Honokiol activates AMP-activated protein kinase in breast cancer cells via an LKB1-dependent pathway and inhibits breast carcinogenesis

• Type of Material: Article
• Authors: Arumugam Nagalingam, Johns Hopkins University School of Medicine; Jack Arbiser, Emory University; Michael Y. Bonner, Emory University; Neeraj K. Saxena, University of Maryland School of Medicine; Dipali Sharma, Johns Hopkins University School of Medicine
• Language: English
• Date: 2012-02-21
• Publisher: BioMed Central
• Publication Version: Final Published Version
• Copyright Statement: © 2012 Sharma et al.; licensee BioMed Central Ltd.
• License: Creative Commons Attribution 2.0 Generic
• Final Published Version (URL): http://breast-cancer-research.com/content/14/1/R35
• Title of Journal or Parent Work: Breast Cancer Research
• ISSN: 1465-5411
• Volume: 14
• Issue: R35
• Start Page: 1
• End Page: 16
• Grant/Funding Information: NKS, NIH K01DK076742 and R03DK089130; DS, NIH R01CA131294 and BCRF; JLA, Emory Skin Disease Research Core Center Grants NIH R01 AR47901 and NIH P30 AR42687, Veterans Administration Hospital Merit Award, funds from Rabinowitch-Davis Foundation for Melanoma Research and the Betty Minsk Foundation for Melanoma Research.
• Research Categories: Health Sciences, Oncology

Abstract

Introduction Honokiol, a small-molecule polyphenol isolated from magnolia species, is widely known for its therapeutic potential as an antiinflammatory, antithrombosis, and antioxidant agent, and more recently, for its protective function in the pathogenesis of carcinogenesis. In the present study, we sought to examine the effectiveness of honokiol in inhibiting migration and invasion of breast cancer cells and to elucidate the underlying molecular mechanisms. Methods Clonogenicity and three-dimensional colony-formation assays were used to examine breast cancer cell growth with honokiol treatment. The effect of honokiol on invasion and migration of breast cancer cells was evaluated by using Matrigel invasion, scratch-migration, spheroid-migration, and electric cell-substrate impedance sensing (ECIS)-based migration assays. Western blot and immunofluorescence analysis were used to examine activation of the liver kinase B1 (LKB1)-AMP-activated protein kinase (AMPK) axis. Isogenic LKB1-knockdown breast cancer cell line pairs were developed. Functional importance of AMPK activation and LKB1 overexpression in the biologic effects of honokiol was examined by using AMPK-null and AMPK-wild type (WT) immortalized mouse embryonic fibroblasts (MEFs) and isogenic LKB1-knockdown cell line pairs. Finally, mouse xenografts, immunohistochemical and Western blot analysis of tumors were used. Results Analysis of the underlying molecular mechanisms revealed that honokiol treatment increases AMP-activated protein kinase (AMPK) phosphorylation and activity, as evidenced by increased phosphorylation of the downstream target of AMPK, acetyl-coenzyme A carboxylase (ACC) and inhibition of phosphorylation of p70S6kinase (pS6K) and eukaryotic translation initiation factor 4E binding protein 1 (4EBP1). By using AMPK-null and AMPK-WT (MEFs), we found that AMPK is required for honokiol-mediated modulation of pACC-pS6K. Intriguingly, we discovered that honokiol treatment increased the expression and cytoplasmic translocation of tumor-suppressor LKB1 in breast cancer cells. LKB1 knockdown inhibited honokiol-mediated activation of AMPK and, more important, inhibition of migration and invasion of breast cancer cells. Furthermore, honokiol treatment resulted in inhibition of breast tumorigenesis in vivo. Analysis of tumors showed significant increases in the levels of cytoplasmic LKB1 and phospho-AMPK in honokiol-treated tumors. Conclusions Taken together, these data provide the first in vitro and in vivo evidence of the integral role of the LKB1-AMPK axis in honokiol-mediated inhibition of the invasion and migration of breast cancer cells. In conclusion, honokiol treatment could potentially be a rational therapeutic strategy for breast carcinoma.