The AGEs/RAGE Transduction Signaling Prompts IL-8/CXCR1/2-Mediated Interaction between Cancer-Associated Fibroblasts (CAFs) and Breast Cancer Cells

Abstract

Advanced glycation end products (AGEs) and the cognate receptor, named RAGE, are involved in metabolic disorders characterized by hyperglycemia, type 2 diabetes mellitus (T2DM) and obesity. Moreover, the AGEs/RAGE transduction pathway prompts a dysfunctional interaction between breast cancer cells and tumor stroma toward the acquisition of malignant features. However, the action of the AGEs/RAGE axis in the main players of the tumor microenvironment, named breast cancer-associated fibroblasts (CAFs), remains to be fully explored. In the present study, by chemokine array, we first assessed that interleukin-8 (IL-8) is the most up-regulated pro-inflammatory chemokine upon AGEs/RAGE activation in primary CAFs, obtained from breast tumors. Thereafter, we ascertained that the AGEs/RAGE signaling promotes a network cascade in CAFs, leading to the c-Fos-dependent regulation of IL-8. Next, using a conditioned medium from AGEs-exposed CAFs, we determined that IL-8/CXCR1/2 paracrine activation induces the acquisition of migratory and invasive features in MDA-MB-231 breast cancer cells. Altogether, our data provide new insights on the involvement of IL-8 in the AGEs/RAGE transduction pathway among the intricate connections linking breast cancer cells to the surrounding stroma. Hence, our findings may pave the way for further investigations to define the role of IL-8 as useful target for the better management of breast cancer patients exhibiting metabolic disorders.

Keywords: AGEs; IL-8; RAGE; breast cancer; cancer-associated fibroblasts.

Figure 1

AGEs trigger rapid responses via RAGE in CAFs. (a) Phosphorylation of ERK1/2 and AKT in CAFs exposed to vehicle (–) and 100 µg/mL AGEs for the indicated times; (b) Phosphorylation of ERK1/2 and AKT in CAFs treated for 30 min with vehicle (–) or 100 µg/mL AGEs alone and in the presence of 1 μM RAGE inhibitor FPS-ZM1; (c) Immunoblots of ERK1/2 and AKT in CAFs transfected with scramble siRNA or siRAGE (10 nM) for 24 h and then treated for 30 min with vehicle (–) and 100 µg/mL AGEs; (d,e) ROS generation in CAFs exposed to vehicle and 100 µg/mL AGEs alone or in the presence of 1 μM RAGE inhibitor FPS-ZM1 and 300 µM free radical scavenger N-acetyl-Lcysteine (NAC), as indicated. The values of fluorescent probe DCF-DA obtained in CAFs treated with vehicle was set as one-fold induction upon which ROS levels induced by AGEs were calculated. Values represent the mean ± SD of three independent experiments performed in triplicate; (f) The activation of ERK1/2 and AKT in CAFs upon 100 µg/mL AGEs exposure for 30 min was abolished using 300 µM free radical scavenger NAC. ERK2 and AKT were used as loading control, as indicated. Side panels show densitometric analysis of the blots normalized to the loading controls. Values represent the mean ± SD of three independent experiments. (*) indicates p < 0.05.

Figure 2

AGEs/RAGE activation upregulates IL-8 expression in CAFs. (a) CAFs were exposed for 8 h to 100 µg/mL AGEs alone and in the presence of 1 μM RAGE inhibitor FPS-ZM1. Gene expression changes of chemokine and related genes were evaluated by TaqMan™ Human Chemokine Array. Values were normalized to 18 S expression; the colors indicate the log2 fold changes of gene expression upon the indicated conditions in relation to the vehicle-treated CAFs. mRNA (b,e) and protein (c,f) expression of IL-8 evaluated, respectively, by real-time PCR and immunoblotting in CAFs treated for 8 h with vehicle (–) or 100 µg/mL AGEs alone and in the presence of 1 μM RAGE inhibitor FPS-ZM1 or in combination with 300 µM free radical scavenger NAC. In RNA experiments, values were normalized to the beta-actin (ACTB) expression and shown as fold changes of IL-8 mRNA expression upon AGEs treatment compared to cells exposed to vehicle; (d) Immunoblots showing IL-8 protein expression in CAFs transfected with scramble siRNA or siRAGE (10 nM) for 24 h and then exposed for 8 h to vehicle (–) or 100 µg/mL AGEs; (g) IL-8 protein expression evaluated by immunoblotting in CAFs treated for 8 h with vehicle (–) or 100 µg/mL AGEs alone and in combination with 100 nM MEK inhibitor trametinib or 1 µM PI3K inhibitor alpelisib. β-actin served as a loading control; (h,j) Evaluation by immunoblotting of IL-8 protein levels in conditioned medium (CM) collected from CAFs treated for 18 h with vehicle (–) or 100 µg/mL AGEs alone and in the presence of 1 μM RAGE inhibitor FPS-ZM1 or in combination with 300 µM free radical scavenger NAC; (i) Immunoblots showing IL-8 protein levels in CM derived from CAFs transfected with scramble siRNA or siRAGE (10 nM) for 24 h and then exposed for 18 h to vehicle (–) or 100 µg/mL AGEs. Ponceau red staining of the membrane was used as a loading control for the CM. Side panels show densitometric analysis of the blots normalized to the loading controls. Values represent the mean ± SD of three independent experiments. (*) indicates p < 0.05.

Figure 3

c-Fos is involved in the upregulation of IL-8 induced by AGEs/RAGE signaling in CAFs. (a) Luciferase activities of IL-8 promoter construct in CAFs treated for 18 h with vehicle or 100 µg/mL AGEs in the presence or absence of 1 μM RAGE inhibitor FPS-ZM1, 300 µM free radical scavenger NAC, 100 nM MEK inhibitor trametinib or 1 µM PI3K inhibitor alpelisib, as indicated; (b) Luciferase activities of c-Fos promoter construct in CAFs upon exposure for 18 h to vehicle or 100 µg/mL AGEs alone and in combination with 1 μM RAGE inhibitor FPS-ZM1, 300 µM free radical scavenger NAC, 100 nM MEK inhibitor trametinib or 1 µM PI3K inhibitor alpelisib, as indicated. The luciferase activities were normalized to the internal transfection control, and values of cells receiving vehicle were set as 1-fold induction upon which the activity induced by AGEs was calculated. Each column represents the mean ± SD of three independent experiments performed in triplicate. mRNA (c,f) and protein (d,g) expression of c-Fos evaluated, respectively, by real-time PCR and immunoblotting in CAFs treated for 4 h with vehicle (–) or 100 µg/mL AGEs alone and in the presence of 1 μM RAGE inhibitor FPS-ZM1 or in combination with 300 µM free radical scavenger NAC. In RNA experiments, values were normalized to the beta-actin (ACTB) expression and shown as fold changes of c-Fos mRNA expression upon AGEs treatment compared to cells exposed to vehicle; (e) Immunoblots showing c-Fos protein expression in CAFs transfected with non-targeting scramble siRNA or siRAGE (10 nM) for 24 h and then exposed for 4 h with vehicle (–) or 100 µg/mL AGEs; (h) c-Fos protein expression evaluated by immunoblotting in CAFs treated for 4 h with vehicle (–) or 100 µg/mL AGEs alone and in combination with 100 nM MEK inhibitor trametinib or 1 µM PI3K inhibitor alpelisib; (i,j) Recruitment of c-Fos to the AP-1 site located within the IL-8 promoter region upon treatment for 4 h with AGEs in CAFs, as assessed by Chromatin Immunoprecipitation (ChIP) assays. Data obtained were normalized to the input and shown as fold changes in relation to nonspecific Immunoglobulin G (IgG). Each column represents the mean ± SD of three independent experiments performed in triplicate; (k) Luciferase activities of IL-8 promoter construct in CAFs transfected for 18 h with an empty vector or a plasmid encoding for a dominant negative form of c-Fos (DN/c-Fos) and then exposed for 18 h to vehicle or 100 µg/mL AGEs. The luciferase activities were normalized to the internal transfection control and values of cells receiving vehicle were set as 1-fold induction upon which the activity induced by treatment was calculated. Each column represents the mean ± SD of three independent experiments performed in triplicate; (l) IL-8 protein expression evaluated by immunoblotting in CAFs transfected with the empty vector or with the DN/c-Fos construct for 18 h and then treated with vehicle (–) or 100 µg/mL AGEs for 8 h. β-actin served as a loading control; (m) Immunoblotting of IL-8 in conditioned medium (CM) collected from CAFs transfected with a vector or with the DN/c-Fos construct and then treated for 8 h with vehicle (–) or 100 µg/mL AGEs. Ponceau red staining of the membrane was used as a loading control for the CM. Side panels show densitometric analysis of the blots normalized to the loading controls. Data shown represent the mean ± SD of three independent experiments. (*) indicates p < 0.05.

Figure 4

IL-8 mediates the acquisition of a spindle-like morphology in MDA-MB-231 cells triggered by conditioned medium (CM) from AGEs-stimulated CAFs. (a) CXCR1/2 mRNA levels according to breast cancer intrinsic molecular subtypes of the integrated Affymetrix cohort; (b) CXCR1/2 expression is associated with a worse relapse-free survival (RFS) of basal breast cancer patients in the Affymetrix dataset. The patients were divided into high and low CXCR1/2 expression levels on the basis of the established cut-point’ (c) MDA-MB-231 cells were incubated for 6 h with CM collected from CAFs previously treated with vehicle or 100 µg/mL AGEs in the presence or absence of 1 μM RAGE inhibitor FPS-ZM1’ (d) MDA-MB-231 cells were cultured for 6 h in CM derived from CAFs previously treated with vehicle or 100 µg/mL AGEs with or without 300 ng/mL IL-8 neutralizing-antibody (Ab IL-8) as well as (e) using 5 μM CXCR1/2 inhibitor reparixin. The spindle-like morphology was quantified as Polarity Index (PI). PI = 1.0 indicates a polygonal shape, conversely a value > 1.0 identified ranges of migratory shapes. Images shown are representative of 10 random fields acquired in three independent experiments. Scale bar = 100 µm. (*) indicates p < 0.05.

Figure 5

The paracrine action of IL-8 promotes the formation of actin stress fibers in MDA-MB-231 cells. (a) MDA-MB-231 cells, which were treated for 6 h with conditioned medium (CM) from CAFs exposed to vehicle or 100 µg/mL AGEs alone and in the presence of 1 μM RAGE inhibitor FPS-ZM1, were stained with FITC-conjugated phalloidin to visualize F-actin stress fibers (green) and DAPI to detect nuclei (blue). The F-actin stress fibers formation in MDA-MB-231 cells promoted by CM collected from CAFs previously treated with 100 µg/mL AGEs, was abrogated using 300 ng/mL IL-8 neutralizing-antibody (Ab IL-8) (b) or 5 μM CXCR1/2 inhibitor reparixin (c). Fluorescence intensities of the number of fibers/cell was quantified by F-actin staining in 10 random fields for each condition; results are expressed as fold change of relative fluorescence units (RFU). Data shown represent the mean ± SD of three independent experiments performed in triplicate. (*) indicates p< 0.05. Enlarged details are shown in the separate boxes. Scale bar 100 μM.

Figure 6

The paracrine activation of IL-8/CXCR1/2 axis promotes cell migration and invasion of MDA-MB-231 cells. Transwell assays were performed to evaluate cell migration (a) and invasion (b) in MDA-MB-231 cells cultured for 6 h in conditioned medium (CM) from CAFs previously treated with vehicle or 100 µg/mL AGEs alone and in combination with 1 μM RAGE inhibitor FPS-ZM1. Cell migration (c) and invasion (d) were assessed in MDA-MB-231 cells cultured for 6 h in conditioned medium (CM) from CAFs previously treated with vehicle or 100 µg/mL AGEs alone and in combination with 300 ng/mL IL-8 neutralizing-antibody (Ab IL-8). The migration (e) and invasion (f) of MDA-MB-231 cells observed upon exposure to CM from CAFs previously treated with 100 µg/mL AGEs were abolished using 5 μM CXCR1/2 inhibitor reparixin. Scale bar = 200 µm. Side panels show the mean ± SD of the number of cells counted in at least 10 random fields of three independent experiments performed in triplicate. (*) indicates p < 0.05.