Macrophage binding to receptor VCAM-1 transmits survival signals in breast cancer cells that invade the lungs

Abstract

Aberrant expression of vascular cell adhesion molecule-1 (VCAM-1) in breast cancer cells is associated with lung relapse, but the role of VCAM-1 as a mediator of metastasis has remained unknown. We report that VCAM-1 provides a survival advantage to breast cancer cells that infiltrate leukocyte-rich microenvironments such as the lungs. VCAM-1 tethers metastasis-associated macrophages to cancer cells via counter-receptor α4-integrins. Clustering of cell surface VCAM-1, acting through Ezrin, triggers Akt activation and protects cancer cells from proapoptotic cytokines such as TRAIL. This prosurvival function of VCAM-1 can be blocked by antibodies against α4-integrins. Thus, newly disseminated cancer cells expressing VCAM-1 can thrive in leukocyte-rich microenvironments through juxtacrine activation of a VCAM-1-Ezrin-PI3K/Akt survival pathway.

Figure 1. VCAM-1 in cancer cells enhances lung metastasis

(A-E) Role of cancer cell VCAM-1 in lung metastasis by mammary tumors. (A) Schematic of the experimental procedure. (B) VCAM-1 protein levels were detected western immunoblotting in control MDA231-LM2 cells (Ctrl) or these cells expressing independent shRNAs targeting human VCAM1 (sh1 and sh2). (C) The indicated cancer cells were injected in the fourth mammary fat pad of mice. Mammary tumor size was measured at the indicated times. (D) Quantification of lung metastasis at week 5 based on bioluminescent signal. (E) Lung sections (week 5) were immunostained with human vimentin. Data are average ± SEM; n = 5 mice. Scale bar, 100 μm. (F-J) Role of cancer cell VCAM-1 in lung colonization from the circulation. (F) Schematic of lung colonization assay by intravenous injection of cancer cells. (G) Indicated MDA231-LM2 cells were intravenously injected in NOD/SCID mice. Lung colonization was quantified by bioluminescence imaging (BLI). (H) Representative images of human vimentin staining of lung sections 6 weeks after MDA-231 cell injection. (I) VCAM-1 protein level as detected by western immunoblotting in control 4T1 cells (Ctrl) or these cells expressing independent shRNAs targeting mouse VCAM1 (sh1 and sh2). 4T1 cells were intravenously injected in BALB/c mice and lung colonization was quantified by BLI 3 weeks after inoculation. Data are average ± SEM; n = 5 mice. Scale bar, 500 μm. See also Figure S1.

Figure 2. VCAM-1 on cancer cells binds tumor-associated macrophages

(A) Schematic illustration of VCAM-1 interactions with leukocyte α4 integrins. (B,C) Adhesion assays of U937 cells on monolayers of parental MDA231 or MDA231-LM2 cells. (B) Representative photomicrographs of adherent TRITC-labeled U937 cells on the monolayer of indicated cancer cells. Scale bar, 100 μm. (C) quantification of the ratio of adherent U937 cells and cancer cells. Data are average ± SEM; n = 10 unit areas. (D) U937 cells treated with anti-α4β1 integrin antibody (anti-α4) or Ig control (-) were tested for binding to monolayers of control or VCAM1-depleted MDA231-LM2. VCAM-1 knockdown was done using two independent shRNAs (sh1, sh2). Data are average ± SEM; n = 10 unit areas. (E) Recombinant VCAM-1 (rVCAM-1) binging assay with different stromal cell populations from lung metastatic nodules, including F4/80⁺ macrophages (Macro), Gr-1⁺ neutrophils (Neutro) and CD31⁺ endothelial cells (Endoth) and GFP⁺ cancer cells. Cell surface binding of bovine serum albumin control (grey closed histogram) or rVCAM-1 (black open histogram) was determined by flow cytometry. (F) Adhesion assays of stromal cells from lung metastatic nodules on monolayers of control or VCAM1-depleted (sh1, sh2) MDA231-LM2 cells. The ratio between adherent F4/80⁺ macrophages to GFP⁺ cancer cells was quantified by flow cytometry. See also Figure S2.

Figure 3. Cancer cell VCAM-1 is dispensable for transendothelial migration or leukocyte recruitment but enhances tumor seeding

(A) MDA231-LM2 cell migration across monolayers of human pulmonary microvascular endothelial cells, with or without activated monocytes in the lower chamber of a transwell. Migrated cancer cells were quantified. Data are average ± SEM; n = 10 unit areas. (B-E) Association of cancer cells with leukocytes in the lungs. (B) Schematic of the experimental procedure. (C) CMFDA-labeled 4T1 cells were intravenously inoculated into BALB/c mice and the association between cancer cells and CD4⁺ and CD8⁺ T cells and CD68⁺ macrophages was analyzed 24 h later. Shown are two representative micrographs per experimental group. Scale bar, 10 μm. (D) Percentage of cancer cells interacting with the indicated leukocytes cells. (E) Quantification of CD68⁺ cells surrounding cancer cells. Data are average ± SEM; n >30 cancer cells. (F-H) Tumor self-seeding assay. (F) Schematic of experimental procedure. GFP/luciferase expressing cancer cells of inoculation of into the arterial circulation of mice bearing primary tumors formed by unlabeled cancer cells. (G) Representative images of mammary tumors seeded with indicated GFP⁺ MDA-231 LM2 cells. GFP⁺ cancer cells and tumor-associated macrophages were stained with anti-GFP and anti-CD68 antibodies in primary tumors (week 4). Scale bar, 10 μm. (H) Seeding of control (Ctrl) or VCAM-1 depleted (sh1 and sh2) MDA231-LM2 cells in mammary tumors after intracardiac inoculation was determined by BLI quantification. Data are average ± SEM; n = 10 mice. See also Figure S3.

Figure 4. VCAM-1 protects cancer cells from apoptosis in the lung microenvironment

(A, B) MDA231-LM2 cells with a doxycycline inducible shVCAM-1 vector were cultured in the presence of doxycycline (A) or were placed in doxycycline-free medium after culturing with doxycycline (B), in order to determine the kinetics of VCAM1 depletion and recovery, respectively. (C) Control or doxycycline-driven VCAM1 shRNA cells were inoculated into mice, and all mice were treated with doxycycline. Lung colonization was assessed by quantitative BLI. (D) BLI quantification of lung colonization after the indicated cancer cells pretreated with doxycycline were injected into mice that were not given doxycycline. The mice were not administrated with drug. Data are average ± SEM; n = 8 mice. (E) TUNEL staining and quantification in the lungs harvested 6 weeks after intravenous injection of cancer cells as indicated in Figure 1E. Representative photomicrographs, and quantification of TUNEL⁺ cells. Data are average ± SEM; n = 10 unit area/mouse, 3 mice/group. Scale bar, 50 μm. (F) The indicated cancer cells were injected into the fourth mammary fat pad of mice and the lungs were harvested as indicated in Figure 1A. Immunostaining for human vimentin and cleaved caspase 3 were performed on consecutive lung tissue sections. Scale bar, 100 μm. See also Figure S4.

Figure 5. The VCAM-1 intracellular domain mediates survival of cancer cells in the lungs

(A) Domain structure of the wild type VCAM-1 with extracellular (EC), transmembrane (TM) and intracellular (IC) domains and VCAM-1 (ΔCyt) construct lacking the IC. WT or ΔCyt VCAM-1 were overexpressed in VCAM1-depleted (by shRNA 1) MDA231-LM2 cells (MDA-LM2-sh1) and CN34-LM2 cells. (B,C) Expression of the wild type VCAM-1, VCAM-1(ΔCyt) or empty vector (Ctrl) in MDA231-LM2 cells that were stably depleted of endogenous VCAM-1 by the expression of shRNA1 and CN34-LM2 cells. (B) VCAM-1 was detected by western immunoblotting in MDA231-LM2 cells. (C) The expression of VCAM-1 on the cell surface was detected by flow cytometry. (C) U937 cell adhesion assays on monolayers of the indicated MDA231 or CN34 cell lines. Data are average ± SEM; n = 10 unit areas. (D, E) The indicated cell lines were intravenously injected into mice. 6 weeks after tumor cell injection, lung BLI was measured (D), and TUNEL⁺ cells were quantified in lung tissue sections (E). Data are average ± SEM; n = 5 mice.

Figure 6. VCAM-1 engagement by α4 integrins triggers anti-apoptotic signals via the cytoplasmic domain

(A) VCAM-1 clustering antibodies (Ab X-linking) were added to the indicated cell lines in the presence or absence of 10 ng/ml TRAIL for 3 h. Caspase-3 activation was detected by western immunoblotting. (B-D) The indicated cancer cells were co-cultured with U937 cells in the presence of TRAIL and α4 integrin antibody (anti-α4) or comparable Ig control (-). (B) After 24 h, TUNEL⁺ (red) and green CMFDA-labeled cancer cells were scored under fluorescent microscope. Arrow indicates a TUNEL⁺/CMFDA⁺ cancer cell and asterisk indicates a (CMFDA-negative) U937 cell. (C) Percentage of TUNEL⁺/CMFDA⁺ cancer cells. Data are average ± SEM; n = 10 unit areas; p value calculated using t test. Scale bar, 20 μm. (D) Cancer cells were quantified after 48 h of co-culture. Data are average ± SEM; n = 4 samples.

Figure 7. VCAM-1 clustering activates PI3K/Akt signaling with the involvement of Ezrin

(A) Time course of Akt phosphorylation at the TORC2 site S473 after addition of VCAM-1 clustering antibodies. The indicated human MDA-231 and CN34 and mouse 4T1 breast cancer cells were used in low-serum media. The ratio of phospho-Akt to total Akt is shown below each lane. (B) Cancer cells overexpressing VCAM-1 were pretreated with PI3K inhibitor LY294002 (LY) or vehicle DMSO (-) for 30 min. VCAM-1 antibody clustering was applied for 10 min and activated Akt (S473) was detected by western blotting. The ratio of phospho-Akt to Akt is shown below each lane. (C) Cancer cells overexpressing VCAM-1 were pretreated with LY294002 (LY) or vehicle (-) for 30 min and VCAM-1 clustering antibodies and TRAIL (10 ng/ml) were added for 3 h as indicated. Caspase-3 activation was detected by western immunoblotting. (D) Time course of Ezrin phosphorylation at Y353 and Ezrin-Akt interaction after addition of VCAM-1 clustering antibodies to the indicated cells. The indicated proteins were analyzed by western immunoblotting of whole cell lysates (input) or anti-Ezrin immunoprecipitates (IP: Ezrin). (E) Time course of Akt association with the cell membrane fraction after addition of VCAM-1 clustering antibodies to the indicated cells. Immunoblotting with antibodies against EGFR and β-actin served as markers of membrane and cytosolic fractions, respectively. (F) Model of VCAM-1 mediated Akt activation. VCAM-1 clustering triggers Ezrin phosphorylation at Y353, which is a known docking site for PI3K. This event is accompanied by Akt binding to Ezrin, Akt association with the cell membrane, and Akt phosphorylation at the TORC2 site S473 for propagation of survival signals. See also Figure S5 and Table S1.

Figure 8. VCAM1 expression is associated with leukocyte content in breast tumors

(A) Relative VCAM1 mRNA level in lung metastatic (LM), brain metastatic (BrM) and bone metastatic (BoM) cells derived from parental MDA-MB-231 or CN34 cell lines. Data are average ± SEM; n = 3. (B) VCAM1 expression in metastatic tissues from cancer patients including samples from lung (n = 18), brain (n = 19) and bone metastasis (n = 17). (C,D) VCAM1 expression in metastatic lesions with high or low leukocyte content. (C) Clustering of 67 metastatic samples of breast cancer patients by leukocyte gene expression signature (LeukoS). (D) VCAM1 expression in leukocyte signature high and low clusters of metastatic lesions. (E) Model of the survival advantage obtained by breast cancer cells that express VCAM-1 in leukocyte-rich tissues such as the lung. On exposure to macrophages and other leukocytes, the engagement of VCAM-1 by α4 integrin counter-receptors triggers Ezrin phosphorylation and activation of PI3K/Akt signaling. PI3K/Akt signaling opposes the effects of death cytokines in the pulmonary microenvironment. See also Figure S6.