The growth of MCF 7 human breast cancer cells is stimulated in vitro by estradiol (E2) and we have previously shown that estrogen-regulated glycoproteins released into the culture medium can partly mimic this effect. In this paper, we evaluate the mitogenic activity of the 52 K glycoprotein, which is a major E2-stimulated protein released by MCF 7 cells. The 52 K protein was purified 600-fold by affinity chromatography on Concanavalin A and an anti-52 K monoclonal antibody Sepharose columns. The 99% purified 52 K protein fraction stimulated the growth of estrogen-deprived MCF 7 cells. A mean 1.7-fold increase was obtained with nanomolar concentrations of seven different preparations of 52 K protein. This stimulation represented 40% of the mitogenic effect of E2. Both the 52 K protein and E2 induced microvilli at the cell surface but the effect of the 52 K protein occurred earlier. Other putative growth factors which are also stimulated by E2 and observed by [35S]cysteine labeling did not comigrate with the purified 52 K protein. Finally, the labeled 52 K protein was found to enter MCF 7 cells and to be processed into an immunoreactive 34 K protein. These data indicate that the E2-regulated 52 K glycoprotein is an autocrine mitogen on MCF 7 cells in culture and support the hypothesis that estrogens stimulate the growth of mammary cancer via this (and possibly other) secreted protein(s) acting as autocrine (and paracrine?) growth factors.

Breast cancers containg eson receptors are responsive to an en treatment and have a better prognosis than e n receptor-negative tumors. The loss of n and progesterone receptors appears to be a id With a p in to less-differentiated tumors. We tfected the human estrogen receLpor into the es n receptor-negative Cell Culture. MDA-MB-231 and MCF7 cells were maintained in monolayer cultures in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10%o fetal bovine serum and 2 mM L-glutamine. MDA-ER (HC1 and HE5) and MDA-pSG1 cells were maintained in the same complete medium but with hygromycin B (200 ,ug/ml).In all experiments, steroids were withdrawn from cells by culture for 6 days in phenol red-free DMEM supplemented with 10% dextran-coated charcoal-treated fetal bovine serum and 2 mM L-glutamine. Tumor cells were harvested with 0.25% trypsin/0.02% EDTA. After 1 min, the monolayers were detached by tapping the flasks and pipetted gently into medium containing fetal bovine serum. The cells intended for mouse inoculations were washed by centrifugation and resuspended in phosphate-buffered saline (PBS). Cell number and viability were determined by staining a cell suspension aliquot with 0.2% trypan blue in saline solution and examining the cells in a hemocytometer.ER Quantification. Confluent cells grown in DMEM containing 10%1o fetal bovine serum were washed twice in chilled PBS. They were collected and pelleted in PBS (700 x g for 5 min at 40(). Cell pellets were suspended in 20 mM Tris HCl buffer (pH 7.5) containing 2 mM dithiothreitol, 20%o (vol/vol) glycerol, and 0.4 M KCI as in ref. 19; the suspension was submitted to a freeze-thaw cycle prior to centrifugation at 10,000 x g for 15 min at 40C in an Eppendorf centrifuge. ERs were assayed in the supernatants with an enzyme immunoassay (Abbott Diagnostic, Rungis, France). Total protein concentrations of the supernatants were measured by the Bradford technique, using albumin for the standard curve.

Numerous scaffold proteins coordinate signals from the environment with actin-based protrusions during shape change and migration. Many scaffolds integrate signals from Rho-family GTPases to effect the assembly of specific actin structures. Here we investigate the mechanism of action MIM-B (missing in metastasis-B) on the actin cytoskeleton. MIM-B binds actin monomer through a WASP homology 2 motif, bundles actin filaments via an IRSp53/MIM domain, and is a long isoform of MIM, a proposed metastasis suppressor. We analysed the activity of MIM-B toward the actin cytoskeleton as well as its potential link to cancer metastasis. Endogenous MIM-B protein is widely expressed and its expression is maintained in various metastatic cell lines. MIM-B induces lamellipodia-like actin-rich protrusions. The IRSp53/MIM domain of MIM-B, as well as Rac activity are required to induce protrusions, but not the WASP homology 2 motif. MIM-B binds and activates Rac via its IRSp53/MIM domain, but this is not sufficient to induce lamellipodia. Finally, our data revealed that actin bundling and Rac-binding properties of MIM-B are not separable. Thus, MIM-B is unlikely to be a metastasis suppressor but acts as a scaffold protein that interacts with Rac, actin and actin-associated proteins to modulate lamellipodia formation.

The aspartyl-protease cathepsin D (cath-D) is overexpressed and hypersecreted by epithelial breast cancer cells and stimulates their proliferation. As tumor epithelial–fibroblast cell interactions are important events in cancer progression, we investigated whether cath-D overexpression affects also fibroblast behavior. We demonstrate a requirement of cath-D for fibroblast invasive growth using a three-dimensional (3D) coculture assay with cancer cells secreting or not pro-cath-D. Ectopic expression of cath-D in cath-D–deficient fibroblasts stimulates 3D outgrowth that is associated with a significant increase in fibroblast proliferation, survival, motility, and invasive capacity, accompanied by activation of the ras–MAPK pathway. Interestingly, all these stimulatory effects on fibroblasts are independent of cath-D proteolytic activity. Finally, we show that pro-cath-D secreted by cancer cells is captured by fibroblasts and partially mimics effects of transfected cath-D. We conclude that cath-D is crucial for fibroblast invasive outgrowth and could act as a key paracrine communicator between cancer and stromal cells, independently of its catalytic activity.