Abstract 1893: Honokiol prevents colonic tumorigenesis and affects stem cell viability by affecting oncogenic YAP1 function
Authors: Subramaniam Dharmalingam , Ponnurangam Sivapriya , Kwatra Deep , Kaushik Gaurav , Ramamoorthy Prabhu , Ramalingam Satish , Tawfik Ossama , Weir Scott J. , Padhye Subhash , Dixon Dan A. , Umar Shahid , Jensen Roy A. , Anant Shrikant 
Published: August 2015

Abstract
Background: Despite advances in early detection, colon cancer remains the second leading cause of death in the United States. We are focused on developing dietary prevention strategies. HNK (HNK) is a biphenolic compound that is used in the traditional Chinese Medicine for treating various ailments. The current study is designed to determine whether HNK affected colon cancer stem cells and to identify a mechanism.
Method: Colon cancer (CRC) cell lines HCT116 and SW480 and normal colon epithelial cells were used in the study. Cell growth was measured by hexoseaminidase and clonogenicity assays. Apoptosis was determined by measuring caspase 3/7 activities. Colosphere formation assay and FACS sorting were used for stem cells. For in vivo effects, we used the AOM/DSS-induced colonic tumorigenesis model. Immunohistochemistry was determined for stem cell markers and Hippo signaling proteins.
Results: HNK induced a significant dose-dependent inhibition of proliferation and colony formation of the two CRC lines, but induced apoptosis. HNK did not affect the normal cells. To demonstrate HNK effects on stem cells, we performed colosphere assays. HNK significantly reduced the number and size of colospheres, suggesting effects on stem cells. In addition, colon stem cell marker proteins DCLK1, LGR5, and CD44 were also decreased. Further proof was obtained by flow cytometry analyses, where HNK reduced the number of DCLK1+ cells. We next determined whether stem cell signaling is affected. For this, we looked at the Hippo signaling pathway, which is active in intestinal stem cells. The key effector protein of this pathway, YAP1 is also oncogenic in many cancer types. In the canonical Hippo signaling pathway, YAP1 function is inhibited. When YAP1 is phosphorylated at Ser127 by the action of upstream Mst1/2 and Lats1/2 kinases, it is sequestered in the cytoplasm where it is degraded, thereby inhibiting downstream gene expression. HNK significantly reduced YAP1 levels. Furthermore, HNK inhibited the expression of YAP interacting proteins TEAD1, TEAD2, and TEAD4. On the other hand, ectopic expression of the TEAD1 partially rescued the cells from HNK-mediated growth suppression. To determine the in vivo effect of HNK on AOM/DSS induced colonic tumorigenesis, HNK were oral gavaged at a dose of 5mg/kg bw for 24 weeks. HNK treatment significantly reduced the colonic tumor numbers and size. Western blot and immunohistochemistry analyses demonstrated significant inhibition in the expression of stem marker proteins, oncogenic YAP1 phosphorylation and TEAD1 in the HNK-treated AOM/DSS colonic tumor tissues.
Conclusion: Together, these data suggest that HNK prevents colonic tumorigenesis that targets stem cells by inhibiting oncogenic YAP1 in Hippo signaling pathway.
Citation Format: Dharmalingam Subramaniam, Sivapriya Ponnurangam, Deep Kwatra, Gaurav Kaushik, Prabhu Ramamoorthy, Satish Ramalingam, Ossama Tawfik, Scott J. Weir, Subhash Padhye, Dan A. Dixon, Shahid Umar, Roy A. Jensen, Shrikant Anant. Honokiol prevents colonic tumorigenesis and affects stem cell viability by affecting oncogenic YAP1 function. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1893. doi:10.1158/1538-7445.AM2015-1893