LincRNA-p21 knockdown reversed tumor-associated macrophages function by promoting MDM2 to antagonize* p53 activation and alleviate breast cancer development

Abstract

Tumor-associated macrophages (TAMs) are important regulators of the complex interplay between immune system and breast cancer. TAMs fuel the cancer progression and metastasis by reprogramming their specific functional phenotype in cancer settings. Therefore, it is important to clarify the mechanisms of shaping specific functional phenotype of macrophages in tumor milieu. LncRNA profiles of TAMs were identified by LncRNA microarray. Flow cytometry was used to detect the surface markers of TAMs. The co-localization among lincRNA-p21, p53 and Mouse Double Minute 2 (MDM2) was identified by FISH probe and immunofluorescence. PyVT-MMTV and BALB/c mice were used for in vivo analysis. In the present work, we found that lincRNA-p21 significantly up-regulated in 4T1 educated macrophages. LincRNA-p21 knockdown facilitated macrophage polarization into pro-inflammatory M1 in tumor microenvironment, which might be caused by MDM2 eliciting proteasome-dependent degradation to p53 and activated NF-κB and STAT3 pathway. TAMs with lincRNA-p21 knockdown induced cancer cell apoptosis, inhibited tumor cell migration and invasion. In vivo, lincRNA-p21 knockdown macrophage adoptive transfer could alleviate breast cancer progression. Our results indicated that lincRNA-p21 was a key regulator of TAMs function in tumor milieu. Our data also shed a light on novel therapeutic targets of tumors characterized by monocytes/macrophages infiltration.

Keywords: Breast cancer; TAMs; Tumor microenvironment; lincRNA-p21; p53.

Fig. 1

LincRNA-p21 up-regulated in TAMs. a TAMs infiltrated in tumor tissue. Representative immunofluorescence microscopy images were shown. Red, CD11b; Green, F4/80; Blue, nuclei. 5 MMTV-PyVT mice were included. b Heat map of lncRNAs profiles in TAMs. Macrophages were treated by 4T1 cultured supernatant (4T1CM) for 48 h compared with control macrophages. Red and blue mean up-regulation and down-regulation, respectively. c–e GO analysis was performed to describe their functions: c biological processes, d cellular components and e molecular functions. f KEGG database was used to analyze pathway, and the significance of differentially expressed genes’ enrichment in each pathway entry. g The expressions of lincRNA-p21, lincRNA-cox2 and lncRNA-A930001c03Rik were detected by RT-qPCR. Macrophages were treated by 4T1CM for 48 h and then collected using for RT-qPCR analysis. H LincRNA-p21 expression increased in LLC supernatant-treated macrophages. Data were obtained from three independent experiments and the representative images shown. *P < 0.05. LLC mean Lewis lung carcinoma cells

Fig. 2

LincRNA-p21 knockdown reversed the functional phenotype of TAMs in tumor milieu. a The transfection efficiency reached 96% (left) and the relative expression of lincRNA-p21 (right). Fluorescent labeled siRNAs were designed to down-regulate lincRNA-p21 in macrophages. The transfection efficiency was observed under fluorescence microscope after 6 h, lincRNA-p21 expression was detected by RT-qPCR after 24 h. Data are presented as mean ± SEM. b CD86 and MHC II up-regulated; conversely, CD206 down-regulated following lincRNA-p21 knockdown in TAMs. The upper panels showed representative images and lower panels showed statistical analysis. c LincRNA-p21 knockdown promoted pro-inflammatory cytokines’ secretion of TAMs. Secretion of IL-4, IL-6, IL-10, IL-12 and TNF-α was assessed by ELISA in the supernatant. d The levels of iNOS up-regulated; however, Arg-1 down-regulated following lincRNA-p21 knockdown in TAMs. Western blotting analysis was employed to assess the levels of iNOS and Arg-1. β-actin was used as a loading control. The left panels show representative blots and right panels show statistical analysis. e LincRNA-p21 knockdown could not increase phagocytosis of TAMs. LincRNA-p21 was knockdown in TAMs, then Escherichia coli (E. coli) was added. After 1 h, the bacteria were sterilized, TAMs were lysed. The number of bacteria phagocytized by TAMs was counted. The left panels show representative images and right panels show statistical analysis. Data are presented as mean ± SEM. All the data were obtained from three independent experiments and the representative images are shown. *P < 0.05 relative to control. N.S. means no significance. SLP1 means lincRNA-p21 downregulated TAMs

Fig. 3

LincRNA-p21 down-regulated TAMs promoted tumor cells apoptosis, inhibited their migration and invasion. a, e LincRNA-p21 down-regulated TAMs promoted tumor cell apoptosis. 4T1 and LLC were co-cultured with lincRNA-p21 down-regulated macrophages for 48 h, respectively. The tumor cells were collected and stained with Annexin-V and 7AAD to detect the apoptosis using flow cytometry, respectively. b LincRNA-p21 down-regulated TAMs inhibited 4T1 cell proliferation. The proliferation of 4T1 cells co-cultured with lincRNA-p21 down-regulated macrophages was measured using Cell Counting Kit-8 (CCK-8) at indicated point. c, f LincRNA-p21 down-regulated TAMs inhibited tumor cells migration. Transwell assays were performed to detect migration ability of tumor cells (4T1 and LLC) co-cultured with lincRNA-p21 down-regulated TAMs for 24 h. The left panels show representative images and right panels show statistical analysis. d LincRNA-p21 down-regulated TAMs inhibited 4T1 cells invasion. Wound-healing assay was used to detect the invasive ability of 4T1 cells co-cultured with lincRNA-p21 down-regulated TAMs. g LincRNA-p21 knockdown could not increase ROS production in TAMs. The left panels show representative images and right panels show statistical analysis. h The expression of Fas on 4T1 cell and FasL on TAMs was assessed by flow cytometry. i R7050 (10 nM) was used to pretreat 4T1 for 6 h and after that 4T1 was co-cultured with lincRNA-p21 down-regulated macrophages for 48 h; 4T1 cells were collected and stained with Annexin V and PI to detect apoptosis. All the data were obtained from three independent experiments and the representative images are shown. *P<0.05 relative to control. N.S. means no significance. LLC and SLP1 mean Lewis lung carcinoma cells and lincRNA-p21 downregulated macrophages

Fig. 4

LincRNA-p21 knockdown inducing functional reversion of TAMs may be caused by promoting MDM2 to antagonize p53 function. a LincRNA-p21 knockdown increased Bcl-2 and decreased Bax expression in TAMs. The left panels show representative blots and right panels show statistical analysis. b LincRNA-p21 and p53 are co-localized in cytoplasm; conversely, MDM2 localized in the nucleus membrane. LincRNA-p21 was labeled with FISH probe and hybridized in TAMs; MDM2 and p53 expressions were detected by immunofluorescence. The nuclei were stained with DAPI. c LincRNA-p21 knockdown activated STAT3 and p65 in TAMs. Macrophages with/without lincRNA-p21 knockdown were treated by 4T1CM and cells were collected at indicated points for using western blotting analysis. The upper panels show representative blots and lower panels show statistical analysis. All data were obtained from three independent experiments and the representative images are shown. SLP1 means lincRNA-p21 downregulated macrophages

Fig. 5

LincRNA-p21 knockdown macrophages’ adoptive transfer could alleviate breast cancer development. 1 × 10⁶ 4T1 cells and 1 × 10⁵ primary macrophages with/without lincRNA-p21 knockdown were subcutaneously (s.c.) inoculated into the backside of 10 BALB/c mice to generate tumors. After tumor inoculation, 1 × 10⁵ macrophages/week with/without lincRNA-p21 knockdown were injected into the solid tumor until the mice were killed. a LincRNA-p21 expression was increased in macrophages from tumors by RT-qPCR. Every dot represents one mouse. b LincRNA-p21 down-regulated macrophages decreased the tumor volumes. c LincRNA-p21 down-regulated macrophages decreased the tumor weights. Data are presented as mean ± SEM. *P<0.05 relative to control

Fig. 6

Schematic of LincRNA-p21 knockdown reverses TAMs function and alleviates breast cancer development