Showing papers by "Joan S. Brugge published in 2004"

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is required for induction of autophagy during lumen formation in vitro

Abstract: 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-XL 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.

Cooperation of the ErbB2 receptor and transforming growth factor β in induction of migration and invasion in mammary epithelial cells

Abstract: 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)β1 and TGFβ3 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 TGFβ 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 TGFβ and ErbB2 on migration and expression of activated Erk kinase was sufficient to cooperate with TGFβ to induce migration and invasion, suggesting that sustained Erk activation is critical for ErbB2/TGFβ cooperation. In addition, we show that costimulation of ErbB2 and TGFβ 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 TGFβ 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.

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

Abstract: Integrins are a family of heterodimeric transmembrane proteins that serve as receptors for extracellular matrix proteins such as fibronectin (FN), laminins, and collagens. Integrins act as important regulators of cell function through their ability to mediate adhesion to extracellular matrices, to induce cytoskeletal rearrangements, and to activate intracellular signaling pathways. The coordinated cellular response to matrix attachment through integrins has been shown to induce a panoply of changes in cell behavior, including alterations in cell survival, proliferation, spreading and migration, gene transcription, and differentiation (15, 18, 20, 21). Many intracellular signaling molecules are activated by integrin engagement, including components of the Ras/Raf/MEK/Erk pathway, the phosphatidylinositol 3′-kinase (PI-3K)/Akt pathway, Src and Abl tyrosine kinases, focal adhesion kinase (FAK), Rho GTPases, the scaffolding proteins Cas, Cbl, and paxillin (and associated signaling molecules), and the serine kinases protein kinase C (PKC), p21-activated kinase (PAK), integrin-linked kinase (ILK), and myosin light chain kinase (MLCK) (51). Many of the signaling molecules activated by integrins are also activated by other receptor-ligand interactions (52). This has raised important issues regarding the basis for signal specificity and whether there is coordination between distinct receptor pathways within the cell. The possibility that integrins can coordinate their activities with other receptors is supported by several recent findings demonstrating interdependence and cross talk between different classes of cellular receptors (12, 34, 52, 54). There are several examples of cross talk between integrins and receptor tyrosine kinase (RTK) pathways (14, 34, 52, 54). Growth factors that activate RTKs can regulate integrin-mediated events such as cell adhesion, cell spreading, and cell migration through alterations in integrin localization and activation (27, 32, 58). Conversely, signals generated by integrins are required for full activation of growth factor signaling pathways. For example, extracellular matrix-mediated adhesion is required for growth factor-induced cell cycle progression in fibroblasts (2, 16, 53). Integrins contribute to fibroblast cell cycle progression by regulating cyclin D1 expression through multiple pathways involving Erk, PI-3K, and the Rho family GTPases Rac, cdc42, and Rho (16, 44). Integrins also lower the levels of the negative cell cycle regulators p21cip1 and p27kip1 (10). More-recent observations provide evidence for a distinct type of cross talk in which integrins can activate RTKs in the absence of exogenously added receptor ligands (35, 41). Several examples of RTKs activated by integrins include epidermal growth factor receptor (EGFR), insulin receptor, platelet-derived growth factor receptor (PDGFR), hepatocyte growth factor receptor (HGFR/Met), vascular endothelial growth factor receptor (VEGFR), and Ron (17, 36, 50, 55, 56, 63). The full implications of these interactions have yet to be fully understood; however, they suggest yet another mechanism for cross talk between integrin- and growth factor-linked pathways. To better understand the role of integrin-induced RTK activation in integrin function, we examined which events triggered by attachment to integrin ligands are dependent on EGFR activation. These studies demonstrate that FN-induced EGFR activity is required for activation of a subset of integrin-induced signaling pathways and that these events are important for regulating early cell cycle events in epithelial cells. In addition, we show that integrin engagement may lead to EGF-independent proliferation under conditions where EGFR is overexpressed.

Use of Three-Dimensional Basement Membrane Cultures to Model Oncogene-Induced Changes in Mammary Epithelial Morphogenesis

Abstract: 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.

Autocrine CSF-1R activation promotes Src-dependent disruption of mammary epithelial architecture

Abstract: 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.

A cytoskeleton-based functional genetic screen identifies Bcl-xL as an enhancer of metastasis, but not primary tumor growth.

Abstract: 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 Mammory Tumor Progression?

Abstract: 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.

Discovery of new gene expression predictors for adjuvant tamoxifen outcome for breast cancer patients

Abstract: 9503 Background: A better understanding of tamoxifen resistance and accurately targeting tamoxifen therapy to responsive patients remain critical challenges in breast cancer treatment. Tamoxifen ac...