[6] It was reported that total and active CREB (p-CREB) significantly increased in HCC, compared to pair-matched normal liver samples.[7] Our previous work also revealed that CREB up-regulates an HCC highly associated long noncoding RNA, HULC expression through interaction with microRNA-372,[8] suggesting the important role of CREB in liver cancer. In the present study, Neratinib cost we highlighted the role of mutual interaction between YAP and CREB in liver tumorigenesis. We found that CREB up-regulated YAP transcription by binding to a novel site in the YAP promoter region. Moreover, we revealed that YAP inhibited the degradation of CREB mediated by mitogen-activated

protein kinase 14 (MAPK14/p38) in HCC cells, thus providing a positive feedback loop to promote cellular YAP and CREB output. Our data also showed that the two proteins were closely correlated in tumor samples, suggesting the important role of their feedback loop in liver cancer. Taken together, this work summarizes a novel link between two major oncoproteins and a potential mechanism for liver tumorigenesis. HepG2, Bel-7402, SMMC-7721, and HEK-293T cells were cultured in Dulbecco’s modified Eagle’s medium. Cells were treated by H89 (20 uM; Cayman Chemical Company, Ann Arbor,

MI), forskolin (50 uM; Cayman), Palbociclib wortmannin (50 uM; Cayman), LY294002 (20 uM; Cell Signaling Technology, Danvers, MA), SB203580 (20 uM; Cell Signaling Technology), SB202190 (5-20 uM; Santa Cruz Biotechnology, Santa Cruz, CA), MG132 (25 uM; Cayman), or human epithelial growth factor (hEGF) (10 ng/mL; Sigma-Aldrich, St Louis, MO) 5-24 hours before harvest. Short hairpin RNAs (shRNAs) were cloned into pLKO.1 lentiviral vectors. Complementary DNA fragments encoding human YAP, CREB, MAPK14/p38, and beta-transducin repeat containing E3 ubiquitin protein ligase (BTRC) were cloned into pGIPZ-based lentiviral vector and pcDNA3.1(+), respectively, and the primers used are listed in Galactosylceramidase Supporting Table 1. shRNA- and

protein-expressing plasmids for phosphatase and tensin homolog (pTEN) were gifts from Dr. Xuqian Fang (Shanghai Jiaotong University, Shanghai, China). YAP-Flag and LATS1-Flag expression plasmids were constructed as described previously.[9] For immunohistochemistry (IHC), human liver cancer tissue microarray (TMA) slides were purchased from U.S. Biomax (Rockville, MD). Slides were incubated in primary antibodies (Abs) against CREB (#1496; Epitomics, Burlingame, CA) and YAP65 (#2060; Epitomics). For immunofluorescence (IF), cells were incubated in primary Abs against YAP (#4912; Cell Signaling Technology, or sc-101199; Santa Cruz Biotechnology), CREB (#9197; Cell Signaling Technology), p-p38 (sc-7973; Santa Cruz Biotechnology), p38 (#9218; Cell Signaling Technology, or sc-271120; Santa Cruz Biotechnology), or BTRC (#4394; Cell Signaling Technology).