Hyaluronan-CD44 Interaction with Leukemia-associated RhoGEF and Epidermal Growth Factor Receptor Promotes Rho/Ras Co-activation, Phospholipase C -Ca Signaling, and Cytoskeleton Modification in Head and Neck Squamous Cell Carcinoma Cells*

In this study we have examined the interaction of CD44 (a major hyaluronan (HA) receptor) with a RhoA-specific guanine nucleotide exchange factor (leukemia-associated RhoGEF (LARG)) in human head and neck squamous carcinoma cells (HNSCC-HSC-3 cell line). Immunoprecipitation and immunoblot analyses indicate that CD44 and the LARG protein are expressed in HSC-3 cells and that these two proteins are physically associated as a complex. HACD44 binding induces LARG-specific RhoA signaling and phospholipase C (PLC ) activity. In particular, the activation of RhoAPLC by HA stimulates inositol 1,4,5-triphosphate production, intracellular Ca mobilization, and the up-regulation of Ca / calmodulin-dependent kinase II (CaMKII), leading to phosphorylation of the cytoskeletal protein, filamin. The phosphorylation of filamin reduces its interaction with filamentous actin, promoting tumor cell migration. The CD44-LARG complex also interacts with the EGF receptor (EGFR). Most importantly, the binding of HA to the CD44-LARG-EGFR complex activates the EGFR receptor kinase, which in turn promotes Ras-mediated stimulation of a downstream kinase cascade including the Raf-1 and ERK pathways leading to HNSCC cell growth. Using a recombinant fragment of LARG (the LARG-PDZ domain) and a binding assay, we have determined that the LARG-PDZ domain serves as a direct linker between CD44 and EGFR. Transfection of the HSC-3 cells with LARG-PDZcDNA significantly reduces LARG association with CD44 andEGFR.Overexpressionof theLARG-PDZdomainalso functionsas a dominant-negative mutant (similar to the PLC/Ca -calmodulindependent kinase II (CaMKII) and EGFR/MAPK inhibitor effects) to block HA/CD44-mediated signaling events (e.g. EGFR kinase activation,Ras/RhoAco-activation,Raf-ERKsignaling, PLC -mediated inositol 1,4,5-triphosphate production, intracellular Ca mobilization, CaMKII activity, filamin phosphorylation, and filamin-actin binding) and to abrogate tumor cell growth/migration. Taken together, our findings suggest that CD44 interaction with LARG and EGFR plays a pivotal role in Rho/Ras co-activation, PLC -Ca signaling, and Raf/ ERK up-regulation required for CaMKII-mediated cytoskeleton function and in head and neck squamous cell carcinoma progression. Human head and neck squamous cell carcinoma (HNSCC) is a very malignant cancer associated with major morbidity and mortality. This deadly disease includes cancers of the lips, oral cavity, pharynx, hypopharynx, larynx, nose, nasal, sinuses, neck, ears, and salivary glands (1–6). Approximately 90% of HNSCCs are very aggressive and rapidly invade the surrounding tissues. Because little is known about themolecular basis underlying the progression of HNSCC to the invasive phenotype, it is very difficult to predict individual tumor aggressiveness and to design effective treatment plans. Thus, there is currently a great need to clarify those aspects of tumor formation underlying the clinical behavior of HNSCC. A number of studies have aimed at identifying the specific molecules expressed in HNSCC that correlate with invasive behavior. Among such candidates are hyaluronan (HA) (7, 8) and its major cell surface receptor, CD44 (9).HA is amajor component of the extracellularmatrix component and is significantly enriched inmany types of tumors (7, 8). HA binds to its specific cell surface receptor, CD44, amultifunctional transmembrane glycoprotein expressed in many cells and tissues including HNSCC cells and carcinoma tissues (10–14). CD44 is often expressed as a variety of variant isoforms, generated by an alternative splicingmechanism (15). The expression of certain CD44 variant (CD44v) isoforms is known to be associated with head and neck cancer progression (10–14). The external portion of CD44 binds HA (9), whereas the intracellular domain interacts with specific signaling molecules such as RhoA-activated Rho kinase (16) and Rho/Rac1-specific guanine nucleotide exchange factors (p115RhoGEF (17), Tiam1 (18), and Vav2 (19)), c-Src kinase (20), protein kinase N (21), IQGAP1 (22), p185 (23), and transforming growth factorreceptors (24)). CD44 also binds directly to cytoskeletal proteins such as ankyrin and ezrin, radixin, and moesin (25–28). The interaction of CD44 with the cytoskeleton and various signalingmolecules plays a pivotal role in promotingmetastatic-specific tumor phenotypes such as matrix metalloproteinase (MMP)-mediated matrix degradation, tumor cell growth, migration, and invasion (10, 16–28). Epidermal growth factor receptor (EGFR) is also overexpressed in