Abstract 3662: Honokiol affects stem cell viability in part through suppression of Hippo signaling pathway.
Authors: Subramaniam Dharmalingam , Ponnurangam Sivapriya , Umar Shahid , Ramalingam Satish , Jensen Roy A. , Anant Shrikant 
Published: August 2014

Abstract
Background: Despite therapeutic advances, colon cancer remains the second leading cause of death in the United States. Cancer stem cells are implicated in resistance to radiation and chemotherapy. Honokiol is a biphenolic compound that has been used in the traditional Chinese Medicine for treating various ailments. The current study is designed to determine whether honokiol affected colon cancer stem cells and to identify a mechanism.
Method: Colon cancer 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, HCT116 xenografts were developed in the flanks of nude mice. Immunohistochemistry was performed for CD31, stem cell markers and Hippo signaling proteins.
Results: Honokiol induced a significant dose-dependent inhibition of proliferation and colony formation of the two colon cancer cell lines, but not that of the normal cells. Honokiol treatment also induced the colon cancer cells to undergo apoptosis. To demonstrate honokiol effects on stem cells, we performed colosphere assays. Honokiol significantly reduced the number and size of colospheres, suggesting effects on stem cells. In addition, colon cancer stem cell marker proteins DCLK1, LGR5, CD44 and SOX-9 were also decreased. Further proof was obtained by flow cytometry analyses, where honokiol reduced the number of DCLK1+ cells. We next determined whether signaling pathways are affected, for this, we tested the effect of honokiol on Hippo signaling pathway, which is an active pathway in intestinal stem cells. Honokiol significantly decreased the phosphorylation of Hippo pathway proteins Mst1/2, Lats1/2 and YAP1. Furthermore, honokiol 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 honokiol-mediated growth suppression. To determine the effect of honokiol on tumor growth in vivo, nude mice harboring HCT116 tumor xenografts in their flanks were administered the compound intraperitoneally every day for 21 days. Honokiol treatment significantly inhibited tumor xenograft growth, with notably lower tumor volume and weight. Microvessel density, based on CD31 staining was also significantly lower in the tumors following honokiol treatment when compared to controls. Western blot and immunohistochemistry analyses demonstrated significant inhibition in the expression of stem marker and Hippo signaling proteins in the honokiol-treated xenograft tissues.
Conclusion: Together, these data suggest that honokiol is a potent inhibitor of colon cancer that targets stem cells by inhibiting Hippo signaling pathway.
Citation Format: Dharmalingam Subramaniam, Sivapriya Ponnurangam, Shahid Umar, Satish Ramalingam, Roy A. Jensen, Shrikant Anant. Honokiol affects stem cell viability in part through suppression of Hippo signaling pathway. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3662. doi:10.1158/1538-7445.AM2013-3662