Abstract 2886: CD44/SMURF1 signaling maintains cancer stem cell-like invasive cells in head and neck squamous cell carcinoma

Abstract

Abstract Head and neck squamous cell carcinoma (HNSCC) is highly invasive and resistant to therapies, where treatments often result in high rates of failure and disease recurrence. A subpopulation of tumor-initiating cancer stem cells (CSC) is thought to be responsible for metastatic invasion and drug resistance in many types of cancer, including HNSCC. CD44 is a known CSC marker in HNSCC but its role in maintaining CSC populations is not well understood. We previously reported that SMAD specific E3 ubiquitin protein ligase 1 (SMURF1) inhibition of bone morphogenetic protein (BMP) signaling is essential for maintaining a CD44-high CSC-like population in HNSCC. In this study, we sought to determine how CD44, a receptor for hyaluronic acid (HA) that constitutes a major component of the tumor stroma, is involved in the regulation of SMURF1 and the maintenance of a HNSCC CSC phenotype. CSC-like cells were enriched from HNSCC cell lines as CD44-high cells. CD44 signaling was stimulated by exogenous HA treatment and inhibited by CD44 knockout (KO) using an inducible CRISPR/Cas9 system. We then assessed changes in SMURF1 protein level, BMP signaling, transwell migration, Matrigel colony formation, and invasion through three-dimensional organotypic culture (OTC) following CD44 modulation. CD44-high cells were found to have increased extracellular HA production. Treatment with exogenous HA reduced BMP signaling, as determined by a reduction in phospho-SMAD1/5/8 levels, and increased transwell migration of CD44-high cells. CD44-KO reduced SMURF1 protein expression and inhibited Matrigel colony formation of an invasive and recurrent HNSCC-derived CD44-high cells but only partially reduced colony formation of a less-invasive cell line derived from a primary HNSCC. Knockout of CD44 expression showed a slight reduction in transwell migration of invasive cells. CD44-high cells also recapitulated an invasive and CSC-like growth pattern in OTC assays. In contrast, CD44-KO inhibited OTC invasion and the epithelial-to-mesenchymal transition (EMT) phenotype, resulting in increased apical epithelial growth and differentiation. CD44 reconstitution appeared to restore the invasive and EMT phenotype. Based on our current findings, CD44 may be crucial for maintaining an anchorage-independent and invasive phenotype of HNSCC but plays a minor role in carcinoma cell migration. Citation Format: Ali Khammanivong, Raj Gopalakrishnan, Erin B. Dickerson. CD44/SMURF1 signaling maintains cancer stem cell-like invasive cells in head and neck squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2886. doi:10.1158/1538-7445.AM2017-2886