ORIGINAL ARTICLE Cancer stem-like cells from head and neck cancers are chemosensitized by the Wnt antagonist, sFRP4, by inducing apoptosis, decreasing stemness, drug resistance and epithelial to mesenchymal transition S Warrier 1,2 , G Bhuvanalakshmi 1 , F Arfuso 2,3 , G Rajan 4 , M Millward 5 and A Dharmarajan 2 Cancer stem cells (CSCs) of head and neck squamous cell carcinoma (HNSCC) are defined by high self-renewal and drug refractory potential. Involvement of Wnt/β-catenin signaling has been implicated in rapidly cycling cells such as CSCs, and inhibition of the Wnt/β-catenin pathway is a novel approach to target CSCs from HNSCC. In this study, we found that an antagonist of FrzB/Wnt, the secreted frizzled-related protein 4 (sFRP4), inhibited the growth of CSCs from two HNSCC cell lines, Hep2 and KB. We enriched the CD44 + CSC population, and grew them in spheroid cultures. sFRP4 decreased the proliferation and increased the sensitivity of spheroids to a commonly used drug in HNSCC, namely cisplatin. Self-renewal in sphere formation assays decreased upon sFRP4 treatment, and the effect was reverted by the addition of Wnt3a. sFRP4 treatment of spheroids also decreased β-catenin, confirming its action through the Wnt/β-catenin signaling pathway. Quantitative PCR demonstrated a clear decrease of the stemness markers CD44 and ALDH, and an increase in CD24 and drug-resistance markers ABCG2 and ABCC4. Furthermore, we found that after sFRP4 treatment, there was a reversal in the expression of epithelial to mesenchymal (EMT) markers with the restoration of the epithelial marker E-cadherin, and depletion of EMT-specific markers twist, snail and N-cadherin. This is the first report demonstrating that the naturally occurring Wnt inhibitor, sFRP4, can be a potential drug to destroy CSC-enriched spheroids from HNSCCs. The repression of EMT and the decrease in stemness profile further strengthen the use of sFRP4 as a potent therapeutic against CSCs. Cancer Gene Therapy (2014) 21, 381–388; doi:10.1038/cgt.2014.42; published online 8 August 2014 INTRODUCTION Head and neck squamous cell carcinoma (HNSCC) is the sixth most prevalent cancer worldwide. 1 Mortality from HNSCC is high because of distant metastases and the emergence of therapy- resistant tumors. Prognosis of HNSCC is determined by the degree of lymphatic invasion and metastasis at diagnosis. 1 The standard of care for patients with HNSCC includes primarily platinum-based chemotherapeutic drugs, surgery and radiotherapy. 2 Despite this, the 5-year survival rate for these patients has still been in the range of 50 - 60% for the last 3 decades. 3 Cancer stem cells (CSCs), a small population of cells in most tumors, support tumor growth through their ability to self-renew and differentiate into heterogeneous tumor tissue. CSCs are typically resistant to radiation therapy and standard cytotoxic agents, which results in their enrichment and in the consequent recurrence of refractory tumors. After initially being identified in leukemia, 4 they have been found in many solid tumors. CSCs have been identified in human HNSCCs using markers such as CD133 and CD44, and by aldehyde dehydrogenase (ALDH) activity. 5 The presence of CSCs in HNSCC has been reported in many studies and has been comprehensively reviewed by Minnelli and Gallo. 6 The HNSCC CSCs reside in perivascular niches in the invasive front, where endothelial cells contribute to their survival and function. Targeting key signaling pathways that are active in CSC self- renewal is one approach to cancer therapy. Abnormal activation of the Wnt/β-catenin signaling pathway has been described in a wide variety of human cancers and in CSCs, and has already been demonstrated in HNSCCs. 7 Wnt dysregulation has a role in inducing epithelial to mesenchymal transition (EMT) transcription factors Twist and Snail. 8–10 CSCs from hepatic carcinoma cell lines exhibited enhanced chemoresistance to cisplatin, which was reversed by lentiviral microRNA knockdown of β-catenin. 11 Similar studies have demonstrated that the Wnt/β-catenin signaling pathway was also able to confer chemoresistance to 5-fluoro- uracil and doxorubicin. 12,13 Activation of the Wnt pathway, by the Wnt receptor frizzled-1, induced expression of the multiple drug- resistance gene MDR1 and promoted resistance to doxorubicin in chemoresistant neuroblastoma cells. 13 In ovarian CSCs, ABCG2 was shown to mediate cisplatin and paclitaxel resistance, which was reversed by β-catenin small interfering RNA knockdown. 14 In this study, we investigated the effect of the naturally occurring protein secreted frizzled-related protein 4 (sFRP4), which is an inhibitor of Wnt signaling, on CSC-enriched spheroid cells 1 Division of Cancer Stem Cells and Cardiovascular Regeneration, Manipal Institute of Regenerative Medicine, Manipal University, Bangalore, India; 2 Faculty of Health Sciences, School of Biomedical Sciences, Curtin University, Perth, Western Australia; 3 School of Anatomy, Physiology and Human Biology, The University of Western Australia, Perth, Western Australia; 4 School of Surgery, The University of Western Australia, Perth, Western Australia and 5 School of Medicine and Pharmacology, The University of Western Australia, Perth, Western Australia. Correspondence: Dr S Warrier or Prof A Dharmarajan, Division of Cancer Stem Cells and Cardiovascular Regeneration, Manipal Institute of Regenerative Medicine, Manipal University, Yelahanka, Bangalore 560065, India. E-mail: sudha.warrier@manipal.edu or a.dharmarajan@curtin.edu.au Received 4 June 2014; accepted 14 June 2014; published online 8 August 2014 Cancer Gene Therapy (2014) 21, 381 – 388 © 2014 Nature America, Inc. All rights reserved 0929-1903/14 www.nature.com/cgt