br Experimental Procedures br Author Contributions

Experimental Procedures
Author Contributions P.A.J. designed and performed majority of the experiments and data analyses. P.D.W. performed limiting dilution assays and facilitated R-SPONDIN1 rescue experiments. N.K. generated and analyzed human breast samples from specific reproductive phases, S.N. assisted with RT-PCR, H.F. maintained mouse strains, M.A.D.G. contributed to in vitro experiments and RANK-Fc administration, H.W.J. assisted with cell preparations, J.M.P. provided Rank-deficient mice, and C.E. facilitated human breast work and advised on study presentation. R.K. directed the study. R.K. and P.A.J. wrote the manuscript with C.E. and N.K.
Acknowledgments
Introduction The cancer stem cell (CSC) model has helped explain why tumor eradication has not been achieved despite advances in treatment. The model suggests that a cellular hierarchy exists in some cancers, with self-renewing CSCs generating progeny constituting the tumor bulk. CSCs possess both tumor and stem cell-like properties (Pardal et al., 2003). Studies have shown that CSCs bear the exclusive ability to regenerate tumors. Treatment of bulk cancer cell populations within tumors with chemotherapy has been shown to select for the outgrowth of therapy-resistant cancer ll-37 Supplier that are more tumorigenic, invasive, and stem-like. Hence, cancer therapies may be rendered ineffective because the bulk of cancer cells within a tumor may be eliminated while leaving behind CSC-enriched cells that proceed to regenerate tumors. This underscores the need for a detailed understanding of the molecular differences between CSCs and non-CSCs to discover cell-state-specific features that may render CSCs susceptible to selective therapeutic intervention. The perpetuation of many cancer types has been suggested to stem from CSCs. We have found HCC to be driven by a liver CSC subset marked by the CD133 phenotype. CD133+ HCC cells display sustained self-renewal, differentiate toward multiple lineages, and phenocopy the original tumor upon xenotransplantation (Ma et al., 2007, 2010). These cells also possess an enhanced ability to resist chemotherapy through activated AKT/BCL-2 (Ma et al., 2008). CD133 is not simply a marker of liver CSCs; it also plays a functional role in regulating HCC tumorigenesis (Tang et al., 2012). Increased CD133 expression in HCC is associated with worse overall survival and higher recurrence rates (Ma et al., 2010). Our results are consistent with studies by other groups where CD133 was also found to be an important risk factor for overall survival of the disease, demonstrating the prominence of CD133 in HCC. Despite our growing understanding of the importance of a CD133+ liver CSC population, the functional paths by which these cells promote hepatocarcinogenesis remains limited. Since the intrinsic molecular mechanisms by which CSCs sustain tumor growth is believed to be inter-related with its tumor microenvironment, our present study aims at investigating the mechanism by which CD133+ liver CSCs mediate tumor formation, self-renewal, and interaction with its niche. Toward this goal, RNA sequencing (RNA-seq) profiling was carried out to compare the differential gene expressions between CD133+ liver CSCs and CD133− differentiated counterparts. Many of the differentially expressed genes common to the two samples encoded for secretory proteins, which we know represent major means of communication between cancer cells and the microenvironment. From our profiling, the most significantly deregulated gene that encodes for a secretory protein is annexin A3 (ANXA3), a gene we now show to be critical in promoting CSC-like properties in CD133+ liver-CSC-driven HCC through both an autocrine and paracrine manner. ANXA3 belongs to the annexin family of Ca2+-dependent phospholipid-binding proteins (Raynal and Pollard, 1994). It has been shown to possess the ability to promote angiogenesis (Park et al., 2005) and rat liver regeneration (Harashima et al., 2008). Upregulation of ANXA3 expression is detected in various tumor types including prostate, ovarian, and lung cancers (Köllermann et al., 2008; Schostak et al., 2009; Liu et al., 2009; Yan et al., 2010). In ovarian cancer, serum ANXA3 levels were significantly upregulated in diseased patients compared with healthy individuals (Yin et al., 2012). Further, overexpression of ANXA3 was found to contribute to platinum resistance in ovarian cancer (Yan et al., 2010). In HCC, ANXA3 was also found to be overexpressed in 5-fluorouracil (5-FU)-resistant cell lines (Yin et al., 2012) and to play a role in promoting tumorigenesis and resistance to chemotherapy (Pan et al., 2013). Nevertheless, the role of endogenous and secretory ANXA3 in the context of CD133+ liver CSCs or HCC and the mechanism by which ANXA3 regulates CSC-like features has not been explored. Here, we investigated the clinical significance, functional role, and therapeutic implications of ANXA3 in CD133+ liver-CSC-driven HCC. We identified caveolin-1-dependent endocytosis to mediate internalization of secretory ANXA3 into HCC cells, thereby activating a dysregulated JNK pathway to promote CSC-like properties. We also developed a monoclonal antibody specific against ANXA3 (anti-ANXA3 mAb) and showed in vivo that the use of this antibody alone or in combination with cisplatin could efficiently lead to a reduced ability of HCC cells to initiate tumor growth and self-renewal, concomitant with a decrease in liver CSC proportions.