👤 Kirankumar Katta

Breast cancer remains a leading cause of cancer-related mortality in women. In recent years, regulation of genes involved in heparan sulphate (HS) biosynthesis have received increased interest as regulators of breast cancer cell adhesion and invasion. The exostosin (EXT) proteins are glycosyltransferases involved in elongation of HS, a regulator of intracellular signaling, cell-cell interactions, and tissue morphogenesis. The EXT family contains five members: EXT1, EXT2, and three EXT-like (EXTL) members: EXTL1, EXTL2, and EXTL3. While the expression levels of these enzymes change in tumor cells, little is known how this changes the structure and function of HS. In the present study, we investigated gene expression profiles of the EXT family members, their glycosyltransferase activities and HS structure in the estrogen receptor (ER), and progesterone receptor (PR) positive MCF7 cells, and the ER, PR, and human epidermal growth factor receptor-2 (HER2) negative MDA-MB-231 and HCC38 epithelial breast carcinoma cell lines. The gene expression profiles for MDA-MB-231 and HCC38 cells were very similar. In both cell lines Show less

Exostosin-1 (EXT1), a member of the EXT protein family, is indispensable for synthesis of heparan sulfate (HS) chains that bind to and modulate the signaling efficiency of numerous growth factor activities. We have previously shown that Ext1 mutated mouse embryonic fibroblasts produce short sulfated HS chains which dramatically influence tumor cell behavior in a 3-dimensional (3D) heterospheroid system composed of tumor cells and fibroblasts. In this study, we have used both 2D co-culture and 3D heterospheroid models, consisting of human A549 carcinoma cells co-cultured with wild-type or Ext1-mutated mouse embryonic fibroblasts. Gene expression profiling of differentially expressed genes in fibroblast/A549 heterospheroids identified P311 as a gene substantially down-regulated in A549 cells co-cultured with Ext1-mutated fibroblasts. In addition, we observed that the Ext1 mutants displayed reduced Tgf-β1 mRNA levels and lower levels of secreted active TGF-β protein. Re-introduction of Ext1 in the Ext1 mutant fibroblasts rescued the levels of Tgf-β1 mRNA, increased the amounts of secreted active TGF-β in these cells, as well as P311 mRNA levels in adjacent A549 cells. Accordingly, small interfering RNAs (siRNAs) against fibroblast Tgf-β1 reduced P311 expression in neighboring A549 tumor cells. Our data raises the possibility that fibroblast Ext1 levels play a role in P311 expression in A549/fibroblast co-culture through TGF-β1. This study considers a possible novel mechanism of Ext1-regulated heparan sulfate structure in modifying tumor-stroma interactions through altering stromal tgf-ß1 expression. Show less

Heparan sulfate proteoglycans are ubiquitously located on cell surfaces and in the extracellular matrices. The negatively charged heparan sulfate chains interact with a multitude of different proteins, thereby influencing a variety of cellular and developmental processes, for example cell adhesion, migration, tissue morphogenesis, and differentiation. The human exostosin (EXT) family of genes contains five members: the heparan sulfate polymerizing enzymes, EXT1 and EXT2, and three EXT-like genes, EXTL1, EXTL2, and EXTL3. EXTL2 has been ascribed activities related to the initiation and termination of heparan sulfate chains. Here we further investigated the role of EXTL2 in heparan sulfate chain elongation by gene silencing and overexpression strategies. We found that siRNA-mediated knockdown of EXTL2 in human embryonic kidney 293 cells resulted in increased chain length, whereas overexpression of EXTL2 in the same cell line had little or no effect on heparan sulfate chain length. To study in more detail the role of EXTL2 in heparan sulfate chain elongation, we tested the ability of the overexpressed protein to catalyze the in vitro incorporation of N-acetylglucosamine and N-acetylgalactosamine to oligosaccharide acceptors resembling unmodified heparan sulfate and chondroitin sulfate precursor molecules. Analysis of the generated products revealed that recombinant EXTL2 showed weak ability to transfer N-acetylgalactosamine to heparan sulfate precursor molecules but also, that EXTL2 exhibited much stronger in vitro N-acetylglucosamine-transferase activity related to elongation of heparan sulfate chains. Show less