Syndecan-1 accumulates in lysosomes of poorly differentiated breast carcinoma cells

Abstract

Expression patterns of syndecan-1, the cell surface heparan sulfate proteoglycan (HSPG) predominant on epithelial cells, were analyzed in tissue samples from 30 infiltrating human breast carcinomas and in 9 human breast carcinoma cell lines. Immunohistochemical staining demonstrates that while a subset of the breast carcinomas lose syndecan-1, this proteoglycan is expressed or overexpressed in a majority of the cases. Interestingly, cells in poor grade tumors contain intracellular syndecan-1, an observation that has not been previously described and was thus further investigated. Examination of cultured breast carcinoma cell lines indicates that they also display the phenotype of the syndecan-1 positive tumors and thereby provide a model system for analysis of intracellular syndecan-1. All cell lines examined express syndecan-1, and poorly differentiated lines such as BT549 cells internalize the proteoglycan from the cell surface where it accumulates as intact HSPG in intracellular vesicles. Colocalization studies using fluorescent markers identify these to be lysosomes. This finding is unexpected, as the accepted mechanism for degradation of syndecan HSPG following endocytosis is fragmentation of the protein core and glycosaminoglycan chains in endosomes, followed by delivery of the fragments to lysosomes. Lysosomal inactivation using ammonium chloride demonstrates that well-differentiated lines such as T47D and MCF-7 cells, which maintain the majority of syndecan-1 on their cell surfaces, also target intact constitutively endocytosed syndecan-1 to lysosomes. Taken together, these results suggest that mammary epithelial cells utilize a previously uncharacterized mechanism for syndecan-1 catabolism. In this pathway the proteoglycan remains intact as it passes through the endosomal system, prior to arriving at its site of intracellular degradation in lysosomes.

Introduction

Heparan sulfate proteoglycans (HSPGs) constitute a superfamily of molecules that are defined by the presence of covalently attached heparan sulfate (HS) glycosaminoglycan chains. Cell surface HSPGs are expressed on adherent cells and function as receptors for extracellular matrix and growth factors, and are thereby involved in the regulation of cellular processes including adhesion, morphology, and growth factor signaling (Park et al., 2000, Tumova et al., 2000). Two major classes of proteoglycans found at the cell surface are glypicans, which are tethered to the plasma membrane via a glycophosphatidylinositol (GPI) linkage, and syndecans, which span the plasma membrane (Bernfield et al., 1999). While many cell surface receptor systems ranging from G-protein coupled receptors to growth factor receptor tyrosine kinases are thought to be modulated by internalization and regulated catabolism (Tsao et al., 2001, Wiley and Burke, 2001), little attention has been given to the degradation mechanisms of these specific proteoglycans following their endocytosis from the cell surface.

Syndecans are a four-member family unified by the presence of short (∼30 amino acid) homologous cytoplasmic domains and highly conserved hydrophobic transmembrane domains, and are distinguished by extracellular domains that vary in amino acid sequence and length (Bernfield et al., 1999, Carey, 1997). Syndecans are expressed in distinct tissue and developmental patterns, raising the possibility that they perform cell type specific functions. Syndecan-4 is expressed in a variety of tissues, while syndecans 1–3 demonstrate more restricted expression patterns (Bernfield et al., 1993, Kim et al., 1994). Syndecan-1 is primarily expressed at the basolateral surface of simple epithelial cells and on keratinocytes, (Rapraeger et al., 1986, Sanderson and Bernfield, 1988), and several lines of evidence suggest that this proteoglycan is important in the function of epithelial cells. For example, syndecan-1 is downregulated during epithelial–mesenchymal interactions associated with changes in epithelial cell shape during development (Sun et al., 1998, Thesleff et al., 1991). Syndecan-1 is also reduced in a number of epithelial malignancies (Inki and Jalkanen, 1996, Sanderson, 2001), raising the possibility that loss of syndecan-1 may contribute to poor epithelial differentiation. Stanley et al. (1999) reported that syndecan-1 expression is reduced on infiltrating ductal carcinomas of the breast and induced in the surrounding stromal tissues. In vitro studies also suggest that syndecan-1 is important for the regulation of epithelial cells, since loss of syndecan-1 disrupts the morphology, growth, and migration of mouse mammary epithelial cells (Kato et al., 1995, Leppa et al., 1992).

Despite the putative importance of syndecan-1 expression in epithelial tissues and particularly in the mammary gland, changes in syndecan-1 expression during the transformation of epithelial cells are poorly understood. Interestingly, the presence of intracellular or cytoplasmic syndecan-1 has been reported in hepatocellular (Matsumoto et al., 1997), pancreatic (Conejo et al., 2000), squamous cell (Bayer-Garner et al., 2001), and breast carcinomas (Stanley et al., 1999). Little characterization or functional analysis of intracellular syndecan exists, however, based in part on the expectation that this proteoglycan will be rapidly degraded upon internalization. Indeed, prior reports on HSPG turnover in rat ovarian granulosa and colon carcinoma cells have described a stepwise catabolic mechanism for trypsin-sensitive proteoglycans, now known to be syndecans (Iozzo, 1987, Yanagishita, 1992, Yanagishita and Hascall, 1992). In this pathway, endocytosed syndecans undergo core protein proteolysis and glycosaminoglycan chain fragmentation in endosomes, followed by complete degradation of the glycosaminoglycan fragments in lysosomes. In contrast, GPI-anchored proteoglycans (glypicans) appear to be degraded exclusively in lysosomes, because partly degraded forms of these HSPG are not detected when lysosomotropic agents such as ammonium chloride and chloroquine are applied.

In the present study, syndecan-1 degradation is investigated in human breast carcinomas and carcinoma cell lines. This report is the first to analyze HSPG turnover in mammary epithelial cells, and to document catabolism of a specific proteoglycan, syndecan-1. Analysis of a panel of 30 infiltrating breast carcinomas reveals that a subset of the tumors lose expression of syndecan-1. In contrast, several of the carcinomas express or overexpress syndecan-1, and all cell lines examined express this proteoglycan. Strikingly, intracellular syndecan-1 is prominent in the most poorly differentiated tumors studied. Poorly differentiated, invasive cell lines mimic this intracellular syndecan-1 phenotype via accumulation of intact syndecan-1 in lysosomes. Lysosomal inactivation demonstrates that both well and poorly differentiated mammary epithelial cells deliver syndecan-1 directly to lysosomes via a non-catabolic route. These findings suggest that in mammary epithelial cells syndecan-1 degradation does not begin until the HSPG arrives in lysosomes, a mechanism distinct from that described in other cell types.