Jesus V. Soriano

Potent blockade of hepatocyte growth factor-stimulated cell motility, matrix invasion and branching morphogenesis by antagonists of Grb2 Src homology 2 domain interactions.

Hepatocyte growth factor (HGF) stimulates mitogenesis, motogenesis, and morphogenesis in a wide range of cellular targets during development, homeostasis and tissue regeneration. Inappropriate HGF signaling occurs in several human cancers, and the ability of HGF to initiate a program of protease production, cell dissociation, and motility has been shown to promote cellular invasion and is strongly linked to tumor metastasis. Upon HGF binding, several tyrosines within the intracellular domain of its receptor, c-Met, become phosphorylated and mediate the binding of effector proteins, such as Grb2. Grb2 binding through its SH2 domain is thought to link c-Met with downstream mediators of cell proliferation, shape change, and motility. We analyzed the effects of Grb2 SH2 domain antagonists on HGF signaling and observed potent blockade of cell motility, matrix invasion, and branching morphogenesis, with ED50 values of 30 nm or less, but only modest inhibition of mitogenesis. These compounds are 1000–10,000-fold more potent anti-motility agents than any previously characterized Grb2 SH2 domain antagonists. Our results suggest that SH2 domain-mediated c-Met-Grb2 interaction contributes primarily to the motogenic and morphogenic responses to HGF, and that these compounds may have therapeutic application as anti-metastatic agents for tumors where the HGF signaling pathway is active.

Roles of Hepatocyte Growth Factor/Scatter Factor and Transforming Growth Factor-β1 in Mammary Gland Ductal Morphogenesis

Epithelial-mesenchymal interactions areresponsible for the unique pattern of ductal branchingmorphogenesis characteristic of the mammary gland. Toinvestigate the factors which control the elongation and branching of lactiferous ducts, wedeveloped an in vitro model of ductal morphogenesis inwhich clonal mouse mammary epithelial cells (TAC-2cells) are grown in collagen gels. In this experimentalsystem, fibroblast conditioned medium (CM)3stimulates the formation of extensively arborizedtubules. The molecule responsible for this tubulogeniceffect was identified as hepatocyte growthfactor/scatter factor (HGF/SF). To determine whether HGF/SF plays arole in mammary gland morphogenesis in vivo, theexpression of HGF/SF and its receptor, cMet, wereanalyzed in the rat mammary gland during pregnancy,lactation, and involution. Levels of HGF/SF and c-Mettranscripts were progressively reduced during pregnancy,were virtually undetectable during lactation, andincreased again during involution. Collectively, these in vitro and in vivo findings suggest thatHGF/SF is a paracrine mediator of mammary gland ductalmorphogenesis. We subsequently investigated the effectof another multifunctional cytokine, namely TGF-beta1, on branching morphogenesis of TAC-2 cells.TGF-β1 had a striking biphasic effect:whereas relatively high concentrations of this cytokineinhibited colony formation, lower concentrationsstimulated extensive elongation and branching of epithelial cords.Taken together, these studies indicate that HGF/SF is astromal-derived paracrine mediator of mammary ductalmorphogenesis, and that when present at lowconcentrations, TGF-β1 can contribute to thisprocess.

REGULATION OF VEGF AND VEGF RECEPTOR EXPRESSION IN THE RODENT MAMMARY GLAND DURING PREGNANCY, LACTATION, AND INVOLUTION

Vascular endothelial growth factors (VEGFs) are endothelial cell‐specific mitogens with potent angiogenic and vascular permeability‐inducing properties. VEGF, VEGF‐C, and VEGFRs ‐1, ‐2, and ‐3 were found to be expressed in post‐pubertal (virgin) rodent mammary glands. VEGF was increased during pregnancy (5‐fold) and lactation (15–19‐fold). VEGF‐C was moderately increased during pregnancy and lactation (2‐ and 3‐fold respectively). VEGF levels were reduced by approximately 75% in cleared mouse mammary glands devoid of epithelial components, demonstrating that although the epithelial component is the major source of VEGF, approximately 25% is derived from stroma. This was confirmed by the findings (a) that VEGF transcripts were expressed predominantly in ductal and alveolar epithelial cells, and (b) that VEGF protein was localized to ductal epithelial cells as well as to the stromal compartment including vascular structures. VEGF was detected in human milk. Finally, transcripts for VEGFRs ‐2 and ‐3 were increased 2–3‐fold during pregnancy, VEGFRs ‐1, ‐2 and ‐3 were increased 2‐4‐fold during lactation, and VEGFRs ‐2 and ‐3 were decreased by 20‐50% during involution. These results point to a causal role for the VEGF ligand‐receptor pairs in pregnancy‐associated angiogenesis in the mammary gland, and suggest that they may also regulate vascular permeability during lactation. Dev Dyn 2000;218:507–524.

Induction of tissue inhibitor of metalloproteinases-3 is a delayed early cellular response to hepatocyte growth factor

Hepatocyte growth factor (HGF) stimulates mitogenic, motogenic, and morphogenic responses in various cell types. We analysed HGF-responsive cells by differential display PCR to identify HGF-induced genes that mediate these biological events. One of the genes identified encoded a member of the tissue inhibitor of metalloproteinases (TIMP) family, TIMP-3. HGF transiently induced TIMP-3 mRNA in keratinocytes as well as kidney and mammary epithelial cells maximally between 4 and 6 h post-stimulation. Increased TIMP-3 protein secretion returned to basal levels within 18 h, while the expression of gelatinases A and B remained unchanged, suggesting that temporary suppression of matrix degradation is a delayed early response to HGF. Ectopic overexpression of TIMP-3 in cultured leiomyosarcoma cells conferred an epithelial morphology, reduced cell growth rate, anchorage-independent growth, and matrix invasion in vitro. Antisense suppression of TIMP-3 was associated with a scattered, fibroblastic cell morphology, as well as enhanced proliferation, anchorage-independent growth, and matrix invasion. A survey of tumor cell lines revealed an inverse relationship between metastatic potential and TIMP-3 expression level. These data suggest that early, transient TIMP-3 expression mediates specific HGF-induced phenotypic changes, and that loss of TIMP-3 expression may enhance the invasion potential of certain tumors.

A role for atm in E-cadherin-mediated contact inhibition in epithelial cells

SummaryAtaxia telangiectasia is a hereditary pleiomorphic syndrome caused by loss of Atm, a phosphoprotein involved in multiple signaling pathways. Here, we propose a novel role for atm in cultured epithelial cells, namely the regulation of cell growth by contact inhibition. We show that atm is upregulated in epithelial cells reaching confluence. Conditional expression of the PI 3-Kinase domain of atm in non-confluent Tac-2 epithelial cells increases the expression of the anti-proliferative gene Tis-21 and downregulates key cell cycle regulator genes, such as cyclins A, B1, B2, E and E2. Finally, we demonstrate that upregulation of atm, and thus Tis-21, in confluent Tac-2 cells can be inhibited by an E-cadherin antibody blocking specifically homophilic E-cadherin interactions between adjacent cell surfaces. Altogether, these results suggest that atm could participate in a molecular pathway linking extracellular signalling to cell cycle control and may help further clarify the role of Atm in epithelial cell biology and carcinogenesis.