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Cellular senescence has been recently shown to play an important role in opposing tumour initiation and promotion. Senescence induced by oncogenes or loss of tumour suppressor genes is thought to critically dependent on the induction of the p19Arf-p53 pathway. The Skp2 E3-ubiquitin ligase can act as a proto-oncogene and its aberrant overexpression is frequently observed in human cancers. Here we show that although Skp2 inactivation on its own does not induce cellular senescence, aberrant proto-oncogenic signals as well as inactivation of tumour suppressor genes do trigger a potent, tumor-suppressive senescence response in mice and cells devoid of Skp2. Notably, Skp2 inactivation and oncogenic stress driven senescence neither elicits activation of the p19Arf-p53 pathway nor DNA damage, but instead depends on ATF4, p27, and p21. We further demonstrate that genetic Skp2 inactivation evokes cellular senescence even in oncogenic conditions in which the p19Arf/p53 response is impaired, whereas a Skp2-SCF complex inhibitor can trigger cellular senescence in p53/PTEN deficient cells and tumour regression in preclinical studies. Our findings therefore provide proof of principle evidence that Skp2 pharmacological inhibition may represent a general approach for cancer prevention and therapy.
Cellular senescence represents an irreversible form of cell-cycle arrest that can be triggered by a variety of insults. Induction of cellular senescence (for example, by oncogenic Ras) results in p19Arf (encoded by the Ink4a/Arf locus, also known as Cdkn2a locus) and p53 accumulation, which is critical for this senescence response. Recent studies suggest that cellular senescence can act as an important tumour-suppressive mechanism to restrict tumour development in vivo1-7.
Inactivation of PTEN functions is frequently observed in human cancers8-10. Although PTEN negatively regulates cell proliferation and survival, we surprisingly discovered that acute Pten inactivation triggers the accumulation of p19Arf–p53 and cellular senescence2. Concomitant inactivation of p53 (also known as Trp53 in mice, and TP53 in humans) and Pten abrogates this senescence response, in turn promoting invasive and lethal prostate cancer2. Although these findings further underscore the critical importance of the cellular senescence Arf–p53 failsafe pathway, the frequent loss or mutation of ARF or P53 in human cancers would compromise the tumour-suppressive efficacy of this response, thereby limiting therapeutic potential.
Skp2 is a critical component of the Skp2–SCF complex, which acts as an E3 ligase to target p27 and other substrates for ubiquitylation and degradation11,12. Recent studies suggest that Skp2 may have oncogenic activity13-16. Notably, SKP2 overexpression is frequently observed in human cancer11,12,17, strongly suggesting that SKP2 overexpression may contribute to tumorigenesis. Skp2-knockout mice are viable and fertile18. Hence, specific inactivation of Skp2 may represent an appealing therapeutic modality. Here we show that Skp2 inactivation profoundly restricts tumorigenesis by eliciting cellular senescence only in oncogenic conditions. Remarkably, this senescence response is triggered in a p19Arf–p53-independent manner. Skp2 pharmacological inactivation may therefore represent a general approach towards a ‘pro-senescence’ therapy for cancer prevention and treatment.
Skp2 deficiency delays cell cycle progression11,12. We therefore asked whether Skp2 deficiency would trigger cellular senescence. We isolated mouse embryonic fibroblasts (MEFs) from wild-type and Skp2−/− mice and determined cellular senescence in these cells by senescence-associated β-galactosidase (SA-β-gal) staining. Although Skp2−/− MEFs proliferated less than wild-type MEFs (Supplementary Fig. 1d)11,12, cellular senescence in Skp2−/− MEFs was comparable to that in the wild-type MEFs (Supplementary Fig. 1a). In contrast, acute inactivation of Pten in MEFs markedly increased cellular senescence as previously reported (Supplementary Fig. 1a)2. Thus, Skp2 deficiency by itself does not elicit cellular senescence.
Ectopic overexpression of proto-oncogenic Ras (H-RasV12) in MEFs elicits cellular senescence through the p19Arf–p53 pathway19,20. Simultaneous co-expression of E1A and Ras in MEFs overcomes Ras-induced senescence by preventing activation of the p19Arf–p53 pathway and resulting in oncogenic transformation20. Thus, E1A enables Ras to overcome the cellular senescence response. As Skp2 also cooperates with oncogenic Ras to induce cell transformation13, it is conceivable that Skp2 might also display its oncogenic activity by antagonizing Ras-induced cellular senescence. On this basis, we tested whether endogenous Skp2 activity is required for cellular transformation induced by Ras and E1A. Although cellular senescence was not observed in wild-type MEFs after Ras and E1A overexpression, Skp2 deficiency triggered cellular senescence (Supplementary Fig. 1b, c). We also found that the ability of Ras to induce cellular senescence was far greater in Skp2−/− MEFs than in wild-type MEFs (Supplementary Fig. 1b, c). It should be noted that the induction of p19Arf and p53 protein levels in Skp2−/− MEFs by Ras was comparable if not lower than that of wild-type MEFs (Supplementary Fig. 1d). Moreover, Skp2 inactivation profoundly restricted cell proliferation and transformation after Ras and E1A overexpression (Supplementary Fig. 1e, f) Thus, Skp2 inactivation triggers cellular senescence in the presence of powerful oncogenic signals, even when the p19Arf–p53 response is evaded.
We assessed whether Skp2 inactivation would trigger cellular senescence even when cells experience loss of major tumour-suppressive networks such as those controlled by Pten and p19Arf–p53. To this end, we crossed Skp2−/− mutants with Pten+/− and Arf−/− mutants. The resulting compound mice were further intercrossed to generate MEFs of different genotypes for cell proliferation and senescence assays. As aforementioned, Skp2−/− MEFs grew much slower than wild-type MEFs, whereas wild-type and Pten+/− MEFs grew comparably (Supplementary Fig. 2a). No obvious cellular senescence was observed in wild-type, Pten+/− and Skp2−/− MEFs (Fig. 1a) Surprisingly, Pten+/− Skp2−/− MEFs had a slower growth rate than Skp2−/− MEFs and exhibited full-blown characteristics of cellular senescence such as flattened large cells and positive SA-β-gal staining (Fig. 1a and Supplementary 2a). We also detected cellular senescence in Pten+/− Skp2−/− MEFs under hypoxic conditions (Supplementary Fig. 2b). We did not see cooperation between Pten inactivation and Skp2 deficiency in triggering apoptosis, although Skp2−/− MEFs had a higher rate of apoptosis than wild-type MEFs (Supplementary Fig. 3a)18. However, the apoptosis rate in the prostate of Pten+/− Skp2−/− mice was higher than in wild-type, Pten+/− and Skp2−/− mice (see later and Supplementary Fig. 3b).
As cellular senescence is largely dependent on activation of the p19Arf–p53 pathway in MEFs2,21,22, we determined whether this pathway is activated in Pten+/− Skp2−/− MEFs. Notably, we found that p19Arf and p53 protein levels in Pten+/− Skp2−/− MEFs were comparable to levels in wild-type MEFs (Fig. 1b and Supplementary Fig. 4), suggesting that the p19Arf–p53 pathway may not be involved in the senescence response in Pten+/− Skp2−/− MEFs. To test this hypothesis further, we exposed MEFs of various genotypes to two well-established p53-inactivating tools: a short hairpin RNA (shRNA) against p53 (ref. 23) or a dominant-negative p53 mutant (p53-DN)24. Notably, in both conditions cell growth was promoted in wild-type and Pten−/− MEFs (Supplementary Fig. 5a, b), but neither of them overcame the cellular senescence nor the growth arrest in Pten+/− Skp2−/− MEFs (Fig. 1c and Supplementary Fig. 5c), suggesting that Skp2-deficiency cooperates with Pten inactivation to trigger a new senescence response by a p19Arf–p53-independent pathway.
p19Arf induction is required for cellular senescence in MEFs in the context of acute Pten inactivation25, whereas loss of p19Arf leads to cell immortalization2,21,22. We investigated whether Skp2 inactivation could elicit cellular senescence in an Arf-deficient genetic background. Notably, Arf−/− Skp2−/− MEFs showed massive cellular senescence similar to Pten+/− Skp2−/− MEFs (Fig. 1d). Moreover, p53 expression was not induced in Arf−/− Skp2−/− MEFs (Supplementary Fig. 6a). This cellular senescence profoundly suppressed the growth of Arf−/− MEFs (Supplementary Fig. 6b). Skp2 deficiency also induced cellular senescence after p53 inactivation (Supplementary Fig. 6c, d).
DNA damage has been recently associated with cellular senescence26-28. However, we found no evidence of DNA-damage-response activation in Pten+/− Skp2−/− MEFs, as determined by the levels of phosphorylated-(S15)-p53 and -γ-H2ax (also known as γ-H2afx) (Supplementary Fig. 4). Collectively, these results support the notion that Skp2 inactivation can trigger a new type of cellular senescence that does not involve DNA damage and can suppress transformation even when the p19Arf–p53 response is impaired.
We next examined the molecular mechanism by which Skp2 deficiency synergizes with oncogenic insults to trigger cellular senescence. Although p53 and p19Arf levels remained unchanged, we found that Skp2 deficiency cooperated with Pten inactivation or Arf loss to induce p27 expression (Fig. 2a, b). p21 expression was also increased in Pten+/− Skp2−/− and Arf−/− Skp2−/− MEFs (Fig. 2a, b). E2F1, cyclin D1 and Cdt1, involved in cell cycle progression and DNA replication, are also targets for Skp2 (refs 12, 29). We found that cyclin D1, but not E2F1 and Cdt1, were induced in Pten+/− Skp2−/− MEFs (Supplementary Fig. 7a). Because cyclin D1 promotes cell cycle progression, its upregulation is unlikely to be involved in mediating senescence in Pten+/− Skp2−/− MEFs.
Endoplasmic reticulum (ER) stress proteins such as BiP (also known as Hspa5 or GRP78), phospho-Perk (p-Perk), and ATF4 are induced after oncogenic insults and have an important role in cellular senescence30. We did not find a significant increase in BiP or p-Perk (Supplementary Fig. 7b and data not shown) in Pten+/− Skp2−/− MEFs. In contrast, ATF4 was markedly induced in Pten+/− Skp2−/− MEFs (Fig. 2a). Likewise, we also observed a marked increase in ATF4 protein levels, but not p-Perk, in Arf−/− Skp2−/− MEFs (Fig. 2b and Supplementary Fig. 7c). The induction of ATF4 protein levels in Pten+/− Skp2−/− MEFs was not accompanied by messenger RNA upregulation, nor by the enhanced ATF4 protein stability (Supplementary Fig. 8 and data not shown). Instead, we observed an increase in phosphorylated eIF2α (p-eIF2α; also known as p-Eif2s1) in Pten+/− Skp2−/− and Arf−/− Skp2−/− MEFs compared to wild-type cells (Fig. 2a, b). Because p-eIF2α positively regulates ATF4 translation31, our results indicate that ATF4 upregulation is probably triggered by the enhancement of p-eIF2α levels.
As p27, p21 and ATF4 were induced in both Pten+/− Skp2−/− and Arf−/− Skp2−/− MEFs, we next determined whether their upregulation contributes to senescence. p27 (also known as cdkn1b) shRNA efficiently abrogated p27 expression and partially rescued growth arrest and cellular senescence in Pten+/− Skp2−/− MEFs (Fig. 2c, d and Supplementary Figs 9a, b and 10a). Similarly, knockdown of Atf4 or p21 (also known as Cdkn1a) in these cells also partially reversed cellular senescence and cell arrest (Fig. 2e–h and Supplementary Figs 9c and 10b, c). Concomitant knockdown of Atf4, p21 and p27 in these cells reversed cellular senescence more efficiently than their individual knockdown (Supplementary Fig. 10d). In contrast, in Skp2−/− MEFs, p27 knockdown accelerated growth whereas Atf4 knockdown did not (Supplementary Fig. 10e, f). These results strongly indicate that the concomitant upregulation of p27, p21and ATF4 is a required and powerful engine for the induction of cellular senescence upon Skp2 inactivation.
We found that inactivation of Skp2 in the presence of an oncogenic stress results in the induction of cellular senescence that opposes transformation in vitro even when the p19Arf–p53 response is impaired. We next determined whether Skp2 loss restricts tumorigenesis in vivo through similar mechanisms and first analysed tumorigenesis in Skp2−/− Pten+/− compound mutants (Supplementary Fig. 11a). Although Pten heterozygous inactivation reduced lifespan in mice, compound Skp2 deficiency prolonged overall survival (Fig. 3a). Pten+/− mice develop lymphadenopathy and adrenal tumours (pheochromacytoma) at complete penetrance32,33. As expected, Pten+/− mice developed adrenal tumours with 100% penetrance by 1 year of age, whereas Skp2 loss remarkably abrogated adrenal tumour formation in compound mutants (P < 0.0001; Fig. 3b, top, c and Supplementary Fig. 11b). Pten protein expression in adrenal tissues was comparable between wild-type, Pten+/− and Pten+/− Skp2−/− mice, before or after tumour occurrence, suggesting that there is no loss of heterozygosity at the Pten locus in the adrenal tissues in any of these mutants and conditions (Supplementary Fig. 11c). Lymphadenopathy after Pten inactivation was also profoundly inhibited by Skp2 loss (P < 0.01; Fig. 3b, top, and Supplementary Fig. 11d). Tumorigenesis was also markedly suppressed in other organs (for example, in the prostate, where the prostatic intraepithelial neoplasia (PIN) incidence was profoundly restricted by Skp2 inactivation; data not shown).
To determine whether Skp2 inactivation along with Pten inactivation would trigger cellular senescence in vivo, we performed SA-β-gal staining in the few hyperplastic lymphoid lesions still identified in Pten+/− Skp2−/− mice (see, for example, Supplementary Fig. 10d). We observed both cellular senescence and p27 induction in the lymphoid tissues from Pten+/− Skp2−/−− mice (Fig. 3d, e and Supplementary Fig. 12), which inversely correlated with cell proliferation (Fig. 3f).
We then examined whether Skp2 inactivation would also restrict tumorigenesis after Arf loss by crossing Skp2−/− with Arf−/− mice (Supplementary Fig. 13). Skp2 inactivation markedly prolonged the overall survival of Arf−/− mice (Fig. 4a). Around 33% of Arf−/− mice developed sarcoma and/or lymphoma within 1 year (Fig. 4b–d)34,35. In contrast, none of the Arf−/− Skp2−/− compound mutant mice showed signs of tumour formation (P < 0.02; Fig. 4b–d).
Complete Pten inactivation in the prostate triggers a tumour-suppressive cellular senescence response2. We therefore examined whether this response could be further potentiated by Skp2 loss and affect tumorigenesis after complete Pten inactivation in the prostate. For prostate-specific inactivation, we made use of Cre-loxP mediated recombination and probasin (Pbsn, also known as PB)-Cre4 transgenic mice expressing the Cre recombinase after puberty in the prostatic epithelium2. We obtained PtenloxP/loxP;PB-Cre4 and PtenloxP/loxPSkp2−/−;PB-Cre4 compound mutant mice, hereafter referred to as Ptenpc−/− and Ptenpc−/− Skp2−/− mice, respectively (Supplementary Fig. 14a). Although complete Pten inactivation in mouse prostates leads to invasive prostate cancers, it does not affect overall survival2. We did not detect a difference in overall survival between Ptenpc−/− and Ptenpc−/− Skp2−/− mice (Supplementary Fig. 14b).
Prostate cancer development in these mice was monitored by magnetic resonance imaging (MRI) and histopathological analysis. Consistent with our previous findings2, MRI analysis showed prostate tumour masses in Ptenpc−/− mice at 6 months of age, which were significantly reduced in Ptenpc−/− Skp2−/− mice (Supplementary Fig. 14c). The average size of the prostate in Ptenpc−/− mice was tenfold larger than in wild-type mice, whereas complete Skp2 loss markedly reduced tumour weight after complete Pten inactivation (Fig. 5a). Histological analysis showed that Skp2 loss inhibited invasive prostate cancer after Pten inactivation, albeit PIN lesions were still observed in Ptenpc−/− Skp2−/− mice (Supplementary Fig. 14d, e). Furthermore, this suppressive effect by Skp2 loss was persistent, as we also observed a profound reduction in tumour weight and invasive prostate cancer in Ptenpc−/− Skp2−/− mice at 15 months of age (Supplementary Fig. 14f, g).
We next investigated, in vivo, the molecular basis for tumour suppression elicited by Skp2 inactivation. We found that p27 protein expression was synergistically induced in prostates from compound mutants, as determined by immunohistochemistry and western blot analysis (Supplementary Fig. 15a, b), whereas p53 expression was comparably induced in Ptenpc−/− and Ptenpc−/− Skp2−/− mice (Supplementary Fig. 15c). Skp2 deficiency profoundly enhanced cellular senescence upon Pten inactivation (Fig. 5b). This was observed at earlier time points and inversely correlated with cell proliferation (Supplementary Fig. 16a, b). Notably, this response was also sustained over time. We could detect massive β-gal positivity in prostates from Ptenpc−/− Skp2−/− mice even at 15 months of age, whereas β-gal positivity was barely detected at that age in prostates from Ptenpc−/− mice (Fig. 5c). Thus, Skp2 inactivation potentiates and sustains over time the senescence response elicited by an oncogenic stimulus, suggesting that pharmacological inhibition of Skp2 may be used as a powerful pro-senescence approach for cancer therapy and chemoprevention.
To corroborate the potential use of such an approach for cancer therapy, we determined whether pharmacological inactivation of the Skp2–SCF complex induces cellular senescence in p53-deficient cells and, importantly, suppresses the growth of the pre-formed tumours. To this end, we took advantage of MLN4924 (ref. 36)—an inhibitor for the neddylation of cullin 1, which is a component of Skp2–SCF complex. We used PC3 prostate cancer cells for this preclinical analysis because these cells are both p53-null and PTEN-null, hence representing one of the most aggressive genetic states encountered in human cancer. Remarkably, treatment of MLN4924 in PC3 cells triggered cellular senescence (Fig. 5d). Moreover, the growth of PC3 tumours treated with MLN4924 in vivo was also suppressed (Fig. 5e). Coherent with these findings, Skp2 silencing in PC3 and in DU145 prostate cancer cells, which have also evaded the p53 response, triggered cellular senescence and cooperated with the DNA-damaging agent doxorubicin to induce cellular senescence and growth arrest (Supplementary Fig. 17). These results demonstrate the critical role of Skp2 inactivation in the induction of cellular senescence not only in mouse cells, but also in human cancer cells experiencing failure of p53 and other major tumour suppressive networks.
On the basis of our results, we propose a working model for the role of Skp2 inactivation-induced cellular senescence in tumour prevention and suppression in vivo (Fig. 5f). This model rests on three new and unexpected findings with important therapeutic implications. First, Skp2 inactivation does not trigger cellular senescence in vivo or in vitro on its own, but rather elicits a senescence response after oncogenic stress. This response is critically dependent on p27, p21 and ATF4 induction. Our results are supported by recent reports showing that acute inactivation of the von Hippel–Lindau (VHL) tumour suppressor in vitro or overexpression of the human T-lymphotropic virus type 1 (HTLV-1) Tax triggers Skp2 downregulation and cellular senescence37,38. Second, we show that cellular senescence driven by Skp2 inactivation along with oncogenic insults takes place without the activation of the p19Arf–p53 failsafe pathway. Although senescence is also observed in p53/PTEN-null cells such as PC3, it will be important to determine the specific genetic states that favour evasion of this failsafe mechanism, also in a cell-type-specific manner. For instance, loss or constitutively low expression of p27, p21 and ATF4 could impair this response. This knowledge will in turn identify new pharmacological nodes of tumour-type-specific intervention. Third, we show that Skp2 deficiency in conjunction with oncogenic signals elicits a senescence response that profoundly restricts tumorigenesis in vivo in numerous mouse models in which tumour suppressor networks are faulty or inactive. Our findings are consistent with a recent report demonstrating that mice transplanted with BCR–ABL-transduced Skp2−/− bone marrow cells show a delayed onset of a myeloproliferative syndrome39.
As Skp2 can in principle be subjected to specific pharmacological inhibition because of its enzymatic activity, our results call for the development and optimization of Skp2 small molecule inhibitors. Skp2 pharmacological inhibition could be particularly appealing and effective in view of the fact that complete Skp2 inactivation in the mouse is compatible with life, whereas cellular senescence is only triggered by Skp2 inactivation in conjunction with oncogenic conditions.
PtenloxP/loxP, Arf−/− and Skp2−/− mice were generated as described previously2,18,35. Female PtenloxP/loxP mice were crossed with male PB-Cre4 transgenic mice for the prostate-specific deletion of Pten. MEFs from wild-type and Skp2−/− mice were prepared as previously described40,41 and cultured in DMEM containing 10% FBS. Cellular senescence was determined by assessing SA-β-gal activity, and the in vivo cell proliferation assay was performed by Ki67 staining on the paraffin tissue sections. The cell transformation assay was determined by the soft agar assay. p53 shRNA is from S. W. Lowe and the pBabe-p53 dominant-negative construct is a gift from M. Oren. MLN4924 was obtained from Millennium Pharmaceuticals.
For retrovirus infection system p27 shRNA (5′-GTGGAATTTCGACTTTCAG-3′), Atf4 shRNA (5′-GAGCATTCCTTTAGTTTAG-3′), and GFP shRNA (5′-GCAAGCTGACCCTGAAGTTC-3′) were subcloned into the pSUPER-puro vector (Oligoengine) according to standard procedures and transfected into Phoenix packaging cells. For lentiviral shRNA infection, 293T cells were co-transfected with p27, Atf4, p21, Skp2 or GFP control shRNA along with packing plasmids (deltaVPR8.9) and envelope plasmid (VSV-G) using Lipofectamine 2000 reagents according to the manufacturer's instructions. Skp2-lentivivral shRNA-1 (5′-GATAGTGTCATGCTAAAGAAT-3′), p27-lentivivral shRNA-1 (5′-CGCAAGTGGAATTTCGACTTT-3′), p27-lentivivral shRNA-2 (5′-CCCGGTCAATCATGAAGAACT-3′), Atf4-lentivivral shRNA-1 (5′-GCGAGTGTAAGGAGCTAGAAA-3′), Atf4-lentivivral shRNA-2 (5′-CGGACAAAGATACCTTCGAGT-3′), p21-lentivivral shRNA-1 (5′ CTGGTGTCTGAGCGGCCTGAA-3′), p21-lentivivral shRNA-2 (5′-GACAGATTTCTATCACTCCAA-3′), and GFP shRNA (5′-GCAAGCTGACCCTGAAGTTC-3′) were transfected with packing plasmids into 293T cells for 2 days, and virus particles containing p27, p21, Atf4, Skp2 or GFP shRNA were used to infect mammalian cells. All the infected cells were cultured in a medium containing the appropriate antibiotics.
Cell lysates were prepared with RIPA buffer (PBS, 1% Nonidet P40, 0.5% sodium deoxycholate, 0.1% SDS and protease inhibitor cocktail (Roche)). The following antibodies were used for western blot analysis: anti-p19Arf (NeoMarkers), anti-p53 (Novocastra), anti-p21 (Santa Cruz), anti-β-actin (Sigma), anti-Hsp90 (BD transduction laboratories), anti-p27 (BD transduction laboratories), anti-α-tubulin (Sigma), anti-phospho-p53 (Ser15) (Cell Signaling), anti-phospho-H2ax (Ser139) (Cell Signaling), anti-phospho-eIF2α (Ser51) (Cell Signaling), anti-eIF2α (Cell Signaling), anti-phospho-Perk (Thr980) (Cell Signaling), anti-cyclin D1 (Santa Cruz), anti-E2F1 (Santa Cruz), anti-Cdt1 (Proteintech Group), anti-Ras (Oncogene), anti-E1A (Neomarkers), and anti-ATF4 (Santa Cruz). For immunohistochemistry, tissues were fixed in 10% formalin and embedded in paraffin in accordance with standard procedures. Sections were stained with anti-p27 (BD transduction laboratories), anti-Ki67 (Novocastra), anti-PTEN (Neomarkers) and anti-p53 (Novocastra) antibodies.
Primary MEFs were isolated from individual embryos of various genotype at passage 2, infected with retroviruses or lentiviruses expressing GFP shRNA, p27 shRNA or Atf4 shRNA for 2 days, selected with 2 μg ml−1 puromycin for 4 days, and plated for the cell proliferation and senescence assay. For cell proliferation assay, 2 × 104 MEFs were seeded in 12 wells in triplicate, collected, and stained with trypan blue at different days. Numbers of viable cells were directly counted under the microscope. To determine cellular senescence, MEFs were plated at 104 cells per well of a 6-well plate in triplicate, and after 4 days SA-β-gal activity was measured using the senescence detection kit (Calbiochem) and quantified (around 100–200 cells per well). For in vivo cellular senescence, frozen sections 6-μm thick were stained for β-gal as described earlier. For in vivo cell proliferation, the paraffin section was used for Ki67 staining, and the percentages of Ki67-positive cells (around 500 cells) from each sample were counted. For transformation assay, wild-type and Skp2−/− MEFs (3 × 104) infected with RasG12V and E1A were suspended in a medium containing 0.3% agar onto solidified 0.6% agar per well of a 6-well plate, and the number of colonies was counted after 21 days.
Primary MEFs of various genotypes of mouse embryos were cultured in 10% FBS for 2 days; cells were collected and labelled with Annexin-V–FITC, followed by a flow cytometry analysis.
Individual mice were subjected to MRI assessment for the detection of prostate tumours as described42.
Nude mice bearing PC3 xenograft tumours (around 300 mm3) were treated with vehicle or 90 mg kg−1 MLN492. Tumour weight was measured at the time of collection after 15 days of treatment with a scheduling regimen of 3 days of treatment followed by 3 days off treatment for a total of three courses.
We are grateful to C. J. Sherr, S. W. Lowe and M. Oren for mice and reagents. We would also like to thank B. Carver, L. DiSantis, J. Clossey and S. Megan for editing and critical reading of the manuscript, J. A. Koutcher, C. Le, C. Matei and M. Lupa for MRI analysis, as well all the members of the Pandolfi laboratory for insightful comments and discussion. We extend our thanks to M. Rolfe, P. G. Smith, and Millennium Pharmaceuticals for discussion and for providing the MLN4924 compound. This work was supported by NIH grants to P.P.P. and M.D. Anderson Trust Scholar Award and DOD Prostate Cancer New Investigator Award to H.K.L.
Supplementary Information is linked to the online version of the paper at www.nature.com/nature.
The authors declare no competing financial interests.