Article Figures & Data
Figures
- Figure 1.
Expression of CNR1 and CNR2 in TCGA HNSCC data. A, Compared with HPV-negative HNSCC, the expression of CNR1 is upregulated in HPV-positive HNSCC samples. B, Compared with HPV-negative HNSCC and normal samples, the expression of CNR2 is upregulated in HPV-positive HNSCC samples. HPV-positive: n = 98; HPV-negative: n = 422; normal: n = 44. Statistical comparisons were determined with the Mann–Whitney U test. *, P < 0.05; ***, P < 0.001; NS, not significant.
- Figure 2.
CNR1 and CNR2 knockdown inhibits HPV-positive HNSCC cell growth. A, qRT-PCR assays validate that the expression of CNR1 is successfully downregulated by pooled siRNA in HPV-positive HNSCC UD-SCC-2, UPCI:SCC090, UM-SCC-47, and 93VU147T cells. B, qRT-PCR assays validate that the expression of CNR2 is successfully downregulated by pooled siRNA in HPV-positive HNSCC UD-SCC-2, UPCI:SCC090, UM-SCC-47, and 93VU147T cells. C, Cell viability is measured after knockdown of the expression of CNR1 in HPV-positive HNSCC cells. Growth is normalized to day 0 and measured over 3 days, and proliferation ratio is calculated relative to day 0. Significant growth inhibition is seen with specific silencing of CNR1 compared with the parental and NC group in UD-SCC-2, UM-SCC-47, and 93VU147T cells. D, Cell viability assay shows significant growth decrease by knockdown of CNR2 in UPCI:SCC090, UM-SCC-47, and 93VU147T cells. Experiments were performed in triplicate and the statistical comparisons were determined with Student t test. *, P < 0.05; **, P < 0.01; ***, P < 0.001; NS, not significant.
- Figure 3.
CNR1 and CNR2 agonists or antagonists affect the proliferation of HPV-positive HNSCC cells. A–C, Treatment with different concentrations (1 nmol/L, 10 nmol/L, 100 nmol/L, and 1 μmol/L) of selective CNR1 agonist ACEA (A), selective CNR2 agonist Hu308 (B), or nonselective cannabinoid receptor agonist THC (C) promotes the proliferation of HPV-positive HNSCC UD-SCC-2 cells. D and E, Treatment with different concentrations (1 nmol/L, 10 nmol/L, 100 nmol/L, and 1 μmol/L) of the selective CNR1 antagonist rimonabant (D) or the selective CNR2 antagonist SR144528 (E) inhibits the proliferation of UD-SCC-2 cells. F, Overview of the effects of cannabinoids on different HPV-positive HNSCC cells growth: red, growth stimulation; blue, growth inhibition; black, nonresponse in growth. Experiments were performed in triplicate, and the statistical comparisons were determined with Student t test. *, P < 0.05; **, P < 0.01.
- Figure 4.
CNR1 and CNR2 agonists or antagonists affect the apoptosis and migration of HPV-positive HNSCC cells. A and B, Treatment with 1 μmol/L ACEA (A) and Hu308 (B) inhibits apoptosis of HPV-positive HNSCC UPCI:SCC090 cells. C, Treatment with 1 μmol/L THC inhibits the apoptosis of HPV-positive HNSCC UM-SCC-47 cells. D and E, Treatment with 1 μmol/L rimonabant (D) and SR144528 (E) induces the apoptosis of HPV-positive HNSCC UM-SCC-47 and HNSCC 93VU147T cells, respectively. F–H, Treatment with 1 μmol/L ACEA (F), Hu308 (G), and THC (H) promotes the migration of HPV-positive HNSCC UD-SCC-2 cells. I and J, Treatment with 1 μmol/L rimonabant (I) and SR144528 (J) inhibits the migration of HPV-positive HNSCC UD-SCC-2 cells. Experiments were performed in triplicate, and the statistical comparisons were determined with Student t test. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
- Figure 5.
Activation of cannabinoid receptor is associated with the p38 MAPK pathway. A–C, Treatment with 1 μmol/L ACEA, Hu308, and THC for 5 minutes (A), 15 minutes (B), and 30 minutes (C) in UPCI:SCC090 cells; in general, the expression of p-p38, p-MAPKAPK2, and p-HSP27 in the p38 MAPK pathway is elevated.
- Figure 6.
Activation of cannabinoid receptor promotes proliferation of HPV-positive HNSCC cells xenografts. A and B, THC-treated tumor xenografts grow substantially faster compared with those in the control group. C, The average weight of the tumors in the THC-treated group is significantly heavier than those of the control group. D, Western blotting shows the p38 MAPK pathway is activated in THC-treated tumor xenografts. E and F, Tumor xenografts grow substantially slower in rimonabant, SR144528, and SB203580-treated group compared with those in control group. G, The average weight of the tumors in the rimonabant and SB203580-treated group is lighter than those of the control group. H and I, CNR1 or CNR2 silenced tumor xenografts grow substantially slower compared with those in EV group. J, The average weight of the tumors in CNR1 or CNR2 knockdown group is significantly lighter than those of the EV group. *, P < 0.05; **, P < 0.01; NS, not significant.
- Figure 7.
The OncoGenome Positioning System map. A, Onco-GPS map of cellular states including all 32 HPV-positive tumor samples. The tumors aggregate roughly into three cellular or oncogenic states, each characterized by a unique pattern of pathway expression. B, Onco-GPS map showing tumor samples derived from patients with cannabinoid exposure (shown in black) and without cannabinoid exposure (shown in gray). Four of the five cannabis-positive samples aggregate into State 0 (purple). Membership in State 0 includes 13 samples overall. C, Onco-GPS shows samples colored according to FGFR1 pathway activity. All tumors with higher enrichment in FGFR1 signaling activity are present in State 0, the majority of these samples deriving from cannabinoid-positive patients. D, Heat map demonstrating ssGSEA enrichment of four selected pathways. FGFR1 signaling (high) and apoptosis (low) represent robust oncogenic characteristics of State 0. Expression patterns corresponding to cannabinoid-positive samples are indicated with a black box.
Additional Files
Supplementary Data
- Figure S1-9 - Figure S1: CNR1 and CNR2 affect the proliferation of HPV positive HNSCC cells. Figure S2: High concentrations of cannabinoids inhibit the growth of HPV positive HNSCC cells. Figure S3: Inhibition of CNR1 and CNR2 attenuates the effect of CNR1 / CNR2 agonists on HPV positive HNSCC cells proliferation. Figure S4: CNR1 and CNR2 agonists or antagonists affect the migration of HPV positive HNSCC cells. Figure S5: Cannabinoid receptor agonist activates p38 MAPK pathway. Figure S6: Inhibition of p38 MAPK attenuates the effect of CNR1 / CNR2 agonists on HPV positive HNSCC cells proliferation. Figure S7: Expression of CNR1 and CNR2 in inducible shRNA stably transfected UM-SCC-47 cells. Figure S8: Survival between patients with and without cannabinoids exposure. Figure S9: Gene set enrichment analysis.
- Table S1 - Expression of cannabinoids and their metabolites in plasma samples of HPV positive HNSCC patients identified by LC-MS/MS.
- Table S2 - Correlations between cannabinoids exposure and clinicopathological parameters.
- Table S3 - List of dysregulated pathways in patients with cannabinoid metabolites compared with patients without cannabinoid metabolites.
- Table S4 - List of relatively enriched pathways for each transcriptional component forming the Onco-GPS map.
- Table S5 - List of relatively enriched pathways corresponding to each oncogenic state forming the Onco-GPS map.
- Supplementary Data Legends - Supplementary Data Legends
Volume 26, Issue 11
