Skp2 expression is associated with high risk and elevated Ki67 expression in gastrointestinal stromal tumours

BACKGROUND: Gastrointestinal stromal tumors (GIST) exhibit an unpredictable clinical course and can rapidly progress to lethality. Predictions about the biological behavior of GIST are based on a number of canonical clinical and pathologic parameters whose validity in distinguishing between a benign and a malignant tumour is still imperfect. The aim of our study was to investigate the role of morphologic parameters and expression of cells cycle regulators as prognosticators in GIST. METHODS: We performed an immunohistochemical analysis for Ki67, p27Kip1, Jab1, and Skp2, on a Tissue Microarray (TMA) containing 94 GIST. Expression of the above proteins was correlated to classically used prognosticators, as well as to risk groups. Clinical significance of histologic and immunohistochemical features were evaluated in 59 patients for whom follow-up information was available. RESULTS: Overexpression of Ki67 and Skp2, and p27Kip1 loss directly correlated with the high risk group (p = 0.03 for Ki67 and Skp2, p = 0.05 for p27Kip1). Jab1 expression did not exhibit correlation with risk. In 59 cases provided with clinical follow-up, high cellularity, presence of necrosis, and Ki67 overexpression were predictive of a reduced overall survival in a univariate model. The same parameters, as well as mitotic rate, tumour size, and p27Kip1 loss were indicative of a shortened relapse free survival interval. High cellularity, and high mitotic rate retained their prognostic significance by multivariate analysis. CONCLUSION: Our data suggest that a number of histologic parameters in combination with immunohistochemical expression of cell cycle regulators can facilitate risk categorization and predict biologic behavior in GIST. Importantly this study demonstrates, for the first time, that Skp2 expression correlates with Ki67 expression and high risk in GIST

The Mechanism of Ubiquitination in the Cullin-RING E3 Ligase Machinery: Conformational Control of Substrate Orientation

In cullin-RING E3 ubiquitin ligases, substrate binding proteins, such as VHL-box, SOCS-box or the F-box proteins, recruit substrates for ubiquitination, accurately positioning and orienting the substrates for ubiquitin transfer. Yet, how the E3 machinery precisely positions the substrate is unknown. Here, we simulated nine substrate binding proteins: Skp2, Fbw7, β-TrCP1, Cdc4, Fbs1, TIR1, pVHL, SOCS2, and SOCS4, in the unbound form and bound to Skp1, ASK1 or Elongin C. All nine proteins have two domains: one binds to the substrate; the other to E3 ligase modules Skp1/ASK1/Elongin C. We discovered that in all cases the flexible inter-domain linker serves as a hinge, rotating the substrate binding domain, optimally and accurately positioning it for ubiquitin transfer. We observed a conserved proline in the linker of all nine proteins. In all cases, the prolines pucker substantially and the pucker is associated with the backbone rotation toward the E2/ubiquitin. We further observed that the linker flexibility could be regulated allosterically by binding events associated with either domain. We conclude that the flexible linker in the substrate binding proteins orients the substrate for the ubiquitin transfer. Our findings provide a mechanism for ubiquitination and polyubiquitination, illustrating that these processes are under conformational control

Cytoplasmic Skp2 Expression Is Increased in Human Melanoma and Correlated with Patient Survival

BACKGROUND: S-phase kinase protein 2 (Skp2), an F-box protein, targets cell cycle regulators via ubiquitin-mediated degradation. Skp2 is frequently overexpressed in a variety of cancers and associated with patient survival. In melanoma, however, the prognostic significance of subcellular Skp2 expression remains controversial. METHODS: To investigate the role of Skp2 in melanoma development, we constructed tissue microarrays and examined Skp2 expression in melanocytic lesions at different stages, including 30 normal nevi, 61 dysplastic nevi, 290 primary melanomas and 146 metastatic melanomas. The TMA was assessed for cytoplasmic and nuclear Skp2 expression by immunohistochemistry. The Kaplan-Meier method was used to evaluate the patient survival. The univariate and multivariate Cox regression models were performed to estimate the hazard ratios (HR) at five-year follow-up. RESULTS: Cytoplasmic but not nuclear Skp2 expression was gradually increased from normal nevi, dysplastic nevi, primary melanomas to metastatic melanomas. Cytoplasmic Skp2 expression correlated with AJCC stages (I vs II-IV, P<0.001), tumor thickness (≤2.00 vs >2.00 mm, P<0.001) and ulceration (P = 0.005). Increased cytoplasmic Skp2 expression was associated with a poor five-year disease-specific survival of patients with primary melanoma (P = 0.018) but not metastatic melanoma (P>0.05). CONCLUSION: This study demonstrates that cytoplasmic Skp2 plays an important role in melanoma pathogenesis and its expression correlates with patient survival. Our data indicate that cytoplasmic Skp2 may serve as a potential biomarker for melanoma progression and a therapeutic target for this disease

PGE2 Induces IL-6 in Orbital Fibroblasts through EP2 Receptors and Increased Gene Promoter Activity: Implications to Thyroid-Associated Ophthalmopathy

BACKGROUND: IL-6 plays an important role in the pathogenesis of Graves' disease and its orbital component, thyroid-associated ophthalmopathy (TAO). Orbital tissues become inflamed in TAO, a process in which prostanoids have been implicated. Orbital fibroblasts both generate and respond to PGE(2), underlying the inflammatory phenotype of these cells. METHODOLOGY/PRINCIPAL FINDINGS: Using cultured orbital and dermal fibroblasts, we characterized the effects of PGE(2) on IL-6 expression. We found that the prostanoid provokes substantially greater cytokine synthesis in orbital fibroblasts, effects that are mediated through cell-surface EP(2) receptors and increased steady-state IL-6 mRNA levels. The pre-translational up-regulation of IL-6 results from increased gene promoter activity and can be reproduced with the PKA agonist, Sp-cAMP and blocked by interrupting the PKA pathway. PGE(2)-induced production of cAMP in orbital fibroblasts was far greater than that in dermal fibroblasts, resulting from higher levels of adenylate cyclase. PGE(2) provokes CREB phosphorylation, increases the pCREB/CREB ratio, and initiates nuclear localization of the pCREB/CREB binding protein/p300 complex (CBP) preferentially in orbital fibroblasts. Transfection with siRNAs targeting either CREB or CBP blunts the induction of IL-6 gene expression. PGE(2) promotes the binding of pCREB to its target DNA sequence which is substantially greater in orbital fibroblasts. CONCLUSION/SIGNIFICANCE: These results identify the mechanism underlying the exaggerated induction of IL-6 in orbital fibroblasts and tie together two proinflammatory pathways involved in the pathogenesis of TAO. Moreover, they might therefore define an attractive therapeutic target for the treatment of TAO

Calpains Mediate Integrin Attachment Complex Maintenance of Adult Muscle in Caenorhabditis elegans

Two components of integrin containing attachment complexes, UNC-97/PINCH and UNC-112/MIG-2/Kindlin-2, were recently identified as negative regulators of muscle protein degradation and as having decreased mRNA levels in response to spaceflight. Integrin complexes transmit force between the inside and outside of muscle cells and signal changes in muscle size in response to force and, perhaps, disuse. We therefore investigated the effects of acute decreases in expression of the genes encoding these multi-protein complexes. We find that in fully developed adult Caenorhabditis elegans muscle, RNAi against genes encoding core, and peripheral, members of these complexes induces protein degradation, myofibrillar and mitochondrial dystrophies, and a movement defect. Genetic disruption of Z-line– or M-line–specific complex members is sufficient to induce these defects. We confirmed that defects occur in temperature-sensitive mutants for two of the genes: unc-52, which encodes the extra-cellular ligand Perlecan, and unc-112, which encodes the intracellular component Kindlin-2. These results demonstrate that integrin containing attachment complexes, as a whole, are required for proper maintenance of adult muscle. These defects, and collapse of arrayed attachment complexes into ball like structures, are blocked when DIM-1 levels are reduced. Degradation is also blocked by RNAi or drugs targeting calpains, implying that disruption of integrin containing complexes results in calpain activation. In wild-type animals, either during development or in adults, RNAi against calpain genes results in integrin muscle attachment disruptions and consequent sub-cellular defects. These results demonstrate that calpains are required for proper assembly and maintenance of integrin attachment complexes. Taken together our data provide in vivo evidence that a calpain-based molecular repair mechanism exists for dealing with attachment complex disruption in adult muscle. Since C. elegans lacks satellite cells, this mechanism is intrinsic to the muscles and raises the question if such a mechanism also exists in higher metazoans

Iron Behaving Badly: Inappropriate Iron Chelation as a Major Contributor to the Aetiology of Vascular and Other Progressive Inflammatory and Degenerative Diseases

The production of peroxide and superoxide is an inevitable consequence of aerobic metabolism, and while these particular "reactive oxygen species" (ROSs) can exhibit a number of biological effects, they are not of themselves excessively reactive and thus they are not especially damaging at physiological concentrations. However, their reactions with poorly liganded iron species can lead to the catalytic production of the very reactive and dangerous hydroxyl radical, which is exceptionally damaging, and a major cause of chronic inflammation. We review the considerable and wide-ranging evidence for the involvement of this combination of (su)peroxide and poorly liganded iron in a large number of physiological and indeed pathological processes and inflammatory disorders, especially those involving the progressive degradation of cellular and organismal performance. These diseases share a great many similarities and thus might be considered to have a common cause (i.e. iron-catalysed free radical and especially hydroxyl radical generation). The studies reviewed include those focused on a series of cardiovascular, metabolic and neurological diseases, where iron can be found at the sites of plaques and lesions, as well as studies showing the significance of iron to aging and longevity. The effective chelation of iron by natural or synthetic ligands is thus of major physiological (and potentially therapeutic) importance. As systems properties, we need to recognise that physiological observables have multiple molecular causes, and studying them in isolation leads to inconsistent patterns of apparent causality when it is the simultaneous combination of multiple factors that is responsible. This explains, for instance, the decidedly mixed effects of antioxidants that have been observed, etc...Comment: 159 pages, including 9 Figs and 2184 reference