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Honokiol activates LKB1-miR-34a axis and antagonizes the oncogenic actions of leptin in breast cancer

Honokiol activates LKB1-miR-34a axis and antagonizes the oncogenic actions of leptin in breast... www.impactjournals.com/oncotarget/ Oncotarget, Vol. 6, No. 30 Honokiol activates LKB1-miR-34a axis and antagonizes the oncogenic actions of leptin in breast cancer 1,* 1,* 3 3,4 Dimiter B. Avtanski , Arumugam Nagalingam , Michael Y. Bonner , Jack L. Arbiser , 2 1 Neeraj K. Saxena , Dipali Sharma Department of Oncology, Johns Hopkins University School of Medicine and the Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, Baltimore MD 21231 Department of Medicine, University of Maryland School of Medicine, Baltimore MD 21201 Department of Dermatology, Emory University School of Medicine, Winship Cancer Institute Atlanta Veterans Administration Medical Center, Atlanta, GA 30322 These authors have contributed equally to this work Correspondence to: Dipali Sharma, e-mail: [email protected] Neeraj Saxena, e-mail: [email protected] Keywords: Honokiol, leptin, LKB1, miR-34a, breast cancer Received: May 13, 2015 Accepted: August 13, 2015 Published: August 24, 2015 ABSTRACT Leptin, a major adipocytokine produced by adipocytes, is emerging as a key molecule linking obesity with breast cancer therefore, it is important to nd fi effective strategies to antagonize oncogenic effects of leptin to disrupt obesity-cancer axis. Here, we examine the potential of honokiol (HNK), a bioactive polyphenol from Magnolia grandiflora , as a leptin-antagonist and systematically elucidate the underlying mechanisms. HNK inhibits leptin-induced epithelial-mesenchymal-transition (EMT), and mammosphere-formation along with a reduction in the expression of stemness factors, Oct4 and Nanog. Investigating the downstream mediator(s), that direct leptin-antagonist actions of HNK; we discovered functional interactions between HNK, LKB1 and miR-34a. HNK increases the expression and cytoplasmic-localization of LKB1 while HNK-induced SIRT1/3 accentuates the cytoplasmic-localization of LKB1. We found that HNK increases miR-34a in LKB1-dependent manner as LKB1-silencing impedes HNK-induced miR-34a which can be rescued by LKB1-overexpression. Finally, an integral role of miR-34a is discovered as miR-34a mimic potentiates HNK-mediated inhibition of EMT, Zeb1 expression and nuclear-localization, mammosphere-formation, and expression of stemness factors. Leptin-antagonist actions of HNK are further enhanced by miR-34a mimic whereas miR-34a inhibitor results in inhibiting HNK’s effect on leptin. These data provide evidence for the leptin-antagonist potential of HNK and reveal the involvement of LKB1 and miR-34a. (increase in cell number), leading to dysregulation of local INTRODUCTION and systemic secretion of biologically active polypeptides, adipocytokines such as leptin [3]. In recent years, leptin Given that one-third of all cancers are attributed has emerged as a key candidate molecule mediating the to obese state, obesity is a well-established risk factor. molecular effects of obesity on cancer [4, 5]. Various Obese state is not only associated with aggressive tumor epidemiological studies have shown that high level of progression, poorer prognosis, increased recurrence and plasma leptin is linked with increased risk and poor poorer survival of obese breast cancer patients, but it prognosis for breast carcinogenesis [6, 7]. Analysis of also impacts tumor initiation [1, 2]. Adipocytes lose their clinical samples showed overexpression of leptin receptor normal physiological size heterogeneity in obese state and in 83% of breast tumor samples whereas no expression undergo hypertrophy (increase in cell size) and hyperplasia www.impactjournals.com/oncotarget 29947 Oncotarget of leptin receptor was observed in normal mammary honokiol-mediated inhibition of breast tumor growth and epithelial cells [8, 9]. Also, leptin overexpression was progression [28]. Recently, we discovered the involvement observed in 92% of breast tumors examined but in none of miR-34a in breast tumor inhibition function of of the cases of normal breast epithelium. Overexpression honokiol. Honokiol treatment inhibited breast tumor of leptin and leptin receptor in breast tumors and its growth in lean and obese-hyperleptinemic mice models association with tumor aggressiveness suggest that leptin in a manner associated with activation of miR-34a [30]. can also influence breast tumor growth and progression via In this report, we specifically investigated the potential an autocrine pathway [9, 10]. of HNK to inhibit leptin-induced epithelial-mesenchymal Various research groups have been studying the transition (EMT) and tumorsphere formation and examine oncogenic role of leptin in cancer. Studies from our the underlying molecular mechanisms. We provide lab and others have established that high leptin levels molecular evidence supporting the regulatory role of (hyperleptinemia) associated with obese state stimulate LKB1, and integral involvement of miR-34a in leptin- breast cancer cell proliferation, invasion, migration, and antagonist potential of HNK. angiogenesis, thereby promoting breast tumor growth and metastasis [11–17]. Leptin also plays a central role RESULTS in the acquisition of mesenchymal characteristics by inducing breast cancer cells to undergo a transition from Honokiol inhibits leptin-induced epithelial- epithelial to spindle-like mesenchymal morphology [13]. mesenchymal transition, mammosphere Leptin has been reported to regulate many signaling formation, and migration of breast cancer cells pathways and transcription factors implicated in breast cancer stem cells (BCSCs). Intact leptin-leptin receptor Epithelial to mesenchymal transition (EMT) of signaling was found to play an integral role in the cancer cells is a crucial early event leading to induction survival of CSC population [18]. It is shown that leptin of cell motility, invasion and distant metastasis. We receptor is a characteristic feature of Tumor initiating recently presented a pivotal role of leptin in acquisition stem cells (TISCs) and participates in the regulation of of mesenchymal characteristics and aggressive behavior core pluripotency-associated transcription factors, Oct4 in breast cancer cells [13]. Here, we specifically examined and Nanog [19, 20]. These studies indicate that leptin not if HNK could inhibit the stimulatory effect of leptin on only plays a significant role in promoting the growth and EMT and metastatic properties of breast cancer cells. metastatic progression of established breast tumors but Following treatment with leptin and HNK, we observed also augments tumor initiation and recurrence. striking morphological differences between MCF7 cells Leptin and leptin-signaling pathway have emerged treated with different combinations. Leptin-treated MCF7 as leading targets for disrupting obesity-breast cancer link; cells exhibited acquisition of fibroblast-like appearance therefore, developing effective, non-endocrine, non-toxic and increased formation of pseudopodia observed agents for the inhibition of neoplastic effects of leptin emanating from the cell membrane. These features is highly important. Current strategies to inhibit leptin signify typical mesenchymal phenotype rather than the pathway such as soluble Leptin receptors (LRs), synthetic normal epithelial phenotype of MCF7 cells, showing that leptin-antagonists, and anti-LR monoclonal antibodies cells have undergone EMT upon leptin treatment. HNK (anti-LR mAbs) [17] are limited by associated toxicities prevented the morphological transition from an epithelial- as well as low efficacy. Recently, active constitutive agents like to mesenchymal-like appearance caused by leptin in natural products used in traditional Asian medicine treatment. HNK alone did not affect the morphology of have shown efficacy as potential cancer preventive as MCF7 cells (Figure 1A). To unequivocally establish that well as therapeutic agents [21, 22]. For years, cones, HNK blocks leptin-induced EMT, we next examined bark and leaves from Magnolia plant species have been the biochemical hallmarks of EMT-reversal including used for their anti-thrombocytic, anti-inflammatory, gain of expression of epithelial markers (occludin, and anxiolytic, anti-depressant, antioxidant, antispasmodic, cytokeratin-18 (CK-18) with a concomitant decrease and antibacterial effects [23–26]. It is now known that in mesenchymal markers (fibronectin, and vimentin) Honokiol (HNK), a natural phenolic compound isolated expression. Leptin treatment resulted in upregulation from an extract of seed cones from Magnolia grandiflora of mesenchymal markers accompanied with a marked [27] is responsible for these medicinal benefits of decrease in the expression of epithelial markers. HNK Magnolia species. Previous studies from our lab have blocked leptin-induced modulation of mesenchymal shown that HNK inhibits breast carcinogenesis in vitro and and epithelial markers leading to decreased expression in vivo [28, 29] thereby establishing HNK as a promising of fibronectin and vimentin and increased expression of bioactive compound against breast carcinogenesis. We also CK-18 and occludin (Figure 1B and 1C, Supplementary found that honokiol treatment increases the expression of Figure 1A). Immunocytochemical analysis provided tumor suppressor LKB1 which plays an integral role in additional evidence to support HNK-mediated leptin- www.impactjournals.com/oncotarget 29948 Oncotarget Figure 1: Honokiol inhibits leptin-induced epithelial-mesenchymal transition and migration of breast cancer cells. A. MCF7 cells were treated with leptin (L) (100 ng/ml) and/or Honokiol (HNK) (5 μM) as indicated. Vehicle treated cells are denoted as (C) Control. Morphological changes associated with EMT are shown in phase-contrast images. The presence of spindle-shaped cells, increased intracellular separation and pseudopodia were noted in leptin-treated cells but not in HNK-treated cells. B. MCF7 cells were treated as in A and total lysates were immunoblotted for Occludin, Snail and Zeb2 expression levels. Actin was used as control. C. MCF7 cells were treated as in A, total RNA was isolated and expression levels of epithelial and mesenchymal marker genes was analyzed. Actin was used as control. D. Breast cancer cells were treated as in A, and subjected to immunofluorescence analysis (1000X magnification) of Zeb1. Bar-graphs show the fold-change in number of cells expressing nuclear Zeb. *p < 0.001, compared with untreated controls. #p < 0.001, compared with leptin-alone treatment. Leptin-induced nuclear translocation of Zeb1 was abrogated by HNK treatment. E. Breast cancer cells were treated as in A, and subjected to immunofluorescence analysis (200X magnification) of E-cadherin, and Occludin. Bar-graphs show the fold-change in number of cells expressing Occludin and E-cadherin. *p < 0.05, compared with untreated controls. #p < 0.01, compared with leptin-alone treatment. F. MDA-MB-231 cells derived tumors were developed in nude mice and treated with leptin and/or HNK (n = 6–8/treatment group). At the end of five weeks of treatment, tumors were collected. Total RNA was isolated from tumor samples and subjected to RT-PCR analysis for the expression of mesenchymal markers and transcription factors. G. Breast cancer cells were treated as in A and subjected to scratch-migration assay. induced EMT reversal showing gain of expression of even in the presence of leptin (Figure 1B, and 1D). In occluding and E-cadherin (Figure 1E). Transcriptional a recent study, we showed that HNK administration repressors for epithelial marker proteins, Zeb1/2 and snail, retarded leptin-induced growth of MDA-MB-231 cells are frequently detected in metastatic cancer cells and are implanted in female athymic mice [30]. We used tumor known to be involved in EMT [31, 32]. We also examined samples from the same study to evaluate the effect of the involvement of these transcription repressors in HNK- HNK on leptin-induced mesenchymal markers by RT- mediated inhibition of leptin-induced EMT. Indeed, leptin PCR analysis. Corroborating in vitro findings, tumors from treatment not only increased the expression of snail, Zeb1 mice co-treated with HNK and leptin showed decreased and Zeb2 but also increased the nuclear translocation of levels of expression of vimentin, fibronectin, Zeb1/2 Zeb1 (Figure 1B, 1C and 1D). Importantly, HNK treatment and slug in comparison to tumors from leptin-treated inhibited leptin-induced expression of snail, Zeb1 and mice (Figure 1F). Since HNK inhibited leptin-induced Zeb2 as well as promoted cytoplasmic retention of Zeb1 EMT, we aimed to examine whether HNK treatment also www.impactjournals.com/oncotarget 29949 Oncotarget blocked induction of migration usually observed in the regulating its activity, we examined the phosphorylation presence of leptin. Significant migration of MCF7, MDA- and expression of AMPK. Honokiol-treatment increased MB-468, MDA-MB-231, SUM149, SUM159 and T47D phosphorylation and expression of AMPK (Figure 3A). breast cancer cells observed in the presence of leptin was Tumor-suppressor function of LKB1 is majorly attributed inhibited in the presence of HNK treatment (Figure 1G, to the cytoplasmic pool of LKB1 as mutant LKB1 lacking Supplementary Figure 1B, 1C). Honokiol treatment does the nuclear localization signal still retains the ability to not affect growth of MCF10A cells while leptin show suppress cell growth [38, 39]. Examining nuclear and only modest effects on MCF10A cells (Supplementary cytoplasmic fractions of breast cancer cells treated Figure 2). with HNK, we found that HNK induced cytoplasmic An emerging hypothesis is that EMT bestows cells localization of LKB1 (Figure 3B). Recent studies have with stem-like characteristics and facilitates an increase reported an important role of Sirtuin-deacetylases, in the subpopulation of CSC (cancer stem cells) [33–35]. SIRT1 and SIRT3 in LKB1 regulation. SIRT1 and SIRT3 Utilizing mammosphere assay that relies on the unique overexpression diminishes lysine acetylation of LKB1 property of breast cancer cells with stem-like potential to and concurrently increases its activity and cytoplasmic/ form large, round, unattached floating spheroid colonies nuclear ratio [40, 41]. We found that breast cancer cells (termed mammosphere), we showed that leptin induced treated with HNK exhibited an increase in the expression mammosphere formation. HNK treatment efficiently of SIRT1 and SIRT3 within 30 minutes post-treatment inhibits leptin-induced EMT as well as migration of (Figure 3C). To examine the effect of SIRT1 and SIRT3 breast cancer cells, therefore, we hypothesize that HNK on HNK-mediated cytoplasmic localization of LKB1, we may also inhibit leptin-induced mammosphere formation. overexpressed SIRT1 and SIRT3 in MCF7 cells followed Indeed, HNK inhibited mammosphere formation even in by HNK treatment. Immunofluorescence analysis showed the presence of leptin (Figure 2A). Given the association that LKB1 is present both in nucleus and cytoplasm in of induced pluripotent stem cell (iPSC) markers, Nanog untreated cells. Corroborating immunoblot analyses and Oct4 with self-renewal and maintenance of stem cell shown in figure 3B, HNK-treated cells exhibited increased fate, we wished to investigate whether iPSC markers are cytoplasmic localization of LKB1 in comparison to affected by leptin treatment. To this end, we assessed the untreated cells which was further enhanced with SIRT1 expression of Oct4, and Nanog in leptin-treated breast and SIRT3 overexpression (Figure 3D, 3F)). Our studies cancer cells and found that the expression of pluripotency show a role of sirtuin-deacetylases in HNK-mediated genes was higher in leptin-treated cells (Figure 2B–2D) functional upregulation of LKB1. with respect to the expression in untreated cells. Cells treated with HNK exhibited reduced levels of iPSC Honokiol upregulates miR-34a in a markers and interestingly, HNK treatment resulted in the LKB1-dependent manner inhibition of leptin-induced expression of Oct4 and Nanog (Figure 2B–2D). Breast cancer cells treated with leptin and Regulatory role of microRNAs (miRNA) in various honokiol were examined for the expression of Cyclin D1 biological and pathological processes, including cancer as a functional control (Supplemental Figure 3). Together, progression and metastasis has been well-recognized in these results show that HNK treatment results in effective recent years. Functionally, miRs are small (~21-mer) inhibition of leptin-induced epithelial-mesenchymal regulatory RNA molecules that exert their regulatory transition, mammosphere formation, stemness and effects by binding to the 3′-untranslated regions (3′-UTR) migration of breast cancer cells. of specific mRNAs triggering mRNA degradation or translational repression [42]. miRNAs have been known to function both as oncogenes, potentiating cancer progression Honokiol induces expression and cytoplasmic and metastasis, and tumor suppressors implicated in localization of tumor suppressor Liver Kinase B1 growth inhibition [43]. One of the important tumor in breast cancer cells suppressor miRNA involved in breast cancer is miR-34a, which is downregulated in aggressive breast tumors [44]. Liver kinase B1 (LKB1), owing to its dual role as a Breast cancer cells treated with HNK exhibited a time- tumor suppressor and an upstream master kinase, serves as dependent increase in miR-34a expression (Figure 4A). a major hub regulating several downstream pathways and Interestingly, leptin treatment decreased the expression of known tumor suppressors (e.g., AMPK-mTOR, JNK and miR-34a in MCF7, SKBR3 and SUM149 breast cancer p53) [36, 37]. We found that HNK increases expression cells (Figure 4B). We raised the question whether LKB1 of LKB1 in breast cancer cells (Figure 3A). Interestingly, plays any role in honokiol-mediated increase of miR-34a. leptin treatment inhibits LKB1 expression whereas shRNA We used LKB1 lentivirus and puromycin to select HNK treatment increases LKB1 expression even in the for stable pools of MCF7 cells with LKB1 depletion. presence of leptin (Figure 3E). Since LKB1 has recently shRNA pLKO.1 and LKB1 stable MCF7 cell pools were been identified as a critical upstream kinase for AMPK www.impactjournals.com/oncotarget 29950 Oncotarget Figure 2: Honokiol abates the stimulatory effect of leptin on mammosphere-formation-potential and acquisition of stem-like properties in breast cancer cells. A. MCF7 and MDA-MB-468 cells were treated with leptin (L) (100 ng/ml) and/or Honokiol (HNK) (5 μM) as indicated and subjected to mammosphere formation. Vehicle-treated cells are denoted as (C) The graph shows the number of mammospheres. *p < 0.001, leptin treatment compared with untreated controls. # p < 0.01, leptin+honokiol compared with leptin-alone treatment. B. MCF7 cells were treated with leptin (100 ng/ml) and/or HNK (5 μM) alone or in combination as indicated, total RNA was isolated and expression levels of Nanog and Oct4 were examined. Actin was used as control. C. Breast cancer cells were treated as in B, and total lysates were immunoblotted for Nanog and Oct4 expression levels. Actin was used as control. D. Bar-graphs show the fold-change in Nanog and Oct4 expression in breast cancer cells treated with leptin and/or honokiol. *p < 0.001, leptin treatment compared with untreated controls. #p < 0.001, leptin+honokiol compared with leptin-alone treatment. www.impactjournals.com/oncotarget 29951 Oncotarget Figure 3: Honokiol induces the expression and cytoplasmic localization of tumor suppressor LKB1 and involvement of SIRT1/3. A. MCF7 cells were treated with 5 μM HNK for various time intervals as indicated, total lysates were immunoblotted for LKB1, phospho-AMPK and total AMPK expression levels. Actin was used as control. B. MCF7 cells were treated with 5 μM HNK for 6 h, nuclear and cytoplasmic fractions were immunoblotted for LKB1 expression. Actin was used as control. C. MCF7 cells were treated with 5 μM HNK for various time intervals as indicated, total lysates were immunoblotted for SIRT1 and SIRT3 expression levels. Actin was used as control. D. MCF7 cells were transfected with SIRT1 and SIRT3 overexpression constructs as indicated followed by 5 μM HNK treatment. Cells were subjected to immunofluorescence analysis of LKB1. E. MCF7 cells were treated with leptin (100 ng/ml) and/or HNK (5 μM) as indicated, cell lysates were immunoblotted for LKB1. F. Bar-graphs show the fold-change in number of cells expressing cytoplasmic localization of LKB1. shRNA analyzed for LKB1 protein expression by immunoblot pLKO.1 and LKB1 cells were treated with HNK analysis. LKB1 protein expression was significantly and expression of miR-34a was determined. Intriguingly, shRNA reduced in LKB1 cells (shRNA1 and shRNA2) displaying a crucial role of LKB1, honokiol treatment did shRNA as compared to pLKO.1 control cells (Figure 4C). not increase miR-34a expression in LKB1 cells while www.impactjournals.com/oncotarget 29952 Oncotarget Figure 4: Honokiol upregulates miR-34a in LKB1-dependent manner in breast cancer cells. A. Expression levels of miR-34a in MCF7 cells treated with 5 μM HNK for various time intervals as indicated. *p < 0.001, compared with untreated controls. B. MCF7, SKBR3 and SUM149 cells were treated with 100 ng/ml leptin and miR-34a expression levels were measured. *p < 0.005, compared shRNA 1–2 with untreated controls. C. Immunoblot analysis of LKB1 in stable pools of LKB1-depleted (LKB1 ) and vector control (pLKO.1) shRNA 1–2 MCF7 cells. LKB1-depleted (LKB1 ) were transfected with LKB1 overexpression vector to create a ‘gain-of-function’ system. shRNA 1–2 D. Expression levels of miR-34a in stable pools of LKB1-depleted (LKB1 ) and vector control (pLKO.1) MCF7 cells. *p < 0.001, compared with untreated controls; **p < 0.001, compared with HNK-treated MCF7-pLKO.1 cells; #p < 0.001, compared with shRNA2 HNK-treated MCF7-pLKO.1 cells. E. LKB1-depleted (LKB1 ) were transfected with LKB1 overexpression vector and expression shRNA2 levels of miR-34a was examined. *p < 0.001, compared with HNK-treated, control-transfected, MCF7- LKB1 cells. pLKO.1 cells exhibited HNK-induced miR-34a expression molecule) further enhanced HNK-mediated inhibition (Figure 4D). As a gain-of-function strategy, LKB1 was of mesenchymal markers Fibronectin and Vimentin shRNA overexpressed in LKB1 cells (Figure 4C), treated with while enhancing the expression of epithelial markers, HNK followed by examination of miR-34a expression. E-cadherin, Occludin and CK-18 (Figure 5A, 5B). As evident in Figure 4E, HNK treatment increased HNK treatment decreased expression of slug and Zeb1, shRNA miR-34a expression in LKB1 cells overexpressing repressors of E-cadherin in comparison to untreated LKB1. These results show that LKB1 plays an important cells. Combination of HNK and miR-34a mimic further role in honokiol-induced miR-34a expression in breast enhanced the inhibition slug and Zeb1 (Figure 5B, 5C). cancer cells. Expression of miR-34a-inhibitor increased Zeb1 expression even in the presence of HNK (Figure 5C). Next, we investigated the effect of miR-34a mimic and Honokiol inhibits EMT, stemness and leptin inhibitor on HNK-induced cytoplasmic retention of function in a miR-34a-dependent manner Zeb1 in breast cancer cells. Breast cancer cells treated We further investigated the importance of with HNK, miR-34a mimic alone and in combination miR-34a in HNK-mediated modulation of EMT markers, exhibited cytoplasmic localization of Zeb1. Treatment stemness factors and inhibition of leptin function. with miR34a inhibitor caused nuclear localization of Zeb1 Ectopic miR-34a expression (in the form of a mimic (Figure 5D). To investigate the involvement of miR-34a www.impactjournals.com/oncotarget 29953 Oncotarget Figure 5: Evidence supporting the involvement of miR-34a in honokiol-mediated modulation of EMT and stemness factors. A. MCF7 cells were transfected with miR-34a mimic followed by treatment with vehicle (C) or HNK (5 μM) as indicated, total RNA was isolated and expression levels of epithelial and mesenchymal marker genes was analyzed. Actin was used as control. B. MCF7 cells were transfected with miR-34a mimic followed by treatment with vehicle C. or HNK (5 μM) as indicated, total protein lysates were immunoblotted for the expression levels of epithelial and mesenchymal marker genes as indicated. Actin was used as control. (C) MCF7 cells were transfected with miR-34a inhibitor or miR-34a mimic followed by treatment with vehicle (C) or HNK (5 μM) as indicated, total RNA was isolated and expression levels of Zeb1 was analyzed. Actin was used as control. Bar-graph shows fold-change in Zeb expression. D. MCF7 cells were transfected with miR-34a inhibitor or miR-34a mimic followed by treatment with vehicle (C) or HNK (5 μM) as indicated, immunofluorescence analysis for Zeb1 was performed. Arrows point the cells showing nuclear localization of Zeb1. Bar-graphs show the fold-change in number of cells expressing nuclear localization of Zeb1. *p < 0.02, HNK treated cells compared with HNK+miR-34a inhibitor treated cells. E. MCF7 cells were transfected with miR-34a mimic followed by treatment with vehicle (C) or HNK (5 μM) as indicated, total RNA was isolated and expression levels of stemness genes (Oct4, Nanog, Sox2) was analyzed. Actin was used as control. F. Breast cancer cells were transfected with miR-34a mimic followed by treatment with vehicle (C) or HNK (5 μM) as indicated, total protein lysates were immunoblotted for the expression levels of stemness genes (Oct4, Nanog, Sox2) genes as indicated. Actin was used as control. www.impactjournals.com/oncotarget 29954 Oncotarget in HNK-mediated inhibition of iPSC factors, we treated our interest in investigating the efficacy of HNK as a breast cancer cells with HNK in the presence of miR34a novel inhibitor of leptin-induced EMT and stemness in mimic or inhibitior. As expected, HNK inhibited the breast cancer. We found that HNK effectively inhibits expression of Oct4, Nanog and Sox2 in breast cancer cells leptin-induced EMT of breast cancer cells as evident which is further decreased by ectopic miR-34a expression from morphological changes and molecular alterations (Figure 5E, 5F). of mesenchymal and epithelial genes. Breast tumors Given our results showing that miR-34a plays an treated with HNK also showed reduced expression of important role in HNK-mediated modulation of EMT mesenchymal markers even in the presence of leptin markers and iPSC factors, we decided to examine whether treatment providing in vivo evidence. Mammosphere miR-34a modulation also affects HNK-mediated inhibition formation induced by leptin was also efficiently abrogated of oncogenic actions of leptin. Expression of miR-34a with HNK treatment along with inhibition of expression mimic potentiated HNK-mediated-inhibition of leptin- of iPSC factors (Oct4, nanog and Sox2). These findings induced clonogenicity, migration and mammosphere imparted convincing evidence supporting the efficacy formation. On the other hand, miR-34a inhibitor interfered of HNK as a novel inhibitor of leptin-induced EMT and with HNK efficacy as a leptin-antagonist resulting in poor stemness warranting further mechanistic investigations. inhibition of leptin-induced clonogenicity, migration and Consequently, we designed further studies to decipher mammosphere formation (Figure 6A, 6B, 6C). Leptin the key nodes of leptin-antagonist function of HNK treatment decreases miR-34a expression (Figure 4B), to facilitate establishing surrogate biomarkers for its therefore, it is interesting to note that miR-34a mimic efficacy and help in clinical development of this bioactive reduced oncogenic effects of leptin while miR-34a molecule as a leptin-antagonist. We found that HNK inhibitor increased leptin’s impact (Figure 6A, 6B, 6C). induces expression and cytoplasmic localization of LKB1. Together, these data provide evidence supporting an HNK also increases expression of Sirtuin-deacetylases, important role of miR-34a in HNK-mediated inhibition of SIRT1/3 and overexpression of both SIRT1 and SIRT3 oncogenic actions of leptin. further increases cytoplasmic localization of LKB1. HNK Collectively, the findings presented here suggest upregulates miR-34a in a LKB1-dependent manner; that HNK inhibits leptin-induced EMT, stemness and miR-34 a plays an important role in HNK-mediated mammosphere formation, and provide evidence for the inhibition of EMT, stemness and leptin-function. involvement of miR-34a as a regulator of HNK-mediated Tumor suppressor LKB1, a key determinant in inhibition of oncogenic actions of leptin, and uncover a Peutz-Jeghers syndrome, has been found to be inactivated novel mechanism of HNK action through activation of in a subset of sporadic lung and pancreatic cancer miR-34a in a LKB1-dependent manner (Figure 7). [37, 45]. LKB1 inactivation is not commonly correlated with human breast carcinoma but interestingly, LKB1 loss has been observed in high-grade DCIS and high- DISCUSSION grade invasive ductal carcinoma [46]. Importantly, LKB1 expression was abrogated only in the DCIS associated Over the last decade, there has been growing with invasion but not in pure DCIS cases indicating that emphasis on the importance of EMT, an essential normal LKB1 loss might promote invasive behavior. In fact, physiological process for embryonic development, tissue low LKB1 protein levels correlate with poor prognosis remodeling and wound healing, now implicated in cancer in breast carcinoma [47]. LKB1 knockdown increases progression. An oncogenic EMT causes epithelial-derived motility and invasiveness of cancer cells, and induces tumors to gain a mesenchymal phenotype facilitating the expression of many mesenchymal marker proteins migration and invasion potential of cancer cells. indicating its possible role in EMT [48, 49]. We show Acquisition of mesenchymal traits not only promotes that HNK increases the expression of LKB1 in breast dissemination of cancer cells from primary tumors, cancer cells. Owing to its N-terminal nuclear localization increasing metastatic progression but is also linked with signal, LKB1 is predominantly located in nucleus other pro-metastatic traits such as increase in tumor- especially when it is not associated with other proteins. initiating cell (TIC)-characteristics including self-renewal, Upon activation, LKB1 translocates to cytoplasm where multipotency and resistance to conventional therapeutics it complexes with STRAD (STE-related adapter) and [33–35]. Recent studies from our lab and others have MO25 (mouse protein 25). SIRT1 and SIRT3 have been shown an important role of leptin in acquisition of shown to deacetylate LKB1 leading to an increase in its mesenchymal characteristics, and survival of cancer cytoplasmic localization, binding with STRAD and MO25 stem cells (CSCs) in vitro and in vivo [13, 20]. Striving and activation of kinase function [40, 41]. We show that to develop effective, clinically viable leptin-antagonists HNK increases the expression of SIRT1 and SIRT3 using bioactive compounds, we recently discovered that and overexpression of SIRT1 and SIRT3 increases the HNK is capable of inhibiting breast cancer growth in cytoplasmic localization of LKB1 in breast cancer cells. hyperleptinemic state [30]. These discoveries sparked www.impactjournals.com/oncotarget 29955 Oncotarget Figure 6: Role of miR-34a in honokiol-mediated inhibition of oncogenic actions of leptin. A. MCF7 cells were transfected with miR-34a inhibitor or miR-34a mimic followed by treatment with vehicle (C), Honokiol (HNK) (5 μM) and/or leptin (L) (100 ng/ml) as indicated and subjected to (A) clonogenicity assay, B. spheroid migration assay and C. mammosphere assay. *p < 0.05, L+HNK+miR-34a mimic treated cells compared with L+HNK+miR-34a inhibitor treated cells. www.impactjournals.com/oncotarget 29956 Oncotarget Figure 7: Schematic representation of the mechanism whereby HNK inhibits leptin-induced EMT and stemness via LKB1 and miR-34a. Leptin treatment inhibits LKB1 expression. Honokiol treatment induces the expression levels of SIRT1/3 and stimulates the expression as well as cytoplasmic localization of LKB1 and also increases the levels of miR-34a leading to the inhibition of EMT and stemness markers. Honokiol treatment results in the inhibition of EMT, migration and mammosphere formation even in the presence of leptin. These results support a role for SIRT1/3 in HNK-mediated either by directly targeting leptin or deactivating leptin LKB1 activation. Although a role of LKB1 in cancer receptor, for example, soluble leptin receptors (LRs), cell EMT has been proposed in cancer cells [49], the leptin peptidomimetics (LPA-2), synthetic leptin- underlying molecular mechanisms remain elusive. We antagonists, and anti-LR monoclonal antibodies (anti-LR discovered that LKB1 plays an important role in HNK- mAbs) [17, 51–53]. Leptin receptor antagonists, Aca 1 mediated upregulation of miR-34a expression in breast (aa 121–129; modifications Nva123, Aca129) and Allo-aca cancer cells. This is an important finding as miR-34a is (aa 121–129; modifications alloThr121, Nva123, Aca129) involved in tumor suppression by inhibiting various genes have been shown to inhibit leptin-dependent growth and regulating cell proliferation, migration, invasion and EMT signaling in various cancer cells [54, 55]. In addition, Stat3 in many cancer types including breast cancer [50]. Our inhibitors (chemical and bioactive) have been shown to study shows that miR-34a is not only vital for HNK- block leptin signaling [56]. It is well-recognized that an mediated inhibition of EMT, and stemness but also plays ideal strategy to inhibit oncogenic effects of leptin should an important role in HNK-mediated inhibition of leptin- be safe, highly efficacious, and should lack toxicity to function. Considering that miRs are promising candidates allow long-term use. Our study showed the potential of as biomarkers owing to their low complexity, stability, HNK as an effective and non-toxic inhibitor of oncogenic and ease of amplification and quantification, miR-34a can effects of leptin. HNK is particularly encouraging as it potentially serve as a biomarker for HNK function. exhibits desirable spectrum of bioavailability in contrast The realization of the impact of hyperleptinemia with many other natural products [57]. While poor associated with obese state on breast cancer incidence, absorption and rapid excretion has marred the development behavior, and prognosis, has spurred a great interest in the of many other polyphenolic agents [58], HNK does not development of effective strategies to inhibit multipartite have these deficiencies. It has been shown that HNK can leptin signaling and oncogenic functions. Various cross the blood-brain barrier and significant systemic levels approaches have been proposed to neutralize leptin activity of HNK can be achieved in preclinical models [59]. Owing www.impactjournals.com/oncotarget 29957 Oncotarget to these qualities, HNK is a promising bioactive agent to Western blotting be developed as leptin-antagonist. Whole cell lysate was prepared by scraping breast In conclusion, we uncovered a novel role of HNK as cancer cells in 250 μl of ice cold modified RIPA buffer an inhibitor of leptin-induced EMT and stemness, which [64]. Equal amount of lysate protein was resolved on involves LKB1 and miR-34a. Our results demonstrate sodium-dodecyl sulfate polyacrylamide gel, transferred the integral role of a previously unrecognized functional to nitrocellulose membrane, and western blot analysis crosstalk between LKB1 and miR-34a in facilitating was performed. Immunodetection was performed using HNK-mediated inhibition of oncogenic actions of leptin. enhanced chemiluminescence (ECL system, Amersham Pharmacia Biotech Inc., Arlington Heights, IL) according MATERIALS AND METHODS to manufacturer’s instructions. Ethics statement RNA isolation, miR, transfection and RT-PCR The investigation was conducted in accordance For RNA isolation and RT-PCR, total cellular with the ethical standards and guidelines approved by RNA was extracted using the TRIzol Reagent (Life the authors’ institutional review board (Johns Hopkins Technologies, Inc., Rockville, MD). RT-PCR was University IACUC). performed using specific sense and antisense PCR primers. Cells were transfected with miR-34a mimic or miR-34a Cell culture and reagents inhibitor or control-miR (Applied Biosystems, Ambion, Austin, TX) using Fugene transfection reagent (Promega The human breast cancer cell lines, MCF7, MDA- Corporation, Madison, WI). Standardization of miR-34a MB-468, SUM149, SUM159, T47D, SKBR3 and MDA- mimic and inhibitor is shown in supplementary figure 4. MB-231 were obtained from the American Type Culture For qRT-PCR detection of miR-34a, miRNA-specific Collection (ATCC, Manassas, VA), resuscitated from RT-primers (assay IDs: hsa-miR-34a, 000426), TaqMan early passage liquid nitrogen vapor stocks as needed miRNA Assay (Applied Biosystems, Ambion, Austin, TX) and cultured according to supplier’s instructions. Cell and Platinum Taq Polymerase Reagents (Invitrogen, Grand line authentication was done by analysis of known Island, NY) were used. Data were calculated by using genetic markers or response (e.g., expression of estrogen the standard ΔΔCt method and microRNA expression receptor and p53 and estrogen responsiveness). Cells was represented as fold-difference of each treatment were cultured for less than 3 months before reinitiating vs. vehicle-treated control. Statistics was performed by cultures and were routinely inspected microscopically using one-way ANOVA and Student’s t-test post-hoc for stable phenotype. For treatment, cells were seeded at 6 analysis. Statistical significance was accepted when p was a density of 1 X 10 /100-mm tissue culture dish. After <0.05. pcDNA3-Flag-LKB1-wild-type (LKB1-WT), 16 hours of serum starvation, the culture media were Flag-SIRT1, pcDNA3.1-Flag-SIRT3 plasmid constructs changed to serum free media containing treatments as were transfected using Fugene 6 (Promega Corporation, indicated. Cells were treated with 100 ng/ml human Madison, WI) transfection reagent. recombinant leptin (Sigma, St. Louis, MO). We extracted Honokiol (HNK) from seed cone of Magnolia grandiflora according to our previously published study [60]. Previous Immunofluorescence and confocal imaging studies from our lab have shown that 25 μM honokiol Breast cancer cells were subjected to immu- inhibits cell viability, and cell proliferation while 5 μM nofluorescence analysis. Fixed and immunofluo - honokiol doesn’t affect cell viability and proliferation. rescently stained cells were imaged using a Zeiss Accordingly, we used 5 μM honokiol to examine the LSM510 Meta (Zeiss) laser scanning confocal system effect of honokiol on mammosphere formation, migration, configured to a Zeiss Axioplan 2 upright microscope. All and invasion [28, 29][30]. Antibodies for Slug (C19G7), experiments were performed multiple times using indepen- Snail (L70G2), Zeb2 (ab25837), Nanog (D73G4), Oct4 dent biological replicates. (#2750), Sox2 (D6D9), LKB1 (D60C5), SIRT1 (C14H4), SIRT3 (C73E3), E-cadherin (24E10), and Occludin (D15G7) were purchased from Cell Signaling Technology Scratch-migration assay (Danvers, MA). β-Actin antibody was purchased from Sigma-Aldrich (St. Louis, MO). Synthetic miRNA To perform migration assays [12, 64]; cells were miR-34a mimic and siRNA were purchased from Applied plated into the 6-well cell culture plate. Cells were allowed Biosystems (Ambion, Austin, TX). pcDNA3-Flag- to grow in DMEM containing 10% FBS to confluence, LKB1-wild-type (LKB1-WT), Flag-SIRT1, pcDNA3.1- and then were washed with serum-free medium and Flag-SIRT3 plasmid constructs were procured from serum starved for 16 h. A 1-mm wide scratch was made Addgene [61–63]. across the cell layer using a sterile pipette tip. Plates were www.impactjournals.com/oncotarget 29958 Oncotarget photographed immediately after scratching. Cells were Zeb1, Zeb2, Slug and actin. All animal studies were treated with human recombinant leptin at 100 ng/ml and/ in accordance with the guidelines of Johns Hopkins or HNK at 5 μM alone and in combination. Plates were University IACUC. photographed after 8 h, 24 h and 48 h at the identical location of the initial image. Statistical analysis All experiments were performed thrice in triplicates. Mammosphere assays were performed as previously Statistical analysis was performed using Microsoft Excel described [34] and spheres (>50 μm) were counted [65]. software. Significant differences were analyzed using student’s t test and two-tailed distribution. Results were Preparation of subcellular fractions considered to be statistically significant if p < 0.05. Results were expressed as mean ± SE between triplicate Cellular cytosolic and nuclear fractions were experiments performed thrice. prepared by incubating cells in 100 μl of ice-cold lysis buffer [10 mM Tris-Hcl (pH 7.4), 10 mM NaCl, 3 mM MgCl , 0.5% NP-40, 2 mM DTT and 0.1 mM PMSF]. ACKNOWLEDGMENTS The lysates were incubated for 5min on ice followed by centrifugation at 4,000g for 10 min at 4 C to precipitate This work was supported by NCI NIH, nuclei. Supernatant was stored as cytoplasmic fraction. 1R21CA185943-01 (to NKS); NCI NIH R01AR47901 (to Nuclear pellet was incubated with 100 μl of ice-cold JLA), NCI NIH R01CA131294, NCI NIH R21CA155686, extraction buffer [20 mM Tris-Hcl (pH 7.9), 0.42M KCl, Avon Foundation, Breast Cancer Research Foundation 0.2 mM EDTA, 10% Glycerol, 2 mM DTT and 0.1 mM (BCRF) 90047965 (to DS). PMSF] for 10min followed by centrifugation at 12,000g for 10min at 4 C to clear the nuclear debris. Total protein was quantified using the Bradford protein assay kit CONFLICTS OF INTEREST (Biorad, Hercules, CA). Equal amount of protein was subjected to western blot analysis. None. LKB1 stable knockdown using lentiviral short REFERENCES hairpin RNA 1. Calle EE, Kaaks R. Overweight, obesity and cancer: epi- Five pre-made lentiviral LKB1 short hairpin RNA demiological evidence and proposed mechanisms. 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Honokiol activates LKB1-miR-34a axis and antagonizes the oncogenic actions of leptin in breast cancer

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www.impactjournals.com/oncotarget/ Oncotarget, Vol. 6, No. 30 Honokiol activates LKB1-miR-34a axis and antagonizes the oncogenic actions of leptin in breast cancer 1,* 1,* 3 3,4 Dimiter B. Avtanski , Arumugam Nagalingam , Michael Y. Bonner , Jack L. Arbiser , 2 1 Neeraj K. Saxena , Dipali Sharma Department of Oncology, Johns Hopkins University School of Medicine and the Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, Baltimore MD 21231 Department of Medicine, University of Maryland School of Medicine, Baltimore MD 21201 Department of Dermatology, Emory University School of Medicine, Winship Cancer Institute Atlanta Veterans Administration Medical Center, Atlanta, GA 30322 These authors have contributed equally to this work Correspondence to: Dipali Sharma, e-mail: [email protected] Neeraj Saxena, e-mail: [email protected] Keywords: Honokiol, leptin, LKB1, miR-34a, breast cancer Received: May 13, 2015 Accepted: August 13, 2015 Published: August 24, 2015 ABSTRACT Leptin, a major adipocytokine produced by adipocytes, is emerging as a key molecule linking obesity with breast cancer therefore, it is important to nd fi effective strategies to antagonize oncogenic effects of leptin to disrupt obesity-cancer axis. Here, we examine the potential of honokiol (HNK), a bioactive polyphenol from Magnolia grandiflora , as a leptin-antagonist and systematically elucidate the underlying mechanisms. HNK inhibits leptin-induced epithelial-mesenchymal-transition (EMT), and mammosphere-formation along with a reduction in the expression of stemness factors, Oct4 and Nanog. Investigating the downstream mediator(s), that direct leptin-antagonist actions of HNK; we discovered functional interactions between HNK, LKB1 and miR-34a. HNK increases the expression and cytoplasmic-localization of LKB1 while HNK-induced SIRT1/3 accentuates the cytoplasmic-localization of LKB1. We found that HNK increases miR-34a in LKB1-dependent manner as LKB1-silencing impedes HNK-induced miR-34a which can be rescued by LKB1-overexpression. Finally, an integral role of miR-34a is discovered as miR-34a mimic potentiates HNK-mediated inhibition of EMT, Zeb1 expression and nuclear-localization, mammosphere-formation, and expression of stemness factors. Leptin-antagonist actions of HNK are further enhanced by miR-34a mimic whereas miR-34a inhibitor results in inhibiting HNK’s effect on leptin. These data provide evidence for the leptin-antagonist potential of HNK and reveal the involvement of LKB1 and miR-34a. (increase in cell number), leading to dysregulation of local INTRODUCTION and systemic secretion of biologically active polypeptides, adipocytokines such as leptin [3]. In recent years, leptin Given that one-third of all cancers are attributed has emerged as a key candidate molecule mediating the to obese state, obesity is a well-established risk factor. molecular effects of obesity on cancer [4, 5]. Various Obese state is not only associated with aggressive tumor epidemiological studies have shown that high level of progression, poorer prognosis, increased recurrence and plasma leptin is linked with increased risk and poor poorer survival of obese breast cancer patients, but it prognosis for breast carcinogenesis [6, 7]. Analysis of also impacts tumor initiation [1, 2]. Adipocytes lose their clinical samples showed overexpression of leptin receptor normal physiological size heterogeneity in obese state and in 83% of breast tumor samples whereas no expression undergo hypertrophy (increase in cell size) and hyperplasia www.impactjournals.com/oncotarget 29947 Oncotarget of leptin receptor was observed in normal mammary honokiol-mediated inhibition of breast tumor growth and epithelial cells [8, 9]. Also, leptin overexpression was progression [28]. Recently, we discovered the involvement observed in 92% of breast tumors examined but in none of miR-34a in breast tumor inhibition function of of the cases of normal breast epithelium. Overexpression honokiol. Honokiol treatment inhibited breast tumor of leptin and leptin receptor in breast tumors and its growth in lean and obese-hyperleptinemic mice models association with tumor aggressiveness suggest that leptin in a manner associated with activation of miR-34a [30]. can also influence breast tumor growth and progression via In this report, we specifically investigated the potential an autocrine pathway [9, 10]. of HNK to inhibit leptin-induced epithelial-mesenchymal Various research groups have been studying the transition (EMT) and tumorsphere formation and examine oncogenic role of leptin in cancer. Studies from our the underlying molecular mechanisms. We provide lab and others have established that high leptin levels molecular evidence supporting the regulatory role of (hyperleptinemia) associated with obese state stimulate LKB1, and integral involvement of miR-34a in leptin- breast cancer cell proliferation, invasion, migration, and antagonist potential of HNK. angiogenesis, thereby promoting breast tumor growth and metastasis [11–17]. Leptin also plays a central role RESULTS in the acquisition of mesenchymal characteristics by inducing breast cancer cells to undergo a transition from Honokiol inhibits leptin-induced epithelial- epithelial to spindle-like mesenchymal morphology [13]. mesenchymal transition, mammosphere Leptin has been reported to regulate many signaling formation, and migration of breast cancer cells pathways and transcription factors implicated in breast cancer stem cells (BCSCs). Intact leptin-leptin receptor Epithelial to mesenchymal transition (EMT) of signaling was found to play an integral role in the cancer cells is a crucial early event leading to induction survival of CSC population [18]. It is shown that leptin of cell motility, invasion and distant metastasis. We receptor is a characteristic feature of Tumor initiating recently presented a pivotal role of leptin in acquisition stem cells (TISCs) and participates in the regulation of of mesenchymal characteristics and aggressive behavior core pluripotency-associated transcription factors, Oct4 in breast cancer cells [13]. Here, we specifically examined and Nanog [19, 20]. These studies indicate that leptin not if HNK could inhibit the stimulatory effect of leptin on only plays a significant role in promoting the growth and EMT and metastatic properties of breast cancer cells. metastatic progression of established breast tumors but Following treatment with leptin and HNK, we observed also augments tumor initiation and recurrence. striking morphological differences between MCF7 cells Leptin and leptin-signaling pathway have emerged treated with different combinations. Leptin-treated MCF7 as leading targets for disrupting obesity-breast cancer link; cells exhibited acquisition of fibroblast-like appearance therefore, developing effective, non-endocrine, non-toxic and increased formation of pseudopodia observed agents for the inhibition of neoplastic effects of leptin emanating from the cell membrane. These features is highly important. Current strategies to inhibit leptin signify typical mesenchymal phenotype rather than the pathway such as soluble Leptin receptors (LRs), synthetic normal epithelial phenotype of MCF7 cells, showing that leptin-antagonists, and anti-LR monoclonal antibodies cells have undergone EMT upon leptin treatment. HNK (anti-LR mAbs) [17] are limited by associated toxicities prevented the morphological transition from an epithelial- as well as low efficacy. Recently, active constitutive agents like to mesenchymal-like appearance caused by leptin in natural products used in traditional Asian medicine treatment. HNK alone did not affect the morphology of have shown efficacy as potential cancer preventive as MCF7 cells (Figure 1A). To unequivocally establish that well as therapeutic agents [21, 22]. For years, cones, HNK blocks leptin-induced EMT, we next examined bark and leaves from Magnolia plant species have been the biochemical hallmarks of EMT-reversal including used for their anti-thrombocytic, anti-inflammatory, gain of expression of epithelial markers (occludin, and anxiolytic, anti-depressant, antioxidant, antispasmodic, cytokeratin-18 (CK-18) with a concomitant decrease and antibacterial effects [23–26]. It is now known that in mesenchymal markers (fibronectin, and vimentin) Honokiol (HNK), a natural phenolic compound isolated expression. Leptin treatment resulted in upregulation from an extract of seed cones from Magnolia grandiflora of mesenchymal markers accompanied with a marked [27] is responsible for these medicinal benefits of decrease in the expression of epithelial markers. HNK Magnolia species. Previous studies from our lab have blocked leptin-induced modulation of mesenchymal shown that HNK inhibits breast carcinogenesis in vitro and and epithelial markers leading to decreased expression in vivo [28, 29] thereby establishing HNK as a promising of fibronectin and vimentin and increased expression of bioactive compound against breast carcinogenesis. We also CK-18 and occludin (Figure 1B and 1C, Supplementary found that honokiol treatment increases the expression of Figure 1A). Immunocytochemical analysis provided tumor suppressor LKB1 which plays an integral role in additional evidence to support HNK-mediated leptin- www.impactjournals.com/oncotarget 29948 Oncotarget Figure 1: Honokiol inhibits leptin-induced epithelial-mesenchymal transition and migration of breast cancer cells. A. MCF7 cells were treated with leptin (L) (100 ng/ml) and/or Honokiol (HNK) (5 μM) as indicated. Vehicle treated cells are denoted as (C) Control. Morphological changes associated with EMT are shown in phase-contrast images. The presence of spindle-shaped cells, increased intracellular separation and pseudopodia were noted in leptin-treated cells but not in HNK-treated cells. B. MCF7 cells were treated as in A and total lysates were immunoblotted for Occludin, Snail and Zeb2 expression levels. Actin was used as control. C. MCF7 cells were treated as in A, total RNA was isolated and expression levels of epithelial and mesenchymal marker genes was analyzed. Actin was used as control. D. Breast cancer cells were treated as in A, and subjected to immunofluorescence analysis (1000X magnification) of Zeb1. Bar-graphs show the fold-change in number of cells expressing nuclear Zeb. *p < 0.001, compared with untreated controls. #p < 0.001, compared with leptin-alone treatment. Leptin-induced nuclear translocation of Zeb1 was abrogated by HNK treatment. E. Breast cancer cells were treated as in A, and subjected to immunofluorescence analysis (200X magnification) of E-cadherin, and Occludin. Bar-graphs show the fold-change in number of cells expressing Occludin and E-cadherin. *p < 0.05, compared with untreated controls. #p < 0.01, compared with leptin-alone treatment. F. MDA-MB-231 cells derived tumors were developed in nude mice and treated with leptin and/or HNK (n = 6–8/treatment group). At the end of five weeks of treatment, tumors were collected. Total RNA was isolated from tumor samples and subjected to RT-PCR analysis for the expression of mesenchymal markers and transcription factors. G. Breast cancer cells were treated as in A and subjected to scratch-migration assay. induced EMT reversal showing gain of expression of even in the presence of leptin (Figure 1B, and 1D). In occluding and E-cadherin (Figure 1E). Transcriptional a recent study, we showed that HNK administration repressors for epithelial marker proteins, Zeb1/2 and snail, retarded leptin-induced growth of MDA-MB-231 cells are frequently detected in metastatic cancer cells and are implanted in female athymic mice [30]. We used tumor known to be involved in EMT [31, 32]. We also examined samples from the same study to evaluate the effect of the involvement of these transcription repressors in HNK- HNK on leptin-induced mesenchymal markers by RT- mediated inhibition of leptin-induced EMT. Indeed, leptin PCR analysis. Corroborating in vitro findings, tumors from treatment not only increased the expression of snail, Zeb1 mice co-treated with HNK and leptin showed decreased and Zeb2 but also increased the nuclear translocation of levels of expression of vimentin, fibronectin, Zeb1/2 Zeb1 (Figure 1B, 1C and 1D). Importantly, HNK treatment and slug in comparison to tumors from leptin-treated inhibited leptin-induced expression of snail, Zeb1 and mice (Figure 1F). Since HNK inhibited leptin-induced Zeb2 as well as promoted cytoplasmic retention of Zeb1 EMT, we aimed to examine whether HNK treatment also www.impactjournals.com/oncotarget 29949 Oncotarget blocked induction of migration usually observed in the regulating its activity, we examined the phosphorylation presence of leptin. Significant migration of MCF7, MDA- and expression of AMPK. Honokiol-treatment increased MB-468, MDA-MB-231, SUM149, SUM159 and T47D phosphorylation and expression of AMPK (Figure 3A). breast cancer cells observed in the presence of leptin was Tumor-suppressor function of LKB1 is majorly attributed inhibited in the presence of HNK treatment (Figure 1G, to the cytoplasmic pool of LKB1 as mutant LKB1 lacking Supplementary Figure 1B, 1C). Honokiol treatment does the nuclear localization signal still retains the ability to not affect growth of MCF10A cells while leptin show suppress cell growth [38, 39]. Examining nuclear and only modest effects on MCF10A cells (Supplementary cytoplasmic fractions of breast cancer cells treated Figure 2). with HNK, we found that HNK induced cytoplasmic An emerging hypothesis is that EMT bestows cells localization of LKB1 (Figure 3B). Recent studies have with stem-like characteristics and facilitates an increase reported an important role of Sirtuin-deacetylases, in the subpopulation of CSC (cancer stem cells) [33–35]. SIRT1 and SIRT3 in LKB1 regulation. SIRT1 and SIRT3 Utilizing mammosphere assay that relies on the unique overexpression diminishes lysine acetylation of LKB1 property of breast cancer cells with stem-like potential to and concurrently increases its activity and cytoplasmic/ form large, round, unattached floating spheroid colonies nuclear ratio [40, 41]. We found that breast cancer cells (termed mammosphere), we showed that leptin induced treated with HNK exhibited an increase in the expression mammosphere formation. HNK treatment efficiently of SIRT1 and SIRT3 within 30 minutes post-treatment inhibits leptin-induced EMT as well as migration of (Figure 3C). To examine the effect of SIRT1 and SIRT3 breast cancer cells, therefore, we hypothesize that HNK on HNK-mediated cytoplasmic localization of LKB1, we may also inhibit leptin-induced mammosphere formation. overexpressed SIRT1 and SIRT3 in MCF7 cells followed Indeed, HNK inhibited mammosphere formation even in by HNK treatment. Immunofluorescence analysis showed the presence of leptin (Figure 2A). Given the association that LKB1 is present both in nucleus and cytoplasm in of induced pluripotent stem cell (iPSC) markers, Nanog untreated cells. Corroborating immunoblot analyses and Oct4 with self-renewal and maintenance of stem cell shown in figure 3B, HNK-treated cells exhibited increased fate, we wished to investigate whether iPSC markers are cytoplasmic localization of LKB1 in comparison to affected by leptin treatment. To this end, we assessed the untreated cells which was further enhanced with SIRT1 expression of Oct4, and Nanog in leptin-treated breast and SIRT3 overexpression (Figure 3D, 3F)). Our studies cancer cells and found that the expression of pluripotency show a role of sirtuin-deacetylases in HNK-mediated genes was higher in leptin-treated cells (Figure 2B–2D) functional upregulation of LKB1. with respect to the expression in untreated cells. Cells treated with HNK exhibited reduced levels of iPSC Honokiol upregulates miR-34a in a markers and interestingly, HNK treatment resulted in the LKB1-dependent manner inhibition of leptin-induced expression of Oct4 and Nanog (Figure 2B–2D). Breast cancer cells treated with leptin and Regulatory role of microRNAs (miRNA) in various honokiol were examined for the expression of Cyclin D1 biological and pathological processes, including cancer as a functional control (Supplemental Figure 3). Together, progression and metastasis has been well-recognized in these results show that HNK treatment results in effective recent years. Functionally, miRs are small (~21-mer) inhibition of leptin-induced epithelial-mesenchymal regulatory RNA molecules that exert their regulatory transition, mammosphere formation, stemness and effects by binding to the 3′-untranslated regions (3′-UTR) migration of breast cancer cells. of specific mRNAs triggering mRNA degradation or translational repression [42]. miRNAs have been known to function both as oncogenes, potentiating cancer progression Honokiol induces expression and cytoplasmic and metastasis, and tumor suppressors implicated in localization of tumor suppressor Liver Kinase B1 growth inhibition [43]. One of the important tumor in breast cancer cells suppressor miRNA involved in breast cancer is miR-34a, which is downregulated in aggressive breast tumors [44]. Liver kinase B1 (LKB1), owing to its dual role as a Breast cancer cells treated with HNK exhibited a time- tumor suppressor and an upstream master kinase, serves as dependent increase in miR-34a expression (Figure 4A). a major hub regulating several downstream pathways and Interestingly, leptin treatment decreased the expression of known tumor suppressors (e.g., AMPK-mTOR, JNK and miR-34a in MCF7, SKBR3 and SUM149 breast cancer p53) [36, 37]. We found that HNK increases expression cells (Figure 4B). We raised the question whether LKB1 of LKB1 in breast cancer cells (Figure 3A). Interestingly, plays any role in honokiol-mediated increase of miR-34a. leptin treatment inhibits LKB1 expression whereas shRNA We used LKB1 lentivirus and puromycin to select HNK treatment increases LKB1 expression even in the for stable pools of MCF7 cells with LKB1 depletion. presence of leptin (Figure 3E). Since LKB1 has recently shRNA pLKO.1 and LKB1 stable MCF7 cell pools were been identified as a critical upstream kinase for AMPK www.impactjournals.com/oncotarget 29950 Oncotarget Figure 2: Honokiol abates the stimulatory effect of leptin on mammosphere-formation-potential and acquisition of stem-like properties in breast cancer cells. A. MCF7 and MDA-MB-468 cells were treated with leptin (L) (100 ng/ml) and/or Honokiol (HNK) (5 μM) as indicated and subjected to mammosphere formation. Vehicle-treated cells are denoted as (C) The graph shows the number of mammospheres. *p < 0.001, leptin treatment compared with untreated controls. # p < 0.01, leptin+honokiol compared with leptin-alone treatment. B. MCF7 cells were treated with leptin (100 ng/ml) and/or HNK (5 μM) alone or in combination as indicated, total RNA was isolated and expression levels of Nanog and Oct4 were examined. Actin was used as control. C. Breast cancer cells were treated as in B, and total lysates were immunoblotted for Nanog and Oct4 expression levels. Actin was used as control. D. Bar-graphs show the fold-change in Nanog and Oct4 expression in breast cancer cells treated with leptin and/or honokiol. *p < 0.001, leptin treatment compared with untreated controls. #p < 0.001, leptin+honokiol compared with leptin-alone treatment. www.impactjournals.com/oncotarget 29951 Oncotarget Figure 3: Honokiol induces the expression and cytoplasmic localization of tumor suppressor LKB1 and involvement of SIRT1/3. A. MCF7 cells were treated with 5 μM HNK for various time intervals as indicated, total lysates were immunoblotted for LKB1, phospho-AMPK and total AMPK expression levels. Actin was used as control. B. MCF7 cells were treated with 5 μM HNK for 6 h, nuclear and cytoplasmic fractions were immunoblotted for LKB1 expression. Actin was used as control. C. MCF7 cells were treated with 5 μM HNK for various time intervals as indicated, total lysates were immunoblotted for SIRT1 and SIRT3 expression levels. Actin was used as control. D. MCF7 cells were transfected with SIRT1 and SIRT3 overexpression constructs as indicated followed by 5 μM HNK treatment. Cells were subjected to immunofluorescence analysis of LKB1. E. MCF7 cells were treated with leptin (100 ng/ml) and/or HNK (5 μM) as indicated, cell lysates were immunoblotted for LKB1. F. Bar-graphs show the fold-change in number of cells expressing cytoplasmic localization of LKB1. shRNA analyzed for LKB1 protein expression by immunoblot pLKO.1 and LKB1 cells were treated with HNK analysis. LKB1 protein expression was significantly and expression of miR-34a was determined. Intriguingly, shRNA reduced in LKB1 cells (shRNA1 and shRNA2) displaying a crucial role of LKB1, honokiol treatment did shRNA as compared to pLKO.1 control cells (Figure 4C). not increase miR-34a expression in LKB1 cells while www.impactjournals.com/oncotarget 29952 Oncotarget Figure 4: Honokiol upregulates miR-34a in LKB1-dependent manner in breast cancer cells. A. Expression levels of miR-34a in MCF7 cells treated with 5 μM HNK for various time intervals as indicated. *p < 0.001, compared with untreated controls. B. MCF7, SKBR3 and SUM149 cells were treated with 100 ng/ml leptin and miR-34a expression levels were measured. *p < 0.005, compared shRNA 1–2 with untreated controls. C. Immunoblot analysis of LKB1 in stable pools of LKB1-depleted (LKB1 ) and vector control (pLKO.1) shRNA 1–2 MCF7 cells. LKB1-depleted (LKB1 ) were transfected with LKB1 overexpression vector to create a ‘gain-of-function’ system. shRNA 1–2 D. Expression levels of miR-34a in stable pools of LKB1-depleted (LKB1 ) and vector control (pLKO.1) MCF7 cells. *p < 0.001, compared with untreated controls; **p < 0.001, compared with HNK-treated MCF7-pLKO.1 cells; #p < 0.001, compared with shRNA2 HNK-treated MCF7-pLKO.1 cells. E. LKB1-depleted (LKB1 ) were transfected with LKB1 overexpression vector and expression shRNA2 levels of miR-34a was examined. *p < 0.001, compared with HNK-treated, control-transfected, MCF7- LKB1 cells. pLKO.1 cells exhibited HNK-induced miR-34a expression molecule) further enhanced HNK-mediated inhibition (Figure 4D). As a gain-of-function strategy, LKB1 was of mesenchymal markers Fibronectin and Vimentin shRNA overexpressed in LKB1 cells (Figure 4C), treated with while enhancing the expression of epithelial markers, HNK followed by examination of miR-34a expression. E-cadherin, Occludin and CK-18 (Figure 5A, 5B). As evident in Figure 4E, HNK treatment increased HNK treatment decreased expression of slug and Zeb1, shRNA miR-34a expression in LKB1 cells overexpressing repressors of E-cadherin in comparison to untreated LKB1. These results show that LKB1 plays an important cells. Combination of HNK and miR-34a mimic further role in honokiol-induced miR-34a expression in breast enhanced the inhibition slug and Zeb1 (Figure 5B, 5C). cancer cells. Expression of miR-34a-inhibitor increased Zeb1 expression even in the presence of HNK (Figure 5C). Next, we investigated the effect of miR-34a mimic and Honokiol inhibits EMT, stemness and leptin inhibitor on HNK-induced cytoplasmic retention of function in a miR-34a-dependent manner Zeb1 in breast cancer cells. Breast cancer cells treated We further investigated the importance of with HNK, miR-34a mimic alone and in combination miR-34a in HNK-mediated modulation of EMT markers, exhibited cytoplasmic localization of Zeb1. Treatment stemness factors and inhibition of leptin function. with miR34a inhibitor caused nuclear localization of Zeb1 Ectopic miR-34a expression (in the form of a mimic (Figure 5D). To investigate the involvement of miR-34a www.impactjournals.com/oncotarget 29953 Oncotarget Figure 5: Evidence supporting the involvement of miR-34a in honokiol-mediated modulation of EMT and stemness factors. A. MCF7 cells were transfected with miR-34a mimic followed by treatment with vehicle (C) or HNK (5 μM) as indicated, total RNA was isolated and expression levels of epithelial and mesenchymal marker genes was analyzed. Actin was used as control. B. MCF7 cells were transfected with miR-34a mimic followed by treatment with vehicle C. or HNK (5 μM) as indicated, total protein lysates were immunoblotted for the expression levels of epithelial and mesenchymal marker genes as indicated. Actin was used as control. (C) MCF7 cells were transfected with miR-34a inhibitor or miR-34a mimic followed by treatment with vehicle (C) or HNK (5 μM) as indicated, total RNA was isolated and expression levels of Zeb1 was analyzed. Actin was used as control. Bar-graph shows fold-change in Zeb expression. D. MCF7 cells were transfected with miR-34a inhibitor or miR-34a mimic followed by treatment with vehicle (C) or HNK (5 μM) as indicated, immunofluorescence analysis for Zeb1 was performed. Arrows point the cells showing nuclear localization of Zeb1. Bar-graphs show the fold-change in number of cells expressing nuclear localization of Zeb1. *p < 0.02, HNK treated cells compared with HNK+miR-34a inhibitor treated cells. E. MCF7 cells were transfected with miR-34a mimic followed by treatment with vehicle (C) or HNK (5 μM) as indicated, total RNA was isolated and expression levels of stemness genes (Oct4, Nanog, Sox2) was analyzed. Actin was used as control. F. Breast cancer cells were transfected with miR-34a mimic followed by treatment with vehicle (C) or HNK (5 μM) as indicated, total protein lysates were immunoblotted for the expression levels of stemness genes (Oct4, Nanog, Sox2) genes as indicated. Actin was used as control. www.impactjournals.com/oncotarget 29954 Oncotarget in HNK-mediated inhibition of iPSC factors, we treated our interest in investigating the efficacy of HNK as a breast cancer cells with HNK in the presence of miR34a novel inhibitor of leptin-induced EMT and stemness in mimic or inhibitior. As expected, HNK inhibited the breast cancer. We found that HNK effectively inhibits expression of Oct4, Nanog and Sox2 in breast cancer cells leptin-induced EMT of breast cancer cells as evident which is further decreased by ectopic miR-34a expression from morphological changes and molecular alterations (Figure 5E, 5F). of mesenchymal and epithelial genes. Breast tumors Given our results showing that miR-34a plays an treated with HNK also showed reduced expression of important role in HNK-mediated modulation of EMT mesenchymal markers even in the presence of leptin markers and iPSC factors, we decided to examine whether treatment providing in vivo evidence. Mammosphere miR-34a modulation also affects HNK-mediated inhibition formation induced by leptin was also efficiently abrogated of oncogenic actions of leptin. Expression of miR-34a with HNK treatment along with inhibition of expression mimic potentiated HNK-mediated-inhibition of leptin- of iPSC factors (Oct4, nanog and Sox2). These findings induced clonogenicity, migration and mammosphere imparted convincing evidence supporting the efficacy formation. On the other hand, miR-34a inhibitor interfered of HNK as a novel inhibitor of leptin-induced EMT and with HNK efficacy as a leptin-antagonist resulting in poor stemness warranting further mechanistic investigations. inhibition of leptin-induced clonogenicity, migration and Consequently, we designed further studies to decipher mammosphere formation (Figure 6A, 6B, 6C). Leptin the key nodes of leptin-antagonist function of HNK treatment decreases miR-34a expression (Figure 4B), to facilitate establishing surrogate biomarkers for its therefore, it is interesting to note that miR-34a mimic efficacy and help in clinical development of this bioactive reduced oncogenic effects of leptin while miR-34a molecule as a leptin-antagonist. We found that HNK inhibitor increased leptin’s impact (Figure 6A, 6B, 6C). induces expression and cytoplasmic localization of LKB1. Together, these data provide evidence supporting an HNK also increases expression of Sirtuin-deacetylases, important role of miR-34a in HNK-mediated inhibition of SIRT1/3 and overexpression of both SIRT1 and SIRT3 oncogenic actions of leptin. further increases cytoplasmic localization of LKB1. HNK Collectively, the findings presented here suggest upregulates miR-34a in a LKB1-dependent manner; that HNK inhibits leptin-induced EMT, stemness and miR-34 a plays an important role in HNK-mediated mammosphere formation, and provide evidence for the inhibition of EMT, stemness and leptin-function. involvement of miR-34a as a regulator of HNK-mediated Tumor suppressor LKB1, a key determinant in inhibition of oncogenic actions of leptin, and uncover a Peutz-Jeghers syndrome, has been found to be inactivated novel mechanism of HNK action through activation of in a subset of sporadic lung and pancreatic cancer miR-34a in a LKB1-dependent manner (Figure 7). [37, 45]. LKB1 inactivation is not commonly correlated with human breast carcinoma but interestingly, LKB1 loss has been observed in high-grade DCIS and high- DISCUSSION grade invasive ductal carcinoma [46]. Importantly, LKB1 expression was abrogated only in the DCIS associated Over the last decade, there has been growing with invasion but not in pure DCIS cases indicating that emphasis on the importance of EMT, an essential normal LKB1 loss might promote invasive behavior. In fact, physiological process for embryonic development, tissue low LKB1 protein levels correlate with poor prognosis remodeling and wound healing, now implicated in cancer in breast carcinoma [47]. LKB1 knockdown increases progression. An oncogenic EMT causes epithelial-derived motility and invasiveness of cancer cells, and induces tumors to gain a mesenchymal phenotype facilitating the expression of many mesenchymal marker proteins migration and invasion potential of cancer cells. indicating its possible role in EMT [48, 49]. We show Acquisition of mesenchymal traits not only promotes that HNK increases the expression of LKB1 in breast dissemination of cancer cells from primary tumors, cancer cells. Owing to its N-terminal nuclear localization increasing metastatic progression but is also linked with signal, LKB1 is predominantly located in nucleus other pro-metastatic traits such as increase in tumor- especially when it is not associated with other proteins. initiating cell (TIC)-characteristics including self-renewal, Upon activation, LKB1 translocates to cytoplasm where multipotency and resistance to conventional therapeutics it complexes with STRAD (STE-related adapter) and [33–35]. Recent studies from our lab and others have MO25 (mouse protein 25). SIRT1 and SIRT3 have been shown an important role of leptin in acquisition of shown to deacetylate LKB1 leading to an increase in its mesenchymal characteristics, and survival of cancer cytoplasmic localization, binding with STRAD and MO25 stem cells (CSCs) in vitro and in vivo [13, 20]. Striving and activation of kinase function [40, 41]. We show that to develop effective, clinically viable leptin-antagonists HNK increases the expression of SIRT1 and SIRT3 using bioactive compounds, we recently discovered that and overexpression of SIRT1 and SIRT3 increases the HNK is capable of inhibiting breast cancer growth in cytoplasmic localization of LKB1 in breast cancer cells. hyperleptinemic state [30]. These discoveries sparked www.impactjournals.com/oncotarget 29955 Oncotarget Figure 6: Role of miR-34a in honokiol-mediated inhibition of oncogenic actions of leptin. A. MCF7 cells were transfected with miR-34a inhibitor or miR-34a mimic followed by treatment with vehicle (C), Honokiol (HNK) (5 μM) and/or leptin (L) (100 ng/ml) as indicated and subjected to (A) clonogenicity assay, B. spheroid migration assay and C. mammosphere assay. *p < 0.05, L+HNK+miR-34a mimic treated cells compared with L+HNK+miR-34a inhibitor treated cells. www.impactjournals.com/oncotarget 29956 Oncotarget Figure 7: Schematic representation of the mechanism whereby HNK inhibits leptin-induced EMT and stemness via LKB1 and miR-34a. Leptin treatment inhibits LKB1 expression. Honokiol treatment induces the expression levels of SIRT1/3 and stimulates the expression as well as cytoplasmic localization of LKB1 and also increases the levels of miR-34a leading to the inhibition of EMT and stemness markers. Honokiol treatment results in the inhibition of EMT, migration and mammosphere formation even in the presence of leptin. These results support a role for SIRT1/3 in HNK-mediated either by directly targeting leptin or deactivating leptin LKB1 activation. Although a role of LKB1 in cancer receptor, for example, soluble leptin receptors (LRs), cell EMT has been proposed in cancer cells [49], the leptin peptidomimetics (LPA-2), synthetic leptin- underlying molecular mechanisms remain elusive. We antagonists, and anti-LR monoclonal antibodies (anti-LR discovered that LKB1 plays an important role in HNK- mAbs) [17, 51–53]. Leptin receptor antagonists, Aca 1 mediated upregulation of miR-34a expression in breast (aa 121–129; modifications Nva123, Aca129) and Allo-aca cancer cells. This is an important finding as miR-34a is (aa 121–129; modifications alloThr121, Nva123, Aca129) involved in tumor suppression by inhibiting various genes have been shown to inhibit leptin-dependent growth and regulating cell proliferation, migration, invasion and EMT signaling in various cancer cells [54, 55]. In addition, Stat3 in many cancer types including breast cancer [50]. Our inhibitors (chemical and bioactive) have been shown to study shows that miR-34a is not only vital for HNK- block leptin signaling [56]. It is well-recognized that an mediated inhibition of EMT, and stemness but also plays ideal strategy to inhibit oncogenic effects of leptin should an important role in HNK-mediated inhibition of leptin- be safe, highly efficacious, and should lack toxicity to function. Considering that miRs are promising candidates allow long-term use. Our study showed the potential of as biomarkers owing to their low complexity, stability, HNK as an effective and non-toxic inhibitor of oncogenic and ease of amplification and quantification, miR-34a can effects of leptin. HNK is particularly encouraging as it potentially serve as a biomarker for HNK function. exhibits desirable spectrum of bioavailability in contrast The realization of the impact of hyperleptinemia with many other natural products [57]. While poor associated with obese state on breast cancer incidence, absorption and rapid excretion has marred the development behavior, and prognosis, has spurred a great interest in the of many other polyphenolic agents [58], HNK does not development of effective strategies to inhibit multipartite have these deficiencies. It has been shown that HNK can leptin signaling and oncogenic functions. Various cross the blood-brain barrier and significant systemic levels approaches have been proposed to neutralize leptin activity of HNK can be achieved in preclinical models [59]. Owing www.impactjournals.com/oncotarget 29957 Oncotarget to these qualities, HNK is a promising bioactive agent to Western blotting be developed as leptin-antagonist. Whole cell lysate was prepared by scraping breast In conclusion, we uncovered a novel role of HNK as cancer cells in 250 μl of ice cold modified RIPA buffer an inhibitor of leptin-induced EMT and stemness, which [64]. Equal amount of lysate protein was resolved on involves LKB1 and miR-34a. Our results demonstrate sodium-dodecyl sulfate polyacrylamide gel, transferred the integral role of a previously unrecognized functional to nitrocellulose membrane, and western blot analysis crosstalk between LKB1 and miR-34a in facilitating was performed. Immunodetection was performed using HNK-mediated inhibition of oncogenic actions of leptin. enhanced chemiluminescence (ECL system, Amersham Pharmacia Biotech Inc., Arlington Heights, IL) according MATERIALS AND METHODS to manufacturer’s instructions. Ethics statement RNA isolation, miR, transfection and RT-PCR The investigation was conducted in accordance For RNA isolation and RT-PCR, total cellular with the ethical standards and guidelines approved by RNA was extracted using the TRIzol Reagent (Life the authors’ institutional review board (Johns Hopkins Technologies, Inc., Rockville, MD). RT-PCR was University IACUC). performed using specific sense and antisense PCR primers. Cells were transfected with miR-34a mimic or miR-34a Cell culture and reagents inhibitor or control-miR (Applied Biosystems, Ambion, Austin, TX) using Fugene transfection reagent (Promega The human breast cancer cell lines, MCF7, MDA- Corporation, Madison, WI). Standardization of miR-34a MB-468, SUM149, SUM159, T47D, SKBR3 and MDA- mimic and inhibitor is shown in supplementary figure 4. MB-231 were obtained from the American Type Culture For qRT-PCR detection of miR-34a, miRNA-specific Collection (ATCC, Manassas, VA), resuscitated from RT-primers (assay IDs: hsa-miR-34a, 000426), TaqMan early passage liquid nitrogen vapor stocks as needed miRNA Assay (Applied Biosystems, Ambion, Austin, TX) and cultured according to supplier’s instructions. Cell and Platinum Taq Polymerase Reagents (Invitrogen, Grand line authentication was done by analysis of known Island, NY) were used. Data were calculated by using genetic markers or response (e.g., expression of estrogen the standard ΔΔCt method and microRNA expression receptor and p53 and estrogen responsiveness). Cells was represented as fold-difference of each treatment were cultured for less than 3 months before reinitiating vs. vehicle-treated control. Statistics was performed by cultures and were routinely inspected microscopically using one-way ANOVA and Student’s t-test post-hoc for stable phenotype. For treatment, cells were seeded at 6 analysis. Statistical significance was accepted when p was a density of 1 X 10 /100-mm tissue culture dish. After <0.05. pcDNA3-Flag-LKB1-wild-type (LKB1-WT), 16 hours of serum starvation, the culture media were Flag-SIRT1, pcDNA3.1-Flag-SIRT3 plasmid constructs changed to serum free media containing treatments as were transfected using Fugene 6 (Promega Corporation, indicated. Cells were treated with 100 ng/ml human Madison, WI) transfection reagent. recombinant leptin (Sigma, St. Louis, MO). We extracted Honokiol (HNK) from seed cone of Magnolia grandiflora according to our previously published study [60]. Previous Immunofluorescence and confocal imaging studies from our lab have shown that 25 μM honokiol Breast cancer cells were subjected to immu- inhibits cell viability, and cell proliferation while 5 μM nofluorescence analysis. Fixed and immunofluo - honokiol doesn’t affect cell viability and proliferation. rescently stained cells were imaged using a Zeiss Accordingly, we used 5 μM honokiol to examine the LSM510 Meta (Zeiss) laser scanning confocal system effect of honokiol on mammosphere formation, migration, configured to a Zeiss Axioplan 2 upright microscope. All and invasion [28, 29][30]. Antibodies for Slug (C19G7), experiments were performed multiple times using indepen- Snail (L70G2), Zeb2 (ab25837), Nanog (D73G4), Oct4 dent biological replicates. (#2750), Sox2 (D6D9), LKB1 (D60C5), SIRT1 (C14H4), SIRT3 (C73E3), E-cadherin (24E10), and Occludin (D15G7) were purchased from Cell Signaling Technology Scratch-migration assay (Danvers, MA). β-Actin antibody was purchased from Sigma-Aldrich (St. Louis, MO). Synthetic miRNA To perform migration assays [12, 64]; cells were miR-34a mimic and siRNA were purchased from Applied plated into the 6-well cell culture plate. Cells were allowed Biosystems (Ambion, Austin, TX). pcDNA3-Flag- to grow in DMEM containing 10% FBS to confluence, LKB1-wild-type (LKB1-WT), Flag-SIRT1, pcDNA3.1- and then were washed with serum-free medium and Flag-SIRT3 plasmid constructs were procured from serum starved for 16 h. A 1-mm wide scratch was made Addgene [61–63]. across the cell layer using a sterile pipette tip. Plates were www.impactjournals.com/oncotarget 29958 Oncotarget photographed immediately after scratching. Cells were Zeb1, Zeb2, Slug and actin. All animal studies were treated with human recombinant leptin at 100 ng/ml and/ in accordance with the guidelines of Johns Hopkins or HNK at 5 μM alone and in combination. Plates were University IACUC. photographed after 8 h, 24 h and 48 h at the identical location of the initial image. Statistical analysis All experiments were performed thrice in triplicates. Mammosphere assays were performed as previously Statistical analysis was performed using Microsoft Excel described [34] and spheres (>50 μm) were counted [65]. software. Significant differences were analyzed using student’s t test and two-tailed distribution. Results were Preparation of subcellular fractions considered to be statistically significant if p < 0.05. Results were expressed as mean ± SE between triplicate Cellular cytosolic and nuclear fractions were experiments performed thrice. prepared by incubating cells in 100 μl of ice-cold lysis buffer [10 mM Tris-Hcl (pH 7.4), 10 mM NaCl, 3 mM MgCl , 0.5% NP-40, 2 mM DTT and 0.1 mM PMSF]. ACKNOWLEDGMENTS The lysates were incubated for 5min on ice followed by centrifugation at 4,000g for 10 min at 4 C to precipitate This work was supported by NCI NIH, nuclei. Supernatant was stored as cytoplasmic fraction. 1R21CA185943-01 (to NKS); NCI NIH R01AR47901 (to Nuclear pellet was incubated with 100 μl of ice-cold JLA), NCI NIH R01CA131294, NCI NIH R21CA155686, extraction buffer [20 mM Tris-Hcl (pH 7.9), 0.42M KCl, Avon Foundation, Breast Cancer Research Foundation 0.2 mM EDTA, 10% Glycerol, 2 mM DTT and 0.1 mM (BCRF) 90047965 (to DS). PMSF] for 10min followed by centrifugation at 12,000g for 10min at 4 C to clear the nuclear debris. Total protein was quantified using the Bradford protein assay kit CONFLICTS OF INTEREST (Biorad, Hercules, CA). Equal amount of protein was subjected to western blot analysis. None. LKB1 stable knockdown using lentiviral short REFERENCES hairpin RNA 1. Calle EE, Kaaks R. Overweight, obesity and cancer: epi- Five pre-made lentiviral LKB1 short hairpin RNA demiological evidence and proposed mechanisms. 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