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. 2015 Jul 9;10(7):e0132285.
doi: 10.1371/journal.pone.0132285. eCollection 2015.

Inhibition of Aerobic Glycolysis Represses Akt/mTOR/HIF-1α Axis and Restores Tamoxifen Sensitivity in Antiestrogen-Resistant Breast Cancer Cells

Yu Mi Woo  1 , Yubin Shin  1 , Eun Ji Lee  1 , Sunyoung Lee  1 , Seung Hun Jeong  2 , Hyun Kyung Kong  1 , Eun Young Park  1 , Hyoung Kyu Kim  2 , Jin Han  2 , Minsun Chang  3 , Jong-Hoon Park  1
Affiliations

Inhibition of Aerobic Glycolysis Represses Akt/mTOR/HIF-1α Axis and Restores Tamoxifen Sensitivity in Antiestrogen-Resistant Breast Cancer Cells

Yu Mi Woo et al. PLoS One. .

Abstract

Tamoxifen resistance is often observed in the majority of estrogen receptor-positive breast cancers and it remains as a serious clinical problem in breast cancer management. Increased aerobic glycolysis has been proposed as one of the mechanisms for acquired resistance to chemotherapeutic agents in breast cancer cells such as adriamycin. Herein, we report that the glycolysis rates in LCC2 and LCC9--tamoxifen-resistant human breast cancer cell lines derived from MCF7--are higher than those in MCF7S, which is the parent MCF7 subline. Inhibition of key glycolytic enzyme such as hexokinase-2 resulted in cell growth retardation at higher degree in LCC2 and LCC9 than that in MCF7S. This implies that increased aerobic glycolysis even under O2-rich conditions, a phenomenon known as the Warburg effect, is closely associated with tamoxifen resistance. We found that HIF-1α is activated via an Akt/mTOR signaling pathway in LCC2 and LCC9 cells without hypoxic condition. Importantly, specific inhibition of hexokinase-2 suppressed the activity of Akt/mTOR/HIF-1α axis in LCC2 and LCC9 cells. In addition, the phosphorylated AMPK which is a negative regulator of mTOR was decreased in LCC2 and LCC9 cells compared to MCF7S. Interestingly, either the inhibition of mTOR activity or increase in AMPK activity induced a reduction in lactate accumulation and cell survival in the LCC2 and LCC9 cells. Taken together, our data provide evidence that development of tamoxifen resistance may be driven by HIF-1α hyperactivation via modulation of Akt/mTOR and/or AMPK signaling pathways. Therefore, we suggest that the HIF-1α hyperactivation is a critical marker of increased aerobic glycolysis in accordance with tamoxifen resistance and thus restoration of aerobic glycolysis may be novel therapeutic _target for treatment of tamoxifen-resistant breast cancer.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Lactate production and glucose consumption were elevated in LCC2 and LCC9 cells.
(A) Cell proliferation rate of MCF7S, LCC2 and LCC9 cells was measured by XTT assay in a time-dependent manner. The data represent the mean ± SD of representative results for 3 independent assays. (B) Viability of MCF7s, LCC2 and LCC9 cells was measured by XTT assay 24 h after the treatment various concentration of 3-BrPA. The data represent the mean ± SD of representative results for 3 independent assays. (C) The amount of 13C-labeled lactate was determined in each cell line by 1H NMR quantification. 13C-labeled lactate derived from glycolysis was traced and quantified for each cell line. Spectra were normalized to the cell dry weight and an NMR scaling factor. (D) The amount of total lactate in the cell extracts was quantified through GC-MS analysis. The vertical axis represents the amount of total lactate (μM) per dry weight of protein (g). (E) 3H-glucose was used to treat each cell line for 2 h. Glucose uptake was expressed as counts per minute per milligram of protein (cpm/min/mg protein). (F) Inhibition of hexokinase-2 (HK-2) repressed cell proliferation, especially in LCC2 and LCC9, compared with MCF7S. Control siRNA (Consi) and HK-2 siRNA (HK2si) were transfected into each cell line with 20 nM for 24 h and cell proliferation was measured after 48 h transfection. The data represent the mean ± SD of 3 independent experiments that were performed in triplicate. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
Fig 2
Fig 2. HIF-1α is upregulated in LCC2 and LCC9 cells.
(A) HIF-1α activity in MCF7S, LCC2 and LCC9 cells was measured by EMSA. (B) Endogenous HIF-1α and LDHA protein levels were measured by western blotting. β-Actin was used as a loading control. (C) The mRNA level of HIF-1α was measured by quantitative RT-PCR using the β-actin gene as an internal control. (D) LDHA was downregulated in MCF7S, LCC2, and LCC9 cells transfected with HIF-1α siRNA. siRNA (20nM) was treated in three cell lines for 48 h. C, control siRNA; H, HIF-1α siRNA (E) HIF-1α was overexpressed by treatment of CoCl2 (50 μM, 24 h) in each cell line. Elevation of HIF-1α induced increase in LDHA expression levels. The data shown are representative results of 3 independent experiments. Band density of each immunoblot was quantified using MultiGauge. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
Fig 3
Fig 3. Increased Akt/mTOR signaling induces HIF-1α activity and aerobic glycolysis.
(A) VHL protein levels, as well as the activity of Akt/mTOR and its downstream _targets measured by western blotting using β-actin as a loading control. (B) After treatment with specific inhibitors of Akt (LY294,002, 40 μM) and mTOR (rapamycin, 30 nM) for 4 and 16 h, respectively, HIF-1α and Akt/mTOR protein expressions were measured by western blotting in LCC2 cells. β-Actin was used as a loading control. Band density of each immunoblot with triplicate was quantified using MultiGauge. (C) Rapamycin at 30 nM was used to treat LCC2 cells for 16 h. HIF-1α activity was measured using EMSA in LCC2 cells. The data represent results of 3 independent experiments. (D) Lactate accumulation was measured by 1H NMR quantification after treatment of rapamycin (30 nM, 16 h). *, P < 0.05.
Fig 4
Fig 4. Akt/mTOR/HIF-1α axis was specifically suppressed by knockdown of hexokinase-2 (HK-2) glycolytic enzyme in LCC2 and LCC9 cells.
(A) Cells were transfected with control siRNA (Consi) or HK-2 siRNA (HK2si) at 20 nM for 48 h followed by treatment of 4-OHT (10 μM, 48 h). (B) Cells were treated with 3-BrPA (50 μM) for 4 h and followed by treatment of 4-OHT (10 μM, 48 h). Western blotting was performed for the lysates prepared from treated cells. β-Actin was served as a loading control. The data shown are representative results for at least 3 independent experiments. Band density was quantified using MultiGauge. *, P < 0.01; **, P < 0.001 vs. Consi transfected cells.
Fig 5
Fig 5. AMPK activation was decreased in LCC2 and LCC9 cells compared to MCF7S cells.
(A) Cells were treated with 1 mM AICAR for 60 min. p-AMPK (Thr172), p-TSC2 (Ser1387), and HIF-1α was detected by western blotting. β-Actin was served as a loading control. The data shown are representative results for at least 3 independent western blot analyses. Band density was quantified using MultiGauge. *, P < 0.05; **, P < 0.01; ***, P < 0.001 vs. no treatment of AICAR and #, P < 0.05; ##, P < 0.01; ###, P < 0.001 vs. MCF7S control cells. (B) AICAR-treated cells were assayed for lactate production. (C) Cells treated with AICAR were treated with 4-OHT (5 μM) for 3 days and cell survival of MCF7S, LCC2, and LCC9 cells was measured by XTT assay. The data represent the mean ± SD of representative results for 3 independent lactate assays. *, P < 0.05; ***, P < 0.001.
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2013R1A2A1A01011908, http://www.nrf.re.kr/nrf_tot_cms/index.jsp. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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