Wei Lei Yang

The E3 Ligase TRAF6 Regulates Akt Ubiquitination and Activation

Regulating Akt The protein kinase Akt is activated in response to receptor-activated generation of the signaling second messenger phosphatidylinositol 3,4,5-trisphosphate and has roles in regulation of diverse processes from metabolism and cell survival to transcription and tumorigenesis. Yang et al. (p. 1134; see the Perspective by Restuccia and Hemmings) report a previously unrecognized mode of regulation of Akt: covalent modification of Akt by linkage to lysine 63 of ubiquitin molecules. Such ubiquitination of Akt promotes localization to the cell membrane and consequent activation in cells stimulated with growth factors. TRAF6 (TNF receptor–associated factor 6) was implicated as the E3 ubiquitin ligase that mediates ubiquitination of Akt. Ubiquitination of Akt may influence its role in cancer cells: A mutant form of Akt associated with human cancer showed increased ubiquitination, and depletion of TRAF6 decreased tumorigenicity of a prostate cancer cell line in a mouse cancer model. Localization and activation of signaling proteins in cancer cells are controlled by ubiquitin labeling. Akt signaling plays a central role in many biological functions, such as cell proliferation and apoptosis. Because Akt (also known as protein kinase B) 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. 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 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.

Skp2 targeting suppresses tumorigenesis by Arf-p53 -independent cellular senescence

Cellular senescence has been recently shown to have an important role in opposing tumour initiation and promotion. Senescence induced by oncogenes or by loss of tumour suppressor genes is thought to critically depend on 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, tumour-suppressive senescence response in mice and cells devoid of Skp2. Notably, Skp2 inactivation and oncogenic-stress-driven senescence neither elicit activation of the p19Arf–p53 pathway nor DNA damage, but instead depend 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 pharmacological inhibition of Skp2 may represent a general approach for cancer prevention and therapy.

The Skp2-SCF E3 ligase regulates Akt ubiquitination, glycolysis, herceptin sensitivity, and tumorigenesis.

Akt kinase plays a central role in cell growth, metabolism, and tumorigenesis. The TRAF6 E3 ligase orchestrates IGF-1-mediated Akt ubiquitination and activation. Here, we show that Akt ubiquitination is also induced by activation of ErbB receptors; unexpectedly, and in contrast to IGF-1 induced activation, the Skp2 SCF complex, not TRAF6, is a critical E3 ligase for ErbB-receptor-mediated Akt ubiquitination and membrane recruitment in response to EGF. 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 and breast cancer metastasis and serves as a marker for poor prognosis in Her2-positive patients. Finally, Skp2 silencing sensitizes Her2-overexpressing tumors to Herceptin treatment. Our study suggests that distinct E3 ligases are utilized by diverse growth factors for Akt activation and that targeting glycolysis sensitizes Her2-positive tumors to Herceptin treatment.

Phosphorylation-dependent regulation of cytosolic localization and oncogenic function of Skp2 by Akt/PKB

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 are critical for Skp2-mediated entry to the cell cycle, and overexpression and cytosolic accumulation of Skp2 have been clearly associated with tumorigenesis, although the functional significance of the latter is still unknown. Here we show that Akt/protein kinase B (PKB) interacts with and directly phosphorylates Skp2. We find that Skp2 phosphorylation by Akt triggers SCF complex formation and E3 ligase activity. 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 14-3-3β-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 activation of Akt correlate with the cytosolic accumulation of Skp2 in human cancer specimens. Our results therefore define a novel proto-oncogenic Akt/PKB-dependent signalling pathway.

Deciphering the transcriptional complex critical for RhoA gene expression and cancer metastasis

The RhoA GTPase is crucial 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 transcriptional complex. Skp2 cooperates with Myc to induce RhoA transcription by recruiting Miz1 and p300 to the RhoA promoter independently of Skp1-Cullin-F-box protein containing complex (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. Overexpression of the Myc–Skp2–Miz1 complex is found in metastatic human cancers and is correlated with RhoA expression. Our study provides 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.

Critical Role of Monoubiquitination of Histone H2AX Protein in Histone H2AX Phosphorylation and DNA Damage Response

DNA damage response is an important surveillance mechanism used to maintain the integrity of the human genome in response to genotoxic stress. Histone variant H2AX is a critical sensor that undergoes phosphorylation at serine 139 upon genotoxic stress, which provides a docking site to recruit the mediator of DNA damage checkpoint protein 1 (MDC1) and DNA repair protein complex to sites of DNA breaks for DNA repair. Here, we show that monoubiquitination of H2AX is induced upon DNA double strand breaks and plays a critical role in H2AX Ser-139 phosphorylation (γ-H2AX), in turn facilitating the recruitment of MDC1 to DNA damage foci. Mechanistically, we show that monoubiquitination of H2AX induced by RING finger protein 2 (RNF2) is required for the recruitment of active ataxia telangiectasia mutated to DNA damage foci, thus affecting the formation of γ-H2AX. Importantly, a defect in monoubiquitination of H2AX profoundly enhances ionizing radiation sensitivity. Our study therefore suggests that monoubiquitination of H2AX is an important step for DNA damage response and may have important clinical implications for the treatment of cancers.

Cycles of Ubiquitination and Deubiquitination Critically Regulate Growth Factor-Mediated Activation of Akt Signaling

The deubiquitinase CYLD suppresses activation of the tumor growth–promoting kinase Akt. DUBbing Akt Growth factors trigger the ubiquitination of the kinase Akt, which facilitates its activation. Yang et al. identified the tumor suppressor CYLD as a deubiquitinase (DUB) that inhibits Akt activity. CYLD associated with Akt under nutrient-deficient conditions and dissociated from Akt after growth factor stimulation. Compared to prostate cancer cells with CYLD, those that were deficient in CYLD showed increased glucose uptake and proliferation, processes that are enhanced by Akt activity, and developed into larger tumors when injected into mice. In individuals with primary prostate tumors, decreased abundance of CYLD correlated with increased activation of Akt. Thus, deubiquitination of Akt is required to reset its activity, and attenuation of Akt may contribute to the tumor-suppressive function of CYLD. K63-linked ubiquitination of Akt is a posttranslational modification that plays a critical role in growth factor–mediated membrane recruitment and activation of Akt. Although E3 ligases involved in growth factor–induced ubiquitination of Akt have been defined, the deubiquitinating enzyme (DUB) that triggers deubiquitination of Akt and the function of Akt deubiquitination remain largely unclear. We showed that CYLD was a DUB for Akt and suppressed growth factor–mediated ubiquitination and activation of Akt. CYLD directly removed ubiquitin moieties from Akt under serum-starved conditions. CYLD dissociated from Akt upon growth factor stimulation, thereby allowing E3 ligases to induce ubiquitination and activation of Akt. CYLD deficiency also promoted cancer cell proliferation, survival, glucose uptake, and, when injected into mice, growth of prostate tumors. Our findings reveal the crucial role of cycles of ubiquitination and deubiquitination of Akt in determining its plasma membrane localization and activation—and further identify CYLD as a molecular switch for these processes.

The role of Skp2 in hematopoietic stem cell quiescence, pool size, and self-renewal

Although the maintenance of HSC quiescence and self-renewal are critical for controlling stem cell pool and transplantation efficiency, the mechanisms by which they are regulated remain largely unknown. Understanding the factors controlling these processes may have important therapeutic potential for BM failure and cancers. Here, we show that Skp2, a component of the Skp2 SCF complex, is an important regulator for HSC quiescence, frequency, and self-renewal capability. Skp2 deficiency displays a marked enhancement of HSC populations through promoting cell cycle entry independently of its role on apoptosis. Surprisingly, Skp2 deficiency in HSCs reduces quiescence and displays increased HSC cycling and proliferation. Importantly, loss of Skp2 not only increases HSC populations and long-term reconstitution ability but also rescues the defect in long-term reconstitution ability of HSCs on PTEN inactivation. Mechanistically, we show that Skp2 deficiency induces Cyclin D1 gene expression, which contributes to an increase in HSC cycling. Finally, we demonstrate that Skp2 deficiency enhances sensitivity of Lin(-) Sca-1(+) c-kit(+) cells and leukemia cells to chemotherapy agents. Our findings show that Skp2 is a novel regulator for HSC quiescence and self-renewal and that targeting Skp2 may have therapeutic implications for BM transplantation and leukemia stem cell treatment.

Skp2-MacroH2A1-CDK8 axis orchestrates G2/M transition and tumorigenesis

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.

Bortezomib enhances radiosensitivity in oral cancer through inducing autophagy-mediated TRAF6 oncoprotein degradation

BackgroundOral squamous cell carcinoma (OSCC) is a malignant tumor that may occur anywhere within the oral cavity. The survival rate of OSCC patients has not improved over the past decades due to its heterogeneous etiology, genetic aberrations, and treatment outcomes. We investigated the role of tumor necrosis factor receptor-associated factor 6 (TRAF6) in OSCC cells treated with bortezomib (a proteasome inhibitor) combined with irradiation (IR) treatment.MethodsThe effects of combined treatment in OSCC cells were investigated using assays of cell viability, autophagy, apoptosis, western blotting, and immunofluorescence staining. The ubiquitination of proteins was analyzed by immunoprecipitation. Stable knockdown of TRAF6 in OSCC cells was constructed with lentivirus. The xenograft murine models were used to observe tumor growth.ResultsWe found synergistic effects of bortezomib and IR on the viability of human oral cancer cells. The combination of bortezomib and IR treatment induced autophagic cell death. Furthermore, bortezomib inhibited IR-induced TRAF6 ubiquitination and inhibited TRAF6-mediated Akt activation. Bortezomib reduced TRAF6 protein expression through autophagy-mediated lysosomal degradation. TRAF6 played an oncogenic role in tumorigenesis of human oral cancer cells and oral tumor growth was suppressed by bortezomib and IR treatment. In addition, OSCC patients with expression of TRAF6 showed a trend towards poorer cancer-specific survival when compared with patients without TRAF6 expression.ConclusionsA combination of a proteasome inhibitor, IR treatment and TRAF6 inhibition could be a novel therapeutic strategy in OSCC.