ECS cells, derived from spheroid cultures, were plated into Millicell chambers (0.8 m pores) coated with matrigel in the presence of 0 or 2 M of the indicated Ezh2 inhibitor. and that treatment with Ezh2 inhibitors may be a strategy for reducing ECS cell survival and suppressing tumor formation. Introduction Epidermal squamous cell carcinoma ranks among the most common forms of cancer. Moreover, due to exposure to environmental irritants and ultraviolet radiation, the incidence continues to increase (1). Early lesions can be removed by surgical excision, but the 5 12 months recurrence rate is still 8% (2). Advanced disease is usually life threatening and there are no effective treatments (3). Moreover, the high rate of skin cancer occurrence in the population means a high cost to society. Recent findings suggest that epidermal squamous cell carcinoma includes a subpopulation of tumor-initiating cells we call epidermal cancer stem cells (ECS cells), which exhibit self-renewal capacity, proliferate infrequently and are required for tumor maintenance and metastasis (4C6). Since the cancer stem cells are thought to give rise to non-stem cancer cells, eliminating the stem cell populace may be necessary to halt tumor formation (7). However, these cells are resistant to the action of traditional anticancer brokers that kill rapidly growing tumor cells (7). On a practical level, stem cells can be identified by the presence of protein epitopes that are associated with stem cells from the corresponding normal tissue. In breast malignancy, the stem cell populace displays a CD44+/CD24? phenotype (8), and CD133 marks cancer stem cells in brain tumors, colorectal carcinoma and pancreatic carcinoma (9C12). In head and neck malignancy, CD44+ cells display malignancy stem cell properties (13), and aldehyde dehydrogenase 1 activity identifies malignancy stem cells in a host of cancer types (14C17). The human epidermis contains multiple stem cell populations (4), including the CD200+/K15+/K19+ hair bulge stem cells (18) and the 6+/1+/CD71? interfollicular stem cells (19,20). CD133 has also been reported to identify human skin malignancy stem cells (5,21,22). Epidermal squamous cell carcinoma cells and tumors are enriched for expression of the polycomb group (PcG) proteins, which are CD81 a conserved family of proteins that act epigenetically to silence tumor suppressor gene expression (6,23,24). These regulators repress gene expression by covalently modifying histones to produce closed chromatin (24C29). PcG proteins operate as two multiprotein chromatin-binding complexespolycomb repressive complex 1 (PRC1) and PRC2 (27). The PRC1 complex includes Bmi-1, Ph1, CBX and Ring 1A/B, whereas the PRC2 multiprotein complex contains Ezh2, EED, Suz12 and RbAp46 (30). As an initial step in regulation, trimethylation of lysine 27 of histone H3 (H3K27me3) occurs via the action of the Ezh2 protein (28,31). In the second step, H3K27me3 serves as a binding site for the chromodomain of the CBX protein of the PRC1 complex (31). Once bound, the PRC1 D-Mannitol complex Ring1B protein ubiquitinates histone H2A at lysine 119 (25,31). The sequential trimethylation and ubiquitination events D-Mannitol result in chromatin condensation leading to gene silencing (27,28). The PcG proteins, by suppressing tumor suppressor expression, have been implicated as important in maintenance of stem cell survival (24,32C36). Indeed, we have shown that several PcG proteins are overexpressed in epidermal squamous cell carcinoma (30,37,38) and that this overexpression enhances epidermal cancer cell survival (6,39C41). Ezh2 is usually a particularly important PcG protein as it is the key catalytic protein in the PRC2 complex and is highly elevated in skin cancer (30). We have shown that Ezh2 is usually overexpressed in ECS cells (6). Moreover, ECS cells form large, aggressive and highly invasive and vascularized tumors following injection of as few as 100 cells in immune compromised mice (6). A key question is usually whether the Ezh2 protein is required for ECS cell survival and tumor formation. In the present study, we show that Ezh2 is required for ECS cell survival, migration and invasion and spheroid and tumor formation. We also show that Ezh2 inhibitors reduced these processes including tumor formation. Materials and methods Antibodies and reagents Dulbecco’s altered Eagle’s medium (11960-077), sodium pyruvate (11360-070), l-glutamine (25030-164), penicillinCstreptomycin answer (15140-122) and 0.25% trypsinCethylenediaminetetraacetic acid (25200-056) were purchased from Gibco (Grand Island, NY). Heat-inactivated fetal calf serum (FCS, F4135) was obtained from Sigma. Antibodies for Ezh2 (612667) D-Mannitol and Oct4 (611203) were obtained from BD transduction laboratories (San.