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1.  Cks1 Is Required for Tumor Cell Proliferation but Not Sufficient to Induce Hematopoietic Malignancies 
PLoS ONE  2012;7(5):e37433.
The Cks1 component of the SCFSkp2 complex is necessary for p27Kip1 ubiquitylation and degradation. Cks1 expression is elevated in various B cell malignancies including Burkitt lymphoma and multiple myeloma. We have previously shown that loss of Cks1 results in elevated p27Kip1 levels and delayed tumor development in a mouse model of Myc-induced B cell lymphoma. Surprisingly, loss of Skp2 in the same mouse model also resulted in elevated p27Kip1 levels but exhibited no impact on tumor onset. This raises the possibility that Cks1 could have other oncogenic activities than suppressing p27Kip1. To challenge this notion we have targeted overexpression of Cks1 to B cells using a conditional retroviral bone marrow transduction-transplantation system. Despite potent ectopic overexpression, Cks1 was unable to promote B cell hyperproliferation or B cell malignancies, indicating that Cks1 is not oncogenic when overexpressed in B cells. Since Skp2 overexpression can drive T-cell tumorigenesis or other cancers we also widened the quest for oncogenic activity of Cks1 by ubiquitously expressing Cks1 in hematopoetic progenitors. At variance with c-Myc overexpression, which caused acute myeloid leukemia, Cks1 overexpression did not induce myeloproliferation or leukemia. Therefore, despite being associated with a poor prognosis in various malignancies, sole Cks1 expression is insufficient to induce lymphoma or a myeloproliferative disease in vivo.
doi:10.1371/journal.pone.0037433
PMCID: PMC3356264  PMID: 22624029
2.  Myc suppression of Nfkb2 accelerates lymphomagenesis 
BMC Cancer  2010;10:348.
Background
Deregulated c-Myc expression is a hallmark of several human cancers where it promotes proliferation and an aggressive tumour phenotype. Myc overexpression is associated with reduced activity of Rel/NF-κB, transcription factors that control the immune response, cell survival, and transformation, and that are frequently altered in cancer. The Rel/NF-κB family member NFKB2 is altered by chromosomal translocations or deletions in lymphoid malignancies and deletion of the C-terminal ankyrin domain of NF-κB2 augments lymphocyte proliferation.
Methods
Precancerous Eμ-Myc-transgenic B cells, Eμ-Myc lymphomas and human Burkitt lymphoma samples were assessed for Nfkb2 expression. The contribution of Nfkb2 to Myc-driven apoptosis, proliferation, and lymphomagenesis was tested genetically in vivo.
Results
Here we report that the Myc oncoprotein suppresses Nfkb2 expression in vitro in primary mouse fibroblasts and B cells, and in vivo in the Eμ-Myc transgenic mouse model of human Burkitt lymphoma (BL). NFKB2 suppression by Myc was also confirmed in primary human BL. Promoter-reporter assays indicate that Myc-mediated suppression of Nfkb2 occurs at the level of transcription. The contribution of Nfkb2 to Myc-driven lymphomagenesis was tested in vivo, where Nfkb2 loss was shown to accelerate lymphoma development in Eμ-Myc transgenic mice, by impairing Myc's apoptotic response.
Conclusions
Nfkb2 is suppressed by c-Myc and harnesses Myc-driven lymphomagenesis. These data thus link Myc-driven lymphomagenesis to the non-canonical NF-κB pathway.
doi:10.1186/1471-2407-10-348
PMCID: PMC2902445  PMID: 20598117
3.  ErbB2 Potentiates Breast Tumor Proliferation through Modulation of p27Kip1-Cdk2 Complex Formation: Receptor Overexpression Does Not Determine Growth Dependency 
Molecular and Cellular Biology  2000;20(9):3210-3223.
Overexpression of the ErbB2 receptor, a major component of the ErbB receptor signaling network, contributes to the development of a number of human cancers. ErbB2 presents itself, therefore, as a target for antibody-mediated therapies. In this respect, anti-ErbB2 monoclonal antibody 4D5 specifically inhibits the growth of tumor cells overexpressing ErbB2. We have analyzed the effect of 4D5-mediated ErbB2 inhibition on the cell cycle of the breast tumor cell line BT474. 4D5 treatment of BT474 cells resulted in a G1 arrest, preceded by rapid dephosphorylation of ErbB2, inhibition of cytoplasmic signal transduction pathways, accumulation of the cyclin-dependent kinase inhibitor p27Kip1, and inactivation of cyclin-Cdk2 complexes. Time courses demonstrated that 4D5 treatment redirects p27Kip1 onto Cdk2 complexes, an event preceding increased p27Kip1 expression; this correlates with the downregulation of c-Myc and D-type cyclins (proteins involved in p27Kip1 sequestration) and the loss of p27Kip1 from Cdk4 complexes. Similar events were observed in ErbB2-overexpressing SKBR3 cells, which exhibited reduced proliferation in response to 4D5 treatment. Here, p27Kip1 redistribution resulted in partial Cdk2 inactivation, consistent with a G1 accumulation. Moreover, p27Kip1 protein levels remained constant. Antisense-mediated inhibition of p27Kip1 expression in 4D5-treated BT474 cells further demonstrated that in the absence of p27Kip1 accumulation, p27Kip1 redirection onto Cdk2 complexes is sufficient to inactivate Cdk2 and establish the G1 block. These data suggest that ErbB2 overexpression leads to potentiation of cyclin E-Cdk2 activity through regulation of p27Kip1 sequestration proteins, thus deregulating the G1/S transition. Moreover, through comparison with an ErbB2-overexpressing cell line insensitive to 4D5 treatment, we demonstrate the specificity of these cell cycle events and show that ErbB2 overexpression alone is insufficient to determine the cellular response to receptor inhibition.
PMCID: PMC85615  PMID: 10757805
4.  S-phase kinase-associated protein-2 (Skp2) promotes vascular smooth muscle cell proliferation and neointima formation in vivo 
Journal of Vascular Surgery  2009;50(5-4):1135-1142.
Objective
Vascular smooth muscle cell (VSMC) proliferation plays an important role in the development of postangioplasty or in-stent restenosis, venous graft failure, and atherosclerosis. Our previous work has demonstrated S-phase kinase-associated protein-2 (Skp2), an F-box subunit of SCFSkp2 ubiquitin ligase, as an important mediator and common final pathway for growth factors, extracellular matrices, and cyclic-nucleotides to regulate VSMC proliferation in vitro. However, whether alteration of Skp2 function also regulates VSMC proliferation in vivo and neointimal thickening postvascular injury remains unclear. We investigated the effect of Skp2 on VSMC proliferation and neointimal formation in vivo.
Methods and Results
Firstly, we demonstrated that Skp2-null mice developed significantly smaller neointimal areas than wild-type mice after carotid ligation. Secondly, to further identify a local rather than a systemic effect of Skp2 alteration, we demonstrated that adenovirus-mediated expression of dominant-negative Skp2 in the balloon-injured rat carotid artery significantly increased medial p27Kip1 levels, inhibited VSMC proliferation, and the subsequent neointimal thickening. Lastly, to determine if Skp2 alone is sufficient to drive VSMC proliferation and lesion development in vivo, we demonstrated that adenovirus-delivery of wild-type Skp2 to the minimally-injured rat carotids is sufficient to downregulate p27Kip1 protein levels, enhanced medial VSMC proliferation, and the neointimal thickening.
Conclusion
This data provides, we believe for the first time, a more comprehensive understanding of Skp2 in the regulation of VSMC proliferation and neointimal formation and suggests that Skp2 is a promising target in the treatment of vasculoproliferative diseases.
Clinical Relevance
This manuscript describes our latest work investigating the role of the Skp2, an F-box protein component of the SCFskp2 ubiquitin-ligase, in promoting VSMC proliferation, and neointima formation in response to vascular injury in vivo. Our previous work has identified a major role for Skp2 as a key target for numerous positive and negative growth regulatory signals in vitro. These signals converge to regulate the expression of Skp2, which then controls cell-cycle progression by promoting degradation of the cyclin-dependent kinase inhibitor, p27Kip1. Until now, there has been no data in the literature on the role played by Skp2 in the regulation of VSMC proliferation and neointima formation in vivo. Our current manuscript describes, we believe for the first time, the important role played by Skp2 in these processes, using both mouse and rat arterial injury models. This is important because proliferation of VSMCs underlies the development of postangioplasty or post-stenting restenosis, venous graft failure, and transplant arteriosclerosis. Our work demonstrates for the first time that Skp2 is a major regulator of VSMC proliferation and neointimal thickening in vivo in response to vascular injury and highlights Skp2 as a potential target for future strategies designed to combat vasculoproliferative diseases.
doi:10.1016/j.jvs.2009.07.066
PMCID: PMC2774860  PMID: 19878790
5.  Cortactin Modulates RhoA Activation and Expression of Cip/Kip Cyclin-Dependent Kinase Inhibitors To Promote Cell Cycle Progression in 11q13-Amplified Head and Neck Squamous Cell Carcinoma Cells ▿ †  
Molecular and Cellular Biology  2010;30(21):5057-5070.
The cortactin oncoprotein is frequently overexpressed in head and neck squamous cell carcinoma (HNSCC), often due to amplification of the encoding gene (CTTN). While cortactin overexpression enhances invasive potential, recent research indicates that it also promotes cell proliferation, but how cortactin regulates the cell cycle machinery is unclear. In this article we report that stable short hairpin RNA-mediated cortactin knockdown in the 11q13-amplified cell line FaDu led to increased expression of the Cip/Kip cyclin-dependent kinase inhibitors (CDKIs) p21WAF1/Cip1, p27Kip1, and p57Kip2 and inhibition of S-phase entry. These effects were associated with increased binding of p21WAF1/Cip1 and p27Kip1 to cyclin D1- and E1-containing complexes and decreased retinoblastoma protein phosphorylation. Cortactin regulated expression of p21WAF1/Cip1 and p27Kip1 at the transcriptional and posttranscriptional levels, respectively. The direct roles of p21WAF1/Cip1, p27Kip1, and p57Kip2 downstream of cortactin were confirmed by the transient knockdown of each CDKI by specific small interfering RNAs, which led to partial rescue of cell cycle progression. Interestingly, FaDu cells with reduced cortactin levels also exhibited a significant diminution in RhoA expression and activity, together with decreased expression of Skp2, a critical component of the SCF ubiquitin ligase that targets p27Kip1 and p57Kip2 for degradation. Transient knockdown of RhoA in FaDu cells decreased expression of Skp2, enhanced the level of Cip/Kip CDKIs, and attenuated S-phase entry. These findings identify a novel mechanism for regulation of proliferation in 11q13-amplified HNSCC cells, in which overexpressed cortactin acts via RhoA to decrease expression of Cip/Kip CDKIs, and highlight Skp2 as a downstream effector for RhoA in this process.
doi:10.1128/MCB.00249-10
PMCID: PMC2953065  PMID: 20805359
6.  Androgen Suppresses the Proliferation of Androgen Receptor-Positive Castration-Resistant Prostate Cancer Cells via Inhibition of Cdk2, CyclinA, and Skp2 
PLoS ONE  2014;9(10):e109170.
The majority of prostate cancer (PCa) patient receiving androgen ablation therapy eventually develop castration-resistant prostate cancer (CRPC). We previously reported that androgen treatment suppresses Skp2 and c-Myc through androgen receptor (AR) and induced G1 cell cycle arrest in androgen-independent LNCaP 104-R2 cells, a late stage CRPC cell line model. However, the mechanism of androgenic regulation of Skp2 in CRPC cells was not fully understood. In this study, we investigated the androgenic regulation of Skp2 in two AR-positive CRPC cell line models, the LNCaP 104-R1 and PC-3AR Cells. The former one is an early stage androgen-independent LNCaP cells, while the later one is PC-3 cells re-expressing either wild type AR or mutant LNCaP AR. Proliferation of LNCaP 104-R1 and PC-3AR cells is not dependent on but is suppressed by androgen. We observed in this study that androgen treatment reduced protein expression of Cdk2, Cdk7, Cyclin A, cyclin H, Skp2, c-Myc, and E2F-1; lessened phosphorylation of Thr14, Tyr15, and Thr160 on Cdk2; decreased activity of Cdk2; induced protein level of p27Kip1; and caused G1 cell cycle arrest in LNCaP 104-R1 cells and PC-3AR cells. Overexpression of Skp2 protein in LNCaP 104-R1 or PC-3AR cells partially blocked accumulation of p27Kip1 and increased Cdk2 activity under androgen treatment, which partially blocked the androgenic suppressive effects on proliferation and cell cycle. Analyzing on-line gene array data of 214 normal and PCa samples indicated that gene expression of Skp2, Cdk2, and cyclin A positively correlates to each other, while Cdk7 negatively correlates to these genes. These observations suggested that androgen suppresses the proliferation of CRPC cells partially through inhibition of Cyclin A, Cdk2, and Skp2.
doi:10.1371/journal.pone.0109170
PMCID: PMC4182885  PMID: 25271736
7.  High-Throughput Screening AlphaScreen Assay for Identification of Small-Molecule Inhibitors of Ubiquitin E3 Ligase SCFSkp2-Cks1 
Journal of biomolecular screening  2013;18(8):910-920.
Decreased levels of cell cycle inhibitor p27Kip1 due to excessive degradation occur in a variety of aggressive human tumors. Since reduced p27Kip1 expression has been associated with a poor prognosis in many human cancers and resistance to certain antitumor therapies, elevation of p27Kip1 expression could improve prognosis and prevent excessive cell proliferation. SCFSkp2 is one of the major ubiquitin E3 ligases responsible for degradation of p27Kip1. Ubiquitination of p27Kip1 also requires a small adaptor protein, Cks1, which facilitates substrate recruitment by bridging the interaction between Skp2 and p27Kip1. It has been shown previously that a direct interaction between Cks1 and Skp2 is required for p27Kip1 degradation. Accordingly, perturbation of the Skp2-Cks1 interaction may represent an attractive target for pharmacological intervention. Here we describe a high-throughput AlphaScreen assay for discovering small-molecule inhibitors of the Skp2-Cks1 protein-protein interaction in vitro. Two compounds (NSC689857 and NSC681152) were identified and validated through a structure-activity relationship analysis. Both compounds were also shown to inhibit p27Kip1 ubiquitination in vitro. These studies demonstrate that disruption of the Skp2-Cks1 interaction provides a viable strategy to prevent p27Kip1 ubiquitination and may potentially be useful for the control of excessive degradation of this cell cycle inhibitor in tumor cells.
doi:10.1177/1087057113485789
PMCID: PMC4168015  PMID: 23589337
E3 ligase; inhibitor; Skp2; Cks1; p27kip1; ubiquitin; proteolysis
8.  CDK inhibitor p57Kip2 is negatively regulated by COP9 signalosome subunit 6 
Cell Cycle  2012;11(24):4633-4641.
Subunit 6 of the COP9 signalosome complex, CSN6, is known to be critical to the regulation of the MDM2-p53 axis for cell proliferation and anti-apoptosis, but its many targets remain unclear. Here we show that p57Kip2 is a target of CSN6, and that CSN6 is a negative regulator of p57Kip2. CSN6 associates with p57Kip2, and its overexpression can decrease the steady-state expression of p57Kip2; accordingly, CSN6 deficiency leads to p57Kip2 stabilization. Mechanistic studies show that CSN6 associates with p57Kip2 and Skp2, a component of the E3 ligase, which, in turn, facilitates Skp2-mediated protein ubiquitination of p57Kip2. Loss of Skp2 compromised CSN6-mediated p57Kip2 destabilization, suggesting collaboration between Skp2 and CSN6 in degradation of p57Kip2. CSN6’s negative impact on p57Kip2 elevation translates into cell growth promotion, cell cycle deregulation and potentiated transformational activity. Significantly, univariate Kaplan-Meier analysis of tumor samples demonstrates that high CSN6 expression or low p57 expression is associated with poor overall survival. These data suggest that CSN6 is an important negative regulator of p57Kip2, and that overexpression of CSN6 in many types of cancer could lead to decreased expression of p57Kip2 and result in promoted cancer cell growth.
doi:10.4161/cc.22887
PMCID: PMC3562308  PMID: 23187808
COP9; CSN6; Skp2; cell cycle; p57
9.  Prognostic implication of p27Kip1, Skp2 and Cks1 expression in renal cell carcinoma: a tissue microarray study 
Background
p27Kip1 plays a major role as a negative regulator of the cell cycle. The regulation of p27Kip1 degradation is mediated by its specific ubiquitin ligase subunits S-phase kinase protein (Skp) 2 and cyclin-dependent kinase subunit (Cks) 1. However, little is known regarding the prognostic utility of p27Kip1, Skp2 and Cks1 expression in renal cell carcinoma.
Methods
Immunohistochemistry was performed for p27Kip1, Skp2 and Cks1 in tissue microarrays of 482 renal cell carcinomas with follow-up. The data were correlated with clinicopathological features. The univariate and multivariate survival analyses were also performed to determine their prognostic significance.
Results
Immunoreactivity of p27Kip1, Skp2 and Cks1 was noted in 357, 71 and 82 patients, respectively. Skp2 and Cks1 expression were not noted in chromophobe cancers. A strong correlation was found between Skp2 and Cks1 expression (P < 0.001), both of which were inversely related to p27Kip1 levels (P = 0.006 and P < 0.001), especially in primary and clear-cell cancers. Low p27Kip1 expression and Skp2 expression were correlated with larger tumor size and higher stage, as well as tumor necrosis. Cks1 expression was only correlated with tumor size. In univariate analysis, low p27Kip1 expression, Skp2 and Cks1 expression were all associated with a poor prognosis, while in multivariate analysis, only low p27Kip1 expression were independent prognostic factors for both cancer specific survival and recurrence-free survival in patients with RCC.
Conclusion
Our results suggest that immunohistochemical expression levels of p27Kip1, Skp2 and Cks1 may serve as markers with prognostic value in renal cell carcinoma.
doi:10.1186/1756-9966-27-51
PMCID: PMC2579281  PMID: 18922157
10.  Mitogenic Signaling by the gep Oncogene Involves the Upregulation of S-Phase Kinase-Associated Protein 2 
Genes & cancer  2010;1(10):1033-1043.
The gep oncogene, defined by the activated mutant of the α-subunit of the G protein G12 (Gα12Q229L or Gα12QL), potently stimulates the proliferation of many different cell types in addition to inducing neoplastic transformation of several fibroblast cell lines. While it has been demonstrated that Gα12QL accelerates G1- to S-phase cell cycle progression, the precise mechanism through which Gα12 communicates to cell cycle machinery is largely unknown. In the present study, we report that the activated—mutational as well as receptor-mediated—Gα12 transmits its proliferative signals to cell cycle machinery by modulating the levels of the S-phase kinase-associated protein 2 (Skp2), an E3 ubiquitin ligase, involved in the regulation of the cyclin-dependent kinase inhibitor (CKI), p27Kip1. Our results show that the expression of Gα12QL leads to an increase in the levels of Skp2 with a correlatable decrease in p27Kip1 levels and subsequent increase in the activities of specific CDKs. By demonstrating that the transient expression of Gα12QL induces an increase in Skp2 levels with resultant downregulation of p27Kip1 in both NIH3T3 and human astrocytoma 1321N1 cells, we establish here that the effect of Gα12 on Skp2/ p27Kip1 is cell type independent. In addition, we demonstrate that LPA-stimulated proliferation and changes in Skp2 and p27Kip1 levels in 1321N1 cells could be inhibited by the expression of a dominant-negative mutant of Gα12, thereby pointing to the critical role of Gα12 in LPA-mediated mitogenic signaling. Our findings also indicate that LPA as well as Gα12-mediated upregulation of Skp2 requires a yet to be characterized mechanism involving JNK. Since Skp2 has been identified as an oncogene, and it is overexpressed in many cancers, our results presented here describe for the first time that Skp2 is a novel target in the cell cycle machinery through which Gα12 and its cognate receptors transmit their oncogenic signals.
doi:10.1177/1947601910390516
PMCID: PMC3083023  PMID: 21533006
[AQ: 1]
11.  Mitogenic Signaling by the gep Oncogene Involves the Upregulation of S-Phase Kinase-Associated Protein 2 
Genes & Cancer  2010;1(10):1033-1043.
The gep oncogene, defined by the activated mutant of the α-subunit of the G protein G12 (Gα12Q229L or Gα12QL), potently stimulates the proliferation of many different cell types in addition to inducing neoplastic transformation of several fibroblast cell lines. While it has been demonstrated that Gα12QL accelerates G1- to S-phase cell cycle progression, the precise mechanism through which Gα12 communicates to cell cycle machinery is largely unknown. In the present study, we report that the activated—mutational as well as receptor-mediated—Gα12 transmits its proliferative signals to cell cycle machinery by modulating the levels of the S-phase kinase-associated protein 2 (Skp2), an E3 ubiquitin ligase, involved in the regulation of the cyclin-dependent kinase inhibitor (CKI), p27Kip1. Our results show that the expression of Gα12QL leads to an increase in the levels of Skp2 with a correlatable decrease in p27Kip1 levels and subsequent increase in the activities of specific CDKs. By demonstrating that the transient expression of Gα12QL induces an increase in Skp2 levels with resultant downregulation of p27Kip1 in both NIH3T3 and human astrocytoma 1321N1 cells, we establish here that the effect of Gα12 on Skp2/p27Kip1 is cell type independent. In addition, we demonstrate that LPA-stimulated proliferation and changes in Skp2 and p27Kip1 levels in 1321N1 cells could be inhibited by the expression of a dominant-negative mutant of Gα12, thereby pointing to the critical role of Gα12 in LPA-mediated mitogenic signaling. Our findings also indicate that LPA as well as Gα12-mediated upregulation of Skp2 requires a yet to be characterized mechanism involving JNK. Since Skp2 has been identified as an oncogene, and it is overexpressed in many cancers, our results presented here describe for the first time that Skp2 is a novel target in the cell cycle machinery through which Gα12 and its cognate receptors transmit their oncogenic signals.
doi:10.1177/1947601910390516
PMCID: PMC3083023  PMID: 21533006
oncogene; G12; LPA; Skp2; JNK
12.  NF-κB/STAT3/PI3K signaling crosstalk in iMycEμ B lymphoma 
Molecular Cancer  2010;9:97.
Background
Myc is a well known driver of lymphomagenesis, and Myc-activating chromosomal translocation is the recognized hallmark of Burkitt lymphoma, an aggressive form of non-Hodgkin's lymphoma. We developed a model that mimics this translocation event by inserting a mouse Myc cDNA gene into the immunoglobulin heavy chain locus, just upstream of the intronic Eμ enhancer. These mice, designated iMycEμ, readily develop B-cell lymphoma. To study the mechanism of Myc-induced lymphoma, we analyzed signaling pathways in lymphoblastic B-cell lymphomas (LBLs) from iMycEμ mice, and an LBL-derived cell line, iMycEμ-1.
Results
Nuclear factor-κB (NF-κB) and signal transducer and activator of transcription 3 (STAT3) were constitutively activated in iMycEμ mice, not only in LBLs but also in the splenic B-lymphocytes of young animals months before tumors developed. Moreover, inhibition of either transcription factor in iMycEμ-1 cells suppressed growth and caused apoptosis, and the abrogation of NF-κB activity reduced DNA binding by both STAT3 and Myc, as well as Myc expression. Inhibition of STAT3 signaling eliminated the activity of both NF-κB and Myc, and resulted in a corresponding decrease in the level of Myc. Thus, in iMycEμ-1 cells NF-κB and STAT3 are co-dependent and can both regulate Myc. Consistent with this, NF-κB and phosphorylated STAT3 were physically associated with one another. In addition, LBLs and iMycEμ-1 cells also showed constitutive AKT phosphorylation. Blocking AKT activation by inhibiting PI3K reduced iMycEμ-1 cell proliferation and caused apoptosis, via downregulation of NF-κB and STAT3 activity and a reduction of Myc levels. Co-treatment with NF-κB, STAT3 or/and PI3K inhibitors led to additive inhibition of iMycEμ-1 cell proliferation, suggesting that these signaling pathways converge.
Conclusions
Our findings support the notion that constitutive activation of NF-κB and STAT3 depends on upstream signaling through PI3K, and that this activation is important for cell survival and proliferation, as well as for maintaining the level of Myc. Together, these data implicate crosstalk among NF-κB, STAT3 and PI3K in the development of iMycEμ B-cell lymphomas.
doi:10.1186/1476-4598-9-97
PMCID: PMC2876994  PMID: 20433747
13.  Epidermal growth factor upregulates Skp2/Cks1 and p27kip1 in human extrahepatic cholangiocarcinoma cells 
AIM: To evaluate the expression status of S-phase kinase-associated protein 2 (Skp2)/cyclin-dependent kinases regulatory subunit 1 (Cks1) and p27kip1, and assess the prognostic significance of Skp2/Cks1 expression with p27kip1 in patients with extrahepatic cholangiocarcinoma.
METHODS: Seventy-six patients who underwent curative resection for histologically confirmed extrahepatic cholangiocarcinoma at our institution from December 1994 to March 2008 were enrolled. Immunohistochemical staining for Skp2, Cks1, p27kip1, and Ki67, along with other relevant molecular biologic experiments, were performed.
RESULTS: By Cox regression analyses, advanced age (> 65 years), advanced AJCC tumor stage, poorly differentiated histology, and higher immunostaining intensity of Skp2 were identified as independent prognostic factors in patients with extrahepatic cholangiocarcinoma. Exogenous epidermal growth factor (EGF, especially 0.1-10 ng/mL) significantly increased the proliferation indices by MTT assay and the mRNA levels of Skp2/Cks1 and p27kip1 in SNU-1196, SNU-1079, and SNU-245 cells. The protein levels of Skp2/Cks1 (from nuclear lysates) and p27kip1 (from cytosolic lysate) were also significantly increased in these cells. There were significant reductions in the protein levels of Skp2/Cks1 and p27kip1 (from nuclear lysate) after the treatment of LY294002. By chromatin immunoprecipitation assay, we found that E2F1 transcription factor directly binds to the promoter site of Skp2.
CONCLUSION: Higher immunostaining intensity of Skp2/Cks1 was an independent prognostic factor for patients with extrahepatic cholangiocarcinoma. EGF upregulates the mRNA and protein levels of Skp2/Cks1 and p27kip1 via the PI3K/Akt pathway and direct binding of E2F1 transcription factor with the Skp2 promoter.
doi:10.3748/wjg.v20.i3.755
PMCID: PMC3921485  PMID: 24574749
S-phase kinase-associated protein 2; Cyclin-dependent kinases regulatory subunit 1; P27kip1; Cholangiocarcinoma; E2F1; PI3K/Akt
14.  Myc interacts with Max and Miz1 to repress C/EBPδ promoter activity and gene expression 
Molecular Cancer  2010;9:92.
Background
"Loss of function" alterations in CCAAT/Enhancer Binding Proteinδ (C/EBPδ) have been reported in a number of human cancers including breast, prostate and cervical cancer, hepatocellular carcinoma and acute myeloid leukemia. C/EBPδ gene transcription is induced during cellular quiescence and repressed during active cell cycle progression. C/EBPδ exhibits tumor suppressor gene properties including reduced expression in cancer cell lines and tumors and promoter methylation silencing.
We previously reported that C/EBPδ expression is inversely correlated with c-Myc (Myc) expression. Aberrant Myc expression is common in cancer and transcriptional repression is a major mechanism of Myc oncogenesis. A number of tumor suppressor genes are targets of Myc transcriptional repression including C/EBPα, p15INK4, p21CIP1, p27KIP1 and p57KIP2. This study investigated the mechanisms underlying Myc repression of C/EBPδ expression.
Results
Myc represses C/EBPδ promoter activity in nontransformed mammary epithelial cells in a dose-dependent manner that requires Myc Box II, Basic Region and HLH/LZ domains. Chromatin Immunoprecipitation (ChIP) assays demonstrate that Myc, Miz1 and Max are associated with the C/EBPδ promoter in proliferating cells, when C/EBPδ expression is repressed. EMSAs demonstrate that Miz1 binds to a 30 bp region (-100 to -70) of the C/EBPδ promoter which contains a putative transcription initiator (Inr) element. Miz1 functions exclusively as a repressor of C/EBPδ promoter activity. Miz1 siRNA expression or expression of a Miz1 binding deficient Myc (MycV394D) construct reduces Myc repression of C/EBPδ promoter activity. Max siRNA expression, or expression of a Myc construct lacking the HLH/LZ (Max interacting) region, also reduces Myc repression of C/EBPδ promoter activity. Miz1 and Max siRNA treatments attenuate Myc repression of endogenous C/EBPδ expression. Myc Box II interacting proteins RuvBl1 (Pontin, TIP49) and RuvBl2 (Reptin, TIP48) enhances Myc repression of C/EBPδ promoter activity.
Conclusion
Myc represses C/EBPδ expression by associating with the C/EBPδ proximal promoter as a transient component of a repressive complex that includes Max and Miz1. RuvBl1 and RuvBl2 enhance Myc repression of C/EBPδ promoter activity. These results identify protein interactions that mediate Myc repression of C/EBPδ, and possibly other tumor suppressor genes, and suggest new therapeutic targets to block Myc transcriptional repression and oncogenic function.
doi:10.1186/1476-4598-9-92
PMCID: PMC2879254  PMID: 20426839
15.  Silencing of KIF14 interferes with cell cycle progression and cytokinesis by blocking the p27Kip1 ubiquitination pathway in hepatocellular carcinoma 
Although it has been suggested that kinesin family member 14 (KIF14) has oncogenic potential in various cancers, including hepatocellular carcinoma (HCC), the molecular mechanism of this potential remains unknown. We aimed to elucidate the role of KIF14 in hepatocarcinogenesis by knocking down KIF14 in HCC cells that overexpressed KIF14. After KIF14 knockdown, changes in tumor cell growth, cell cycle and cytokinesis were examined. We also examined cell cycle regulatory molecules and upstream Skp1/Cul1/F-box (SCF) complex molecules. Knockdown of KIF14 resulted in suppression of cell proliferation and failure of cytokinesis, whereas KIF14 overexpression increased cell proliferation. In KIF14-silenced cells, the levels of cyclins E1, D1 and B1 were profoundly decreased compared with control cells. Of the cyclin-dependent kinase inhibitors, the p27Kip1 protein level specifically increased after KIF14 knockdown. The increase in p27Kip1 was not due to elevation of its mRNA level, but was due to inhibition of the proteasome-dependent degradation pathway. To explore the pathway upstream of this event, we measured the levels of SCF complex molecules, including Skp1, Skp2, Cul1, Roc1 and Cks1. The levels of Skp2 and its cofactor Cks1 decreased in the KIF14 knockdown cells where p27Kip1 accumulated. Overexpression of Skp2 in the KIF14 knockdown cells attenuated the failure of cytokinesis. On the basis of these results, we postulate that KIF14 knockdown downregulates the expression of Skp2 and Cks1, which target p27Kip1 for degradation by the 26S proteasome, leading to accumulation of p27Kip1. The downregulation of Skp2 and Cks1 also resulted in cytokinesis failure, which may inhibit tumor growth. To the best of our knowledge, this is the first report that has identified the molecular target and oncogenic effect of KIF14 in HCC.
doi:10.1038/emm.2014.23
PMCID: PMC4044675  PMID: 24854087
cell cycle; cytokinesis; hepatocellular carcinoma; KIF14; p27kip1; ubiquitination
16.  The expression of the ubiquitin ligase subunit Cks1 in human breast cancer 
Breast Cancer Research  2005;7(5):R737-R744.
Introduction
Loss of the cell-cycle inhibitory protein p27Kip1 is associated with a poor prognosis in breast cancer. The decrease in the levels of this protein is the result of increased proteasome-dependent degradation, mediated and rate-limited by its specific ubiquitin ligase subunits S-phase kinase protein 2 (Skp2) and cyclin-dependent kinase subunit 1 (Cks1). Skp2 was recently found to be overexpressed in breast cancers, but the role of Cks1 in these cancers is unknown. The present study was undertaken to examine the role of Cks1 expression in breast cancer and its relation to p27Kip1 and Skp2 expression and to tumor aggressiveness.
Methods
The expressions of Cks1, Skp2, and p27Kip1 were examined immunohistochemically on formalin-fixed, paraffin-wax-embedded tissue sections from 50 patients with breast cancer and by immunoblot analysis on breast cancer cell lines. The relation between Cks1 levels and patients' clinical and histological parameters were examined by Cox regression and the Kaplan–Meier method.
Results
The expression of Cks1 was strongly associated with Skp2 expression (r = 0.477; P = 0.001) and inversely with p27Kip1 (r = -0.726; P < 0.0001). Overexpression of Cks1 was associated with loss of tumor differentiation, young age, lack of expression of estrogen receptors and of progesterone receptors, and decreased disease-free (P = 0.0007) and overall (P = 0.041) survival. In addition, Cks1 and Skp2 expression were increased by estradiol in estrogen-dependent cell lines but were down-regulated by tamoxifen.
Conclusion
These results suggest that Cks1 is involved in p27Kip1 down-regulation and may have an important role in the development of aggressive tumor behavior in breast cancer.
doi:10.1186/bcr1278
PMCID: PMC1242136  PMID: 16168119
17.  The Amelioration of Renal Damage in Skp2-Deficient Mice Canceled by p27 Kip1 Deficiency in Skp2−/− p27−/− Mice 
PLoS ONE  2012;7(4):e36249.
SCF-Skp2 E3 ubiquitin ligase (Skp2 hereafter) targets several cell cycle regulatory proteins for degradation via the ubiquitin-dependent pathway. However, the target-specific physiological functions of Skp2 have not been fully elucidated in kidney diseases. We previously reported an increase in Skp2 in progressive nephropathy and amelioration of unilateral ureteral obstruction (UUO) renal injury associated with renal accumulation of p27 in Skp2−/− mice. However, it remains unclear whether the amelioration of renal injury in Skp2−/− mice is solely caused by p27 accumulation, since Skp2 targets several other proteins. Using Skp2−/−p27−/− mice, we investigated whether Skp2 specifically targets p27 in the progressive nephropathy mediated by UUO. In contrast to the marked suppression of UUO renal injury in Skp2−/− mice, progression of tubular dilatation associated with tubular epithelial cell proliferation and tubulointerstitial fibrosis with increased expression of collagen and α-smooth muscle actin were observed in the obstructed kidneys in Skp2−/−p27−/− mice. No significant increases in other Skp2 target proteins including p57, p130, TOB1, cyclin A and cyclin D1 were noted in the UUO kidney in Skp2−/− mice, while p21, c-Myc, b-Myb and cyclin E were slightly increased. Contrary to the ameliorated UUO renal injure by Skp2-deficiency, the amelioration was canceled by the additional p27-deficiency in Skp2−/−p27−/− mice. These findings suggest a pathogenic role of the reduction in p27 targeted by Skp2 in the progression of nephropathy in UUO mice.
doi:10.1371/journal.pone.0036249
PMCID: PMC3338689  PMID: 22558406
18.  Estrogen and Progesterone Regulate p27kip1 Levels via the Ubiquitin-Proteasome System: Pathogenic and Therapeutic Implications for Endometrial Cancer 
PLoS ONE  2012;7(9):e46072.
The levels of proteins that control the cell cycle are regulated by ubiquitin-mediated degradation via the ubiquitin-proteasome system (UPS) by substrate-specific E3 ubiquitin ligases. The cyclin-dependent kinase inhibitor, p27kip1 (p27), that blocks the cell cycle in G1, is ubiquitylated by the E3 ligase SCF-Skp2/Cks1 for degradation by the UPS. In turn, Skp2 and Cks1 are ubiquitylated by the E3 ligase complex APC/Cdh1 for destruction thereby maintaining abundant levels of nuclear p27. We previously showed that perpetual proteasomal degradation of p27 is an early event in Type I endometrial carcinogenesis (ECA), an estrogen (E2)-induced cancer. The present studies demonstrate that E2 stimulates growth of ECA cell lines and normal primary endometrial epithelial cells (EECs) and induces MAPK-ERK1/2-dependent phosphorylation of p27 on Thr187, a prerequisite for p27 ubiquitylation by nuclear SCF-Skp2/Cks1 and subsequent degradation. In addition, E2 decreases the E3 ligase [APC]Cdh1 leaving Skp2 and Cks1 intact to cause p27 degradation. Furthermore, knocking-down Skp2 prevents E2-induced p27 degradation and growth stimulation suggesting that the pathogenesis of E2-induced ECA is dependent on Skp2-mediated degradation of p27. Conversely, progesterone (Pg) as an inhibitor of endometrial proliferation increases nuclear p27 and Cdh1 in primary EECs and ECA cells. Pg, also increases Cdh1 binding to APC to form the active E3ligase. Knocking-down Cdh1 obviates Pg-induced stabilization of p27 and growth inhibition. Notably, neither E2 nor Pg affected transcription of Cdh1, Skp2, Cks1 nor p27. These studies provide new insights into hormone regulation of cell proliferation through the UPS. The data implicates that preventing nuclear p27 degradation by blocking Skp2/Cks1-mediated degradation of p27 or increasing Cdh1 to mediate degradation of Skp2-Cks1 are potential strategies for the prevention and treatment of ECA.
doi:10.1371/journal.pone.0046072
PMCID: PMC3459846  PMID: 23029392
19.  High-Throughput Screening Reveals Alsterpaullone, 2-Cyanoethyl as a Potent p27Kip1 Transcriptional Inhibitor 
PLoS ONE  2014;9(3):e91173.
p27Kip1 is a cell cycle inhibitor that prevents cyclin dependent kinase (CDK)/cyclin complexes from phosphorylating their targets. p27Kip1 is a known tumor suppressor, as the germline loss of p27Kip1 results in sporadic pituitary formation in aged rodents, and its presence in human cancers is indicative of a poor prognosis. In addition to its role in cancer, loss of p27Kip1 results in regenerative phenotypes in some tissues and maintenance of stem cell pluripotency, suggesting that p27Kip1 inhibitors could be beneficial for tissue regeneration. Because p27Kip1 is an intrinsically disordered protein, identifying direct inhibitors of the p27Kip1 protein is difficult. Therefore, we pursued a high-throughput screening strategy to identify novel p27Kip1 transcriptional inhibitors. We utilized a luciferase reporter plasmid driven by the p27Kip1 promoter to transiently transfect HeLa cells and used cyclohexamide as a positive control for non-specific inhibition. We screened a “bioactive” library consisting of 8,904 (4,359 unique) compounds, of which 830 are Food and Drug Administration (FDA) approved. From this screen, we successfully identified 111 primary hits with inhibitory effect against the promoter of p27Kip1. These hits were further refined using a battery of secondary screens. Here we report four novel p27Kip1 transcriptional inhibitors, and further demonstrate that our most potent hit compound (IC50 = 200 nM) Alsterpaullone 2-cyanoethyl, inhibits p27Kip1 transcription by preventing FoxO3a from binding to the p27Kip1 promoter. This screen represents one of the first attempts to identify inhibitors of p27Kip1 and may prove useful for future tissue regeneration studies.
doi:10.1371/journal.pone.0091173
PMCID: PMC3960108  PMID: 24646893
20.  PD-1 inhibits T cell proliferation by upregulating p27 and p15 and suppressing Cdc25A 
Cell Cycle  2012;11(23):4305-4309.
The programmed cell death-1 (PD)-1 receptor (CD279) is a potent T cell inhibitor with a critical role in peripheral tolerance, but it can also compromise anti-viral and antitumor T cell responses. The effects of PD-1 on the cell cycle leading to inhibition of T cell expansion are poorly understood. Recently, we examined the effects of PD-1 on the molecular control of the cell cycle machinery and on TCR-activated signaling pathways that regulate these downstream outcomes. Our studies showed that PD-1 blocks cell cycle progression in the G1 phase. PD-1 did not alter the expression of G1 phase cyclins or cyclin-dependent kinases (Cdks) but, instead, suppressed the transcription of SKP2, the substrate recognition component of the SCFSkp2 ubiquitin ligase that leads p27kip1 to degradation and resulted in accumulation of p27kip1. Subsequently, T cells receiving PD-1 signals displayed impaired Cdk2 activation and failed to phosphorylate two critical Cdk2 substrates, the retinoblastoma gene product (Rb) and the TGFβ-specific transcription factor Smad3, leading to suppression of E2F target genes but enhanced Smad3 transactivation. These events resulted in upregulation of the Cdk4/6 inhibitor p15INK4B and repression of the Cdk-activating phosphatase Cdc25A. The suppressive effect of PD-1 on Skp2 expression was mediated by inhibition of both PI3K/Akt and Ras/MEK/Erk pathways and was only partially reversed by IL-2, which restored activation of MEK/Erk but not Akt. Thus, PD-1 targets Ras and PI3K/Akt signaling to inhibit transcription of Skp2 and to activate Smad3 as an integral component of a pathway that regulates blockade of cell cycle progression in T lymphocytes. Here, we discuss the detailed sequence of these signaling events and their implications in mediating cell-intrinsic and -extrinsic mechanisms that inhibit proliferation of T effector cells in response to PD-1-mediated signaling.
doi:10.4161/cc.22135
PMCID: PMC3552912  PMID: 23032366
Cdc25; Cdk2; PD-1; Smad3; T cells
21.  Skp2 Regulates G2/M Progression in a p53-dependent Manner 
Molecular Biology of the Cell  2008;19(11):4602-4610.
Targeted proteasomal degradation mediated by E3 ubiquitin ligases controls cell cycle progression, and alterations in their activities likely contribute to malignant cell proliferation. S phase kinase-associated protein 2 (Skp2) is the F-box component of an E3 ubiquitin ligase complex that targets p27Kip1 and cyclin E1 to the proteasome. In human melanoma, Skp2 is highly expressed, regulated by mutant B-RAF, and required for cell growth. We show that Skp2 depletion in melanoma cells resulted in a tetraploid cell cycle arrest. Surprisingly, co-knockdown of p27Kip1 or cyclin E1 failed to prevent the tetraploid arrest induced by Skp2 knockdown. Enhanced Aurora A phosphorylation and repression of G2/M regulators cyclin B1, cyclin-dependent kinase 1, and cyclin A indicated a G2/early M phase arrest in Skp2-depleted cells. Furthermore, expression of nuclear localized cyclin B1 prevented tetraploid accumulation after Skp2 knockdown. The p53 status is most frequently wild type in melanoma, and the tetraploid arrest and down-regulation of G2/M regulatory genes were strongly dependent on wild-type p53 expression. In mutant p53 melanoma lines, Skp2 depletion did not induce cell cycle arrest despite up-regulation of p27Kip1. These data indicate that elevated Skp2 expression may overcome p53-dependent cell cycle checkpoints in melanoma cells and highlight Skp2 actions that are independent of p27Kip1 degradation.
doi:10.1091/mbc.E07-11-1137
PMCID: PMC2575176  PMID: 18716061
22.  Androgen Suppresses Proliferation of Castration-Resistant LNCaP 104-R2 Prostate Cancer Cells via Androgen Receptor, Skp2, and c-Myc 
Cancer science  2011;102(11):2022-2028.
Summary
Androgen ablation therapy is the primary treatment for metastatic prostate cancer. However, this therapy is associated with several undesired side-effects, including increased risk of cardiovascular diseases. To study if termination of long-term androgen ablation and restoring testosterone level may suppress the growth of relapsed hormone-refractory prostate tumors, we implanted testosterone pellets in castrated nude mice carrying androgen receptor (AR)-positive LNCaP 104-R2 cells, which relapsed from androgen-dependent LNCaP 104-S cells after long-term androgen deprivation. 104-R2 tumor xenografts regressed after testosterone pellets implant. 24 out of 33 tumors adapted to elevation of testosterone level and relapsed as androgen-insensitive tumors. Relapsed tumors (R2Ad) expressed less AR and prostate-specific antigen (PSA). We then study the molecular mechanism lying underneath the androgenic regulation of prostate cancer cell proliferation. Androgen suppresses proliferation of 104-R2 by inducing G1 cell cycle arrest via reduction of Skp2 and c-Myc, and induction of p27Kip1. 104-R2 cells adapted to androgen treatment and the adapted cells, R2Ad, were androgen-insensitive cells with slower growing rate and low protein level of AR, high levels of c-Myc and Skp2, and low levels of p27Kip1. Nuclear AR and PSA expression is present in 104-R2 cells but not R2Ad cells when androgen is absent. Overexpression of AR in R2Ad cells regenerated an androgen-repressed phenotype, while knockdown of AR in 104-R2 cells generated an androgen-insensitive phenotype. Overexpression of Skp2 and c-Myc in 104-R2 cells blocked the growth inhibition caused by androgens. We concluded that androgens cause growth inhibition in LNCaP 104-R2 prostate cancer cells via AR, Skp2, and c-Myc.
doi:10.1111/j.1349-7006.2011.02043.x
PMCID: PMC3200457  PMID: 21781227
23.  A component of the mir-17-92 polycistronic oncomir promotes oncogene-dependent apoptosis 
eLife  2013;2:e00822.
mir-17-92, a potent polycistronic oncomir, encodes six mature miRNAs with complex modes of interactions. In the Eμ-myc Burkitt’s lymphoma model, mir-17-92 exhibits potent oncogenic activity by repressing c-Myc-induced apoptosis, primarily through its miR-19 components. Surprisingly, mir-17-92 also encodes the miR-92 component that negatively regulates its oncogenic cooperation with c-Myc. This miR-92 effect is, at least in part, mediated by its direct repression of Fbw7, which promotes the proteosomal degradation of c-Myc. Thus, overexpressing miR-92 leads to aberrant c-Myc increase, imposing a strong coupling between excessive proliferation and p53-dependent apoptosis. Interestingly, miR-92 antagonizes the oncogenic miR-19 miRNAs; and such functional interaction coordinates proliferation and apoptosis during c-Myc-induced oncogenesis. This miR-19:miR-92 antagonism is disrupted in B-lymphoma cells that favor a greater increase of miR-19 over miR-92. Altogether, we suggest a new paradigm whereby the unique gene structure of a polycistronic oncomir confers an intricate balance between oncogene and tumor suppressor crosstalk.
DOI: http://dx.doi.org/10.7554/eLife.00822.001
eLife digest
The role of genes, in very simple terms, is to be transcribed into messenger RNA molecules, which are then translated into strings of amino acids that fold into proteins. Each of these steps is extremely complex, and a wide range of other molecules can speed up, slow down, stop or otherwise disrupt the expression of genes as protein products. Genes can also code for nucleic acids that are not translated into proteins, such as microRNAs. These are small RNA molecules that can reduce the production of proteins by repressing the translation step and/or by partially degrading the messenger RNA molecules.
mir-17-92 is a gene that exemplifies much of this complexity. It codes for six different microRNAs in a single primary transcript, and has been implicated in a number of cancers, including lung cancer, Burkitt’s lymphoma and other forms of lymphomas and leukemia. One of six microRNAs has a longer evolutionary history than the remaining five: mir-92 is found in vertebrates, chordates and invertebrates, whereas the other five are only found in vertebrates. However, it is not known how or why the mir-17-92 gene evolved to code for multiple different microRNAs.
Olive et al. have studied how these mir-17-92 microRNAs functionally interact in mice with Burkitt’s lymphoma, a form of cancer that is associated with a gene called c-Myc being over-activated. Mutations in this gene promote the proliferation of cells, and in cooperation with other genetic lesions, this ultimately leads to cancer. mir-17-92 is implicated in this cancer because it represses the process of programmed cell death (which is induced by the protein c-Myc) that the body employs to stop tumors growing.
Olive et al. found that deleting one of the six microRNAs, miR-92, increased the tendency of the mir-17-92 gene to promote Burkitt’s lymphoma. By repressing an enzyme called Fbw7, miR-92 causes high levels of c-Myc to be produced. While this leads to the uncontrolled proliferation of cells that promotes cancer, it also increases programmed cell death, at least in part, by activating the p53 pathway, a well-known tumor suppression pathway. The experiments also revealed that the action of miR-92 and that of one of the other microRNAs, miR-19, were often opposed to each other. These findings have revealed an unexpected interaction among different components within a single microRNA gene, which acts to maintain an intricate balance between pathways that promote and suppress cancer.
DOI: http://dx.doi.org/10.7554/eLife.00822.002
doi:10.7554/eLife.00822
PMCID: PMC3796314  PMID: 24137534
microRNAs; c-Myc; Eμ-myc lymphoma; apoptosis; p53; Mouse
24.  Lack of Cyclin-Dependent Kinase 4 Inhibits c-myc Tumorigenic Activities in Epithelial Tissues 
Molecular and Cellular Biology  2004;24(17):7538-7547.
The proto-oncogene c-myc encodes a transcription factor that is implicated in the regulation of cellular proliferation, differentiation, and apoptosis and that has also been found to be deregulated in several forms of human and experimental tumors. We have shown that forced expression of c-myc in epithelial tissues of transgenic mice (K5-Myc) resulted in keratinocyte hyperproliferation and the development of spontaneous tumors in the skin and oral cavity. Although a number of genes involved in cancer development are regulated by c-myc, the actual mechanisms leading to Myc-induced neoplasia are not known. Among the genes regulated by Myc is the cyclin-dependent kinase 4 (CDK4) gene. Interestingly, previous studies from our laboratory showed that the overexpression of CDK4 led to keratinocyte hyperproliferation, although no spontaneous tumor development was observed. Thus, we tested the hypothesis that CDK4 may be one of the critical downstream genes involved in Myc carcinogenesis. Our results showed that CDK4 inhibition in K5-Myc transgenic mice resulted in the complete inhibition of tumor development, suggesting that CDK4 is a critical mediator of tumor formation induced by deregulated Myc. Furthermore, a lack of CDK4 expression resulted in marked decreases in epidermal thickness and keratinocyte proliferation compared to the results obtained for K5-Myc littermates. Biochemical analysis of the K5-Myc epidermis showed that CDK4 mediates the proliferative activities of Myc by sequestering p21Cip1 and p27Kip1 and thereby indirectly activating CDK2 kinase activity. These results show that CDK4 mediates the proliferative and oncogenic activities of Myc in vivo through a mechanism that involves the sequestration of specific CDK inhibitors.
doi:10.1128/MCB.24.17.7538-7547.2004
PMCID: PMC506988  PMID: 15314163
25.  AMPKα2 Deletion Exacerbates Neointima Formation by Upregulating Skp2 in Vascular Smooth Muscle Cells 
Circulation research  2011;109(11):1230-1239.
Rationale
Adenosine monophosphate-activated protein kinase (AMPK), a metabolic and redox sensor, is reported to suppress cell proliferation of non-malignant and tumor cells. Whether AMPKα alters vascular neointima formation induced by vascular injury is unknown.
Objective
The aim of this study was to determine the roles of AMPKα in the development of vascular neointima hyperplasia and to elucidate the underlying mechanisms.
Methods and Results
Vascular smooth muscle cells (VSMCs) proliferation and neointimal hyperplasia were evaluated in cultured VSMCs and wire-injured mouse carotid arteries from wild-type (WT, C57BL/6J), AMPKα2−/−, and AMPKα1−/− VSMCs. Mouse VSMCs derived from aortas of AMPKα2−/− mice exhibited increased proliferation compared to either WT or AMPKα1−/− VSMCs. Further, deletion of AMPKα2, but not AMPKα1, reduced the level of p27Kip1, acyclin-dependent kinase inhibitor, and increased the level of S-phase kinase-associated protein 2 (Skp2), a known E3 ubiquitin ligase for p27Kip1, via activation of p52 nuclear factor kappa B (NF-κB)-2. Moreover, either pharmacological (i.e., via compound C) or genetical (i.e., via AMPKα2-specific siRNA) inhibition of AMPK decreased p27Kip1 levels, but increased the abundance of Skp2 in human VSMCs. Furthermore, gene silencing of Skp2 reversed the levels of p27Kip1 and VSMCs proliferation. Finally, neointima formation after mechanical arterial injury was increased in AMPKα2−/−, but not AMPKα1−/−, mice.
Conclusions
These findings indicate that deletion of AMPKα2 via p52-Skp2-mediated ubiquintination and degradation of p27Kip1 accentuates neointimal hyperplasia in response to wire injury.
doi:10.1161/CIRCRESAHA.111.250423
PMCID: PMC3235405  PMID: 21980125
Neointima formation; AMPK; VSMC; NF-κB; Skp2

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