Skp2-MacroH2A1-CDK8 axis orchestrates G2/M transition, polyploidy and tumourigenesis

Understanding the mechanism by which cell growth, migration, polyploidy, and tumourigenesis are regulated may provide important therapeutic strategies for cancer therapy. Here we identify the Skp2-macroH2A1 (mH2A1)-CDK8 axis as a critical pathway for these processes, and deregulation of this pathway is associated with human breast cancer progression and patient survival outcome. We showed that mH2A1 is a new substrate of Skp2 SCF complex whose degradation by Skp2 promotes CDK8 gene and protein expression. Strikingly, breast tumour suppression upon Skp2 deficiency can be rescued by mH2A1 knockdown or CDK8 restoration using mouse tumour models. We further show that CDK8 regulates p27 protein expression by facilitating Skp2-mediated p27 ubiquitination and degradation. Our study establishes a critical role of Skp2-mH2A1-CDK8 axis in breast cancer development and targeting this pathway offers a promising strategy for breast cancer therapy.


Introduction
Maintaining genomic integration and proper cell cycle entry is a critical failsafe mechanism for preventing cancer initiation and progression [1][2] . Cancer cells often display aberrant cell cycle and higher degree of aneuploidy and/or polyploidy 3 . How these events occur during the cancer progression remains largely unclear. Understanding the regulatory mechanism that orchestrates these processes may provide new therapeutic strategies for cancer prevention and treatment.
Skp2 is an F-box protein that forms an SCF complex with Skp1 and Cullin-1 to constitute E3 ligase and plays a critical role in cell cycle regulation, maintenance of genomic integrity and cancer development [4][5][6][7] . As a consequence, primary Skp2 deficient mouse embryonic fibroblasts (MEFs) display upregulated p27 expression and higher percentage of G2/M phase and polyploidy. Notably, loss of p27 can revere G2/M arrest and polyploidy in primary Skp2 deficient MEFs, suggesting that p27 is a relevant substrate for Skp2 and is responsible for G2/M arrest and polyploidy upon Skp2 deficiency [8][9][10][11] . However, recent study reveals that p27 silencing does not rescue G2/M arrest and polyploidy in p53-mutant melanoma cells upon Skp2 knockdown 12 . Accordingly, these results imply that in addition to p27, Skp2 may also impact other pathways to orchestrate G2/M arrest and polyploidy under distinct genetic contexts.
Using affinity purification in conjunction with mass spectrometry analysis, we identified MacroH2A1 (mH2A1) also known as H2AFY as a novel binding partner for Skp2. mH2A is an epigenetic factor that primarily localizes in the heterochromatin and cooperates with HP1 to induce transcriptional repression, thereby leading to X chromosome inactivation 13 . mH2A consists of mH2A1 (mH2A 1.1 and mH2A1.2) and mH2A2 isoforms, which may share similar functions [14][15][16] . A recent study revealed that mH2A1 is a potential tumour suppressor whose expression is lost in advanced melanoma due to its promoter hypermethylation, and that its overexpression leads to the suppression of melanoma development 17 . Although the promoter hypermethylation of mH2A1 is proposed as a potential mechanism by which mH2A1 expression is lost in melanoma, how its activity and protein stability are regulated remain completely puzzled.
In this study, we unravel that Skp2 is a novel E3 ligase for mH2A1 and triggers mH2A1 ubiquitination and degradation, in turn leading to CDK8 gene expression. Notably, we find that upregulation of mH2A1 and subsequent CDK8 downregulation contributes to growth defect, G2/M arrest, polyploidy and tumour suppression upon Skp2 loss. Finally, we show that CDK8 is a novel regulator of p27 by facilitating Skp2-mediated ubiquitination and degradation of p27. Our study not only reveals novel insights into how mH2A1 stability is regulated, but also establishes a new role of mH2A1-CDK8 in Skp2-mediated cell cycle regulation, genomic stability and tumourigenesis.

Skp2 interacts with mH2A1
To define the novel mechanism by which Skp2 regulates G2/M arrest and polyploidy, we performed a systematic approach to identify novel Skp2 binding partners. To this end, we generated 293T cells stably overexpressed with Xpress-Skp2 (XP-Skp2), and the total cell lyates from these cells were immunoprecipitated with Xpress to pull down XP-Skp2 and Skp2 interacting proteins were identified by mass spectrometry analysis. Using this approach, we were able to obtain several peptides existed in Skp2 immunocomplex that correspond to mH2A1 (Fig. 1a). These peptides can be found in mH2A1.1 and mH2A1.2. mH2A is an epigenetic factor that regulates X chromosome inactivation and serves as a potential tumour suppressor for melanoma development 13,17 .
To validate the interaction between Skp2 and mH2A1, we performed the reciprocal coimmunoprecipitation experiments and found that endogenous Skp2 interacted with endogenous mH2A1 (Fig. 1b, c). In vitro GST pull down assay revealed that Skp2 could interact with mH2A1 (Fig. 1d). We further demonstrated that C-terminal of Skp2 (aa 201-424) and N-terminal region of mH2A1 are required for the interaction between Skp2 and mH2A1 by using Co-IP assays (Fig. 1e, f and Supplementary Fig.1a). Immunofluorescence assay further revealed that Skp2 was co-localized with mH2A1.1 and mH2A1.2 in nucleus in MDA-MB-231 breast cancer cells ( Supplementary Fig. 1b).
Skp2 SCF directly triggers ubiquitination and degradation of its protein substrates. Since Skp2 normally binds to its protein substrates though Skp2 C-terminal LRR domain, it is highly possible that mH2A1 is a novel Skp2 substrate. To test this hypothesis, we conducted in vivo ubiquitination assay to determine whether Skp2 promotes mH2A1 ubiquitination. Notably, overexpression of Skp2, but not of E3 ligase-dead Skp2-LRR mutant, promoted ubiquitination of mH2A1.1 in the presence of proteosome inhibitor MG132 (Fig. 2a), suggesting that Skp2 SCF is likely an E3 ligase for mH2A1. Similar results were also obtained for mH2A1.2 ( Supplementary Fig.1c). To corroborate the notion that Skp2 SCF is a direct E3 ligase for mH2A1, we performed in vitro ubiquitination assay and found that Skp2 SCF could induce mH2A1 ubiquitination in vitro (Fig. 2b). Notably, we found that ubiquitination of mH2A1.1 and mH2A1.2 was profoundly reduced upon Skp2 knockdown ( Fig. 2c and Supplementary Fig. 1d), suggesting that Skp2 is required for ubiquitination of mH2A1.1 and mH2A1.2. We further demonstrated that Skp2 triggered K48-linked ubiquitination of mH2A1.1, while it induced K63-linked ubiquitnaiton of Akt (Fig. 2d). Accordingly, these results suggest that mH2A1 is a novel substrate for Skp2.
K48-linked ubiquitination is linked to proteosome-dependent degradation 18 . The finding that Skp2 promotes K48-linked ubiquitination of mH2A1 in a proteosome-dependent manner suggests that Skp2 may regulate mH2A1 protein level and stability. Consistent with this notion, we found that mH2A1 protein level was upregulated in primary Skp2 −/− MEFs compared to wild-type (Wt) MEFs ( Supplementary Fig. 1e). We also confirm this result by demonstrating that Skp2 knockdown in various cancer cells induced mH2A1 protein expression ( Supplementary Fig. 2a,b), correlated with a reduction in ubiquitination of mH2A1.1 and mH2A1.2 upon Skp2 knockdown ( Fig. 1c and Supplementary Fig. 1d). In accordance with its protein expression, mH2A1 protein stability was also higher in Skp2 −/− MEFs and Skp2 knockdown breast cancer cells ( Fig. 2e and Supplementary Fig. 3a). Moreover, we showed that Skp2 overexpression in breast cancer cells reduced mH2A1 protein levels ( Supplementary Fig. 2c) and protein stability ( Supplementary Fig. 3b). Our results therefore suggest that Skp2 SCF is the E3 ligase for mH2A1, which promotes mH2A1 ubiquitination and degradation.

mH2A1 regulates G2/M arrest and polyploidy upon Skp2 loss
Primary Skp2 −/− MEFs display profound reduction in cell proliferation and migration, but higher percentage of G2/M phase and polyploidy 11,[19][20] . The finding that mH2A1 protein expression is induced in Skp2 −/− MEFs prompted us to test whether mH2A1 overexpression recapitulates the phenotypes in Skp2 deficient cells. Indeed, we found that mH2A1.1 overexpression induced growth arrest, G2/M arrest, and polyploidy, mirroring the phenotypes upon Skp2 deficiency in MEFs (Fig. 3a-d). We next determined whether mH2A1 upregulation is responsible for these phenotypes observed in Skp2 −/− MEFs. To achieve this goal, we knocked down mH2A1 in Skp2 −/− MEFs (Fig. 4a) and determined whether the phenotypes from Skp2 −/− MEFs are rescued. FACS analysis revealed that while primary Skp2 −/− MEFs exhibited much higher percentage of G2/M phase and polyploidy compared to WT MEFs, mH2A1 knockdown could significantly rescue these phenotypes ( Fig. 4b-d), suggesting that mH2A1 upregulation contributes to G2/M arrest and polyploidy in Skp2 −/− MEFs.
Notably, we found that growth defect in Skp2 −/− MEFs could be partially rescued by mH2A1 knockdown (Fig. 4e). Similarly, mH2A1 knockdown also rescued the growth defect of two breast cancer cell lines with Skp2 silencing ( Supplementary Fig. 4a,b), as determined cell proliferation assay and colony forming assay ( Supplementary Fig. 4c). Moreover, we found that cell migration defect upon Skp2 knockdown in breast cancer cells could be also rescued by mH2A1 knockdown (Supplementary Fig. 4d).

mH2A1 mediates tumour suppressive effect upon Skp2 knockdown
Recent studies reveal that Skp2 overexpression promotes cancer development, but its deficiency leads to tumour suppression [4][5][6][19][20] . We next sought to determine whether mH2A upregulation accounts for breast tumour suppression upon Skp2 silencing. Remarkably, while Skp2 silencing markedly reduced breast cancer development, mH2A1 knockdown reversed such tumour suppressive effect (Fig. 4f). Consistent with results in cell models, breast tumour with Skp2 deficiency also displayed enhanced mH2A1 protein expression ( Supplementary Fig. 4e). It should be noted that mH2A1 knockdown promoted breast cancer development (Fig. 4f). The immunohistochemistry (IHC) staining in breast tumours revealed that Skp2 knockdown reduced breast cancer cell proliferation in vivo, as determined by Ki-67 staining, and such suppression effect could be rescued by mH2A1 knockdown (Fig. 4g). However, there was no obvious difference in apoptosis between Skp2 knockdown versus both Skp2 and mHA1 knockdown groups ( Supplementary Fig. 4f). Accordingly, these results suggest that mH2A1 induction serves as a tumour suppressive signal responsible for inhibition of cell growth, cell migration and breast cancer development upon Skp2 deficiency.

CDK8 restoration rescues the phenotypes upon Skp2 loss
To understand how Skp2-mediated mH2A1 ubiquitination and degradation regulates G2/M arrest, polyploidy, cell migration and tumourigenesis, we performed the systematically nonbiased microarray analysis in primary WT and Skp2 deficient cells. We found that around 3000 genes were upregulated, but 4,000 genes were downregulated upon Skp2 deficiency (data were deposited to GEO as GSE23064). As mH2A1 is a transcription repressor, upregulation of mH2A1 observed in Skp2 deficiency cells should lead to gene repression. We therefore focused on the genes downregulated in Skp2 deficient cells. Among those genes downregulated upon Skp2 deficiency, we found that CDK8 (cyclin-dependent kinase 8) was the gene whose mRNA expression was profoundly reduced in Skp2 deficient cells (Supplementary Table. 1). We selected CDK8 for further study because of its important role in cancer regulation [21][22] . CDK8 is a component of the Mediator complex consisting of CDK8, Cyclin C, Med12 and Med13, which can serve as either a transcription activator or repressor dependently of distinct promoters of its target genes. Interestingly, CDK8 can also indirectly regulate gene expression through directly phosphorylating its protein substrates, such as receptor activated Smads and E2F1, and likely independently of Mediator complex formation [23][24][25] . CDK8 displays an oncogenic activity in melanoma and colon cancer [21][22] . To confirm our microarray results, we performed real time PCR analysis to determine the mRNA level of CDK8 expression in various cancer cells upon Skp2 overexpression or knockdown. Consistently, we found that Skp2 overexpression promoted mRNA level of CDK8, while its knockdown reduced it (Fig. 5a). In accordance with the real time PCR analysis, Western blot analysis revealed that Skp2 overexpressed breast cancer cells displayed higher CDK8 protein expression, whereas Skp2 knockdown cells exhibited less CDK8 protein expression (Fig. 5b, c). Accordingly, Skp2 is therefore a novel upstream regulator for CDK8 gene and protein expression.
CDK8 gene expression was negatively regulated by mH2A 17 . Given Skp2 promotes mH2A1 ubiquitination and degradation, it is highly possible that Skp2 regulates CDK8 expression by promoting mH2A1 degradation. To test this hypothesis, we silenced mH2A1 expression in the context of Skp2 knockdown to assess CDK8 expression. As expected, while Skp2 knockdown markedly reduced CDK8 protein expression, mH2A1 silencing rescued this effect (Fig. 5d), suggesting that Skp2 induces CDK8 expression acting through mH2A1 degradation. We next investigated whether CDK8 is a critical downstream player that executes Skp2-medaied G2/M transition, polyploidy, and cell proliferation. Notably, we found that CDK8 restoration partially rescued defect in cell growth, G2/M arrest and polyploidy observed in Skp2 −/− MEFs ( Fig. 5e-h). It should be noted that the effect of Skp2, mH2A1 and CDK8 on G2/M arrest and polyploidy can be seen in primary MEFs, but not in breast cancer cells such as MDA-MB-231 cells. Since MDA-MB-231 display diverse genetic alterations like p53 mutations, it is likely that some of genetic alterations may affect the effect of Skp2, mH2A1 and CDK8 on G2/M arrest and polyploidy. Altogether, these results suggest that Skp2 promotes mH2A1 degradation and CDK8 expression, in turn regulating cell growth, G2/M transition and polyploidy in primary MEFs.

CDK8 rescues breast cancer development upon Skp2 loss
To understand whether CDK8 mediated Skp2's oncogenic activity, we restored CDK8 expression in Skp2 knockdown breast cancer cells for cell proliferation and cell migration assays. Strikingly, while Skp2 knockdown inhibited breast cancer cell proliferation and migration, restoration of CDK8 in Skp2 knockdown cancer cells rescued these defects ( Supplementary Fig. 5a, b), to the extent similar to that regulated by mH2A1 knockdown (Supplementary Fig. 4a). As CDK8 rescues defect in breast cancer cell growth upon Skp2 knockdown, we examined whether CDK8 may regulate cell cycle profile in breast cancer cells. FACS analysis revealed that CDK8 knockdown did not obviously affect cell cycle phases ( Supplementary Fig. 5c).
Strikingly, we found that CDK8 restoration rescued the defect in breast cancer development upon Skp2 knockdown (Fig. 6a). Skp2 knockdown reduced breast cancer cell proliferation in vivo and such suppression effect could be rescued by CDK8 restoration ( Supplementary  Fig. 5d). However, no obvious difference in apoptosis was observed between groups. Altogether, these results suggest that Skp2 promotes CDK8 expression to regulate cancer cell growth, migration and breast cancer development.

The correlations of Skp2, mH2A1 and CDK8 in human breast cancer samples
To understand the role of Skp2-mH2A1-CDK8 axis and their clinical correlations in human breast cancer, we performed IHC to determine the expression of these proteins in human breast cancer samples (Fig. 6b) Notably, we found that all Skp2, mH2A1.1, mH2A1.2 and CDK8 expression significantly predicted disease-specific (DSS) and metastasis-free survival (MeFS) ( Table-3, together with pT status, pN status and stage, Fig. 6f-h, and Supplementary Fig. 6c-h). In the multivariate analysis, only Skp2 overexpression remained prognostically significant for DSS. Of note, both Skp2 and CDK8 expression significantly predicted worse MeFS (Table-4). These results along with the data from mouse tumour models suggest that Skp2-mH2A1-CDK8 axis serves as a critical signal for breast caner development and represents an important biomarker for poor survival outcome of breast cancer patients.

CDK8 promotes p27 ubiquitination and degradation
p27 is shown to be a critical substrate of Skp2 and is responsible for Skp2-mediaed cell cycle progression, polyploidy and tumourigenesis. The finding that mH2A1-CDK8 axis also regulates Skp2-mediated cell cycle progression, polyploidy and tumourigenesis raises the possibility that CDK8 may function to cross-talk with p27. To test this hypothesis, we examined whether CDK8 regulates p27 expression. Although CDK8 knockdown failed to affect mRNA level of p27, it significantly induced p27 protein expression (Supplementary Fig. 7a, b). Conversely, CDK8 overexpression reduced p27 protein expression, but not p27 mRNA level (Supplementary Fig. 7c-e). Thus, CDK8 is a novel regulator for p27 protein expression.
Notably, the ability of CDK8 to promote p27 ubiquitination was compromised upon Skp2 knockdown, suggesting that CDK8 may function upstream of Skp2 to facilitate Skp2mediated ubiquitination and degradation of p27. Indeed, we found that the ability of Skp2 to promote p27 ubiquitination was also compromised upon CDK8 knockdown (Fig. 7c). However, CDK8 overexpression did not affect Akt ubiquitination by SCF-Skp2 ( Supplementary Fig. 7a). These results suggest that CDK8 cooperates with Skp2 to regulate p27 ubiquitination and degradation.
CDK8 not only serves as a transcription regulator, but also a protein kinase. The finding that CDK8 only regulates p27 protein expression and stability ( Supplementary Fig. 7b-d and Supplementary Fig. 8a,b), but not p27 mRNA level ( Supplementary Fig. 7e), suggests that CDK8 may functions as a kinase for p27 and in turn regulates p27 ubiquitination and degradation. To test this hypothesis, we determined whether CDK8 interacts with p27. CO-IP assay revealed that endogenous CDK8 could interact with exogenous and endogenous p27 ( Supplementary Fig. 8c, d). We next determined whether CDK8 phosphorylates p27 and found that CDK8 overexpression induced p27 phosphorylation at T187 residue (Fig. 7d), while CDK8 silencing reduced it (Fig. 7e). In vitro kinase assay further showed that CDK8, but not kinase dead CDK8 (CDK8-KD), could induce p27 phosphorylation at T187 (Fig.  7F).
Phosphorylation of p27 at T187 by CDK2 is thought to be critical for Skp2-mediated p27 ubiquitination and degradation. Our finding that CDK8 is likely a kinase to phosphorylate p27 at T187 raises the possibility that CDK8 may trigger p27 phosphorylation at T187, in turn facilitating p27 ubiquitination and degradation. This was indeed a case, since CDK8 promoted ubiquitination of wild-type p27, but not ubiquitination of p27-T187A mutant (Fig.  7g). Accordingly, our results suggest that CDK8 is may be a kinase for p27 and that phosphorylation of p27 at T187 induced by CDK8 is required for CDK8-mediated ubiquitination of p27.

Discussion
Our study has revealed several unexpected findings with important clinical implications. First, our study provides the first evidence of how mH2A1 protein abundance and stability is regulated. The exchange of the canonical H2A histones for the mH2A variant is one of the most striking epigenetic alterations at the level of the nucleosome 26 . There is an increasing interest in analyzing the role of histone variant mH2A1 in cancer, but little is known about the molecular mechanism that control mH2A1 protein levels. In the present study, we unequivocally showed Skp2 SCF is an E3 ligase for mH2A1, which triggers K48-linked ubiquitination and degradation of mH2A1. Surprisingly, while Skp2 triggers K48-linked ubiquitination of mH2A1, it also induces K63-linked ubiquitination of mH2A1 (Fig. 2d). Since K63-linked ubiquitination normally does not cause protein degradation, it is likely that Skp2 may also negatively regulate mH2A1 transcriptional activity through nonproteolytic K63-linked ubiquitination of mH2A1. As Skp2 expression was negatively associated with mH2A1.1 (r=−0.624, p<0.001) and mH2A1.2 (r=−0.578, p<0.001) in clinical samples, the deregulation of mH2A1 may represent an oncogenic event. This result also implicates that Skp2 overexpression may contribute to breast cancer pathogenesis by abolishing of mH2A1, which can alter cancer cell proliferation 27 . This notion is indeed supported by our results using animal model showing that mH2A1 silencing rescues tumour suppressive effect upon Skp2 loss (Fig. 4f).
Our data also suggest a new approach to increase mH2A1 levels by Skp2 targeting, which may has important clinical usage for the treatment of cancer and certain diseases. For example, it is found that elevated mH2A1 expression facilitates the sensitivity of Huntington's disease to the treatment of histone deacetylase inhibitors 28 . It will be interesting to see if the combination of Skp2 inhibition by a specific Skp2 inhibitor 29 along with histone deacetylase inhibitors yields better efficacy for Huntington's disease and/or cancer.
Second, we have identified the new role of mH2A1 and CDK8 in orchestrating G2/M checkpoint and polyploidy in primary MEFs. We showed that upregulation of mH2A1 or downregulation of CDK8 in primary MEFs not only regulates cell growth and migration, but also causes G2/M arrest and polyploidy, similar to Skp2 deficiency does. While exactly how polypolidy is regulated by Skp2, mH2A1 and CDK8, it is likely that defect in mitosis may be one of the mechanisms accounting for polyploidy as it was shown that defect in mitosis can link to polyploidy 30 . However, such assumption requires further investigation.
Third, our study also provides importantly novel mechanism by which oncogenic Skp2 regulates G2/M arrest, polyploidy, cell growth, cell migration and tumourigenesis. We showed upregulation of mH2A1 that leads to the downregulation of CDK8 is responsible for these phenotypes regulated by Skp2 deficiency. While G2/M arrest and polypoloidy has impact on tumourigenesis, they are not very likely involved in Skp2-mH2A1-CDK8mediated tumourigenicity of MDA-MB-231 cells, since G2/M arrest and polyploidy regulated by Skp2, mH2A1 and CDK8 can be seen in primary MEFs, but not in MDA-MB-231 cells where p53 is mutated. Thus, the p53 status may have great impact on the role of these proteins in G2/M arrest and polyploidy. This phenomenon is also consistent with the previous study showing that Skp2 silencing induces G2/M arrest in p53-intact, but not in p53-mutant melanoma cells 12 . Therefore, it is possible that Skp2-mH2A-CDK8 axis regulates tumourigenicity of MDA-MB-231 cells by modulating cell growth and cell migration.
Recent study reveals that mH2A1 is downregulation melanoma and lung cancers 17,31 . It also has been reported that CDK8 expression is upregulated in melanoma and colorectal cancers 17,21 . However, the expression of these two markers and their prognosis value in breast cancer remain undefined. We showed that Skp2 overexpression, mH2A1 downregulation and CDK8 overexpression are detected in advanced breast cancer and all predict poor survival outcome for breast cancer patients. Our study demonstrates that the expression of mH2A1 and CDK8 in protein expression level can predict the survival outcome of breast cancer patients. Importantly, Skp2 expression positively correlated with CDK8 expression, but inversely associates with mH2A1 expression. It suggests that mH2A1 is a novel tumour suppressor, while CDK8 can serve as an oncogene in breast cancer.
Finally, our study reveals that CDK8 may be a kinase for p27. CDK8 is shown to drives phosphorylation of p27 at T187 residue, leading to facilitating Skp2-mediated p27 ubiquitination and degradation. Previous studies reveal that CyclinE/CDK2 or CyclinA/ CDK2 is also a kinase for T187 phosphorylation of p27. While CDK8 may serve as a direct kinase for p27 and trigger T187 phophorylation, it may also regulate p27 T187 phosphorylation indirectly through modulating CyclinA/CDK2 activity [32][33] . In support of this notion, we found that CDK8 knockdown in breast cancer cells reduces Cyclin A, but not CDK2 and Cyclin E, protein expression, accompanied by a reduction in pRb phosphorylation, a readout for CDK2 kinase activity ( Supplementary Fig. 5c). Thus, our results suggest that CDK8 may regulate p27 T187 phosphorylaiton and degradation by either serving as a kinase for p27 or regulating CyclinA/CDK2 kinase activity.
Notably, we show that CDK8 and Skp2 functionally rely on each other to regulate ubiquitination and degradation of p27. The rationalized model to explain this phenomenon is that CDK8 firstly regulates p27 phosphorylation at T187, which may provide a priming site for Skp2 and allow Skp2 to promote ubiquitination and degradation of p27. Given that Skp2 is a positive regulator for CDK8 gene expression, it is likely that Skp2 may also orchestrate p27 T187 phosphorylation by up-regulating CDK8 expression, thereby providing a positive feedback loop to further facilitate p27 ubiquitination and degradation. As previous studies reveal that p27 is an important Skp2 downstream substrate that mediates Skp2's biological functions 10,34 , it is very likely that CDK8 may converge on the p27 pathway to regulate Skp2-mediated cell cycle progression, polyploidy and tumourigenesis.
In summary, our study uncovers a molecular mechanism controlling mH2A1 stability and identifies the Skp2-mH2A1-CDK8 axis as a key pathway for regulating G2/M transition, polyploidy, cell proliferation, cell migration, and breast cancer development (Fig. 7h). We also show that CDK8 promotes phosphorylation of p27 at T187 and then induces p27 ubiquitination and degradation in a Skp2-dependent manner. Given that Skp2 and CDK8 are overexpressed in breast cancer and their overexpression promotes breast cancer development in mouse models, pharmacological inactivation of Skp2 and CDK8 is therefore a potential strategy for those cancers with Skp2 and CDK8 overexpression.

Western blotting
Western blotting was performed as previously described 20 Supplementary Fig 9-17.

Mass Spectrometry and Protein Identification
293T cells stably transfected with vector or Xpress-Skp2 (XP-Skp2) were lysed in E1A buffer (250 mM NaCl, 50 mM HEPES pH 7.5, 0.1% Nonidet P40, 5 mM EDTA), and 8 mg of protein lysates were immunopreicipated with XP antibody, which is not coupled to other matrix, washed by E1A buffer, and subjected to SDS-PAGE. Protein bands were excised from silver-stained gels, and destained by SilverQuestTM (invitrogen) and dried in a SpeedVac concentrator. The gel pieces were then swollen in 30 µl of 20 mM ammonium bicarbonate buffer containing 0.01mg/mL modified sequencing grade trypsin (Promega) and incubated overnight at 37°C. The resulting peptides were extracted sequentially with 1% TFA and 0.1% TFA/60% ACN and the combined extracts lyophilized and analyzed using a Qstar XL Q-TOF mass spectrometer (Applied Biosystems) coupled to an UltiMate™ Nano LC system (Dionex/LC Packings, Amsterdam, Netherlands). Proteins were identified by MASCOT software (Matrix Science, Boston, MA) against the NCBI protein database. The following search parameters were used: trypsin was used as the digest enzyme, mass tolerance for monoisotopic peptide window was set to ± 1.0 Da, the MS/MS tolerance window was set to ± 0.5 Da, one missed cleavage was allowed, and carbamidomethyl, deamidated and oxidized methionine were chosen as variable modifications, respectively. The utilized matching criteria were as follows: each identified peptide had to score more than 20, and at least two unique peptides had to be specific to the identified protein.

Ubiquitylation assay in vitro and in vivo
In vivo ubiquitination assays were performed as described 35 . In brief, 293T cells were transfected with the indicated plasmids for 48 hr and lysed by the denatured buffer (6M guanidine-HCl, 0.1M Na2HPO4/NaH2PO4, 10mM imidazole). The supernatants will incubate with nickel beads for 3 hr, washed, and subjected to immunoblotting analysis. For in vitro ubiquitination assays, Flag-Skp2 and Flag-mH2A1 proteins purified from the 293T cells were incubated with for at 37 °C for 3 hr in 20 ml of reaction buffer (20 mM HEPES [pH 7.4], 10 mM MgCl2, 1 mM DTT, 59 mM ubiquitin, 50 nM E1, 850 nM of Ubc13/ Uev1a, 1 mM ATP, 30 mM creatine phosphate, and 1 U of creatine kinase). Proteins were eluted in SDS-sample buffer and immunoblotted with anti-mH2A1 antibody.

MTT cell growth Assay and cell migration assay
For MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium Bromide) cell growth assay, cells were planted in 96-well plates with a concentration of 1000 cells per well. 20 µl of 5 mg/ml MTT was added into the media and cultured for 4 h. Then the media were removed, DMSO was added to dissolve the deposits. At last, the absorbance at 550 nm can be measured by the microplate reader. Cell migration assay was carried out in 24 well transwell plate with 8-µm polyethylene terephalate membrane filters [Falcon cell culture insert (Becton-Dickinson)] separating the lower and upper culture chambers. In brief, Approximately 1 ×10 5 cells were seeded into in the upper chamber with serum-free DMEM medium. The bottom chamber contained DMEM with 10% FBS. After the incubation period, the filter was removed and non-migrant cells on the upper side of the filter were detached using a cotton swab. Cells that migrated to the lower side of the membrane were stained with 0.1% crystal violet for 20 min and counted from three random fields.

Flow Cytometry
For DNA content studies, cells were harvested, washed with phosphate-buffered saline (PBS), and fixed in ice-cold 70% ethanol. Cells were then stained with 0.01% propidium iodide solution for 30 minutes. The ethanol was removed by spinning down for 5 minutes, and cells were analyzed with a FACSCan flow cytometer.

In vitro kinase assay
CDK8-WT and CDK8-KD proteins were synthesized in vitro using the TNT High-Yield Protein Expression System (Promega). Flag-p27 was obtained by immunoprecipitation using anti-Flag agarose from the lysates of 293 cells overexpressing Flag-p27. Then, the samples were incubated with 3 µg of CDK8-WT and CDK8-KD in 50 µl of kinase buffer (Cell Signaling Technology) containing 10 µM ATP at 30°C for 30 minutes. Subsequently, the samples were subjected to SDS-PAGE, followed by immunoblotting.

Microarray analysis
WT and Skp2 −/− LT-HSCs (long-term-hematopoietic stem cells) were isolated from WT and Skp2 −/− mice as described 36 . Total RNAs from WT and Skp2 −/− LT-HSCs were subjected to microarray analysis. Microarray experiments were carried out using the "Human Whole Genome Oligo Microarray Kit" from Agilent (Agilent Technologies, Alto, CA) and the manufacturer's protocol was followed. Detailed protocol can be found at Agilent website: www.Agilent.com. Briefly, 200ng of total RNA from each sample was used and labeled with either Cy3-or Cy5-CTP and after 17hr hybridization at 65°C, the arrays were washed and scanned with Agilent's dual-laser based scanner. Then, Feature Extraction software GE2-v5_95 was used to link a feature to a design file and determine the relative fluorescence intensity between the two samples. Gene lists were created using p-value information from the internal replicates within the microarray. The result was shown as a ratio between Skp2 −/− and WT HSCs (Skp2 −/− verses WT).

In vivo tumourigenesis assay
For in vivo tumourigenesis assays, MDA-MB-231 stable cells mixed with matrigel (1:1) were subcutaneously injected into the left flank of 6-week-old female nude mice. Tumour size was measured weekly using a caliper, and Tumour volume was determined with the standard formula: L×W 2 ×0.52, where L is the longest diameter and W is the shortest diameter. The animal study was approved by the Institutional Animal Care and Use Committee (IACUC) from MD Anderson Cancer Center.

Patients and human materials
The institutional review board of Chi-Mei Medical Center had approved the study by using formalin-fixed tissue from BioBank (IRB9812-012). As a rule set by MOHW (Taiwan), informed consent had been obtained for every case enrolled into BioBank. Immunohistochemical analysis was performed on 189 consecutively treated breast carcinomas underwent modified radical mastectomy between 1997 and 2003. No adjuvant chemotherapy was given in these patients. Pathological staging was assigned based on the7 th edition of American Joint Committee on Cancer (AJCC) system.

Immunohistochemistry and scoring
The procedures of immunohistochemical studies were performed as previously described (Cell. 2012 May 25;149(5):1098-111.). In brief, sections were cut onto an adhesive-coated glass slides at 3-µm thickness. For staining in human samples, the slides were incubated with primary antibodies against Skp2 (Invitrogen, 1:100), mH2A1.1 (Cell signaling, 1:50), mH2A1.2 (1:100), CDK8 (Santa Cruz, 1:100), polyclonal cleaved caspase-3 (Cell Signaling, 1:100) and Ki-67 antibody (Epitomics, 1:160). Primary antibodies were detected using the ChemMateDAKO EnVision kit (DAKO, K5001). The slides were incubated with the secondary antibody for 30 minutes and developed with 3,3-diaminobenzidine for 5 minutes. Incubation without the primary antibody was used as a negative control. Immunoexpression of cleaved caspase-3 was scored by two experienced pathologists using a multiheaded microscope to reach a consensus for each case. The staining was evaluated based on a combination of both the percentage and intensity of positively stained Tumor cells to generate an H-score, which was calculated using the following equation: H-score = ΣPi (i + 1), where i is the intensity of the stained Tumor cells (0 to 4 +), and Pi is the percentage of stained Tumor cells for each intensity. The final score = extent score × intensity score: -=negative immunostaining: score 0-2; + = moderate immunostaining; score 3-5; ++ = strong immunostaining; score =>6. In order to evaluate the stained sections descriptively with Ki-67 antibodies, the numbers of positive cells were counted. Cell nuclei for Ki-67 with bright brown color were considered as positive cells. Cell counts were made at x40 magnification with conventional light microscope in ten randomly selected fields, counting minimum of 1,000 cells. Labeling Index (LI) = Number of IHC positive cells × 100/Total number of cells observed.

Statistical analysis
Statistical analyses were performed using the SPSS 14 software package. The Kruskal-Wallis H test and Mann-Whitney U test were used to assess the differential expression level of Skp2, mH2A1.1, mH2A1.2 and CDK8 expression in relation to important clinicopathologic variables. The Pearson's correlation coefficient was used to clarify the association between Skp2, mH2A1.1, mH2A1.2 and CDK8 expression levels. The endpoint analyzed was disease-specific (DSS) and distal metastasis-free survivals (MeFS), calculated from the starting date of surgery to the date disease-related death or metastatic events. The median period of follow-up was 107 months (range, 6 to 144). Univariate survival analysis was performed using the Cox proportional hazards model. Survival curves were plotted using the Kaplan-Meier method, and log-rank tests were performed to evaluate prognostic differences between groups for categorical variables. A multivariate model was performed using Cox proportionalhazards regression, including parameters with univariate p<0.05. Since primary Tumour status (pT) and nodal status (pN) are component factors of Tumour stage, only stage was enrolled into multivariate analysis. For all analyses, two-sided tests of significance were used with p=< 0.05 considered significant.

Supplementary Material
Refer to Web version on PubMed Central for supplementary material.       Flag-p27-187A were immunoprecipitated with Flag antibody and subjected to the immunoblotting assay. p27 phosphorylation at T187 was detected by using an antibody against phosphorylated p27 at T187. (e) Cell lysates from Luc or CDK8 knockdown 293T cells transfected with vector or Flag-p27 were immunoprecipitated with Flag antibody and subjected to the immunoblotting assay. p27 phosphorylation at T187 was detected by using an antibody against phosphorylated p27 at T187. (f) CDK8 phosphorylates p27 in vitro. Immunopurified Flag-p27 proteins were incubated with or without recombinant active CDK8 or CDK8-KD proteins and subjected to the immunoblotting assay. p27 phosphorylation at T187 was detected by using an antibody against phosphorylated p27 at T187. (g) In vivo ubiquitination assay of 293T cells transfected with Flag-p27 WT or Flag-p27 T187A along with other constructs. (h) The working model for Skp2 E3 ligase-mediated cell growth, cell migration, cell cycle checkpoint, polyploidy and tumourigenesis. Skp2 interacts with mH2A1 and induces its ubiquitination and degradation. mH2A1 degradation induced by Skp2 leads to gene and protein expression of CDK8, which may serve as a kinase for p27 or regulate CyclinA/CDK2 to drive p27 phosphorylation at T187, leading to promoting Skp2-mediated p27 ubiquitination and degradation, which may contribute to cell growth, cell migration, G2/M arrest, polyploidy and tumourigenesis. Alternatively, Skp2-mH2A1-CDK8 axis may regulate these processes independently of p27 regulation.   Table 3 Univariate log-rank analyses.