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. 2009 Feb;174(2):602-12.
doi: 10.2353/ajpath.2009.080636. Epub 2009 Jan 15.

The chemokine receptor CXCR4 and the metalloproteinase MT1-MMP are mutually required during melanoma metastasis to lungs

Rubén A Bartolomé  1 , Sergio Ferreiro, María E Miquilena-Colina, Lorena Martínez-Prats, María L Soto-Montenegro, David García-Bernal, Juan J Vaquero, Reuven Agami, Rafael Delgado, Manuel Desco, Paloma Sánchez-Mateos, Joaquin Teixidó
Affiliations

The chemokine receptor CXCR4 and the metalloproteinase MT1-MMP are mutually required during melanoma metastasis to lungs

Rubén A Bartolomé et al. Am J Pathol. 2009 Feb.

Abstract

Melanoma is the most aggressive skin cancer once metastasis begins; therefore, it is important to characterize the molecular players involved in melanoma dissemination. The chemokine receptor CXCR4 and the membrane-bound metalloproteinase MT1-MMP are expressed on melanoma cells and represent candidate molecules for the control of metastasis. Using human melanoma transfectants that either overexpress or silence CXCR4 or MT1-MMP, or that have a combination of overexpression and interference of these proteins, we show that CXCR4 and MT1-MMP coordinate their activities at different steps along melanoma cell metastasis into the lungs. Results from in vivo xenograft mouse models of melanoma lung colonization and mice survival and short-term, homing nested polymerase chain reaction experiments from lung samples indicated that CXCR4 is required at early phases of melanoma cell arrival in the lungs. In contrast, MT1-MMP is not needed for these initial steps but promotes subsequent invasion and dissemination of the tumor with CXCR4. Investigation of potential cross talk between CXCR4 and MT1-MMP revealed that MT1-MMP accumulates intracellularly after melanoma cell stimulation with the CXCR4 ligand CXCL12, and that this process involves the activation of the Rac-Erk1/2 pathway. Subsequent to cell contact with specific basement membrane proteins, MT1-MMP redistributes to the cell membrane in a phosphatidylinositol 3-kinase-dependent manner. These results suggest that combination therapies that target CXCR4 and MT1-MMP should improve the limitations of the current therapies for metastatic melanoma.

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Figures

Figure 1
Figure 1
In vitro functional characterization of human melanoma transfectants with stable interference and overexpression of CXCR4 and MT1-MMP. A: Flow cytometry analyses of CXCR4 and MT1-MMP expression on BLM melanoma transfectants. Note the 3-decade log scale starting at 0 and ending at 103. B: Western blotting analyses of CXCR4 and MT1-MMP expression on BLM melanoma transfectants. Loading controls were assessed with anti-RhoA antibodies. Shown is a representative result of three independent experiments (top) and densitometer analyses in arbitrary units showing fold induction of expression related to mock transfectants (bottom). C: Transfectants were subjected to Matrigel invasion assays toward CXCL12 or medium alone. Invasion was significantly up-regulated, *P < 0.05, or significantly inhibited, ΔP < 0.05, with respect to mock transfectant invasion. D: Transfectants (1.2 × 105) were incubated in Dulbecco’s modified Eagle’s medium/1% fetal bovine serum, without (medium) or with CXCL12, and cell number was determined after the indicated times.
Figure 2
Figure 2
Growth and lung metastasis of subcutaneously-inoculated CXCR4 and MT1-MMP melanoma transfectants. Mice (n = 9) were inoculated into the lateral thoracic wall with the indicated melanoma transfectants. Plotted are the days when tumors reached 2.5 cm3, together with statistical significance (N.S., nonsignificant) (A), and percentage of mice displaying lung metastases (B). Shown is a representative result from three independent experiments.
Figure 3
Figure 3
CXCR4 and MT1-MMP are mutually required for in vivo melanoma metastasis. A: Survival curves of mice (n = 10) inoculated into the tail vein with the indicated transfectants, and statistics from the different curves (N.S., nonsignificant). B: Degree of lung colonization by melanoma transfectants (left), and representative lung metastases from CXCR4hi (34 days, massive metastasis) and CXCR4lo (70 days) mice. Arrow indicates a metastatic tumor node. C: Melanoma transfectants were transiently transfected with pEGFP-C1 vector, and subsequently intravenously inoculated into SCID mice. After the indicated times, GFP expression in lungs was determined by nested PCR. Negative controls with nontransfected cells are also shown. D, left: SCID mice were inoculated with CXCR4hi or CXCR4lo melanoma transfectants and subjected to PET-CT analyses. Co-registered PET and CT studies were superimposed. Coronal sections from a CXCR4hi mouse at days 23 and 30 are shown. Intersections between lines correspond to the center of lung metastases (LM) (R, right; L: left). D, right: Data indicate number of mice with lung metastases, mostly one to two tumor nodes.
Figure 4
Figure 4
MT1-MMP accumulates intracellularly on CXCL12 stimulation and redistributes to the cell surface after melanoma cell incubation with basement membrane proteins. A: Melanoma transfectants were incubated for 24 hours with CXCL12 and subjected to immunoblotting with anti-MT1-MMP mAb. Control loading was assessed with β-actin antibodies (top). Then cells were plated for 45 minutes on Matrigel (1.5 μg/mm2) or incubated in invasion medium alone (control), and after detachment with PBS/EDTA they were analyzed for MT1-MMP cell surface expression by flow cytometry (bottom). Mock transfectants were plated for the indicated times either on Matrigel (B), on the indicated ECM proteins, or incubated with transforming growth factor-β1 or epidermal growth factor (C), and subsequently analyzed by flow cytometry for MT1-MMP cell surface expression. Note the 3-decade log scale starting at 0 and ending at 103.
Figure 5
Figure 5
Rac-Erk1/2 signaling controls up-regulation by CXCL12 of MT1-MMP expression. A: Cells were incubated for 24 hours with CXCL12 in the absence (control) or presence of LY (20 μmol/L) or UO126 (5 μmol/L), and tested with Western blot analyses for MT1-MMP expression. BLM melanoma cells were transfected with control or Rac1 siRNA (B), or with GFP-fused wild-type (wt) or constitutively activated (CA) forms of Rac (C). Transfectants were subsequently incubated for 24 hours with CXCL12 and subjected to Western blotting for expression of MT1-MMP, phospho-Erk1/2, total Erk1/2, and Rac1. D: Transfectants expressing Rac wt or Rac CA were subjected to Matrigel invasion assays toward CXCL12, in the absence or presence of UO126 and control or anti-MT1-MMP mAb (left). Supernatants from these invasions were tested by gelatinolytic zymography for MMP-2 activity (right).
Figure 6
Figure 6
Role of phosphatidylinositol 3-kinase and Erk1/2 on melanoma cell surface expression of MT1-MMP and cell invasion. A: Mock cells were incubated for 24 hours with or without CXCL12, in the absence or presence of UO126, SB203580 (13 μmol/L), or JNK inhibitor II (30 μmol/L), whereas LY was added for the last 2.5 hours of this incubation. Subsequently, cells were exposed to Matrigel or medium alone, and subjected to flow cytometry to determine MT1-MMP expression. A representative result from three independent experiments is shown. B: BLM cells were subjected to Matrigel invasion assays in the presence of the indicated inhibitors. Invasion was significantly inhibited, **P < 0.01 (n = 3). C: Mock cells were subjected to adhesion assays to Matrigel, in the presence of blocking anti-β1 mAb Lia1/2.1 or control nonblocking anti-α5 mAb. Basal adhesion to BSA is also shown. Adhesion was significantly inhibited, ***P < 0.001. D: Mock cells were incubated for 30 minutes with or without Matrigel, in the presence of anti-β1 or control anti-α5 mAb, and subsequently collected and tested by immunoblotting with antibodies against phospho-Akt or Akt.
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