Crosstalk between Chemokine Receptor CXCR4 and Cannabinoid Receptor CB2 in Modulating Breast Cancer Growth and Invasion

Cannabinoids bind to cannabinoid receptors CB(1) and CB(2) and have been reported to possess anti-tumorigenic activity in various cancers. However, the mechanisms through which cannabinoids modulate tumor growth are not well known. In this study, we report that a synthetic non-psychoactive cannabinoid that specifically binds to cannabinoid receptor CB(2) may modulate breast tumor growth and metastasis by inhibiting signaling of the chemokine receptor CXCR4 and its ligand CXCL12. This signaling pathway has been shown to play an important role in regulating breast cancer progression and metastasis.We observed high expression of both CB(2) and CXCR4 receptors in breast cancer patient tissues by immunohistochemical analysis. We further found that CB(2)-specific agonist JWH-015 inhibits the CXCL12-induced chemotaxis and wound healing of MCF7 overexpressing CXCR4 (MCF7/CXCR4), highly metastatic clone of MDA-MB-231 (SCP2) and NT 2.5 cells (derived from MMTV-neu) by using chemotactic and wound healing assays. Elucidation of the molecular mechanisms using various biochemical techniques and confocal microscopy revealed that JWH-015 treatment inhibited CXCL12-induced P44/P42 ERK activation, cytoskeletal focal adhesion and stress fiber formation, which play a critical role in breast cancer invasion and metastasis. In addition, we have shown that JWH-015 significantly inhibits orthotopic tumor growth in syngenic mice in vivo using NT 2.5 cells. Furthermore, our studies have revealed that JWH-015 significantly inhibits phosphorylation of CXCR4 and its downstream signaling in vivo in orthotopic and spontaneous breast cancer MMTV-PyMT mouse model systems.This study provides novel insights into the crosstalk between CB(2) and CXCR4/CXCL12-signaling pathways in the modulation of breast tumor growth and metastasis. Furthermore, these studies indicate that CB(2) receptors could be used for developing innovative therapeutic strategies against breast cancer

CB₂ agonist inhibits CXCL12-induced focal adhesions and stress fibres formation.

<p>Confocal microscopic visualization of (A) MCF-7/CXCR4 for focal adhesion (stained for Vinculin) or (B) SCP2 cells for stress fibers (stained for phalloidin). The cells were pretreated with vehicle or JWH-015 (20 µm) overnight and then stimulated with CXCL12 (100 ng/ml; 30 min).</p

Expression of CXCR4 and CB₂ receptors in primary breast cancer tissues and cell lines.

<p>(A) Representative photomicrographs of immunohistochemical staining of primary breast cancer tissues showing CXCR4 and CB₂ staining. Scale bars 50 µm and 200 µm. (B). FACS analysis of cell surface expression of CXCR4 and CB₂ in MCF-7/CXCR4, SCP2 and MMTV-<i>neu</i> (NT 2.5) cells.</p

CB₂ agonist inhibits tumor growth in syngenic mouse models.

<p>NT 2.5 cells (2×10⁶ in 100 ul PBS) were implanted orthotopically into mammary gland (#4) of mice. Experimental mice (n = 5) were treated peritumorally either with JWH-015 (5 mg/kg body wt) or vehicle on alternate days for 28 days starting 14 days after injection of the cells. (A) Tumors were measured every wk with external calipers and tumor volume was calculated according to the formula <i>V</i> = 0.52×<i>a</i>²×<i>b</i>, where <i>a</i> is the smallest superficial diameter and <i>b</i> is the largest superficial diameter. (B) After 28 days, the tumors were excised and weighed. (C) A representative photograph of mice showing tumors dissected from vehicle or JWH-015 treated groups. (D) Representative photomicrographs of immunostaining with Ki67 (proliferation marker) of tumors extracted from JWH-015 treated mice compared to vehicle treated. Scale bars, 50 µm. (E) Tumor lysates from mice treated with JWH or vehicle were analyzed for Phospho-CXCR4 (pCXCR4), CXCR4, Phospho-ERK (pERK), ERK and GAPDH by Immunoblotting. (F) Tumor lysates derived from PyMT transgenic mice treated with JWH or vehicle and analyzed for pERK, ERK, pCXCR4 and CXCR4 by Immunoblotting. *<i>P</i><0.05 vs. vehicle. Veh: Vehicle.</p

CB₂ agonist inhibits CXCL12-induced cell migration in breast cancer cells.

<p>Breast cancer cells (A) MCF-7/CXCR4-WT and (B) MDA-MB231 (SCP2) were pretreated overnight with vehicle or JWH-015 (20 µM) and 1×10⁵ cells were plated on the top chamber of 8 µm pore polycarbonate membrane filters and medium in absence or presence of CXCL12 (100 ng/ml) was placed in the lower chamber. After 12 hours of incubation, cells that migrated across the filter towards medium with or without CXCL12 (100 ng/ml) were fixed, stained and counted by bright-field microscopy in five random fields. Data represent the mean ± SD, representative experiments (n = 3) are shown. *<i>P</i><0.05 vs. vehicle. Veh: Vehicle.</p

CB₂ agonist inhibits CXCL12-induced wound healing in breast cancer cells.

<p>Breast cancer cells (A) MCF-7/CXCR4-WT, (B) MDA-MB231 (SCP2), and (C) MMTV-<i>neu</i> (NT 2.5, murine mammary cells) were grown for confluence in complete medium in six-well plates and then scratched with a 200 µl pipette tip to make wounds. The cells were treated with vehicle or JWH-015 (20 µm) and the closure of the wounds was monitored in presence or absence of CXCL12 (100 ng/ml) by microscopy after 18 or 24 hrs. Quantitative analysis of % wound closure as shown in left panels was determined my ImageJ software. Data represent the mean ± SD, representative experiments (n = 3) are shown. *<i>P</i><0.05 vs. vehicle; **<i>P</i><0.005 vs. vehicle. Veh: Vehicle.</p

CB₂ agonist inhibits CXCL12-induced ERK phosphorylation.

<p>MDA-MB231 (SCP2) cells were treated overnight with vehicle or JWH-015 (20 µm) and stimulated with CXCL12 (100 ng/ml) for different time periods. Cell lysates were analyzed for Phospho-ERK (p-ERK), ERK and GAPDH by Immunoblotting.</p