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PDF file - 1574K, YAP1 and SOX9 expression in EC cell lines.

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PDF file - 1015K, YAP1 Induces SOX9 promoter activity in 293T cells and primer design for SOX9 ChIP-PCR.

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PDF file - 127K, SOX9 is elevated in mouse liver tumors with inactivating Hippo pathway mutations.

Cancer stem cells are proposed to initiate and maintain tumor growth. Dysregulation of normal stem cell signaling may lead to the generation of cancer stem cells (CSCs), however, the molecular determinants of this process remain poorly understood. Here we show that the transcriptional co-activator YAP1 through direct regulation of SOX9 promotes the generation of CSCs and that the inhibition of YAP1 and SOX9 attenuates CSC formation. SOX9 transcripts and expression are upregulated upon YAP1 activation and several lines of evidence indicate that SOX9 is a direct target of YAP1. The Chromatin Immunoprecipitation analysis and luciferase assays demonstrate direct binding of YAP1 to the SOX9 promoter through a conserved TEAD binding site. Mutation of this site abrogates transcriptional regulation of SOX9 by YAP1 and Tead2. Functional studies demonstrate that YAP1 regulation of SOX9 is necessary and sufficient to confer CSC properties and tumorigenesis in vitro and in vivo. The small molecule inhibitor of YAP1, Verteporfin (VP) significantly blocks CSC self-renewal properties in cells with high YAP1 and a high proportion of the CSC marker aldehyde dehydrogenase 1 (ALDH1) indicating that VP targets the CSC population. These data identify YAP1 as a driver of esophageal cancer (EC) stem cells, in part, by regulation of SOX9 and suggest that pharmacological inhibition of YAP1 may be an effective means of specifically targeting EC stem cells. Citation Format: Shumei Song, Jaffer A. Ajani, Soichiro Honjo, Dipen M. Maru, Qiongrong Chen, Jiankang Jin, Ailing W. Scott, Todd R. Heallen, Lianchun Xiao, Wayne L. Hofstetter, Brian Weston, Jeffrey H. Lee, Roopma Wadhwa, Kazuki Sudo, James F. Martin, John R. Stroehlein, Mien-Chie Hung, Randy L. Johnson. The Hippo coactivator YAP1 upregulates SOX9 and endows cancer stem cell properties in non-transformed cells and esophageal cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3896. doi:10.1158/1538-7445.AM2014-3896

Norcantharidin (NCTD) is the demethylated analog of cantharidin, a naturally occurring toxin isolated from Mylabris. NCTD has been used to treat hepatocellular carcinoma with clinical efficacy and limited side effects. However, the underlying mechanism of its antitumor effects remains elusive. We have previously shown that TR3 expression is significantly decreased in metastatic melanomas and it is implicated in regulation of melanoma cell apoptosis. In this study, we showed that NCTD inhibited melanoma cell viability and induced apoptosis in vitro. NCTD induced translocation of TR3 from nucleus to mitochondria where it colocalized with Bcl-2 in melanoma cells. NCTD also increased Cytochome c release from mitochondria to the cytoplasm. There was also increased expression of Bax and cleaved caspase-3, accompanied with decreased expression of Bcl2 and Nf-kB2. The effects of NCTD were inhibited by knockdown TR3 expression in melanoma cells. To study its function in vivo, we treated Tyr::cre;BRAFCa/+;Ptenlox/lox mice with NCTD. Temozolomide and PBS were used as controls, and ten mice were used in each group. Temozolomide modestly increased mice survival, whereas NCTD significantly improved the survival of treated mice. Our data indicates that NCTD inhibits melanoma growth by inducing cell apoptosis via activation of a TR3 dependent pathway. These results suggest that NCTD is a potential therapeutic agent for melanoma. Financial support was provided by CA-093372, CA-116103 to XX. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5043. doi:10.1158/1538-7445.AM2011-5043