PML tumor suppressor protein is required for HCV production

PML tumor suppressor protein, which forms discrete nuclear structures termed PML-nuclear bodies, has been associated with several cellular functions, including cell proliferation, apoptosis and antiviral defense. Recently, it was reported that the HCV core protein colocalizes with PML in PML-NBs and abrogates the PML function through interaction with PML. However, role(s) of PML in HCV life cycle is unknown. To test whether or not PML affects HCV life cycle, we examined the level of secreted HCV core and the infectivity of HCV in the culture supernatants as well as the level of HCV RNA in HuH-7-derived RSc cells, in which HCV-JFH1 can infect and efficiently replicate, stably expressing short hairpin RNA targeted to PML. In this context, the level of secreted HCV core and the infectivity in the supernatants from PML knockdown cells was remarkably reduced, whereas the level of HCV RNA in the PML knockdown cells was not significantly affected in spite of very effective knockdown of PML. In fact, we showed that PML is unrelated to HCV RNA replication using the subgenomic HCV-JFH1 replicon RNA, JRN/3-5B. Furthermore, the infectivity of HCV-like particle in the culture supernatants was significantly reduced in PML knockdown JRN/3-5B cells expressing core to NS2 coding region of HCV-JFH1 genome using the trans-packaging system. Finally, we also demonstrated that INI1 and DDX5, the PML-related proteins, are involved in HCV production. Taken together, these findings suggest that PML is required for HCV production.     

Docking-dependent regulation of the Rb tumor suppressor protein by Cdk4

Phosphorylation of target proteins by cyclin D1-Cdk4 requires both substrate docking and kinase activity. In addition to the ability of cyclin D1-Cdk4 to catalyze the phosphorylation of consensus sites within the primary amino acid sequence of a substrate, maximum catalytic activity requires the enzyme complex to anchor at a site remote from the phospho-acceptor site. A novel Cdk4 docking motif has been defined within a stretch of 19 amino acids from the C-terminal domain of the Rb protein that are essential for Cdk4 binding. Mutation or deletion of the docking motif prevents Cdk4-dependent phosphorylation of full-length Rb protein or C-terminal Rb fragments in vitro and in cells, while a peptide encompassing the Cdk4 docking motif specifically inhibits Cdk4-dependent phosphorylation of Rb. Cyclin D1-Cdk4 can overcome the growth-suppressive activity of Rb in both cell cycle progression and colony formation assays; however, while mutants of Rb in which the Cdk4 docking site has been either deleted or mutated retain growth suppressor activity, they are resistant to inactivation by cyclin D1-Cdk4. Finally, binding of Cdk4 to its docking site can inhibit cleavage of exogenous and endogenous Rb in response to distinct apoptotic signals. The Cdk4 docking motif in Rb gives insight into the mechanism by which enzyme specificity is ensured and highlights a role for Cdk4 docking in maintaining the Rb protein in a form that favors cell survival rather than apoptosis.     

CK2 mediates phosphorylation and ubiquitin-mediated degradation of the PML tumor suppressor

The PML tumor suppressor controls growth suppression, induction of apoptosis, and cellular senescence. PML loss occurs frequently in hematopoietic and solid tumors. PML loss often correlates with tumor progression. Casein kinase 2 (CK2) is a stress-activated serine/threonine protein kinase that is oncogenic and frequently overexpressed in human tumor of multiple histological origins. In addition, CK2 overexpression due to gene amplification has been reported to be an adverse prognostic factor in non-small cell lung cancer. At the 5th International Conference on Protein Kinase CK2 in Padova, Italy, we reviewed our recent findings that PML undergoes ubiquitin/proteasome-mediated degradation in immortalized and tumor derived cell lines. PML degradation depends on direct CK2 phosphorylation of PML Ser517. PML mutants that are resistant to CK2 phosphorylation display increased tumor suppressive functions in assays measuring apoptosis, replicative senescence, and in xenograft models. More significantly, CK2 pharmacological inhibition enhances PML tumor suppressive property. These data identify a key post-translational mechanism that controls PML protein levels in cancer cells and suggest that CK2 inhibitors may be beneficial anti-cancer drugs.     

CK2 mediates phosphorylation and ubiquitin-mediated degradation of the PML tumor suppressor

Molecular and Cellular Biochemistry -     

A CK2-dependent mechanism for degradation of the PML tumor suppressor

The PML tumor suppressor controls key pathways for growth suppression, induction of apoptosis, and cellular senescence. PML loss occurs frequently in human tumors through unknown posttranslational mechanisms. Casein kinase 2 (CK2) is oncogenic and frequently upregulated in human tumors. Here we show that CK2 regulates PML protein levels by promoting its ubiquitin-mediated degradation dependent on direct phosphorylation at Ser517. Consequently, PML mutants that are resistant to CK2 phosphorylation display increased tumor-suppressive functions. In a faithful mouse model of lung cancer, we demonstrate that Pml inactivation leads to increased tumorigenesis. Furthermore, CK2 pharmacological inhibition enhances the PML tumor-suppressive property in vivo. Importantly, we found an inverse correlation between CK2 kinase activity and PML protein levels in human lung cancer-derived cell lines and primary specimens. These data identify a key posttranslational mechanism that controls PML protein levels and provide therapeutic means toward PML restoration through CK2 inhibition.     

The protein tyrosine phosphatase receptor D,a broadly inactivated tumor suppressor regulating STAT function

Comment on: Veeriah S, Brennan C, Meng S, Singh B, Fagin JA, Solit DB, et al. The tyrosine phosphatase PTPRD is a tumor suppressor that is frequently inactivated and mutated in glioblastoma and other human cancers. Proc Natl Acad Sci USA 2009; 106:9435-40.     

The antioxidant function of the p53 tumor suppressor

It is widely accepted that the p53 tumor suppressor restricts abnormal cells by induction of growth arrest or by triggering apoptosis. Here we show that, in addition, p53 protects the genome from oxidation by reactive oxygen species (ROS), a major cause of DNA damage and genetic instability. In the absence of severe stresses, relatively low levels of p53 are sufficient for upregulation of several genes with antioxidant products, which is associated with a decrease in intracellular ROS. Downregulation of p53 results in excessive oxidation of DNA, increased mutation rate and karyotype instability, which are prevented by incubation with the antioxidant N-acetylcysteine (NAC). Dietary supplementation with NAC prevented frequent lymphomas characteristic of Trp53-knockout mice, and slowed the growth of lung cancer xenografts deficient in p53. Our results provide a new paradigm for a nonrestrictive tumor suppressor function of p53 and highlight the potential importance of antioxidants in the prophylaxis and treatment of cancer.     

14-3-3 Mediated regulation of the tumor suppressor protein, RASSF1A

Death receptor-dependent apoptosis is an important mechanism of growth control. It has been demonstrated that Ras association domain family protein 1A (RASSF1A) is a tumor suppressor protein involved in death receptor-dependent apoptosis. However, it is unclear how RASSF1A-mediated cell death is initiated. We have now detailed 14-3-3 dependent regulation of RASSF1A-mediated cell death. We demonstrate that basal association of RASSF1A with 14-3-3 was lost following stimulation with tumor necrosis factor alpha (TNFα) or TNFα related apoptosis inducing ligand (TRAIL). Subsequent to the loss of 14-3-3 association, RASSF1A associated with modulator of apoptosis (MOAP-1) followed by death receptor association with either TNFα receptor 1 (TNF-R1) or TRAIL receptor 1 (TRAIL-R1). 14-3-3 association required basal phosphorylation by the serine/threonine kinase, glycogen synthase kinase 3β (GSK-3β), on serine 175, 178, and 179. Mutation of these critical serines resulted in the loss of 14-3-3 association and earlier recruitment of RASSF1A to MOAP-1, TNF-R1, and TRAIL-R1. Furthermore, stable cells containing a triple serine mutant of RASSF1A [serine (S) 175 to alanine (A) [S175A], S178A, and S179A] resulted in increased basal cell death, enhanced Annexin V staining and enhanced cleavage of poly (ADP-ribose) polymerase (PARP) following TNFα stimulation when compared to stable cells containing wild type RASSF1A. RASSF1A-mediated cell death is, therefore, tightly controlled by 14-3-3 association.     

A novel radioprotective function for the mitochondrial tumor suppressor protein Fus1

FUS1/TUSC2 is a mitochondrial tumor suppressor with activity to regulate cellular oxidative stress by maintaining balanced ROS production and mitochondrial homeostasis. Fus1 expression is inhibited by ROS, suggesting that individuals with a high level of ROS may have lower Fus1 in normal tissues and, thus, may be more prone to oxidative stress-induced side effects of cancer treatment, including radiotherapy. As the role of Fus1 in the modulation of cellular radiosensitivity is unknown, we set out to determine molecular mechanisms of Fus1 involvement in the IR response in normal tissues. Mouse whole-body irradiation methodology was employed to determine the role for Fus1 in the radiation response and explore underlying molecular mechanisms. Fus1^−/− mice were more susceptible to radiation compared with Fus1^+/+ mice, exhibiting increased mortality and accelerated apoptosis of the GI crypt epithelial cells. Following untimely reentrance into the cell cycle, the Fus1^−/− GI crypt cells died at accelerated rate via mitotic catastrophe that resulted in diminished and/or delayed crypt regeneration after irradiation. At the molecular level, dysregulated dynamics of activation of main IR response proteins (p53, NFκB, and GSK-3β), as well as key signaling pathways involved in oxidative stress response (SOD2, PRDX1, and cytochrome c), apoptosis (BAX and PARP1), cell cycle (Cyclins B1 and D1), and DNA repair (γH2AX) were found in Fus1^−/− cells after irradiation. Increased radiosensitivity of other tissues, such as immune cells and hair follicles was also detected in Fus1^−/− mice. Our findings demonstrate a previously unknown radioprotective function of the mitochondrial tumor suppressor Fus1 in normal tissues and suggest new individualized therapeutic approaches based on Fus1 expression.     

The bladder tumor suppressor protein TERE1 (UBIAD1) modulates cell cholesterol: implications for tumor progression

Convergent evidence implicates the TERE1 protein in human bladder tumor progression and lipid metabolism. Previously, reduced TERE1 expression was found in invasive urologic cancers and inhibited cell growth upon re-expression. A role in lipid metabolism was suggested by TERE1 binding to APOE, a cholesterol carrier, and to TBL2, a candidate protein in triglyceride disorders. Natural TERE1 mutations associate with Schnyder's corneal dystrophy, characterized by lipid accumulation. TERE1 catalyzes menaquinone synthesis, known to affect cholesterol homeostasis. To explore this relationship, we altered TERE1 and TBL2 dosage via ectopic expression and interfering RNA and measured cholesterol by Amplex red. Protein interactions of wild-type and mutant TERE1 with GST-APOE were evaluated by binding assays and molecular modeling. We conducted a bladder tumor microarray TERE1 expression analysis and assayed tumorigenicity of J82 cells ectopically expressing TERE1. TERE1 expression was reduced in a third of invasive specimens. Ectopic TERE1 expression in J82 bladder cancer cells dramatically inhibited nude mouse tumorigenesis. TERE1 and TBL2 proteins inversely modulated cellular cholesterol in HEK293 and bladder cancer cells from 20% to 50%. TERE1 point mutations affected APOE interactions, and resulted in cholesterol levels that differed from wild type. Elevated tumor cell cholesterol is known to affect apoptosis and growth signaling; thus, loss of TERE1 in invasive bladder cancer may represent a defect in menaquinone-mediated cholesterol homeostasis that contributes to progression.     

Redox regulation of the tumor suppressor PTEN by glutathione

Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) expressed in Saccharomyces cerevisiae was reversibly oxidized by hydrogen peroxide and reduced by cellular reductants. Reduction of hPTEN was delayed in each of S. cerevisiae gsh1Δ and gsh2Δ mutants. Expression of γ-glutamylcysteine synthetase Gsh1 in the gsh1Δ mutant rescued regeneration rate of hPTEN. Oxidized hPTEN was reduced by glutathione in a concentration- and time-dependent manner. Glutathionylated PTEN was detected. Incubation of 293T cells with BSO and knockdown expression of GCLc in HeLa cells by siRNA resulted in the delay of reduction of oxidized PTEN. Also, in HeLa cells transfected with GCLc siRNA, stimulation with epidermal growth factor resulted in the increase of oxidized PTEN and phosphorylation of Akt. These results suggest that the reduction of oxidized hPTEN is mediated by glutathione.     

The tumor suppressor function of mitochondria: Translation into the clinics

Recently, the inevitable metabolic reprogramming experienced by cancer cells as a result of the onset of cellular proliferation has been added to the list of hallmarks of the cancer cell phenotype. Proliferation is bound to the synchronous fluctuation of cycles of an increased glycolysis concurrent with a restrained oxidative phosphorylation. Mitochondria are key players in the metabolic cycling experienced during proliferation because of their essential roles in the transduction of biological energy and in defining the life–death fate of the cell. These two activities are molecularly and functionally integrated and are both targets of commonly altered cancer genes. Moreover, energetic metabolism of the cancer cell also affords a target to develop new therapies because the activity of mitochondria has an unquestionable tumor suppressor function. In this review, we summarize most of these findings paying special attention to the opportunity that translation of energetic metabolism into the clinics could afford for the management of cancer patients. More specifically, we emphasize the role that mitochondrial β-F1-ATPase has as a marker for the prognosis of different cancer patients as well as in predicting the tumor response to therapy.     

The interaction of the von Hippel-Lindau tumor suppressor and heterochromatin protein 1

Inactivation of the von Hippel-Lindau (VHL) tumor suppressor is associated with renal carcinoma, hemangioblastoma and pheochromocytoma. The VHL protein is a component of a ubiquitin ligase complex that ubiquitinates and degrades hypoxia inducible factor-α (HIF-α). Degradation of HIF-α by VHL is proposed to suppress tumorigenesis and tumor angiogenesis. Several lines of evidence also suggest important roles for HIF-independent VHL functions in tumor suppression and other biological processes. Using GST-VHL pull-down experiment and mass spectrometry, we detected an interaction between VHL and heterochromatin protein 1 (HP1). We identified a conserved HP1-binding motif (PXVXL) in the β domain of VHL, which is disrupted in a renal carcinoma-associated P81S mutant. We show that the VHL P81S mutant displays reduced binding to HP1, yet retains the ability to interact with elongin B, elongin C, and cullin 2 and is fully capable of degrading HIF-α. We also demonstrate that HP1 increases the chromatin association of VHL. These results suggest a role for the VHL-HP1 interaction in VHL chromatin targeting.