ADIPOQ/adiponectin induces cytotoxic autophagy in breast cancer cells through STK11/LKB1-mediated activation of the AMPK-ULK1 axis

Abstract

ADIPOQ/adiponectin, an adipocytokine secreted by adipocytes in the breast tumor microenvironment, negatively regulates cancer cell growth hence increased levels of ADIPOQ/adiponectin are associated with decreased breast cancer growth. However, its mechanisms of action remain largely elusive. We report that ADIPOQ/adiponectin induces a robust accumulation of autophagosomes, increases MAP1LC3B-II/LC3B-II and decreases SQSTM1/p62 in breast cancer cells. ADIPOQ/adiponectin-treated cells and xenografts exhibit increased expression of autophagy-related proteins. LysoTracker Red-staining and tandem-mCherry-GFP-LC3B assay show that fusion of autophagosomes and lysosomes is augmented upon ADIPOQ/adiponectin treatment. ADIPOQ/adiponectin significantly inhibits breast cancer growth and induces apoptosis both in vitro and in vivo, and these events are preceded by macroautophagy/autophagy, which is integral for ADIPOQ/adiponectin-mediated cell death. Accordingly, blunting autophagosome formation, blocking autophagosome-lysosome fusion or genetic-knockout of BECN1/Beclin1 and ATG7 effectively impedes ADIPOQ/adiponectin induced growth-inhibition and apoptosis-induction. Mechanistic studies show that ADIPOQ/adiponectin reduces intracellular ATP levels and increases PRKAA1 phosphorylation leading to ULK1 activation. AMPK-inhibition abrogates ADIPOQ/adiponectin-induced ULK1-activation, LC3B-turnover and SQSTM1/p62-degradation while AMPK-activation potentiates ADIPOQ/adiponectin's effects. Further, ADIPOQ/adiponectin-mediated AMPK-activation and autophagy-induction are regulated by upstream master-kinase STK11/LKB1, which is a key node in antitumor function of ADIPOQ/adiponectin as STK11/LKB1-knockout abrogates ADIPOQ/adiponectin-mediated inhibition of breast tumorigenesis and molecular analyses of tumors corroborate in vitro mechanistic findings. ADIPOQ/adiponectin increases the efficacy of chemotherapeutic agents. Notably, high expression of ADIPOQ receptor ADIPOR2, ADIPOQ/adiponectin and BECN1 significantly correlates with increased overall survival in chemotherapy-treated breast cancer patients. Collectively, these data uncover that ADIPOQ/adiponectin induces autophagic cell death in breast cancer and provide in vitro and in vivo evidence for the integral role of STK11/LKB1-AMPK-ULK1 axis in ADIPOQ/adiponectin-mediated cytotoxic autophagy.

Keywords: AMPK; STK11/LKB1; ULK1; adiponectin; autophagy; breast cancer.

Figure 1.

ADIPOQ/adiponectin inhibits breast cancer growth and induces autophagosome accumulation. (A) Breast cancer cells were treated with 5 µg/ml ADIPOQ/adiponectin and subjected to soft-agar colony-formation assay for 3 wk. Histogram represents average number of colonies counted (in 6 microfields). *, P < 0.001, compared with untreated controls. Vehicle-treated cells, denoted with the letter “C.” (B) Clonogenicity of breast cancer cells treated with 5 µg/ml ADIPOQ/adiponectin as indicated. (C) MDA-MB-231 cell-derived xenografts were developed in nude mice and treated with control-adenoviral (Ad-Luc) and ADIPOQ/adiponectin-adenoviral (Ad-ADIPOQ) (10⁸ pfu). Tumor growth was monitored by measuring the tumor volume for 6 wk. (n = 8 mice per group). Ad-ADIPOQ/adiponectin treatment reduced tumor size as compared with Ad-Luc, *P < 0.001. (D) Tumors from vehicle (V) and ADIPOQ/adiponectin-treated mice were subjected to immunohistochemical analysis using MKI67 antibodies. Scale bar: 100 µm. Bar diagrams show quantification of immunohistochemical analysis. *P < 0.001, compared with control. (E) TUNEL-positive cells in tumor sections were counted. Each bar represents the mean apoptotic cell rate (n = 6–8). *, P < 0.01, compared with untreated controls. (F) MCF7 cells were treated with 5 µg/ml ADIPOQ/adiponectin as indicated and visualized under an electron microscope. Scale bar: 2 µm. Top pictures are shown with approximately 7,400x magnifications. Double-membrane autophagosomes were counted in randomly selected ∼100 cells. The number of autophagosomes was counted from randomly selected fields. Cells with >2 autophagosomes were counted. (G) MCF7 and MDA-MB-231 cells were treated with 5 µg/ml ADIPOQ/adiponectin and subjected to immunocytochemistry using LC3B antibody. Scale bar: 20 µm. Representative immunofluorescence images are shown.

Figure 2.

ADIPOQ/adiponectin induces autophagy in breast cancer cells. (A) Breast cancer cells were treated with 5 µg/ml ADIPOQ/adiponectin, and total cell lysates were immunoblotted for LC3B and SQSTM1/p62 expression. ACTB was used as a loading control. Bar diagram shows quantification of western blot signals from multiple independent experiments. (B) Immunoblot analysis of BECN1, ATG5, ATG7 and ATG12 in breast cancer cells treated with 5 µg/ml ADIPOQ/adiponectin as indicated. ACTB was used as a loading control. (C) Total protein lysates from tumors from control-adenoviral (Ad-Luc) and ADIPOQ/adiponectin -adenoviral (Ad-ADIPOQ)-treated mice were examined for the expression of BECN1, ATG7, LC3B and SQSTM1/p62. ACTB was used as a loading control. (D) Tumors from vehicle (V) and ADIPOQ/adiponectin-treated mice were subjected to immunohistochemical (IHC) analysis using BECN1, ATG5, SQSTM1/p62 and LC3B antibodies. Scale bar: 100 µm. Bar diagrams show quantification of IHC analysis. *P < 0.01, compared with control. (E,F) Breast cancer cells were transfected with an LC3B-encoding plasmid, treated with 5 µg/ml ADIPOQ/adiponectin and stained with LysoTracker Red. Scale bar: 20 µm. Representative immunofluorescence images are shown.

Figure 3.

Inhibition of autophagy inhibits ADIPOQ/adiponectin-mediated reduction in cell-survival and apoptosis-induction in breast cancer cells. (A) MCF7 and MDA-MB-231 cells were treated with 5 µg/ml ADIPOQ/adiponectin alone or in combination with 200 nM Baf and 2 mM 3-MA as indicated and subjected to XTT assay. *P < 0.001, compared with control; ^#P < 0.005, compared with ADIPOQ/adiponectin-treated cells. (B) Breast cancer cells were treated as in (A) and subjected to DNA fragmentation assay. *P < 0.01, compared with control; ^#P < 0.01, compared with ADIPOQ/adiponectin -treated cells. (C) MCF7 and MDA-MB-231 cells were treated with 5 µg/ml ADIPOQ/adiponectin and 2 mM 3-MA and total cell lysates were immunoblotted for cleaved PARP1 (cPARP1), PARP1 and ACTB as indicated. (D) BECN1 and ATG7 were knocked out in MCF7 cells using CRISPR/Cas9 and total cell lysates were immunoblotted for BECN1 and ATG7. ACTB was used as loading control. (E) Control, BECN1-KO and ATG7-KO MCF7 cells were treated with 5 µg/ml ADIPOQ/adiponectin and total cell lysates were immunoblotted for cleaved-PARP1 and total-PARP1 expression levels. (F) Clonogenicity of control, BECN1-KO and ATG7-KO MCF7 cells treated with 5 µg/ml ADIPOQ/adiponectin as indicated.

Figure 4.

ADIPOQ/adiponectin induces energy depletion and AMPK-activation in breast cancer cells and inhibition of AMPK hinders ADIPOQ/adiponectin-mediated modulation of autophagy markers. (A) Intracellular ATP production was measured in MCF7, MDA-MB-231, MDA-MB-468 and SUM149 cells treated with ADIPOQ/adiponectin (5 µg/ml) for the indicated times. Relative ATP levels are expressed as fold change with respect to control. *P < 0.01, compared with control. (B) Total cell lysates from MCF7 and MDA-MB-231 cells treated with 5 µg/ml ADIPOQ/adiponectin for the indicated times were immunoblotted for phospho-PRKAA1 (p-PRKAA1), and total PRKAA1. ACTB was used as a loading control. (C) Total protein lysates from tumors from control-adenoviral (Ad-Luc) and ADIPOQ/adiponectin-adenoviral (Ad-ADIPOQ)-treated mice were examined for the expression of p-PRKAA1 and PRKAA1. ACTB was used as a loading control. (D) Tumors from vehicle (V) and ADIPOQ/adiponectin-treated mice were subjected to immunohistochemical analysis using p-PRKAA1 antibodies. Scale bar: 100 µm. (E) MCF7 and MDA-MB-231 cells were treated with ADIPOQ/adiponectin (5 µg/ml), compound C (CC) and AICAR alone and in combination as indicated, and total cell lysates were immunoblotted for LC3B and SQSTM1/p62 expression. ACTB was used as a loading control. (F) Prkaa1 wild-type (Prkaa1-WT) and prkaa1 knockout (prkaa1-KO) MEFs were treated with 5 µg/ml ADIPOQ/adiponectin and total cell lysates were immunoblotted for LC3B and SQSTM1/p62 expression. ACTB was used as a loading control.

Figure 5.

ADIPOQ/adiponectin increases ULK1 expression via AMPK in breast cancer cells. (A) MCF7 and MDA-MB-231 cells were treated with 5 µg/ml ADIPOQ/adiponectin for various time intervals as indicated and total cell lysates were immunoblotted for ULK1 and phospho-ULK1 (p-ULK1) expression. ACTB is used as a loading control. (B) Total protein lysates from tumors from control-adenoviral (Ad-Luc) (denoted as C) and ADIPOQ/adiponectin-adenoviral (Ad-ADIPOQ)-treated mice were examined for the expression of ULK1 and p-ULK1. ACTB was used as a loading control. (C) Tumors from vehicle (V) and ADIPOQ/adiponectin-treated mice were subjected to immunohistochemical analysis using ULK1 and p-ULK1 antibodies. Scale bar: 100 µm. (D, E) MCF7 and MDA-MB-231 cells were treated with ADIPOQ/adiponectin (5 µg/ml), compound C and AICAR alone and in combination as indicated, and total cell lysates were immunoblotted for ULK1 and p-ULK1 expression. ACTB was used as a loading control. (F) Prkaa1 wild-type (Prkaa1-WT) and prkaa1 knockout (prkaa1-KO) MEFs were treated with 5 µg/ml ADIPOQ/adiponectin and total cell lysates were immunoblotted for ULK1 and p-ULK1 expression. ACTB was used as control.

Figure 6.

STK11/LKB1 plays an important role in ADIPOQ/adiponectin-mediated cytotoxic-autophagy and inhibition of tumor growth. (A) Total protein lysates of MCF7 and MDA-MB-231 cells infected with STK11/LKB1 shRNA (STK11/LKB1^sh) and vector-pLKO.1 control (vector) were immunoblotted for the expression of STK11/LKB1. MCF7 cells containing STK11/LKB1 shRNA and MCF7 cells containing vector were treated with 5 µg/ml ADIPOQ/adiponectin and total protein lysates were examined for p-PRKAA1 and PRKAA1 expression in an immunoblot assay. (B) MCF7 cells containing STK11/LKB1 shRNA, MCF7 cells containing vector, MDA-MB-231 cells containing STK11/LKB1 shRNA, and MDA-MB-231 cells containing vector were treated with 5 µg/ml ADIPOQ/adiponectin and total protein lysates were examined for LC3B and BECN1 expression in an immunoblot assay. (C) MCF7 cells with STK11/LKB1 shRNA, MCF7 cells with vector, MDA-MB-231 cells with STK11/LKB1 shRNA, and MDA-MB-231 cells with vector were treated with 5 µg/ml ADIPOQ/adiponectin and subjected to a clonogenicity assay. (D) Tumors derived from MDA-MB-231 cells with STK11/LKB1 shRNA and MDA-MB-231 cells with vector-pLKO.1 control (vector) were developed in nude mice and treated with control-adenoviral-Ad-Luc (C) and ADIPOQ/adiponectin-adenoviral (Ad-ADIPOQ) (10⁸ pfu). Tumor growth was monitored by measuring the tumor volume for 5 weeks (n = 8–10); (P < 0.001), vector + ADIPOQ/adiponectin compared with STK11/LKB1 shRNA⁺ ADIPOQ/adiponectin. (E-G) Tumors from vehicle (V) and ADIPOQ/adiponectin-treated mice were subjected to immunohistochemical analysis using MKI67, ATG5 and BECN1 antibodies. Scale bar: 100 µm. Bar diagrams show quantification of IHC-analysis. *P < 0.01, compared with control.

Figure 7.

ADIPOQ/adiponectin sensitizes breast cancer cells to a variety of chemotherapy drugs. (A, B) MCF7 and MDA-MB-231 cells were treated with 5 µg/ml ADIPOQ/adiponectin, carboplatin (Car.), paclitaxel (Pac.) or doxorubicin (Dox.) either alone or in combination and subjected to clonogenicity. *P < 0.01, compared with control; **P < 0.001, compared with control; ^#P < 0.05, compared with cells treated with carboplatin, paclitaxel and doxorubicin alone. (C) MCF7 and MDA-MB-231 cells were treated as in (A) and subjected to TUNEL assay. *P < 0.05, compared with control; **P < 0.01, compared with carboplatin alone; ^#P < 0.01, compared with paclitaxel alone; ^##P < 0.01, compared with doxorubicin alone. (D) MCF7 cells were treated with 5 µg/ml ADIPOQ/adiponectin, carboplatin (Car.), paclitaxel (Pac.) and or doxorubicin (Dox.) either alone or in combination and total cell lysates were subjected to immunoblot analysis using cleaved-PARP1, total-PARP1 antibodies. ACTB was included as a loading control.

Figure 8.

Higher expression of ADIPOQ/adiponectin-receptor and BECN1 correlates with increased overall survival. (A, B) Chemotherapy-treated patients (n = 416 in the Metabric cohort and n = 791 in the Affymetrix cohort) were included in a survival analysis. In the Metabric data set, higher expression of ADIPOR2 (adiponectin receptor 2) and BECN1 correlated with better survival (HR = 0.68, 95% CI = 0.51–0.90, P = 0.0076 and HR = 0.68, 95% CI = 0.50–0.91, P = 0.0087, respectively). (C) ADIPOQ/adiponectin levels were correlated with survival in the Affymetrix cohort but the results were not statistically significant (HR = 0.83, 95% CI = 0.62–1.11, P = 0.22). (D) Schematic representation of ADIPOQ/adiponectin-mediated activation of cytotoxic autophagy via the STK11/LKB1 axis. ADIPOQ/adiponectin treatment increases expression and cytoplasmic localization of STK11/LKB1 and reduces negative phosphorylation of STK11/LKB1 via reduction of phospho-MAPK1/ERK2. ADIPOQ-induced STK11/LKB1 leads to AMPK-activation, which in turn increases ULK1 expression and phosphorylation leading to cytotoxic autophagy and tumor growth inhibition.