Twist1 induces CCL2 and recruits macrophages to promote angiogenesis - PubMed (original) (raw)

Twist1 induces CCL2 and recruits macrophages to promote angiogenesis

Janine M Low-Marchelli et al. Cancer Res. 2013.

Abstract

The transcription factor Twist1 induces epithelial-mesenchymal transition and extracellular matrix degradation to promote tumor metastasis. Although Twist1 also plays a role in embryonic vascular development and tumor angiogenesis, the molecular mechanisms that underlie these processes are not as well understood. Here, we report a novel function for Twist1 in modifying the tumor microenvironment to promote progression. We found that expression of Twist1 in human mammary epithelial cells potently promoted angiogenesis. Surprisingly, Twist1 expression did not increase the secretion of the common proangiogenic factors VEGF and basic fibroblast growth factor but rather induced expression of the macrophage chemoattractant CCL2. Attenuation of endogenous Twist1 in vivo blocked macrophage recruitment and angiogenesis, whereas exogenous CCL2 rescued the ability of tumor cells lacking Twist1 to attract macrophages and promote angiogenesis. Macrophage recruitment also was essential for the ability of Twist1-expressing cells to elicit a strong angiogenic response. Together, our findings show that how Twist1 recruits stromal macrophages through CCL2 induction to promote angiogenesis and tumor progression. As Twist1 expression has been associated with poor survival in many human cancers, this finding suggests that anti-CCL2 therapy may offer a rational strategy to treat Twist1-positive metastatic cancers.

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Figures

Figure 1

Figure 1. Twist1 promotes angiogenesis in a chicken chorioallantoic membrane angiogenesis assay

A) Lysates from HMLE-Ctrl and HMLE-Twist1 cells were analyzed by SDS-PAGE and probed for Twist1 and β-actin (full-length blot, Supplementary Fig. S1). B) Cells were suspended in collagen and resulting onplants were grafted onto the CAMs of 10-day embryos. Live angiogenic scoring was performed 72 hrs later using a dissection microscope. Shown are representative images of onplants with new vessel growth. C) Angiogenesis quantification for one representative experiment. Angiogenic index is the percentage of vessel positive grids in each onplant. Each data point represents the angiogenic index in one of approximately 25 onplants distributed across 4–6 embryos per cell type, * p<0.05 by Student’s T-test. D) Quantification of average angiogenesis for 11 independent experiments. Error bars are SEM, *p<0.05 by Student’s T-test.

Figure 2

Figure 2. Twist1 is necessary and sufficient to induce CCL2 expression

A) Analysis of secreted cytokines by HMLE-Ctrl and HMLE-Twist1 cells by cytokine expression array. Conditioned media were collected for 48 hr and incubated with array membranes to measure 174 human cytokines. CCL2 levels are highlighted on one set of membranes. B) Levels of CCL2 protein were measured by ELISA in conditioned media from indicated HMLE cells. C) Levels of CCL2 mRNA were measured by QPCR analysis inindicated HMLE cells. D) Levels of CCL2 mRNA were measured by QPCR analysis in HMLE-Twist1-ER cells (solid line) or HMLE cells (dashed line) treated with 20 nM 4-hydroxy-tamoxifen. E) Lysates from 168FARN cells expressing two shRNAs against Twist1 or a control shRNA were analyzed by SDS-PAGE and probed for Twist1 and α-Tubulin (full-length blots Supplementary Fig. S2D). F) The levels of CCL2 were measured by ELISA in conditioned media from indicated 168FARN cells. G) Levels of CCL2 mRNA as measured by QPCR analysis in indicated 168FARN cells.

Figure 3

Figure 3. CCL2 mediates Twist1-induced angiogenesis in the CAM assay

A) QPCR analysis of CCL2 mRNA levels in HMLE-Ctrl and HMLE-Twist1 cells with indicated shRNAs. B) The levels of CCL2 protein were measured by ELISA in conditioned media from indicated HMLE cells. C) Lysates from indicated HMLE cells were analyzed by SDS-PAGE and probed for Twist1, E-cadherin, fibronectin, and α-tubulin (full-length blots, SFig.3A). D) Quantification of angiogenesis from three independent CAM angiogenesis experiments. For each experiment, 5–8 onplants were evenly distributed across 4–6 ten-day-old embryos per cell type tested, *p<0.05 versus HMLE-Ctrl, #p<0.05 versus HMLE-Twist1 shCtrl by ANOVA followed by Tukey Test.

Figure 4

Figure 4. Twist1 expression in mammary epithelial cells promotes macrophage attraction in a CCL2-dependent manner

A) Cell surface expression of CCR2 on isolated mouse BMDM as measured by flow cytometry analysis using anti-CCR2 (open) versus Control IgG (shaded) antibody staining. B) BMDM were subjected to Boyden Chamber migration assays toward conditioned media from HMLE-Ctrl or HMLE-Twist1 cells. Representative images of migrated macrophages stained with crystal violet (top) and quantification of relative migration (bottom) for one representative experiment, *p<0.05 by Student’s T-test. C) Macrophage migration toward conditioned media from 168FARN cells with indicated shRNAs for one representative experiment, *p<0.05 versus shCtrl by ANOVA followed by Tukey Test. D) Macrophage migration toward conditioned media from 168FARN cells with shRNAs against Twist1, coincubated with either PBS or rCCL2 (10 ng/mL) for one representative experiment, *p<0.05 by Student’s T-test. E) Macrophage migration toward conditioned media from HMLE-Twist1 cells expressing indicated shRNAs for one representative experiment, *p<0.05 versus shCtrl by ANOVA followed by Tukey Test.

Figure 5

Figure 5. Twist1 expression promotes angiogenesis and macrophage infiltration in the Matrigel plug assay

A) A mixture of Matrigel and 168FARN mouse mammary tumor cells was subcutaneously injected into 4–5 mice/group. Resulting plugs were harvested 5 days later and processed for immunohistochemical staining. Top row, macroscopic images of plugs; middle row, images of tissue sections stained for blood vessel marker CD31(brown); bottom row, images of tissue sections stained for mature macrophage marker F4/80 (brown). Nuclei were stained with hematoxylin (blue). Insets show magnification for staining detail, scale bar = 2 mm. B and C) Quantification of stains in (A), 5 random high-powered fields per section were counted for number of CD31 stained vessels or were scored for F4/80 staining intensity and distribution; 7–10 plugs/group and 1–2 sections/plug were analyzed. Scale bar = 50 µm, error bars are SEM, *p<0.05 versus shCtrl by ANOVA followed by Tukey Test.

Figure 6

Figure 6. CCL2 and macrophage recruitment are essential for Twist1-induced angiogenesis

A) A mixture of Matrigel and 168FARN cells was subcutaneously injected mice either previously depleted of macrophages by clodronate-encapsulated liposomes (9 mice) or treated with Control liposomes (6 mice). Resulting plugs were harvested 5 days later and processed for immunohistochemical staining. Top row, macroscopic images of plugs; middle row, images of tissue sections stained for blood vessel marker CD31(brown); bottom row, images of tissue sections stained for mature macrophage marker F4/80 (brown). Nuclei were stained with hematoxylin (blue). Scale bar = 2 mm. B and C) Quantification of stains in (A), 5 random high-powered fields per section were counted for number of CD31 stained vessels or were scored for F4/80 staining intensity and distribution; 10–15 plugs/group and 1–2 sections/plug were analyzed. Scale bar = 50 µm, error bars are SEM. *p<0.05 by Student’s T-test. D) Matrigel plug assays were performed using 168FARN mouse mammary tumor cells and supplemented with recombinant CCL2 (10 ng/mL final concentration) or PBS using at least 3 mice/group. Scale bar = 2 mm. E and F) Quantification of stains in (D), with 6–8 plugs/group and 1–2 sections/plug analyzed. Scale bar = 50 µm, error bars are SEM, *p<0.05 by Student’s T-test.

Figure 7

Figure 7. Proposed model for Twist1-induced angiogenesis through CCL2 induction and macrophage recruitment

Carcinoma cells expressing Twist1 upregulate CCL2 transcript leading to increased CCL2 protein in the tumor microenvironment. The CCL2 gradient attracts macrophages, which then promote angiogenesis.

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References

    1. Martin TA, Goyal A, Watkins G, Jiang WG. Expression of the transcription factors snail, slug, and twist and their clinical significance in human breast cancer. Ann Surg Oncol. 2005;12:488–496. - PubMed
    1. Gomez I, Pena C, Herrera M, Munoz C, Larriba MJ, Garcia V, et al. TWIST1 is expressed in colorectal carcinomas and predicts patient survival. PLoS One. 2011;6:e18023. - PMC - PubMed
    1. Xie F, Li K, Ouyang X. Twist, an independent prognostic marker for predicting distant metastasis and survival rates of esophageal squamous cell carcinoma patients. Clin Exp Metastasis. 2009;26:1025–1032. - PubMed
    1. Yang J, Mani SA, Donaher JL, Ramaswamy S, Itzykson RA, Come C, et al. Twist, a master regulator of morphogenesis, plays an essential role in tumor metastasis. Cell. 2004;117:927–939. - PubMed
    1. Casas E, Kim J, Bendesky A, Ohno-Machado L, Wolfe CJ, Yang J. Snail2 is an essential mediator of Twist1-induced epithelial mesenchymal transition and metastasis. Cancer Res. 2011;71:245–254. - PMC - PubMed

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