Activation of tumor suppressor LKB1 by honokiol abrogates cancer stem-like phenotype in breast cancer via inhibition of oncogenic Stat3

Abstract

Tumor suppressor and upstream master kinase Liver kinase B1 (LKB1) plays a significant role in suppressing cancer growth and metastatic progression. We show that low-LKB1 expression significantly correlates with poor survival outcome in breast cancer. In line with this observation, loss-of-LKB1 rendered breast cancer cells highly migratory and invasive, attaining cancer stem cell-like phenotype. Accordingly, LKB1-null breast cancer cells exhibited an increased ability to form mammospheres and elevated expression of pluripotency-factors (Oct4, Nanog and Sox2), properties also observed in spontaneous tumors in Lkb1^-/- mice. Conversely, LKB1-overexpression in LKB1-null cells abrogated invasion, migration and mammosphere-formation. Honokiol (HNK), a bioactive molecule from Magnolia grandiflora increased LKB1 expression, inhibited individual cell-motility and abrogated the stem-like phenotype of breast cancer cells by reducing the formation of mammosphere, expression of pluripotency-factors and aldehyde dehydrogenase activity. LKB1, and its substrate, AMP-dependent protein kinase (AMPK) are important for HNK-mediated inhibition of pluripotency factors since LKB1-silencing and AMPK-inhibition abrogated, while LKB1-overexpression and AMPK-activation potentiated HNK's effects. Mechanistic studies showed that HNK inhibited Stat3-phosphorylation/activation in an LKB1-dependent manner, preventing its recruitment to canonical binding-sites in the promoters of Nanog, Oct4 and Sox2. Thus, inhibition of the coactivation-function of Stat3 resulted in suppression of expression of pluripotency factors. Further, we showed that HNK inhibited breast tumorigenesis in mice in an LKB1-dependent manner. Molecular analyses of HNK-treated xenografts corroborated our in vitro mechanistic findings. Collectively, these results present the first in vitro and in vivo evidence to support crosstalk between LKB1, Stat3 and pluripotency factors in breast cancer and effective anticancer modulation of this axis with HNK treatment.

Figure 1

Low LKB1 associates with poor prognosis and LKB1-silencing increases migration and invasion of breast cancer cells. (a) Kaplan– Meier plot of breast cancer patients in the TCGA BRCA data set binarized into high and low LKB1 expression using the median LKB1 expression as threshold. Patients with high LKB1 expression have longer overall survival (HR = 0.52, P < 0.01). (b) A statistically significant inverse relationship between tumor grade and LKB1 expression is observed in the VDX and Schmidt data sets suggesting that downregulation of LKB1 may play a role in increased tumor aggressiveness. (c) MCF7 and MDA-MB-231 cells were stably silenced for LKB1. Immunoblot analysis shows LKB1 expression levels in stable pools of LKB1-depleted (LKB1^{shRNA 1–2}) and vector control (pLKO.1) MCF7 and MDA-MB-231 cells. (d) MDA-MB-231-LKB1^shRNA1 and MDA-MB-231-pLKO.1 cells were subjected to spheroid-migration assay. (e) MDA-MB-231-LKB1^shRNA1, MDA-MB-231-pLKO.1, MCF7-LKB1^shRNA1 and MCF7-pLKO.1 cells were tested in scratch-migration assay. Bar diagram represents fold-change in migration. *P < 0.001, compared with pLKO.1 cells. (f) MDA-MB-231-LKB1^shRNA1, MDA-MB-231-pLKO.1, MCF7-LKB1^shRNA1 and MCF7-pLKO.1 cells were subjected to matrigel-invasion assay. Bar diagram represents % of LKB1^shRNA-cells invaded through matrigel in comparison to pLKO.1 cells. *P < 0.005, compared with pLKO.1 cells. HR, hazard ratio; TCGA, The Cancer Genome Atlas.

Figure 2

Silencing of LKB1 increases the expression of Oct4, Nanog and Sox2 in breast cancer cells. (a) MDA-MB-231-LKB1^shRNA1–2 and MDA-MB-231-pLKO.1 cells were subjected to mammosphere assay. *P < 0.001, compared with pLKO.1 cells. (b) Human embryonic stem cell score (hESC Score) calculated from a set of 68 embryonic stem-related genes is inversely correlated with LKB1 expression (Pearson’s correlation coefficient = − 0.40, P < 0.001). (c, d) Total protein was isolated from MDA-MB-231-LKB1^shRNA1–2, MDA-MB-231-pLKO.1, MCF7-LKB1^shRNA1–2 and MCF7-pLKO.1 cells followed by immunoblot analysis for proteins as indicated. Actin was used as control. Bar diagram shows quantitation of western blot signals from multiple independent experiments. RDU (relative density units). (e) Total RNA was isolated from MDA-MB-231-LKB1^shRNA, MDA-MB-231-pLKO.1, MCF7-LKB1^shRNA and MCF7-pLKO.1 cells followed by real-time PCR analysis for Oct4, Nanog and Sox2 expression. *P < 0.001, compared with pLKO.1 cells. (f) Tumor lysates from Lkb1^lox/lox (LKB1-WT) and LKB1^−/− (LKB1-null) mice were subjected to immunoblot analysis. Expression of LKB1, Oct4, Nanog and Sox2 was examined as indicated. Actin was used as control. (g) Tumors from LKB1^lox/lox and LKB1^−/− mice were subjected to immunohistochemical (IHC) analysis using Oct4, Nanog and Sox2 antibodies.

Figure 3

LKB1 overexpression inhibits migration, invasion and mammosphere-formation of breast cancer cells. Honokiol treatment activates LKB1 and inhibits motility of breast cancer cells. (a–c) LKB1-depleted (LKB1^shRNA) breast cancer cells were transfected with vector-control (V) or full length-LKB1 plasmid (LKB1^O/E), and subjected to matrigel-invasion (a), scratch-migration (b) and mammosphere (c) assays. *P < 0.001, compared with vector-transfected cells. (d) Breast cancer cells were treated with 5 μM Honokiol (HNK) for indicated time-intervals. Total protein lysates were immunoblotted for LKB1 expression. β-actin was used as a control. (e) Total RNA was isolated from MCF7 and MDA-MB-231 cells treated with 5 μM Honokiol (HNK) for 24 h and expression level of LKB1 was analyzed. β-actin was used as loading-control. Bar graph shows fold change in LKB1 expression. *P < 0.05, C vs HNK treatment. (f) MCF7 and MDA-MB-231 breast cancer cells were treated with 5 μM Honokiol (HNK) for indicated time-intervals. Total lysates were immunoblotted for phospho-AMPK (pAMPK-Thr172) and total-AMPK expression. β-actin was used as a loading-control. (g) Breast cancer cells were treated with 5 μM Honokiol (HNK) and cell lysates were examined for catalytic activity towards LKBtide peptide. *P < 0.01, C vs HNK treatment. (h) MDA-MB-231 xenograft tumors from mice treated with vehicle and honokiol (HNK) were subjected to immunohistochemical (IHC) analysis using LKB1 antibodies. (i) Bar diagrams show quantitation of IHC-analysis of LKB1. Columns, mean (n = 8); bar, s.d. * significantly different (P < 0.005) compared with control. HNK suppresses MDA-MB-231 breast cancer cell migration in microfluidic device. The speed (j), persistence (k) and velocity (l) of MDA-MB-231 cells migrating through channels of prescribed widths are shown in the presence of HNK (5 μM) or vehicle control (VC). Micrographs indicate cells, shown by an arrowhead, migrating through 20 μm (m, n) and 10 μm (o, p) in width channels in the presence of HNK or VC. Data represent the mean ± s.e. of cells from n = 4 independent experiments. *P < 0.05 determined by paired t-test.

Figure 4

Honokiol inhibits stem-like characteristics of breast cancer cells. (a) Breast cancer cells were treated with 5 μM honokiol (HNK) and subjected to mammosphere formation. Vehicle treated cells are denoted as (c). The graph shows the number of mammospheres. *P < 0.05, compared with untreated controls. (b) Breast cancer cells were grown as mammospheres in the absence or presence of 5 μM honokiol, the activity of ALDH was analyzed by Aldeflour assay. The panels show the representative 2D plots of Aldeflour assay with or without HNK treatment and the graph shows the ALDH-positive population. *P < 0.001, compared with untreated controls. (c) MDA-MB-231 and MCF7 cells were treated with 5 μM honokiol (HNK) for indicated time-intervals. Total RNA was isolated and expression levels of Oct4, Nanog, Sox2 and c-myc were examined. β-actin was used as loading-control. (d) MDA-MB-231 and MCF7 cells were treated with 5 μM honokiol (HNK) for indicated time-intervals. Total protein lysates were immunoblotted for Oct4, Nanog and Sox2 expression. β-actin was used as a control. (e) Breast cancer cells were treated with vehicle control (control), TGFβ+TNFα (10 ng/ml of each), 5 μM honokiol (HNK) or TGFβ+TNFα +HNK for 24 h, and subjected to immunofluorescence analysis of Oct4, Nanog and Sox2. Nuclei were stained with DAPI. (f) Total protein lysates were isolated from MDA-MB-231 xenograft tumors from mice treated with vehicle and honokiol (HNK) and subjected to immunoblot analysis. Expression of Oct4, Nanog and Sox2 was examined as indicted. (g) MDA-MB-231 xenograft tumors from mice treated with vehicle and honokiol (HNK) were subjected to immunohistochemical (IHC) analysis using Oct4, Nanog and Sox2 antibodies. (h) Bar diagrams show quantitation of IHC-analysis of Oct4, Nanog and Sox2. Columns, mean (n = 8); bar, s.d. * significantly different (P < 0.05) compared with control.

Figure 5

Honokiol decreases stemness in vivo. (a) Schematic outline of the tumorigenicity assays. (b) Tumor volume of MDA-MB-231 secondary xenografts established with 5 × 10⁵ cells. Plots show the values of tumor volume (open circles) and mean tumor volume (diamonds). Red-Vehicle, Blue-Honokiol. (c) Plots show Kaplan–Meier curves (Blue-Honokiol, Red-Vehicle) for time to detect tumors (100 mm³ volume). (d) Honokiol decreases tumor incidence at limiting dilution. Tumor incidence at weeks 2, 3 and 4 of secondary transplants of MDA-MB-231 at limiting dilutions. The tumors/numbers of mice/group are shown. The bottom row indicates the estimated breast tumor-initiating/stem cell (SC) frequencies.

Figure 6

LKB1-AMPK axis plays an important role in honokiol-mediated inhibition of stemness transcription factors. (a) LKB1-depleted (LKB1^{shRNA 1–2}) and vector control (pLKO.1) MDA-MB-231 cells were treated with 5 μM honokiol (HNK). Total RNA was examined for the expression of Oct4, Nanog and Sox2 as indicted. β-actin was used as control. (b) Total protein lysates of LKB1-depleted (LKB1^{shRNA 1–2}) and vector control (pLKO.1) breast cancer cells treated with 5 μM honokiol (HNK) were analyzed for the expression of Nanog and Sox2 as indicated. β-actin was used as loading-control. (c) LKB1-depleted (LKB1^shRNA1–2) breast cancer cells were transfected with vector-control (V) or full length-LKB1 plasmid (LKB1^O/E); total protein lysates were examined for LKB1 expression as indicated using immunoblot analysis. (d) LKB1-depleted (LKB1^shRNA1) breast cancer cells were transfected with vector-control (V) or full length-LKB1 plasmid (LKB1^O/E) followed by treatment with 5 μM honokiol (HNK). Total protein lysates were examined for Oct4 and Nanog. β-actin was used as loading-control. (e) Bar diagram shows quantitation of western blot signals. *P < 0.001, compared with untreated controls. (f) MCF7 and MDA-MB-231 breast cancer cells were treated with 5 μM honokiol (HNK) in combination with AICAR (100 μM) or compound C (CC, 20 μM). Total RNA was examined for the expression of Oct4, Nanog and Sox2. *P < 0.005, compared with untreated controls; ^##P < 0.05, compared with HNK-treated cells; **P < 0.001, compared with HNK-treated cells. AICAR, 5-aminoimidazole-4-carboxamide ribonucleotide.

Figure 7

Stat3 gets recruited to the promoters of Oct4, Nanog and Sox2 and honokiol inhibits stemness transcription factors via inhibiting Stat3. (a) Breast cancer cells were treated with 5 μM Honokiol (HNK) for indicated time-intervals. Total protein lysates were immunoblotted for phospho-Stat3 and total Stat3 expression. β-actin was used as a control. (b) MCF7 cells were transfected with Stat3-overexpression (Stat3 O/E) followed by 5 μM Honokiol (HNK) treatment and co-treatment with HNK and Stattic as indicated. Total RNA was analyzed for the expression of Oct4, Nanog and Sox2. Bar graph shows fold change in gene expression. *P < 0.05, C vs HNK treatment; **P < 0.01, HNK+Stat3O/E vs HNK alone; ^#P < 0.005, HNK+Stattic vs HNK alone. (c, d) MCF7 cells were transfected with vector or constitutively active Stat3 overexpression plasmid (CA-Stat3) followed by treatment with 5 μM Honokiol (HNK). MCF7 cells were treated with 5 μM Honokiol (HNK) with or without co-treatment with Stattic. Total protein lysates were analyzed for the expression of Oct4, Nanog and Sox2 as indicated. β-actin was used as control. Bar diagram shows relative density units of the western signals.

Figure 8

LKB1 is important for honokiol-mediated inhibition of Stat3 expression and recruitment to Oct4, Nanog and Sox2 in breast cancer cells. (a) STAT3-score calculated from a set of 24 STAT3-activated genes compiled by Azare et al. is inversely correlated with LKB1 expression (Pearson correlation coefficient = − 0.44, P < 0.001). (b) LKB1-depleted (LKB1^shRNA) and vector control (pLKO.1) breast cancer cells were treated with 5 μM honokiol (HNK). Total protein lysates were examined for the expression of LKB1, phospho-Stat3 and total Stat3 as indicted. β-actin was used as loading-control. (c) LKB1-depleted (LKB1^shRNA) breast cancer cells were transfected with vector-control (V) or full length-LKB1 plasmid (LKB1^O/E), total protein lysates were examined for LKB1, phospho-Stat3 and total Stat3 expression as indicated using immunoblot analysis. β-actin was used as loading-control. (d) Bar diagram shows quantitation of western blot signals from multiple independent experiments. Cells were treated as indicated *P < 0.001, compared with untreated controls. (e, f) Soluble chromatin was prepared from LKB1-depleted (LKB1^shRNA), vector control (pLKO.1), LKB1-transfected LKB1^shRNA (LKB1^shRNA+LKB1O/E) MCF7 breast cancer cells treated with vehicle (c) or 5 μM honokiol (HNK). Chromatin immunoprecipitation assay was performed using pStat3 antibody. IgG antibody was included as control. The purified DNA was analyzed by real-time quantitative PCR using primers spanning the Stat3-binding sites at Oct4, Nanog and Sox2 promoter. PCR products were also analyzed on agarose gels.

Figure 9

LKB1 is integral for honokiol-mediated inhibition of mammosphere and breast tumor growth. (a, b) LKB1-depleted (LKB1^{shRNA 1–2}) and vector control (pLKO.1) MCF7 and MDA-MB-231 breast cancer cells were treated with 5 μM honokiol (HNK) and subjected to mammosphere assay. *P < 0.001, compared with untreated controls. (c) LKB1-depleted (LKB1^shRNA) and vector control (pLKO.1) MDA-MB-231 cells derived tumors were developed in nude mice and treated with vehicle and Honokiol (HNK). Tumor growth was monitored by measuring the tumor volume for 6 weeks. (n = 8–10); (P < 0.001), pLKO.1+HNK compared with LKB1^shRNA+HNK. (d) Tumors from LKB1^shRNA+Vehicle, pLKO.1+Vehicle, LKB1^shRNA+HNK, and pLKO.1 +HNK groups were subjected to immunohistochemical (IHC) analysis using Ki-67 antibodies. Bar diagram shows the quantitation of Ki-67 expression in tumors from each treatment group. Columns, mean (n = 5); *P < 0.005, compared with control. (e) Tumors from LKB1^shRNA+Vehicle, pLKO.1+Vehicle, LKB1^shRNA+HNK, and pLKO.1 +HNK groups were subjected to IHC analysis using phospho-Stat3 (pStat3) antibodies. Bar diagram shows the quantitation of pStat3 expression in tumors from each treatment group. Columns, mean (n = 5); *P < 0.005, compared with control. (f) Total protein lysates from vector control (pLKO.1) and LKB1 null (LKB1^shRNA) tumors were examined for the expression of pStat3 and Stat3 as indicated. β-actin was used as loading-control. (g) Schematic representation. Honokiol treatment increases the expression of tumor suppressor LKB1 which in turn activates AMPK phosphorylation. Increased LKB1 abrogates Stat3 phosphorylation and inhibits recruitment of Stat3 on Oct4, Nanog and Sox2 resulting in inhibition of the expression of Oct4, Nanog and Sox2 leading to inhibition of stemness, mammosphere and breast tumor growth.