orexin agonist br Experimental Procedures br Author Contribu

Experimental Procedures
Author Contributions
Acknowledgments This work was funded by CRUK (grant A10065). A.P. and H.W. thank Worldwide Cancer Research for support (grant 13-0146).
Introduction Cancer stem orexin agonist (CSCs) generally account for a rare subpopulation of cells within tumors; however, some reports showed that up to 25% of cancer cells within certain tumors display the characteristics of CSCs (Kelly et al., 2007; Quintana et al., 2008). CSCs have been defined according to their ability to drive tumor growth in xenografted animals accompanied by self-renewal and differentiation (Clarke et al., 2006). Moreover, CSCs have been reported to be highly associated with therapy resistance, recurrence, and metastasis (Dean et al., 2005; Meacham and Morrison, 2013). During the process of tumor initiation and progression, tumor cells must escape immunologic detection and elimination (Dunn et al., 2002). Given these unique properties of CSCs, these cells may have a stronger capability than differentiated tumor cells of evading various host immune surveillance mechanisms. The complement system, a main component of innate immunity, circulates to conduct immune surveillance and discriminate invading pathogens and cell debris from healthy host tissues (Morgan et al., 2005; Ricklin et al., 2010). After activation, complement components are cleaved into different fragments with multiple functions: C3a/C5a primes inflammation, C3b/iC3b induces opsono-phagocytosis, and C5b-9(n) (membrane attack complex, MAC) provokes rapid cell death (Dunkelberger and Song, 2010). To protect host cells from bystander complement attack, several membrane complement regulatory proteins (mCRPs) have evolved to restrict complement activation at diverse stages. CD46 acts as a cofactor for the inactivation of cell-bound C4b and C3b by serum factor I, CD55 inactivates C3 and C5 convertases by accelerating the decay of these proteases, and CD59 is the sole mCRP to prevent MAC formation (Zhou et al., 2008). Various endogenous (autologous antibodies, C1q, pentraxins, ficolins, etc.) (Ricklin et al., 2010) and exogenous (therapeutic antibodies, such as rituximab for B lymphoma [Zhou et al., 2008] and cetuximab for certain solid tumors [Hsu et al., 2010]) pattern recognition molecules can substantially activate complement in tumor microenvironment, which is critical in tumor cells, especially CSCs, for eventual survival from complement-mediated elimination (Ricklin et al., 2010). Numerous studies, including ours, have demonstrated that high expression of mCRPs, mainly CD46, CD55, and CD59, confer tumor cell resistance to antibody-based cancer therapy by preventing complement cascade amplification or MAC formation; therefore, functional inhibition of mCRPs may unleash the resistance (Goswami et al., 2016; Hu et al., 2011; Macor et al., 2015; Wang et al., 2010). Compared with other mCRPs, CD59 has been considered the most effective mCRP to protect tumor cells from complement-mediated lysis (Fishelson, 2003; Zhou et al., 2008). However, there are few reports on CSC evasion of complement-mediated elimination. In addition, normal stem cells may similarly encounter frequent complement attack, which requires high expression of mCRPs. Therefore, mCd59b (Genbank: NM_181858.1) deficiency, but not mCd59a (Genbank: NM_001111060.2) deficiency, could induce male infertility associated with fewer sperm cells (Qin et al., 2003). However, the underlying mechanisms for stem cells escaping complement surveillance remain largely unclear.
Results
Discussion CSCs account for a tiny subset of cancer cells; however, as “cancer seeds,” these cells have been considered a major obstacle to curing cancer due to their characteristics of distinctive surface proteins, self-renewal, differentiation, slow-cycling state, and high association with therapy resistance and metastasis (Clarke et al., 2006; Dean et al., 2005; Meacham and Morrison, 2013). Therefore, many efforts have been made to develop small molecules or antibodies that are currently in different clinical phases and that target various signaling pathways in CSCs (Kaiser, 2015). However, it is strongly suggested that CSC-specific therapy should be combined with traditional therapy to quickly eradicate whole tumors (Kaiser, 2015). Therefore, a bispecific target against differentiated and stem cancer cells may hold great potential for cancer therapy. Herein, we demonstrated that CD59 is upregulated by SOX2 in CSCs and that CD59 silencing completely eliminated tumors in a mouse model implanted with stem-like cancer cells.