Search

Inhibitors of the mechanistic target of rapamycin (mTOR) are currently used to treat advanced metastatic breast cancer. However, whether an aggressive phenotype is sustained through adaptation or resistance to mTOR inhibition remains unknown. Here, complementary studies in human tumors, cancer models and cell lines reveal transcriptional reprogramming that supports metastasis in response to mTOR inhibition. This cancer feature is driven by EVI1 and SOX9. EVI1 functionally cooperates with and positively regulates SOX9, and promotes the transcriptional upregulation of key mTOR pathway components (REHB and RAPTOR) and of lung metastasis mediators (FSCN1 and SPARC). The expression of EVI1 and SOX9 is associated with stem cell-like and metastasis signatures, and their depletion impairs the metastatic potential of breast cancer cells. These results establish the mechanistic link between resistance to mTOR inhibition and cancer metastatic potential, thus enhancing our understanding of mTOR targeting failure.

Abnormal tauphosphorylation and deposition in neurones and glial cells is one of the major features in taupathies. The present study examines the involvement of the Ras/MEK/ERK pathway of tauphosphorylation in Alzheimer disease (AD), Pick's disease (PiD), progressive supranuclear palsy (PSP) and corticobasal degeneration (CBD), by Western blotting, single and double‐labelling immunohistochemistry, and p21Ras activation assay. Since this pathway is also activated in several paradigms of cell death and cell survival, activated ERK expression is also analysed with double‐labelling immunohistochemistry and in situend‐labelling of nuclear DNA fragmentation to visualise activated ERK in cells with increased nuclear DNA vulnerability. The MEK1 antibody recognises one band of 45 kD that identifies phosphorylation‐independent MEK1, whose expression levels are not modified in diseased brains. The ERK antibody recognises one band of 42 kD corresponding to the molecular weight of phosphorylation‐independent ERK2; the expression levels, as well as the immunoreactivity of ERK in individual cells, is not changed in AD, PiD, PSP and CBD. The antibody MAPK‐P distinguishes two bands of 44 kD and 42 kD that detect phosphorylated ERK1 and ERK2. MAPK‐P expression levels, as seen with Western blotting, are markedly increased in AD, PiD, PSP and CBD. Moreover, immunohistochemistry discloses granular precipitates in the cytoplasm of neurones in AD, mainly in a subpopulation of neurones exhibiting early taudeposition, whereas neurones with developed neurofibrillary tangles are less commonly immunostained. MAPK‐P also decorates neurones with Pick bodies in PiD, early taudeposition in neurones in PSP and CBD, and cortical achromatic neurones in CBD. In addition, strong MAPK‐P immunoreactivity is found in large numbers of tau‐positive glial cells in PSP and CBD, as seen with double‐labelling immunohistochemistry. Yet no co‐localisation of enhanced phosphorylated ERK immunoreactivity and nuclear DNA fragmentation is found in AD, PiD, PSP and CBD. Finally, activated Ras expression levels are increased in AD cases when compared with controls. These results demonstrate increased phosphorylated (active) ERK expression in association with early taudeposition in neurones and glial cells in taupathies, and suggest activated Ras as the upstream activator of the MEK/ERK pathway of tauphosphorylation in AD.

In this work, we analyzed the role of the PI3K-p70 S6 kinase (S6K) signaling cascade in the stimulation of endothelial cell proliferation. We found that inhibitors of the p42/p44 MAPK pathway (PD98059) and the PI3K-p70 S6K pathway (wortmannin, Ly294002, and rapamycin) all block thymidine incorporation stimulated by fetal calf serum in the resting mouse endothelial cell line 1G11. The action of rapamycin can be generalized, since it completely inhibits the mitogenic effect of fetal calf serum in primary endothelial cell cultures (human umbilical vein endothelial cells) and another established capillary endothelial cell line (LIBE cells). The inhibitory effect of rapamycin is only observed when the inhibitor is added at the early stages of G(0)-G(1) progression, suggesting an inhibitory action early in G(1). Rapamycin completely inhibits growth factor stimulation of protein synthesis, which perfectly correlates with the inhibition of cell proliferation. In accordance with its inhibitory action on protein synthesis, activation of cyclin D1 and p21 proteins by growth factors is also blocked by preincubation with rapamycin. Expression of a p70 S6K mutant partially resistant to rapamycin reverses the inhibitory effect of the drug on DNA synthesis, indicating that rapamycin action is via p70 S6K. Thus, in vascular endothelial cells, activation of protein synthesis via p70 S6K is an essential step for cell cycle progression in response to growth factors.

Glucose constitutes a major fuel for the heart, and high glucose uptake during fetal development is coincident with the highest level of expression of the glucose transporter GLUT-1 during life. We have previously reported that GLUT-1 is repressed perinatally in rat heart, and GLUT-4, which shows a low level of expression in the fetal stage, becomes the main glucose transporter in the adult. Here, we show that the perinatal expression of GLUT-1 and GLUT-4 glucose transporters in heart is controlled directly at the level of gene transcription. Transient transfection assays show that the -99/-33 fragment of the GLUT-1 gene is sufficient to drive transcriptional activity in rat neonatal cardiomyocytes. Electrophoretic mobility shift assays demonstrate that the transcription factor Sp1, a trans-activator of GLUT-1 promoter, binds to the -102/-82 region of GLUT-1 promoter during the fetal state but not during adulthood. Mutation of the Sp1 site in this region demonstrates that Sp1 is essential for maintaining a high transcriptional activity in cardiac myocytes. Sp1 is markedly down-regulated both in heart and in skeletal muscle during neonatal life, suggesting an active role for Sp1 in the regulation of GLUT-1 transcription. In all, these results indicate that the expression of GLUT-1 and GLUT-4 in heart during perinatal development is largely controlled at a transcriptional level by mechanisms that might be related to hyperplasia and that are independent from the signals that trigger cell hypertrophy in the developing heart. Furthermore, our results provide the first functional insight into the mechanisms regulating muscle GLUT-1 gene expression in a live animal.

Vascular Endothelial Growth Factor (VEGF) is a potent mitogen for vascular endothelial cells that has been implicated in tumor neovascularization. We show that, in hamster fibroblasts (CCL39 cells), VEGF mRNAs are expressed at low levels in serum-deprived or exponentially growing cells, whereas it is rapidly induced after stimulation of quiescent cells with serum. CCL39 derivatives, transformed with Polyoma virus or with active members of the p42/p44 mitogen-activated protein (MAP) kinase pathway, Gly/Val point mutant of Ras at position 12 (Ras-Val12), MKK1 in which Ser218 and Ser222 were mutated to Asp (MKK1-SS/DD)), express very high levels of VEGF mRNA. To analyze the contribution of the p42/p44MAP kinase in this induction, we used the CCL39-derived cell line (Raf-1:ER) expressing an estradiol-activable Raf-1. We show a time and an estradiol dose-dependent up-regulation of VEGF mRNA clearly detectable after 2 h of stimulation. The induction of VEGF mRNA in response to conditioned activation of Raf-1 is reverted by an inhibitor of MKK1, PD 098059, highlighting a specific role for the p42/p44 MAP kinase pathway in VEGF expression. Interestingly, hypoxia has an additive effect on VEGF induction in CCL39 cells stimulated by serum or in Raf-1:ER cells stimulated by estradiol. In contrast to VEGF, the isoforms VEGF-B and VEGF-C are poorly regulated by growth and oncogenic factors. We have identified a GC-rich region of the VEGF promoter between -88 and -66 base pairs which contains all the elements responsible of its up-regulation by constitutive active Ras or MKK1-SS/DD. By mutation of the putative binding sites and electrophoretic mobility supershift experiments, we showed that the GC-rich region constitutively binds Sp1 and AP-2 transcription factors. Furthermore, following activation of the p42/p44 MAP kinase module, the binding of Sp1 and AP-2 is increased in the complexes formed in this region of the promoter. Altogether, these data suggest that hypoxia and p42/p44 MAP kinase independently play a key role in the regulation of the VEGF expression.

Skeletal muscle differentiation involves myoblast alignment, elongation, and fusion into multinucleate myotubes, together with the induction of regulatory and structural muscle-specific genes. Here we show that two phosphatidylinositol 3-kinase inhibitors, LY294002 and wortmannin, blocked an essential step in the differentiation of two skeletal muscle cell models. Both inhibitors abolished the capacity of L6E9 myoblasts to form myotubes, without affecting myoblast proliferation, elongation, or alignment. Myogenic events like the induction of myogenin and of glucose carrier GLUT4 were also blocked and myoblasts could not exit the cell cycle, as measured by the lack of mRNA induction of p21 cyclin-dependent kinase inhibitor. Overexpresssion of MyoD in 10T1/2 cells was not sufficient to bypass the myogenic differentiation blockade by LY294002. Upon serum withdrawal, 10T1/2-MyoD cells formed myotubes and showed increased levels of myogenin and p21. In contrast, LY294002-treated cells exhibited none of these myogenic characteristics and maintained high levels of Id, a negative regulator of myogenesis. These data indicate that whereas phosphatidylinositol 3-kinase is not indispensable for cell proliferation or in the initial events of myoblast differentiation, i.e. elongation and alignment, it appears to be essential for terminal differentiation of muscle cells.

In this study we have used wortmannin, a highly specific inhibitor of phosphatidylinositol (PI) 3-kinase, to assess the role of this enzyme on GLUT1 glucose carrier distribution and glucose transport activity in myoblasts from two skeletal-muscle cell lines, L6E9 and Sol8. As detected in L6E9 cells, myoblasts exhibited basal and insulin-stimulated PI 3-kinase activities. Incubation of intact myoblasts with wortmannin resulted in a marked inhibition of both basal and insulin-stimulated PI 3-kinase activities. L6E9 and Sol8 myoblasts showed basal and insulin-stimulated glucose transport activities, both of them inhibited by wortmannin in a dose-dependent manner (IC50 approximately 10-20 nM). Concomitantly, immunofluorescence analysis revealed that 1 h treatment with wortmannin led to a dramatic intracellular accumulation of GLUT1 carriers (the main glucose transporter expressed in L6E9 and Sol8 myoblasts) in both cell systems. The effect of wortmannin on GLUT1 cellular redistribution was independent of the presence of insulin. The cellular distribution of two structural plasma-membrane components such as beta 1-integrin or the alpha 1 subunit of the Na(+)-K(+)-ATPase were unaffected by wortmannin in both the absence and the presence of insulin. As a whole, our results indicate that PI 3-kinase is necessary to basal and insulin-stimulated glucose transport in L6E9 and Sol8 myoblasts. Moreover, immunofluorescence assays suggest that in both cellular models there is a constitutive GLUT 1 trafficking pathway (independent of insulin) that involves PI 3-kinase and which, when blocked, locks GLUT1 in a perinuclear compartment.

Fluoride is a nucleophilic reagent which has been reported to inhibit a variety of different enzymes such as esterases, asymmetrical hydrolases and phosphatases. In this report, we demonstrate that fluoride inhibits tyrosine kinase activity of insulin receptors partially purified from rat skeletal muscle and human placenta. Fluoride inhibited in a similar dose-dependent manner both beta-subunit autophosphorylation and tyrosine kinase activity for exogenous substrates. This inhibitory effect of fluoride was not due to the formation of complexes with aluminum and took place in the absence of modifications of insulin-binding properties of the insulin receptor. Fluoride did not complete with the binding site for ATP or Mn2+. Fluoride also inhibited the autophosphorylation and tyrosine kinase activity of receptors for insulin-like growth factor I from human placenta. Addition of fluoride to the pre-phosphorylated insulin receptor produced a slow (time range of minutes) inhibition of receptor kinase activity. Furthermore, fluoride inhibited tyrosine kinase activity in the absence of changes in the phosphorylation of prephosphorylated insulin receptors, and the sensitivity to fluoride was similar to the sensitivity of the unphosphorylated insulin receptor. The effect of fluoride-on tyrosine kinase activity was markedly decreased when insulin receptors were preincubated with the copolymer of glutamate/tyrosine. Prior exposure of receptors to free tyrosine or phosphotyrosine also prevented the inhibitory effect of fluoride. However, the protective effect of tyrosine or phosphotyrosine was maximal at low concentrations, suggesting the interaction of these compounds with the receptor itself rather than with fluoride. These data suggest: (i) that fluoride interacts directly and slowly with the insulin receptor, which causes inhibition of its phosphotransferase activity; (ii) that the binding site of fluoride is not structurally modified by receptor phosphorylation; and (iii) based on the fact that fluoride inhibits phosphotransferase activity in the absence of alterations in the binding of ATP, Mn2+ or insulin, we speculate that fluoride binding might affect the transfer of phosphate from ATP to the tyrosine residues of the beta-subunit of the insulin receptor and to the tyrosine residues of exogenous substrates.

Muscle cell differentiation caused a reduction of glucose transport, GLUT1 glucose transporter expression, and GLUT1 mRNA levels. A fragment of 2.1 kilobases of the rat GLUT1 gene linked to chloramphenicol acetyltransferase drove transcriptional activity in myoblasts, and differentiation caused a decrease in transcription. Transient transfection of 5' and 3' deletion constructs showed that the fragment -99/-33 of the GLUT1 gene drives transcriptional activity of the GLUT1 gene and participates in the reduced transcription after muscle differentiation. Electrophoretic mobility shift assays showed the binding of Sp1 protein to the fragment -102/-37 in the myoblast state but not in myotubes, and Sp1 was found to transactivate the GLUT1 promoter. Western blot analysis indicated that Sp1 was drastically down-regulated during myogenesis. Furthermore, the forced over-expression of MyoD in C3H10T1/2 cells mimicked the effects observed during myogenesis, Sp1 down-regulation and reduced transcriptional activity of the GLUT1 gene promoter. In all, these data suggest a regulatory model in which MyoD activation during myogenesis causes the down-regulation of Sp1, which contributes to the repression of GLUT1 gene transcription and, therefore, leads to the reduction in GLUT1 expression and glucose transport.

In this study, the relationship between the concentration of extracellular insulin, insulin binding and insulin action was evaluated in skeletal muscle. Initially we investigated the dose-response relationship of insulin action using three different experimental models that are responsive to insulin, i.e. the isolated perfused rat hindquarter, incubated strips of soleus muscle, and insulin receptors partially affinity-purified from skeletal muscle. We selected as insulin-sensitive parameters glucose uptake in the perfused hindquarter, lactate production in the incubated muscle preparation, and tyrosine receptor kinase activity in the purified receptor preparation. Our results showed that the dose-response curves obtained in the perfused hindquarter and in the incubated muscle were superimposable. In contrast, the dose-response curve for insulin-stimulated receptor tyrosine kinase activity in partially purified receptors was displaced to the left compared with the curves obtained in the perfused hindquarter and in the incubated muscle. The differences between the dose-response curve for receptor tyrosine kinase and those for glucose uptake and lactate production were not explained by a substantial insulin concentration gradient between medium and interstitial space. Thus the medium/interstitial insulin concentration ratio, when assayed in the incubated intact muscle at 5 degrees C, was close to 1. We also compared the dose-response curve of insulin-stimulated receptor tyrosine kinase with the pattern of insulin-binding-site occupancy. The curve of insulin-stimulated receptor kinase activity fitted closely with the occupancy of high-affinity binding sites. In summary, assuming that the estimation of the medium/interstitial insulin concentration ratio obtained at 5 degrees C reflects the actual ratio under more physiological conditions, our results suggest that maximal insulin action is obtained in skeletal muscle at insulin concentrations which do allow full occupancy of high-affinity binding sites. Therefore our data provide evidence for a lack of spare high-affinity insulin receptors in skeletal muscle.