Abstract 3759: SMURF1 modulates BMP signaling and maintenance of HNSCC cancer stem cells.
Authors: Khammanivong Ali , Mulligan Miranda J. , Brady Nicholas J. , Gopalakrishnan Raj , Dickerson Erin B. 
Published: August 2014

Abstract
Cancer stem cells (CSCs) possess capabilities for long-term survival (quiescence), self-renewal and differentiation. In addition, they are highly implicated in drug resistance and tumor recurrence, which are associated with treatment failures for many malignancies, including head and neck squamous cell carcinoma (HNSCC). Bone morphogenetic protein (BMP) signaling pathways are involved in orchestrating diverse biological functions, as well as regulation of embryogenesis, cell differentiation, proliferation, migration, and apoptosis. BMP signaling is regulated by SMAD specific E3 ubiquitin protein ligase 1 (SMURF1), which has been implicated in invasiveness in several malignancies. The roles of BMP signaling and SMURF1 regulation in HNSCC CSC phenotypes and chemoresistance are not known. Objectives: In this study, our objectives were to enrich CSC populations from HNSCC cell lines and to characterize molecular mechanisms promoting CSC survival and drug resistance. Methods: Enrichment of HNSCC CSCs was performed using long-term anchorage-independent sphere forming growth under serum-free and low oxygen conditions. Sphere-forming cells were then characterized for CSC-like phenotypes using qRT-PCR and immunoblotting for known CSC markers. Sphere cells also were tested for CSC-like characteristics, including long-term survival and self-renewal using a sphere reformation assay, ability to undergo adipogenesis, and chemoresistance. Results: We determined that sphere-forming cells mimic characteristics previously attributed to CSCs, showing increased expression of CD44 and BMI1. Sphere cells also showed markedly higher SMURF1 expression than their monolayer counterparts cultured under standard anchorage-dependent differentiating growth conditions, suggesting that these sphere-forming cells may have reduced BMP signaling, differentiation, and growth. Consistently, BMP receptor downstream signal transduction was inactivated, as indicated by decreased phosphorylation of SMAD1/5/8 and down-regulation of downstream BMP target genes, including ID1 and ID2. Other BMP signaling inhibitors, BMPER and BAMBI, were upregulated in sphere cells. Resistance to the anti-cancer agent, cisplatin, was also higher in sphere cells. Knockdown of SMURF1 expression by shRNA did not affect initial sphere formation and resistance to cisplatin, but it reduced long-term sphere renewal (or maintenance) and enhanced adipogenesis. Sphere cells with high SMURF1 expression showed low or no adipogenic differentiation. Conclusion: Our data suggest that SMURF1 inhibits BMP signaling in HNSCC to maintain the CSC niche by controlling stemness and quiescence of the CSC population, but it has no direct effect on chemoresistance. Targeting SMURF1 may therefore provide a long-term benefit for HNSCC treatment to reduce resistant CSC populations and disease recurrence.
Citation Format: Ali Khammanivong, Miranda J. Mulligan, Nicholas J. Brady, Raj Gopalakrishnan, Erin B. Dickerson. SMURF1 modulates BMP signaling and maintenance of HNSCC cancer stem cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3759. doi:10.1158/1538-7445.AM2013-3759