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. 2013 Sep 1;19(17):4706-16.
doi: 10.1158/1078-0432.CCR-13-0011. Epub 2013 May 20.

The ubiquitin-CXCR4 axis plays an important role in acute lung infection-enhanced lung tumor metastasis

Libo Yan  1 , Qingchun Cai, Yan Xu
Affiliations

The ubiquitin-CXCR4 axis plays an important role in acute lung infection-enhanced lung tumor metastasis

Libo Yan et al. Clin Cancer Res. .

Abstract

Purpose: Our goals were to test the effect of acute lung infection on tumor metastasis and to investigate the underlying mechanisms.

Experimental design: We combined bacteria-induced and lipopolysaccharide (LPS)-induced acute lung injury/inflammation (ALI) mouse models with mouse metastatic models to study the effect of acute inflammation on lung metastasis in mice. The mechanisms were investigated in ex vivo, in vitro, and in vivo studies.

Results: Both bacteria- and LPS-induced ALI significantly enhanced lung metastasis of four tail vein-injected mouse tumor cell lines. Bacteria also enhanced lung metastasis when 4T1 cells were orthotopically injected. The bronchoalveolar lavage fluid (BALF) from LPS- or bacteria-injected mice stimulated migration of tumor cells. In vivo tracking of metastatic RM-9 cells showed that bacterial injection enhanced early dissemination of tumor cells to the lung. The majority of the BALF migratory activity could be blocked by AMD3100, a chemokine receptor 4 (CXCR4) inhibitor. All tested cell lines expressed CXCR4. The levels of extracellular ubiquitin, but not stromal cell-derived factor-1, in BALF were significantly increased by LPS. Ubiquitin was able to induce AMD3100-sensitive migration of tumor cells. Finally, the antibacterial agent amoxicillin and the CXCR4 inhibitor AMD3100 blocked the enhancement effect of bacterial infection on tumor metastasis.

Conclusions: Acute lung infection dramatically increased cancer cell homing to the lung and lung metastasis. This change may be due to an alteration of the lung microenvironment and preparation of a favorable metastatic "niche." This effect was seen in multiple cancer types and thus may have broad applications for cancer patients in prevention and/or treatment of metastasis.

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Conflict of interest statement

Authors declare that there is no conflict of interest.

Figures

Figure 1
Figure 1. DH5α- or LPS-induced lung inflammation promoted lung metastasis of tumor cells
A. Top panel: representative images of the lungs from mice 14 days after treatment with PBS, LPS, or DH5α followed by i.v injection of B16-F10 melanoma cells. Quantitative summary of the lung weights and the numbers of metastases (n = 5 mice per group). Bottom panel: the effect of timing of LPS-administration in relation to B16-F10 cell injection (at 0 hr) on lung metastasis are shown by representative images of the lungs, and the quantitative assessments of the lung metastases (the lungs were collected 14 days after tumor cell injection). The control group had PBS injected at −6 hr. B. Effect of LPS-induced ALI on metastasis to the lungs for three additional cell lines. Data is shown as representative lung images and quantitative summaries of the lung weights (n = 5 mice per group; lungs were collected 20 days after tumor cell injection). C. Effect of bacterial pneumonia on lung metastases of 4T1 and EGFP-RM-9 cells. Left panel: representative images of lungs from mice treated with DH5α followed by 4T1 or EGFP-RM-9 i.v injection. The EGFP-RM-9 lung metastases images were taken using a Nikon dissecting fluorescence microscope SMZ1000 (Nikon, Melville, NY, USA). Right panel: quantitative summary of the lung weights (n = 5 mice per group; lungs were collected 20 days after tumor cell injection). D. Effect of bacterial pneumonia on 4T1 orthotopic metastasis model. Top left panel: representative images of lungs from mice orthotopically injected with 4T1 followed by DH5α challenges. Top right panel: quantitative summary of the lung weights (n = 6 mice per group; lungs were collected 28 days after tumor cell injection). The bottom panels showed that bacterial pneumonia did not affect primary tumor growth. **, P < 0.01; ***, P < 0.001.
Figure 2
Figure 2. Bronchoalveolar lavage fluid (BALF) promoted tumor cell migration
A. BALF from LPS- or DH5α-challenged mice induced significantly higher migration of B16 and 4T1 cells when compared to PBS control BALF in vitro. Medium control (RPMI 1640), LPS (5 μg/ml), 6 hr-PBS-BALF, 6 hr-LPS-BALF, 7 days-LPS-BALF and 6 hr-DH5α-BALF were tested for their effect on tumor cell migration. B. Comparison of chemokinetic vs. chemotactic effect of 6 hr-LPS-BALF on cell migration. LPS-BALF was added to the upper chamber (with the cells) or the lower chamber of the transwells. C. Left: Representative images of the frozen lung sections from mice treated with PBS or DH5α followed by i.v. injection of EGFP-RM-9 tumor cells (2×105), arrow: EGFP positive tumor cells. Right: Quantitative summary of the numbers of EGFP positive cells in each field (n = 5 mice per group). **, P < 0.01; ***, P < 0.001.
Figure 3
Figure 3. The Ub-CXCR4 axis played an important role in LPS-BALF induced tumor cell migration
A. Left: The expression of CXCR4 in tumor cells was determined by Western blot. Right: The expression of CXCR4 in tumor cells surface was determined by FACS. Arrow-marked histogram represents rat IgG2b K isotype control, arrowhead-marked histogram represents CXCR4. B. Pre-incubation with anti-mouse CXCR4 antibody (10 μg/ml) and CXCR4 inhibitor (AMD3100, 1 μg/ml) reduced 6 hr-LPS-BALF-induced 4T1 and RM-9 tumor cell migration. C. The concentrations of SDF-1 and extracellular Ub in PBS-BALF, 6 hr-LPS-BALF, 7 days-LPS-BALF and 6 hr-DH5α-BALF were detected by ELISA assays. The right panel:the effect on tumor cell migration of Ub (10μg/ml) when it was added to the upper or the lower chamber of the transwells. D. The effect of AMD3100 (1 μg/ml; 30 min preincubation) on Ub (10 μg/ml)-induced B16-F10, 4T1 and RM-9 tumor cell migration. **, P < 0.01; ***, P < 0.001.
Figure 4
Figure 4. The effect of AKT activation on Ub-induced tumor cell migration
A. Effect of PTX (100 ng/ml, 16 hr pre-incubation), LY294002 (10 μM, 30 min pre-incubation), MK-2206 (1 μM, 30 min pre-incubation), and PD98059 (30 μM, 30 min pre-incubation) on Ub (10 μg/ml)-induced 4T1 cell migration. B. Effect of dominant negative (DN) and constitutively active (CA)-AKT transfection on Ub (10 μg/ml)-induced 4T1 cell migration. The 4T1 cell migration assays were conducted 48 hr after transfection. C. The effect of Ub on phosphorylation of AKT and FAK was detected by Western blot. The B16-F10 and 4T1 cells were treated with Ub (10μg/ml) for 10 min or 15 min. D. The effect of Ub on phosphorylation of ERK was tested by Western blot. ***, P < 0.001.
Figure 5
Figure 5. AMD3100 and AMO treatment reduced bacterial-enhanced lung metastasis
A. Representative lung images from mice challenged with DH5α followed by EGFP-RM-9 (2×105) i.v. injection and treatment with AMO (0.4 mg/mouse) or AMD3100 (5 mg/kg) daily. The lungs were collected 20 days post-tumor cell injection. Right lower: Quantitative summary of the lung weights of EGFP-RM-9 injected mice (n = 5 mice per group). B. Representative images of the lungs from mice challenged with DH5α followed by B16-F10 (2×105 cells) i.v. injection and treated with amoxicillin (AMO) (0.4mg/mouse) or AMD3100 (5 mg/kg) daily. The lungs were collected 20 days post-tumor cells injection. Right: Quantitative summary of the lung weights of B16-F10 injected mice (n = 5 mice per group). *, P < 0.05. ***, P < 0.001.
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