Honokiol, a small-molecule polyphenol isolated from magnolia species, is widely known for its therapeutic potential as an anti-inflammatory, anti-thrombosis, anti-oxidant 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 elucidate the underlying molecular mechanisms.

Clonogenicity and 3D-colony formation assays were used to examine breast cancer cell growth upon honokiol treatment. The effect of honokiol on invasion and migration of breast cancer cells was evaluated by utilizing 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 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 biological 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.

Analysis of the underlying molecular mechanisms revealed that honokiol treatment increases AMP-activated protein kinase (AMPK) phosphorylation and activity as evident by increased phosphorylation of 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). 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 gene LKB1 in breast cancer cells. LKB1 knockdown inhibited honokiol-mediated activation of AMPK and more importantly, 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 increase in the levels of cytoplasmic LKB1 and phospho-AMPK in honokiol-treated tumors.

Taken together, these data provide first in vitro and in vivo evidence of the integral role of LKB1-AMPK axis in honokiol mediated inhibition of invasion and migration of breast cancer cells. In conclusion, honokiol treatment could potentially be a rational therapeutic strategy for breast carcinoma.

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