VPS34 regulates TSC1/TSC2 heterodimer to mediate RheB and mTORC1/S6K1 activation and cellular transformation.

VPS34 is reported to activate S6K1 and is implicated in regulating cell growth, the mechanisms of which remain elusive. Here, we describe novel mechanisms by which VPS34 upregulates mTOR/S6K1 activity via downregulating TSC2 protein and activating RheB activity. Specifically, upregulation of VPS34 lipid kinase increases local production of ptdins(3)p in the plasma membrane, which recruits PIKFYVE, a FYVE domain containing protein, to ptdins(3)p enriched regions of the plasma membrane, where VPS34 forms a protein complex with PIKFYVE and TSC1. This in turn disengages TSC2 from the TSC1/TSC2 heterodimer, leading to TSC2 ubiquitination and degradation. Downregulation of TSC2 promotes the activation of RheB and mTOR/S6K1. When VPS34 lipid kinase activity is increased by introduction of an H868R mutation, ptdins(3)p production at the plasma membrane is dramatically increased, which recruits more PIKFYVE and TSC1 molecules to the plasma membrane. This results in the enhanced TSC2 ubiquitination and degradation, and subsequent activation of RheB and mTORC1/S6K1, leading to oncogenic transformation. The role played by VPS34 in regulating mTOR/S6K1 activity and cellular transformation is underscored by the fact that the VPS34 kinase dead mutant blocks VPS34-induced recruitment of PIKFYVE and TSC1 to the plasma membrane. This study provides mechanistic insight into the cellular function of VPS34 in regulating oncogenic transformation and important indications for identifying VPS34 specific mutations in human cancers.

Trastuzumab-induced recruitment of Csk-homologous kinase (CHK) to ErbB2 receptor is associated with ErbB2-Y1248 phosphorylation and ErbB2 degradation to mediate cell growth inhibition.

The inhibitory effect of trastuzumab, a humanized monoclonal antibody directed against the extracellular domain of ErbB2, is associated with its ability to induce ErbB2-Y1248 phosphorylation, and the status of phosphorylated ErbB2-Y1248 (ErbB2-pY1248) may correlate with the sensitivity of breast cancers to trastuzumab. The mechanisms of which remain unclear. Here, we show that binding of trastuzumab to ErbB2 activates ErbB2 kinase activity and enhances ErbB2-Y1248 phosphorylation in trastuzumab-sensitive breast cancer cells. This in turn increases the interaction between ErbB2 and non-receptor Csk-homologous kinase (CHK), leading to growth inhibition of breast cancer cells. Overexpression of CHK mimics trastuzumab treatment to mediate ErbB2-Y1248 phosphorylation, Akt downregulation, and growth inhibition of trastuzumab-sensitive breast cancer cells. CHK overexpression combined with trastuzumab exerts an additive effect on cell growth inhibition. We further demonstrate that positive ErbB2-pY1248 staining in ErbB2-positive breast cancer biopsies correlates with the increased trastuzumab response in trastuzumab neoadjuvant settings. Collectively, this study highlights an important role for ErbB2-pY1248 in mediating trastuzumab-induced growth inhibition and trastuzumab-induced interactions between CHK and ErbB2-pY1248 is identified as a novel mechanism of action that mediates the growth inhibition of breast cancer cells. The novel mechanistic insights into trastuzumab action revealed by this study may impact the design of next generation of therapeutic monoclonal antibodies targeting receptor tyrosine kinases, as well as open new avenues to identify novel targets for the treatment of ErbB2-positive cancers.

Insulin activation of vacuolar protein sorting 34 mediates localized phosphatidylinositol 3-phosphate production at lamellipodia and activation of mTOR/S6K1.

The class III phosphatidylinositol 3-kinase, VPS34, phosphorylates the D3 hydroxyl of inositol generating phosphatidylinositol 3-phosphate (ptdins(3)p). Initial studies suggested that ptdins(3)p solely functioned as a component of vesicular and endosomal membranes and that VPS34 did not function in signal transduction. However, VPS34 has recently been shown to be required for insulin-mediated activation of S6 kinase 1 (S6K1). Whether VPS34 activity is directly regulated by insulin is unclear. It is also not known whether VPS34 activity can be spatially restricted in response to extracellular stimuli. Data presented here demonstrate that in response to insulin, VPS34 is activated and translocated to lamellipodia where it produces ptdins(3)p. The localized production of ptdins(3)p is dependent on Src phosphorylation of VPS34. In cells expressing VPS34 with mutations at Y231 or Y310, which are Src-phosphorylation sites, insulin-stimulated VPS34 translocation to the plasma membrane and lamellipodia formation are blocked. mTOR also colocalizes with VPS34 and ptdins(3)p at lamellipodia following insulin-stimulation. In cells expressing the VPS34-Y231F mutant, which blocks lamellipodia formation, mTOR localization at the plasma membrane and insulin-mediated S6K1 activation are reduced. This suggests that mTOR localization at lamellipodia is important for full activation of S6K1 induced by insulin. These data demonstrate that insulin can spatially regulate VPS34 activity through Src-mediated tyrosine phosphorylation and that this membrane localized activity contributes to lamellipodia formation and activation of mTOR/S6K1signaling.

Trastuzumab alters the expression of genes essential for cardiac function and induces ultrastructural changes of cardiomyocytes in mice.

Treatment with trastuzumab, a humanized monoclonal antibody directed against the extracellular domain of Human Epidermal Growth Factor Receptor 2 (HER2), very successfully improves outcomes for women with HER2-positive breast cancer. However, trastuzumab treatment was recently linked to potentially irreversible serious cardiotoxicity, the mechanisms of which are largely elusive. This study reports that trastuzumab significantly alters the expression of myocardial genes essential for DNA repair, cardiac and mitochondrial functions, which is associated with impaired left ventricular performance in mice coupled with significant ultrastructural alterations in cardiomyocytes revealed by electron microscopy. Furthermore, trastuzumab treatment also promotes oxidative stress and apoptosis in myocardium of mice, and elevates serum levels of cardiac troponin-I (cTnI) and cardiac myosin light chain-1 (cMLC1). The elevated serum levels of cMLC1 in mice treated with trastuzumab highlights the potential that cMLC1 could be a useful biomarker for trastuzumab-induced cardiotoxicity.

Small GTPase Rho regulates R-cadherin through Dia1/profilin-1.

R-cadherin is a member of the classical cadherins. Though much is known about E-cadherin in adherens junction formation in epithelial cells, the role of R-cadherin in epithelial cells remains elusive. This study examines regulation of R-cadherin adherens junctions by the small GTPase Rho and its downstream effectors in MDA-MB-231 breast cancer cells, MDA-MB-231 cells stably expressing the N-terminus of c-Cbl, and MCF10A normal breast epithelial cells. We find that the small GTPase Rho regulates R-cadherin adherens junction formation via Dia1 (also known as p140mDia) and profilin-1-mediated signaling pathway. The role played by Rho in regulating R-cadherin is underscored by the fact that constitutively active RhoA(Q63L) induces R-cadherin junction formation in MDA-MB-231 cells. Importantly, R-cadherin adherens junction formation facilitates a mesenchymal to epithelial-like transition in MDA-MB-231 cells. Additionally, our data suggest an inverse relationship between EGFR signaling and R-cadherin adherens junction formation. Taken together, results from this study indicate that R-cadherin is a critical regulator of epithelial phenotype.

Trastuzumab regulates IGFBP-2 and IGFBP-3 to mediate growth inhibition: implications for the development of predictive biomarkers for trastuzumab resistance.

Activation of insulin-like growth factor-I receptor (IGF-IR) signaling is an important mechanism for trastuzumab resistance. IGF-binding proteins (IGFBP) modulate IGF-IR signaling and play important roles in the control of breast cancer progression. In this article, we report that trastuzumab treatment enhances the expression and secretion of IGFBP-3 in SKBR3 cells, a trastuzumab-sensitive breast cancer cell line, and that this upregulation of IGFBP-3 induced by trastuzumab correlates with trastuzumab-mediated growth inhibition. We describe a new role for IGFBP-3 in the regulation of IGF-I-mediated cross-talk between IGF-IR and ErbB2 signaling pathways. In particular, treatment of SKBR3 cells with recombinant IGFBP-3 blocks IGF-I-induced activation of IGF-IR and ErbB2, and stable expression of IGFBP-3 inhibits SKBR3 cell growth. We find an inverse relationship in the levels of secreted IGFBP-3 such that high levels of IGFBP-3 are associated with trastuzumab-sensitive breast cancer cells (SKBR3 and BT-474), whereas low levels of IGFBP-3 are found in trastuzumab-resistant cells (clone 3 and JIMT-1). In contrast to IGFBP-3, the secretion and expression of IGFBP-2 are upregulated in trastuzumab-resistant SKBR3 cells. Furthermore, we show that IGFBP-2 stimulates activation of ErbB2 and that trastuzumab reduces IGFBP-2-stimulated ErbB2 activation. Based on our data, we propose a novel mechanism of action whereby trastuzumab enhances the expression and secretion of IGFBP-3, which interferes with IGF-I-mediated mitogenic signaling via autocrine and paracrine mechanisms and reduces IGFBP-2-induced ErbB2 activation to mediate growth inhibition. Changes in secretion profiles of IGFBP-2 and IGFBP-3 in trastuzumab-sensitive and trastuzumab-resistant cells may promote the development of IGFBP-2 and IGFBP-3 as predictive biomarkers for trastuzumab resistance.

pp60c-Src phosphorylates and activates vacuolar protein sorting 34 to mediate cellular transformation.

Vacuolar protein sorting 34 (VPS34) contributes to the regulation of the mammalian target of rapamycin complex 1/S6 kinase 1 pathway downstream of nutrient signaling. However, intracellular mechanisms leading to VPS34 activation remain unclear. Here, we report that Src directly phosphorylates VPS34, and that this phosphorylation activates VPS34 lipid kinase activity, leading to Src-Y527F-mediated cellular transformation. Silencing endogenous VPS34 specifically inhibits Src-Y527F-induced colony formation in soft agar, but not Ras-G12V-induced colony formation. We have identified two novel hVPS34 mutations, which either eliminate lipid kinase activity (kinase-dead mutant) or reduce tyrosine phosphorylation by Src-Y527F. When kinase-dead mutant of hVPS34 is stably expressed in Src-Y527F-transformed cells, transformation activities are blocked, indicating that the lipid kinase activity of hVPS34 is essential for Src-mediated cellular transformation. Furthermore, stable expression of this hVPS34 kinase-dead mutant causes an increased number of binucleate and multinucleate cells, suggesting that the kinase activity of hVPS34 is also required for cytokinesis. Moreover, when the hVPS34 mutant that has reduced tyrosine phosphorylation by Src is stably expressed in Src-Y527F-transformed cells, Src-Y527F-stimulated colony formation is also reduced. Data presented here provide important evidence that VPS34 lipid kinase activity could be positively regulated by Src-mediated tyrosine phosphorylation in mammalian cells. This finding highlights a previously unappreciated relationship between VPS34, a class III phosphatidylinositol-3-kinase, and Src non-receptor tyrosine kinase. Additionally, we find that the levels of VPS34 expression and tyrosine phosphorylation are correlated with the tumorigenic activity of human breast cancer cells, indicating that Src to VPS34 signaling warrants further investigation as a pathway contributing to the development and progression of human cancers.

Rac1 contributes to trastuzumab resistance of breast cancer cells: Rac1 as a potential therapeutic target for the treatment of trastuzumab-resistant breast cancer.

Although treatment with trastuzumab improves outcomes for women with ErbB2-positive breast cancer, many patients who achieve an initial response to trastuzumab subsequently acquire resistance within 1 year. Rac1, a Ras-like small GTPase, has been implicated in the control of cell growth and morphology and is believed to be associated with breast cancer progression and metastasis. Here, we show that when parental SKBR3 cells become resistant to trastuzumab, Rac1 activity is increased, leading to altered cell morphology, which is accompanied by significant cytoskeleton disorganization. Furthermore, both trastuzumab-mediated down-regulation of ErbB2 and epidermal growth factor-induced down-regulation of epidermal growth factor receptor are impaired in the trastuzumab-resistant SKBR3 cells, indicating that the endocytic down-regulation of ErbB receptors is compromised in the resistant cells. This results in an aberrant accumulation of ErbB2 on the cell surface and enhanced ErbB2 and extracellular signal-regulated kinase activity in trastuzumab-resistant SKBR3 cells. Additionally, overexpression of constitutively active Rac1G12V in parental SKBR3 cells reduces sensitivity to trastuzumab. After reduction of Rac1 activity by NSC23766, a specific Rac1 inhibitor, trastuzumab-resistant SKBR3 cells display a cellular morphology similar to parental SKBR3 cells. Moreover, we show that NSC23766 restores trastuzumab-mediated endocytic down-regulation of ErbB2 and reduces extracellular signal-regulated kinase activity in resistant SKBR3 cells. Our findings highlight an important role for Rac1 in trastuzumab resistance of human breast cancer cells and identify the impaired trastuzumab-mediated endocytic down-regulation of ErbB2 as a novel mechanism of trastuzumab resistance. The significant effects of NSC23766 on trastuzumab-resistant SKBR3 cells warrant further study of NSC23766 as a potential treatment of trastuzumab-resistant breast cancers.

Cdc42 regulates E-cadherin ubiquitination and degradation through an epidermal growth factor receptor to Src-mediated pathway.

E-cadherins play an essential role in maintaining epithelial polarity by forming Ca2+-dependent adherens junctions between epithelial cells. Here, we report that Ca2+ depletion induces E-cadherin ubiquitination and lysosomal degradation and that Cdc42 plays an important role in regulating this process. We demonstrate that Ca2+ depletion induces activation of Cdc42. This in turn up-regulates epidermal growth factor receptor (EGFR) signaling to mediate Src activation, leading to E-cadherin ubiquitination and lysosomal degradation. Silencing Cdc42 blocks activation of EGFR and Src induced by Ca2+ depletion, resulting in a reduction in E-cadherin degradation. The role of Cdc42 in regulating E-cadherin ubiquitination and degradation is underscored by the fact that constitutively active Cdc42(F28L) increases the activity of EGFR and Src and significantly enhances E-cadherin ubiquitination and lysosomal degradation. Furthermore, we found that GTP-dependent binding of Cdc42 to E-cadherin is critical for Cdc42 to induce the dissolution of adherens junctions. Our data support a model that activation of Cdc42 contributes to mesenchyme-like phenotype by targeting of E-cadherin for lysosomal degradation.

Src-dependent phosphorylation of ASAP1 regulates podosomes.

Invadopodia are Src-induced cellular structures that are thought to mediate tumor invasion. ASAP1, an Arf GTPase-activating protein (GAP) containing Src homology 3 (SH3) and Bin, amphiphysin, and RVS161/167 (BAR) domains, is a substrate of Src that controls invadopodia. We have examined the structural requirements for ASAP1-dependent formation of invadopodia and related structures in NIH 3T3 fibroblasts called podosomes. We found that both predominant splice variants of ASAP1 (ASAP1a and ASAP1b) associated with invadopodia and podosomes. Podosomes were highly dynamic, with rapid turnover of both ASAP1 and actin. Reduction of ASAP1 levels by small interfering RNA blocked formation of invadopodia and podosomes. Podosomes were formed in NIH 3T3 fibroblasts in which endogenous ASAP1 was replaced with either recombinant ASAP1a or ASAP1b. ASAP1 mutants that lacked the Src binding site or GAP activity functioned as well as wild-type ASAP1 in the formation of podosomes. Recombinant ASAP1 lacking the BAR domain, the SH3 domain, or the Src phosphorylation site did not support podosome formation. Based on these results, we conclude that ASAP1 is a critical target of tyrosine kinase signaling involved in the regulation of podosomes and invadopodia and speculate that ASAP1 may function as a coincidence detector of simultaneous protein association through the ASAP1 SH3 domain and phosphorylation by Src.

Cdc42: an effector and regulator of ErbB1 as a strategic target in breast cancer therapy.

ErbB1 and ErbB2 are often overexpressed in breast cancer. Overexpression of these receptors is correlated with poor prognosis. ErbB receptor-targeted therapies have been developed for the treatment of human breast cancer. While ErbB2 overexpression is usually caused by gene amplification, the mechanism for ErbB1 overexpression remains elusive. An important mechanism for the downregulation of ErbB1 is via Cbl-mediated receptor ubiquitination and degradation. Increasing evidence suggests that loss of Cbl-regulated ErbB1 degradation contributes to ErbB1 overexpression in cancer cells. Cdc42 is overexpressed in some breast cancers and evidence is accumulating that activated Cdc42 contributes to the accumulation of ErbB1 in cells through the regulation of c-Cbl function. Different therapeutic strategies targeting ErbB receptors and Cdc42 will be reviewed and discussed.

Growth and motility inhibition of breast cancer cells by epidermal growth factor receptor degradation is correlated with inactivation of Cdc42.

Overexpression of epidermal growth factor receptor (EGFR) contributes to increased cell proliferation and migration in breast cancer. However, mechanisms of EGFR overexpression remain elusive and often cannot be attributed to gene amplification. In NIH3T3 fibroblasts, active Cdc42 inhibits c-Cbl-regulated EGFR degradation to induce cellular transformation. Here, we use two EGFR-overexpressing breast cancer cell lines, MDA-MB-231 and BT20, as models to test the hypothesis that up-regulated Cdc42 activity impairs c-Cbl-mediated EGFR degradation and contributes to EGFR overexpression. We show that silencing Cdc42 significantly reduces protein levels of EGFR, leading to a marked reduction in cell proliferation and migration, and c-Cbl knockdown increases the levels of EGFR. Expression of c-Cbl-N480, a c-Cbl mutant that is not regulated by Cdc42 and blocks Cdc42-induced transformation but still binds and ubiquitinates EGFR, enhances the rate of EGFR degradation and subsequently inhibits cell proliferation. Moreover, down-regulated EGFR signaling induced by c-Cbl-N480 decreased activity of Cdc42 and Rac1, resulting in inhibition of cell migration. These findings indicate that Cdc42 and c-Cbl are critical components involved in the regulation of EGFR protein levels and that restoration of proper EGFR degradation by disrupting Cdc42 regulation of c-Cbl can reduce cell proliferation and migration in MDA-MB-231 and BT20 cells.

A BAR domain in the N terminus of the Arf GAP ASAP1 affects membrane structure and trafficking of epidermal growth factor receptor.

BACKGROUND: Arf GAPs are multidomain proteins that function in membrane traffic by inactivating the GTP binding protein Arf1. Numerous Arf GAPs contain a BAR domain, a protein structural element that contributes to membrane traffic by either inducing or sensing membrane curvature. We have examined the role of a putative BAR domain in the function of the Arf GAP ASAP1. RESULTS: ASAP1's N terminus, containing the putative BAR domain together with a PH domain, dimerized to form an extended structure that bound to large unilamellar vesicles containing acidic phospholipids, properties that define a BAR domain. A recombinant protein containing the BAR domain of ASAP1, together with the PH and Arf GAP domains, efficiently bent the surface of large unilamellar vesicles, resulting in the formation of tubular structures. This activity was regulated by Arf1*GTP binding to the Arf GAP domain. In vivo, the tubular structures induced by ASAP1 mutants contained epidermal growth factor receptor (EGFR) and Rab11, and ASAP1 colocalized in tubular structures with EGFR during recycling of receptor. Expression of ASAP1 accelerated EGFR trafficking and slowed cell spreading. An ASAP1 mutant lacking the BAR domain had no effect. CONCLUSIONS: The N-terminal BAR domain of ASAP1 mediates membrane bending and is necessary for ASAP1 function. The Arf dependence of the bending activity is consistent with ASAP1 functioning as an Arf effector.

Arf GAPs: multifunctional proteins that regulate membrane traffic and actin remodelling.

The ADP-ribosylation factor (Arf) Arf GTPase-activating proteins (GAPs) are a family of proteins that induce hydrolysis of GTP bound to Arf. A conserved domain containing a zinc finger motif mediates catalysis. The substrate, Arf.GTP, affects membrane trafficking and actin remodelling. Consistent with activity as an Arf regulator, the Arf GAPs affect both of these pathways. However, the Arf GAPs are likely to have Arf-independent activities that contribute to their cellular functions. Structures of the Arf GAPs are diverse containing catalytic, protein-protein interaction and lipid interaction domains in addition to the Arf GAP domain. Some Arf GAPs have been identified and characterized on the basis of activities other than Arf GAP. Here, we describe the Arf GAP family, enzymology of some members of the Arf GAP family and known functions of the proteins. The results discussed illustrate roles for both Arf-dependent and -independent activities in the regulation of cellular architecture.

Arf and its many interactors.

Arf GTP-binding proteins regulate membrane traffic and actin remodeling. Similar to other GTP-binding proteins, a complex of Arf-GTP with an effector protein mediates Arf function. Arf interacts with at least three qualitatively different types of effectors. First, it interacts with structural proteins, the vesicle coat proteins. The second type of effector is lipid-metabolizing enzymes, and the third comprises those proteins that bind to Arf-GTP but whose biochemical or biological functions are not yet clearly defined. Arf interacts with two other families of proteins, the exchange factors and the GTPase-activating proteins. Recent work examining the functional relationships among the diverse Arf interactors has led to reconsideration of the prevailing paradigms for Arf action.

Arf1 dissociates from the clathrin adaptor GGA prior to being inactivated by Arf GTPase-activating proteins.

The effectors of monomeric GTP-binding proteins can influence interactions with GTPase-activating proteins (GAPs) in two ways. In one case, effector and GAP binding to the GTP-binding protein is mutually exclusive. In another case, the GTP-binding protein bound to an effector is the substrate for the GTPase-activating protein. Here predictions for these two mechanisms were tested for the Arf1 effector GGA and ASAP family Arf GAPs. GGA inhibited Arf GAP activity of ASAP1, AGAP1, ARAP1, and Arf GAP1 and inhibited binding of Arf1.GTPgammaS to AGAP1 with K(i) values correlating with the K(d) for the GGA.Arf1 complex. ASAP1 blocked Arf1.GTPgammaS binding to GGA with a K(i) similar to the K(d) for the ASAP.Arf1.GTPgammaS complex. No interaction of GGA with ASAP1 was detected. Consistent with GGA sequestering Arf from GAPs, overexpression of GGA slowed the rate of Arf dissociation from the Golgi apparatus following treatment with brefeldin A. Mutational analysis revealed the amino-terminal alpha-helix and switch I of Arf1 contributed to interaction with both GGA and GAPs. These data exclude the mechanism previously documented for Arf GAP1/coatomer in which Arf1 is inactivated in a tripartite complex. Instead, termination of Arf1 signals mediated through GGA require that Arf1.GTP dissociates from GGA prior to interaction with GAP and consequent hydrolysis of GTP.

AGAP1, an endosome-associated, phosphoinositide-dependent ADP-ribosylation factor GTPase-activating protein that affects actin cytoskeleton.

We have identified three members of the AGAP subfamily of ASAP family ADP-ribosylation factor GTPase-activating proteins (Arf GAPs). In addition to the Arf GAP domain, these proteins contain GTP-binding protein-like, ankyrin repeat and pleckstrin homology domains. Here, we have characterized the ubiquitously expressed AGAP1/KIAA1099. AGAP1 had Arf GAP activity toward Arf1>Arf5>Arf6. Phosphatidylinositol 4,5-bisphosphate and phosphatidic acid synergistically stimulated GAP activity. As found for other ASAP family Arf GAPs, the pleckstrin homology domain was necessary for activity. Deletion of the GTP-binding protein-like domain affected lipid dependence of Arf GAP activity. In vivo effects of AGAP1 were distinct from other ASAP family Arf GAPs. Overexpressed AGAP1 induced the formation of and was associated with punctate structures containing the endocytic markers transferrin and Rab4. AP1 was redistributed from the trans-Golgi to the punctate structures. Like other ASAP family members, AGAP1 overexpression inhibited the formation of PDGF-induced ruffles. However, distinct from other ASAP family members, AGAP1 also induced the loss of actin stress fibers. Thus, AGAP1 is a phosphoinositide-dependent Arf GAP that impacts both the endocytic compartment and actin.