Cannabinoids receptor type 2, CB2, expression correlates with human colon cancer progression and predicts patient survival

Abstract

Many studies have demonstrated that the endocannabinoid system (ECS) is altered in different tumor types, including colon cancer. However, little is known about the role of the ECS in tumor progression. Here we report the correlation between CB 2 expression and pathological data in a series of 175 colorectal cancer patients, as well as the response of the HT29 colon cancer-derived cell line upon CB 2 activation. CB 2 mRNA was detected in 28.6% of samples tested. It was more frequent in N+ patients and predicts disease free survival and overall survival in colon cancer. In positive samples, CB 2 was expressed with great intensity in tumor epithelial cells and correlated with tumor growth. Treatment of HT29 with CB 2 agonist revealed membrane loss of E-cadherin and SNAIL1 overexpression. A direct correlation between CB 2 and SNAIL1 expression was also found in human tumors. CB 2 receptor expression is a poor prognostic marker for colon cancer and the activation of this receptor, with non-apoptotic doses of agonists, could be collaborating with disease progression. These results raise the question whether the activation of CB 2 should be considered as anti-tumoral therapy.

Keywords: CB2; Colorectal cancer; Disease free survival; overall survival; prognosis marker.

Figure 1. Kaplan-Meier curves and p values for DFS (left panels) and OS (right panels) regarding CB₂ mRNA expression for the complete colorectal cancer series, CRC; specific for colon cancer series, CC; and for rectal cancer series, RC

Patients with tumor in stage IV are not included in the DFS analysis. Number of patients for each cohort is indicated in the graph. Discontinuous line, patients with positive expression of CB₂. Continuous line, patients with negative expression of CB₂.

Figure 2. Kaplan-Meier curves and p values for DFS regarding CB₂ mRNA expression in CC patients without lymph node involvement, N−; patients with lymph node involvement, N+; patients with vascular invasion negative, VI−; and patients with vascular invasion positive, VI+

Patients with tumor in stage IV are not included. Number of patients for each cohort is indicated in the graph. Discontinuous line, patients with positive expression of CB2. Continuous line, patients with negative expression of CB₂.

Figure 3. Left panel: Immunohistochemical staining of CB₂ receptor in human colon from a colorectal cancer patient

The representative section contained both normal and tumoral tissue. CB₂ was expressed with greater intensity in support cells (infiltrating lymphocytes, etc.) and transformed epithelial cells. Normal epithelial cells showed low or negative staining for CB₂ protein. The intensity of signal is color coded: red arrow indicate high positive staining, green arrow show moderate staining, and black arrow depict negative staining. Right panel: Comparative immunohistochemical analysis of CB₂, and Ki67 in samples from two patients with high (upper) or low (bottom) expression levels of CB₂. Normal colonic biopsies (A and D) showed very low staining for CB₂ in epithelial cells in both cases. Tumoral sections from colorectal cancer patients with high CB₂ expression (B) showed high Ki67 levels (C). In contrast tumor sample with low CB₂ expression levels (E) showed low levels of Ki67 (F).

Figure 4. Cytotoxicity of different concentrations of JWH-133 on HT29 cells

Cells were incubated in low-FCS medium (0.5% FCS) for 24 hours in the presence of the vehicle (DMSO, 0.06%) or different concentrations of JWH-133 ranging from 5 to 30 μmol/L. Cell viability in JWH-133- and vehicle-treated cells is expressed as mean ± SD of three independent experiments respect no-treated cells (wt, 100%).

Figure 5. Left panel: Confocal microscopy analysis of E-cadherine

HT29 cells were incubated with vehicle (DMSO, 0.02%) (A) or 7.5 μmo/L JWH-133 (B-E) in low-FCS medium (0.5% FCS) for 48h. Blue, cell nuclei; red, E-cadherine. Right panel: SNAIL1 expression levels in HT29 cells incubated for 48h with different concentrations of JWH-133. Changes in SNAIL1 levels are expressed as a fold change compared with the control (DMSO-treated cells). RNA was analyzed by quantitative (real-time) RT-PCR as described in Material and Methods section. Data are expressed as mean±SD of three independent experiments. * p < 0,05 ** p < 0,005 *** p < 0,001.