The number of differentially expressed genes, as determined by Bootstrap t test, varied from 2,041 genes in KMCH to 2,731 genes in Huh7 and 4,133 genes in WRL68, underlining the heterogeneity of CSCs in liver cancer cell lines (Supporting Fig. 4A). Pathway enrichment analysis revealed that each individual cell line was characterized by activation of unique oncogenic pathways with known associations to HCC, such

as EGFR (Huh7), MYC (WRL68), and SRC (KMCH) (Supporting Fig. 4B). Furthermore, all cell lines demonstrated ubiquitous activation of nuclear factor κB (NF-κB) signaling, suggesting that CSCs exhibit a generalized increase in stress resistance and survival. To more specifically identify genes related to liver CSCs, we looked for commonly dysregulated genes across all three cell ABT-263 in vivo lines. We found 1,259 differentially expressed genes between SP-ZEB and non–SP-ZEB cells, with 617 genes displaying more than 1.5-fold expression changes (Fig. 5, Supporting Table 4). This 617-gene set very efficiently separated SP-ZEB and non–SP-ZEB and is therefore referred to as the common SP-ZEB signature (Fig. 5A). Gene set enrichment analysis (GSEA) demonstrated

that the SP-ZEB gene signature overlapped with two previously published gene sets associated with adult stem cells and hepatoblasts (Fig. 5B).23, 24 Conversely, non–SP-ZEB cells showed a negative correlation, suggesting a more differentiated BAY 73-4506 concentration phenotype.23 Again, the common signature was characterized by activation of medchemexpress NF-κB as well as interleukin-6 and Wnt/β-catenin signaling pathways, which are known to be involved in cancer

(Supporting Fig. 5A,B,D). Notably, we found that genes centered around the core hepatocyte nuclear factor 4α network, critical for hepatocyte differentiation, were consistently down-regulated (Fig. 5C; Supporting Fig. 5C). Ingenuity Pathway Analysis (IPA) revealed that in addition to genes dysregulated in various cancers (AXIN2, EDN1, EP400, RICTOR, ADAMTS1, HOXA13, AGGF1, CCND1) and/or during invasion and metastasis (SNAI2, TIMP4, MMP25, RHOB, CCL20, CTAG2, CGN, CX3CL1, LGR4), the common SP-ZEB signature also contained genes associated with stem/progenitor and liver development (CK19, FOXA2, CLDN2, SOX9, SOX4, DMBT1, MED12, AMD1). GSEA further identified an abundance of gene sets involved in cytoskeleton architecture, vascular development, and c-Jun N-terminal kinase signaling in SP cells (Supporting Table 3). Selected targets of the generated signature (SOX4 and p-NF-κB) were validated using human tissue microarray of 95 HCC and 10 normal livers.25, 26 Significant enrichment of both markers was found during malignant progression (p-NF-κB, P < 0.001; SOX4, P < 0.05) (Supporting Fig. 5E,F).