Myc and Skp2 overexpression promotes p27 ubiquitination and degradation in Ewing Sarcoma
Yuta Kubota, Masanori Kawano, Ichiro Itonaga, Nobuhiro Kaku, Kazuhiro Tanaka
TL;DR
This study shows that Myc and Skp2 proteins drive Ewing Sarcoma growth by speeding up cell division and breaking down a tumor-suppressing protein called p27.
Contribution
The paper reveals a novel mechanism where Myc and Skp2 promote Ewing Sarcoma progression through p27 ubiquitination and CCNE/CDK2 complex activation.
Findings
Myc and Skp2 overexpression correlates with reduced p27 levels and increased cell proliferation in Ewing Sarcoma.
Knockdown of Myc and Skp2 leads to G1 cell cycle arrest and reduced tumor growth in mouse models.
The CCNE/CDK2 complex activates Rb phosphorylation and p27 degradation via Skp2-mediated ubiquitination.
Abstract
Ewing sarcoma remains partially uncontrollable even after treatment with chemotherapy, surgery, and radiation therapy due to its high malignancy. To explore genes that drive Ewing sarcoma cell proliferation, we conducted a comprehensive analysis of mRNA expression. Based on cDNA array results, we identified consistently elevated expression of Myc and S-phase kinase-associated protein 2 (Skp2) across all five Ewing sarcoma cell lines examined. The functional roles of Myc and Skp2 were assessed by siRNA-mediated knockdown and overexpression assays, followed by cell proliferation, cell cycle, and protein expression analyses. Ubiquitination of p27 and activation of the CCNE/CDK2 complex were evaluated by immunoprecipitation and western blotting. Finally, the in vivo relevance of Myc and Skp2 knockdown was validated using a xenograft mouse model. Knockdown (KD) using siRNAs specific for…
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Fig 7- —http://dx.doi.org/10.13039/501100001691Japan Society for the Promotion of Science
- —http://dx.doi.org/10.13039/501100001691Japan Society for the Promotion of Science
- —http://dx.doi.org/10.13039/501100001691Japan Society for the Promotion of Science
- —http://dx.doi.org/10.13039/100009619Japan Agency for Medical Research and Development
- —http://dx.doi.org/10.13039/100009619Japan Agency for Medical Research and Development
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Taxonomy
TopicsSarcoma Diagnosis and Treatment · Cancer-related Molecular Pathways · Acute Myeloid Leukemia Research
1. Introduction
Ewing sarcoma, composed predominantly of undifferentiated small round cells, is a primary malignant bone tumor that primarily affects children and young adults. We performed comprehensive mRNA expression analyses in Ewing sarcoma cell lines and commonly identified increased expression of S-phase kinase-associated protein 2 (Skp2) and Myc.
Myc is a transcription factor recognized as an oncogene, with diverse functions [1]. We specifically observed changes in the expression of cell cycle-related genes such as cyclin D [2] and TGFBR2 [3], influenced by Myc. S-phase kinase-associated protein 2 (Skp2) is part of the Skp1–Cullin–F-box (SCF) complex, an E3 ubiquitin ligase. Since many of Skp2’s targets inhibit cell cycle progression and act as tumor suppressors, Skp2 is considered oncogenic in various cancers [4–6].
Skp2 facilitates protein ubiquitination using specific post-translational modifications, such as phosphorylation at Thr187 of p27, as a signal [7]. Myc suppresses p21 expression by promoting the transcription factor AP4 [8]. Reduced p21 levels result in increased cyclin E (CCNE) expression, which activates CDK2 through complex formation [9]. The CCNE/CDK2 complex phosphorylates p27 at Thr187 [10], marking it for ubiquitination by Skp2. Additionally, this complex promotes Rb phosphorylation at Ser807/808 [11].
Myc functions as an oncogene by regulating a range of target genes and is also known to increase Skp2 expression [12]. Phosphorylated Rb activates E2F1, which subsequently upregulates its target genes, including CCNE, CDK2, and Myc. The newly expressed CCNE and CDK2 proteins enhance each other’s activity under conditions of reduced p21. De novo Myc may further upregulate AP4 and Skp2 expressions.
In summary, Ewing sarcoma cell lines with constitutively high Myc expression demonstrate increased Skp2 levels induced by Myc, reinforcing a positive feedback loop involving CCNE, which may augment p27 ubiquitination.
In this study, we investigated the upregulation of Myc, which promotes activation of the CCNE/CDK2 complex and Thr187 phosphorylation of p27, leading to its degradation in Ewing sarcoma cells. We elucidate the underlying mechanism, focusing on the excessive degradation of cell cycle regulatory proteins.
2. Materials and methods
2.1. Cell lines
Ewing sarcoma cell lines (SKES-1, RDES, SKNMC, and SCCH) were purchased from the JCRB Cell Bank (Tokyo, Japan), and WE68 was kindly provided by Dr. Frans van Valen (Westfälische-Wilhelms University, Münster, Germany). Human mesenchymal stem cells (hMSCs) were obtained from TaKaRa Biotechnology (Otsu, Japan). Each cell line was authenticated by the source company and maintained under the conditions described previously [13].
2.2. Analysis of mRNA expression by cDNA arrays
Gene expression profiling was performed using the GeneChip® Human Genome U133 Plus 2.0 Array (Affymetrix, Santa Clara, CA, USA). RNA was extracted from SKES-1, RDES, SKNMC, SCCH, WE68, and hMSCs, and analyzed as described previously [14]. Probe sets showing ≥2-fold differences between groups were considered significantly differentially expressed.
2.3. Reverse transcriptase quantitative PCR
Total RNA extraction and reverse transcriptase quantitative PCR (RT-qPCR) was previously described [15]. DNA synthesis was performed using the PrimeScript RT Reagent Kit (Takara, Japan). Quantitative PCR was carried out with SYBR Green Master Mix (Thermo Fisher Scientific, Waltham, MA, USA). Primer sequences: Myc-forward: 5’-GCT GCC AAG AGG GTC A-3’, Myc-reverse: 5’-CGC ACA AGA GTT CCG TAG-3’, Skp2-forward: 5’-CTG TCT CAA GGG GTG ATT GC-3’, Skp2-reverse: 5’-TTC GAT AGG TCC ATG TGC TG-3’, GAPDH-forward: 5’-TGC CTC CTG CAC CAC CAA CT-3’, GAPDH-reverse: 5’-CCC GTT CAG CTC AGG GAT GA-3’.
2.4. Oligonucleotide and expression vector transfection
siRNA transfection and cell proliferation assay were performed as previously described [16]. Briefly, siRNA transfection was performed using Lipofectamine™ 2000 (Thermo Fisher Scientific, Cat. #11668−019) in antibiotic-free Opti-MEM (Thermo Fisher Scientific) following the manufacturer’s protocol. The following reagents were used: Myc-siRNA, Skp2-siRNA, and negative control siRNA (Invitrogen; Thermo Fisher Scientific) at 10–160 nM. AP4-siRNA (Cat. #sc-37690) and p21-siRNA (Cat. #sc-29427), purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Expression vectors: AP4 (12101), Skp2 (89142), and Myc (26818) were obtained from Addgene (Watertown, MA, USA). Recombinant cyclin E/CDK2 complex (Abcam, Cat. #ab85639) and control IgG (Funakoshi, Japan; Cat. #Ab00178–10.2) were used in the phosphorylation assay.
2.5. Apoptosis assay
Apoptotic markers were detected by Western blotting using the following antibodies (Cell Signaling Technology, Tokyo, Japan): PARP (#9542), cleaved PARP (#9541), caspase-3 (#9662), cleaved caspase-3 (#9661), and GAPDH (#5174).
Three independent experiments were performed.
2.6. Cell cycle analysis
Cell cycle distribution was evaluated using the FITC BrdU Flow Kit (BD Biosciences, Bedford, MA, USA) according to the manufacturer’s protocol. Cells were serum-starved in DMEM with 1% FBS for 48 h, then pulsed with 10% FBS and 100 µM BrdU for 6 h. Data were collected using a FACS Fortessa cytometer (BD Biosciences) and analyzed with FlowJo software (version 10.8).
2.7. Western blot
Western blotting was performed as described previously [17]. Primary antibodies were obtained from Cell Signaling Technology (Tokyo, Japan) unless otherwise indicated: Myc (#18583), Skp2 (#2652), p21 (#2947), p27 (#3686), CDK2 (#2546), p-CDK2 (Thr160, #2561), CCNE (#4129), Lamin B1 (#13435), and GAPDH (#5174). Additional antibodies: p-p27 (Thr187, Abcam #ab75908) and AP4 (Thermo Fisher Scientific #A302-760A). Peroxidase-conjugated anti-rabbit IgG secondary antibody (Jackson ImmunoResearch, West Grove, PA, USA; 1:2000 dilution) was used.
2.8. Immunoprecipitation
Cell lysates were prepared using Pierce IP Lysis Buffer (Thermo Fisher Scientific, Cat. #87788). After centrifugation, supernatants were incubated with anti-p27 antibody (3686, CST) at 4 °C overnight, followed by Protein G Sepharose 4 Fast Flow beads (GE Healthcare, Chicago, IL, USA) for 2 h. After washing, immunocomplexes were analyzed by SDS-PAGE and immunoblotted with anti-Ubiquitin antibody (3936, CST).
2.9. Immunohistochemistry staining
Formalin-fixed, paraffin-embedded tissues were sectioned (5 µm), subjected to heat-induced antigen retrieval in citrate buffer (pH 6.0), and stained with primary antibodies against p27 (#3686, CST) and CCNE (Abcam, #ab74276). Secondary antibody: HRP-conjugated anti-rabbit IgG (Jackson ImmunoResearch). Signal detection: DAB Substrate Kit (Vector Laboratories, Newark, CA, USA) followed by hematoxylin counterstaining.
2.10. Animal experiments
BALB/c nu/nu mice (6–8 weeks old, female; Kyudo Laboratory, Tosu, Japan) were used for xenograft assays. Mice were housed under SPF conditions with 12 h light/dark cycles and ad libitum access to food and water. SKES-1 cells (1 × 10⁶ cells in 100 µL PBS) were injected subcutaneously into the left flank. Tumor volumes were measured 2–3 times weekly. All experimental protocols were approved by the Medical Ethics of Oita University. All methods were carried out in accordance with relevant guidelines and regulations. All animal protocols were approved by the Oita University Medical Ethics Committee (No. 182403) and complied with ARRIVE guidelines. After quarantine, all mice were kept in a pathogen-free environment on a standard 12 hr-day/12 hr-night cycle and were fed a standard sterilized pellet diet and water ad libitum. SKES-1 cells (1 × 10^6^ cells per mouse) were injected subcutaneously into the left flank of each mouse. Tumors were measured by caliper two or three times per week, and the volume was calculated as previously described [18]. Tumors were allowed to grow until they reached 30–50 mm^3^ and the mice were randomly distributed and treated. The maximum tumor diameter permitted under the relevant animal protocols is 25 mm. Animals were monitored two or three times per week for tumor size and general health. When the tumor volume reached the predetermined limit, the observation period was immediately terminated, and the animal was euthanized without delay to minimize suffering. This limit was not exceeded in any experiments. 28 days after tumor inoculation, the 15 mice were sacrificed by cervical dislocation without prior anesthesia and/or analgesia in order to minimize any potential suffering or distress to the animals. Staff members who are involved in animals completed the animal handling training program required by the Oita University Faculty of Medicine and managed the animals in strict accordance with its regulations.
2.11. Statistical analysis
All data are presented as mean ± SD or SEM, as indicated. Each experiment was independently repeated at least three times (n = 3). Statistical analyses were performed using two-tailed Student’s t-test for comparisons between two groups and ANOVA with Scheffé’s post hoc test for multiple comparisons. Exact p values are reported in the figure panels whenever possible, and differences were considered statistically significant at p < 0.05. All statistical analyses were performed using IBM SPSS Statistics version 25 (IBM Japan, Tokyo, Japan) [18].
3. Results
3.1. A microarray analysis showed increased Myc and Skp2 in Ewing sarcoma cell lines.
We analyzed mRNA expression using RNA extracted from Ewing sarcoma cell lines. The results showed increased expression of Myc and the ubiquitin ligase Skp2 in all five Ewing sarcoma cell lines compared to hMSCs (S1A Fig).
3.2. Changes in mRNA and protein expression of Myc and Skp2 by siRNA transfection
In order to clarify the disease specificity, we included human mesenchymal stem cells (hMSCs) as non-malignant control (Fig 1A). The expression levels of Myc and Skp2 were markedly higher in SKES1 cells than in hMSCs, whereas p27 expression showed the opposite tendency (S1B Fig).
Decreased expression of target mRNAs and proteins by knockdown of Myc and Skp2 using siRNA.(A) Western blot analysis of Myc, Skp2, and p27 expression in human mesenchymal stem cells (hMSCs) and Ewing sarcoma cells (SKES1) treated with specific siRNAs or negative control siRNA. GAPDH was used as a loading control. The inclusion of hMSCs as a non-malignant comparator clarifies disease-specific expression differences. There were decreases in each mRNA by Myc-siRNA (20 nM) and (B) Skp2-siRNA (20 nM) (B). The expression of Myc protein was analyzed by (C) immunoblotting and (D) densitometric quantification. The effect of Skp2-siRNA on Skp2 protein expression was analyzed by (E) immunoblotting and (F) quantified accordingly.
To verify the influence of Myc and Skp2 on mRNA and protein expression, we performed knockdown (KD) using siRNA targeting Myc and Skp2, respectively. Myc mRNA expression significantly decreased at 20 nM in the Myc-siRNA group (p = 0.007). Skp2 mRNA expression also significantly decreased at 20 nM (p = 0.009) (Fig 1B). We then verified whether the decrease in mRNA caused by Myc-siRNA was reflected at the protein level (Fig 1C). A decrease in Myc protein was observed following the introduction of Myc-siRNA (20 nM) (Fig 1D). Similarly, we examined whether Skp2-siRNA caused a decrease in protein expression (Fig 1E). Skp2-siRNA was introduced, and a decrease in Skp2 protein expression was observed at 20 nM (Fig 1F).
3.3. Effects on cell growth and apoptosis following siRNA introduction
To analyze whether downregulation of Myc and Skp2 affect cell growth, we performed a cell growth assay following siRNA-mediated knockdown of Myc and Skp2 (Fig 2A). Introduction of Myc-siRNA into SKES-1 (p = 0.007) and RDES cells (p = 0.006) significantly suppressed cell growth. Similarly, knockdown using Skp2-siRNA significantly suppressed cell growth in both SKES-1 (p = 0.009) and RDES cells (p = 0.009) (Fig 2B). Although we investigated whether this reduction in growth was caused by apoptosis (Fig 2C), no expression of cleaved PARP or cleaved caspase 3 was detected (Fig 2D). These results indicate that apoptosis is not induced during cell growth suppression.
*Influence of Myc and Skp2 on cell growth and apoptosis.(A) The cell growth of SKES and RDES cells was significantly decreased by Myc-KD. (B) Similarly, cell growth in both cell lines was suppressed by Skp2-KD. (C) The possibility that the decrease in cell growth was caused by apoptosis was evaluated; (D) Apoptosis was not induced under conditions where cell growth suppression was observed. The results are represented as the mean ± SEM (n.s., no significance). *, p < 0.01 versus the related untreated groups.
3.4. Myc and Skp2 accelerate cell cycle turnover
Since Myc and Skp2 were found to promote cell proliferation, their effects on the cell cycle were evaluated (Fig 3A). Myc knockdown cells showed significant decreases in the S and G2/M phases and an increase in the G1 phase population (p = 0.008). In the Skp2 knockdown group, similar changes were observed. In cells where both Myc and Skp2 were simultaneously knocked down, the decreases in S and G2/M phases and the increase in G1 phase were more pronounced, indicating the greatest delay in cell cycle progression in the double knockdown group (Fig 3B).
*Myc and Skp2 accelerate cell cycle progression.(A) The effect of Myc and Skp2 knockdown on the cell cycle was examined to determine whether growth suppression was due to cell cycle delay. In Myc-KD, Skp2-KD, and simultaneous Myc- and Skp2-KD groups, the S phase and G2/M phase populations decreased, while the G1 phase population increased. (B) These populations were quantified. The simultaneous knockdown group showed the most prominent changes. The results are represented as the mean ± SEM (n.s., no significance). *, p < 0.01 versus the related control groups.
3.5. Myc induces AP4 to suppress p21 expression
The mRNA (Fig 4A) and protein expression levels (Fig 4B) of AP4 were investigated following introduction of the Myc expression vector. A significant increase in AP4 mRNA and protein expression (Supplemental. S2A Fig) was observed at 12 hours (p = 0.006) and 24 hours (p = 0.003), respectively, after Myc vector induction. Next, we examined whether the increase in AP4 induced by Myc would transcriptionally target p21. Introduction of AP4-siRNA decreased AP4 expression and simultaneously increased p21 expression (Fig 4C) (Supplemental. S2B Fig). Conversely, introduction of the AP4 expression vector reduced p21 expression significantly (p = 0.007) (Fig 4D). The AP4 vector had no effect on Myc expression (Fig 4E). When AP4 was forcibly expressed, p21 levels were inversely proportional to AP4 expression, while Myc levels remained unaffected (Supplemental. S2C Fig). This indicates that AP4 is a target gene of Myc, and that Myc suppresses p21 expression through AP4 in Ewing sarcoma cells.
*AP4 is a target of Myc and decreases p21 expression.(A) The effect of Myc-siRNA on AP4 mRNA expression was evaluated. (B) A time-course analysis showed a correlation between Myc and AP4 protein expression. (C) Downregulation of AP4 led to upregulation of p21. (D) AP4 regulates p21 mRNA expression. The results are represented as the mean ± SEM (n.s., no significance). *, p < 0.01 versus the related control groups. (E) A time-course analysis of AP4 KD showed that p21 expression increased inversely with AP4 levels, while Myc remained unaffected. (F) Changes in p21 expression influenced the phosphorylation of CDK2 and Rb. It was found that p21 reduces phosphorylation of both CDK2 and Rb. (G) The presence of p21 inhibits binding between CDK2 and CCNE.
3.6. Decreased p21 expression promotes activation of the CCNE/CDK2 complex and modification of p27 (Thr187)
We investigated the effects of p21 expression on the expression and phosphorylation of CCNE, CDK2, and p27 proteins (Fig 4F). Knockdown of p21 by siRNA had no effect on the protein expression levels of CCNE, CDK2, or Rb. However, p21 decreased the level of phosphorylated pCDK2 (Thr160) (Supplemental. S2D Fig). Phosphorylation of Rb is inhibited by p21. This result revealed that p21 protein decreased the phosphorylation of CDK2 (Thr160) and Rb (S807). The interaction strength between p21 and the CCNE**/**CDK2 complex was inversely proportional (Fig 4G) (Supplemental. S2E Fig).
3.7. The CCNE/CDK2 complex induces Rb phosphorylation and E2F1 activation
We examined whether CCNE/CDK2 complex-induced phosphorylation of Rb would enhance E2F1 transcriptional activity and expression of its target genes. Cells treated with the CCNE/CDK2 complex showed phosphorylation of Rb (Ser807/811) and p27 (Thr187) (Fig 5A), along with increased expression of Myc, CCNE, CCNA, and CDK2 proteins (Fig 5B). Additionally, the CCNE/CDK2 complex increased expression of Myc, CCNE, CCNA, and CDK2 proteins (Supplemental. S3A Fig). These findings suggest that phosphorylation of Rb (Ser807/811) by the CCNE/CDK2 complex activates E2F1, leading to transcription of E2F1 target genes: Myc, CCNE, CCNA, and CDK2, suggesting the positive feedback mechanism of Myc expression.
The influence of p21 on the phosphorylation of CDK2 and Rb protein was examined.Changes in the (A) phosphorylation levels of p27 and Rb protein, and (B) protein levels of Myc, CCNE, CCNA, and CDK2 by the CCNE/CDK2 complex were observed. (C) The effects of the CCNE/CDK2 complex on Rb and E2F1 binding were investigated using immunoprecipitation. The CCNE/CDK2 complex inhibited the binding of Rb to E2F1.
Next, the effect of the CCNE/CDK2 complex on Rb and E2F1 binding was examined (Fig 5C). Comparable amounts of Rb and E2F1 proteins were extracted from the nucleus, regardless of the presence or absence of the CCNE/CDK2 complex. In immunoprecipitation (IP) with anti-E2F1 antibodies, CCNE/CDK2 reduced Rb levels (Supplemental. S3B Fig). Similarly, in IP with Rb antibodies, CCNE/CDK2 reduced E2F1 levels. These results suggest that the CCNE/CDK2 complex weakens the binding between Rb and E2F1.
3.8. Myc promotes p27 degradation by increasing the expression of ubiquitin ligase components
We investigated whether Myc targets Skp2 and E2F1 and contributes to the formation of the ubiquitin ligase complex (Fig 6A). Myc expression via vector increased the expression of Skp2, E2F1, Cul1, and CKS1, all of which were reduced by Myc-siRNA (Supplemental. S4A Fig). Increased Myc expression enhanced p27 ubiquitination, whereas Myc-siRNA reduced it (Fig 6B) (Supplemental. S4B Fig). This is because Myc increases the expression of components of the ubiquitin ligase complex, promoting ubiquitination of p27 (Thr187) as a marker.
Myc-induced changes in the expression of E2F1, Skp2, and ubiquitination complex components.(A) The expression of ubiquitin ligase complex components was compared between Myc-overexpressing and siRNA knockdown groups. (B) Changes in p27 ubiquitination were also compared between these groups.
3.9. Changes in tumor volume and expression of p27 and CCNE in mouse tumor tissue
In a mouse model, tumor growth was significantly suppressed in the Myc and Skp2 knockdown groups compared to the control group (Fig 7A). Immunostaining of tumor tissue (Fig 7B) significantly showed increased p27 expression (p = 0.006) and decreased CCNE expression (p = 0.007) in the Myc-reduced group compared to the control (Fig 7C). A summary diagram of this experiment is presented. We demonstrate that Myc expression weakens the binding of CCNE/CDK2 through AP4, ultimately promoting the degradation of p27 (Fig 7D).
*Effects of Myc and Skp2 in Ewing sarcoma cell lines using a mouse model.(A) Tumor growth was compared across groups after tumor tissues were transplanted subcutaneously into mice. (B) The expression of p27 and CCNE in mouse tumor tissues was analyzed by immunohistochemistry. Original magnification, × 400; Scale bars: 50 μm. (C) The proportion of immunohistochemically positive cells was quantified. Data represent mean ± SEM of three independent experiments. *, p < 0.01 versus the related control groups. (D) A schematic summary of the study was created, demonstrating that Myc enhances the expression of AP4 and Skp2, ultimately leading to p27 ubiquitination.
4. Discussion
Ewing sarcoma is a primary malignant bone tumor that typically affects adolescents. It is treated with standard chemotherapy [19] and surgical resection, but in many cases, these are associated with severe adverse effects [20]. Anticancer agents used in chemotherapy, as well as molecular biological approaches using Ewing sarcoma cells, are essential for the development of molecularly targeted drugs for this disease.
We have attempted to identify factors closely associated with the degree of malignancy by performing comprehensive RNA analysis of Ewing sarcoma [3,18]. During this process, we found that the mRNA levels of Myc and Skp2 are commonly elevated in Ewing sarcoma cells. Myc is a transcription factor, and its expression is frequently increased in malignant tumors [21–23]. Skp2 is a ubiquitin ligase that plays a role in protein degradation by tagging target proteins for ubiquitination following specific phosphorylation events [24–26].
It has been reported that Myc directly induces the expression of AP-4 by binding to the E-box motif in the AP-4 promoter as a transcription factor [27]. Furthermore, AP-4, once expressed, functions as a repressor by binding to the E-box motif in the promoter region of p21, thereby downregulating p21 expression [28].
Previous studies have reported that Myc induces Skp2 transcription, identifying Skp2 as a direct downstream target gene of Myc [5]. Moreover, Myc has been shown to strongly enhance E2F1 expression [29,30], and E2F1 has been experimentally confirmed to upregulate Skp2 expression [12]. To date, however, no study has examined the Myc–E2F1–Skp2 regulatory axis, which warrants further investigation. There is strong evidence that AP4 represses the transcription or expression of p21 (CDKN1A) [31]. Moreover, studies using both mouse and human cell systems have shown that AP4 suppresses the expression of p16 and p21. Activation of AP4 (by inducing AP4-ER with 4-OHT) decreased p21 mRNA and protein levels, and direct binding of AP4 to the p21 promoter region was also confirmed [8].
Although we did not directly investigate the relationship between AP4 and p21 in our experiments, these previous findings suggest that AP4 negatively regulates p21 expression. Functionally, p21 inhibits the activity of CDK2 [32], which forms a complex with CCNE to become activated. Increased phosphorylation of Rb then leads to cell cycle progression [33]. Therefore, AP4-induced suppression of p21 facilitates CDK2 activation. Activated CDK2 phosphorylates p27 at Thr187, which serves as a signal for Skp2-mediated ubiquitination. The CCNE/CDK2 complex also plays an essential role in Rb protein phosphorylation. Phosphorylated Rb activates transcription of E2F1, which in turn upregulates Myc as one of its target genes, forming a positive feedback loop.
Skp2 has been identified as a direct target gene of Myc, and it has been reported that Skp2 suppresses p27 expression [34]. These findings align with our observations that changes in Myc expression positively correlate with Skp2 expression and inversely correlate with p27 levels. In Ewing sarcoma cell lines, Myc and Skp2 genes are constitutively overexpressed, although the detailed mechanisms remain unclear. Importantly, our xenograft experiments further validated these findings in vivo, demonstrating that suppression of Myc and Skp2 led to slower tumor growth and reciprocal regulation of p27 and CCNE expression. These results support the biological relevance of our proposed mechanism in a living system, beyond vitro cell line experiments. If an inhibitor targeting any point in this mechanism can be identified, it may suppress p27 degradation and potentially contribute to the development of effective therapies. Previous studies have reported that both MYC and Skp2 are overexpressed in various human malignancies, including oral squamous cell carcinoma [35], breast cancer [6], and prostate cancer [26], and that high Skp2 expression is associated with decreased p27 levels, enhanced malignancy, recurrence, and poor prognosis. Moreover, MYC has been shown to directly induce Skp2 expression through transcriptional activation, establishing a MYC–Skp2 regulatory axis that promotes tumor cell proliferation [5]. These findings indicate that dysregulation of the MYC–Skp2 pathway represents a common oncogenic mechanism across multiple tumor types. In the present study, we demonstrated that this pathway is also activated in Ewing sarcoma cells, suggesting that MYC and Skp2 overexpression in Ewing sarcoma may reflect not only disease-specific features but a shared oncogenic mechanism observed in many cancers.
It has been more than two decades since the standard chemotherapy regimen for Ewing sarcoma was established [19]. From a translational viewpoint, the Myc–CDK2–Skp2–p27 pathway includes several druggable targets that could be combined with existing therapies. Small-molecule Skp2 inhibitors such as SZL-P1-41 have been shown to restore p27 and suppress tumor growth in preclinical models [36]. Upstream, selective CDK2 inhibitors including BLU-222 and PF-07104091 are now in early clinical trials and may synergize with standard chemotherapy [37]. Moreover, BET inhibitors such as JQ1 can indirectly suppress MYC and have demonstrated promising activity in Ewing sarcoma models [38]. Therefore, combined inhibition of MYC, CDK2, and Skp2, together with conventional regimens, represents a rational therapeutic strategy that warrants further investigation.
Our results revealed a multistep mechanism initiated by Myc, involving Ap4 and CCNE/CDK2, that mediates p27 degradation. Since this regulatory cascade proceeds through sequential activation of three transcription factors—Myc, Ap4, and E2F1—there is potential to intervene in this pathway, offering a promising strategy to disrupt the positive feedback loop.
In this study, we clarified the feedback loop mechanism that occurs in Ewing sarcoma cells. We believe that our findings may contribute to the development of new therapeutic strategies for Ewing sarcoma.
Supporting information
S1 FigUp regulation of Myc and skp2 expression in ES cell lines.(A) The cDNA array analysis demonstrated that the expressions of 3043 mRNAs were significantly changed between five ES cell lines and hMSCs. We found that 1062 genes were significantly up-regulated, whereas 1884 genes were significantly down regulated and the remaining 97 genes were up or down regulated in five ES cell lines compared to hMSCs. (B) Protein quantification of Myc, Skp2, and p27 in hMSCs and SKES-1 cells. siRNA-mediated knockdown resulted in a statistically significant decrease in the expression levels of these proteins. **, p < 0.01 versus the related control groups.(TIF)
S2 FigQuantification of protein expression by western blotting.(A) Time-course changes of AP4 protein expression under Myc overexpression. (B) p21 expression upon AP4 knockdown. (C) Quantification of AP4 and Myc protein expression. (D) Analysis of p21 and Myc in response to AP4 reduction. (E) Quantification of the binding ability between CDK and CCNE proteins by immunoprecipitation. The results are represented as the mean ± SEM (n.s., no significance). **, p < 0.01 versus the related control groups.(TIF)
S3 FigThe impact of CDK2/CCNE expression on downstream pathway factors was analyzed by western blotting.(A) Analysis of changes in phosphorylation of Rb and p27, along with changes in Myc, CCNE, CCNA, and CDK2 expression. (B) Quantification of the interaction between Rb and CCNE under the presence of CCNE/CDK2 using immunoprecipitation. The results are represented as the mean ± SEM (n.s., no significance). **, p < 0.01 versus the related control groups.(TIF)
S4 FigThe effect of altering Myc expression on Skp2, E2F1, and components of the ubiquitin ligase complex was investigated.(A) Investigation of the protein expression levels of Skp2 and ubiquitin ligase complex components. (B) Examination and quantification of ubiquitin bound to p27 by western blotting. The results are represented as the mean ± SEM (n.s., no significance). **, p < 0.01 versus the related control groups.(TIF)
S1 FileRaw data gel image.Uncropped and unedited original gel images corresponding to the western blot experiments presented in Figures 1, 2, 4–6.(PDF)
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