Cyclooxygenase-2 in tumor-associated macrophages promotes metastatic potential of breast cancer cells through Akt pathway

Abstract

Tumor-associated macrophages (TAMs) promote cancer development and progression by releasing various cytokines and chemokines. Previously, we have found that the number of COX-2⁺ TAMs was associated with lymph node metastasis in breast cancer. However, the mechanism remains enigmatic. In this study, we show that COX-2 in breast TAMs enhances the metastatic potential of breast cancer cells. COX-2 in TAMs induces MMP-9 expression and promotes epithelial-mesenchymal transition (EMT) in breast cancer cells. In addition, COX-2/PGE₂ induces IL-6 release in macrophages. Furthermore, we find that the activation of Akt pathway in cancer cells is crucial for the pro-metastatic effect of COX-2⁺ TAMs by regulating MMP-9 and EMT. These findings indicate that TAMs facilitate breast cancer cell metastasis through COX-2-mediated intercellular communication.

Keywords: Tumor microenvironment; breast cancer; cyclooxygenase-2.; macrophages.

Figure 1

COX-2 in TAMs promotes migration and invasion of breast cancer cells. (A) The expression of COX-2 in TAMs transfected with adenoviral COX-2 or siRNA COX-2 was checked by western blot. β-actin was used as an internal loading control. The blots shown are representative of six independent experiments. (B) For the wound healing assay, confluent monolayers of MCF-7 and MDA-MB-468 cells co-cultured with or without (Alone) TAMs transfected with adenoviral COX-2 or siRNA COX-2 for 7 days were scarred. The migration of the cells into the wound zone was monitored by microscopy over a 12-h period. The upper panel shows the wound healing assay in MCF-7 cells. White lines indicate the scraped zone. (C) and (D) The transwell invasion assay. After MCF-7 and MDA-MB-468 cells were co-cultured with or without (Alone) TAMs (C) or MDMs (D) transfected with adenoviral COX-2 or siRNA COX-2 for 7 days, the cells that migrated to the lower chamber or invaded through the Matrigel were fixed, stained, and counted using a light microscope. Four random fields per filter were scanned for the presence of cells on the lower side of the membrane. The upper panel shows the cell invasion assay in MCF-7 cells (original magnification, ×100). All experiments were performed thrice in triplicate. The data are presented as the mean ± SD. *p < 0.05 and **p < 0.01 (versus Alone group).

Figure 2

COX-2 in TAMs induces MMP-9 expression in breast cancer cells. (A) Template showing the location of the MMP/TIMP antibodies spotted onto the RayBio Human MMP Array Kit. POS: positive; NEG: negative. (B) TAMs-induced modulation of MMP/TIMP proteins in MCF-7 cells. After MCF-7 cells were co-cultured with or without TAMs for 7 days, the cell lysate was applied to the antibody array. The pixel density was measured, and the data are presented as ratios (compared to the Alone). Protein exhibiting a ratio>2 is indicated with black box. (C) MMP-9 expression in MCF-7 and MDA-MB-468 cells co-cultured with or without (Alone) TAMs for 7 days was assessed by Western blot. β-actin was used as an internal loading control. The blots shown are representative of six independent experiments. (D) MMP-9 expression in MCF-7 and MDA-MB-468 cells co-cultured with or without (Alone) TAMs transfected with adenoviral COX-2 or siRNA COX-2 for 7 days was assessed by Western blot. β-actin was used as an internal loading control. The blots shown are representative of six independent experiments. (E) MMP-9 activity in supernatant collected from different cancer cells was detected using an active human MMP-9 fluorescence assay. The experiments were performed thrice in triplicate. The data are presented as the mean ± SD. **p < 0.01 (versus Alone group).

Figure 3

COX-2 in TAMs enhances TAMs-induced EMT in breast cancer cells. (A) EMT markers in MCF-7 and MDA-MB-468 cells were assessed by Western blot. β-actin was used as an internal loading control. The blots shown are representative of six independent experiments. (B) Immunostaining showed the downregulation of E-cadherin and upregulation of Vimentin in MCF-7 and MDA-MB-468 cells (original magnification: 400×). The nucleus is stained with DAPI (blue), E-cadherin is stained with DyLight 549 (red), and Vimentin is stained with DyLight 488 (green). The experiments were performed thrice in triplicate. The data are presented as the mean ± SD. **p<0.01 (versus Alone group). (C) EMT markers in MCF-7 and MDA-MB-468 cells co-cultured with or without (Alone) TAMs for 7 days were assessed by Western blot. β-actin was used as an internal loading control. The blots shown are representative of six independent experiments. (D) Correlation of COX-2⁺ TAMs and Vimentin in breast cancer tissues (n = 160) was analyzed by Pearson's correlation analysis.

Figure 4

COX-2 induces PGE₂ and IL-6 release in macrophages and regulates MMP-9 and EMT by activating Akt pathway in breast cancer cells. (A) The level of PGE₂ in MDMs or TAMs transfected with adenoviral COX-2 or siRNA COX-2 was detected by EIA. The experiments were performed thrice in triplicate. The data are presented as the mean ± SD. **p < 0.01. (B) The level of IL-6 in MDMs or TAMs transfected with adenoviral COX-2 or siRNA COX-2, or treated with PGE₂ (1 μM) was detected by ELISA. The experiments were performed thrice in triplicate. The data are presented as the mean ± SD. **p < 0.01. (C) Inhibiting Akt1 in breast cancer cells attenuated cell invasion induced by COX-2 in TAMs. Breast cancer cells transfected with adenoviral siRNA Akt1 or Akt1 were co-cultured with or without (Alone) TAMs transfected with adenoviral COX-2 or siRNA COX-2. The transwell invasion assay was then carried out as described before. (D) Inhibiting Akt1 resulted in the suppression of MMP-9 and snail in breast cancer cells co-cultured with COX-2⁺ TAMs. The expression of related proteins in breast cancer cells was detected by Western blot. β-actin was used as an internal loading control, and the blots shown are representative of six independent experiments.

Figure 5

COX-2 in TAMs regulates p-Akt, MMP-9 and EMT in vivo. The animal model was constructed by injecting 4T1 cells alone or with RAW264.7-derived TAMs transfected with adenoviral COX-2 or siRNA COX-2 into the mammary fat pads of NOD/SCID mice. p-Akt, MMP-9, E-cadherin and Vimentin in the tumor tissues were evaluated by IHC (original magnification, ×400). The number of stained and unstained cells was counted to generate the percentage of positive cells in each group. All the data were presented as the mean ± SD, n = 10, *p < 0.05 and **p < 0.01.

Figure 6

Proposed mechanism showing COX-2⁺ TAMs-mediated cell metastasis in breast cancer.