Coordinate Interactions of Csk, Src, and Syk Kinases with [alpha]IIb[beta]3 Initiate Integrin Signaling to the Cytoskeleton

The Journal of Cell Biology. Apr, 2002  |  Pubmed ID: 11940607

Integrins regulate cell adhesion and motility through tyrosine kinases, but initiation of this process is poorly understood. We find here that Src associates constitutively with integrin alphaIIbbeta3 in platelets. Platelet adhesion to fibrinogen caused a rapid increase in alphaIIbbeta3-associated Src activity, and active Src localized to filopodia and cell edges. Csk, which negatively regulates Src by phosphorylating Tyr-529, was also constitutively associated with alphaIIbbeta3. However, fibrinogen binding caused Csk to dissociate from alphaIIbbeta3, concomitant with dephosphorylation of Src Tyr-529 and phosphorylation of Src activation loop Tyr-418. In contrast to the behavior of Src and Csk, Syk was associated with alphaIIbbeta3 only after fibrinogen binding. Platelets multiply deficient in Src, Hck, Fgr, and Lyn, or normal platelets treated with Src kinase inhibitors failed to spread on fibrinogen. Inhibition of Src kinases blocked Syk activation and inhibited phosphorylation of Syk substrates (Vav1, Vav3, SLP-76) implicated in cytoskeletal regulation. Syk-deficient platelets exhibited Src activation upon adhesion to fibrinogen, but no spreading or phosphorylation of Vav1, Vav3, and SLP-76. These studies establish that platelet spreading on fibrinogen requires sequential activation of Src and Syk in proximity to alphaIIbbeta3, thus providing a paradigm for initiation of integrin signaling to the actin cytoskeleton.

Sensing the Environment: a Historical Perspective on Integrin Signal Transduction

Nature Cell Biology. Apr, 2002  |  Pubmed ID: 11944041

Cell adhesion mediated by integrin receptors has a critical function in organizing cells in tissues and in guiding haematopoietic cells to their sites of action. However, integrin adhesion receptors have broader functions in regulating cell behaviour through their ability to transduce bi-directional signals into and out of the cell and to engage in reciprocal interactions with other cellular receptors. This historical perspective traces the key findings that have led to our current understanding of these important functions of integrins.

Direct Phosphorylation of Focal Adhesion Kinase by C-Src: Evidence Using a Modified Nucleotide Pocket Kinase and ATP Analog

Biochemical and Biophysical Research Communications. Jun, 2002  |  Pubmed ID: 12051709

A single mutation in the nucleotide binding pocket of select protein kinases allows for use of a bulky, substituted-ATP analog not used by the wild-type kinase [1]. Using this approach with the protein tyrosine kinase c-Src, we have generated a mutant T338G and expressed it in Src/Yes/Fyn null fibroblasts (SYF1) at near endogenous levels. T338G Src exhibits high specificity for a substituted ATP analog N(6)-2-phenyl ethyl ATP (peATP), which is not used by wild-type c-Src in autophosphorylation nor substrate phosphorylation assays. By employing the T338G Src mutant and [gamma-(32)P]peATP analog, we demonstrate that c-Src can directly phosphorylate focal adhesion kinase (Fak) in vitro. We also show that incubation of permeabilized, T338 Src-expressing cells with peATP causes an increase in Fak tyrosine phosphorylation not observed in wild-type Src cells. Taken together, these data provide evidence that Src directly phosphorylates Fak and demonstrates the limitations of using this modified ATP strategy for analysis of direct substrates of protein kinases in permeabilized cells.

The Role of Apoptosis in Creating and Maintaining Luminal Space Within Normal and Oncogene-expressing Mammary Acini

Cell. Oct, 2002  |  Pubmed ID: 12372298

We have utilized in vitro three-dimensional epithelial cell cultures to analyze the role of apoptosis in the formation and maintenance of a hollow glandular architecture. Lumen formation is associated with the selective apoptosis of centrally located cells; this apoptosis follows apicobasal polarization and precedes proliferative suppression during acinar development. Notably, either inhibiting apoptosis (by exogenously expressing antiapoptotic Bcl family proteins) or enhancing proliferation (via Cyclin D1 or HPV E7 overexpression) does not result in luminal filling, suggesting glandular architecture is resistant to such isolated oncogenic insults. However, the lumen is filled when oncogenes that enhance proliferation are coexpressed with those that inhibit apoptosis, or when ErbB2, which induces both activities, is activated by homodimerization. Hence, apoptosis can counteract increased proliferation to maintain luminal space, suggesting that tumor cells must restrain apoptosis to populate the lumen.

Overview of the Alliance for Cellular Signaling

Nature. Dec, 2002  |  Pubmed ID: 12478301

The Alliance for Cellular Signaling is a large-scale collaboration designed to answer global questions about signalling networks. Pathways will be studied intensively in two cells--B lymphocytes (the cells of the immune system) and cardiac myocytes--to facilitate quantitative modelling. One goal is to catalyse complementary research in individual laboratories; to facilitate this, all alliance data are freely available for use by the entire research community.

Morphogenesis and Oncogenesis of MCF-10A Mammary Epithelial Acini Grown in Three-dimensional Basement Membrane Cultures

Methods (San Diego, Calif.). Jul, 2003  |  Pubmed ID: 12798140

The three-dimensional culture of MCF-10A mammary epithelial cells on a reconstituted basement membrane results in formation of polarized, growth-arrested acini-like spheroids that recapitulate several aspects of glandular architecture in vivo. Oncogenes introduced into MCF-10A cells disrupt this morphogenetic process, and elicit distinct morphological phenotypes. Recent studies analyzing the mechanistic basis for phenotypic heterogeneity observed among different oncogenes (e.g., ErbB2, cyclin D1) have illustrated the utility of this three-dimensional culture system in modeling the biological activities of cancer genes, particularly with regard to their ability to disrupt epithelial architecture during the early aspects of carcinoma formation. Here we provide a collection of protocols to culture MCF-10A cells, to establish stable pools expressing a gene of interest via retroviral infection, as well as to grow and analyze MCF-10A cells in three-dimensional basement membrane culture.

Integrins and EGFR Coordinately Regulate the Pro-apoptotic Protein Bim to Prevent Anoikis

Nature Cell Biology. Aug, 2003  |  Pubmed ID: 12844146

Epithelial cells must adhere to the extracellular matrix (ECM) for survival, as detachment from matrix triggers apoptosis or anoikis. Integrins are major mediators of adhesion between cells and ECM proteins, and transduce signals required for cell survival. Recent evidence suggests that integrin receptors are coupled to growth factor receptors in the regulation of multiple biological functions; however, mechanisms involved in coordinate regulation of cell survival are poorly understood and mediators responsible for anoikis have not been well characterized. Here, we identify the pro-apoptotic protein Bim as a critical mediator of anoikis in epithelial cells. Bim is strongly induced after cell detachment and downregulation of Bim expression by RNA interference (RNAi) inhibits anoikis. Detachment-induced expression of Bim requires a lack of beta(1)-integrin engagement, downregulation of EGF receptor (EGFR) expression and inhibition of Erk signalling. Overexpressed EGFR was uncoupled from integrin regulation, resulting in the maintenance of Erk activation in suspension, and a block in Bim expression and anoikis. Thus, Bim functions as a key sensor of integrin and growth factor signals to the Erk pathway, and loss of such coordinate regulation may contribute to tumour progression.

The PHD Finger of the Chromatin-associated Protein ING2 Functions As a Nuclear Phosphoinositide Receptor

Cell. Jul, 2003  |  Pubmed ID: 12859901

Phosphoinositides (PtdInsPs) play critical roles in cytoplasmic signal transduction pathways. However, their functions in the nucleus are unclear, as specific nuclear receptors for PtdInsPs have not been identified. Here, we show that ING2, a candidate tumor suppressor protein, is a nuclear PtdInsP receptor. ING2 contains a plant homeodomain (PHD) finger, a motif common to many chromatin-regulatory proteins. We find that the PHD fingers of ING2 and other diverse nuclear proteins bind in vitro to PtdInsPs, including the rare PtdInsP species, phosphatidylinositol 5-phosphate (PtdIns(5)P). Further, we demonstrate that the ING2 PHD finger interacts with PtdIns(5)P in vivo and provide evidence that this interaction regulates the ability of ING2 to activate p53 and p53-dependent apoptotic pathways. Together, our data identify the PHD finger as a phosphoinositide binding module and a nuclear PtdInsP receptor, and suggest that PHD-phosphoinositide interactions directly regulate nuclear responses to DNA damage.

Akt Activation Disrupts Mammary Acinar Architecture and Enhances Proliferation in an MTOR-dependent Manner

The Journal of Cell Biology. Oct, 2003  |  Pubmed ID: 14568991

Activation of the serine/threonine kinase Akt/PKB positively impacts on three cellular processes relevant to tumor progression: proliferation, survival, and cell size/growth. Using a three-dimensional culture model of MCF-10A mammary cells, we have examined how Akt influences the morphogenesis of polarized epithelial structures. Activation of a conditionally active variant of Akt elicits large, misshapen structures, which primarily arise from the combined effects of Akt on proliferation and cell size. Importantly, Akt activation amplifies proliferation during the early stages of morphogenesis, but cannot overcome signals suppressing proliferation in late-stage cultures. Akt also cooperates with oncoproteins such as cyclin D1 or HPV E7 to promote proliferation and morphogenesis in the absence of growth factors. Pharmacological inhibition of the Akt effector, mammalian target of rapamycin (mTOR), with rapamycin prevents the morphological disruption elicited by Akt activation, including its effect on cell size and number, and the cooperative effect of Akt on oncogene-driven proliferation, indicating that mTOR function is required for the multiple biological effects of Akt activation during morphogenesis.

Src Kinase Activation by Direct Interaction with the Integrin Beta Cytoplasmic Domain

Proceedings of the National Academy of Sciences of the United States of America. Nov, 2003  |  Pubmed ID: 14593208

Src tyrosine kinases transmit integrin-dependent signals pivotal for cell movement and proliferation. Here, we establish a mechanism for Src activation by integrins. c-Src is shown to bind constitutively and selectively to beta3 integrins through an interaction involving the c-Src SH3 domain and the carboxyl-terminal region of the beta3 cytoplasmic tail. Clustering of beta3 integrins in vivo activates c-Src and induces phosphorylation of Tyr-418 in the c-Src activation loop, a reaction essential for adhesion-dependent phosphorylation of Syk, a c-Src substrate. Unlike c-Src, Hck, Lyn, and c-Yes bind more generally to beta1A, beta2, and beta3 cytoplasmic tails. These results invoke a model whereby Src is primed for activation by direct interaction with an integrin beta tail, and integrin clustering stabilizes activated Src by inducing intermolecular autophosphorylation. The data provide a paradigm for integrin regulation of Src and a molecular basis for the similar functional defects of osteoclasts or platelets from mice lacking beta3 integrins or c-Src.

Cooperation of the ErbB2 Receptor and Transforming Growth Factor Beta in Induction of Migration and Invasion in Mammary Epithelial Cells

Proceedings of the National Academy of Sciences of the United States of America. Feb, 2004  |  Pubmed ID: 14739340

MCF10A mammary epithelial cells form growth-arrested structures when cultured in three-dimensional basement membrane gels. Activation of the receptor tyrosine kinase ErbB2 induces formation of proliferative structures that share properties with noninvasive early stage lesions. We conducted a genetic screen to identify cDNAs that can cooperate with ErbB2 to induce migration in these cells, with the hypothesis that they would represent candidate "second hits" in the development of invasive breast carcinomas. We found that expression of transforming growth factor (TGF)beta1 and TGFbeta3 in cells expressing activated ErbB2 induces migration in transwell chambers and invasive behavior in both basement membrane cultures and invasion chambers. The ability of ErbB2 to cooperate with TGFbeta correlated with sustained, elevated activation of extracellular signal-regulated kinase (Erk)-mitogen-activated protein kinase. Pharmacological reduction of Erk activity inhibited the cooperative effect of TGFbeta and ErbB2 on migration and expression of activated Erk kinase was sufficient to cooperate with TGFbeta to induce migration and invasion, suggesting that sustained Erk activation is critical for ErbB2/TGFbeta cooperation. In addition, we show that costimulation of ErbB2 and TGFbeta induces autocrine secretion of factors that are sufficient to induce migration, but not invasion, by means of both epidermal growth factor receptor-dependent and -independent processes. These results support the role of TGFbeta as a pro-invasion factor in the progression of breast cancers with activated ErbB2 and suggest that activation of the Erk and epidermal growth factor receptor pathways are key in mediating these events.

Tumor Necrosis Factor-related Apoptosis-inducing Ligand (TRAIL) is Required for Induction of Autophagy During Lumen Formation in Vitro

Proceedings of the National Academy of Sciences of the United States of America. Mar, 2004  |  Pubmed ID: 14993595

The molecular events regulating the elimination of cells to create a hollow lumen during tissue development are poorly understood. By using an in vitro morphogenesis model in which MCF-10A human mammary epithelial cells form hollow acini-like structures, we have observed both caspase-mediated apoptosis and autophagy associated with cells that are lost during lumen formation. Here, we show that the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) mediates induction of autophagic processes associated with lumen formation. TRAIL is up-regulated during morphogenesis of MCF-10A mammary epithelial cells in 3D basement-membrane cultures and inhibition of TRAIL signaling during morphogenesis blocks the formation of autophagic vacuoles. In addition, treatment with exogenous TRAIL induces extensive autophagy in monolayer and 3D cultures. When combined with inhibition of caspase 3 activity (by Bcl-X(L) overexpression), inhibition of TRAIL-induced autophagy results in luminal filling. Thus, TRAIL regulates an autophagic program during acinar morphogenesis, which together with caspase-mediated apoptotic events, results in lumen formation during MCF-10A morphogenesis.

A Cytoskeleton-based Functional Genetic Screen Identifies Bcl-xL As an Enhancer of Metastasis, but Not Primary Tumor Growth

Oncogene. Jun, 2004  |  Pubmed ID: 15064711

Many mouse models of breast cancer form large primary tumors that rarely metastasize. Models with aggressive metastasis express oncoproteins that simultaneously affect growth and apoptosis pathways. To define the role of apoptotic resistance and to model a challenge faced by tumor cells during metastatic dissemination, we focused on apoptosis induced by cell shape change. Inhibiting actin polymerization with Latrunculin-A causes cell rounding and death within hours in nontumorigenic human 10A-Ras mammary epithelial cells. In contrast, MDA-MB-231 metastatic breast tumor cells resist LA-induced death, and survive for days despite cell rounding. Infecting 10A-Ras cells with a MDA-MB-231 retroviral expression library, and selecting with Latrunculin-A repeatedly identified Bcl-xL as a suppressor of cytoskeleton-dependent death. Although Bcl-xL enhances the spread of metastatic breast tumor cell lines, the distinct effects of apoptotic resistance on tumor growth in the mammary gland and during metastasis have not been compared directly. We find that Bcl-xL overexpression in mouse mammary epithelial cells does not induce primary tumor formation or enhance MEK-induced tumorigenesis within the mammary gland environment. However, it strongly enhances metastatic potential. These results with Bcl-xL provide novel evidence that isolated apoptotic resistance can increase metastatic potential, but remain overlooked by assays based on breast tumor growth.

ErbB2 and TGF-beta: a Cooperative Role in Mammary Tumor Progression?

Cell Cycle (Georgetown, Tex.). May, 2004  |  Pubmed ID: 15107620

Amplification and overexpression of ErbB2 (HER2/Neu) is one of the most common alterations associated with breast cancer. Activation of ErbB2 via homodimerization in a non-transformed human mammary epithelial cell line, MCF-10A, in basement membrane cultures leads to formation of proliferative structures that share properties with non-invasive early stage lesions. Recently, we have shown that activation of ErbB2 homodimers combined with expression of transforming growth factor (TGF)-beta induces invasive and migratory activity in MCF-10A cells. In this system, migration requires inputs from numerous cellular pathways. We discuss this data and a model for migration induced by ErbB2 and TGF-beta. Concurrent studies by other groups have also shown that ErbB2 and TGF-beta can cooperate to increase metastatic and invasive behavior in murine mammary tumors. Here we discuss these studies and the potential implications of this research on breast cancer therapeutics.

Autocrine CSF-1R Activation Promotes Src-dependent Disruption of Mammary Epithelial Architecture

The Journal of Cell Biology. Apr, 2004  |  Pubmed ID: 15117969

Elevated coexpression of colony-stimulating factor receptor (CSF-1R) and its ligand, CSF-1, correlates with invasiveness and poor prognosis of a variety of epithelial tumors (Kacinski, B.M. 1995. Ann. Med. 27:79-85). Apart from recruitment of macrophages to the tumor site, the mechanisms by which CSF-1 may potentiate invasion are poorly understood. We show that autocrine CSF-1R activation induces hyperproliferation and a profound, progressive disruption of junctional integrity in acinar structures formed by human mammary epithelial cells in three-dimensional culture. Acini coexpressing receptor and ligand exhibit a dramatic relocalization of E-cadherin from the plasma membrane to punctate intracellular vesicles, accompanied by its loss from the Triton-insoluble fraction. Interfering with Src kinase activity, either by pharmacological inhibition or mutation of the Y561 docking site on CSF-1R, prevents E-cadherin translocation, suggesting that CSF-1R disrupts cell adhesion by uncoupling adherens junction complexes from the cytoskeleton and promoting cadherin internalization through a Src-dependent mechanism. These findings provide a mechanistic basis whereby CSF-1R could contribute to invasive progression in epithelial cancers.

Vav GEFs Are Required for Beta2 Integrin-dependent Functions of Neutrophils

The Journal of Cell Biology. Jul, 2004  |  Pubmed ID: 15249579

Integrin regulation of neutrophils is essential for appropriate adhesion and transmigration into tissues. Vav proteins are Rho family guanine nucleotide exchange factors that become tyrosine phosphorylated in response to adhesion. Using Vav1/Vav3-deficient neutrophils (Vav1/3ko), we show that Vav proteins are required for multiple beta2 integrin-dependent functions, including sustained adhesion, spreading, and complement-mediated phagocytosis. These defects are not attributable to a lack of initial beta2 activation as Vav1/3ko neutrophils undergo chemoattractant-induced arrest on intercellular adhesion molecule-1 under flow. Accordingly, in vivo, Vav1/3ko leukocytes arrest on venular endothelium yet are unable to sustain adherence. Thus, Vav proteins are specifically required for stable adhesion. beta2-induced activation of Cdc42, Rac1, and RhoA is defective in Vav1/3ko neutrophils, and phosphorylation of Pyk2, paxillin, and Akt is also significantly reduced. In contrast, Vav proteins are largely dispensable for G protein-coupled receptor-induced signaling events and chemotaxis. Thus, Vav proteins play an essential role coupling beta2 to Rho GTPases and regulating multiple integrin-induced events important in leukocyte adhesion and phagocytosis.

Epidermal Growth Factor Receptor-dependent Regulation of Integrin-mediated Signaling and Cell Cycle Entry in Epithelial Cells

Molecular and Cellular Biology. Oct, 2004  |  Pubmed ID: 15367678

Integrin-mediated adhesion of epithelial cells to extracellular matrix (ECM) proteins induces prolonged tyrosine phosphorylation and partial activation of epidermal growth factor receptor (EGFR) in an integrin-dependent and EGFR ligand-independent manner. Integrin-mediated activation of EGFR in epithelial cells is required for multiple signal transduction events previously shown to be induced by cell adhesion to matrix proteins, including tyrosine phosphorylation of Shc, Cbl, and phospholipase Cgamma, and activation of the Ras/Erk and phosphatidylinositol 3'-kinase/Akt signaling pathways. In contrast, activation of focal adhesion kinase, Src, and protein kinase C, adhesion to matrix proteins, cell spreading, migration, and actin cytoskeletal rearrangements are induced independently of EGFR kinase activity. The ability of integrins to induce the activation of EGFR and its subsequent regulation of Erk and Akt activation permitted adhesion-dependent induction of cyclin D1 and p21, Rb phosphorylation, and activation of cdk4 in epithelial cells in the absence of exogenous growth factors. Adhesion of epithelial cells to the ECM failed to efficiently induce degradation of p27, to induce cdk2 activity, or to induce Myc and cyclin A synthesis; subsequently, cells did not progress into S phase. Treatment of ECM-adherent cells with EGF, or overexpression of EGFR or Myc, resulted in restoration of late-G(1) cell cycle events and progression into S phase. These results indicate that partial activation of EGFR by integrin receptors plays an important role in mediating events triggered by epithelial cell attachment to ECM; EGFR is necessary for activation of multiple integrin-induced signaling enzymes and sufficient for early events in G(1) cell cycle progression. Furthermore, these findings suggest that EGFR or Myc overexpression may provoke ligand-independent proliferation in matrix-attached cells in vivo and could contribute to carcinoma development.

Use of Three-dimensional Basement Membrane Cultures to Model Oncogene-induced Changes in Mammary Epithelial Morphogenesis

Journal of Mammary Gland Biology and Neoplasia. Oct, 2004  |  Pubmed ID: 15838601

The development of breast carcinomas involves a complex set of phenotypic alterations in breast epithelial cells and the surrounding microenvironment. While traditional transformation assays provide models for investigating certain aspects of the cellular processes associated with tumor initiation and progression, they do not model alterations in tissue architecture that are critically involved in tumor development. In this review, we provide examples of how three-dimensional (3D) cell culture models can be utilized to dissect the pathways involved in the development of mammary epithelial structures and to elucidate the mechanisms responsible for oncogene-induced phenotypic alterations in epithelial behavior and architecture. Many normal mammary epithelial cell lines undergo a stereotypic morphogenetic process when grown in the presence of exogenous matrix proteins. This 3D morphogenesis culminates in the formation of well-organized, polarized spheroids, and/or tubules that are highly reminiscent of normal glandular architecture. In contrast, transformed cell lines isolated from mammary tumors exhibit significant deviations from normal epithelial behavior in 3D culture. We describe the use of 3D models as a method for both reconstructing and deconstructing the cell biological and biochemical events involved in mammary neoplasia.

Vav Family GEFs Link Activated Ephs to Endocytosis and Axon Guidance

Neuron. Apr, 2005  |  Pubmed ID: 15848800

Ephrin signaling through Eph receptor tyrosine kinases can promote attraction or repulsion of axonal growth cones during development. However, the mechanisms that determine whether Eph signaling promotes attraction or repulsion are not known. We show here that the Rho family GEF Vav2 plays a key role in this process. We find that, during axon guidance, ephrin binding to Ephs triggers Vav-dependent endocytosis of the ligand-receptor complex, thus converting an initially adhesive interaction into a repulsive event. In the absence of Vav proteins, ephrin-Eph endocytosis is blocked, leading to defects in growth cone collapse in vitro and significant defects in the ipsilateral retinogeniculate projections in vivo. These findings suggest an important role for Vav family GEFs as regulators of ligand-receptor endocytosis and determinants of repulsive signaling during axon guidance.

Bim Regulation of Lumen Formation in Cultured Mammary Epithelial Acini is Targeted by Oncogenes

Molecular and Cellular Biology. Jun, 2005  |  Pubmed ID: 15899862

Epithelial cells organize into cyst-like structures that contain a spherical monolayer of cells that enclose a central lumen. Using a three-dimensional basement membrane culture model in which mammary epithelial cells form hollow, acinus-like structures, we previously demonstrated that lumen formation is achieved, in part, through apoptosis of centrally localized cells. We demonstrate that the proapoptotic protein Bim may selectively trigger apoptosis of the centrally localized acinar cells, leading to temporally controlled lumen formation. Bim is not detectable during early stages of three-dimensional mammary acinar morphogenesis and is then highly upregulated in all cells of acini, coincident with detection of apoptosis in the centrally localized acinar cells. Inhibition of Bim expression by RNA interference transiently blocks luminal apoptosis and delays lumen formation. Oncogenes that induce acinar luminal filling, such as ErbB2 and v-Src, suppress expression of Bim through a pathway dependent on Erk-mitogen-activated protein kinase; however, HPV 16 E7, an oncogene that stimulates cell proliferation but not luminal filling, is unable to reduce Bim expression. Thus, Bim is a critical regulator of luminal apoptosis during mammary acinar morphogenesis in vitro and may be an important target of oncogenes that disrupt glandular epithelial architecture.

G1/S Cell Cycle Arrest Provides Anoikis Resistance Through Erk-mediated Bim Suppression

Molecular and Cellular Biology. Jun, 2005  |  Pubmed ID: 15923641

Proper attachment to the extracellular matrix is essential for cell survival. Detachment from the extracellular matrix results in an apoptotic process termed anoikis. Anoikis induction in MCF-10A mammary epithelial cells is due not only to loss of survival signals following integrin disengagement, but also to consequent downregulation of epidermal growth factor (EGFR) and loss of EGFR-induced survival signals. Here we demonstrate that G(1)/S arrest by overexpression of the cyclin-dependent kinase inhibitors p16(INK4a), p21(Cip1), or p27(Kip1) or by treatment with mimosine or aphidicolin confers anoikis resistance in MCF-10A cells. G(1)/S arrest-mediated anoikis resistance involves suppression of the BH3-only protein Bim. Furthermore, in G(1)/S-arrested cells, Erk phosphorylation is maintained in suspension and is necessary for Bim suppression. Following G(1)/S arrest, known proteins upstream of Erk, including Raf and Mek, are not activated. However, retained Erk activation under conditions in which Raf and Mek activation is lost is observed, suggesting that G(1)/S arrest acts at the level of Erk dephosphorylation. Thus, anoikis resistance by G(1)/S arrest is mediated by a mechanism involving Bim suppression through maintenance of Erk activation. These results provide a novel link between cell cycle arrest and survival, and this mechanism could contribute to the survival of nonreplicating, dormant tumor cells that avert apoptosis during early stages of metastasis.

Activation of NF-kappaB Following Detachment Delays Apoptosis in Intestinal Epithelial Cells

Oncogene. Sep, 2005  |  Pubmed ID: 16007176

We reported earlier that IL-1beta, an NF-kappaB-regulated cytokine, was made by intestinal epithelial cells during detachment-induced apoptosis (anoikis) and that IL-1 was antiapoptotic for detached cells. Since surviving anoikis is a prerequisite for cancer progression and metastases, we are further exploring the link between anoikis and cytokines. Here we determined that multiple genes are expressed following detachment including a number of NF-kappaB-regulated products and therefore aimed to determine whether NF-kappaB signalling plays any role in regulating apoptosis. Using Western blotting, we detected that IkappaBalpha becomes phosphorylated immediately following detachment and that levels of phospho-IkappaBalpha peaked within 20 min. Phosphorylation of IkappaBalpha was followed by Rel A (p65) nuclear translocation. Increased NF-kappaB activity following detachment was confirmed using the detection of NF-kappaB-promoted luciferase gene expression delivered by adenovirus infection. Infection of cells with adenovirus expressing a super-repressor IkappaBalpha protein and pharmacological inhibitors of NF-kappaB resulted in the failure to phosphorylate IkappaBalpha, a more rapid activation of caspases and earlier apoptosis. We also detected that IkappaB kinase alpha (IKKalpha) and not IKKbeta became phosphorylated following detachment. Since IKKalpha is activated by NF-kappaB-inducing kinase (NIK), we overexpressed native NIK using an adenovirus vector that resulted in enhanced phospho-IkappaBalpha and nuclear p65 in detached cells compared to control detached cells but did not result in a significantly greater number of cells surviving to 24 h. We conclude that detachment directly activates NF-kappaB, which, in addition to launching an inflammatory cytokine wave, contributes to a delay in apoptosis in intestinal epithelial cells.

Modelling Glandular Epithelial Cancers in Three-dimensional Cultures

Nature Reviews. Cancer. Sep, 2005  |  Pubmed ID: 16148884

Little is known about how the genotypic and molecular abnormalities associated with epithelial cancers actually contribute to the histological phenotypes observed in tumours in vivo. 3D epithelial culture systems are a valuable tool for modelling cancer genes and pathways in a structurally appropriate context. Here, we review the important features of epithelial structures grown in 3D basement membrane cultures, and how such models have been used to investigate the mechanisms associated with tumour initiation and progression.

Breast Cancer-associated PIK3CA Mutations Are Oncogenic in Mammary Epithelial Cells

Cancer Research. Dec, 2005  |  Pubmed ID: 16322248

Activation of the phosphoinositide 3-kinase (PI3K) pathway has been implicated in the pathogenesis of a variety of cancers. Recently, mutations in the gene encoding the p110alpha catalytic subunit of PI3K (PIK3CA) have been identified in several human cancers. The mutations primarily result in single amino acid substitutions, with >85% of the mutations in either exon 9 or 20. Multiple studies have shown that these mutations are observed in 18% to 40% of breast cancers. However, the phenotypic effects of these PIK3CA mutations have not been examined in breast epithelial cells. Herein, we examine the activity of the two most common variants, E545K and H1047R, in the MCF-10A immortalized breast epithelial cell line. Both variants display higher PI3K activity than wild-type p110alpha yet remain sensitive to pharmacologic PI3K inhibition. In addition, expression of p110alpha mutants in mammary epithelial cells induces multiple phenotypic alterations characteristic of breast tumor cells, including anchorage-independent proliferation in soft agar, growth factor-independent proliferation, and protection from anoikis. Expression of these mutant p110alpha isoforms also confers increased resistance to paclitaxel and induces abnormal mammary acinar morphogenesis in three-dimensional basement membrane cultures. Together, these data support the notion that the cancer-associated mutations in PIK3CA may significantly contribute to breast cancer pathogenesis and represent attractive targets for therapeutic inhibition.

Novel Role for PDEF in Epithelial Cell Migration and Invasion

Cancer Research. Dec, 2005  |  Pubmed ID: 16357167

Cell migration and invasion are two critical cellular processes that are often deregulated during tumorigenesis. To identify factors that contribute to oncogenic progression by stimulating cell migration, we conducted a powerful retroviral based migration screen using an MCF7 cDNA library and the immortalized human breast epithelial cell line MCF-10A. We identified prostate derived Ets factor (PDEF), an Ets transcription factor that is overexpressed in both prostate and breast carcinoma, as a candidate promigratory gene from this screen. Whereas PDEF induced limited motility of MCF-10A cells, coexpression of PDEF with the receptor tyrosine kinases (RTK) ErbB2 and colony-stimulating factor receptor (CSF-1R)/CSF-1 significantly enhanced MCF-10A motility. Furthermore, cells coexpressing PDEF with either ErbB2 or CSF-1R/CSF-1 induced a dramatic invasive phenotype in three-dimensional cultures. Constitutive activation of the extracellular signal-regulated kinase (ERK) pathway also enhanced PDEF-induced motility and invasion, suggesting that activation of the ERK/mitogen-activated protein kinase by ErbB2 and CSF-1R/CSF-1 can cooperate with PDEF to promote motility and invasion. Furthermore, PDEF promoted anchorage-independent growth of ErbB2 and CSF-1R/CSF-1-expressing cells. Using laser capture microdissection, we also found that PDEF mRNA is overexpressed in breast tumor epithelia throughout tumor progression. Taken together, these findings suggest that the transcription factor PDEF may play an important role in breast tumorigenesis and that PDEF overexpression may be particularly significant in tumors that exhibit activation of oncogenic RTKs such as ErbB2 and CSF-1R.

Distinct Roles of Akt1 and Akt2 in Regulating Cell Migration and Epithelial-mesenchymal Transition

The Journal of Cell Biology. Dec, 2005  |  Pubmed ID: 16365168

The Akt family of kinases are activated by growth factors and regulate pleiotropic cellular activities. In this study, we provide evidence for isoform-specific positive and negative roles for Akt1 and -2 in regulating growth factor-stimulated phenotypes in breast epithelial cells. Insulin-like growth factor-I receptor (IGF-IR) hyperstimulation induced hyperproliferation and antiapoptotic activities that were reversed by Akt2 down-regulation. In contrast, Akt1 down-regulation in IGF-IR-stimulated cells promoted dramatic neomorphic effects characteristic of an epithelial-mesenchymal transition (EMT) and enhanced cell migration induced by IGF-I or EGF stimulation. The phenotypic effects of Akt1 down-regulation were accompanied by enhanced extracellular signal-related kinase (ERK) activation, which contributed to the induction of migration and EMT. Interestingly, down-regulation of Akt2 suppressed the EMT-like morphological conversion induced by Akt1 down-regulation in IGF-IR-overexpressing cells and inhibited migration in EGF-stimulated cells. These results highlight the distinct functions of Akt isoforms in regulating growth factor-stimulated EMT and cell migration, as well as the importance of Akt1 in cross-regulating the ERK signaling pathway.

Functional Proteomics Approach to Investigate the Biological Activities of CDNAs Implicated in Breast Cancer

Journal of Proteome Research. Mar, 2006  |  Pubmed ID: 16512675

Functional proteomics approaches that comprehensively evaluate the biological activities of human cDNAs may provide novel insights into disease pathogenesis. To systematically investigate the functional activity of cDNAs that have been implicated in breast carcinogenesis, we generated a collection of cDNAs relevant to breast cancer, the Breast Cancer 1000 (BC1000), and conducted screens to identify proteins that induce phenotypic changes that resemble events which occur during tumor initiation and progression. Genes were selected for this set using bioinformatics and data mining tools that identify genes associated with breast cancer. Greater than 1000 cDNAs were assembled and sequence verified with high-throughput recombination-based cloning. To our knowledge, the BC1000 represents the first publicly available sequence-validated human disease gene collection. The functional activity of a subset of the BC1000 collection was evaluated in cell-based assays that monitor changes in cell proliferation, migration, and morphogenesis in MCF-10A mammary epithelial cells expressing a variant of ErbB2 that can be inducibly activated through dimerization. Using this approach, we identified many cDNAs, encoding diverse classes of cellular proteins, that displayed activity in one or more of the assays, thus providing insights into a large set of cellular proteins capable of inducing functional alterations associated with breast cancer development.

Requirements for Vav Guanine Nucleotide Exchange Factors and Rho GTPases in FcgammaR- and Complement-mediated Phagocytosis

Immunity. Mar, 2006  |  Pubmed ID: 16546099

Vav guanine nucleotide exchange factors (GEFs) have been implicated in cell adhesion by integrin and immune response receptors through the regulation of Rho GTPases. Here, we examine the role of Vav and Rho GTPases in phagocytosis by using primary murine macrophages. The genetic deletion of Rac1 and Rac2 prevents phagocytosis mediated by integrin and Fcgamma receptors (FcgammaR), whereas the genetic deletion of Vav1 and Vav3 only prevents integrin-mediated phagocytosis through the complement receptor alpha(M)beta(2). In addition, a Rac1/2 or Vav1/3 deficiency blocks Arp2/3 recruitment and actin polymerization at the complement-induced phagosome, indicating that these proteins regulate early steps in phagocytosis. Moreover, constitutively active Rac is able to rescue actin polymerization and complement-mediated phagocytosis in Vav-deficient macrophages. These studies indicate that Rac is critical for complement- and FcgammaR-mediated phagocytosis. In contrast, Vav is specifically required for complement-mediated phagocytosis, suggesting that Rac is regulated by GEFs other than Vav downstream of the FcgammaR.

An Active Form of Vav1 Induces Migration of Mammary Epithelial Cells by Stimulating Secretion of an Epidermal Growth Factor Receptor Ligand

Cell Communication and Signaling : CCS. 2006  |  Pubmed ID: 16709244

Vav proteins are guanine nucleotide exchange factors (GEF) for Rho family GTPases and are activated following engagement of membrane receptors. Overexpression of Vav proteins enhances lamellipodium and ruffle formation, migration, and cell spreading, and augments activation of many downstream signaling proteins like Rac, ERK and Akt. Vav proteins are composed of multiple structural domains that mediate their GEF function and binding interactions with many cellular proteins. In this report we examine the mechanisms responsible for stimulation of cell migration by an activated variant of Vav1 and identify the domains of Vav1 required for this activity.

P63 Regulates an Adhesion Programme and Cell Survival in Epithelial Cells

Nature Cell Biology. Jun, 2006  |  Pubmed ID: 16715076

p63 is critical for epithelial development yet little is known about the transcriptional programmes it regulates. By characterising transcriptional changes and cellular effects following modulation of p63 expression, we have defined a vital role for p63 in cellular adhesion. Knockdown of p63 expression caused downregulation of cell adhesion-associated genes, cell detachment and anoikis in mammary epithelial cells and keratinocytes. Conversely, overexpression of the TAp63gamma or deltaNp63alpha isoforms of p63 upregulated cell adhesion molecules, increased cellular adhesion and conferred resistance to anoikis. Apoptosis induced by loss of p63 was rescued by signalling downstream of beta4 integrin. Our results implicate p63 as a key regulator of cellular adhesion and survival in basal cells of the mammary gland and other stratified epithelial tissues.

Transforming Properties of YAP, a Candidate Oncogene on the Chromosome 11q22 Amplicon

Proceedings of the National Academy of Sciences of the United States of America. Aug, 2006  |  Pubmed ID: 16894141

In a screen for gene copy-number changes in mouse mammary tumors, we identified a tumor with a small 350-kb amplicon from a region that is syntenic to a much larger locus amplified in human cancers at chromosome 11q22. The mouse amplicon contains only one known gene, Yap, encoding the mammalian ortholog of Drosophila Yorkie (Yki), a downstream effector of the Hippo(Hpo)-Salvador(Sav)-Warts(Wts) signaling cascade, recently identified in flies as a critical regulator of cellular proliferation and apoptosis. In nontransformed mammary epithelial cells, overexpression of human YAP induces epithelial-to-mesenchymal transition, suppression of apoptosis, growth factor-independent proliferation, and anchorage-independent growth in soft agar. Together, these observations point to a potential oncogenic role for YAP in 11q22-amplified human cancers, and they suggest that this highly conserved signaling pathway identified in Drosophila regulates both cellular proliferation and apoptosis in mammalian epithelial cells.

SePARating Polarity and Proliferation in ErbB2 Oncogenesis

Nature Cell Biology. Nov, 2006  |  Pubmed ID: 17077856

BIM Regulates Apoptosis During Mammary Ductal Morphogenesis, and Its Absence Reveals Alternative Cell Death Mechanisms

Developmental Cell. Feb, 2007  |  Pubmed ID: 17276340

The adult, virgin mammary gland is a highly organized tree-like structure formed by ducts with hollowed lumen. Although lumen formation during pubertal development appears to involve apoptosis, the molecular mechanisms that regulate this process are not known. Here, we demonstrate that disruption of the BH3-only proapoptotic factor Bim in mice prevents induction of apoptosis in and clearing of the lumen in terminal end buds during puberty. However, cells that fill the presumptive luminal space are eventually cleared from the adjacent ducts by a caspase-independent death process. Within the filled Bim(-/-) ducts, epithelial cells are deprived of matrix attachment and undergo squamous differentiation prior to clearing. Similarly, we also detect squamous differentiation in vitro when immortalized mammary epithelial cells are detached from the matrix. These data provide important mechanistic information on the processes involved in sculpting the mammary gland and demonstrate that BIM is a critical regulator of apoptosis in vivo.

P63, Cell Adhesion and Survival

Cell Cycle (Georgetown, Tex.). Feb, 2007  |  Pubmed ID: 17297292

The development of stratified epithelia and their derivatives is a complex process requiring a multifaceted transcriptional program. p63, the p53-related transcription factor, is fundamental to this process. However, the underlying mechanisms by which p63 exerts its influence on stratified epithelial development and integrity remain elusive. Recent work from our laboratories has demonstrated that p63 mediates its effects on stratified epithelial function at least in part via its ability to regulate multiple aspects of epithelial cellular adhesion and survival. The identification of cell-cell and cell-matrix adhesion subprograms downstream of p63 provides an initial understanding of p63's role in epithelial development, integrity and homeostasis.

Functional Role and Oncogene-regulated Expression of the BH3-only Factor Bmf in Mammary Epithelial Anoikis and Morphogenesis

Proceedings of the National Academy of Sciences of the United States of America. Mar, 2007  |  Pubmed ID: 17360431

The formation of a lumen in three-dimensional mammary epithelial acinar structures in vitro involves selective apoptosis of centrally localized cells that lack matrix attachment. Similarly, apoptosis is induced by forced detachment of mammary epithelial cells from matrix, a process referred to as anoikis. Through microarray analysis, we found that mRNA levels of the proapoptotic BH3-only protein Bmf are up-regulated during both anoikis and acinar morphogenesis. Importantly, down-regulation of Bmf expression by small interfering RNAs is sufficient to prevent anoikis and acinar cell death and promote anchorage-independent growth to a similar extent as down-regulation of another BH3-only protein, Bim, which was previously shown to be required for these processes. Knockdown of the BH3-only proteins Bad or Bid does not suppress anoikis or luminal apoptosis or promote anchorage-independent growth, but protects from other defined apoptotic stimuli, indicating specificity of BH3-only function. Bmf mRNA is significantly up-regulated upon loss of matrix attachment or disruption of the actin cytoskeleton, but not in response to several other stresses. Interestingly, constitutive activation of the Mek/Erk or phosphatidylinositol 3-kinase/Akt pathways suppresses the transcriptional up-regulation of Bmf during anoikis. Thus, Bmf is a central mediator of anoikis in mammary cells and a target of oncogenes that contribute to the progression of glandular epithelial tumors. Finally, Bmf is expressed during involution of the mouse mammary gland, suggesting that Bmf may also critically contribute to developmental processes in vivo.

Common Effector Processing Mediates Cell-specific Responses to Stimuli

Nature. Aug, 2007  |  Pubmed ID: 17637676

The fundamental components of many signalling pathways are common to all cells. However, stimulating or perturbing the intracellular network often causes distinct phenotypes that are specific to a given cell type. This 'cell specificity' presents a challenge in understanding how intracellular networks regulate cell behaviour and an obstacle to developing drugs that treat signalling dysfunctions. Here we apply a systems-modelling approach to investigate how cell-specific signalling events are integrated through effector proteins to cause cell-specific outcomes. We focus on the synergy between tumour necrosis factor and an adenoviral vector as a therapeutically relevant stimulus that induces cell-specific responses. By constructing models that estimate how kinase-signalling events are processed into phenotypes through effector substrates, we find that accurate predictions of cell specificity are possible when different cell types share a common 'effector-processing' mechanism. Partial-least-squares regression models based on common effector processing accurately predict cell-specific apoptosis, chemokine release, gene induction, and drug sensitivity across divergent epithelial cell lines. We conclude that cell specificity originates from the differential activation of kinases and other upstream transducers, which together enable different cell types to use common effectors to generate diverse outcomes. The common processing of network signals by downstream effectors points towards an important cell biological principle, which can be applied to the understanding of cell-specific responses to targeted drug therapies.

Tensins: a New Switch in Cell Migration

Developmental Cell. Sep, 2007  |  Pubmed ID: 17765673

Epidermal Growth Factor (EGF) is an important regulator of normal epithelial and carcinoma cell migration. The mechanism by which EGF induces cell migration is not fully understood. A recent report in Nature Cell Biology (Katz et al., 2007) demonstrates that EGF regulates migration through a switch in the expression of two tensin isoforms, weakening the association of beta1 integrin with the actin cytoskeleton in focal adhesions.

Engineering Tumors with 3D Scaffolds

Nature Methods. Oct, 2007  |  Pubmed ID: 17767164

Microenvironmental conditions control tumorigenesis and biomimetic culture systems that allow for in vitro and in vivo tumor modeling may greatly aid studies of cancer cells' dependency on these conditions. We engineered three-dimensional (3D) human tumor models using carcinoma cells in polymeric scaffolds that recreated microenvironmental characteristics representative of tumors in vivo. Strikingly, the angiogenic characteristics of tumor cells were dramatically altered upon 3D culture within this system, and corresponded much more closely to tumors formed in vivo. Cells in this model were also less sensitive to chemotherapy and yielded tumors with enhanced malignant potential. We assessed the broad relevance of these findings with 3D culture of other tumor cell lines in this same model, comparison with standard 3D Matrigel culture and in vivo experiments. This new biomimetic model may provide a broadly applicable 3D culture system to study the effect of microenvironmental conditions on tumor malignancy in vitro and in vivo.

A Nonapoptotic Cell Death Process, Entosis, That Occurs by Cell-in-cell Invasion

Cell. Nov, 2007  |  Pubmed ID: 18045538

Epithelial cells require attachment to extracellular matrix (ECM) to suppress an apoptotic cell death program termed anoikis. Here we describe a nonapoptotic cell death program in matrix-detached cells that is initiated by a previously unrecognized and unusual process involving the invasion of one cell into another, leading to a transient state in which a live cell is contained within a neighboring host cell. Live internalized cells are either degraded by lysosomal enzymes or released. We term this cell internalization process entosis and present evidence for entosis as a mechanism underlying the commonly observed "cell-in-cell" cytological feature in human cancers. Further we propose that entosis is driven by compaction force associated with adherens junction formation in the absence of integrin engagement and may represent an intrinsic tumor suppression mechanism for cells that are detached from ECM.

IL-6 Involvement in Epithelial Cancers

The Journal of Clinical Investigation. Dec, 2007  |  Pubmed ID: 18060028

In this issue of the JCI, two reports provide intriguing new information on the role of the inflammatory cytokine IL-6 in breast and lung cancer. The study by Sansone et al. implicates IL-6 in the instigation of malignant properties in breast cancer stem cells (see the related article beginning on page 3988). The study by Gao et al. identifies mutant variants of EGFR as inducers of IL-6 in lung adenocarcinomas (see the related article beginning on page 3846). These studies add to our understanding of potential roles for IL-6 in cancer and further motivate investigations of IL-6-targeted chemotherapeutics.

Circulating Colony Stimulating Factor-1 and Breast Cancer Risk

Cancer Research. Jan, 2008  |  Pubmed ID: 18172291

Colony stimulating factor-1 (CSF1) and its receptor (CSF1-R) are important in mammary gland development and have been implicated in breast carcinogenesis. In a nested case-control study in the Nurses' Heath Study of 726 breast cancer cases diagnosed between June 1, 1992, and June 1, 1998, and 734 matched controls, we prospectively evaluated whether circulating levels of CSF1 (assessed in 1989-1990) are associated with breast cancer risk. The association varied by menopausal status (P(heterogeneity) = 0.009). CSF1 levels in the highest quartile (versus lowest) were associated with an 85% reduced risk of premenopausal breast cancer [relative risk (RR), 0.15; 95% confidence interval (95% CI), 0.03-0.85; P(trend) = 0.02]. In contrast, CSF1 levels in the highest quartile conferred a 33% increased risk of postmenopausal breast cancer (RR, 1.33; 95% CI, 0.96-1.86; P(trend) = 0.11), with greatest risk for invasive (RR, 1.45; 95% CI, 1.02-2.07; P(trend) = 0.06) and ER+/PR+ tumors (RR, 1.72; 95% CI, 1.11-2.66; P(trend) = 0.04). Thus, the association of circulating CSF1 levels and breast cancer varies by menopausal status.

Lumen Formation During Mammary Epithelial Morphogenesis: Insights from in Vitro and in Vivo Models

Cell Cycle (Georgetown, Tex.). Jan, 2008  |  Pubmed ID: 18196964

The mammary gland undergoes a morphogenetic program during embryogenesis and puberty that leads to the development of hollow ductal system terminating in acinar units. It later expands to generate an elaborate network to deliver milk to newborn progeny. Previous studies in our laboratory using three-dimensional basement membrane cultures of mammary epithelial cells, in which acini-like structures form from single cells, have indicated that lumen formation requires clearance of the cells in the center of the acini by apoptosis. This apoptotic program in vitro requires the pro-death mediator BIM. Recently we found that BIM is also required in the mouse mammary gland for apoptosis during lumen formation, which correlates the 3D acinar model to mammary morphogenesis in vivo. Herein we put into perspective the relevance of our in vitro and in vivo findings to discuss luminal space formation and maintenance during mammary morphogenesis.

[Entosis, a Cell Death Process Related to Cell Cannibalism Between Tumor Cells]

Médecine Sciences : M/S. Mar, 2008  |  Pubmed ID: 18334170

Regulation of Cell Adhesion and Collective Cell Migration by Hindsight and Its Human Homolog RREB1

Current Biology : CB. Apr, 2008  |  Pubmed ID: 18394891

Cell movements represent a major driving force in embryonic development, tissue repair, and tumor metastasis [1]. The migration of single cells has been well studied, predominantly in cell culture [2, 3]; however, in vivo, a greater variety of modes of cell movement occur, including the movements of cells in clusters, strands, sheets, and tubes, also known as collective cell migrations [4, 5]. In spite of the relevance of these types of movements in both normal and pathological conditions, the molecular mechanisms that control them remain predominantly unknown. Epithelial follicle cells of the Drosophila ovary undergo several dynamic morphological changes, providing a genetically tractable model [6]. We found that anterior follicle cells, including border cells, mutant for the gene hindsight (hnt) accumulated excess cell-cell adhesion molecules and failed to undergo their normal collective movements. In addition, HNT affected border cell cluster cohesion and motility via effects on the JNK and STAT pathways, respectively. Interestingly, reduction of expression of the mammalian homolog of HNT, RREB1, by siRNA inhibited collective cell migration in a scratch-wound healing assay of MCF10A mammary epithelial cells, suppressed surface activity, retarded cell spreading after plating, and led to the formation of immobile, tightly adherent cell colonies. We propose that HNT and RREB1 are essential to reduce cell-cell adhesion when epithelial cells within an interconnected group undergo dynamic changes in cell shape.

The Cell Biology of Cell-in-cell Structures

Nature Reviews. Molecular Cell Biology. Oct, 2008  |  Pubmed ID: 18784728

For decades, authors have described unusual cell structures, referred to as cell-in-cell structures, in which whole cells are found in the cytoplasm of other cells. One well-characterized process that results in the transient appearance of such structures is the engulfment of apoptotic cells by phagocytosis. However, many other types of cell-in-cell structure have been described that involve viable non-apoptotic cells. Some of these structures seem to form by the invasion of one cell into another, rather than by engulfment. The mechanisms of cell-in-cell formation and the possible physiological roles of these processes will be discussed.

Identification of Genes That Regulate Epithelial Cell Migration Using an SiRNA Screening Approach

Nature Cell Biology. Sep, 2008  |  Pubmed ID: 19160483

To provide a systematic analysis of genes that regulate epithelial cell migration, we performed a high throughput wound healing screen with MCF-10A breast epithelial cells, using siRNAs targeting 1,081 human genes encoding phosphatases, kinases and proteins predicted to influence cell migration and adhesion. The primary screen identified three categories of hits: those that accelerate, those that inhibit and those that impair migration with associated effects on cell proliferation or metabolism. Extensive validation of all the hits yielded 66 high confidence genes that, when downregulated, either accelerated or impaired migration; 42 of these high confidence genes have not been previously associated with motility or adhesion. Time-lapse video microscopy revealed a broad spectrum of phenotypic changes involving alterations in the extent and nature of disruption of cell-cell adhesion, directionality of motility, cell polarity and shape, and protrusion dynamics. Informatics analysis highlighted three major signalling nodes, beta-catenin, beta1-integrin and actin, and a large proportion of the genes that accelerated migration impaired cell-cell adhesion.

Fibroblast Growth Factor Receptor 1-transformed Mammary Epithelial Cells Are Dependent on RSK Activity for Growth and Survival

Cancer Research. Mar, 2009  |  Pubmed ID: 19258500

Fibroblast growth factor receptor 1 (FGFR1) is frequently amplified and highly expressed in lobular carcinomas of the breast. In this report, we evaluated the biological activity of FGFR1 in a wide range of in vitro assays. Conditional activation of FGFR1 in the nontransformed MCF10A human mammary cell line, MCF10A, resulted in cellular transformation marked by epidermal growth factor-independent cell growth, anchorage-independent cell proliferation and survival, loss of cell polarity, and epithelial-to-mesenchymal transition. Interestingly, small-molecule or small interfering RNA inhibition of ribosomal S6 kinase (RSK) activity induced death of the FGFR1-transformed cells, but not of the parental MCF10A cell line. The dependence of FGFR1-transformed cells on RSK activity was further confirmed in cell lines derived from mouse and human lobular carcinomas that possess high FGFR1 activity. Taken together, these results show the transforming activity of FGFR1 in mammary epithelial cells and identify RSK as a critical component of FGFR1 signaling in lobular carcinomas, thus implicating RSK as a candidate therapeutic target in FGFR1-expressing tumors.

YB-1 Translational Control of Epithelial-mesenchyme Transition

Cancer Cell. May, 2009  |  Pubmed ID: 19411064

Transitions between epithelial and mesenchmal phenotypes play critical roles in normal development and cancer progression. In this issue of Cancer Cell, Evdokimova et al. demonstrate that YB-1 regulates epithelial-mesenchyme transition (EMT) by inducing cap-independent translation of mRNAs encoding EMT-promoting factors and suppressing cap-dependent translation of mRNAs encoding growth-promoting factors.

Antioxidant and Oncogene Rescue of Metabolic Defects Caused by Loss of Matrix Attachment

Nature. Sep, 2009  |  Pubmed ID: 19693011

Normal epithelial cells require matrix attachment for survival, and the ability of tumour cells to survive outside their natural extracellular matrix (ECM) niches is dependent on acquisition of anchorage independence. Although apoptosis is the most rapid mechanism for eliminating cells lacking appropriate ECM attachment, recent reports suggest that non-apoptotic death processes prevent survival when apoptosis is inhibited in matrix-deprived cells. Here we demonstrate that detachment of mammary epithelial cells from ECM causes an ATP deficiency owing to the loss of glucose transport. Overexpression of ERBB2 rescues the ATP deficiency by restoring glucose uptake through stabilization of EGFR and phosphatidylinositol-3-OH kinase (PI(3)K) activation, and this rescue is dependent on glucose-stimulated flux through the antioxidant-generating pentose phosphate pathway. Notably, we found that the ATP deficiency could be rescued by antioxidant treatment without rescue of glucose uptake. This rescue was found to be dependent on stimulation of fatty acid oxidation, which is inhibited by detachment-induced reactive oxygen species (ROS). The significance of these findings was supported by evidence of an increase in ROS in matrix-deprived cells in the luminal space of mammary acini, and the discovery that antioxidants facilitate the survival of these cells and enhance anchorage-independent colony formation. These results show both the importance of matrix attachment in regulating metabolic activity and an unanticipated mechanism for cell survival in altered matrix environments by antioxidant restoration of ATP generation.

YAP-dependent Induction of Amphiregulin Identifies a Non-cell-autonomous Component of the Hippo Pathway

Nature Cell Biology. Dec, 2009  |  Pubmed ID: 19935651

The Hippo signalling pathway regulates cellular proliferation and survival, thus has profound effects on normal cell fate and tumorigenesis. The pivotal effector of this pathway is YAP (yes-associated protein), a transcriptional co-activator amplified in mouse and human cancers, where it promotes epithelial to mesenchymal transition (EMT) and malignant transformation. So far, studies of YAP target genes have focused on cell-autonomous mediators; here we show that YAP-expressing MCF10A breast epithelial cells enhance the proliferation of neighbouring untransfected cells, implicating a non-cell-autonomous mechanism. We identify the gene for the epidermal growth factor receptor (EGFR) ligand amphiregulin (AREG) as a transcriptional target of YAP, whose induction contributes to YAP-mediated cell proliferation and migration, but not EMT. Knockdown of AREG or addition of an EGFR kinase inhibitor abrogates the proliferative effects of YAP expression. Suppression of the negative YAP regulators LATS1 and 2 (large tumour suppressor 1 and 2) is sufficient to induce AREG expression, consistent with physiological regulation of AREG by the Hippo pathway. Genetic interaction between the Drosophila YAP orthologue Yorkie and Egfr signalling components supports the link between these two highly conserved signalling pathways. Thus, YAP-dependent secretion of AREG indicates that activation of EGFR signalling is an important non-cell-autonomous effector of the Hippo pathway, which has implications for the regulation of both physiological and malignant cell proliferation.

Transcriptional Regulation of Metastatic [Id]entity by KLF17

Genome Biology. 2009  |  Pubmed ID: 19951400

A novel in vivo screening approach has identified KLF17 as a key metastasis suppressor gene that acts through regulation of Id1 transcription factor-dependent induction of the epithelial-to-mesenchymal transition.

A Stiff Blow from the Stroma: Collagen Crosslinking Drives Tumor Progression

Cancer Cell. Dec, 2009  |  Pubmed ID: 19962663

Matrix stiffness is an important microenvironmental cue that regulates cell growth, motility, and differentiation. In a recent Cell report, Weaver and colleagues implicate lysyl-oxidase-mediated collagen crosslinking as a contributor to tumor matrix stiffening, which leads to enhanced integrin signaling and invasive behavior in tumors.

Tumor Self-seeding: Bidirectional Flow of Tumor Cells

Cell. Dec, 2009  |  Pubmed ID: 20064369

Circulating tumor cells are responsible for seeding metastatic growth at distant sites. Kim et al. (2009) now discover that circulating tumor cells can reinfiltrate tumors at their primary organs and promote tumor progression.

Mutant P53 Drives Invasion by Promoting Integrin Recycling

Cell. Dec, 2009  |  Pubmed ID: 20064378

p53 is a tumor suppressor protein whose function is frequently lost in cancers through missense mutations within the Tp53 gene. This results in the expression of point-mutated p53 proteins that have both lost wild-type tumor suppressor activity and show gain of functions that contribute to transformation and metastasis. Here, we show that mutant p53 expression can promote invasion, loss of directionality of migration, and metastatic behavior. These activities of p53 reflect enhanced integrin and epidermal growth factor receptor (EGFR) trafficking, which depends on Rab-coupling protein (RCP) and results in constitutive activation of EGFR/integrin signaling. We provide evidence that mutant p53 promotes cell invasion via the inhibition of TAp63, and simultaneous loss of p53 and TAp63 recapitulates the phenotype of mutant p53 in cells. These findings open the possibility that blocking alpha5/beta1-integrin and/or the EGF receptor will have therapeutic benefit in mutant p53-expressing cancers.

MYC Regulation of a "poor-prognosis" Metastatic Cancer Cell State

Proceedings of the National Academy of Sciences of the United States of America. Feb, 2010  |  Pubmed ID: 20133671

Gene expression signatures are used in the clinic as prognostic tools to determine the risk of individual patients with localized breast tumors developing distant metastasis. We lack a clear understanding, however, of whether these correlative biomarkers link to a common biological network that regulates metastasis. We find that the c-MYC oncoprotein coordinately regulates the expression of 13 different "poor-outcome" cancer signatures. In addition, functional inactivation of MYC in human breast cancer cells specifically inhibits distant metastasis in vivo and invasive behavior in vitro of these cells. These results suggest that MYC oncogene activity (as marked by "poor-prognosis" signature expression) may be necessary for the translocation of poor-outcome human breast tumors to distant sites.

Dose-dependent Induction of Distinct Phenotypic Responses to Notch Pathway Activation in Mammary Epithelial Cells

Proceedings of the National Academy of Sciences of the United States of America. Mar, 2010  |  Pubmed ID: 20194747

Aberrant activation of Notch receptors has been implicated in breast cancer; however, the mechanisms contributing to Notch-dependent transformation remain elusive because Notch displays dichotomous functional activities, promoting both proliferation and growth arrest. We investigated the cellular basis for the heterogeneous responses to Notch pathway activation in 3D cultures of MCF-10A mammary epithelial cells. Expression of a constitutively active Notch-1 intracellular domain (NICD) was found to induce two distinct types of 3D structures: large, hyperproliferative structures and small, growth-arrested structures with reduced cell-to-matrix adhesion. Interestingly, we found that these heterogeneous phenotypes reflect differences in Notch pathway activation levels; high Notch activity caused down-regulation of multiple matrix-adhesion genes and inhibition of proliferation, whereas low Notch activity maintained matrix adhesion and provoked a strong hyperproliferative response. Moreover, microarray analyses implicated NICD-induced p63 down-regulation in loss of matrix adhesion. In addition, a reverse-phase protein array-based analysis and subsequent loss-of-function studies identified STAT3 as a dominant downstream mediator of the NICD-induced outgrowth. These results indicate that the phenotypic responses to Notch are determined by the dose of pathway activation; and this dose affects the balance between growth-stimulative and growth-suppressive effects. This unique feature of Notch signaling provides insights into mechanisms that contribute to the dichotomous effects of Notch during development and tumorigenesis.

Identifying Single-cell Molecular Programs by Stochastic Profiling

Nature Methods. Apr, 2010  |  Pubmed ID: 20228812

Cells in tissues can be morphologically indistinguishable yet show molecular expression patterns that are remarkably heterogeneous. Here we describe an approach to comprehensively identify co-regulated, heterogeneously expressed genes among cells that otherwise appear identical. The technique, called stochastic profiling, involves repeated, random selection of very small cell populations via laser-capture microdissection followed by a customized single-cell amplification procedure and transcriptional profiling. Fluctuations in the resulting gene-expression measurements are then analyzed statistically to identify transcripts that are heterogeneously coexpressed. We stochastically profiled matrix-attached human epithelial cells in a three-dimensional culture model of mammary-acinar morphogenesis. Of 4,557 transcripts, we identified 547 genes with strong cell-to-cell expression differences. Clustering of this heterogeneous subset revealed several molecular 'programs' implicated in protein biosynthesis, oxidative-stress responses and NF-kappaB signaling, which we independently confirmed by RNA fluorescence in situ hybridization. Thus, stochastic profiling can reveal single-cell heterogeneities without the need to measure expression in individual cells.

Unregulated ARF6 Activation in Epithelial Cysts Generates Hyperactive Signaling Endosomes and Disrupts Morphogenesis

Molecular Biology of the Cell. Jul, 2010  |  Pubmed ID: 20462959

Tumor development in glandular tissues is associated with structural alterations in the hollow ducts and spherical structures that comprise such tissues. We describe a signaling axis involving sustained activation of the GTP-binding protein, ARF6, that provokes dramatic changes in the organization of epithelial cysts, reminiscent of tumorigenic glandular phenotypes. In reconstituted basement membrane cultures of renal epithelial cysts, enhanced ARF6 activation induces the formation of cell-filled glandular structures with multiple lumens and disassembled cadherin-based cell-cell contacts. All of these alterations are accompanied by growth factor receptor internalization into signaling endosomes and reversed by blocking ARF6 activation or receptor endocytosis. Receptor localization in signaling endosomes results in hyperactive extracellular signal-regulated kinase signaling leading to Bcl-2 stabilization and aberrant cysts. Similarly, formation of hyperproliferative and disorganized mammary acini induced by chronic stimulation of colony-stimulating factor 1 receptor is coupled to endogenous ARF6 activation and constitutive receptor internalization and is reversed by ARF6 inhibition. These findings identify a previously unrecognized link between ARF6-regulated receptor internalization and events that drive dramatic alterations in cyst morphogenesis providing new mechanistic insight into the molecular processes that can promote epithelial glandular disruption.

PTK6 Regulates IGF-1-induced Anchorage-independent Survival

PloS One. 2010  |  Pubmed ID: 20668531

Proteins that are required for anchorage-independent survival of tumor cells represent attractive targets for therapeutic intervention since this property is believed to be critical for survival of tumor cells displaced from their natural niches. Anchorage-independent survival is induced by growth factor receptor hyperactivation in many cell types. We aimed to identify molecules that critically regulate IGF-1-induced anchorage-independent survival.

Profiling Y561-dependent and -independent Substrates of CSF-1R in Epithelial Cells

PloS One. 2010  |  Pubmed ID: 21049007

Receptor tyrosine kinases (RTKs) activate multiple downstream cytosolic tyrosine kinases following ligand stimulation. SRC family kinases (SFKs), which are recruited to activated RTKs through SH2 domain interactions with RTK autophosphorylation sites, are targets of many subfamilies of RTKs. To date, there has not been a systematic analysis of the downstream substrates of such receptor-activated SFKs. Here, we conducted quantitative mass spectrometry utilizing stable isotope labeling (SILAC) analysis to profile candidate SRC-substrates induced by the CSF-1R tyrosine kinase by comparing the phosphotyrosine-containing peptides from cells expressing either CSF-1R or a mutant form of this RTK that is unable to bind to SFKs. This analysis identified previously uncharacterized changes in tyrosine phosphorylation induced by CSF-1R in mammary epithelial cells as well as a set of candidate substrates dependent on SRC recruitment to CSF-1R. Many of these candidates may be direct SRC targets as the amino acids flanking the phosphorylation sites in these proteins are similar to known SRC kinase phosphorylation motifs. The putative SRC-dependent proteins include known SRC substrates as well as previously unrecognized SRC targets. The collection of substrates includes proteins involved in multiple cellular processes including cell-cell adhesion, endocytosis, and signal transduction. Analyses of phosphoproteomic data from breast and lung cancer patient samples identified a subset of the SRC-dependent phosphorylation sites as being strongly correlated with SRC activation, which represent candidate markers of SRC activation downstream of receptor tyrosine kinases in human tumors. In summary, our data reveal quantitative site-specific changes in tyrosine phosphorylation induced by CSF-1R activation in epithelial cells and identify many candidate SRC-dependent substrates phosphorylated downstream of an RTK.

Mechanistic Biology in the Next Quarter Century

Molecular Biology of the Cell. Nov, 2010  |  Pubmed ID: 21079017

A Non-genetic Route to Aneuploidy in Human Cancers

Nature Cell Biology. Mar, 2011  |  Pubmed ID: 21336303

Aneuploidy is common in human tumours and is often indicative of aggressive disease. Aneuploidy can result from cytokinesis failure, which produces binucleate cells that generate aneuploid offspring with subsequent divisions. In cancers, disruption of cytokinesis is known to result from genetic perturbations to mitotic pathways or checkpoints. Here we describe a non-genetic mechanism of cytokinesis failure that occurs as a direct result of cell-in-cell formation by entosis. Live cells internalized by entosis, which can persist through the cell cycle of host cells, disrupt formation of the contractile ring during host cell division. As a result, cytokinesis frequently fails, generating binucleate cells that produce aneuploid cell lineages. In human breast tumours, multinucleation is associated with cell-in-cell structures. These data define a previously unknown mechanism of cytokinesis failure and aneuploid cell formation that operates in human cancers.

Uncovering a Tumor Suppressor for Triple-negative Breast Cancers

Cell. Mar, 2011  |  Pubmed ID: 21376226

"Triple-negative" breast cancers are aggressive malignancies that respond poorly to treatments. Now Sun et al. (2011) find that the activity of the protein tyrosine phosphatase PTPN12 is lost in a large percentage of this breast cancer subtype, offering molecular drivers and possible therapeutic targets for this heterogeneous and intractable cancer.

Psidin, a Conserved Protein That Regulates Protrusion Dynamics and Cell Migration

Genes & Development. Apr, 2011  |  Pubmed ID: 21406550

Dynamic assembly and disassembly of actin filaments is a major driving force for cell movements. Border cells in the Drosophila ovary provide a simple and genetically tractable model to study the mechanisms regulating cell migration. To identify new genes that regulate cell movement in vivo, we screened lethal mutations on chromosome 3R for defects in border cell migration and identified two alleles of the gene psidin (psid). In vitro, purified Psid protein bound F-actin and inhibited the interaction of tropomyosin with F-actin. In vivo, psid mutations exhibited genetic interactions with the genes encoding tropomyosin and cofilin. Border cells overexpressing Psid together with GFP-actin exhibited altered protrusion/retraction dynamics. Psid knockdown in cultured S2 cells reduced, and Psid overexpression enhanced, lamellipodial dynamics. Knockdown of the human homolog of Psid reduced the speed and directionality of migration in wounded MCF10A breast epithelial monolayers, whereas overexpression of the protein increased migration speed and altered protrusion dynamics in EGF-stimulated cells. These results indicate that Psid is an actin regulatory protein that plays a conserved role in protrusion dynamics and cell migration.

The Myosin-II-responsive Focal Adhesion Proteome: a Tour De Force?

Nature Cell Biology. Mar, 2011  |  Pubmed ID: 21423174

The formation and maturation of focal adhesions involves significant changes in protein composition and requires acto-myosin contractility. A mass spectrometry approach reveals changes to the focal adhesion proteome on myosin inhibition, providing a valuable resource for the cell adhesion field.

The Myosin-II-responsive Focal Adhesion Proteome: a Tour De Force?

Nature Cell Biology. Apr, 2011  |  Pubmed ID: 21460805

The formation and maturation of focal adhesions involves significant changes in protein composition and requires acto-myosin contractility. A mass spectrometry approach reveals changes to the focal adhesion proteome on myosin inhibition, providing a valuable resource for the cell adhesion field.

Phosphoglycerate Dehydrogenase Diverts Glycolytic Flux and Contributes to Oncogenesis

Nature Genetics. Sep, 2011  |  Pubmed ID: 21804546

Most tumors exhibit increased glucose metabolism to lactate, however, the extent to which glucose-derived metabolic fluxes are used for alternative processes is poorly understood. Using a metabolomics approach with isotope labeling, we found that in some cancer cells a relatively large amount of glycolytic carbon is diverted into serine and glycine metabolism through phosphoglycerate dehydrogenase (PHGDH). An analysis of human cancers showed that PHGDH is recurrently amplified in a genomic region of focal copy number gain most commonly found in melanoma. Decreasing PHGDH expression impaired proliferation in amplified cell lines. Increased expression was also associated with breast cancer subtypes, and ectopic expression of PHGDH in mammary epithelial cells disrupted acinar morphogenesis and induced other phenotypic alterations that may predispose cells to transformation. Our findings show that the diversion of glycolytic flux into a specific alternate pathway can be selected during tumor development and may contribute to the pathogenesis of human cancer.

Erk Regulation of Pyruvate Dehydrogenase Flux Through PDK4 Modulates Cell Proliferation

Genes & Development. Aug, 2011  |  Pubmed ID: 21852536

Loss of extracellular matrix (ECM) attachment leads to metabolic impairments that limit cellular energy production. Characterization of the metabolic alterations induced by ECM detachment revealed a dramatic decrease in uptake of glucose, glutamine, and pyruvate, and a consequent decrease in flux through glycolysis, the pentose phosphate pathway, and the tricarboxylic acid (TCA) cycle. However, flux through pyruvate dehydrogenase (PDH) is disproportionally decreased, concomitant with increased expression of the PDH inhibitory kinase, PDH kinase 4 (PDK4), and increased carbon secretion. Overexpression of ErbB2 maintains PDH flux by suppressing PDK4 expression in an Erk-dependent manner, and Erk signaling also regulates PDH flux in ECM-attached cells. Additionally, epidermal growth factor (EGF), a potent inducer of Erk, positively regulates PDH flux through decreased PDK4 expression. Furthermore, overexpression of PDK4 in ECM-detached cells suppresses the ErbB2-mediated rescue of ATP levels, and in attached cells, PDK4 overexpression decreases PDH flux, de novo lipogenesis, and cell proliferation. Mining of microarray data from human tumor data sets revealed that PDK4 mRNA is commonly down-regulated in tumors compared with their tissues of origin. These results identify a novel mechanism by which ECM attachment, growth factors, and oncogenes modulate the metabolic fate of glucose by controlling PDK4 expression and PDH flux to influence proliferation.

Intersection of FOXO- and RUNX1-mediated Gene Expression Programs in Single Breast Epithelial Cells During Morphogenesis and Tumor Progression

Proceedings of the National Academy of Sciences of the United States of America. Oct, 2011  |  Pubmed ID: 21873240

Gene expression networks are complicated by the assortment of regulatory factors that bind DNA and modulate transcription combinatorially. Single-cell measurements can reveal biological mechanisms hidden by population averages, but their value has not been fully explored in the context of mRNA regulation. Here, we adapted a single-cell expression profiling technique to examine the gene expression program downstream of Forkhead box O (FOXO) transcription factors during 3D breast epithelial acinar morphogenesis. By analyzing patterns of mRNA fluctuations among individual matrix-attached epithelial cells, we found that a subset of FOXO target genes was jointly regulated by the transcription factor Runt-related transcription factor 1 (RUNX1). Knockdown of RUNX1 causes hyperproliferation and abnormal morphogenesis, both of which require normal FOXO function. Down-regulating RUNX1 and FOXOs simultaneously causes widespread oxidative stress, which arrests proliferation and restores normal acinar morphology. In hormone-negative breast cancers lacking human epidermal growth factor receptor 2 (HER2) amplification, we find that RUNX1 down-regulation is strongly associated with up-regulation of FOXO1, which may be required to support growth of RUNX1-negative tumors. The coordinate function of these two tumor suppressors may provide a failsafe mechanism that inhibits cancer progression.

Ovarian Cancer Spheroids Use Myosin-generated Force to Clear the Mesothelium

Cancer Discovery. Jul, 2011  |  Pubmed ID: 22303516

Dissemination of ovarian tumors involves the implantation of cancer spheroids into the mesothelial monolayer on the walls of peritoneal and pleural cavity organs. Biopsies of tumors attached to peritoneal organs show that mesothelial cells are not present under tumor masses. We have developed a live, image-based in vitro model in which interactions between tumor spheroids and mesothelial cells can be monitored in real time to provide spatial and temporal understanding of mesothelial clearance. Here we provide evidence that ovarian cancer spheroids utilize integrin - and talin - dependent activation of myosin and traction force to promote mesothelial cells displacement from underneath a tumor cell spheroid. These results suggest that ovarian tumor cell clusters gain access to the sub-mesothelial environment by exerting force on the mesothelial cells lining target organs, driving migration and clearance of the mesothelial cells.

ErbB2 Stabilizes Epidermal Growth Factor Receptor (EGFR) Expression Via Erk and Sprouty2 in Extracellular Matrix-detached Cells

The Journal of Biological Chemistry. Jan, 2011  |  Pubmed ID: 20956544

Epithelial cells are dependent on extracellular matrix (ECM) attachment for maintenance of metabolic activity and suppression of apoptosis. Here we show that loss of ECM attachment causes down-regulation of epidermal growth factor receptor (EGFR) and β1 integrin protein and mRNA expression and that ErbB2, which is amplified in 25% of breast tumors, reverses these effects of ECM deprivation. ErbB2 rescue of β1 integrin mRNA and protein in suspended cells is dependent on EGFR, however, the rescue of EGFR expression does not require β1 integrin. We show that there is a significant decrease in the stability of EGFR in ECM-detached cells that is reversed by ErbB2 overexpression. Rescue of both EGFR and β1 integrin protein by ErbB2 is dependent on Erk activity and induction of its downstream target Sprouty2, a protein known to regulate EGFR protein stability. Interestingly, expression of EGFR and β1 integrin protein is more dependent on Erk/Sprouty2 in ECM-detached ErbB2-overexpressing cells when compared with ECM-attached cells. These results provide further insight into the ErbB2-driven anchorage independence of tumor cells and provide a new mechanism for regulation of EGFR and β1 integrin expression in ECM-detached cells.

Outgrowth of Single Oncogene-expressing Cells from Suppressive Epithelial Environments

Nature. Feb, 2012  |  Pubmed ID: 22318515

Tumorigenesis is a clonal evolution process that is initiated from single cells within otherwise histologically normal tissue. It is unclear how single, sporadic mutant cells that have sustained oncogenic alterations evolve within a tightly regulated tissue environment. Here we investigated the effects of inducing oncogene expression in single cells in organotypic mammary acini as a model to elucidate the processes by which oncogenic alterations initiate clonal progression from organized epithelial environments. Sporadic cells induced to overexpress oncogenes that specifically perturb cell-cycle checkpoints (for example, E7 from human papilloma virus 16, and cyclin D1), deregulate Myc transcription or activate AKT signalling remained quiescent within growth-arrested acini. By contrast, single cells that overexpress ERBB2 initiated a cellular cascade involving cell translocation from the epithelial layer, as well as luminal outgrowth that is characteristic of neoplastic progression in early-stage epithelial tumours. In addition, ERBB2-mediated cell translocation to the lumen was found to depend on extracellular-regulated kinase and matrix metalloproteinase activities, and genetic alterations that perturb local cell-matrix adhesion drove cell translocation. We also provide evidence that luminal cell translocation may drive clonal selection by promoting either the death or the expansion of quiescent oncogene-expressing cells, depending on whether the pre-existing alterations allow anchorage-independent survival and growth. Our data show that the initial outgrowth of single oncogene-expressing cells from organized epithelial structures is a highly regulated process, and we propose that a cell translocation mechanism allows sporadic mutant cells to evade suppressive micro-environments and elicits clonal selection for survival and proliferative expansion outside the native niches of these cells.