Modulation of the tumor microenvironment and inhibition of EGF/EGFR pathway: novel anti-tumor mechanisms of Cannabidiol in breast cancer

Abstract

The anti-tumor role and mechanisms of Cannabidiol (CBD), a non-psychotropic cannabinoid compound, are not well studied especially in triple-negative breast cancer (TNBC). In the present study, we analyzed CBD's anti-tumorigenic activity against highly aggressive breast cancer cell lines including TNBC subtype. We show here -for the first time-that CBD significantly inhibits epidermal growth factor (EGF)-induced proliferation and chemotaxis of breast cancer cells. Further studies revealed that CBD inhibits EGF-induced activation of EGFR, ERK, AKT and NF-kB signaling pathways as well as MMP2 and MMP9 secretion. In addition, we demonstrated that CBD inhibits tumor growth and metastasis in different mouse model systems. Analysis of molecular mechanisms revealed that CBD significantly inhibits the recruitment of tumor-associated macrophages in primary tumor stroma and secondary lung metastases. Similarly, our in vitro studies showed a significant reduction in the number of migrated RAW 264.7 cells towards the conditioned medium of CBD-treated cancer cells. The conditioned medium of CBD-treated cancer cells also showed lower levels of GM-CSF and CCL3 cytokines which are important for macrophage recruitment and activation. In summary, our study shows -for the first time-that CBD inhibits breast cancer growth and metastasis through novel mechanisms by inhibiting EGF/EGFR signaling and modulating the tumor microenvironment. These results also indicate that CBD can be used as a novel therapeutic option to inhibit growth and metastasis of highly aggressive breast cancer subtypes including TNBC, which currently have limited therapeutic options and are associated with poor prognosis and low survival rates.

Keywords: Cannabidiol; EGFR; Triple negative breast cancer; Tumor microenvironment.

Figure 1

CBD inhibits proliferation of breast cancer cells. SUM159 (A), 4T1.2 (B) and SCP2 (C) cells were treated with CBD (3–15) μM for 48 h and then subjected to MTT assay. Measurements were plotted as % of control.

Figure 2

CBD inhibits EGF induced breast cancer EGF‐induced cell proliferation, colony formation, migration and invasion. . SUM159 (A) or 4T1.2 (B) cells were treated with vehicle or CBD (3 and 6) μM with or without EGF (100 ng/ml) for 48 h and subjected to MTT assay. Data represent fold change of proliferation after 48 h (Day 2) relative to basal level of proliferation (Day 0). Colony forming assay was performed for SUM159 (C) or 4T1.2 (D) cells that were treated with vehicle or CBD with or without EGF (100 ng/ml). SUM159 (E) or 4T1.2 (F) cells were treated with CBD 6 μM for 48 h and subjected to transwell migration assay. SUM159 (G) or 4T1.2 (H) cells were treated with CBD 6 μM for 48 h and subjected to transwell invasion assay. Number of migrated or invaded cells were counted and plotted as % of control.

Figure 3

CBD inhibits EGF/EGFR signaling. (A) SUM159 cells were treated with vehicle or CBD 6 μM in the presence or absence of EGF and subjected to NF‐kB luciferase reporter assay. (B) SUM159 cells were treated with CBD 6 μM, stimulated with EGF for 15 or 30 min then cell lysates were used for western blot analysis for the indicated proteins. Relative expression of p‐EGFR/EGFR, p‐AKT/AKT and p‐ERK/ERK has been quantified by image‐j software and provided as numbers (indicated in red color) (C) Confocal microscopy visualization of SUM159 cells treated with vehicle or CBD 6 μM and stimulated with EGF (100 ng/ml) and stained for phalloidin (red), vinculin (green) and DAPI (blue).

Figure 4

CBD inhibits breast tumor growth in different mouse model systems. Tumor volume measurements of 4T1.2 (A) or MVT‐1 (B) mouse models were assessed every week for control and treated groups. Tumor weight of various experimental groups was determined in 4T1.2 (C) or MVT‐1 (D) mouse models. Representative photographs showing tumors dissected from various experimental groups of 4T1.2 (E) or MVT‐1 (F) mouse models. Representative photomicrographs of immunostaining with Ki67, CD31 and phospho‐EGFR (p‐EGFR) of tumors of control and CBD‐treated groups in 4T1.2 (G) or MVT‐1 (H) mouse models. Western blot images of 4T1.2 (I) or MVT‐1 (J) tumors showing the expression of phospho‐ERK or AKT (p‐ERK, p‐AKT) and total ERK and AKT (ERK, AKT) proteins in control and CBD‐treated groups.

Figure 5

CBD inhibits lung metastasis in different mouse model systems. Representative lung images and H&E staining photomicrographs were taken of control and CBD‐treated groups of 4T1.2 (A) and MVT‐1 (B) mouse models. The number of metastatic lung nodules was counted for 4T1.2 (C) and MVT‐1 (D) mouse models of control and CBD‐treated groups. Total lung weight was determined for 4T1.2 (E) and MVT‐1 (F) mouse models of control and CBD‐treated groups. RT‐PCR quantification was performed for MMP‐9 and MMP‐2 in 4T1.2 (G) and MVT‐1 (H) mouse models in control and CBD‐treated tumors 18S rRNA primers were used for loading control purpose.

Figure 6

CBD inhibits macrophage recruitment to the breast tumor microenvironment. Representative IHC photomicrographs of control and CBD‐treated tumors of 4T1.2 (A) and MVT‐1 B) mouse models using F4/80 and Arginase‐I antibodies. Percentages of CD11b+/F4/80+ (C) and CD206+/F4/80+ (D) cells in control and CBD‐treated tumors in MVT‐1 mouse model. Representative IHC photomicrographs of lungs of control and CBD‐treated groups of 4T1.2 (E) and MVT‐1 (F) mouse models using F4/80 and Arginase‐I antibodies.

Figure 7

CBD inhibits macrophage recruitment through modulation of breast cancer cell cytokine profile. (A) 4T1.2 cells were treated with vehicle or CBD for 48 h and conditioned media were used for cytokine profiling. (B) Quantification of cytokine array data using Image‐J software for the affected cytokines. Data represent protein levels relative to loading controls. (C) Relative RAW 264.7 cells migration towards SFM (Con) or the conditioned media of vehicle‐treated (CM) and CBD‐treated (CM‐CBD) 4T1.2 tumor cells (D) A diagram shows a putative anti‐proliferative and anti‐metastatic mechanism of action of CBD.