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.


| INTRODUC TI ON
Head and neck cancer (HNC) was the world's seventh most common cancer in 2018, with most cases being squamous cell carcinoma arising from mucosal surfaces of four major anatomical sites: the oral cavity, sinonasal cavity, larynx, and pharynx. 1 Head and neck squamous cell carcinoma (HNSCC) is a global health burden due to its high incidence and poor prognosis. There are approximately 600,000 new cases worldwide, with 40-50% mortality. 2,3 Current primary modalities for the treatment of HNSCC are surgery, radiotherapy, and concurrent chemoradiation. Accounting for distinct anatomical subgroups, invasive properties, and cervical lymph node metastasis of the tumors, the prognosis of this malignancy is still frustrating despite employment of combined treatment. 4,5 Therefore, further investigation into the underlying mechanism is of great help to improve the prognosis of HNSCC patients.
Ecto-5′-nucleotidase (CD73), encoded by the NT5E gene, is a 70-kD glycosylphosphatidylinositol (GPI) protein anchored on the cell surface that can function in enzymatic or nonenzymatic pathways. 6,7 As an AMP hydrolyzing enzyme, CD73 is involved in the conversion of AMP into phosphate and adenosine, which play important roles in immunomodulation in the tumor microenvironment (TME). 7,8 In addition to enzymatic function, CD73 interacts directly with extracellular matrix (ECM) components and is engaged in T-cell signal transduction and cell adhesion. 9 CD73 has been reported to influence cancer progression in both enzymatic and nonenzymatic pathways. 7 Ample evidence has shown that CD73 is overexpressed in many cancers, such as breast cancer, non-small cell lung cancer, gastric cancer, colorectal cancer, ovarian cancer, and gallbladder cancer. Furthermore, CD73 has been found to be linked to the clinical characteristics and prognosis of cancer patients and plays a critical role during tumor progression. 6,[10][11][12][13] Recently, it was reported that the expression of CD73 is upregulated in clinical HNSCC tissues and that CD73 expression in HNSCC correlated positively with tumor stage and poor prognosis. However, whether CD73 could facilitate HNSCC progression and the underlying molecular mechanism still needs further elucidation. 10,14 Metastasis is the major reason for cancer-related death. 15 Invasion and penetration of ECM, or other tissue barriers, is a prerequisite in cancer metastasis. 16 Invadopodia, dynamic actin-rich protrusions in cancer cells, specialize in degrading ECM, thus facilitating tumor metastasis. 15,17 Signaling, such as growth factor and integrins, could stimulate F-actin assembly, which is modulated by structural proteins, such as N-WASP, TKS4, TKS5, and cortactin, priming the formation of invadopodia. Proteases, such as MT1-MMP, MMP2, and MMP9, are especially enriched in invadopodia and released to drill the ECM. 17 Mitogen-activated protein kinase (MAPK) signaling pathways constitute a complex and delicate network modulating cellular biological processes such as proliferation, differentiation, and cell survival. 18,19 As a result, aberrant MAPK signal transduction is involved in a variety of human diseases, especially cancer. 20 The MAPK cascade mainly consists of three crucial kinases, MAPK3K, MAPKK, and MAPK, which work through sequential phosphorylation. 20 There are three distinctive cascades of MAPKs: extracellular signal-regulated kinases (ERKs), Jun amino terminal kinases (JNKs), and stressactivated protein kinases (p38/SAPKs). ERKs tend to be activated by growth factors or mitogens and are responsible for proliferation, survival, differentiation, metabolism, nervous system development, and immune response, while p38 MAPKs can respond to multiple inflammatory and stressful factors and participate in inflammation, proliferation, differentiation, apoptosis, and invasion. Similarly, JNKs are usually activated by stress factors such as oxidative stress and radiation and are responsible for inflammation, apoptosis, cytokine secretion, and metabolism. [21][22][23] In this study, we analyzed the clinical implication of CD73 in HNSCC. Additionally, we performed GSEA and GSVA for CD73 to predict the biological function of CD73 and the corresponding signaling pathway, which was further verified by in vitro and in vivo experiments.

| MATERIAL S AND ME THODS
The materials and methods are described in Appendix S1.

| CD73/NT5E expression was significantly upregulated in HNSCC and predicted poor prognosis
To explore the expression difference of CD73/NT5E between HNSCC tissues and adjacent normal tissues, we first extracted the mRNA quantification of NT5E in the transcriptomic sequence dataset of our JKLOD cohort, including 81 pairs of oral squamous cell carcinoma tissues and adjacent normal tissues. The results showed that NT5E was significantly upregulated in oral squamous cell carcinoma tissues ( Figure 1A, p < 0.0001). The high expression of NT5E in HNSCC tissue was also confirmed in the TCGA-HNSCC cohort, which included 502 HNSCC tissues and 44 adjacent normal tissues ( Figure 1B p < 0.0001). In addition to transcriptome analysis, we also found that the protein level of CD73 was robustly upregulated in HNSCC tissues in 10 pairs of normal and tumor tissues ( Figure 1C), which was further verified by IHC ( Figure 1D). short progression-free survival ( Figure 1H, HR = 1.66; log-rank p = 2.4 × 10 −5 ). The clinical feature of CD73 was also investigated in the JKLOD cohort. It turned out that higher CD73 expression is associated with later pathological staging and it may be correlated with later T staging or N staging, while the sample size could not make the difference significant ( Figure S1). Multivariate Cox regression analysis involving clinical features further indicated F I G U R E 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 that CD73/NT5E is an independent prognostic factor for HNSCC ( Figure 1I, HR = 3.01, p < 0.001).

| CD73 promotes the proliferation, migration, and invasion of HNSCC cells
To investigate the functional role of CD73 in HNSCC progression, cells also demonstrated compromised proliferation and EMT phenotypes ( Figure 2L). Meanwhile, we also overexpressed CD73 in CAL27 and HN4 and repeated all the above phenotype experiment.
Taken together, these data strongly indicated that CD73 could promote the proliferation, migration, and invasion of HNSCC cells.

| CD73 promotes invadopodia formation in HNSCC cells
With the role of CD73 in HNSCC cell malignancy confirmed, we next investigated the specific biological processes in which CD73 is involved. First, a transcriptome sequence dataset of 502 tumor tissues in the TCGA-HNSCC cohort was downloaded. Utilizing the GSVA algorithm, we scored 9996 gene sets of biological processes for each sample. Then, Spearman correlation analysis was employed to calculate the correlation between NT5E/CD73 expression and the score of each biological process. The results showed that "invadopodium" and "invadopodium membrane" were among the top 10 correlated biological processes ( Figure 3A). In addition, we divided the 502 samples into two groups based on the expression of NT5E/ CD73 and analyzed the biological processes enriched in the group with high NT5E/CD73 expression. Invadopodium-and cytoskeletal regulation-related biological processes were highly enriched ( Figure 3B).
To test the correlation between CD73 and invadopodia formation, we performed Western blotting to detect the changes in key proteins involved in invadopodia formation upon CD73 knockdown, including WASP, NWASP, and MMP14 ( Figure 3C). 17 Interestingly, all invadopodia-related markers were downregulated after suppression of CD73 in CAL27 and HN4 cells. Then, we performed double fluorescence staining with phalloidin to label F-actin and anti-cortactin antibody for si-NC-or si-CD73-transfected CAL27 and HN4 cells. 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 HN4. It turned out that CAL27 and HN4 with higher CD73 expression showed even more vigorous invadopodia ( Figure S4).
These results further indicated that CD73 may boost HNSCC malignancy by facilitating invadopodia formation.

| CD73 could activate the MAPK signaling pathway
Our results support that CD73 can promote HNSCC progression in many ways; however, the potential molecular mechanism is largely unknown. First, bioinformatic analysis was employed to predict the possible relevant pathways. RNA sequencing data of the TCGA-HNSCC cohort were divided into two groups according to the mRNA expression of CD73/NT5E. KEGG pathways enriched in the NT5E_ high group were calculated utilizing the GSEA algorithm. The results showed that "Pathways in cancer" and "MAPK signaling pathway" were significantly enriched ( Figure 4A,B). In addition, genes related to the MAPK signaling pathway showed differential expression between the NT5E_high and NT5E_low groups ( Figure 4C). Then, we detected by immunoblotting the expression of key proteins in the MAPK signaling pathway affected by CD73 interference. Our results showed that knockdown of CD73 could inhibit the phosphorylated forms of p38 MAPK, MEK1/2, and ERK1/2 but barely influence the expression level of total p38 MAPK, MEK1/2, and ERK1/2 in CAL27 and HN4 cells ( Figure 4D,E). Additionally, the changes in these markers of the MAPK signaling pathway could be reversed by LM22B, which could activate the MAPK signaling pathway. Therefore, we concluded that CD73 could activate the MAPK signaling pathway ( Figure 4F,G).

| CD73 boosts HNSCC malignancy via the MAPK signaling pathway
As the MAPK signaling pathway could be activated by CD73, we and HN4 cell lines ( Figure 5G,H). Finally, the immunoblotting assay was employed to detect the change in specific markers upon Lenti-shControl or Lenti-shCD73 transfection and LM22B treatment.
Lenti-shCD73, similar to si-CD73, increased the expression of E-cadherin and decreased the expression of N-cadherin, vimentin, Snail, Ki67, PCNA, WASP, NWASP, and MP-MMP, indicating compromised EMT phenotype, proliferation ratio, and invadopodia formation, while these markers showed opposite changes upon LM22B treatment compared with Lenti-shCD73 transfection. Furthermore, LM22B treatment reversed the change in these markers caused by lenti-shCD73 ( Figure 5I,J). In view of the above, we concluded that CD73 could boost HNSCC malignancy via the MAPK signaling pathway.

| Knockdown of CD73 inhibits tumor growth and lung metastasis in vivo
To determine the effect of CD73 in vivo, we established xenograft and lung metastasis models with Lenti-shControl and Lenti-shCD73 To predict the specific biological processes in which CD73 is involved, we analyzed the correlation between the expression of CD73 and the GSVA score of all the biological processes. Interestingly, "invadopodium" was among the top 10 correlated biological processes according to the Spearman correlation coefficient, which was also F I G U R E 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 confirmed by GSEA. There is evidence that CD73 is involved in cytoskeletal regulation and could influence the invasion properties of gastric cancer cells. 32 WASP and NWASP are key proteins regulating pseudopodia and invadopodia assembly, and MT1-MMP denotes the maturation and invasion properties of invadopodia. Western blot analysis showed that WASP, NWAP, and MT1-MMP were significantly downregulated after CD73 knockdown. Double fluorescence staining with phalloidin to label F-actin and anti-cortactin showed decreased pseudopodia, and a fluorescent gelatin degradation assay showed decreased invaded gelatin by invadopodia after CD73 suppression. Thus, we conclude that CD73 is involved in invadopodia formation in HNSCC.
Furthermore, we investigated the potential molecular mechanism through which CD73 boosts the malignancy of HNSCC. GSEA revealed that the "MAPK signaling pathway" was significantly upregulated in tumor tissues with higher NT5E expression. Previous studies showed that CD73 could activate the MEK/ERK and p38 MAPK signaling pathways, which are two major branches of the MAPK signaling pathway. 38,39 Interestingly, MAPK signaling could also promote the expression of CD73 via transcriptional regulation. 40 Additionally, ERK signaling is responsible for invadopodia formation. 14,41,42 As expected, the phosphorylated forms of p38 MAPK, MEK1/2, and ERK1/2 were inhibited by knockdown of CD73. Furthermore, functional recovery experiments indicated that activating the MAPK signaling pathway could reverse the effect of CD73 knockdown on cell proliferation, migration, invasion, and invadopodia formation. Thus, we speculated that CD73 could promote the proliferation, migration, invasion and invadopodia formation of HNSCC cells, while the specific mechanisms still need to be clarified further.
In summary, CD73/NT5E was highly expressed in HNSCC tissues and predicted poor prognosis, indicating its potential as a diagnostic and prognostic biomarker. It could also promote proliferation, migration, invasion, and invadopodia formation via the MAPK signaling pathway. Moreover, CD73 depletion of HNSCC cells effectively suppressed proliferation and metastasis in a xenograft or lung metastasis nude mouse model, suggesting CD73 as a promising therapeutic target through nonimmune pathways.

ACK N OWLED G M ENTS
This work was supported in part by National Natural Science

D I SCLOS U R E
The authors have declared that no competing interests exist.

E TH I C S S TATEM ENT
The study was authorized by the research ethics committee of Stomatological Hospital of Jiangsu Province.

I N FO R M ED CO N S ENT
All the subjects signed informed consent on sample acquisition and privacy protection.

A N I M A L S TU D I E S
The animal studies were implemented according to the guidelines of the Committee of Nanjing Medical University for Animal Resources (Approval ID 2102024).