Hyaluronan Constitutively Regulates Activation of Multiple Receptor Tyrosine Kinases in Epithelial and Carcinoma Cells

Abstract

Hyaluronan (HA) is enriched in the pericellular matrices of many malignant human tumors, and manipulations of HA interactions have strong effects on tumor progression in animal models. Increased HA production stimulates ERBB2 activation, leading to increased cell survival activities and several malignant cell properties. On the other hand, inhibition of constitutive HA-tumor cell interactions in malignant cells inhibits these properties. We have now investigated the role of HA in activation of several additional receptor tyrosine kinases (RTKs), i.e. IGF1R-␤, PDGFR-␤, EGFR and c-MET, in colon, prostate, and breast carcinoma cells. In each case we show that antagonists of endogenous HA interactions inhibit their tyrosine phosphorylation, i.e. activation. On the other hand, we show that these RTKs are activated in phenotypically normal or relatively benign tumor cells by experimentally increasing HA production. We also investigated the role of HA in constitutive versus ligand-induced activation of RTKs. In HCA7 colon and C4-2 prostate carcinoma cells, ERBB2 is constitutively activated in a ligand-independent manner, whereas IGF1R-␤ and PDGFR-␤ require ligand interaction for activation. We show that both constitutive activation of ERBB2 and ligand-mediated activation of IGF1R-␤ and PDGFR-␤ are reversed by co-treatment of the cells with a HA antagonist. We conclude that HA serves a general function in RTK activation.Aberrant activities of several types of RTKs 3 have been implicated in the genesis of a significant proportion of human cancers, including breast, colon, and prostate carcinomas. Increased RTK activity can arise from gene amplification, activating mutations, or altered regulation, e.g. by cross-talk between RTKs, integrins, and other receptors or by autocrine and paracrine stimulation by various regulatory factors. These changes lead in turn to enhanced tumor cell growth, motility, survival, and resistance to therapies (1-4).HA is a very large, anionic polysaccharide that is localized at the cell surface and in the extracellular matrix of numerous tissues, wherein it plays an important structural role under homeostatic conditions. However, when cells proliferate or migrate, e.g. in embryonic processes, tissue remodeling, inflammation, and diseases such as cancer and atherosclerosis, HA-induced signaling is activated (5-10). High levels of HA in tumors are a prognostic factor in several malignancies (5), and manipulations of HA production or interaction with cell surface receptors strongly influence tumor growth and metastasis (7, 10). CD44 is a major cell surface receptor for HA and also plays an important role in tumor progression (11, 12). We and others have shown that interaction of HA with CD44 induces formation of complexes containing CD44 and ERBB2 or EGFR in a variety of tumor cells (13-16). We have shown that HA constitutively regulates formation of a signaling complex containing phosphorylated ERBB2, CD44, ezrin, PI3K, and the chaperone molecules HSP90 and CDC37 (16,17). In this work we have use...

Expression of Versican V3 by Arterial Smooth Muscle Cells Alters Tumor Growth Factor β (TGFβ)-, Epidermal Growth Factor (EGF)-, and Nuclear Factor κB (NFκB)-dependent Signaling Pathways, Creating a Microenvironment That Resists Monocyte Adhesion

“…Hyaluronan synthesis is regulated by a number of pathways, including the EGFR and NFB signaling pathways (71). Hyaluronan triggers the CD44-EGFR signaling pathway, which up-regulates hyaluronan synthesis in a positive feedback mechanism (33)(34)(35)(36)(37). Moreover, hyaluronan synthases have NFB binding regions in their promoters and are up-regulated by NFB agonists, such as IL-1␤ and TNF␣ (25, 38 -40).…”

“…Expression of V3 Attenuates EGFR-and NFB-dependent Signaling Pathways-Hyaluronan synthesis is regulated by a number of signaling pathways, including the EGFR-PI3K/ERK signaling network (33)(34)(35)(36)(37). Moreover, hyaluronan synthases have NFB binding regions in their promoters that are regulated by NFB agonists, such as interleukin 1␤ (IL-1␤) and tumor necrosis factor ␣ (TNF␣) (25, 38 -40).…”

Fluorescence Resonance Energy Transfer (FRET) and Proximity Ligation Assays Reveal Functionally Relevant Homo- and Heteromeric Complexes among Hyaluronan Synthases HAS1, HAS2, and HAS3

“…A genetic defect in hyaluronan synthesis leads to a lethal failure in heart development (4). Recent research has also assigned a number of critical signaling functions to hyaluronan, mediated by its cell surface receptors (CD44 and RHAMM) (5-7) and common growth factor receptors like those of the ErbB (8), PDGF (9), and TGF␤ families (10). There is now considerable interest in the metabolism of hyaluronan as a potential target of therapy in pathological processes such as cancer (11), arterial disease (12), diabetes (13), and various forms of inflammation (14).…”

Delivery of CD44 shRNA/Nanoparticles within Cancer Cells

“…Increased HA in these cells regulates expression and enzymatic activity of COX-2, activation of ErbB2 and AKT, and translocates ␤-catenin to the nucleus (30,31). To explore the mechanism of constitutive HA-CD44 interaction and the consequent outcomes in cancer cells, we have shown that all four types of reagents, namely HA-oligosaccharides, anti-CD44 antibody, soluble CD44, or CD44 siRNA, block signaling responses in a variety of tumor cell types and also block activation of receptor tyrosine kinases (30,31,33,34). Although the anti-tumor activity of HA-oligosaccharides is effective in sensitizing tumor cells to chemotherapeutic agents in cell culture models (34 -36) and in reducing tumor growth in a subcutaneous in vivo xenograft model (37), HA-oligosaccharides alone are not effective in reducing tumor progression in an Apc Min/ϩ mice in vivo model.…”

“…HA oligomers and overexpression of the ectodomain of CD44 (soluble CD44) (32) that acts as a competitive decoy by binding to endogenous HA, inhibit cell survival pathway activities, including activation of several receptor tyrosine kinases, namely ERBB2, EGFR, IGF1R␤, c-MET, and PDGFR␤, in several types of malignant colon, breast, and prostate carcinoma cells (33,34). In our recent study we demonstrated that elevated HA in normal intestinal epithelial cells (HIEC6-HAS2) regulates several properties required for the transformed phenotype.…”