Akt signaling plays a central role in many biological functions, such as cell proliferation and apoptosis. Since Akt resides primarily in the cytosol, it is not known how these signaling molecules are recruited to the plasma membrane and subsequently activated by growth factor stimuli. Here, we found that the protein kinase Akt undergoes lysine 63 chain ubiquitination, which is important for Akt membrane localization and phosphorylation. TRAF6 was found to be a direct E3 ligase for Akt and was essential for Akt ubiquitination, membrane recruitment, and phosphorylation upon growth-factor stimulation. The human cancer-associated Akt mutant (E17K) displayed an increase in Akt ubiquitination, in turn contributing to the enhancement of Akt membrane localization and phosphorylation. Thus, Akt ubiquitination is an important step for oncogenic Akt activation.

Cellular senescence has been recently shown to play an important role in opposing tumour initiation and promotion. Senescence induced by oncogenes or loss of tumour suppressor genes is thought to critically dependent on the induction of the p19Arf-p53 pathway. The Skp2 E3-ubiquitin ligase can act as a proto-oncogene and its aberrant overexpression is frequently observed in human cancers. Here we show that although Skp2 inactivation on its own does not induce cellular senescence, aberrant proto-oncogenic signals as well as inactivation of tumour suppressor genes do trigger a potent, tumor-suppressive senescence response in mice and cells devoid of Skp2. Notably, Skp2 inactivation and oncogenic stress driven senescence neither elicits activation of the p19Arf-p53 pathway nor DNA damage, but instead depends on ATF4, p27, and p21. We further demonstrate that genetic Skp2 inactivation evokes cellular senescence even in oncogenic conditions in which the p19Arf/p53 response is impaired, whereas a Skp2-SCF complex inhibitor can trigger cellular senescence in p53/PTEN deficient cells and tumour regression in preclinical studies. Our findings therefore provide proof of principle evidence that Skp2 pharmacological inhibition may represent a general approach for cancer prevention and therapy.

Skp2 E3 ligase is overexpressed in numerous human cancers and plays a critical role in cell cycle progression, senescence, metabolism, cancer progression and metastasis. In the present study, we identified a specific Skp2 inhibitor using high-throughput in silico screening of large and diverse chemical libraries. This Skp2 inhibitor selectively suppresses Skp2 E3 ligase activity, but not activity of other SCF complexes. It also phenocopies the effects observed upon genetic Skp2 deficiency, such as suppressing survival, Akt-mediated glycolysis as well as triggering p53-independent cellular senescence. Strikingly, we discovered a critical function of Skp2 in positively regulating cancer stem cell populations and self-renewal ability through genetic and pharmacological approaches. Notably, Skp2 inhibitor exhibits potent anti-tumor activities in multiple animal models and cooperates with chemotherapeutic agents to reduce cancer cell survival. Our study thus provides pharmacological evidence that Skp2 is a promising target for restricting cancer stem cell and cancer progression.

Skp2 is an F-box protein that forms the SCF complex with Skp1 and Cullin-1 to constitute an E3 ligase for ubiquitylation. Ubiquitylation and degradation of the p27 is critical for Skp2-mediated cell cycle entry, and overexpression and cytosolic accumulation of Skp2 have been clearly associated with tumorigenesis although the functional significance of the latter has remained elusive. Here we show that the Akt/PKB interacts with and directly phosphorylates Skp2. We find that Skp2 phosphorylation by Akt triggers SCF complex formation and E3 ligase activity. Importantly, a phosphorylation-defective Skp2 mutant is drastically impaired in its ability to promote cell proliferation and tumorigenesis. Furthermore, we show that Akt-mediated phosphorylation triggers 14-3-3-β-dependent Skp2 relocalization to the cytosol, and we attribute a specific role to cytosolic Skp2 in the positive regulation of cell migration. Finally, we demonstrate that high levels of Akt activation correlate with Skp2 cytosolic accumulation in human cancer specimens. Our results therefore define a novel proto-oncogenic Akt/PKB-dependent signaling pathway. NIH Public Access Author ManuscriptNat Cell Biol. Author manuscript; available in PMC 2010 March 2. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptThe ubiquitin-proteasome system regulates the cell cycle through control of protein ubiquitylation and degradation 1,2 . One of the key ubiquitin ligases (E3 ligase) in this process is the Skp1/Cul-1/F-box (SCF) complex, which consists of Skp1, Cullin-1 (Cul-1), RBX1, as well as an F-box protein, all required for its E3 ubiquitin ligase activity. Disruption of this complex severely ablates its enzymatic activity 1,2 .Skp2 (S-phase kinase associated protein-2) is a SCF F-box protein and is responsible for substrate recognition 1,2 . It binds to p27 and targets it for ubiquitylation and degradation [3][4][5] .Overexpression of Skp2 induces cell cycle entry, and the degradation of p27 is required for Skp2-mediated cell cycle progression 6,7 . Skp2 deficiency displays elevated p27 protein levels and a profound impairment in proliferation accompanied by nuclear enlargement, polypoidy, and centrosome overduplication 8,9 . Overexpression of Skp2 is frequently observed in human cancers of diverse histology, while in most human cancers reduced level of p27 represents an adverse prognostic marker 1,2 . Skp2 cooperates with H-Ras G12V to transform primary rodent fibroblasts 10 . Overexpression of Skp2 in the T-cell compartment cooperates with N-Ras to induce T cell lymphomas 11 , while prostate specific expression of Skp2 leads to prostatic intraepithelial neoplasia (PIN) 12 . These observations suggest that Skp2 overexpression may contribute to tumorigenesis.Although substantial advances have been made in understanding the mechanisms that control its levels of expression, by contrast, the molecular mechanisms by which Skp2 activity within the SCF complex and its subcellular localization are regulated are currently unknown. Thi...

Akt kinase plays a central role in cell growth, metabolism and tumorigenesis. Although TRAF6 E3 ligase orchestrates IGF-1-mediated Akt ubiquitination and activation, it is unclear whether TRAF6 is involved in Akt activation by other growth factor receptors as well. Here we show that Akt ubiquitination is also induced by activation of ErbB receptors; unexpectedly, Skp2 SCF complex, but not TRAF6, is a critical E3 ligase for ErbB receptor-mediated Akt ubiquitination and membrane recruitment. Interestingly, Skp2 deficiency impairs Akt activation, Glut1 expression, glucose uptake and glycolysis, and breast cancer progression in various tumor models. Moreover, Skp2 overexpression correlates with Akt activation, breast cancer metastasis, and serves as a marker for poor prognosis in Her2-positive patients. Finally, we showed that Skp2 silencing sensitizes Her2-overexpressing tumors to Herceptin treatment. Our study suggests that distinct E3 ligases are utilized by diverse growth factors for Akt ubiquitination and activation.

RhoA GTPase plays a crucial role in numerous biological functions and is linked to cancer metastasis. However, the understanding of the molecular mechanism responsible for RhoA transcription is still very limited. Here we show that RhoA transcription is orchestrated by the Myc/Skp2/Miz1/p300 transcription complex. Skp2 cooperates with Myc to induce RhoA transcription by recruiting Miz1 and p300 to the RhoA promoter independently of SCF-Skp2 E3 ligase activity. Deficiency of this complex results in impairment in RhoA expression, cell migration, invasion, and breast cancer metastasis, recapitulating the phenotypes observed in RhoA knockdown, and RhoA restoration rescues the defect in cell invasion. Strikingly, the overexpression of Myc/Skp2/Miz1 complex is found in metastatic human cancers and correlated with RhoA expression. Our study provides great insight into how oncogenic Skp2 and Myc coordinate to induce RhoA transcription and establishes a novel SCF-Skp2 E3 ligase-independent function for oncogenic Skp2 in transcription and cancer metastasis.

Histone methyltransferases and demethylases reversibly modulate histone lysine methylation, which is considered a key epigenetic mark associated with gene regulation. Recently, aberrant regulation of gene expression by histone methylation modifiers has emerged as an important mechanism for tumorigenesis. However, it remains largely unknown how histone methyltransferases and demethylases co-regulate transcriptional profiles for cancer cell characteristics. Here, we show that in breast cancer cells, the histone H3 lysine 27 (H3K27) demethylase UTX (also known as KDM6A) positively regulates gene expression programs associated with cell proliferation and invasion. The majority of UTX-controlled genes, including a cohort of oncogenes and pro-metastatic genes, are co-regulated by the H3K4 methyltransferase mixed lineage leukemia 4 (MLL4, also called ALR, KMT2D, and MLL2). UTX interacted with a C-terminal region of MLL4. UTX knockdown resulted in significant decreases in the proliferation and invasiveness of breast cancer cells in vitro and in a mouse xenograft model. Such defective cellular characteristics of UTX-depleted cells were phenocopied by MLL4 knockdown cells. UTX-catalyzed demethylation of trimethylated H3K27 and MLL4-mediated trimethylation at H3K4 occurred inter-dependently at co-target genes of UTX and MLL4. Clinically, high levels of UTX or MLL4 were associated with poor prognosis in breast cancer patients. Taken together, these findings uncover that coordinated regulation of gene expression programs by a histone methyltransferase and a histone demethylase is coupled to the proliferation and invasion of breast cancer cells.

Epigenetic dysregulation has emerged as a major contributor to tumorigenesis. Histone methylation is a wellestablished mechanism of epigenetic regulation that is dynamically modulated by histone methyltransferases and demethylases. The pathogenic role of histone methylation modifiers in non-small cell lung cancer (NSCLC), which is the leading cause of cancer deaths worldwide, remains largely unknown. Here, we found that the histone H3 lysine 36 (H3K36) demethylase KDM2A (also called FBXL11 and JHDM1A) is frequently overexpressed in NSCLC tumors and cell lines. KDM2A and its catalytic activity were required for in vitro proliferation and invasion of KDM2A-overexpressing NSCLC cells. KDM2A overexpression in NSCLC cells with low KDM2A levels increased cell proliferation and invasiveness. KDM2A knockdown abrogated tumor growth and invasive abilities of NSCLC cells in mouse xenograft models. We identified dual-specificity phosphatase 3 (DUSP3) as a key KDM2A target gene and found that DUSP3 dephosphorylates ERK1/2 in NSCLC cells. KDM2A activated ERK1/2 through epigenetic repression of DUSP3 expression via demethylation of dimethylated H3K36 at the DUSP3 locus. High KDM2A levels correlated with poor prognosis in NSCLC patients. These findings uncover an unexpected role for a histone methylation modifier in activating ERK1/2 in lung tumorigenesis and metastasis, suggesting that KDM2A may be a promising therapeutic target in NSCLC.