CD44 interacts with EGFR and promotes head and neck squamous cell carcinoma initiation and progression

Summary

Objectives

CD44 is a promising target for therapy in head and neck squamous cell carcinoma (HNSCC) and has two defined roles in tumorigenesis: it is a cancer stem cell (CSC) marker and it promotes migration and proliferation through interaction with many signaling molecules. The purpose of this study was to investigate the role of CD44 in HNSCC carcinogenesis.

Materials and methods

The effects of CD44 in cell proliferation, migration, apoptosis and cisplatin resistance were studied by its overexpression in HNSCC cells. We also evaluated the effect of CD44 on tumor progression by siRNA methodology, immunohistochemistry (IHC) and western blot analysis. CD44 and EGFR colocalization were examined in CAL 27 cells by laser scanning confocal microscopy. The interaction between CD44 and EGFR was analyzed by immunoprecipation.

Results

Overexpression of CD44 enhances cell proliferation and migration and correlates with increased cisplatin resistance and apoptosis inhibition in SCC25 cells. Downregulation of CD44 in CAL27 cells inhibited constitutive EGFR phosphorylation and significantly reduced tumor growth in nude mice. CD44 and EGFR colocalized in CAL 27 cells. CD44 coimmunoprecipated with EGFR in CAL 27 cells, indicating that these proteins interact with each other.

Conclusion

CD44 therapy in HNSCC may target the CSC population and alter EGFR signaling.

Introduction

Head and neck squamous cell carcinoma (HNSCC) is a devastating illness affecting 50,000 people in the United States and 600,000 worldwide each year.[1], [2] The main risk factors include tobacco and alcohol use and HPV virus infection.[3], [4] Most of these patients present with late stage disease when cure rates reach only 30%. Despite aggressive treatments with combinations of surgery, radiation and chemotherapy, little progress has been made towards better outcomes.

Several tumor markers are overexpressed in HNSCC including epidermal growth factor receptor (EGFR), one of four members of the ErbB family. The ErbB family members form homo and heterodimers that, when disregulated, initiate tumorigenic signaling cascades.⁵ EGFR appears to be the most influential ErbB receptor in HNSCC and signals through the Ras-MAPK, PI3K-PTEN-AKT, and phospholipase C pathways.⁵ Surprisingly, agents against EGFR result in modest response rates for HNSCC. The mechanisms of resistance are still not clear. Point mutations are reported at low frequencies⁵ though a truncated, constitutively active mutant EGFRvIII has been reported in 42% of HNSCC analyzed⁵ and may help explain resistance. Other mechanisms of resistance include inability to internalize EGFR and cross-activation of the EGFR intracellular tyrosine kinase or downstream molecular pathways such as Ras-MAPK or PI3-PTEN-AKT.[5], [6], [7]

Additional targets are clearly needed for HNSCC treatment. Our work showed that soluble CD44, a breakdown product of CD44, is overexpressed in oral rinses from HNSCC patients and distinguishes HNSCC from controls with 62–79% sensitivity and 75–100% specificity.[8], [9], [10] CD44 was also recently identified as a tumor initiating marker in HNSCC.¹¹ While cetuximab may increase populations of CD44+ and CD133+ tumor initiating cells,¹² resulting in resistance, anti-CD44 therapy would be expected to eliminate those resistant populations. CD44 consists of a family of transmembrane glycoproteins with a common domain and a variable region of alternatively spliced exons (exons 5–14).¹³ The common domain includes an extracellular region that interacts with growth factors and hyaluronan (HA), and a cytoplasmic moiety capable of interacting with cytoskeletal components including ezrin, radixin, moesin (ERM) and merlin.[14], [15], [16], [17] Interactions between CD44, HA, and ErbB family members promote tumorigenesis[18], [19], [20] and may represent a pathway to EGFR inhibitor resistance by cross-activation of related pathways.

To evaluate the role of CD44 in HNSCC, we have overexpressed it in a CD44 low cell line, SCC25, that forms a differentiating cyst in nude mice²¹ and investigated its effects on tumor growth, migration, apoptosis and sensitivity to cisplatin. Then we downregulated CD44 in an overexpressing cell line, CAL 27 and examined effects on xenograft tumor growth and EGFR signaling. We also showed that EGFR and CD44 colocalize and complex together, further implying a signaling interaction. Based on these studies, targeting CD44 may inhibit tumor initiation and alter EGFR signaling.