CD73 facilitates invadopodia formation and boosts malignancy of head and neck squamous cell carcinoma via the MAPK signaling pathway

Abstract

Elevated adenosine generated by CD73 (ecto-5'-nucleotidase; NT5E) could boost immunosuppressive responses and promote immune evasion in the tumor microenvironment. However, despite the immune response, CD73 could also promote tumor progression in a variety of cancers, and the nonimmunologic role and corresponding molecular mechanism of CD73 involved in head and neck squamous cell carcinoma (HNSCC) progression are not well characterized. Here, we demonstrated that CD73/NT5E is overexpressed in HNSCC tissues and predicts poor prognosis. Suppression of CD73 inhibited the proliferation, migration, and invasion of HNSCC cell lines (CAL27 and HN4) in vitro and in vivo. Gene set variation analysis (GSVA) and gene set enrichment analysis (GSEA) predicted that CD73 may be involved in invadopodia formation and MAPK signaling activation. As expected, knockdown of CD73 inhibited the MAPK signaling pathway, and the suppressive effect of CD73 knockdown on proliferation, migration, invasion, and invadopodia formation was reversed by a MAPK signaling activator. Our results suggest that CD73 could promote the proliferation, migration, invasion, and invadopodia formation of HNSCC via the MAPK signaling pathway and provide new mechanistic insights into the nonimmunological role of CD73 in HNSCC.

Keywords: CD73; EMT; HNSCC; MAPK signaling pathway; invadopodia.

FIGURE 1

CD73/NT5E is overexpressed and predicts poor prognosis in head and neck squamous cell carcinoma (HNSCC). A, Expression of NT5E in HNSCC tissues (n = 81) compared with paired adjacent normal tissues (n = 81) in the Jiangsu Key Laboratory of Oral Disease (JKLOD) cohort and the significance of difference was evaluated by paired t test. B, Expression of NT5E in HNSCC tissues (n = 502) compared with adjacent normal tissues (n = 44) in the TCGA‐HNSCC cohort and the significance of difference was evaluated by unpaired t test. C, Western blot detecting the expression of CD73 in 10 pairs of HNSCC tissues and adjacent normal tissues. D, Immunohistochemistry (IHC) staining of CD73 in HNSCC tissues and adjacent normal tissues. E, F, Kaplan‐Meier curves for the overall survival and relapse‐free survival of 502 patients in the TCGA‐HNSCC cohort divided into NT5E_high and NT5E_low groups according to NT5E expression. HR, hazard ratio. G, H, Kaplan‐Meier curves for the overall survival and disease‐free survival of 122 patients divided into NT5E_high and NT5E_low groups according to CD73 expression by IHC staining. I, Multivariate Cox regression analysis of CD73 by IHC score along with clinical prognostic parameters for 122 HNSCC patients. ns, not significant; *p < 0.05, **p < 0.01, and ***p < 0.001

FIGURE 2

Depletion of CD73 inhibits the proliferation, migration, and invasion of head and neck squamous cell carcinoma (HNSCC) cells. A, Western blot detecting the expression of CD73 in HNSCC cell lines and HOK cells. B, Western blot detecting the expression of CD73 in CAL27 and HN4 cells transfected with si‐NC and si‐CD73. C, D, CCK‐8 assay estimating the viability of CAL27 and HN4 cells after transfection with si‐NC and si‐CD73. E, F, EdU assay estimating the proliferation ratio of CAL27 and HN4 cells after transfection with si‐NC and si‐CD73. G, Colony‐formation assay estimating the colony‐formation ability of CAL27 and HN4 cells after transfection with si‐NC and si‐CD73. H, J, Wound‐healing assay estimating the migration ability of CAL27 and HN4 cells after transfection with si‐NC and si‐CD73. I, K, Transwell migration and invasion assays estimating the migration and invasion ability of CAL27 and HN4 cells after transfection with si‐NC and si‐CD73. L, Western blot detecting the expression of EMT‐related and proliferation‐related markers in CAL27 and HN4 cells after transfection with si‐NC and si‐CD73. ns, not significant; *p < 0.05, **p < 0.01, and ***p < 0.001

FIGURE 3

Depletion of CD73 inhibits invadopodia formation in head and neck squamous cell carcinoma (HNSCC) cells. A, Correlation analysis between NT5E expression and gene set variation analysis (GSVA) scores of biological processes. The top 10 correlated biological processes with Spearman correlation coefficients are shown in the left panel, and the correlation dot plot of NT5E and GSVA score of “invadopodium” is shown in the right panel. B, Gene set enrichment analysis (GSEA) results of invadopodia‐related biological processes. C, Western blot detecting the expression of invadopodia‐related markers in CAL27 and HN4 cells transfected with si‐NC and si‐CD73. D, Double fluorescence staining with phalloidin to label F‐actin and anti‐cortactin antibody for si‐NC– or si‐CD73–transfected CAL27 and HN4 cells. E, Fluorescent gelatin degradation assay evaluating the invasion ability of invadopodia in si‐NC– or si‐CD73–transfected CAL27 and HN4 cells. ns, not significant; *p < 0.05, **p < 0.01, and ***p < 0.001

FIGURE 4

CD73 activates MAPK signaling pathway. A, B, Gene set enrichment analysis (GSEA) plots showing that “pathway in cancer” and “MAPK signaling pathway” were highly enriched in the NT5E_high groups. C, Heatmap showing the expression of genes in the MAPK signaling pathway in the NT5E_high and NT5E_low groups. D, E, Western blot analysis of the expression of markers in the MAPK signaling pathway in CAL27 and HN4 cells transfected with si‐NC and si‐CD73. F, G, Western blot detecting the expression of markers in the MAPK signaling pathway in CAL27 and HN4 cells with combined treatment of CD73 knockdown and LM22B

FIGURE 5

CD73 promotes proliferation, migration, invasion, and invadopodia formation via the MAPK signaling pathway. A, B, Colony‐formation assay estimating the colony‐formation ability of CAL27 and HN4 cells with combined treatment of CD73 knockdown and LM22B. C, D,) Wound‐healing assay estimating the migration ability of CAL27 and HN4 cells with combined treatment of CD73 knockdown and LM22B. E, F,) Transwell migration and invasion assays estimating the migration and invasion ability of CAL27 and HN4 cells with combined treatment of CD73 knockdown and LM22B. G, H, Fluorescent gelatin degradation assay evaluating the invasion ability of invadopodia in CAL27 and HN4 cells with combined treatment of CD73 knockdown and LM22B. I, J, Western blot detecting the expression of markers related to EMT phenotype, proliferation ratio, and invadopodia formation in CAL27 and HN4 cells with combined treatment of CD73 knockdown and LM22B. ns, not significant; *p < 0.05, **p < 0.01, and ***p < 0.001

FIGURE 6

Depletion of CD73 inhibits the proliferation and metastasis of head and neck squamous cell carcinoma (HNSCC) cells in vivo. A, General images of subcutaneous tumors of lenti‐shNC– or lenti‐shCD73–transfected CAL27 cells from a xenograft nude mouse model. B, Volume of tumors in lenti‐shNC or lenti‐shCD73 groups at indicated time. E, The weights of the tumors are measured. D‐F, White light and H&E staining images of pulmonary metastasis of lenti‐shNC– or lenti‐shCD73–transfected CAL27 cells from a lung metastasis model. The number of metastases and weight of the metastatic lung were measured. G, Immunohistochemistry (IHC) staining of Ki67, E‐cadherin, vimentin, and NWASP. H, The protein expression of Ki67, E‐cadherin, vimentin, and NWASP was measured. ns, not significant; *p < 0.05, **p < 0.01, and ***p < 0.001