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Address reprint request to Marcelo L. Rodriguez-Puebla, Ph.D., North Carolina State University, College of Veterinary Medicine, Molecular Biomedical Sciences, 4700 Hillsborough St, Raleigh, NC 27606
Center for Comparative Medicine and Translational Research, North Carolina State University, Raleigh, North CarolinaDepartment of Molecular Biomedical Sciences, North Carolina State University, Raleigh, North Carolina
The proto-oncogene c-Myc encodes a transcription factor that is implicated in the regulation of cellular proliferation, differentiation, and apoptosis. Myc accelerates the rate of cell proliferation, at least in part, through its ability to down-regulate the expression of the cell cycle inhibitor p27Kip1. Moreover, p27Kip1 protein levels are regulated by ubiquitin-mediated turnover, leading to destruction by the E3 ubiquitin ligase SCFSkp2. Therefore, we hypothesize that a lack of Skp2 expression should lead to increased p27Kip1 levels and further inhibition of Myc-mediated proliferation and tumorigenesis. Myc expression in epithelial tissues of transgenic mice (K5-Myc) led to increased keratinocyte proliferation and the development of spontaneous tumors within the oral cavity. We generated K5-Myc–transgenic mice in an Skp2-null background. Consistent with our hypothesis, we found that Myc-mediated keratinocyte hyperproliferation was abolished by the loss of Skp2. However, Skp2 ablation did not affect Myc-driven tumorigenesis because the incidence, latency, and degree of differentiation of oral tumors were identical between K5-Myc/Skp2+/+ and K5-Myc/Skp2−/− mice. Altogether, these findings suggest that Skp2 and p27Kip1 are critical for Myc-driven keratinocyte proliferation; however, Myc-mediated tumorigenesis in the oral epithelium is independent of the Skp2-p27Kip1 axis.
The proteasome pathway involves ubiquitin modification and degradation of substrates by the proteasome complex. Ubiquitin-mediated protein turnover is regulated by E3 ubiquitin ligases, such as SCF and anaphase-promoting complexes. The E3 SCFSkp2 complex is composed of four subunits: S-phase kinase-associated protein 1 (Skp1), cullin, a ring-finger protein, and Skp2, which is a member of a large family of F-box adaptor proteins.
Skp2 targets several cell cycle regulators for ubiquitination, including the Cip/Kip family of cyclin-dependent kinase inhibitors (p21Cip1, p27Kip1, and p57Kip2), cyclin D1, p130, and Myc (alias c-myc).
Skp2 contributes to G2/M progression by mediating the degradation of p27Kip1, which is responsible for most of the phenotypes exhibited by Skp2-null mice.
In addition, Skp2 also targets other important regulators of survival and/or apoptosis for degradation. For instance, Skp2 suppresses p53-dependent apoptosis by antagonizing the interaction between CREB-binding protein/p300 and p53.
Consistent with its role in tumor development, Skp2 is overexpressed in many experimental and human tumors and has a transforming capacity; therefore, it is classified as an oncogene.
Notably, elevated levels of Skp2 in human tumors correlate with low p27Kip1 levels; overexpression of Skp2 in prostate epithelium decreases p27Kip1 levels and induces proliferation.
The Myc proto-oncogenes are members of short-lived transcription factors, a family that plays an important role in cell proliferation, apoptosis, and cancer development.
The role of Myc in cell proliferation is partly mediated through its ability to down-regulate the expression of p27Kip1. Myc regulates p27Kip1 levels through several mechanisms, such as protein level,
Myc induces E2f1, which promotes cyclin E transcription and further activation of cyclin E–CDK-2 complexes, which, in turn, phosphorylate p27Kip1 on Thr187, allowing its recognition by SCFSkp2.
Thus, Myc contributes to p27Kip1 protein degradation, which is a key regulator of Myc-induced proliferation and tumorigenesis. More important, Myc mediates p27Kip1 degradation by inducing Skp2 in B cells and fibroblasts, although Skp2 deficiency had a modest effect on Myc-induced proliferation and lymphomagenesis.
The fact that both Myc and Skp2 oncogenic activities seem to be partly mediated by p27Kip1 down-regulation led us to hypothesize that a lack of Skp2 should increase p27Kip1 protein levels, thus blocking Myc-mediated tumorigenesis. To test this prediction, we developed the K5-Myc/Skp2−/− compound mouse. K5-Myc and other transgenic mouse models have shown that Myc overexpression in the basal and suprabasal cell layers of stratified epithelia leads to hyperplasia, increased epidermal thickness, and keratinocyte proliferation.
As expected, Myc-mediated epidermal proliferation was abolished in K5-Myc/Skp2−/− mice. However, the incidence, latency, and degree of differentiation of oral tumors were identical between K5-Myc and K5-Myc/Skp2−/− mice. Collectively, these findings suggest that Skp2 plays an important role in Myc-induced keratinocyte proliferation. However, similar to the modest effects observed in Myc-induced lymphomagenesis,
K5-Myc–transgenic mice were bred with mice heterozygous for Skp2 (Skp2+/−) to generate K5-Myc/Skp2+/−. These mice were bred with Skp2+/− mice to generate K5-Myc–transgenic and nontransgenic mice that were homozygous, heterozygous, or nullizygous for Skp2.
Transgene-Specific PCR
Genomic DNA was extracted from mouse tail clips and used for genotyping with PCR. K5-Myc–positive mice were determined with upstream (5′-CTGACCAGCAGTACGAATG-3′) and downstream (5′-GAGTCCAATCACGTCCAAG-3′) primers specific for the β-globin intron sequence, which renders a 450-bp product. The Skp2 wild-type allele was amplified with upstream (5′-GCATCGCCTTCTATCGCCTTCTTG-3′) and downstream (5′-CCCGTGGAGGGAAAAAGAGGGACG-3′) primers that produce a 430-bp band, and the Skp2-null allele was amplified with upstream (5′-AGAGTGGAAGAACCCAGGCAGGAC-3′) and downstream (5′-TTCCCACCCCCACATCCAGTCATT-3′) primers that produce a 500-bp band.
Western Blot Analysis and Kinase Assays
Mice were sacrificed, and the dorsal surface of each animal was shaved, treated with a depilatory agent for 1 minute, and then rinsed with tap water. The dorsal surfaces were then excised, and the epidermal tissues were scraped off with a razor blade. The epidermis was then harvested in homogenization buffer [50 mmol/L HEPES (pH 7.5), 150 mmol/L NaCl, 2.5 mmol/L EGTA, 1 mmol/L ethylenediaminetetraacetic acid, 0.1% Tween-20, 1 mmol/L dithiothreitol, 0.1 mmol/L phenylmethylsulfonyl fluoride, 0.2 U/mL aprotinin, 10 mmol/L β-glycerophosphate, 0.1 mmol/L sodium vanadate, and 1 mmol/L NaF] and homogenized with a manual homogenizer. The epidermal homogenate was centrifuged at 14,000 rpm at 4°C to collect the supernatant, which was used directly for Western blotting analysis or stored at −80°C. The protein concentration was determined using a protein assay system (Bio-Rad Laboratories, Richmond, CA). Protein lysates (30 μg from each sample) were electrophoresed on 12% acrylamide gels and electrophoretically transferred onto nitrocellulose membranes. After being blocked with 5% nonfat powdered milk in Dulbecco's PBS, the membranes were incubated with 1 μg/mL of the specific antibodies. Polyclonal antibodies were used against the following proteins: p21 (H-164), p27 (M-197), and p27 (C-19) (Santa Cruz Biotechnology, Santa Cruz, CA). Peroxidase-conjugated anti-mouse and anti-rabbit polyclonal secondary antibodies, followed by enhanced chemiluminescence (ECL detection kit; GE Healthcare, Piscataway, NJ), were used for immunoblotting detection. To study the kinase activities, 500 μg of fresh protein was extracted and immunoprecipitated in NP-40 lysis buffer [Tris (pH 7.5), 150 mmol/L NaCl, 0.5% NP-40, 50 mmol/L NaF, 1 mmol/L Na3VO4, 1 mmol/L dithiothreitol, and 1 mmol/L phenylmethylsulfonyl fluoride] with precoated antibodies against CDK2 and CDK4 for 2 hours at 4°C. Beads were washed twice each with NP-40 buffer and once with kinase buffer [50 mmol/L HEPES (pH 7), 10 mmol/L MgCl2, and 5 mmol/L MnCl2]. Then, 30 μL of kinase buffer, 1 μg of pRb or histone H1 (Upstate Biotechnology Inc., Charlottesville, VA) substrate, 5 μCi of [γ-32P] ATP (6000 Ci/mmol), 1 mmol/L dithiothreitol, and 5 μmol/L ATP were added to the bead pellet and incubated for 30 minutes at 30°C. SDS sample buffer was added, and each sample was boiled for 3 minutes to stop the reaction and electrophoresed through polyacrylamide gels. Western blot and kinase assay bands were quantified using gel software (UN-SCANT IT) for Windows.
Immunostaining
For immunofluorescence, tissue cross sections of formalin-fixed, parafin-embedded mouse skin and heads were permeabilized using citrate antigen retrieval buffer (H-3300; Vector Laboratories Inc., Burlingame, CA), blocked with 10% normal goat serum (S-1000; Vector Laboratories Inc.), and stained with antibodies against p27Kip1(C-19; Santa Cruz Biotechnology), followed by incubation with a conjugated secondary antibody (Alexa Fluor; Invitrogene, Carlsbad, CA). ImageJ software (http://rsb.info.nih.gov/ij) was used to quantify the accumulation and localization of p27Kip1.
Epithelial cell proliferation was measured by i.p. injection of 60 μg/g of 5-bromodeoxyuridine (BrdU) 30 minutes before the mice were sacrificed using CO2 asphyxiation. BrdU incorporation was detected using immunohistochemical (IHC) staining of paraffin-embedded skin sections with a mouse anti-BrdU (ab-2) monoclonal antibody (Calbiochem, EMB Biosciences, San Diego, CA), a biotin-conjugated anti-mouse antibody (Vector Laboratories Inc.), and an avidin-biotin peroxidase kit (Vectastain Elite; Vector Laboratories Inc.), with diaminobenzidine as the chromogen. Apoptotic cells were determined using terminal deoxynucleotidyl transferase–mediated dUTP nick-end labeling assays with a kit (FragEL DNA Fragmentation Detection kit and Colorimetric-TdT enzyme; Calbiochem, EMB Biosciences Inc.), following the manufacturer's instructions. The cells were counterstained with methyl green to quantify normal and apoptotic cells. The number of apoptotic cells in the tumors was determined in 250-μm2 sections using a reticule grid. Apoptotic keratinocytes in the interfollicular and follicular epidermis were quantified in 1-cm sections. Hair follicles carrying at least one apoptotic cell in the bulge were counted as positive to determine the incidence of apoptosis in the follicles. In all cases, 12 fields were counted per section in 10 paraffin-embedded sections, representing five mice per genotype.
Statistical Analysis
Statistical analysis was performed using computer software (GraphPad Prism 4 Software; GraphPad Software, San Diego).
Results
Lack of Skp2 Reduces Epidermal Thickness and Proliferation
The lack of Skp2 induces the accumulation of p27Kip1 and cyclin E, resulting in a decrease in the rate of proliferation of mouse embryo fibroblasts and T lymphocytes.
These data suggest that a reduced rate of cell growth may cause the small body size of Skp2−/− mice. The effect of Skp2 ablation in mouse epidermis has not been previously evaluated; therefore, we determined the role of Skp2 in keratinocyte proliferation. An analysis of paraffin-embedded skin tissue revealed that Skp2−/− mice exhibited hypoplastic epidermis (20% decrease in the number of interfollicular keratinocytes) and reduced keratinocyte proliferation, as observed by an 87% decrease in BrdU incorporation compared with wild-type littermates (P < 0.001, Tukey-Kramer multiple-comparison test; Figure 1). In agreement, the epidermal thickness was also reduced twofold in Skp2−/− mice (Figure 1B). Next, we determined whether p27Kip1 protein levels were altered in Skp2−/− keratinocytes. Western blot analysis of epidermal lysates from Skp2−/− mice showed a twofold increase in p27Kip1 protein levels compared with wild-type siblings (Figure 2A). This result is in agreement with previously published data using Skp2−/− mouse embryo fibroblasts and primary hepatocytes.
As previously mentioned, there was an 87% reduction in BrdU-positive S-phase keratinocytes in the Skp2−/− epidermis. Therefore, to study p27Kip1 accumulation in S-phase keratinocytes, we induced rapid entry into the S phase using a topical application of 12-O-tetradecanoylphorbol-13-acetate on mouse epidermis. Multiple applications of 12-O-tetradecanoylphorbol-13-acetate drive 90% of keratinocytes into the S phase within 24 hours of treatment. Accordingly, we analyzed mouse skin treated with three consecutive applications of 12-O-tetradecanoylphorbol-13-acetate at 24-hour intervals. Western blot analysis of S-phase Skp2−/− keratinocytes revealed a 10-fold increase in p27Kip1 protein levels compared with wild-type epidermis (Figure 2A). More important, other putative Skp2 substrates, such as p21Cip1 (Figure 2), D-type cyclins, and cyclin E (data not shown), did not accumulate in the Skp2−/− epidermis. Therefore, these results suggest that degradation of p27Kip1 by Skp2 plays a major role in normal keratinocyte proliferation and skin homeostasis. To determine whether p27Kip1 accumulation affects CDK activities, we studied the CDK2 and CDK4 in vitro activities in mouse epidermis lysates. Surprisingly, we did not detect changes in CDK2 activities between wild-type and Skp2−/− mice (Figure 2B); however, a 2.5-fold reduction of CDK4 activity was observed on the Skp2−/− epidermis compared with wild-type siblings (Figure 2B). These results are consistent with the previous observation that lack of p27Kip1 increases CDK4 activity.
Altogether, these results suggest that Skp2 ablation leads to diminished keratinocyte proliferation, likely through p27Kip1-mediated CDK4 inhibition.
Figure 1K5-Myc/Skp2−/− epidermis phenotype. A: Representative paraffin-embedded sections of skin stained with H&E from wild-type, K5-Myc, Skp2−/−, and K5-Myc/Skp2−/− mice. B: Quantification of keratinocyte proliferation and epidermal thickness. Epidermal proliferation was quantified by counting the total number of cells in the epidermis and BrdU-positive cells in the basal layer of the interfollicular epidermis of wild-type, K5-Myc, Skp2−/−, and K5-Myc/Skp2−/− littermates. Epidermal thickness was determined in 10 consecutive sections for each sample and is presented as the average thickness (μm) for each group. The error bars represent the SEM.
Figure 2Cell cycle protein analysis from mouse epidermis. A: Protein lysates of normal and 12-O-tetradecanoylphorbol-13-acetate (TPA)–treated (asterisk) epidermis from wild-type and Skp2−/− mice were separated by SDS-PAGE and blotted onto a nitrocellulose membrane. A primary antibody against p27Kip1 was used for immunoblotting analysis. B: Protein lysates of normal epidermis from wild-type and Skp2−/− mice were immunoprecipitated with antibodies against CDK2 (IP/cdk2) and CDK4 (IP/cdk4), and in vitro protein kinase assays were performed with a pRb peptide (Rb) or histone H1 (H1) as the substrate. Co indicates that protein lysates from wild-type epidermis were immunoprecipitated with unrelated antibodies; No Sub, protein lysates from wild-type epidermis were immunoprecipitated with antibodies against CDK2 and CDK4, and kinase assays were developed in the absence of the respective substrates. C: Protein lysates of normal epidermis from K5-Myc, K5-Myc/Skp2−/−, Skp2−/−, and wild-type mice were separated by SDS-PAGE and blotted onto a nitrocellulose membrane. Primary antibodies against Skp2, p27Kip1, and p21Cip1 were used for immunoblotting analysis. β-Actin was used as the loading control, and protein levels were quantified with a densitometer.
have previously reported that the skin of K5-Myc–transgenic mice shows epidermal hyperplasia, a feature that contributes to the increased epidermal thickness observed in these mice, and increased proliferative keratinocytes in the basal cell layer (Figure 1). To investigate whether genetic inhibition of Skp2 could affect Myc-mediated epidermal proliferation, we developed K5-Myc–transgenic mice lacking Skp2 (K5-Myc/Skp2−/− mice). Histological analysis of epidermal cross sections of K5-Myc/Skp2−/− mice showed a complete reversion of the increased epidermal thickness observed in K5-Myc mice (Figure 1). Quantification of the epidermal thickness revealed a significant reduction in K5-Myc/Skp2−/− mice compared with K5-Myc siblings (13 versus 18 μm; P < 0.01, Tukey-Kramer multiple-comparison test; Figure 1B). Moreover, Skp2 ablation in K5-Myc mice led to a twofold reduction in proliferative keratinocytes (BrdU labeling index) compared with K5-Myc mice (P < 0.0001). More important, the number of keratinocytes in the S phase was reduced by 30% in K5-Myc/Skp2−/− mice compared with wild-type littermates (Figure 1B), suggesting that Skp2 plays an essential role in regulating keratinocyte proliferation.
Myc-induced proliferation is partly mediated by p27Kip1 down-regulation.
Consistent with these data, epidermal lysates from K5-Myc mice showed a threefold reduction in p27Kip1 protein levels compared with wild-type littermates (Figure 2C, lanes 1 and 2). In agreement with the reversion of Myc-induced proliferation, the lack of Skp2 in K5-Myc mice led to a fivefold increase in p27Kip1 protein levels compared with K5-Myc littermates (Figure 2C, lanes 1 and 3). Analysis of other putative Skp2 targets, such as p21Cip1, did not show relevant changes among the four analyzed genotypes.
Overall, our results are consistent with the recently reported suppression of p27Kip1 in B cells and lymphomas that arise in Eμ-Myc–transgenic mice.
Therefore, these results strongly suggest that p27Kip1 accumulation on Skp2 ablation counteracts the Myc action in the mouse epidermis, leading to a reversion of Myc-induced keratinocyte proliferation and hyperplasia.
Ablation of Skp2 Does Not Inhibit Myc-Induced Oral Tumorigenesis
Overexpression of the murine Myc gene in the basal cell layer of stratified epithelia results in a high incidence of spontaneous tumors in the oral mucosa of transgenic mice,
but the mechanism leading to this malignant phenotype is not fully understood. We demonstrated that Skp2 and p27Kip1 are key regulators of Myc-induced proliferation in mouse epidermis. Moreover, loss of p27Kip1 accelerates lymphoma development in the Eμ-Myc model of human Burkitt lymphoma.
Therefore, we reasoned that lack of Skp2, and the consequent accumulation of p27Kip1, would counteract the effect of Myc expression, blocking tumorigenesis. To test this hypothesis, we analyzed the onset of oral tumors in K5-Myc–transgenic mice lacking Skp2 (K5-Myc/Skp2−/− mice), along with their K5-Myc/Skp2+/−, K5-Myc/Skp2+/+, Skp2+/−, Skp2−/−, and wild-type siblings. Histological examination of mice bearing the Myc transgene with one, two, or no functional Skp2 alleles revealed no significant differences in the onset of tumors in the oral mucosa among these genotypes. These tumors were classified as squamous cell carcinomas consisting of islands (ie, cords of neoplastic epithelial cells showing a variable degree of squamous differentiation, with occasional keratin pearls in the centers). The invasive margins of the tumors were surrounded by proliferative fibrous connective tissue (Figure 3). The tumors were observed in mice as early as 10 weeks old, with an incidence of approximately 90% in K5-Myc, K5-Myc/Skp2+/−, and K5-Myc/Skp2−/− mice (Figure 3). No differences were found among latency, incidence, differentiation, or level of proliferation among K5-Myc, K5-Myc/Skp2−/−, and K5-Myc/Skp2+/− mice (Figure 3G). Nontransgenic Skp2−/−, Skp2+/−, and Skp2+/+ mice were analyzed up to the age of 12 months; and no signs of spontaneous tumor development in the oral cavity were detected. In contrast to the epidermis, no hypoplasia was observed in the oral epithelium of Skp2−/− mice compared with wild-type mice (Figure 3, A and D). Moreover, histological analysis did not show any significant differences in the number of cells or the structure of the oral epithelium between K5-Myc and K5-Myc/Skp2−/− mice (Figure 3). Consequently, immunostaining analysis of oral tumors showed similar levels of p27Kip1 in both K5-Myc and K5-Myc/Skp2−/− tumors (data not shown).
Figure 3Odontogenic tumors in K5-Myc and K5-Myc/Skp2−/− mice. Representative paraffin-embedded sections of the oral cavity (A and D) and odontogenic tumors (B and E) were obtained from K5-Myc (A–C) and K5-Myc/Skp2−/− (D–F) mice stained with H&E. C and F: Magnification of the insets from B and E, respectively. G: Histopathological analysis of K5-Myc, K5-Myc/Skp2+/−, and K5-Myc/Skp2−/− siblings. The percentage of mice with odontogenic tumors classified as squamous cell carcinomas is given. Latency was determined as weeks of tumor onset. None of the Skp2+/−, Skp2−/−, and wild-type mice developed tumors. H: Immunofluorescence analysis of p27Kip1 expression on oral cavity epithelium from wild-type (Wt), Skp2−/−, K5-Myc, and K5-Myc/Skp2−/− mice. NC indicates negative control (Skp2−/− section without a specific primary antibody).
Histopathological analysis of the oral epithelium showed no significant differences in cell proliferation (BrdU incorporation) and apoptosis among K5-Myc, K5-Myc/Skp2+/−, and K5-Myc/Skp2−/− tumors (data not shown). Consistent with these results, semiquantitative analysis of p27Kip1 immunofluorescence (intensity per cell) showed twofold increases in the oral epithelium of Skp2−/− compared with wild-type mice; however, no significant differences were detected among K5-Myc, K5-Myc/Skp2−/−, and Skp2−/− mice (Figure 3H). Therefore, we conclude that the elevated p27Kip1 levels observed in the oral cavity do not affect the onset of Myc-driven tumorigenesis. Altogether, these results suggest that the increased p27Kip1 levels do not effectively block Myc-induced oral tumorigenesis.
Discussion
We hypothesized that a lack of Skp2 expression might counteract the actions of Myc, leading to a decrease in cell proliferation and, likely, reduced tumorigenesis. Herein, we report that Skp2 ablation in mouse epidermis results in epidermal hypoplasia and reduced keratinocyte proliferation. More important, the development of epidermal hypoplasia appears to be mainly mediated by increases in p27Kip1 because we did not observe changes in other putative substrates of Skp2, such as cyclin D1, p21Cip1, or p57Kip.
More important, in vitro analysis of CDK activities shows that CDK4, but not CDK2, inhibits in the Skp2−/− epidermis. The Cip/Kip family members were initially described as general CDK inhibitors; however, several groups
have shown that p21Cip1 and p27Kip1 bind, but do not interfere, with CDK4/CDK6 activities. There is no clear consent about the role of Cip/Kip members on CDK4 activity. Ray et al
recently reported two independent models by which p27Kip1 might inhibit CDK4. According to these models, the absence of the specific tyrosine phosphorylation of p27Kip1 results in the binding and inhibition of CDK4 by p27Kip1. Consistent with this model, lack of CDK4, but not of CDK2, has reduced Myc-induced keratinocyte proliferation
have shown that the ablation of CDK2 in a p27Kip1-null background did not mitigate the phenotypes of p27Kip1 deficiency, clearly showing that p27Kip1 can act independently of CDK2. Thus, whether CDK4 is the main target for p27Kip1 inhibition in mouse epidermis warrants future investigations.
Transgenic mice overexpressing Myc in the basal cell layer of the stratified epithelium experienced severe epidermal hyperplasia, hypertrophy, and, in some cases, tumor development.
We found that forced expression of the Myc oncogene led to decreased p27Kip1 protein levels in mouse epidermis. Supporting our initial hypothesis, genetic inhibition of Skp2, and the consequent p27Kip1 accumulation, severely crippled the ability of the Myc oncogene to drive keratinocyte proliferation and further induce epidermal hyperplasia. Consistent with these data, biochemical analysis of K5-Myc/Skp2−/− mouse epidermis showed that lack of Skp2 expression reversed the reduced levels of p27Kip1 observed in the K5-Myc epidermis. The Myc oncogene also affects p27Kip1 activity by inducing CDK4 expression, leading to p27Kip1 sequestration by the CDK4/D-type cyclin complexes.
Therefore, changes in p27Kip1 protein levels play an important role in Myc-induced hyperplasia and Skp2–/– hypoplasia. Interesting, Myc-mediated p27Kip1 down–regulation was dependent on the elevated levels of Skp2 in B cells from the transgenic Eμ-Myc mouse.
have shown that low levels of p27Kip1 and/or p27Kip1 sequestration, on CDK4 expression in the K5-Myc epidermis, results in CDK2 activation. However, CDK2 ablation does not reverse the hyperproliferative phenotype of the K5-Myc epidermis.
Thus, it is tempting to hypothesize that Myc activity deregulates keratinocyte proliferation through mechanisms other than CDK2 activation. Altogether, these results suggest that Skp2 plays a central role in the deregulation of keratinocyte proliferation triggered by Myc overexpression.
Deregulation of Myc expression plays a causal role in the genesis of several types of human and experimental malignancies.
The gene ratios c-MYC: cyclin-dependent kinase (CDK)N2A and CCND1:CDKN2A correlate with poor prognosis in squamous cell carcinoma of the head and neck.
Herein, we showed that Skp2 ablation does not inhibit Myc-induced oral tumorigenesis. In fact, we did not find any differences in the latency, incidence, differentiation, or level of proliferation among K5-Myc, K5-Myc/Skp2−/−, and K5-Myc/Skp2+/− mice. As expected, we observed accumulation of the p27Kip1 protein in the Skp2−/− oral epithelium, although high p27Kip1 levels were also observed in K5-Myc and K5-Myc/Skp2−/− mice. It is not clear why Myc expression does not reduce p27Kip1 levels in the oral cavity, but this observation warrants further investigation. The analysis of p27Kip1 in oral tumors from K5-Myc and K5-Myc/Skp2−/− mice showed no significant differences between both genotypes (data not shown). Thus, we speculate that lack of Skp2 results in a tissue-specific effect in mouse epidermis and oral epithelium, blocking keratinocyte proliferation, likely through CDK4 inhibition but not oral epithelium cell proliferation. In support of a tissue-specific role of p27Kip1 levels, a clear discrepancy was observed between the phenotype of epidermis and the oral epithelium of these mouse models. The Skp2−/− mice showed no signs of developing epithelial hypoplasia in the oral cavity, as observed in their epidermis. Also, the oral epithelium of K5-Myc mice showed no development of the hyperplasia observed in the epidermis. Therefore, we hypothesized that Myc-induced oral tumorigenesis does not depend on p27Kip1 levels, resulting in the unsuccessful blocking of tumor development in K5-Myc/Skp2−/− mice. In support of our finding, Skp2 deficiency had a modest or no effect on Myc-induced lymphomagenesis in Eμ-Myc–transgenic mice.
showed that Skp2 inactivation on its own does not induce cellular senescence. However, aberrant oncogenic signals and/or inactivation of tumor suppressor genes triggers a potent suppressive senescence response in mice devoid of Skp2.
In addition, Skp2 targets regulators of survival and/or apoptosis for degradation. For instance, Skp2 suppresses p53-dependent apoptosis by regulating CBP/p300 protein levels and antagonizing the interaction between CBP/p300 and p53.
We did not observe changes in apoptosis levels in K5-Myc/Skp2−/− and K5-Myc tumors; thus, our studies predict that p27Kip1 accumulation, senescence, and/or apoptosis does not play an important role in Myc-induced oral tumorigenesis. Skp2 is also a strong stimulator of Myc's transcriptional activities,
suggesting that ablation of Skp2 may reduce cell proliferation and/or tumor development. Conversely, Skp2 participates in Myc proteasomal degradation, predicting that ablation of Skp2 would result in Myc stabilization and increase tumor development. Our results show that the stimulation of Myc transcriptional activity by Skp2
is dispensable for Myc oncogenic activities. In this scenario, ablation of Skp2 stabilizes p27Kip1 and c-myc protein levels, allowing K5-Myc to behave as a potent oncogene, even in the presence of elevated levels of p27Kip1 protein.
Collectively, the data presented in this study and recent reports from other researchers
suggest that Skp2 is an efficient regulator of normal proliferation through the regulation of p27Kip1 protein levels, but it is inefficient in alleviating Myc-induced tumorigenesis. Therefore, the suitability of Skp2 as a target for therapeutic intervention must be considered in a tissue-dependent manner and in the context of the particular oncogenic pathway affected.
Acknowledgments
We thank Paula Miliani de Marval for her critical review of the manuscript and the Laboratory Animal Resources (College of Veterinary Medicine, North Carolina State University, Raleigh).
References
Gregory M.A.
Hann S.R.
c-Myc proteolysis by the ubiquitin-proteasome pathway: stabilization of c-Myc in Burkitt's lymphoma cells.
The gene ratios c-MYC: cyclin-dependent kinase (CDK)N2A and CCND1:CDKN2A correlate with poor prognosis in squamous cell carcinoma of the head and neck.
Supported by a grant from the National Cancer Institute/NIH (RO1CA116328).
Current address of C.S., Department of Molecular Biomedical Sciences, North Carolina State University, Raleigh, North Carolina; of E.M., Dell Pediatric Research Institute, College of Pharmacy, The University of Texas, Austin, Texas.
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