The Hippo pathway terminal effector TAZ/WWTR1 mediates oxaliplatin sensitivity in p53 proficient colon cancer cells.

YAP and TAZ, the Hippo pathway terminal transcriptional activators, are frequently upregulated in cancers. In tumor cells, they have been mainly associated with increased tumorigenesis controlling different aspects from cell cycle regulation, stemness, or resistance to chemotherapies. In fewer cases, they have also been shown to oppose cancer progression, including by promoting cell death through the action of the p73/YAP transcriptional complex, in particular after chemotherapeutic drug exposure. Using HCT116 cells, we show here that oxaliplatin treatment led to core Hippo pathway down-regulation and nuclear accumulation of TAZ. We further show that TAZ was required for the increased sensitivity of HCT116 cells to oxaliplatin, an effect that appeared independent of p73, but which required the nuclear relocalization of TAZ. Accordingly, Verteporfin and CA3, two drugs affecting the activity of YAP and TAZ, showed antagonistic effects with oxaliplatin in co-treatments. Importantly, using several colorectal cell lines, we show that the sensitizing action of TAZ to oxaliplatin is dependent on the p53 status of the cells. Our results support thus an early action of TAZ to sensitize cells to oxaliplatin, consistent with a model in which nuclear TAZ in the context of DNA damage and p53 activity pushes cells towards apoptosis.

Mechanisms underlying the cooperation between loss of epithelial polarity and Notch signaling during neoplastic growth in Drosophila.

Aggressive neoplastic growth can be initiated by a limited number of genetic alterations, such as the well-established cooperation between loss of cell architecture and hyperactive signaling pathways. However, our understanding of how these different alterations interact and influence each other remains very incomplete. Using Drosophila paradigms of imaginal wing disc epithelial growth, we have monitored the changes in Notch pathway activity according to the polarity status of cells (scrib mutant). We show that the scrib mutation impacts the direct transcriptional output of the Notch pathway, without altering the global distribution of Su(H), the Notch-dedicated transcription factor. The Notch-dependent neoplasms require, however, the action of a group of transcription factors, similar to those previously identified for Ras/scrib neoplasm (namely AP-1, Stat92E, Ftz-F1 and basic leucine zipper factors), further suggesting the importance of this transcription factor network during neoplastic growth. Finally, our work highlights some Notch/scrib specificities, in particular the role of the PAR domain-containing basic leucine zipper transcription factor and Notch direct target Pdp1 for neoplastic growth.

Mechanical Control of Cell Migration by the Metastasis Suppressor Tetraspanin CD82/KAI1.

The plasma membrane is a key actor of cell migration. For instance, its tension controls persistent cell migration and cell surface caveolae integrity. Then, caveolae constituents such as caveolin-1 can initiate a mechanotransduction loop that involves actin- and focal adhesion-dependent control of the mechanosensor YAP to finely tune cell migration. Tetraspanin CD82 (also named KAI-1) is an integral membrane protein and a metastasis suppressor. Its expression is lost in many cancers including breast cancer. It is a strong inhibitor of cell migration by a little-known mechanism. We demonstrated here that CD82 controls persistent 2D migration of EGF-induced single cells, stress fibers and focal adhesion sizes and dynamics. Mechanistically, we found that CD82 regulates membrane tension, cell surface caveolae abundance and YAP nuclear translocation in a caveolin-1-dependent manner. Altogether, our data show that CD82 controls 2D cell migration using membrane-driven mechanics involving caveolin and the YAP pathway.

MAGI1 inhibits the AMOTL2/p38 stress pathway and prevents luminal breast tumorigenesis.

Alterations to cell polarization or to intercellular junctions are often associated with epithelial cancer progression, including breast cancers (BCa). We show here that the loss of the junctional scaffold protein MAGI1 is associated with bad prognosis in luminal BCa, and promotes tumorigenesis. E-cadherin and the actin binding scaffold AMOTL2 accumulate in MAGI1 deficient cells which are subjected to increased stiffness. These alterations are associated with low YAP activity, the terminal Hippo-pathway effector, but with an elevated ROCK and p38 Stress Activated Protein Kinase activities. Blocking ROCK prevented p38 activation, suggesting that MAGI1 limits p38 activity in part through releasing actin strength. Importantly, the increased tumorigenicity of MAGI1 deficient cells is rescued in the absence of AMOTL2 or after inhibition of p38, demonstrating that MAGI1 acts as a tumor-suppressor in luminal BCa by inhibiting an AMOTL2/p38 stress pathway.

The apical scaffold big bang binds to spectrins and regulates the growth of Drosophila melanogaster wing discs.

During development, cell numbers are tightly regulated, ensuring that tissues and organs reach their correct size and shape. Recent evidence has highlighted the intricate connections between the cytoskeleton and the regulation of the key growth control Hippo pathway. Looking for apical scaffolds regulating tissue growth, we describe that Drosophila melanogaster big bang (Bbg), a poorly characterized multi-PDZ scaffold, controls epithelial tissue growth without affecting epithelial polarity and architecture. bbg-mutant tissues are smaller, with fewer cells that are less apically constricted than normal. We show that Bbg binds to and colocalizes tightly with the ß-heavy-Spectrin/Kst subunit at the apical cortex and promotes Yki activity, F-actin enrichment, and the phosphorylation of the myosin II regulatory light chain Spaghetti squash. We propose a model in which the spectrin cytoskeleton recruits Bbg to the cortex, where Bbg promotes actomyosin contractility to regulate epithelial tissue growth.

Characterization of the Akt2 domain essential for binding nuclear p21cip1 to promote cell cycle arrest during myogenic differentiation.

The binding of the cdk inhibitor p21cip1 to Akt2 in the nucleus is an essential component in determining the specific role of Akt2 in the cell cycle arrest that precedes myogenic differentiation. Here, through a combination of biochemical and cell biology approaches, we have addressed the molecular basis of this binding. Using amino-terminal truncation of Akt2, we show that p21cip1 binds at the carboxy terminal of Akt2 since deletion of the first 400 amino acids did not affect the interaction between Akt2 and p21cip1. Pull down using carboxy terminal-truncated Akt2 protein revealed the importance of the region between amino acids 400 and 445 for the binding to p21cip1. Since Akt2_400-445 and Akt2_420-445 peptides could both bind p21cip1, this refines the binding domain on Akt2 between amino acids 420 and 445. In order to confirm these data in living cells, we developed a protocol to synchronize myoblasts at the cell cycle exit point when p21cip1 expression is induced by MyoD before myogenic differentiation. When a synthetic Akt2 peptide spanning the region (410-437) was microinjected in p21-expressing myoblasts, p21cip1 no longer localized exclusively in the nucleus, instead being redistributed throughout the cell, thus showing that injected peptide 410-437 acts to compete with the binding of endogenous Akt2 to p21cip1. Taken together, our data suggest that this 27 amino acid sequence on Akt2 is necessary and sufficient to bind p21cip1 both in vitro and in living cells.

Akt1 and Akt2: differentiating the aktion.

Kinases of the Akt family are integral and essential components in growth factor signaling pathways activated downstream of the membrane bound phospho-inositol-3 kinase. In light of strong homologies in the primary amino acid sequence, the three Akt kinases were long surmised to play redundant and overlapping roles in insulin signaling across the spectra of cell and tissue types. Over the last 10 years, work using mouse knockout models, cell specific inactivation, and more recently targeted gene inactivation, has brought into question the redundancy within Akt kinase isoforms and instead pointed to isoform specific functions in different cellular events and diseases. Here we concentrate on the differential roles played by Akt1 and Akt2 in a variety of cellular processes and in particular during cancer biogenesis. In this overview, we illustrate that while Akt1 and 2 are often implicated in many aspects of cellular transformation, the two isoforms frequently act in a complementary opposing manner. Furthermore, Akt1 and Akt2 kinases interact differentially with modulating proteins and are necessary in relaying roles during the evolution of cancers from deregulated growth into malignant metastatic killers. These different actions of the two isoforms point to the importance of treatments targeting isoform specific events in the development of effective approaches involving Akt kinases in human disease.

Distinct pools of cdc25C are phosphorylated on specific TP sites and differentially localized in human mitotic cells.

BACKGROUND: The dual specificity phosphatase cdc25C was the first human cdc25 family member found to be essential in the activation of cdk1/cyclin B1 that takes place at the entry into mitosis. Human cdc25C is phosphorylated on Proline-dependent SP and TP sites when it becomes active at mitosis and the prevalent model is that this phosphorylation/activation of cdc25C would be part of an amplification loop with cdk1/cyclin B1. METHODOLOGY/PRINCIPAL FINDINGS: Using highly specific antibodies directed against cdc25C phospho-epitopes, pT67 and pT130, we show here that these two phospho-forms of cdc25C represent distinct pools with differential localization during human mitosis. Phosphorylation on T67 occurs from prophase and the cdc25C-pT67 phospho-isoform closely localizes with condensed chromosomes throughout mitosis. The phospho-T130 form of cdc25C arises in late G2 and associates predominantly with centrosomes from prophase to anaphase B where it colocalizes with Plk1. As shown by immunoprecipitation of each isoform, these two phospho-forms are not simultaneously phosphorylated on the other mitotic TP sites or associated with one another. Phospho-T67 cdc25C co-precipitates with MPM2-reactive proteins while pT130-cdc25C is associated with Plk1. Interaction and colocalization of phosphoT130-cdc25C with Plk1 demonstrate in living cells, that the sequence around pT130 acts as a true Polo Box Domain (PBD) binding site as previously identified from in vitro peptide screening studies. Overexpression of non-phosphorylatable alanine mutant forms for each isoform, but not wild type cdc25C, strongly impairs mitotic progression showing the functional requirement for each site-specific phosphorylation of cdc25C at mitosis. CONCLUSIONS/SIGNIFICANCE: These results show for the first time that in human mitosis, distinct phospho-isoforms of cdc25C exist with different localizations and interacting partners, thus implying that the long-standing model of a cdc25C/cdk1 multi-site auto amplification loop is implausible.

Impaired muscle regeneration and myoblast differentiation in mice with a muscle-specific KO of IGF-IR.

IGF-I and its receptor IGF-IR are seen as critical effectors of muscle hypertrophy, a notion recently questioned. Using MKR transgenic mice that express a dominant negative IGF-IR only in skeletal muscle, we have examined the role of the IGF-IR signaling in differentiation and repair of muscle fibers after damage-induced muscle regeneration. This process is impaired in MKR muscle, with incomplete regeneration, persistence of infiltrating cells and sustained expression of differentiation markers. Analysis of MKR and WT muscle-derived progenitor stem cells and myoblasts showed twice as many such cells in MKR muscle and an incomplete in vitro differentiation, that is, despite similar levels of myogenin expression, the level of fusion of MKR myoblasts was significantly reduced in comparison to WT myoblasts. These data show IGF-IR signaling is not only required at early hyperplasia stages of muscle differentiation, but also for late stages of myofiber maturation and hypertrophy.

Akt2 is implicated in skeletal muscle differentiation and specifically binds Prohibitin2/REA.

Akt1 and Akt2 are the major isoforms of Akt expressed in muscle cells and muscle tissue. We have performed siRNA silencing of Akt1 and Akt2 in C2 myoblasts to characterize their specific implication in muscle differentiation. Whereas silencing Akt2, and not Akt1, inhibited cell cycle exit and myoblast differentiation, Akt2 overexpression led to an increased proportion of differentiated myoblasts. In addition, we demonstrate that Akt2 is required for myogenic conversion induced by MyoD overexpression in fibroblasts. We show Akt2, but not Akt1, binds Prohibitin2/Repressor of Estrogen Activator, PHB2/REA, a protein recently implicated in transcriptionnal repression of myogenesis. Co-immunoprecipitation experiments on endogenous proteins showed the Akt2-REA complex does not contain Prohibitin1. We have analyzed expression and localization of PHB2/REA during proliferation and differentiation of mouse and human myoblasts. PHB2/REA shows punctated nuclear staining which partially co-localizes with Akt2 in differentiated myotubes and PHB2 levels decrease at the onset of myogenic differentiation concomitant with an increase in Akt2. There appears to be an inverse correlation between Akt2 and PHB2 protein levels where cells silenced for Akt2 expression show increased level of PHB2/REA and overexpression of Akt2 resulted in decreased Prohibitin2/REA. Taken together, these results, along with our previous observations, clearly show that Akt2 and not Akt1 plays a major and early role in cell cycle exit and myogenic differentiation and this function involves its specific interaction with PHB2/REA.

Only Akt1 is required for proliferation, while Akt2 promotes cell cycle exit through p21 binding.

Protein kinase B (PKB/Akt) is an important modulator of insulin signaling, cell proliferation, and survival. Using small interfering RNA duplexes in nontransformed mammalian cells, we show that only Akt1 is essential for cell proliferation, while Akt2 promotes cell cycle exit. Silencing Akt1 resulted in decreased cyclin A levels and inhibition of S-phase entry, effects not seen with Akt2 knockdown and specifically rescued by microinjection of Akt1, not Akt2. In differentiating myoblasts, Akt2 knockout prevented myoblasts from exiting the cell cycle and showed sustained cyclin A expression. In contrast, overexpression of Akt2 reduced cyclin A and hindered cell cycle progression in M-G1 with increased nuclear p21. p21 is a major target in the differential effects of Akt isoforms, with endogenous Akt2 and not Akt1 binding p21 in the nucleus and increasing its level. Accordingly, Akt2 knockdown cells, and not Akt1 knockdown cells, showed reduced levels of p21. A specific Akt2/p21 interaction can be reproduced in vitro, and the Akt2 binding site on p21 is similar to that in cyclin A spanning T145 to T155, since (i) prior incubation with cyclin A prevents Akt2 binding, (ii) T145 phosphorylation on p21 by Akt1 prevents Akt2 binding, and (iii) binding Akt2 prevents phosphorylation of p21 by Akt1. These data show that specific interaction of the Akt2 isoform with p21 is key to its negative effect on normal cell cycle progression.

Muscle-specific overexpression of CD36 reverses the insulin resistance and diabetes of MKR mice.

Insulin resistance is one of the primary characteristics of type 2 diabetes. Mice overexpressing a dominant-negative IGF-I receptor specifically in muscle (MKR mice) demonstrate severe insulin resistance with high levels of serum and tissue lipids and eventually develop type 2 diabetes at 5-6 wk of age. To determine whether lipotoxicity plays a role in the progression of the disease, we crossed MKR mice with mice overexpressing a fatty acid translocase, CD36, in skeletal muscle. The double-transgenic MKR/CD36 mice showed normalization of the hyperglycemia and the hyperinsulinemia as well as a marked improvement in liver insulin sensitivity. The MKR/CD36 mice also exhibited normal rates of fatty acid oxidation in skeletal muscle when compared with the decreased rate of fatty acid oxidation in MKR. With the reduction in insulin resistance, beta-cell function returned to normal. These and other results suggest that the insulin resistance in the MKR mice is associated with increased muscle triglycerides levels and that whole-body insulin resistance can be, at least partially, reversed in association with a reduction in muscle triglycerides levels, although the mechanisms are yet to be determined.

Protection against hypoxic-ischemic injury in transgenic mice overexpressing Kir6.2 channel pore in forebrain.

The role of the K-ATP channel pore-forming subunit Kir6.2 on protection from cerebral hypoxic-ischemic injury was assessed in transgenic mice overexpressing normal Kir6.2 or a dominant negative form (AFA) of this subunit in the forebrain. The resulting mice overexpress either the Kir6.2 or the AFA transgene mainly in the cerebral cortex and hippocampus. The Kir6.2 transgenic mice are resistant to hypoxic-ischemic injury showing a decreased region of cortical damage as compared to the dominant negative AFA and the wild-type mice. Moreover, the overexpression of Kir6.2 allowed an important silencing of the neurons present in forebrain regions thus protecting them from ischemic injury. Interestingly, the phenotype observed in Kir6.2 transgenic mice was observed without increased sulfonylurea binding. Taken together, these results indicate that the transgenic overexpression of Kir6.2 in forebrain significantly protects mice from hypoxic-ischemic injury and neuronal damage seen in stroke.

Peroxisome proliferator-activated receptor-alpha agonist treatment in a transgenic model of type 2 diabetes reverses the lipotoxic state and improves glucose homeostasis.

Abnormalities in insulin action are the characteristics of type 2 diabetes. Dominant-negative muscle-specific IGF-I receptor (MKR) mice exhibit elevated lipid levels at an early age and eventually develop type 2 diabetes. To evaluate the role of elevated lipids in the progression of the diabetic state, MKR mice were treated with WY14,643, a peroxisome proliferator-activated receptor (PPAR)-alpha agonist. WY14,643 treatment markedly reduced serum fatty acid and triglyceride levels within a few days, as well as muscle triglyceride levels, and subsequently normalized glucose and insulin levels in MKR mice. Hyperinsulinemic-euglycemic clamp analysis showed that WY14,643 treatment enhanced muscle and adipose tissue glucose uptake by improving whole-body insulin sensitivity. Insulin suppression of endogenous glucose production by the liver of MKR mice was also improved. The expression of genes involved in fatty acid oxidation was increased in liver and skeletal muscle, whereas gene expression levels of hepatic gluconeogenic enzymes were decreased in WY14,643-treated MKR mice. WY14,643 treatment also improved the pattern of glucose-stimulated insulin secretion from the perfused pancreata of MKR mice and reduced the beta-cell mass. Taken together, these findings suggest that the reduction in circulating or intracellular lipids by activation of PPAR-alpha improved insulin sensitivity and the diabetic condition of MKR mice.

Insulin-like growth factor I induces MDM2-dependent degradation of p53 via the p38 MAPK pathway in response to DNA damage.

In many tissues, the insulin-like growth factor I (IGF-I) receptor (IGF-IR) is known to functionally oppose apoptosis. Recently, we demonstrated a direct role for the IGF-IR in the rescue of DNA-damaged fibroblasts by activating a DNA repair pathway (Héron-Milhavet, L., Karas, M., Goldsmith, C. M., Baum, B. J., and LeRoith, D. (2001) J. Biol. Chem. 276, 18185-18192). p53 is a nuclear transcription factor that can block progression of the cell cycle, modulate DNA repair, and trigger apoptosis. In this work, we tested the effect of IGF-I on the regulation of the p53 signaling cascade. The DNA-damaging agent 4-nitroquinoline 1-oxide was applied to NIH-3T3 cells overexpressing normal IGF-IRs (NWTb3 cells). We showed that after 4-nitroquinoline 1-oxide-induced DNA damage, IGF-I induced exclusion of the p53 protein from the nucleus and led to its degradation in the cytoplasm, whereas p53 mRNA was unaffected. Degradation of the p53 protein was associated with an increase in MDM2, an upstream modulator of the half-life and activity of the p53 protein. p53 degradation was also associated with down-regulation of p21. We further showed that the effects of IGF-I on mdm2 transcription and on MDM2/p19 ARF association were mediated by the p38 MAPK pathway. In conclusion, we describe a novel role for IGF-I in the regulation of the MDM2/p53/p21 signaling pathway during DNA damage.