Tumor-associated macrophages secrete CC-chemokine ligand 2 and induce tamoxifen resistance by activating PI3K/Akt/mTOR in breast cancer

Li, Dongbo; Ji, Hongfei; Niu, Xingjian; Yin, Lei; Wang, Yiran; Gu, Yucui; Wang, Jinlu; Zhou, Xiaoping; Zhang, Han; Zhang, Qingyuan

As a well-known chemokine abundantly present in macrophages, CCL2 is responsible for the recruitment of monocytes. [37] [38] [39] [40] [41] [42] 46 The concentration of CCL2 is positively correlated with its chemotaxispromoting potential. We have already determined that the levels of CCL2 secreted by MΦ, MS, and MR are different. The next question is whether the chemotaxis of THP-1 cells induced by these three F I G U R E 2 CC-chemokine ligand 2 (CCL2) secreted by tumor-associated macrophages (TAM) activates the PI3K/Akt/mTOR pathway to induce endocrine resistance in breast cancer cells. A, RT-PCR shows the transcription of seven cytokines in three macrophages, MΦ, TAM from a tamoxifen-sensitive tumor microenvironment (TME) (MS), and TAM from tamoxifen-resistant TME (MR). Only the change trend of CCL2 is consistent with the previous experiment results, and the difference was statistically significant. B, CCL2 levels in the conditioned medium (CM) of tamoxifen-sensitive MCF7 breast cancer cells (MCF7-S), tamoxifen-resistant MCF7 breast cancer cells (MCF7-R), MΦ, MS, and MR cells. CCL2 was mainly produced by macrophages rather than breast cancer cells. C, Relative viability of MCF7 and MCF7 + CCL2 (100 nm/mL) cells treated with 5 μmol/L tamoxifen. D, Relative viability of T47D and T47D + CCL2 (100 nm/mL) cells treated with 5 μmol/L tamoxifen. E, Apoptosis of MCF7 and MCF7 + CCL2 (100 nm/mL) cells was analyzed by flow cytometry at 24 h after adding 5 μmol/L tamoxifen. F, Apoptosis of T47D and T47D + CCL2 (100 nm/mL) cells was analyzed by flow cytometry at 24 h after adding 5 μmol/L tamoxifen. G, Western blot analysis of the PI3K/Akt/mTOR pathway. CCL2 and CM of MR could activate the PI3K/AKT/mTOR pathway in MCF7 cells. When MR was treated with Bindarit for 24 h, activation of the PI3K/AKT/mTOR pathway by CM decreased. *P < .05, **P < .01, ***P < .001 F I G U R E 3 Tamoxifen-sensitive MCF7 breast cancer cells (MCF7-S) and tamoxifen-resistant MCF7 breast cancer cells (MCF7-R) secrete tumor necrosis factor (TNF)α and induce activation of the nuclear factor kappa B (NF-κB) and mTORC1-FOXK1 pathways in tumor-associated macrophages (TAM). A, TNFα levels in conditioned medium (CM) from MCF7-S, MCF7-R, MΦ, TAM from a tamoxifen-sensitive tumor microenvironment (TME) (MS), and TAM from tamoxifen-resistant TME (MR) cells. Level of TNFα in the CM of TAM was higher, but the difference between MS and MR was not significant (NS). B, Western blot analysis of the NF-κB pathway. CM of MCF7-S and MCF7-R could activate the NF-κB pathway of TAM. C, Western blot analysis of the mTORC1-FOXK1 pathway. CM of MCF7-S and MCF7-R could activate the mTORC1-FOXK1 pathway of TAM. Dephosphorylation of MR was more obvious than that of MS. Therefore, CM of MCF7-R can promote activation of the mTORC1-FOXK1 pathway more than MCF7-S. *P < .05, **P < .01 macrophages coincides with the secretion of CCL2. Results of the chemotaxis assay confirmed that the chemotaxis-inducing effect of TAM toward THP-1 cells was stronger than that of macrophages, the chemotaxis-inducing effect of MR was stronger than that of MS, and Bindarit the inhibitor of CCL2 reverses the enhanced chemotaxis ability of MR ( Figure 4A ). To further validate this phenomenon in vitro, we analyzed tissue samples from 100 patients with ER-positive breast cancer. Immunohistochemistry of serial pathological sections showed that a high expression of CCL2 in the paraneoplastic stroma was correlated with infiltration of