Loss of cannabinoid receptor 1 accelerates intestinal tumor growth

Abstract

Although endocannabinoid signaling is important for certain aspects of gastrointestinal homeostasis, the role of the cannabinoid receptors (CB) in colorectal cancer has not been defined. Here we show that CB1 expression was silenced in human colorectal cancer due to methylation of the CB1 promoter. Our genetic and pharmacologic studies reveal that loss or inhibition of CB1 accelerated intestinal adenoma growth in Apc(Min/+) mice whereas activation of CB1 attenuated intestinal tumor growth by inducing cell death via down-regulation of the antiapoptotic factor survivin. This down-regulation of survivin by CB1 is mediated by a cyclic AMP-dependent protein kinase A signaling pathway. These results indicate that the endogenous cannabinoid system may represent a potential therapeutic target for prevention or treatment of colorectal cancer.

Fig. 1

CB1 and CB2 expression in human colorectal tumors. (A-B) Cnr1 mRNA expression in 19 pairs of human tumors with matched normal tissues (A) and in 10 CRC cell lines (B) was measured as described the Method. In (A), the relative expression of CB1 is the average of triplicate samples normalized against the transcript levels of h-β-actin; in (B), data are the means + SE of the relative expression from three independent experiments. (C) CB1 protein levels in these human samples were determined by western blotting. (D) Treatment with 5-aza-dC restored the expression of Cnr1 mRNA (left panel) and CB1 protein (right panel) in CRC cells lines as measured by quantitative real-time PCR and western blotting.

Fig. 2

Methylation status of the CB1 promoter in human colorectal tumors. Bisulphite sequencing PCR was used to determine the methylation status of all 39 of the CpG sites in the Cnr1 promoter region (−212 to +140) in first 13 sets of paired human samples used in Fig. 1 and in four CRC cell lines. Numbers represent CpG sites relative to the transcription start site. The black regions indicate percentages of methylation exceeding 30%. The threshold for methylation was set at 30% (the upper limit of normal).

Fig. 3

The effect of Cnr1 deletion on intestinal polyp burden. (A-B) Male mice with different genotypes were killed at 13 weeks of age and polyp numbers and sizes were measured in the small intestine (A) and colon (B). Data are expressed as means ± SEM (*P < 0.05, Bonferroni test). (C) Representative H&E-stained sections of intestines from Cnr1^−/−/Apc^Min/+ and Cnr1^+/+/Apc^Min/+ mice are shown (scale bar = 500 μm).

Fig. 4

The effect of CB1 antagonist and agonist on intestinal polyp growth. (A-B) In the CB1 antagonist treatments, 6-week-old male Cnr1^+/+/Apc^Min/+ mice were treated with 0.5% CMC with or without AM251. (C-D) In the CB1 agonist experiments, 6-week-old male Cnr1^+/+/Apc^Min/+ (C) and Cnr1^−/−/Apc^Min/+ (D) mice were treated with PBS with or without R-1. At the end of the experiments, the number and size of polyps in the small intestine (A, C, and D) and colon (B, C, and D) were quantified.

Fig. 5

Activation of CB1 induces apoptosis and downregulates survivin. (A) SW-480 and LS-174T cells were treated with the R-1 for 24 hours and percentages of apoptotic cells were determined by flow cytometry as described in the Methods. (B) SW-480 cells transfected with either a negative control (siRNAC) or a CB1-selective siRNA (siRNACB1) were treated with R-1 and the relative expression of Cnr1 mRNA (left panel) in these cells was determined by quantitative real-time PCR as described in Fig. 1B, and the apoptotic rate (right panel) was measured as noted above. (C) SW-480 cells were treated with R-1 and levels of indicated protein targets were detected by western blotting as described in the Methods (top panel). Survivin levels were also determined from the experiments in panel B by western blotting (lower panel). (D) SW-480 cells overexpressing survivin or containing vector were treated with R-1 and the apoptotic rate in these cells was measured as noted above. The panel C is a representative of three different experiments with similar results.

Fig. 6

A cAMP-dependent PKA pathway mediates pro-apoptotic effects of CB1. (A) The levels of PKA kinase activity in SW-480 cells treated with R-1 were determined as described in the Methods. (B) SW-480 cells treated with H-89 were subjected to examine apoptosis rate (left panel) and survivin expression (right panel) described in the Method. (C) SW-480 cells were pretreated with 8-AHA-cAMP and then treated with R-1; and the apoptotic rate (left panel) and survivin expression (right panel) were determined as described the Method. (D) SW-480 cells were pretreated with a PI3K inhibitor (LY294002) or a MAPK inhibitor (PD98059) for 1 h and then treated with R-1 in serum-free medium for 1 day after serum starvation 24 h. The survivin expression was determined by western blotting.