Simultaneous Activation of Induced Heterodimerization between CXCR4 Chemokine Receptor and Cannabinoid Receptor 2 (CB2) Reveals a Mechanism for Regulation of Tumor Progression

Abstract

The G-protein-coupled chemokine receptor CXCR4 generates signals that lead to cell migration, cell proliferation, and other survival mechanisms that result in the metastatic spread of primary tumor cells to distal organs. Numerous studies have demonstrated that CXCR4 can form homodimers or can heterodimerize with other G-protein-coupled receptors to form receptor complexes that can amplify or decrease the signaling capacity of each individual receptor. Using biophysical and biochemical approaches, we found that CXCR4 can form an induced heterodimer with cannabinoid receptor 2 (CB2) in human breast and prostate cancer cells. Simultaneous, agonist-dependent activation of CXCR4 and CB2 resulted in reduced CXCR4-mediated expression of phosphorylated ERK1/2 and ultimately reduced cancer cell functions such as calcium mobilization and cellular chemotaxis. Given that treatment with cannabinoids has been shown to reduce invasiveness of cancer cells as well as CXCR4-mediated migration of immune cells, it is plausible that CXCR4 signaling can be silenced through a physical heterodimeric association with CB2, thereby inhibiting subsequent functions of CXCR4. Taken together, the data illustrate a mechanism by which the cannabinoid system can negatively modulate CXCR4 receptor function and perhaps tumor progression.

Keywords: CXC chemokine receptor type 4 (CXCR-4); G protein-coupled receptor (GPCR); cannabinoid receptor; dimerization; metastasis.

FIGURE 1.

Simultaneous SDF1α/AM1241 treatment inhibited internalization of CXCR4. One million MDA-MB-231 cells were serum-starved for 24 h prior to treatment with SDF1α, AM1241, or SDF1α/AM1241 simultaneously for 10 min. Cells were detached by incubation with 1× citric saline at 37 °C, washed in 1× PBS, centrifuged, then resuspended, and fixed in 4% cold paraformaldehyde. Cells were blocked in 1% BSA in PBS on ice for 60 min prior to incubation with a CXCR4-specific antibody at 4 °C overnight in blocking buffer. The next day, cells were washed, centrifuged at maximal speed, and then incubated with a FITC-conjugated secondary antibody for 30 min at 4 °C. After washes in 1× PBS, cells were stored in 0.1% BSA in PBS until analyzed by FACS. Statistical analysis is described under “Materials and Methods.” *, p < 0.001. Experiments were performed at least thrice. Error bars represent S.E.

FIGURE 2.

SDF1α and AM1241 did not compete for binding to CXCR4. MDA-MB-231 (A and B) or 293T cells (A) were plated in 96-well puncher plates and grown to a density of 75% prior to the experiment. To assign absolute affinity of each ligand for CXCR4 receptor, a competitive displacement assay was used as discussed by Misra et al. (62) using cold ligand AM1241 as a test compound along with [^99mTc-MAS₃]SDF1α and [^99mTc-MAS₃]AM1241 as radiotracers on the surface of MDA-MB-231 (CXCR4-positive) and 293T (CXCR4-negative) cell lines. Cells were washed twice with ice-cold 1× PBS, pH 7.4, and incubated for 20 min at 4 °C with 0.5 μCi of radiotracer in the presence or absence of the test compound. Cells were then washed thrice with 1× PBS, and the well contents were transferred directly to plastic tubes in γ counter racks. Well contents were counted on a Wallac Wizard 1470 γ counter. Experiments were performed at least thrice. Error bars represent S.E.

FIGURE 3.

Simultaneous SDF1α/AM1241 treatment induced physical association between CXCR4 and CB2. A, PC3 cells were treated for 15 min with SDF1α, AM1241, or SDF1α/AM1241 simultaneously. Cells were lysed and then incubated with anti-CXCR4 antibody for 1 h at 4 °C prior to adding Protein A/G PLUS-agarose beads and further incubating overnight at 4 °C. Immunoprecipitates were separated by 10% SDS-PAGE and then immunoblotted for CB2 protein expression. CB2 Eluent represents immunoprecipitated proteins attached to Protein A/G beads; CB2 Supernatant represents protein lysate after immunoprecipitation and the first centrifugal spin; CXCR4 Input represents lysate prior to immunoprecipitation. B, MDA-MB-231 cells were harvested as described above. Immunoprecipitates were separated by 10% SDS-PAGE and then immunoblotted for CB2 or EP-2 protein expression. C, PC3 cells were harvested as described above, and immunoprecipitates were separated by 10% SDS-PAGE and then immunoblotted for CB2 or melanocortin-5 (MC5). Each experiment was performed at least twice. IP, immunoprecipitation; WB, Western blotting.

FIGURE 4.

Endogenous CXCR4 and CB2 formed heterodimers in cancer cells. Heterodimerization of CXCR4 and CB2 was recognized by incubating cells with primary antibodies raised in two different species and secondary antibodies linked to different DNA oligomers, one designated PLUS and one designated MINUS. If the two receptors were close enough, the two different antibody-DNA probes were able to ligate and hybridize. Following an amplification process, the presence of fluorescently tagged nucleotides allowed detection of a punctate fluorescence signal by confocal microscopy. PC3 (A) and MDA-MB-231 (B) cells were each plated on coverslips; serum-starved for 24 h; and then treated with SDF1α, AM1241, or SDF1α/AM1241 simultaneously for 1 min prior to fixing with methanol (PC3) or 2% paraformaldehyde (MDA-MB-231). Cells were prepared as described above (Duolink). A CXCR4/CB2 heterodimer was detected via positive ligation of PLA probes on a rhodamine filter (excitation, 571 nm; emission, 590 nm), and nuclei were stained with DAPI. Images were reviewed and captured using a Zeiss LSM 700 confocal microscope. Scale bars, 50 μm. B, merged images of MDA-MD-231 cells only; colocalization and heterodimerization in merged images are indicated by punctate foci and white arrows. Designated PC3 (C) or MDA-MBA-231 (D) cells were pretreated with AMD3100 (1 μg/ml) prior to treatment with SDF1α and/or AM1241. Cells were later harvested for Duolink as described above. Magnification, 20×; scale bars, 50 μm. Each experiment was performed at least twice.

FIGURE 4.

Endogenous CXCR4 and CB2 formed heterodimers in cancer cells. Heterodimerization of CXCR4 and CB2 was recognized by incubating cells with primary antibodies raised in two different species and secondary antibodies linked to different DNA oligomers, one designated PLUS and one designated MINUS. If the two receptors were close enough, the two different antibody-DNA probes were able to ligate and hybridize. Following an amplification process, the presence of fluorescently tagged nucleotides allowed detection of a punctate fluorescence signal by confocal microscopy. PC3 (A) and MDA-MB-231 (B) cells were each plated on coverslips; serum-starved for 24 h; and then treated with SDF1α, AM1241, or SDF1α/AM1241 simultaneously for 1 min prior to fixing with methanol (PC3) or 2% paraformaldehyde (MDA-MB-231). Cells were prepared as described above (Duolink). A CXCR4/CB2 heterodimer was detected via positive ligation of PLA probes on a rhodamine filter (excitation, 571 nm; emission, 590 nm), and nuclei were stained with DAPI. Images were reviewed and captured using a Zeiss LSM 700 confocal microscope. Scale bars, 50 μm. B, merged images of MDA-MD-231 cells only; colocalization and heterodimerization in merged images are indicated by punctate foci and white arrows. Designated PC3 (C) or MDA-MBA-231 (D) cells were pretreated with AMD3100 (1 μg/ml) prior to treatment with SDF1α and/or AM1241. Cells were later harvested for Duolink as described above. Magnification, 20×; scale bars, 50 μm. Each experiment was performed at least twice.

FIGURE 5.

Diminution of endogenous SDF1α did not inhibit endogenous CXCR4/CB2 heterodimerization. MDA-MB-231 (A) and PC3 (E) cells were transfected with siRNA (Santa Cruz Biotechnology; 100 nm) targeting SDF1α prior to harvesting for Duolink. Transfected siRNA reduced endogenous SDF1α production in MDA-MB-231 (D) and PC3 (G) cells as well as secretion into the medium in MDA-MB-231 cells (C) (Bioneer; 100 nm). α-Tubulin was used as a loading control. Homodimerization of MDA-MB-231 (B) and PC3 (F) cells was analyzed to ensure that the siRNA did not alter receptor function. Magnification, 40×; scale bars, 20 μm. Each experiment was performed at least twice.

FIGURE 6.

CXCR4/CB2 heterodimer modulated ERK1/2 phosphorylation. A, serum-starved MDA-MB-231 cells were treated with SDF1α, AM1241, or SDF1α/AM1241 simultaneously for 15 min. Whole protein lysates were isolated prior to separation by 10% SDS-PAGE and transferred to PVDF membranes. Protein-bound membranes were processed for Western blotting analysis as described under “Materials and Methods.” B, PC3 cells were treated with ligands for 15 min prior to harvesting as described above. α-Tubulin was used as a loading control. Each experiment was performed at least twice.

FIGURE 7.

Interaction between CXCR4 and CB2 inhibited CXCR4-mediated functions. Serum-starved PC3 (A) or MDA-MB-231 (B) cells, both preincubated with calcium dye for 1 h, were treated with SDF1α, AM1241, AMD3100, or SDF1α/AM1241 simultaneously at 37 °C for 15 min (A) or 30 s (B). Calcium flux was determined at an excitation of 490 nm and emission of 525 nm on a microplate reader. C, serum-starved MDA-MB-231 cells were treated with SDF1α, AM1241, or SDF1α/AM1241 simultaneously for a 1 h prior to detaching with 1× citric saline. Transwell plates were prepared for migration by placing RPMI 1640 medium alone, SDF1α, AM1241, or SDF1α/AM1241 simultaneously in the lower (bottom) chamber. Equal cell numbers (∼ 2 × 10⁴) were seeded in the upper well of 24-well transmigration chambers to allow migration/chemotaxis toward ligand solutions in the bottom chamber at 37 °C for 4 h. Chambers were cleaned, stained, and counted on a light microscope. D–G, serum-starved MDA-MB-231 cells were seeded in the upper chamber of Transwell inserts, and various combinations of RPMI 1640 medium alone, SDF1α (D), AM1241 (D, E, and G), CXCL1 (100 ng/ml) (E), or JWH-015 (1 μm) and AM630 (1 μm) (F) were added to the lower (bottom) chambers. Cells were allowed to migrate toward ligand combinations at 37 °C prior to harvesting as described above. The migration index was calculated as the x-fold change in migration observed over untreated treated cells or cells that migrated to SDF1α alone. Statistical analysis is described under “Materials and Methods.” ***, p < 0.05; **, p < 0.01; *, p < 0.001. Scale bar, 50 μm. Each experiment above was performed at least thrice. Error bars represent S.E. A graphical representation of the restoration of CXCR4-mediated migration in the presence of AMD630 is shown in the bottom panel.

FIGURE 7.

Interaction between CXCR4 and CB2 inhibited CXCR4-mediated functions. Serum-starved PC3 (A) or MDA-MB-231 (B) cells, both preincubated with calcium dye for 1 h, were treated with SDF1α, AM1241, AMD3100, or SDF1α/AM1241 simultaneously at 37 °C for 15 min (A) or 30 s (B). Calcium flux was determined at an excitation of 490 nm and emission of 525 nm on a microplate reader. C, serum-starved MDA-MB-231 cells were treated with SDF1α, AM1241, or SDF1α/AM1241 simultaneously for a 1 h prior to detaching with 1× citric saline. Transwell plates were prepared for migration by placing RPMI 1640 medium alone, SDF1α, AM1241, or SDF1α/AM1241 simultaneously in the lower (bottom) chamber. Equal cell numbers (∼ 2 × 10⁴) were seeded in the upper well of 24-well transmigration chambers to allow migration/chemotaxis toward ligand solutions in the bottom chamber at 37 °C for 4 h. Chambers were cleaned, stained, and counted on a light microscope. D–G, serum-starved MDA-MB-231 cells were seeded in the upper chamber of Transwell inserts, and various combinations of RPMI 1640 medium alone, SDF1α (D), AM1241 (D, E, and G), CXCL1 (100 ng/ml) (E), or JWH-015 (1 μm) and AM630 (1 μm) (F) were added to the lower (bottom) chambers. Cells were allowed to migrate toward ligand combinations at 37 °C prior to harvesting as described above. The migration index was calculated as the x-fold change in migration observed over untreated treated cells or cells that migrated to SDF1α alone. Statistical analysis is described under “Materials and Methods.” ***, p < 0.05; **, p < 0.01; *, p < 0.001. Scale bar, 50 μm. Each experiment above was performed at least thrice. Error bars represent S.E. A graphical representation of the restoration of CXCR4-mediated migration in the presence of AMD630 is shown in the bottom panel.