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.
In this study, we have analyzed the expression of TRIM24/TIF-1␣, a negative regulator of various transcription factors (including nuclear receptors and p53) at the genomic, mRNA, and protein levels in human breast tumors. In breast cancer biopsy specimens, TRIM24/TIF-1␣ mRNA levels (assessed by Real-Time Quantitative PCR or microarray expression profiling) were increased as compared to normal breast tissues. At the genomic level, array comparative genomic hybridization analysis showed that the TRIM24/TIF-1␣ locus (7q34) exhibited both gains and losses that correlated with mRNA levels. By re-analyzing a series of 238 tumors, high levels of TRIM24/TIF-1␣ mRNA significantly correlated with various markers of poor prognosis (such as the molecular subtype) and were associated with worse overall survival. By using a rabbit polyclonal antibody for immunochemistry, the TRIM24/TIF-1␣ protein was detected in nuclei of normal luminal epithelial breast cells, but not in myoepithelial cells. Tissue microarray analysis confirmed that its expression was increased in epithelial cells from normal to breast infiltrating duct carcinoma and correlated with worse overall survival. Altogether, this work is the first study that shows that overexpression of the TRIM24/TIF-1␣ gene in breast cancer is associated with poor prognosis and worse survival, and it suggests that this transcription coregulator may play a role in mammary carcinogenesis and represent a novel prognostic marker.
In an attempt to understand the mechanism by which estrogens stimulate cell proliferation and mammary carcinogenesis, metastatic human breast cancer cell lines (MCF7, ZR75-1) were found to secrete a 52,000 dalton (52K) protein under estrogen stimulation. Following its purification to homogeneity, the 52K protein was identified as a secreted procathepsin-D-like aspartyl protease bearing mannose-6-phosphate signals. This precursor displays an in vitro autocrine mitogenic activity on estrogen-deprived MCF7 cells and is able to degrade basement membrane and proteoglycans following its autoactivation. The total protease (52K + 48K and 34K) was detected and assayed by monoclonal antibodies and was found to be highly concentrated in proliferative and cystic mastopathies. In breast cancer, its cytosolic concentration appears to be correlated more to tumor invasiveness than to hormone responsiveness. The mRNA of the 52K protease accumulates rapidly following estradiol treatment, as was shown by Northern blot analysis with cloned cDNA. The 52K cathepsin-D-like protease is the first example of a lysosomal protease induced by estrogens in cancer cells. Results obtained using different approaches suggest that two cysteinyl cathepsins are also related to cell transformation and invasiveness. It has been proposed that cathepsin-B is involved in breast cancer and metastatic melanoma, and its regulation by estrogen has been shown in the rat uterus. Cathepsin-L corresponds to the major excreted protein (MEP) whose synthesis and secretion are markedly increased by transformation of NIH 3T3 cells with Ki ras and are regulated by several growth factors. In addition to secreted autocrine growth factors and to other proteases (plasminogen activator, collagenase), lysosomal cathepsins may therefore play an important role in the process of tumor growth and invasion as long as their precursor is secreted abundantly.
Abstract. We have studied the posttranslational modifications of the 52-kD protein, an estrogenl~gulated autocrine mitogen secreted by several human breast cancer cells in culture (Westley, B., and H. Rochefort, 1980, Cell, 20:353-362). The secreted 52-kD protein was found to be phosphorylated mostly (94%) on high-mannose N-linked oligosaccharide chains, and mannose-6-phosphate signals were identified. The phosphate signal was totally removed by alkaline phosphatase hydrolysis. The secreted 52-kD protein was partly taken up by MCF7 cells via mannose-6-phosphate receptors and processed into 48-and 34-kD protein moieties as with lysosomal hydrolases. By electron microscopy, immunoperoxidase staining revealed most of the reactive proteins in lysosomes. After complete purification by immunoaffinity chromatography, we identified both the secreted 52-kD protein and its processed cellular forms as aspartic and acidic proteinases specifically inhibited by pepstatin. The 52-kD protease is secreted in breast cancer cells under its inactive proenzyme form, which can be autoactivated at acidic pH with a slight decrease of molecular mass. The enzyme of breast cancer cells, when compared with cathepsin D(s) of normal tissue, was found to be similar in molecular weight, enzymatic activities (inhibitors, substrates, specific activities), and immunoreactivity. However, the 52-kD protein and its cellular processed forms of breast cancer cells were totally sensitive to endo-13-N-acetylglucosaminidase H (Endo H), whereas several cellular cathepsin D(s) of normal tissue were partially Endo H-resistant. This difference, in addition to others concerning tissue distribution, mitogenic activity and hormonal regulation, strongly suggests that the 52-kD cathepsin D-like enzyme of breast cancer cells is different from previously described cathepsin D(s). The 52-kD estrogen-induced lysosomal proteinase may have important functions in facilitating the mammary cancer cells to proliferate, migrate, and metastasize.
Earlier studies indicated that density-arrested cancer cells released an unidentified growth inhibitor whose secretion was prevented by overexpression of the lysosomal protease cathepsin D (cath D). In this study, this growth inhibitor was purified by affinity chromatography and identified as the heat shock cognate 70 protein (hsc70) based on its peptide microsequencing and specific antibody recognition. Among intracellular proteins, including other heat shock proteins, only constitutive hsc70 was secreted in response to the high-cell density. Moreover, hsc70 secretion from cancer cells was generated by serum deprivation, whereas its cellular concentration did not change. Prevention of Hsc70 secretion by cath D overexpression was associated with the formation of multilayer cell cultures, thus indicating a loss of contact inhibition. In addition, we showed that supplementing the culture medium with purified hsc70 inhibited cell proliferation in the nanomolar range. Conversely, removal of this extracellular hsc70 from the medium by either retention on ADP-agarose or competition at the Hsc70 binding site restored cell proliferation. Hsc70 appears active in human breast cancer cells and hypersecreted by direct cath D inhibition. These results suggest a new role of this secreted hsc70 chaperone in cell proliferation that might account for the higher tumor growth of cancer cells overexpressing cath D.
Estrogen receptors (ERs) are overexpressed in human breast cancers (BCs) and associated with differentiated tumors and with a more favorable prognosis. Paradoxically, ERs mediate the mitogenic action of estrogens in human BC cells and the efficacy of antiestrogens in adjuvant therapy of primary tumors. The exact mechanism underlying the ER protection against cancer progression to metastasis remains to be investigated. Herein, we show that ERs decrease invasiveness of BC cells. Detailed studies revealed that the unliganded and the E2-activated ERs decrease cancer cell invasion in vitro through two distinct mechanisms. In the presence of ligand, ERalpha inhibits invasion through a mechanism requiring the functional ERalpha domains involved in the transcriptional activation of target genes. Moreover, using different approaches, we found that cell-cell contacts were markedly increased by 17beta-estradiol (E2) treatment and decreased by the pure antiestrogen, ICI182,780. This cell-cell adhesion was associated with an increase of the major intercellular junctions, desmosomes. Conversely, in the absence of ligand, ERalpha also inhibits invasion through a distinct mechanism involving protein-protein interaction with the region of the first zinc finger of ERalpha. The relationship of these data with clinical studies and their potential therapeutic consequences will be discussed.
The breast and ovarian cancer susceptibility gene BRCA1 encodes a tumor suppressor. BRCA1 protein, which is involved in DNA damage response , has been thought to be found primarily in cell nuclei. In the present investigation , immunohistological studies of BRCA1 protein in frozen breast cancer tissue and MCF7 and HeLa cell lines revealed BRCA1 expression in both nucleoli and nucleoplasmic foci. The majority of known cancer-causing BRCA1 mutations induce protein truncation, highlighting a requirement for the BRCA1 C-terminal domain repeats in mediating BRCA1 tumor suppressor function. However, somatic mutations in BRCA1 have not been found in sporadic breast cancer tumor tissue. 5 Instead it is thought that BRCA1 participates in the tumorigenesis of sporadic breast cancer through reduction in BRCA1 mRNA and protein levels, as compared with normal tissue. -10Functionally, BRCA1 participates in many signaling pathways involved in transcription and checkpoint control, and is recruited for the formation of DNA repair complexes, in association with proteins such as Mre11-Nbs1-Rad50, and BRCA2.11 Cell cycle studies have shown that BRCA1 protein is found in nuclear foci (dots) during S-phase, and after ␥-irradiation BRCA1 colocalizes with BRCA1-associated ring domain and Rad51-containing foci. 12Our immunohistological studies of frozen tissue sections from breast carcinomas and transmission electron microscopic studies of estrogen-stimulated MCF7 cells have shown nuclear, nucleolar, and cytoplasmic BRCA1 protein staining. 13,14 With transmission electron microscopy, we found the BRCA1 nuclear staining on the periphery of dots, around nucleoli, and also in the cytoplasm in
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