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

Abstract

Leptin, a major adipocytokine produced by adipocytes, is emerging as a key molecule linking obesity with breast cancer therefore, it is important to find 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.

Keywords: Honokiol; LKB1; breast cancer; leptin; miR-34a.

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.

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.

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.

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 with untreated controls. C. Immunoblot analysis of LKB1 in stable pools of LKB1-depleted (LKB1^{shRNA 1–2}) and vector control (pLKO.1) MCF7 cells. LKB1-depleted (LKB1^{shRNA 1–2}) were transfected with LKB1 overexpression vector to create a ‘gain-of-function’ system. D. Expression levels of miR-34a in stable pools of LKB1-depleted (LKB1^{shRNA 1–2}) 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 HNK-treated MCF7-pLKO.1 cells. E. LKB1-depleted (LKB1^shRNA2) were transfected with LKB1 overexpression vector and expression levels of miR-34a was examined. *p < 0.001, compared with HNK-treated, control-transfected, MCF7- LKB1^shRNA2 cells.

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.

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.

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.