Hyperactivation of HER2-SHCBP1-PLK1 axis promotes tumor cell mitosis and impairs trastuzumab sensitivity to gastric cancer.

Trastuzumab is the backbone of HER2-directed gastric cancer therapy, but poor patient response due to insufficient cell sensitivity and drug resistance remains a clinical challenge. Here, we report that HER2 is involved in cell mitotic promotion for tumorigenesis by hyperactivating a crucial HER2-SHCBP1-PLK1 axis that drives trastuzumab sensitivity and is targeted therapeutically. SHCBP1 is an Shc1-binding protein but is detached from scaffold protein Shc1 following HER2 activation. Released SHCBP1 responds to HER2 cascade by translocating into the nucleus following Ser273 phosphorylation, and then contributing to cell mitosis regulation through binding with PLK1 to promote the phosphorylation of the mitotic interactor MISP. Meanwhile, Shc1 is recruited to HER2 for MAPK or PI3K pathways activation. Also, clinical evidence shows that increased SHCBP1 prognosticates a poor response of patients to trastuzumab therapy. Theaflavine-3, 3'-digallate (TFBG) is identified as an inhibitor of the SHCBP1-PLK1 interaction, which is a potential trastuzumab sensitizing agent and, in combination with trastuzumab, is highly efficacious in suppressing HER2-positive gastric cancer growth. These findings suggest an aberrant mitotic HER2-SHCBP1-PLK1 axis underlies trastuzumab sensitivity and offer a new strategy to combat gastric cancer.

Nonphagocytic Activation of NOX2 Is Implicated in Progressive Nonalcoholic Steatohepatitis During Aging.

BACKGROUND AND AIMS: Older patients with obesity/type II diabetes mellitus frequently present with advanced NASH. Whether this is due to specific molecular pathways that accelerate fibrosis during aging is unknown. Activation of the Src homology 2 domain-containing collagen-related (Shc) proteins and redox stress have been recognized in aging; however, their link to NASH has not been explored. APPROACH AND RESULTS: Shc expression increased in livers of older patients with NASH, as assessed by real time quantitative PCR (RT-qPCR) or western blots. Fibrosis, Shc expression, markers of senescence, and nicotinamide adenine dinucleotide phosphate, reduced form oxidases (NOXs) were studied in young/old mice on fast food diet (FFD). To inhibit Shc in old mice, lentiviral (LV)-short hairpin Shc versus control-LV were used during FFD. For hepatocyte-specific effects, floxed (fl/fl) Shc mice on FFD were injected with adeno-associated virus 8-thyroxine-binding globulin-Cre-recombinase versus control. Fibrosis was accelerated in older mice on FFD, and Shc inhibition by LV in older mice or hepatocyte-specific deletion resulted in significantly improved inflammation, reduction in senescence markers in older mice, lipid peroxidation, and fibrosis. To study NOX2 activation, the interaction of p47phox (NOX2 regulatory subunit) and p52Shc was evaluated by proximity ligation and coimmunoprecipitations. Palmitate-induced p52Shc binding to p47phox , activating the NOX2 complex, more so at an older age. Kinetics of binding were assessed in Src homology 2 domain (SH2) or phosphotyrosine-binding (PTB) domain deletion mutants by biolayer interferometry, revealing the role of SH2 and the PTB domains. Lastly, an in silico model of p52Shc/p47phox interaction using RosettaDock was generated. CONCLUSIONS: Accelerated fibrosis in the aged is modulated by p52Shc/NOX2. We show a pathway for direct activation of the phagocytic NOX2 in hepatocytes by p52Shc binding and activating the p47phox subunit that results in redox stress and accelerated fibrosis in the aged.

Targeting the Shc-EGFR interaction with indomethacin inhibits MAP kinase pathway signalling.

Receptor tyrosine kinase (RTK)-mediated hyperactivation of the MAPK/Erk pathway is responsible for a large number of pathogenic outcomes including many cancers. Considerable effort has been directed at targeting this pathway with varying degrees of long term therapeutic success. Under non-stimulated conditions Erk is bound to the adaptor protein Shc preventing aberrant signalling by sequestering Erk from activation by Mek. Activated RTK recruits Shc, via its phosphotyrosine binding (PTB) domain (ShcPTB), precipitating the release of Erk to engage in a signalling response. Here we describe a novel approach to inhibition of MAP kinase signal transduction through attempting to preserve the Shc-Erk complex under conditions of activated receptor. A library of existing drug molecules was computationally screened for hits that would bind to the ShcPTB and block its interaction with the RTKs EGFR and ErbB2. The primary hit from the screen was indomethacin, a non-steroidal anti-inflammatory drug. Validation of this molecule in vitro and in cellular efficacy studies in cancer cells provides proof of principle of the approach to pathway down-regulation and a potential optimizable lead compound.

ShcA Protects against Epithelial-Mesenchymal Transition through Compartmentalized Inhibition of TGF-ß-Induced Smad Activation.

Epithelial-mesenchymal transition (EMT) is a normal cell differentiation event during development and contributes pathologically to carcinoma and fibrosis progression. EMT often associates with increased transforming growth factor-ß (TGF-ß) signaling, and TGF-ß drives EMT, in part through Smad-mediated reprogramming of gene expression. TGF-ß also activates the Erk MAPK pathway through recruitment and Tyr phosphorylation of the adaptor protein ShcA by the activated TGF-ß type I receptor. We found that ShcA protects the epithelial integrity of nontransformed cells against EMT by repressing TGF-ß-induced, Smad-mediated gene expression. p52ShcA competed with Smad3 for TGF-ß receptor binding, and down-regulation of ShcA expression enhanced autocrine TGF-ß/Smad signaling and target gene expression, whereas increased p52ShcA expression resulted in decreased Smad3 binding to the TGF-ß receptor, decreased Smad3 activation, and increased Erk MAPK and Akt signaling. Furthermore, p52ShcA sequestered TGF-ß receptor complexes to caveolin-associated membrane compartments, and reducing ShcA expression enhanced the receptor localization in clathrin-associated membrane compartments that enable Smad activation. Consequently, silencing ShcA expression induced EMT, with increased cell migration, invasion, and dissemination, and increased stem cell generation and mammosphere formation, dependent upon autocrine TGF-ß signaling. These findings position ShcA as a determinant of the epithelial phenotype by repressing TGF-ß-induced Smad activation through differential partitioning of receptor complexes at the cell surface.

Sirtuin 1-mediated inhibition of p66shc expression alleviates liver ischemia/reperfusion injury.

OBJECTIVES: Ischemia/reperfusion is a leading cause of liver damage after surgical intervention, trauma, and transplantation. It has been reported that the nicotinamide adenine dinucleotide-dependent deacetylase sirtuin 1 attenuates myocardial, cerebral, and renal ischemia/reperfusion damage. This study aimed to investigate the involvement of sirtuin 1-mediated p66shc inhibition in liver ischemia/reperfusion and explore the effect of carnosic acid and ischemic preconditioning on liver ischemia/reperfusion-induced damage. DESIGN: Laboratory investigation. SETTING: University laboratory. SUBJECTS: Male Sprague-Dawley rats and HepG2 cells. INTERVENTIONS: The rats were subjected to 45 minutes of ischemia to 70% of the liver, followed by 3-hour reperfusion. The HepG2 cells were subjected to hypoxia/reoxygenation-induced injury. MEASUREMENTS AND MAIN RESULTS: In the rats with liver ischemia/reperfusion injury, carnosic acid pretreatment and ischemic preconditioning dramatically reduced the serum aminotransferase activity and proinflammatory chemokine levels and improved the liver histological evaluations. Carnosic acid and ischemic preconditioning also increased manganese superoxide dismutase and Bcl-xL, but down-regulated cleaved caspase-3. Interestingly, the protective effect of carnosic acid and ischemic preconditioning was positively associated with sirtuin 1 activation. By contrast, p66shc, a kinase that promotes oxidative injury and apoptosis, was inhibited by carnosic acid and ischemic preconditioning. Sirtuin 1 small interfering RNA knockdown experiments confirmed that carnosic acid increased sirtuin 1-mediated repression of p66shc in HepG2 cells and that the protective effect of carnosic acid against hypoxia/reoxygenation injury was inhibited by the sirtuin 1 inhibitor nicotinamide. These results suggest that carnosic acid protects hepatocytes from hypoxia/reoxygenation damage through sirtuin 1-mediated p66shc suppression. To support this notion, we further demonstrated that the sirtuin 1 activator resveratrol achieved a protective effect similar to that of carnosic acid against hypoxia/reoxygenation injury, whereas sirtuin 1 small interfering RNA and nicotinamide had the opposite effect. CONCLUSIONS: Carnosic acid and ischemic preconditioning protect against ischemia/reperfusion-induced liver injury. Mechanistically, the protective effect involves the sirtuin 1-mediated inhibition of p66shc, suggesting that this pathway is a novel potential therapeutic target for protecting the liver from ischemia/reperfusion injury.

TNFα signals via p66(Shc) to induce E-Selectin, promote leukocyte transmigration and enhance permeability in human endothelial cells.

Endothelial cells participate in inflammatory events leading to atherogenesis by regulating endothelial cell permeability via the expression of VE-Cadherin and ß-catenin and leukocyte recruitment via the expression of E-Selectins and other adhesion molecules. The protein p66(Shc) acts as a sensor/inducer of oxidative stress and may promote vascular dysfunction. The objective of this study was to investigate the role of p66(Shc) in tumor necrosis factor TNFα-induced E-Selectin expression and function in human umbilical vein endothelial cells (HUVEC). Exposure of HUVEC to 50 ng/ml TNFα resulted in increased leukocyte transmigration through the endothelial monolayer and E-Selectin expression, in association with augmented phosphorylation of both p66(Shc) on Ser(36) and the stress kinase c-Jun NH2-terminal protein kinase (JNK)-1/2, and higher intracellular reactive oxygen species (ROS) levels. Overexpression of p66(Shc) in HUVEC resulted in enhanced p66(Shc) phosphorylation on Ser(36), increased ROS and E-Selectin levels, and amplified endothelial cell permeability and leukocyte transmigration through the HUVEC monolayer. Conversely, overexpression of a phosphorylation-defective p66(Shc) protein, in which Ser(36) was replaced by Ala, did not augment ROS and E-Selectin levels, nor modify cell permeability or leukocyte transmigration beyond those found in wild-type cells. Moreover, siRNA-mediated silencing of p66(Shc) resulted in marked reduction of E-Selectin expression and leukocyte transmigration. In conclusion, p66(Shc) acts as a novel intermediate in the TNFα pathway mediating endothelial dysfunction, and its action requires JNK-dependent phosphorylation of p66(Shc) on Ser(36).

Mitochondria-targeted antioxidant peptide SS31 prevents hypoxia/reoxygenation-induced apoptosis by down-regulating p66Shc in renal tubular epithelial cells.

BACKGROUND/AIMS: Ischemia/reperfusion injury plays a crucial role in renal transplantation and represents a significant risk factor for acute kidney injury and delayed graft function. Mitochondria-targeted antioxidant peptide SS31 has been shown to attenuate ischemia/reperfusion injury by inhibiting oxidative stress. The present study was carried out to investigate whether the pretreatment of SS31 could reduce hypoxia/reoxygenation (H/R)-induced injury by inhibiting p66Shc. METHODS: The cultured rat renal proximal tubular cell line NRK52E cells were exposed to 24 h hypoxia (5% CO2, 1% O2, 94% N2) followed by 6 h reoxygenation (5% CO2, 21% O2, 74% N2). SS31 was added to the culture medium 4 h prior to the treatment. Then the cell viability, apoptosis, and oxidative stress levels were determined. In addition, western blot analysis was performed to determine the expression of p66Shc, p-p66Shc, cytochrome c, and caspase-3. RESULTS: H/R induced apoptotic cell death, accompanied with activation of total and p-p66Shc in NRK52E cells. Pretreatment with SS31 or overexpression of a dominantnegative Ser36 mutant p66Shc (p66Shc S36A) or p66Shc siRNA prevented cell death, whereas the protection effect of SS31 was completely blocked by overexpression of wild-type p66Shc. Furthermore, SS31 pretreatment reduced H/R-induced intracellular oxidative stress, cytochrome c translocation to the cytoplasm, and caspase-3 activation through inhibiting p66Shc. CONCLUSION: This study revealed that SS31 pretreatment serves a protective role against H/R-induced apoptosis of human renal tubular epithelial cells, and the mechanism is related to suppression of p66Shc.

Downregulated adaptor protein p66(Shc) mitigates autophagy process by low nutrient and enhances apoptotic resistance in human lung adenocarcinoma A549 cells.

Macroautophagy or autophagy is a lysosome-dependent process in which enzymatic degradation and recycling of cytosolic components occur in stressful contexts. The mechanisms underlying the signaling from starvation to the regulation of autophagy are not fully understood. We previously showed that the Src family member p66(Shc) (focal adhesion-associated 66 kDa isoform of the Src homology and collagen) promotes anoikis and suppresses tumor metastasis via k-Ras-dependent control of proliferation and survival. However, the role of p66(Shc) in low-nutrient-induced autophagy-related pathways remains elusive. In this work, human lung adenocarcinoma A549 cells were used to further investigate the biological effects of p66(Shc) on autophagy and apoptotic resistance. Here, we show that deficiency of p66(Shc) mitigates the low-nutrient-induced autophagy process in the levels of microtubule-associated protein 1A light chain protein 3B (LC3B) conversion, in the number of autophagic vacuoles and in p62/sequestosome 1 protein degradation. However, autophagy-related protein Beclin 1 was not significantly changed during low-nutrient treatment. Furthermore, we found that prolonged phosphorylation of extracellular signaling-regulated kinase (Erk)1/2, but not phosphorylation of Akt is significantly sustained when p66(Shc) expression is inhibited by shRNA. In addition, cleavage of caspase 7 and poly(ADP-ribose) polymerase, but not caspase 6 and 9 are retarded with this effect compared to the shRNA control cells. Together, these findings suggest the possibility that p66(Shc) plays a pivotal role in coordinately regulating autophagy process and apoptotic resistance in A549 cells under nutrient-limited conditions.

Thrombin-induced NF-κB activation and IL-8/CXCL8 release is mediated by c-Src-dependent Shc, Raf-1, and ERK pathways in lung epithelial cells.

In addition to its functions in thrombosis and hemostasis, thrombin also plays an important role in lung inflammation. Our previous report showed that thrombin activates the protein kinase C (PKC)α/c-Src and Gßγ/Rac1/PI3K/Akt signaling pathways to induce IκB kinase α/ß (IKKα/ß) activation, NF-κB transactivation, and IL-8/CXCL8 expressions in human lung epithelial cells (ECs). In this study, we further investigated the mechanism of c-Src-dependent Shc, Raf-1, and extracellular signal-regulated kinase (ERK) signaling pathways involved in thrombin-induced NF-κB activation and IL-8/CXCL8 release. Thrombin-induced increases in IL-8/CXCL8 release and κB-luciferase activity were inhibited by the Shc small interfering RNA (siRNA), p66Shc siRNA, GW 5074 (a Raf-1 inhibitor), and PD98059 (a mitogen-activated protein kinase (MAPK) kinase (MEK) inhibitor). Treatment of A549 cells with thrombin increased p66Shc and p46/p52Shc phosphorylation at Tyr239/240 and Tyr317, which was inhibited by cell transfection with the dominant negative mutant of c-Src (c-Src DN). Thrombin caused time-dependent phosphorylation of Raf-1 and ERK, which was attenuated by the c-Src DN. Thrombin-induced IKKα/ß phosphorylation was inhibited by GW 5074 and PD98059. Treatment of cells with thrombin induced Gßγ, c-Src, and p66Shc complex formation in a time-dependent manner. Taken together, these results show for the first time that thrombin activates Shc, Raf-1, and ERK through Gßγ, c-Src, and Shc complex formation to induce IKKα/ß phosphorylation, NF-κB activation, and IL-8/CXCL8 release in human lung ECs.

SnoN/SkiL expression is modulated via arsenic trioxide-induced activation of the PI3K/AKT pathway in ovarian cancer cells.

SnoN/SkiL (TGFß regulator) is dysregulated in ovarian cancer, a disease associated with acquired drug-resistance. Arsenic trioxide (As2O3, used in treating APL) induces SnoN to oppose the apoptotic response in ovarian cancer cells. We now report that As2O3 increases phosphorylation of EGFR/p66ShcA and EGFR degradation. As2O3 activates Src(Y416) whose activity (inhibited by PP2) modulates EGFR activation, its interaction with Shc/Grb2, and p-AKT. Inhibition of PI3K reduces SnoN and cell survival. Although EGFR or MAPK1 siRNA did not alter SnoN expression, As2O3-induced cleaved PARP was reduced together with increased XIAP. Collectively, As2O3 mediates an initial rise in pY-Src(416) to regulate the PI3K/AKT pathway which increases SnoN and cell survival; these early events may counter the cell death response associated with increased pY-EGFR/MAPK activation.

Targeting MCT-1 oncogene inhibits Shc pathway and xenograft tumorigenicity.

Overexpression of Shc adaptor proteins is associated with mitogenesis, carcinogenesis and metastasis. Multiple copies in T-cell malignancy 1 (MCT-1) oncoprotein promotes cell proliferation, survival and tumorigenic effects. Our current data show that MCT-1 is a novel regulator of Shc-Ras-MEK-ERK signaling and MCT-1 is significantly co-activated with Shc gene in human carcinomas. The knockdown of MCT-1 enhances apoptotic cell death accompanied with the activation of caspases and cleavage of caspase substrates under environmental stress. The cancer cell proliferation, chemo-resistance and tumorigenic capacity are proved to be effectively suppressed by targeting MCT-1. Accordingly, an important linkage between MCT-1 oncogenicity and Shc pathway in tumor development has now been established. Promoting MCT-1 expression by gene hyperactivation may be recognized as a tumor marker and MCT-1 may serve as a molecular target of cancer therapy.

Mitochondrial dysfunction accounts for aldosterone-induced epithelial-to-mesenchymal transition of renal proximal tubular epithelial cells.

Epithelial-mesenchymal transition (EMT) plays a pivotal role in the pathogenesis of renal tubulointerstitial fibrosis. We previously demonstrated that aldosterone (Aldo)-induced EMT is dependent on mitochondrial-derived oxidative stress. This study investigated whether mitochondrial dysfunction (MtD) is involved in the pathogenesis of EMT and whether peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α), a major regulator of oxidative metabolism and mitochondrial function, prevents EMT by improving MtD. Aldo decreased PGC-1α expression while increasing its acetylation and induced MtD, as evidenced by oxidative stress, mitochondrial membrane potential collapse, mitochondrial DNA damage, and mitochondrial complex activity reduction. Aldo time-dependently induced p66Shc phosphorylation and expression. Mineralocorticoid receptor antagonist eplerenone and p66Shc short interfering RNA prevented Aldo-induced MtD and EMT, as evidenced by downregulation of α-smooth muscle actin and upregulation of E-cadherin. Mitochondrial DNA depletion by ethidium bromide or mitochondrial transcription factor A inhibitory RNA (RNAi) induced MtD, further promoting EMT. RNAi-mediated suppression of PGC-1α induced MtD and EMT, whereas overexpression of PGC-1α prevented Aldo-induced MtD and inhibited EMT. Similarly, overexpression of silent mating type information regulation 2 homolog 1 (SIRT1), a gene upstream of PGC-1α, or the SIRT1 activator resveratrol restored Aldo-induced MtD and EMT by upregulating PGC-1α. These findings, which implicate a role for MtD in EMT and suggest that SIRT1 and PGC-1α coordinate to improve mitochondrial function and EMT, may guide us in therapeutic strategies for renal tubulointerstitial fibrosis.

Repression of P66Shc expression by SIRT1 contributes to the prevention of hyperglycemia-induced endothelial dysfunction.

RATIONALE: Inactivation of the p66Shc adaptor protein confers resistance to oxidative stress and protects mice from aging-associated vascular diseases. However, there is limited information about the negative regulating mechanisms of p66Shc expression in the vascular system. OBJECTIVE: In this study, we investigated the role of SIRT1, a class III histone deacetylase, in the regulation of p66Shc expression and hyperglycemia-induced endothelial dysfunction. METHODS AND RESULTS: Expressions of p66Shc gene transcript and protein were significantly increased by different kinds of class III histone deacetylase (sirtuin) inhibitors in human umbilical vein endothelial cells and 293A cells. Adenoviral overexpression of SIRT1 inhibited high-glucose-induced p66Shc upregulation in human umbilical vein endothelial cells. Knockdown of SIRT1 increased p66Shc expression and also increased the expression levels of plasminogen activator inhibitor-1 expression, but decreased manganese superoxide dismutase expression in high-glucose conditions. However, knockdown of p66Shc significantly reversed the effects of SIRT1 knockdown. In addition, p66Shc overexpression significantly decreased manganese superoxide dismutase expression and increased plasminogen activator inhibitor-1 expression in high-glucose conditions, which were recovered by SIRT1 overexpression. Moreover, compared to streptozotocin-induced wild-type diabetic mice, endothelium-specific SIRT1 transgenic diabetic mice had decreased p66Shc expression at both the mRNA and the protein levels, improved endothelial function, and reduced accumulation of nitrotyrosine and 8-OHdG (markers of oxidative stress). We further found that SIRT1 was able to bind to the p66Shc promoter (-508 bp to -250 bp), resulting in a decrease in the acetylation of histone H3 bound to the p66Shc promoter region. CONCLUSION: Our findings indicate that repression of p66Shc expression by SIRT1 contributes to the protection of hyperglycemia-induced endothelial dysfunction.

Hyperglycemia-induced p66shc inhibits insulin-like growth factor I-dependent cell survival via impairment of Src kinase-mediated phosphoinositide-3 kinase/AKT activation in vascular smooth muscle cells.

Hyperglycemia has been shown to induce the p66shc expression leading to increased reactive oxygen species (ROS) generation and apoptosis. In the present study, we demonstrated that hyperglycemia induced p66shc expression in vascular smooth muscle cells. This induction was associated with an increase in apoptosis as assessed by the increase of capspase-3 enzymatic activity, cleaved caspase-3 protein, and the number of dead cells. The ability of IGF-I to inhibit apoptosis was also attenuated. Further studies showed that hyperglycemia-induced p66shc inhibited IGF-I-stimulated phosphoinositide (PI)-3 kinase and AKT activation. Mechanistic studies showed that knockdown of p66shc enhanced IGF-I-stimulated SHPS-1/p85, p85/SHP-2, and p85/Grb2 association, all of which are required for PI-3 kinase/AKT activation. These responses were attenuated by overexpression of p66shc. IGF-I-stimulated p85 and AKT recruitment to the cell membrane fraction was altered in the same manner. Disruption of p66shc-Src interaction using either a blocking peptide or by expressing a p66shc mutant that did not bind to Src rescued IGF-I-stimulated PI-3 kinase/AKT activation as well as IGF-I-dependent cell survival. Although the highest absolute level of ROS was detected in p66shc-overexpressing cells, the relative increase in ROS induced by hyperglycemia was independent of p66shc expression. Taken together, our data suggest that the increase in p66shc that occurs in response to hyperglycemia is functioning to inhibit IGF-I-stimulated signaling and that the incremental increase in SMC sensitivity to IGF-I stimulation that occurs in response to p66shc induction of ROS is not sufficient to overcome the inhibitory effect of p66shc on Src kinase activation.

Essential role of the redox-sensitive kinase p66shc in determining energetic and oxidative status and cell fate in neuronal preconditioning.

Ischemic preconditioning is a phenomenon in which low-level stressful stimuli upregulate endogenous defensive programs, resulting in subsequent resistance to otherwise lethal injuries. We previously observed that signal transduction systems typically associated with neurodegeneration such as caspase activation are requisite events for the expression of tolerance and induction of HSP70. In this work, we sought to determine the extent and duration of oxidative and energetic dysfunction as well as the role of effector kinases on metabolic function in preconditioned cells. Using an in vitro neuronal culture model, we observed a robust increase in Raf and p66(Shc) activation within 1 h of preconditioning. Total ATP content decreased by 25% 3 h after preconditioning but returned to baseline by 24 h. Use of a free radical spin trap or p66(shc) inhibitor increased ATP content whereas a Raf inhibitor had no effect. Phosphorylated p66(shc) rapidly relocalized to the mitochondria and in the absence of activated p66(shc), autophagic processing increased. The constitutively expressed chaperone HSC70 relocalized to autophagosomes. Preconditioned cells experience significant total oxidative stress measured by F(2)-isoprostanes and neuronal stress evaluated by F(4)-neuroprostane measurement. Neuroprostane levels were enhanced in the presence of Shc inhibitors. Finally, we found that inhibiting either p66(shc) or Raf blocked neuroprotection afforded by preconditioning as well as upregulation of HSP70, suggesting both kinases are critical for preconditioning but function in fundamentally different ways. This is the first work to demonstrate the essential role of p66(shc) in mediating requisite mitochondrial and energetic compensation after preconditioning and suggests a mechanism by which protein and organelle damage mediated by ROS can increase HSP70.

Involvement of the p66Shc protein in glucose transport regulation in skeletal muscle myoblasts.

The p66(Shc) protein isoform regulates MAP kinase activity and the actin cytoskeleton turnover, which are both required for normal glucose transport responses. To investigate the role of p66(Shc) in glucose transport regulation in skeletal muscle cells, L6 myoblasts with antisense-mediated reduction (L6/p66(Shc)as) or adenovirus-mediated overexpression (L6/p66(Shc)adv) of the p66(Shc) protein were examined. L6/(Shc)as myoblasts showed constitutive activation of ERK-1/2 and disruption of the actin network, associated with an 11-fold increase in basal glucose transport. GLUT1 and GLUT3 transporter proteins were sevenfold and fourfold more abundant, respectively, and were localized throughout the cytoplasm. Conversely, in L6 myoblasts overexpressing p66(Shc), basal glucose uptake rates were reduced by 30% in parallel with a approximately 50% reduction in total GLUT1 and GLUT3 transporter levels. Inhibition of the increased ERK-1/2 activity with PD98059 in L6/(Shc)as cells had a minimal effect on increased GLUT1 and GLUT3 protein levels, but restored the actin cytoskeleton, and reduced the abnormally high basal glucose uptake by 70%. In conclusion, p66(Shc) appears to regulate the glucose transport system in skeletal muscle myoblasts by controlling, via MAP kinase, the integrity of the actin cytoskeleton and by modulating cellular expression of GLUT1 and GLUT3 transporter proteins via ERK-independent pathways.

Distinct role of ShcC docking protein in the differentiation of neuroblastoma.

The biological and clinical heterogeneity of neuroblastoma is closely associated with signaling pathways that control cellular characteristics such as proliferation, survival and differentiation. The Shc family of docking proteins is important in these pathways by mediating cellular signaling. In this study, we analysed the expression levels of ShcA and ShcC proteins in 46 neuroblastoma samples and showed that a significantly higher level of ShcC protein is observed in neuroblastomas with poor prognostic factors such as advanced stage and MYCN amplification (P<0.005), whereas the expression level of ShcA showed no significant association with these factors. Using TNB1 cells that express a high level of ShcC protein, it was demonstrated that knockdown of ShcC by RNAi caused elevation in the phosphorylation of ShcA, which resulted in sustained extracellular signal-regulated kinase activation and neurite outgrowth. The neurites induced by ShcC knockdown expressed several markers of neuronal differentiation suggesting that the expression of ShcC potentially has a function in inhibiting the differentiation of neuroblastoma cells. In addition, marked suppression of in vivo tumorigenicity of TNB1 cells in nude mice was observed by stable knockdown of ShcC protein. These findings indicate that ShcC is a therapeutic target that might induce differentiation in the aggressive type of neuroblastomas.