RU486 Metabolite Inhibits CCN1/Cyr61 Secretion by MDA-MB-231-Endothelial Adhesion

Abstract

Successful adhesion of circulating tumor cells (CTCs) to microvascular endothelium of distant metastatic tissue is the key starting step of metastatic cascade that could be effectively chemoprevented as we demonstrated previously. Here, we hypothesize that the hetero-adhesion may produce secretory biomarkers that may be important for both premetastatic diagnosis and chemoprevention. We show that co-incubation of triple-negative breast cancer (TNBC) cell line MDA-MB-231 with human pulmonary microvascular endothelial monolayers (HPMEC) secretes Cyr61 (CCN1), primarily from MDA-MB-231. However, addition of metapristone (RU486 metabolite) to the co-incubation system inhibits Cyr61 secretion probably via the Cyr61/integrin αvβ1 signaling pathway without significant cytotoxicity on both MDA-MB-231 and HPMEC. Transfection of MDA-MB-231 with Cyr61-related recombinant plasmid or siRNA enhances or reduces Cyr61 expression, accordingly. The transfection significantly changes hetero-adhesion and migration of MDA-MB-231, and the changed bioactivities by overexpressed CYR61 could be antagonized by metapristone in vitro. Moreover, the circulating MDA-MB-231 develops lung metastasis in mice, which could be effectively prevented by oral metapristone without significant toxicity. The present study, for the first time, demonstrates that co-incubation of MDA-MB-231 with HPMEC secrets CYR61 probably via the CYR61/integrin α_vβ₁ signaling pathway to promote adhesion-invasion of TNBC (early metastatic step). Metapristone, by interfering the adhesion-invasion process, prevents metastasis from happening.

Keywords: CYR61; MDA-MB-231/HPMEC co-culture; integrin αvβ1; metapristone; metastasis chemoprevention.

Figure 1

Secretion of Cyr61 from MDA-MB-231 and human pulmonary microvascular endothelial cells co-culture system and inhibition by metapristone of the secretion. (A) Effect of metapristone on MDA-MB-231 viability. (B) Effect of metapristone on MCF-7 viability. (C) Effect of metapristone on HPMEC viability. (D) Flow chart showing that MDA-MB-231 and HPMEC were co-cultured in the presence and absence of metapristone for 24 h followed by trypsin digestion and iTRAQ pharmacoproteomic analysis; The Cyr61 proteomic signal was shown at its corresponding m/z. (E) Confocal microscopy images showing the morphological changes by metapristone (50 µM) in the same spots where the two cell lines were co-cultured. (F) inhibition by metapristone of Cyr61 secretion. (G) time course of Cyr61 secretion (ELISA assay) under different conditions: 1) MDA-MB-231+ HPMEC; 2) MDA-MB-231+ HPMEC+ metapristone (50 µM); 3) MDA-MB-231; 4) MDA-MB-231+ metapristone (50 µM); 5) HPMEC; 6) HPMEC+ metapristone (50 µM). The data represent the mean± SEM (n = 3). *, P < 0.05, **, P < 0.01 and ***, P < 0.001 vs. the untreated control.

Figure 2

Cyr61-related cell adhesion and migration were mediated by metapristone. MDA-MB-231 cells were transfected with pcDNA3.1, pcDNA3.1-Cyr61 recombinant plasmid, or Ram-siRNA, Cyr61-siRNA for 36–48 h. (A) Upper panel: inhibition by metapristone of MDA-MB-231 cell adhesion to human pulmonary microvascular endothelial cells; note, the adhesion between pcDNA3.1-Cyr61 cancer cells and HPMECs was significantly enhanced. Whereas, the adhesion between Cyr-61-siRNA cancer cells and HPMECs was reduced in comparison with their controls. Metapristone caused concentration-dependent inhibition of the adhesion. Middle and lower panels: changes by metapristone in cell migration rate following the scratch assay. (B) Quantitative analysis of the adhesion between Cyr61-transfected cancer cells and HPMECs. (C) The MDA-MB-231 cells were transfected and treated differently, and the migration rate following the scratch assay was quantitatively determined; The data represent mean± SEM (n = 3), *, P < 0.05; **, P < 0.01; and ***, P < 0.001 vs. the controls.

Figure 3

Formation of Cyr61/integrin αvβ1 complex in MDA-MB-231 cells. (A) Co-immunoprecipitation evidence: MDA-MB-231 cell lysates were added to aminolink-coupling resin columns that were individually coupled with anti-Cyr61 antibody (land 1 and 3), anti-integrin αv antibody (land 2), or anti-integrin β1 antibody (land 4). The bound complexes were eluted with the washing buffer and tested by western blotting with anti-integrin αv antibody (land 1), anti-Cyr61 antibody (land 2 and 4), or anti-integrin β1 antibody, individually. Samples of Lysis (before column; left), IgG, and Target (column elutes; right) after western blotting showed that the anti-Cyr61 antibody elute was integrin αv positive (land 1), integrin β1 positive (land 3), and Cyr61 positive (land 2 and 4). (B) Cyr61/integrin αvβ1 co-localization evidence: MDA-MB-231 cells were immunofluorescent-stained with Cyr61 (green), integrin αv (the upper two panels), or integrin β1 (the lower two panels; both in dark red) and DAPI (in blue for nuclear) dyes followed by confocal microscopic examination at amplification×20 (top panel) or ×40 (bottom panel). The merged images showed co-localization of Cyr61 with integrin αv and β1.

Figure 4

Inhibition by metapristone of expressions of Cyr61 and integrin αvβ1 in MDA-MB-231 cells. Western blot images (A) and the related quantitative analysis, (B) and quantitative ELISA analysis (C) of inhibition by metapristone of expressions of Cyr61 and integrin αvβ1 in MDA-MB-231 cells. (D) mRNA expression of Cyr61, integrin αv and β1 was inhibited by metapristone in a concentration-dependent manner. The data are expressed as the mean± SEM (n = 3). *, P < 0.05; **, P < 0.01, and ***, P < 0.001 vs. the untreated controls.

Figure 5

Metapristone inhibits lung metastasis of MDA-MB-231 cells via decreasing levels of Cyr61 and integrin αvβ1. (A) photography of mouse lung metastasis after six weeks of MDA-MB-231 inoculation via tail vein injection. The mice were pretreated with oral metapristone for three days before the inoculation followed by 6-week oral administration of metapristone. Control, drug vehicle; (B) quantitative comparison in mouse lung tumor nodules between the control and metapristone groups (n = 5/group); **P < 0.01, ***P < 0.001. (C) inhibition rate of mouse lung tumor metastasis after metapristone treatment; (D) hematoxylin–eosin staining of the lungs (amplification× 5); the arrows indicate metastatic foci that were significantly reduced by metapristone; (E) lung immunostaining with antibodies against Cyr61, integrin αv and integrin β1; the staining showed reduction in Cyr61/integrin avβ1 formation by metapristone.

Figure 6

(A) possible mechanism of Cyr61 secretion and its inhibition by metapristone. Activated CTCs adhere to endothelial cells in the metastatic microenvironment, and the adhesion induces hetero-cellular communication and the resultant secretion of Cyr61 from CTCs, which forms Cyr61/integrin avβ1 complex to advance the adhesion/invasion metastasis. Metapristone inhibits the Cyr61 secretion, and the related adhesion/invasion process; (B) quantitative comparison in mouse lung tumor nodules between the control and metapristone groups (n = 5/group); (C) inhibition rate of mouse lung tumor metastasis after metapristone treatment; (D) hematoxylin–eosin staining of the lungs (amplification× 5); the arrows indicate metastatic foci that were significantly reduced by metapristone; (E) lung immunostaining with antibodies against Cyr61, integrin αv and integrin β1; the staining showed reduction in Cyr61/integrin avβ1 formation by metapristone.