br Results br Discussion Frequent tumor relapse in multiple

Results
Discussion Frequent tumor relapse in multiple tumor types has now been attributed to the presence of residual CSCs after conventional treatments. We and others have previously identified CD133 to mark a liver CSC subpopulation in HCC (Ma et al., 2007, 2010). Yet, the functional paths by which these p-gp inhibitors promote hepatocarcinogenesis remain limited, significantly impeding our efforts in developing CSC-specific therapies. We characterized the mRNA transcriptome of CD133+ and CD133− subpopulations in HCC by RNA-seq. Pathway enrichment analysis found the CD133+ subset to be tightly associated with an activated MAPK pathway, which is in concordant with our previous study, where we found CD133 to promote angiogenesis through IL-8-activated ERK (Tang et al., 2012). In addition, our CD133− non-CSC subset was also found to be enriched for genes critical in hepatocyte differentiation. This result is also consistent with our finding where CD133− cells were found to be unable to differentiate into skeletal and cardiac lineages, while in contrast, CD133+ liver CSCs could efficiently differentiate into non-hepatocyte-like, angiomyogenic-like cells following cell-directed differentiation in vitro (Ma et al., 2007), suggesting that CD133− cells exist in a terminally differentiated state. Of interest, one-third of the differentially expressed genes identified were found to encode for secretory proteins. There is accumulating data to show that maintenance of cancer and stemness properties is dependent on the microenvironment in which deregulated secreting factors can communicate through paracrine or autocrine signaling. Given the importance of secretory factors in modulating CSC features and that they can be found accessible in the conditioned media of cells or serum in patients, thus making them prime druggable targets for novel therapy development, we focused our studies in this area. The focus of ANXA3 research in the past has centered on its expression in various diseases. A number of studies have found ANXA3 to be frequently overexpressed in ovarian, breast, colon, lung, gastric, gallbladder, testicular, and urothelial cancers (Köllermann et al., 2008; Schostak et al., 2009; Liu et al., 2009; Yan et al., 2010; Wu et al., 2013). In contrast, ANXA3 was also found to be downregulated in prostate and papillary thyroid cancers (Wu et al., 2013). In recent years, a small number of studies have identified ANXA3 to be secreted, where low levels of ANXA3 present in the urine were found to have diagnostic significance for early prostate cancer (Schostak et al., 2009; Yin et al., 2012) and secretion of ANXA3 from ovarian cancer cells is associated with platinum resistance. Here, we found ANXA3 to represent the most significantly upregulated gene that encoded for a secretory protein in the CD133+ liver CSC subset. Haraguchi et al. also likewise found ANXA3 to be preferentially expressed in the side population (SP) isolated from Huh7 cells (Haraguchi et al., 2006). SP cells from HCC were subsequently characterized to also express CD133. Specifically in HCC, Tong et al. found ANXA3 to be upregulated in 5-FU-resistant HCC cells and that silencing of ANXA3 by RNAi resulted in enhanced sensitivity of HCC cells to chemotherapy (Tong et al., 2012). By mass-spectrometry-based profiling, Tsai et al. identified several altered proteins, including ANXA3, to be highly expressed in Huh7 CD133+ liver CSCs (Tsai et al., 2012). Pan et al. also reported the preferential upregulation of endogenous ANXA3 in CD133+ cells isolated from Huh7 and that ANXA3 is required for sphere formation, tumor initiation, migration, invasion, and chemoresistance in HCC cells via a deregulated HIF1α/NOTCH pathway (Pan et al., 2013, 2015). These studies do suggest the importance of ANXA3 in CD133+ liver CSCs and in HCC. However, since the authors did not perform in vivo transplantation and serial propagations at limited dilutions, which remains the gold standard to test tumorigenicity and self-renewal, the reports are at best suggestive. Further, their work only looked at cytoplasmic ANXA3. Our present study definitively reports the functional role of ANXA3 in mediating CSC-like properties in HCC. Our work also demonstrates the clinical relevance and functional significance of secretory ANXA3 in HCC. Whether ANXA3 can be developed as a standalone biomarker or used in combination with AFP for clinical use warrants further investigation in a larger patient sample cohort. Our functional studies identified the role of endogenous and exogenous ANXA3 in conferring CSC-like properties.