The Crosstalk between GPR81/IGFBP6 Promotes Breast Cancer Progression by Modulating Lactate Metabolism and Oxidative Stress

Abstract

Breast cancer is the most frequent tumor and the leading cause of cancer deaths in women. In recent years, lactate metabolism and, in particular, its receptor GPR81 have been shown to play a vital role in cancer biology. GPR81 is upregulated in breast cancer and promotes tumor growth by tumor cell-derived lactate. Therefore, the search for possible crosstalk and the involvement of new molecules capable of generating this pathology is always in continuous development. In this study, the relationship between GPR81 and IGFBP6 protein in tumor growth and oxidative stress in the human breast cancer cell line MDA-MB-231 was studied. Cells were treated with lactate or the GPR81 receptor agonist and antagonist 3,5-DHBA and 3-OBA, respectively. In addition, oxidative stress and proliferation were also evaluated in cells challenged with the recombinant IGFBP6 protein. Our data showed that lactate induced cell proliferation and wound healing of the MDA-231 breast cancer cell through the overexpression of both the lactate receptor GPR81 and IGFBP6. The increase in IGFBP6 was able, in turn, to improve the mitochondrial fitness and redox state, as suggested by the reduced levels of mitochondrial ROS production after IGFBP6 treatment, presumably mediated by the increase in the ROS detoxifying genes HMOX1, GSTK1 and NQO1. In conclusion, our data highlight a novel axis between GPR81 and IGFBP6 in MDA-231 cells able to modulate lactate metabolism and oxidative stress. This complex signaling may represent a new therapeutic target for breast cancer.

Keywords: GPR81; IGFBP6; breast cancer; lactate; oxidative stress.

Figure 1

GPR81 stimulation promotes breast cancer cell growth. Effect of lactate (20 mM), 3,5-DHBA (150 μM, GPR81 agonist) and 3-OBA (3 mM, GPR81 antagonist) on cell proliferation (A,B), colony formation capacity (C) and wound healing (D–F). Data are expressed as mean ± SD of at least four independent experiments. (*** p < 0.001; **** p < 0.0001).

Figure 2

GPR81 modulates mitochondrial metabolism gene expression. Evaluation of relative mRNA expression levels of (A) PGC1alpha, (B) SIRT1, (C) COX IV, (D) COX II, (E) ATPsyn, (F) MCT1, following 24 h of lactate (20 mM), 3,5-DHBA (150 μM) and 3-OBA (3 mM) exposition, analyzed by real-time PCR. The calculated value of 2^−ΔΔCt in untreated controls is 1. Data are expressed as mean ± SD of at least four independent experiments. * p < 0.05; ** p < 0.005; *** p < 0.001; **** p < 0.0001.

Figure 3

GPR81/IGFBP6 axis in breast cancer cells. Effect of lactate (20 mM) (A), 3,5-DHBA (150 μM) (B) and 3-OBA (3 mM) (C) on IGFBP6 mRNA expression levels, following 24 h of treatment. Effect of IGFBP6 treatment (800 ng/mL) on lactate production (D), GPR81 mRNA expression levels (E) and GPR81 protein expression (F), following 24 h of treatment. Evaluation of relative mRNA expression levels of LDHA, MCT4 and MCT1 (G) in IGFBP6-exposed (400 ng/mL) cells. The calculated value of 2^-ΔΔCt in untreated controls is 1. Data are expressed as mean ± SD of at least four independent experiments. ** p < 0.005; *** p < 0.001; **** p < 0.0001.

Figure 4

IGFBP6 modulates mitochondrial metabolism and promotes breast cancer cell proliferation. Evaluation of relative mRNA expression levels of PGC1 alpha, SIRT1, COX IV, COX II and ATPsyn (A), following 24 h of IGFBP6 (400 ng/mL) treatment. The calculated value of 2^-ΔΔCt in untreated controls is 1. Effect of IGFBP6 exposure (800 ng/mL) on cell proliferation (B), colony formation capacity (C) and wound healing (D). Data are expressed as mean ± SD of at least four independent experiments. **** p < 0.0001.

Figure 5

IGFBP6 induces an antioxidant response in MDA-MB-231 cells. Evaluation of relative mRNA expression levels of HMOX1 (A), GSTK1 (B) and NQ01 (C), following 24 h of IGFBP6 (800 ng/mL) treatment. The calculated value of 2^−ΔΔCt in untreated controls is 1. Data are expressed as mean ± SD of at least four independent experiments. **** p < 0.0001. Representative plots of ROS (D) and mitochondrial ROS (E) production using DCF and MitoSox probe, following 24 h of IGFBP6 exposure. Representative plot of mitochondrial membrane potential (F) evaluated with DiOC2 staining, following 24 h of IGFBP6 treatment. Plots are representative of at least four independent experiments.

Figure 6

Graphical representation of GPR81 and IGFBP6 axis.