Honokiol activates AMP-activated protein kinase in breast cancer cells via an LKB1-dependent pathway and inhibits breast carcinogenesis
© Sharma et al.; licensee BioMed Central Ltd. 2012
Received: 27 July 2011
Accepted: 21 February 2012
Published: 21 February 2012
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.
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.
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.
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.
Breast cancer is one of the most common cancers and the second leading cause of cancer-related mortality in women. About 226,870 new cases of invasive breast cancer and about 63,300 new cases of carcinoma in situ will be diagnosed in 2012, according to the latest estimates for breast cancer in the United States by American Cancer Society. Despite major advances in screening programs and development of various targeted therapeutic approaches, mortality related to breast cancer still remains at a staggering high level, with approximately 1 in 35 women dying of breast cancer. Available therapies, including radiation, endocrine, and conventional chemotherapy, are often limited by high toxicity, lower efficacy, therapeutic resistance, and therapy-related morbidity. Therefore, more-effective therapeutic strategies are clearly needed to combat breast cancer and to reduce morbidity and mortality.
The importance of active constitutive agents in natural products has become increasingly apparent, owing to their potential cancer preventive as well as therapeutic properties [1, 2]. In traditional Asian medicine, root and stem bark of Magnolia species have been used for centuries to treat anxiety, nervous disorders, fever, gastrointestinal symptoms, and stroke . Therapeutic benefits of Magnolia species have been attributed to honokiol, a natural phenolic compound isolated from an extract of seed cones from Magnolia grandiflora [3, 4]. Honokiol has shown antithrombocytic, antibacterial, antiinflammatory, antioxidant, and anxiolytic effects, and it may prove beneficial against hepatotoxicity, neurotoxicity, thrombosis, and angiopathy . Two pioneering studies showing the remarkable inhibitory effects of honokiol on mouse skin-tumor promotion and demonstrating efficacy of honokiol against established tumors in mice [5, 6] ascertained the anticancer potential of honokiol. Subsequent studies showed the anticancer activities of honokiol in many cancer cell lines and tumor models [7–11].
Honokiol has been found to alter many cellular processes and to modulate molecular targets that are known to affect apoptosis, growth, and survival of tumor cells. A review of previous studies suggests that the mechanism by which honokiol causes growth arrest and cell death may be cell-line/tumor-type specific and involve many signaling pathways. For instance, Bax upregulation has been observed in some but not in other cellular systems [7, 12]. Honokiol decreases phosphorylation of ERK, Akt, and c-Src to induce apoptosis effectively in SVR angiosarcoma cells , inhibits the ERK signaling pathway to exert antiangiogenesis activity , but activates ERK in cortical neurons to induce neurite outgrowth [14, 15]. In chronic lymphocytic leukemia (CLL), honokiol causes apoptosis through activation of caspase 8, followed by caspase 9 and 3 activation . Honokiol-mediated increased cleavage of Mcl-1 and downregulation of XIAP as well as BAD upregulation is observed in multiple myeloma, whereas Bid, p-Bad, Bak, Bax, Bcl-2, and Bcl-xL remain unchanged . Honokiol also inhibits the NF-κB signaling pathway, thus affecting expression of many downstream genes in endothelial cells, human monocytes, lymphoma, embryonic kidney cells, promyelocytic leukemia, multiple myeloma, breast cancer, cervical cancer, and head and neck cancer [16–19]. Thus, honokiol elicits several cellular responses and modulates multiple facets of signal transduction.
In the present study, we specifically investigated the effect of honokiol on the malignant properties of breast cancer cells, including migration and invasion, and also examined the underlying molecular mechanisms. Intriguingly, we discovered that honokiol increases the expression of tumor-suppressor LKB1 to modulate the signaling pathway involving the AMPK-pS6K axis. We directly tested the requirement of AMPK and LKB1 in honokiol-mediated inhibition of malignant properties of breast cancer cells. Our results showed that LKB1 and AMPK are integral molecules required for honokiol-mediated modulation of 4EBP1-pS6K and inhibition of migration and invasion of breast cancer cells.
Materials and methods
Cell culture and reagents
The human breast cancer cell lines, MCF7 and MDA-MB-231, were obtained from the American Type Culture Collection and maintained in DMEM supplemented with 10% fetal bovine serum (FBS) (Gemini Bioproducts, Woodland, CA, USA) and 2 μM L-glutamine (Invitrogen, Carlsbad, CA, USA). Cell-line authentication was done by analysis of known genetic markers or response (for example, expression of estrogen receptor and p53 and estrogen responsiveness) . AMPK-null and AMPK-WT immortalized MEFs were kindly provided by Dr. Keith R. Laderoute (SRI International, Menlo Park, CA, USA) . Honokiol is a natural product extracted from seed cone of Magnolia grandiflora, as previously described . Antibodies for p-AMPK (phospho-AMPK), AMPK, ACC, p-ACC (phospho-ACC), pS6K, p-pS6K (phospho-S6K), 4EBP1, p-4EBP1 (phospho-4EBP1), p-Akt (phospho-Akt), Akt, and LKB1 (3047) were purchased from Cell Signaling Technology (Danvers, MA, USA).
LKB1 stable knockdown using lentiviral short-hairpin RNA
Five pre-made lentiviral LKB1 short-hairpin RNA (shRNA) constructs and a negative control construct created in the same vector system (pLKO.1) were purchased from Open Biosystems (Huntsville, AL, USA). Paired LKB1 stable knockdown cells (MCF7 and MDA-MB-231) were generated by following our previously published protocol .
Cell-viability assay was performed by estimating the reduction of XTT (2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxyanilide), by using a commercially available kit (Roche) . Breast cancer cells were treated with honokiol as indicated.
For colony-formation assay , MCF7 and MDA-MB-231 cells were treated with honokiol as indicated for 10 days; colonies containing > 50 normal-appearing cells were counted.
Anchorage-independent soft-agar growth assay
Anchorage-independent growth of MCF7 and MDA-MB-231 cells in the presence of honokiol treatment was determined by colony formation on soft agar . Colonies were counted in five randomly selected fields at ×10 magnification by using Olympus IX50 inverted microscope.
Migration assay was performed according to our published protocol . Cells were treated with honokiol as indicated. Plates were photographed after 24 and 48 hours at the identical location of the initial image.
Electric cell-substrate impedance sensing (ECIS) wound-healing assay
Wound-healing assay was performed by using the ECIS (Applied BioPhysics, Troy, NY, USA) technology and following our previously established protocol .
Spheroid migration assay
MDA-MB-231 and MCF7 cells (1.5 × 104) were seeded in 0.5% agar-coated plates and cultured on an orbital shaker (100 rpm) for 48 hours in a humidified atmosphere containing 5% CO2 at 37°C. Intact tumor spheroids were selected and transferred to six-well plates. The spheroids were treated with honokiol, as indicated. After 48 hours of incubation, spheroids were fixed with 10% buffered formalin in PBS and stained with crystal violet. The migration of cells from spheroids was observed under a light microscope.
For an in vitro model system of metastasis, Matrigel invasion assay  was performed by using a Matrigel invasion chamber from BD Biocoat Cellware (San Jose, CA, USA). The slides were coded to prevent counting bias, and the number of invaded cells on representative sections of each membrane were counted under light microscope. The number of invaded cells for each experimental sample represents the average of triplicate wells.
Whole cell lysate  was prepared by scraping MCF7 and MDA-MB-231 cells in 250 μl of ice-cold modified RIPA buffer. An equal amount of protein was resolved on sodium dodecylsulfate polyacrylamide gel, transferred to nitrocellulose membrane, and Western blot analysis was performed. Immunodetection was performed by using enhanced chemiluminescence (ECL system; Amersham Pharmacia Biotech, Arlington Heights, IL, USA) according to manufacturer's instructions.
Immunoprecipitation of LKB1 was performed by following the previously published protocol  by using anti-LKB1 antibody followed by immunoblotting with anti-STRAD antibody.
Immunofluorescence and confocal imaging
Breast cancer cells (5 × 105 cells/well) were plated in four-well chamber slides (Nunc, Rochester, NY, USA) followed by treatment with honokiol and subjected to immunofluorescence analysis as described . Fixed and immunofluorescently stained cells were imaged by using a Zeiss LSM510 Meta (Zeiss) laser scanning confocal system configured to a Zeiss Axioplan 2 upright microscope with a 63XO (NA 1.4) plan-apochromat objective. All experiments were performed multiple times by using independent biologic replicates.
Breast tumorigenesis assay
MDA-MB-231 (5 × 106) cells in 0.1 ml of HBSS were injected subcutaneously into the right gluteal region of 4- to 6-week-old female athymic nude mice. Two weeks after initial implantation, the animals were placed into two experimental groups. Mice were treated with intraperitoneal injections of (a) control (saline and Intralipidor (b) honokiol, at 3 mg/mouse/day in 20% Intralipid (Baxter Healthcare, Deerfield, IL, USA), 3 times per week for the duration of the experiment. Tumors were measured by using vernier calipers, with tumor volume calculated by using the formula (V = a/2 × b2), where V is the tumor volume in cubic millimeters, and a and b are the largest and smallest diameters in millimeters, respectively. All animals were killed after 4 weeks of treatment. Tumors were collected; weighed, fixed in 10% neutral-buffered formalin; and subjected to further analysis with immunohistochemistry.
We used tumor sections to determine the effect of honokiol on expression of p-AMPK, LKB1, and Ki-67 by immunohistochemistry. Immunohistochemistry was performed essentially as described by us previously for other proteins [25, 26]. At least four nonoverlapping representative images from each tumor section from five mice of each group were captured by using ImagePro software for quantitation of p-AMPK, LKB1, and Ki-67 expression. Total cell lysates were prepared from tumor samples and subjected to immunoblot analysis. All animal studies were conducted in accordance with the guidelines of University ACUC.
All experiments were performed thrice in triplicates. Statistical analysis was performed by using Microsoft Excel software. Significant differences were analyzed by using the Student t test and two-tailed distribution. Data were considered to be statistically significant if P < 0.05. Data were expressed as mean ± SEM between triplicate experiments performed thrice.
Honokiol treatment inhibits clonogenicity, migration, and invasion of breast cancer cells
Honokiol-induced AMPK activation plays an integral role in honokiol-mediated inhibition of mTOR activity and migration potential of cells
mTOR, a key regulator of cell growth and proliferation, exists in two structurally and functionally distinct multiprotein complexes, mTORC1 and mTORC2. mTORC1 is known to activate protein synthesis and cell growth through regulating pS6K and 4E-BP1 activity, whereas mTORC2 phosphorylates Akt on Ser-473, activating cell growth, proliferation, and survival [43, 44]. We found that honokiol increases AMPK activation and inhibits mTORC1 function, as evidenced by inhibition of pS6K and 4E-BP1 phosphorylation.
We next determined whether honokiol treatment modulates mTORC2 function. mTORC2 phosphorylates Akt on Ser-473. Therefore, to determine whether mTORC2 is also inhibited by honokiol under similar conditions, breast cancer cells were treated with honokiol, and the phosphorylation of Akt was determined. Honokiol did not alter Akt phosphorylation on Ser-473 in breast cancer cells (Additional file 3). These results provide evidence that honokiol only inhibits mTORC1 in breast cancer cells. Contrasting findings have been reported previously, showing reduction in Akt phosphorylation in response to honokiol treatment. Of note, MDA-MB-231 cells were treated with much higher concentrations of honokiol (60, 80, and 100 μM) in this study . Hence, the observed decrease in Akt phosphorylation may be due to the treatment with higher concentrations of honokiol. Honokiol inhibits breast cancer growth in a concentration-dependent manner, with higher concentrations much more inhibitory than lower concentrations (Figure 1).
We next asked whether AMPK is directly involved in honokiol-mediated inhibition of migration. AMPK-WT MEFs exhibited inhibition of migration in response to honokiol treatment in scratch migration as well as ECIS-based migration assay. Interestingly, honokiol treatment could not inhibit migration of AMPK-null MEFs (Figure 4c; Additional file 4). AMPK knockdown also inhibited the antiproliferative effect of honokiol (Figure 4e). These results showed that AMPK is an integral molecule in mediating the negative effects of honokiol on the mTOR axis and migration potential of cells.
Inhibition of LKB1 abrogates honokiol-mediated modulation of AMPK and inhibition of migration and invasion of breast cancer cells
We raised the question whether LKB1 plays an important regulatory role in honokiol- mediated modulation of AMPK and inhibition of migration and invasion of breast cancer cells. To address these questions, we used LKB1shRNA lentivirus and puromycin to select for stable pools of MCF7 and MDA-MB-231 cells with LKB1 depletion. We analyzed pLKO.1 and LKB1shRNA stable MCF7 and MDA-MB-231 cell pools for LKB1 protein expression with immunoblot analysis and found that LKB1 protein expression was significantly reduced in LKB1shRNA cells (shRNA1 and shRNA2) as compared with pLKO.1 control cells (Figure 5d). pLKO.1 and LKB1shRNA cells were treated with honokiol, and phosphorylation of AMPK was determined by using Western blot analysis. We found that honokiol increased phosphorylation of AMPK in pLKO.1 cells. Intriguingly, displaying a crucial role of LKB1, honokiol treatment did not change the phosphorylation levels of AMPK in LKB1shRNA cells (Figure 5e). Invasion and migration are the key biologic features of malignant behavior of carcinoma cells . In addition to examining the effect of LKB1 depletion on honokiol-induced modulation of AMPK, we also examined the requirement of LKB1 in honokiol-mediated inhibition of metastatic properties of breast cancer cells. As evident from Figure 5f, honokiol treatment efficiently inhibited migration of pLKO.1 cells, whereas untreated pLKO.1 cells showed increased migration. Our results showed that LKB1shRNA cells exhibited increased migration in the absence of honokiol treatment. Interestingly, honokiol treatment did not inhibit the migration of LKB1shRNA cells (Figure 5f). We next examined the effect of honokiol on invasion potential of pLKO.1 and LKB1shRNA cells and found that honokiol inhibited invasion of pLKO.1 cells, whereas LKB1shRNA cells were not affected by honokiol treatment (Figure 5g). These results collectively show that honokiol-induced LKB1 overexpression is indeed a crucial component of the signaling machinery used by honokiol in modulating the AMPK-S6K axis and inhibiting the metastatic properties of breast cancer cells.
Honokiol treatment inhibits breast-tumor progression in athymic nude mice
The antitumor activity of honokiol, a natural product derived from magnolia plant and used in traditional Asian medicine, has been reported in various preclinical models . In the current study, we investigated the potential of honokiol in the inhibition of migration and invasion of breast cancer cells and the underlying molecular mechanisms. The following novel findings are reported in this study: (i) honokiol treatment inhibits malignant properties such as invasion and migration of breast cancer cells; (ii) honokiol stimulates AMPK phosphorylation and activity while reducing mTOR activity, as evidenced by reduced phosphorylation of pS6K and 4EBP1; (iii) AMPK protein is required for honokiol-mediated inhibition of pS6K and 4EBP1; (iv) honokiol increases the expression and cytosolic localization of tumor suppressor LKB1, which is an essential effector molecule to mediate the honokiol effect on the AMPK-pS6K axis and inhibition of invasion and migration of breast cancer cells; and (v) honokiol inhibits breast tumor growth and modulates the LKB1-AMPK-pS6K axis in vivo. Our results show that honokiol treatment significantly inhibits malignant properties of breast cancer cells through modulation of the LKB1-AMPK-pS6K axis; thus using honokiol may be a suitable therapeutic strategy for metastatic breast cancer.
Many bioactive molecules and their synthetic analogues have been reported to demonstrate activity against breast cancer [68–71]. Although the lower toxicity associated with bioactive molecules is a much desired quality, their limited bioavailability hinders further development. Honokiol exhibits a desirable spectrum of bioavailability, in contrast with many other natural products . The development of other polyphenolic agents has been obstructed by poor absorption and rapid excretion . Honokiol does not have this disability, as significant systemic levels of honokiol can be obtained in preclinical models, and it can cross the blood-brain barrier . These qualities of honokiol make it a promising small-molecular-weight natural anticancer agent. Indeed, honokiol has been found to alter many molecular targets in various cancer models to inhibit tumor cell growth and survival [3, 6, 9, 10, 12, 19]. One of the major findings of this study is that the LKB1-AMPK pathway plays a major role in mediating the effect of honokiol effect on migration and invasion of breast cancer cells.
AMPK, a master sensor of cellular energy balance in mammalian cells, regulates glucose and lipid metabolism . Biochemical regulation of serine/threonine protein kinase AMPK activation occurs through multiple mechanisms . AMPK undergoes a conformational change in response to direct binding of AMP to its nucleotide-binding domain, exposing the activation loop of the catalytic kinase subunit. LKB1 phosphorylates a critical threonine in this activation loop to activate AMPK. Dephosphorylation by protein phosphatases also plays an important role in regulating AMPK activity . Genetic depletion of LKB1 in mouse embryonic fibroblasts (MEFs) results in a loss of AMPK activation after energy stresses that increase AMP , showing the requirement of LKB1 in AMPK activation. We found that honokiol increases AMPK activation, which can be efficiently inhibited by the silencing of LKB1. AMPK represents a pivotal point in the mTOR pathway regulating a vast range of cellular activities, including transcription, translation, cell size, mRNA turnover, protein stability, ribosomal biogenesis, and cytoskeletal organization . Besides being directly activated by tumor-suppressor LKB1, AMPK itself regulates the activation of two other tumor suppressors, TSC1 and TSC2, which are critical regulators of Rheb and mTOR . We found that AMPK knockdown inhibits honokiol-mediated mTOR inhibition. Honokiol-mediated inhibition of mTOR also suggests that honokiol and its derivatives may prove excellent candidates as targeted therapies for carcinomas characterized by hyperactive mTOR signaling.
LKB1 kinase is a tumor suppressor and a key determinant in the Peutz-Jeghers syndrome, an inherited susceptibility to gastrointestinal, lung, pancreatic, and breast cancer [47, 74]. Inactivation of the LKB1 gene has been shown in a subset of sporadic lung and pancreatic cancer. Although the loss of LKB1 expression is not commonly observed in human breast carcinoma, it certainly correlates with high-grade DCIS and high-grade invasive ductal carcinoma . It is important to note that LKB1 expression was not abrogated in pure DCIS cases but only in the DCIS associated with invasion, indicating that loss of LKB1 could potentially promote invasion. Supporting this notion, low LKB1 protein levels have been reported to correlate with poor prognosis in breast carcinoma . Our studies show that honokiol treatment increases the expression and cytosolic localization of LKB1 in breast xenograft tumors and inhibits tumor growth. LKB1 is localized predominantly in the nucleus, translocating to the cytosol, either by forming a heterotrimeric complex with STRAD (ste20-related adaptor protein) and MO25 (mouse protein 25) or by associating with LIP1 (LKB1-interacting protein), to exert its biologic functions [50, 52, 55, 75, 76]. The cytoplasmic pool of LKB1 plays an important role in mediating its tumor-suppressor properties. Wild-type LKB1, when co-expressed with STRAD and MO25, exhibits increased cytoplasmic localization, whereas mutant LKB1, unable to interact with STRAD and MO25, remains in the nucleus [75, 77]. Promotion of cytosolic translocation of LKB1 is a common mechanism to activate downstream LKB1 functions, as AMPK activation by metformin, peroxynitrile, or adiponectin also involves LKB1 cytosolic translocation [22, 64, 78–80]. Honokiol treatment increases LKB1-STRAD complex formation in addition to overexpression of LKB1, thus increasing the functional pool of LKB1. Our study shows for the first time that honokiol stimulates the cytosolic translocation of LKB1 in breast cancer cells.
We uncovered a novel mechanism by which honokiol inhibits invasion and migration of breast cancer cells, which involves enhanced expression and cytosolic localization of LKB1 and AMPK activation. We also demonstrated the requirement of LKB1 and AMPK in honokiol-mediated inhibition of migration and invasion of breast cancer cells. Our results thus provide new insight into the mechanisms by which honokiol, a promising anticancer agent, inhibits breast carcinogenesis.
AN, MYB, NKS, and DS declare no conflict of interest. JLA is listed as an inventor on patents filed by Emory University. Emory has licensed its honokiol technologies to Naturopathic Pharmacy. JLA has received stock in Naturopathic Pharmacy, which, to the best of our knowledge, is not publically traded.
4E binding protein 1
acetyl-coenzyme A carboxylase
AMP-activated protein kinase
electric cell-substrate impedance sensing
extracellular signal-regulated kinase
liver kinase B1
mouse embryonic fibroblasts
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.
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