as a control. Actin immunoblotting served as a loading control.

FIGURE 2

Myc regulation of Skp2 is indirect. A. SYBR-green real-time PCR analysis of Odc and Skp2 RNA levels in primary early passage MEFs infected with pBabe-Myc-ER^TAM-IRES-Puro (Myc-ER, black bars) or pBabe-IRES-Puro (Puro control) retroviruses (white bars). Puromycin-resistant cells were treated with 2 μM 4-HT to activate the Myc-ER^TAM transgene. Levels of mRNA were standardized to the expression of Ubiquitin (Ub). B. SYBR-green real-time PCR analysis of Skp2 and p27 RNA levels in primary early passage MEFs infected with pBabe-Myc-ER^TAM-IRES-Puro (Myc-ER) or pBabe-IRES-Puro (Puro control) retroviruses. Puromycin-resistant cells were pre-treated with Chx for 30 min and were then treated with 2μm 4-HT for the times indicated. RNA was isolated from the cells and analyzed by real-time PCR. Levels of Skp2 and p27 mRNA were standardized to the expression of Ubiquitin (Ub). C. Primary early passage MEFs were infected with MSCV-IRES-Puro (Puro) or MSCV-Myc-IRES-Puro (Myc) retroviruses, Puromycin-selected, and treated with Chx (10 μg/ml) for the indicated time. Proteins levels were then assessed by immunoblotting.

FIGURE 3

Myc regulation of Skp2 is independent of E2f1. A. SYBR-green real-time PCR analysis of Skp2 and p27 RNA expression in splenic B220⁺ B cells from 4 week-old non-transgenic and Eμ-Myc transgenic mice of the indicated E2f1 genotypes. Levels of RNA were standardized to Ubiquitin (Ub). B. Immunoblot analyses of the levels of c-Myc, Skp2 and actin in FACS sorted GFP-expressing primary E2f1^+/+ and E2f1^−/− MEFs infected with MSCV-Myc-IRES-GFP (Myc) or MSCV-IRES-GFP (GFP) retroviruses.

FIGURE 4

The effects of the Skp2 deficiency on Myc-induced proliferation. A. Pre-cancerous (4 week-old) Eμ-Myc transgenic mice of the indicated Skp2 genotype were analyzed for white blood counts (WBC, left panel), and lymphocyte numbers in the peripheral blood (middle panel), and for weights of their spleens (right panel). * indicates p<0.05. B. B cells from ex vivo cultured bone marrow of Eμ-Myc;Skp2^+/+ and Eμ-Myc;Skp2^−/− mice were assessed for their spontaneous apoptotic index (n = 3). C. BrdU-incorporation into DNA (S phase) was used to assess the S-phase indices of B cells cultured ex vivo from the bone marrow of pre-cancerous Eμ-Myc;Skp2^+/+ versus Eμ-Myc;Skp2^−/− mice (n = 3; bars indicate mean ± SEM; * indicates p<0.05). D. Eμ-Myc;Skp2^+/+ and Eμ-Myc;Skp2^−/− littermates were injected with BrdU, and cells from bone marrow (BM) and spleen were harvested after 12 hr. BrdU-incorporation was then determined by FACS. The bars show the mean ± SEM of three independent experiments. The differences between Eμ-Myc;Skp2^+/+ and Eμ-Myc;Skp^−/− genotypes were not statistically significant.

FIGURE 5

Skp2 loss has no significant effect on Myc-driven lymphomagenesis as compared to wild type controls. The survival of Eμ-Myc transgenic littermates of the indicated Skp2 genotypes is shown. The differences in the rates of tumor incidence between the Skp2^+/+ and the Skp2^−/− group are not statistically significant (Cox-Regression analysis; significance 0.405, Hazard ratio 0.61, 95% confidence interval: 0.33–1.16). A statistically significant difference arises when all three cohorts are included in a Log Rank (Mantel-Cox) test (p=0.017). Numbers of animals per group are given in parentheses.

FIGURE 6

Skp2 is required for Myc-mediated suppression of p27^Kip1. A. Immunoblot analyses of Myc and p27^Kip1 protein levels in pre-cancerous (4 week-old) splenic B220⁺ B cells from non-transgenic (wt) and Eμ-Myc transgenics of the indicated Skp2 genotypes. B. Immunoblot analysis of Myc and p27^Kip1 levels in ex vivo cultured B cells from pre-cancerous Eμ-Myc;Skp2^+/+ versus Eμ-Myc;Skp2^−/− mice. Two separate experiments of B cells cultured from different paired littermates are shown. C. SYBR-green real-time PCR analysis of p27 mRNA levels in splenic B cells from pre-cancerous Eμ-Myc;Skp2^+/+, Eμ-Myc;Skp2^+/−, and Eμ-Myc;Skp2^−/− mice compared to a non-transgenic wild type littermate (wt). Levels of mRNAs were standardized to the expression of Ubiquitin (Ub).