An atlas of altered expression of deubiquitinating enzymes in human cancer.

BACKGROUND: Deubiquitinating enzymes (DUBs) are proteases that process ubiquitin (Ub) or ubiquitin-like gene products, remodel polyubiquitin(-like) chains on target proteins, and counteract protein ubiquitination exerted by E3 ubiquitin-ligases. A wealth of studies has established the relevance of DUBs to the control of physiological processes whose subversion is known to cause cellular transformation, including cell cycle progression, DNA repair, endocytosis and signal transduction. Altered expression of DUBs might, therefore, subvert both the proteolytic and signaling functions of the Ub system. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we report the first comprehensive screening of DUB dysregulation in human cancers by in situ hybridization on tissue microarrays (ISH-TMA). ISH-TMA has proven to be a reliable methodology to conduct this kind of study, particularly because it allows the precise identification of the cellular origin of the signals. Thus, signals associated with the tumor component can be distinguished from those associated with the tumor microenvironment. Specimens derived from various normal and malignant tumor tissues were analyzed, and the "normal" samples were derived, whenever possible, from the same patients from whom tumors were obtained. Of the ∼90 DUBs encoded by the human genome, 33 were found to be expressed in at least one of the analyzed tissues, of which 22 were altered in cancers. Selected DUBs were subjected to further validation, by analyzing their expression in large cohorts of tumor samples. This analysis unveiled significant correlations between DUB expression and relevant clinical and pathological parameters, which were in some cases indicative of aggressive disease. CONCLUSIONS/SIGNIFICANCE: The results presented here demonstrate that DUB dysregulation is a frequent event in cancer, and have implications for therapeutic approaches based on DUB inhibition.

Alterations of the Notch pathway in lung cancer.

Notch signaling regulates cell specification and homeostasis of stem cell compartments, and it is counteracted by the cell fate determinant Numb. Both Numb and Notch have been implicated in human tumors. Here, we show that Notch signaling is altered in approximately one third of non-small-cell lung carcinomas (NSCLCs), which are the leading cause of cancer-related deaths: in approximately 30% of NSCLCs, loss of Numb expression leads to increased Notch activity, while in a smaller fraction of cases (around 10%), gain-of-function mutations of the NOTCH-1 gene are present. Activation of Notch correlates with poor clinical outcomes in NSCLC patients without TP53 mutations. Finally, primary epithelial cell cultures, derived from NSCLC harboring constitutive activation of the Notch pathway, are selectively killed by inhibitors of Notch (gamma-secretase inhibitors), showing that the proliferative advantage of these tumors is dependent upon Notch signaling. Our results show that the deregulation of the Notch pathway is a relatively frequent event in NSCLCs and suggest that it might represent a possible target for molecular therapies in these tumors.

Transforming growth factor beta facilitates beta-TrCP-mediated degradation of Cdc25A in a Smad3-dependent manner.

Ubiquitin-dependent degradation of Cdc25A is a major mechanism for damage-induced S-phase checkpoint. Two ubiquitin ligases, the Skp1-cullin-beta-TrCP (SCFbeta-TrCP) complex and the anaphase-promoting complex (APCCdh1), are involved in Cdc25A degradation. Here we demonstrate that the transforming growth factor beta (TGF-beta)-Smad3 pathway promotes SCF(beta-TrCP)-mediated Cdc25A ubiquitination. Cells treated with TGF-beta, as well as cells transfected with Smad3 or a constitutively active type I TGF-beta receptor, exhibit increased ubiquitination and markedly shortened half-lives of Cdc25A. Furthermore, Cdc25A is stabilized in cells transfected with Smad3 small interfering RNA (siRNA) and cells from Smad3-null mice. TGF-beta-induced ubiquitination is associated with Cdc25A phosphorylation at the beta-TrCP docking site (DS82G motif) and physical association of Cdc25A with Smad3 and beta-TrCP. Cdc25A mutant proteins deficient in DS82G phosphorylation are resistant to TGF-beta-Smad3-induced degradation, whereas a Cdc25A mutant protein defective in APCCdh1 recognition undergoes efficient degradation. Smad3 siRNA inhibits beta-TrCP-Cdc25A interaction and Cdc25A degradation in response to TGF-beta. beta-TrCP2 siRNA also inhibits Smad3-induced Cdc25A degradation. In contrast, Cdh1 siRNA had no effect on Cdc25A down-regulation by Smad3. These data suggest that Smad3 plays a key role in the regulation of Cdc25A ubiquitination by SCFbeta-TrCP and that Cdc25A stabilization observed in various cancers could be associated with defects in the TGF-beta-Smad3 pathway.

EBP1 is a nucleolar growth-regulating protein that is part of pre-ribosomal ribonucleoprotein complexes.

EBP1 was identified as a protein that interacts with the ErbB-3 receptor and possibly contributes to transducing growth regulatory signals. The existence of EBP1 homologs across species from simple eukaryotes to humans and its wide tissue expression pattern suggest that EBP1 acts as a general signaling molecule. We provide evidence that EBP1 is localized to the cytoplasm and to the nucleolus, and that its nucleolar localization requires amino-acid sequences present at both the amino- and carboxy-terminus of the molecule. We also show that EBP1 overexpression inhibits proliferation of human fibroblasts, and that this effect is linked to its nucleolar localization. Using mass spectrometry we demonstrate that EBP1 is part of ribonucleoprotein complexes and associates with different rRNA species. It is becoming clear that cell growth and proliferation are actively coordinated with rRNA processing and ribosome assembly. Our findings indicate that EBP1 is a nucleolar growth-regulating protein, and we propose that it could represent a new link between ribosome biosynthesis and cell proliferation.

Cdc25A phosphatase: combinatorial phosphorylation, ubiquitylation and proteolysis.

In eukaryotic cells, control mechanisms of cell-cycle progression have evolved to accurately monitor the integrity of genetic information to be transferred to the progeny. Cdc25A phosphatase is an essential activator of cell-cycle progression and is targeted by checkpoint signals. Ubiquitylation regulates Cdc25A activity through fine tuning of its protein levels. Two different ubiquitin ligases (APC/C and SCF complex) are involved in Cdc25A turnover. While APC/C is involved in regulating Cdc25A at the exit of mitosis, SCF regulates the abundance of Cdc25A in S phase and G2. In response to DNA damage or to stalled replication, the activation of the ATM and ATR protein kinases leads to Chk1 and Chk2 activation and to Cdc25A hyperphosphorylation. These events stimulate SCF-mediated ubiquitylation of Cdc25A and its proteolysis. This contributes to delaying cell-cycle progression, thereby preventing genomic instability. Based on recent findings, we discuss the role of Cdc25A ubiquitylation and degradation in cell-cycle progression and in response to DNA damage. Moreover, we discuss the role of phosphorylation at multiple sites in triggering ubiquitylation signals.

Hierarchical order of phosphorylation events commits Cdc25A to betaTrCP-dependent degradation.

We have recently demonstrated that regulation of Cdc25A protein abundance during S phase and in response to DNA damage is mediated by SCF(betaTrCP) activity. Based on sequence homology of known betaTrCP substrates, we found that Cdc25A contains a conserved motif (DSG), phosphorylation of which is required for interaction with betaTrCP.1 Here, we show that phosphorylation at Ser 82 within the DSG motif anchors Cdc25A to betaTrCP and that Chk1-dependent phosphorylation at Ser 76 affects this interaction as well as betaTrCP-dependent degradation. We propose that a hierarchical order of phosphorylation events commits Cdc25A to betaTrCP-dependent degradation. According to our model, phosphorylation at Ser 76 is a "priming" step required for Ser 82 phosphorylation, which in turn allows recruitment of Cdc25A by betaTrCP and subsequent betaTrCP-dependent degradation.

Degradation of Cdc25A by beta-TrCP during S phase and in response to DNA damage.

The Cdc25A phosphatase is essential for cell-cycle progression because of its function in dephosphorylating cyclin-dependent kinases. In response to DNA damage or stalled replication, the ATM and ATR protein kinases activate the checkpoint kinases Chk1 and Chk2, which leads to hyperphosphorylation of Cdc25A. These events stimulate the ubiquitin-mediated proteolysis of Cdc25A and contribute to delaying cell-cycle progression, thereby preventing genomic instability. Here we report that beta-TrCP is the F-box protein that targets phosphorylated Cdc25A for degradation by the Skp1/Cul1/F-box protein complex. Downregulation of beta-TrCP1 and beta-TrCP2 expression by short interfering RNAs causes an accumulation of Cdc25A in cells progressing through S phase and prevents the degradation of Cdc25A induced by ionizing radiation, indicating that beta-TrCP may function in the intra-S-phase checkpoint. Consistent with this hypothesis, suppression of beta-TrCP expression results in radioresistant DNA synthesis in response to DNA damage--a phenotype indicative of a defect in the intra-S-phase checkpoint that is associated with an inability to regulate Cdc25A properly. Our results show that beta-TrCP has a crucial role in mediating the response to DNA damage through Cdc25A degradation.

Regulating mammalian checkpoints through Cdc25 inactivation.

Precise monitoring of DNA replication and chromosome segregation ensures that there is accurate transmission of genetic information from a cell to its daughters. Eukaryotic cells have developed a complex network of checkpoint pathways that sense DNA lesions and defects in chromosome segregation, spindle assembly and the centrosome cycle, leading to an inhibition of cell-cycle progression for the time required to remove the defect and thus preventing genomic instability. The activation of checkpoints that are responsive to DNA damage or incomplete DNA replication ultimately results in the inhibition of cyclin-dependent kinases. This review focuses on our understanding of the biochemical mechanisms that specifically inactivate Cdc25 (cell division cycle 25) phosphatases to achieve this. The evidence for links between checkpoint deregulation and oncogenesis is discussed.

Control of meiotic and mitotic progression by the F box protein beta-Trcp1 in vivo.

SCF ubiquitin ligases, composed of three major subunits, Skp1, Cul1, and one of many F box proteins (Fbps), control the proteolysis of important cellular regulators. We have inactivated the gene encoding the Fbp beta-Trcp1 in mice. beta-Trcp1(-/-) males show reduced fertility correlating with an accumulation of methaphase I spermatocytes. beta-Trcp1(-/-) MEFs display a lengthened mitosis, centrosome overduplication, multipolar metaphase spindles, and misaligned chromosomes. Furthermore, cyclin A, cyclin B, and Emi1, an inhibitor of the anaphase promoting complex, are stabilized in mitotic beta-Trcp1(-/-) MEFs. Indeed, we demonstrate that Emi1 is a bona fide substrate of beta-Trcp1. In contrast, stabilization of beta-catenin and IkappaBalpha, two previously reported beta-Trcp1 substrates, does not occur in the absence of beta-Trcp1 and instead requires the additional silencing of beta-Trcp2 by siRNA. Thus, beta-Trcp1 regulates the timely order of meiotic and mitotic events.

Effect of apoptogenic stimuli on colon carcinoma cell lines with a different c-myc expression level.

We have recently demonstrated that a high c-myc endogenous amplification level confers an apoptosis-prone phenotype to serum-deprived colon carcinoma SW613-S cells. The aim of this study was to gain new insights into the features of c-myc-dependent apoptosis, by extending our analysis to different apoptogenic stimuli. The study was carried out on clones, derived from the human colon carcinoma SW613-S cell line, which harbor different levels of endogenous c-myc amplification, and on isogenic cell lines with an enforced c-myc overexpression. Our results indicate that cells with endogenous or transfected exogenous c-myc overexpression (SW613-12A1 and -2G1mycP2Tu1 cell lines, respectively), activate the apoptotic machinery in response to the treatment with etoposide, doxorubicin and vitamin D3, which induce apoptosis through the death receptor Fas. The low levels of c-myc expression present in SW613-B3 and -B3mycC5, seem to be unable to activate Fas-mediated apoptosis, thus suggesting that only a high c-myc expression can bypass the lack of Fas receptor. Apoptosis induction mediated by DNA damage and long-term culture was independent of c-myc expression. A pathway of apoptosis characterized by the activation of the enzyme L-DNase II, was observed in both 12A1 and B3 cell lines.

Dual mode of degradation of Cdc25 A phosphatase.

The Cdc25 dual-specificity phosphatases control progression through the eukaryotic cell division cycle by activating cyclin-dependent kinases. Cdc25 A regulates entry into S-phase by dephosphorylating Cdk2, it cooperates with activated oncogenes in inducing transformation and is overexpressed in several human tumors. DNA damage or DNA replication blocks induce phosphorylation of Cdc25 A and its subsequent degradation via the ubiquitin-proteasome pathway. Here we have investigated the regulation of Cdc25 A in the cell cycle. We found that Cdc25 A degradation during mitotic exit and in early G(1) is mediated by the anaphase-promoting complex or cyclosome (APC/C)(Cdh1) ligase, and that a KEN-box motif in the N-terminus of the protein is required for its targeted degradation. Interestingly, the KEN-box mutated protein remains unstable in interphase and upon ionizing radiation exposure. Moreover, SCF (Skp1/Cullin/F-box) inactivation using an interfering Cul1 mutant accumulates and stabilizes Cdc25 A. The presence of Cul1 and Skp1 in Cdc25 A immunocomplexes suggests a direct involvement of SCF in Cdc25 A degradation during interphase. We propose that a dual mechanism of regulated degradation allows for fine tuning of Cdc25 A abundance in response to cell environment.