Cannabinoids Promote Progression of HPV-Positive Head and Neck Squamous Cell Carcinoma via p38 MAPK Activation

Abstract

Purpose: Human papillomavirus (HPV)-related head and neck squamous cell carcinoma (HNSCC) is associated with daily marijuana use and is also increasing in parallel with increased marijuana use in the United States. Our study is designed to define the interaction between cannabinoids and HPV-positive HNSCC.

Experimental design: The expression of cannabinoid receptors CNR1 and CNR2 was analyzed using The Cancer Genome Atlas (TCGA) HNSCC data. We used agonists, antagonists, siRNAs, or shRNA-based models to explore the roles of CNR1 and CNR2 in HPV-positive HNSCC cell lines and animal models. Cannabinoid downstream pathways involved were determined by Western blotting and analyzed in a primary HPV HNSCC cohort with single-sample gene set enrichment analysis (ssGSEA) and the OncoGenome Positioning System (Onco-GPS).

Results: In TCGA cohort, the expression of CNR1 and CNR2 was elevated in HPV-positive HNSCC compared with HPV-negative HNSCC, and knockdown of CNR1/CNR2 expression inhibited proliferation in HPV-positive HNSCC cell lines. Specific CNR1 and CNR2 activation as well as nonselective cannabinoid receptor activation in cell lines and animal models promoted cell growth, migration, and inhibited apoptosis through p38 MAPK pathway activation. CNR1/CNR2 antagonists suppressed cell proliferation and migration and induced apoptosis. Using whole-genome expression analysis in a primary HPV HNSCC cohort, we identified specific p38 MAPK pathway activation signature in tumors from HPV HNSCC patients with objective measurement of concurrent cannabinoid exposure.

Conclusions: Cannabinoids can promote progression of HPV-positive HNSCC through p38 MAPK pathway activation.

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. The statistical comparisons were determined with 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 down-regulated 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 down-regulated 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 the expression of CNR1 in HPV positive HNSCC cells. Growth is normalized to day zero and measured over 3 days, and proliferation ratio is calculated relative to day zero. Significant growth inhibition is seen with specific silencing of CNR1 compared with parental and negative control (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’s 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 nM, 10 nM, 100 nM and 1 μM) of selective CNR1 agonist ACEA (A), selective CNR2 agonist Hu308 (B) or non-selective cannabinoid receptor agonist THC (C) promotes the proliferation of HPV positive HNSCC UD-SCC-2 cells. (D–E) Treatment with different concentrations (1 nM, 10 nM, 100 nM and 1 μM) of selective CNR1 antagonist Rimonabant (D) or 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, non-response in growth. Experiments were performed in triplicate and the statistical comparisons were determined with Student’s 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, B) Treatment with 1 μM ACEA (A) and Hu308 (B) inhibits apoptosis of HPV positive HNSCC UPCI:SCC090 cells. (C) Treatment with 1 μM THC inhibits the apoptosis of HPV positive HNSCC UM-SCC-47 cells. (D, E) Treatment with 1 μM 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 μM ACEA (F), Hu308 (G) and THC (H) promotes the migration of HPV positive HNSCC UD-SCC-2 cells. (I–J) Treatment with 1 μM 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’s t test. *: P < 0.05, **: P < 0.01, ***: P < 0.001.

Figure 5. Activation of cannabinoid receptor is associated with p38 MAPK pathway

(A–C) Treatment with 1 μM ACEA, Hu308 and THC for 5 min (A), 15 min (B) and 30 min (C) in UPCI:SCC090 cells, in general, the expression of p-p38, p-MAPKAPK2 and p-HSP27 in p38 MAPK pathway is elevated.

Figure 6. Activation of cannabinoid receptor promotes proliferation of HPV positive HNSCC cells xenografts

(A, B) THC treated tumor xenografts grow substantially faster compared to those in control group. (C) The average weight of the tumors in THC treated group is significantly heavier than those of the control group. (D) Western blotting shows p38 MAPK pathway is activated in THC treated tumor xenografts. (E, F) Tumor xenografts grow substantially slower in Rimonabant, SR144528 and SB203580 treated group compared to those in control group. (G) The average weight of the tumors in Rimonabant and SB203580 treated group is lighter than those of the control group. (H, I) CNR1 or CNR2 silenced tumor xenografts grow substantially slower compared to those in empty vector group. (J) The average weight of the tumors in CNR1 or CNR2 knockdown group is significantly lighter than those of empty vector 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 grey). Four of the five cannabis positive 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) Heatmap 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.