Autocrine Growth Stimulation of the MCF 7 Breast Cancer Cells by the Estrogen-Regulated 52 K Protein*

Abstract

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.

Cathepsin D is partly endocytosed by the LRP1 receptor and inhibits LRP1-regulated intramembrane proteolysis

“…We checked these results by investigating the effect of recombinant proteolytically inactive D231N pro-cath-D on the production of endogenous LRP1b-CTF in HMF .1(-) plasmids using Lipofectamine (Gibco-BRL, Life Technologies SAS, Villebon sur Yvette, France). pcDNA3.1(-)cath-D expression plasmid encoding human pre-pro-cath-D has previously been described (Vignon et al, 1986;Glondu et al, 2001Glondu et al, , 2002Berchem et al, 2002;Laurent-Matha et al, 2005;Hu et al, 2008). fibroblasts ( Figure 3C, panel a).…”

“…Human cath-D is synthesized as a 52-kDa precursor that is rapidly converted in the endosomes to form an active, 48-kDa, single-chain intermediate, and then in the lysosomes into the fully active mature protease, composed of a 34-kDa heavy chain and a 14-kDa light chain (Gieselmann et al, 1985). The overexpression of cath-D in breast cancer cells leads to the hypersecretion of the 52-kDa pro-cath-D into the extracellular environment (Vignon et al, 1986;Capony et al, 1989;Fusek and Vetvicka, 1994). Cath-D affects both the cancer cells and the stromal cells of the tumor microenvironment.…”

“…Inhibition of cath-D expression in breast cancer cells decreases tumor growth and metastasis Ohri et al, 2007;Vashishta et al, 2007). Human secreted pro-cath-D stimulates breast cancer cell proliferation (Vignon et al, 1986;Fusek and Vetvicka, 1994;Vetvicka et al, 1994;Ohri et al, 2008), fibroblast outgrowth (Laurent-Matha et al, 2005) and angiogenesis (Hu et al, 2008). We have shown that a mutated catalytically inactive version of cath-D (D231N) is still mitogenic for tumor cells and fibroblasts (Glondu et al, 2001;Berchem et al, 2002;Laurent-Matha et al, 2005).…”

Relationship between cathepsin-D content and disease-free survival in node-negative breast cancer patients: a meta-analysis

“…Cathepsin-D, firstly identified as a 52-kDa oestrogen-regulated glycoprotein (Westley et al, 1970), displays both proteolytic activity in culture and an autocrine mitogenic activity in breast cancer cells (Vignon et al, 1986). The involvement of cathepsin-D in cancer invasion is also supported by the demonstration that transfection of cathepsin-D cDNA into rat tumorigenic cells increases their metastatic potential in nude mice (Garcia et al, 1990).…”

“…To date, several biological factors have been identified and proposed as potential prognostic indexes in human breast cancer. Among these, particular attention has been focused on proteolytic enzymes, such as cathepsin-D and urokinase-type plasminogen activator, which are involved in basement membrane/extracellular matrix degradation and tumour invasiveness and metastasis (Liotta et al, 1991).Cathepsin-D, firstly identified as a 52-kDa oestrogen-regulated glycoprotein (Westley et al, 1970), displays both proteolytic activity in culture and an autocrine mitogenic activity in breast cancer cells (Vignon et al, 1986). The involvement of cathepsin-D in cancer invasion is also supported by the demonstration that transfection of cathepsin-D cDNA into rat tumorigenic cells increases their metastatic potential in nude mice (Garcia et al, 1990).…”