Endocytosis of pro-cathepsin D into breast cancer cells is mostly independent of mannose-6-phosphate receptors.

Cathepsin D trafficking is altered in cancer cells, leading to increased secretion of the pro-enzyme, which can be reinternalized by the same cancer cells and by stromal cells. We studied pro-cathepsin D endocytosis in two human breast cancer cell lines (MDA-MB231, MCF-7) and in human normal fibroblasts. Pro-enzyme uptake was studied indirectly through immunofluorescence analysis of anti-pro-cathepsin D monoclonal antibodies internalized in living cells. Both cancer cell lines internalized the pro-cathepsin D-antibody complex into endosomal compartments in the presence of 10 mM mannose-6-phosphate. Non-malignant fibroblasts, which do not secrete pro-cathepsin D, only internalized anti-cathepsin D antibody when purified pro-cathepsin D was added and this endocytosis was totally inhibited by mannose-6-phosphate. Cathepsin D endocytosis in cancer cells was not mediated by lectins or another receptor binding the cathepsin profragment. It was not due to fluid endocytosis, since another protein pS2 secreted by MCF-7 was not endocytosed with its antibody in the same conditions. Double-immunofluorescence and confocal microscopy analyses revealed that antibodies specific to pro-cathepsin D (M2E8) and to the mannose-6-phosphate/IGFII receptor were co-internalized independently in non-permeabilized MDA-MB231 cells and MCF-7 cells, but not in fibroblasts. Moreover, when metabolically labelled pro-cathepsin D secreted by MCF-7 or MDA-MB231 cells was incubated with homologous or heterologous non-radioactive cells, the time-dependent uptake and maturation of the pro-enzyme into fibroblasts were totally inhibited by mannose-6-phosphate, whereas they were not in the two breast cancer cell lines. The percentage of mannose-6-phosphate-independent binding of radioactively labelled pro-cathepsin D to MDA-MB231 cells at 16 degrees C was higher (7-8%) at low pro-cathepsin D concentration than at high concentration (1.5%), indicating the presence of saturable binding site(s) at the cell surface that are different from the mannose-6-phosphate receptors. We conclude that, in contrast to fibroblasts, breast cancer cells can endocytose the secreted pro-cathepsin D by a cell surface receptor that is different from the mannose-6-phosphate receptors or other lectins. The nature of this alternative receptor and its significance in the action of secreted pro-cathepsin D remain to be elucidated.

Adenomatous transformation of the human anterior pituitary is associated with alterations in integrin expression.

Integrins are heterodimeric transmembrane molecules that mediate cell-cell and cell-substratum adhesion. Because alterations in the adhesive properties of tumor cells influence tumor growth and progression, the distribution of different alpha and beta integrin subunits was studied in both the parenchyma and the connective tissue in 6 normal and 25 adenomatous human anterior pituitaries. All normal parenchymal cells expressed the alpha3beta1 and alpha6beta4 integrins. By contrast, in adenomatous parenchymal cells the expression of alpha3beta1 was down-regulated and that of alpha6beta4 abrogated. Neoexpression of alphavbeta3 Occurred in the parenchyma of a subset of adenomas. All normal connective tissue cells expressed the alpha1 and beta1 subunits, a third subunit (alpha5) being present in the normal endothelium. By comparison, all adenomatous stromal cells expressed many more integrin subunits (alpha1, alpha3, alpha5, alphav, beta1 and beta3), adenomatous endothelial cells bearing additional subunits (alpha6, beta4 and beta5). Vitronectin, absent from the normal connective tissue, was constantly observed in the adenomatous stroma. To conclude, compared with cells of the normal gland, adenomatous anterior pituitary cells display a decreased expression of integrins whereas the adenomatous stroma expresses a rich repertoire of integrins. These changes are not related to the secretory type, grade or invasiveness of the adenoma. The resulting alterations in the adhesive properties of adenomatous cells could facilitate their dissemination. Enrichment of the integrin repertoire expressed by the adenomatous vasculature is indicative of its dual nature, systemic and tumoral.

Fibronectin isoforms are differentially expressed in normal and adenomatous human anterior pituitaries.

The expression of fibronectin (FN) isoforms containing the extradomains A and B (ED-A+ and ED-B+ FNs) as well as a differentially O-glycosylated oncofetal form of the protein (onf-FN) was investigated in 6 normal human anterior pituitaries and 25 human pituitary adenomas. In normal tissue, immunohistochemical experiments showed the presence of FN molecules lacking the extradomains A and B (ED-A- and ED-B- FNs) without onf-FN immunoreactivity. These proteins were localized in the connective tissue compartment and especially in the vessel walls. Analysis of FN mRNA demonstrated an in situ synthesis of ED-A- and ED-B- FNs in the normal anterior pituitary. By contrast, in the adenomas, immunoreactivity for ED-A+ FN was observed in all cases. ED-B+ and onf-FN immunoreactivities were observed in 14 and 8 adenomas, respectively, regardless of the type, grade or invasiveness of the adenomas. ED-A+ FN mRNA was expressed in all adenomas studied, and ED-B+ FN mRNA was present in ED-B+ immunoreactive cases only. In pituitary adenomas, these 3 forms of FN were specifically associated with the endothelium and vascular smooth-muscle cells. Our results demonstrate that the processes of remodelling of the connective tissue compartment that occur in adenoma angiogenesis are associated with pre- and post-translational alterations of FN synthesis leading to the expression of ED-A+, ED-B+ and oncofetal FNs.

Immunohistochemical localization of different laminin isoforms in human normal and adenomatous anterior pituitary.

BACKGROUND: Laminin (LM) is an integral component of basement membranes (BM), with important roles in various aspects of cell biology. Several different isoforms of LM have been described, and each comprises a molecule consisting of three subunit polypeptides, the A, B1, B2, M or S chain. EXPERIMENTAL DESIGN: The distribution of different LM subunits was studied in human nontumoral anterior pituitaries obtained postmortem and in pituitary adenomas by immunocytochemical methods using specific monoclonal antibodies. RESULTS: In normal tissue, the A, B1, and B2 chains had a ubiquitous localization in both parenchymatous and vascular BMs and in the pericapillary connective tissue space also. The S chain seemed to have principally a vascular localization, in contrast to the M chain that was mostly localized in the parenchymatous BM. The same antigens were investigated in 23 human pituitary adenomas of different secretory type, grade and invasiveness. In all cases, the five monoclonal antibodies gave a positive staining. The presence or the localization of LM chains did not display a specific pattern in the adenomas according to their secretory type, grade or extent of local invasion. In the adenomas, the anti-A, -B1, and -B2 monoclonal antibodies stained the stroma and in particular the vascular BMs as well as the sparse elements of parenchymatous BMs when they were present. The staining with anti-S antibody was localized in the stroma and around all the blood vessels. In contrast, the immunoreactive material to anti-M antibody was associated to the sparse fragments of parenchymatous BMs and it delineated the boundary of adenoma cells and stroma. Within the stroma, the anti-M antibody immunoreactivity was regularly associated with the arterial walls but rarely with the venule walls. CONCLUSIONS: The human normal and tumoral anterior pituitary express all five LM subunits and thus contain several LM isoforms with different patterns of localization. The most striking difference between the human normal and tumoral anterior pituitary concerns the peculiar expression of LM isoforms by adenomatous neovessels.

Interactions between normal and tumoral tissues at the boundary of human anterior pituitary adenomas. An immunohistochemical study.

We studied the boundary between adenoma and peritumoral anterior pituitary tissues in order to understand their mutual interactions during tumour progression. We selected 18 adenomas of different secretory type, grade and invasiveness in which fragments of peritumoral anterior pituitary were still attached to the adenoma. Immunohistochemistry was performed on serial sections with markers of the basement membranes (type IV collagen), the hormone-producing cells of the normal and neoplastic anterior pituitary, and the folliculo-stellate cells (S-100 protein). In passing from tumour to gland, localized areas of passive compression of the normal gland were seen in only 3 cases. In all the tumours, the boundary consisted partly or solely of a transitional zone characterized by the presence of enlarged cell-cords. Openings in the basement membrane of these enlarged cell-cords were seen in contact with the tumour tissue. Normal and neoplastic cells intermingled in the transitional zone. Normal residual cells could be seen in the central area of the tumour but no adenomatous cells were observed in the gland around the tumour. Folliculo-stellate cells were concentrated in the vicinity of the transition zone. These findings favour the existence of an active process of adenoma expansion within the normal parenchyma, without noticeable infiltration of tumour cells into surrounding gland.

Specific alterations of the basement membrane and stroma antigens in human pituitary tumours in comparison with the normal anterior pituitary. An immunocytochemical study.

Our report is the first immunocytochemical study of the principal elements of the basement membrane (BM) and connective tissue in normal and adenomatous human anterior pituitaries. In normal tissues, both the parenchymatous BM limiting the endocrine cell cords and the endothelial BM around the capillaries were continuous and were stained with anti-laminin (LM), anti-type IV collagen (CIV) and anti-fibronectin (FN) antisera. Antiserum to type I collagen (CI) stained the connective tissue only. The same antigens were investigated in 23 human pituitary adenomas, 6 of them having been diagnosed as locally invasive by the radiologist and the neurosurgeon. In all cases a lack of cordal structure was observed and the parenchymatous BM was completely absent (9 cases) or fragmented (14 cases). No correlation could be established between the extent of parenchymatous BM alterations and the invasive behaviour of the tumour. In contrast, a continuous endothelial BM was observed around the blood vessels in all cases and its presence was confirmed in double immunofluorescence experiments using anti-von Willebrand factor and anti-LM or anti-CIV antisera. Anti-FN and CI also stained the wall of the vessels. The tumours showed arterial development, in addition to the capillaries found in normal tissue. The present results favour the hypothesis of a decreased synthesis of parenchymatous BM by human adenomatous pituitary cells in comparison with normal cells and show that these tumours are the site of an active arterial neovascularization.