Background & Aims Cancer cells often lose contact inhibition to undergo anchorage-independent proliferation and become resistant to apoptosis by inactivating the Hippo signaling pathway, resulting in activation of the transcriptional co-activator yes-associated protein (YAP). However, the oncogenic mechanisms of YAP are unclear. Methods Using cross-species analysis of expression data, the Notch ligand Jagged-1 (Jag-1) was identified as downstream target of YAP in hepatocytes and hepatocellular carcinoma (HCC) cells. We analyzed the functions of YAP in HCC cells via overexpression and RNA silencing experiments. We used transgenic mice that overexpressed a constitutively activated form of YAP (YAPS127A), and measured protein levels in HCC and colorectal and pancreatic tumor samples from patients. Results Human HCC cell lines and mouse hepatocytes that overexpress YAPS127A upregulated Jag-1, leading to activation of the Notch pathway and increased proliferation. Induction of Jag-1, activation of Notch, and cell proliferation required binding of YAP to its transcriptional partner TEAD4; TEAD4 binding required Mst1/2, but not WNT-β-catenin signaling. Levels of YAP correlated with Jag-1 expression and Notch signaling in human tumor samples and shorter survival times of patients with HCC or colorectal cancer. Conclusion The transcriptional regulator YAP upregulates Jag-1 to activate Notch signaling in HCC cells and mouse hepatocytes. YAP-dependent activity of Jag-1 and Notch correlate in human HCC and colorectal tumor samples with patient survival times, suggesting the use of YAP and Notch inhibitors as therapeutics for gastrointestinal cancer.

Background & Aims The cancer stem cells (CSCs) have important therapeutic implications for multi-resistant cancers including hepatocellular carcinoma (HCC). Among the key pathways frequently activated in liver CSCs is NF-kB signaling. Methods We evaluated the CSCs-depleting potential of NF-kB inhibition in liver cancer achieved by the IKK inhibitor curcumin, RNAi and specific peptide SN50. The effects on CSCs were assessed by analysis of Side Population (SP), sphere formation and tumorigenicity. Molecular changes were determined by RT-qPCR, global gene expression microarray, EMSA, and Western blotting. Results HCC cell lines exposed to curcumin exhibited differential responses to curcumin and were classified as sensitive and resistant. In sensitive lines, curcumin-mediated induction of cell death was directly related to the extent of NF-kB inhibition. The treatment also led to a selective CSC-depletion as evidenced by a reduced SP size, decreased sphere formation, down-regulation of CSC markers and suppressed tumorigenicity. Similarly, NF-kB inhibition by SN50 and siRNA against p65 suppressed tumor cell growth. In contrast, curcumin-resistant cells displayed a paradoxical increase in proliferation and expression of CSC markers. Mechanistically, an important component of the CSC-depleting activity of curcumin could be attributed to a NF-kB-mediated HDAC inhibition. Co-administration of the class I/II HDAC inhibitor trichostatine sensitized resistant cells to curcumin. Further, integration of a predictive signature of curcumin sensitivity with human HCC database indicated that HCCs with poor prognosis and progenitor features are most likely to benefit from NF-kB inhibition. Conclusions These results demonstrate that blocking NF-kB can specifically target CSC populations and suggest a potential for combined inhibition of NF-kB and HDAC signaling for treatment of liver cancer patients with poor prognosis.

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