Search

Integrin β4 (ITGB4) is a member of the integrin family, which plays a crucial role in mediating cell adhesion to the extracellular matrix. Recent studies have demonstrated that ITGB4 is involved in tumorigenesis and metastasis during the development of cancer. However, the role of ITGB4 in oral squamous cell carcinoma (OSCC) remains unclear. A Multiplex immunohistochemistry (OPAL™, mIHC) assay was employed to stain ITGB4, ALDH1, PD-L1, cytokeratin (CK), CD8 and PD-1 in a human OSCC tissue microarray, containing 26 normal oral epithelium samples, 21 oral epithelium dysplasia samples and 76 OSCC samples. The expression pattern and clinicopathological characteristics of ITGB4 were analyzed and compared with those of PD-1, PD-L1, ALDH1 and CD8. The correlation between subgroups of tumor cells, including ITGB4+PD-L1+ and ITGB4+ALDH1+, and subgroups of T cells, including CD8+ and CD8+PD-1+, was evaluated using two-tailed Pearson’s statistics. A Kaplan–Meier curve was built, and a log-rank test was performed to analyze the survival rate of different subgroups. The mIHC staining results show that ITGB4 was mostly expressed in the tumor cells, with a significant increase in the OSCC specimens compared with normal oral epithelium and oral epithelium dysplasia. The paired analysis, conducted between the OSCC tumor tissue and normal paracancer mucosa, confirmed the results. The study further revealed that ITGB4+PD-L1+ cancer cells, but not ITGB4+ALDH1+ cancer cells, were significantly associated with the infiltration of CD8+ T cells (positivity p = 0.005, positive number p = 0.03). Additionally, ITGB4+PD-L1+ tumor cells were positively correlated with CD8+PD-1+ T cells (positivity p = 0.02, positive number p = 0.03). Most intriguingly, the subgroup of ITGB4/PD-L1high with CD8/PD-1high displayed the best prognosis compared with the other considered subgroups. The results show that the expression of ITGB4 was increased in OSCC compared with normal oral mucosa. Furthermore, a specific subgroup with high levels of expression of ITGB4/PD-L1 and CD8/PD-1 was found to have a relatively better prognosis compared with the other subgroups. Ultimately, this study sheds light on the potential role of ITGB4 in OSCC and provides a basis for further investigation.

Immature myeloid cells including myeloid‐derived suppressor cells (MDSCs) and tumour‐associated macrophages (TAMs) promote tumour growth and metastasis by facilitating tumour transformation and angiogenesis, as well as by suppressing antitumour effector immune responses. Therefore, strategies designed to reduce MDSCs and TAMs accumulation and their activities are potentially valuable therapeutic goals. In this study, we show that negative immune checkpoint molecule B7‐H3 is significantly overexpressed in human head and neck squamous cell carcinoma (HNSCC) specimen as compared with normal oral mucosa. Using immunocompetent transgenic HNSCC models, we observed that targeting inhibition of B7‐H3 reduced tumour size. Flow cytometry analysis revealed that targeting inhibition of B7‐H3 increases antitumour immune response by decreasing immunosuppressive cells and promoting cytotoxic T cell activation in both tumour microenvironment and macroenvironment. Our study provides direct in vivo evidence for a rationale for B7‐H3 blockade as a future therapeutic strategy to treat patients with HNSCC.

T‐cell immunoglobulin mucin 3 (TIM3) contributes to immune suppression during progression of many cancers, but the precise role of TIM3 in head and neck squamous cell carcinoma (HNSCC) is not clearly understood. In this study, we report that TIM3 expression was significantly up‐regulated in patients with HNSCC and associated with lymph node metastasis. Additionally, TIM3 expression was increased in patients with recurrent HNSCC and patients with preradiotherapy or prechemotherapy. We also characterized CD8+ T cells and CD11b+ CD33+ myeloid‐derived suppressor cells (MDSCs) in human HNSCC, and found that their expression was positively correlated with TIM3 expression. To determine the underlying mechanism of TIM3 in immune response during HNSCC progression, we utilized the Tgfbr1/Pten 2cKO HNSCC mouse model with TIM3 overexpression. Treatment with anti‐TIM3 monoclonal antibody effectively suppressed tumor growth through restoring effector T‐cell function by targeting CD4+ TIM3+ cells and CD8+ TIM3+ cells and decreasing MDSCs. Our findings demonstrate TIM3 expression in patients with HNSCC and suggest anti‐TIM3 immunotherapy as a novel therapeutic approach for effective treatment of HNSCC.

// Teng-Fei Fan 1, * , Tian-Fu Wu 1, * , Lin-Lin Bu 1 , Si-Rui Ma 1 , Yi-Cun Li 1 , Liang Mao 1 , Zhi-Jun Sun 1, 2 , Wen-Feng Zhang 1, 2 1 The State Key Laboratory Breeding Base of Basic Science of Stomatology & Key Laboratory of Oral Biomedicine Ministry of Education, Wuhan University, Wuhan, China 2 Department of Oral Maxillofacial-Head Neck Oncology, School and Hospital of Stomatology, Wuhan University, Wuhan, China * These authors have contributed equally to this work Correspondence to: Zhi-Jun Sun, email: zhijundejia@163.com Wen-Feng Zhang, email: zhangwf59@whu.edu.cn Keywords: dihydromyricetin, autophagy, apoptosis, reactive oxygen species, head and neck squamous cell carcinoma Received: April 15, 2016 Accepted: July 10, 2016 Published: July 25, 2016 ABSTRACT Chemotherapy is an effective weapon in the battle against cancer, but numerous cancer patients are either not sensitive to chemotherapy or develop drug resistance to current chemotherapy regimens. Therefore, an effective chemotherapy mechanism that enhances tumor sensitivity to chemotherapeutics is urgently needed. The aim of the present study was to determine the antitumor activity of dihydromyricetin (DHM) on head and neck squamous cell carcinoma (HNSCC) and its underlying mechanisms. We demonstrated that DHM can markedly induce apoptotic cell death and autophagy in HNSCC cells. Meanwhile, increased autophagy inhibited apoptosis. Pharmacological or genetic inhibition of autophagy further sensitized the HNSCC cells to DHM-induced apoptosis. Mechanistic analysis showed that the antitumor of DHM may be due to the activation phosphorylation of signal transducer and activator of transcription 3 (p-STAT3), which contributed to autophagy. Importantly, DHM triggered reactive oxygen species (ROS) generation in the HNSCC cells and the levels of ROS decreased with N-acetyl-cysteine (NAC), a ROS scavenger. Moreover, NAC abrogated the effects of DHM on STAT3-dependent autophagy. Overall, the following critical issues were observed: first, DHM increased the p-STAT3-dependent autophagy by generating ROS-signaling pathways in head and neck squamous cell carcinoma. Second, inhibiting autophagy could enhance DHM-induced apoptosis in head and neck squamous cell carcinoma.

To assess the role of transcription factor μE3 (TFE3) in the tumorigenesis of head and neck squamous cell carcinoma (HNSCC), human HNSCC tissue arrays were investigated for TFE3 expression. Human HNSCC tissues with neoadjuvant inductive chemotherapey (docetaxel, cisplatin and fluorouracil, TPF) and mice HNSCC tissues from transgenic mice model were evaluated for TFE3 expression and the hypoxia pathway. The roles of EGF/EGFR mediated hypoxia in TFE3 nuclear expression were analyzed in vitro and in vivo. TFE3 expression was higher in human HNSCC tissues compared with that in normal oral mucosa. Moreover, high TFE3 expression was related to HIF-1α, PAI-1, and EGFR, which demonstrated the activation of the hypoxia pathway in HNSCC tissues. Furthermore, elevated TFE3 expression was observed in HNSCC after cisplatin-based chemotherapy, and high TFE3 expression may indicate poor response to TPF inductive chemotherapy. Furthermore, similar changes with increased TFE3 were observed in HNSCC of the transgenic mouse HNSCC model. Hypoxic culture in the human HNSCC cell line increased TFE3 expression, which promoted cell survival under hypoxia. EGFR inhibiton by cetuximab could attenuate hypoxia-induced TFE3 in the HNSCC cell line and transgenic mouse HNSCC model. These findings indicated that TFE3 was an important hypoxia-induced transcriptional factor in HNSCC. TFE3 could be regarded as a durgable therapeutic oncotarget by EGFR inhibition.

Signaling transducer and activator 3 (STAT3) and cancer stem cells (CSCs) have garnered huge attention as a therapeutic focus, based on evidence that they may represent an etiologic root of tumor initiation and radio-chemoresistance. Here, we investigated the high phosphorylation status of STAT3 (p-STAT3) and its correlation with self-renewal markers in head neck squamous cell carcinoma (HNSCC). Over-expression of p-STAT3 was found to have increased in post chemotherapy HNSCC tissue. We showed that blockade of p-STAT3 eliminated both bulk tumor and side population (SP) cells with characteristics of CSCs in vitro. Inhibition of p-STAT3 using small molecule S3I-201 significantly delayed tumorigenesis of spontaneous HNSCC in mice. Combining blockade of p-STAT3 with cytotoxic drugs cisplatin, docetaxel, 5-fluorouracil (TPF) enhanced the antitumor effect in vitro and in vivo with decreased tumor sphere formation and SP cells. Taken together, our results advocate blockade of p-STAT3 in combination with conventional chemotherapeutic drugs enhance efficacy by improving CSCs eradication in HNSCC.

Head and neck squamous cell carcinoma (HNSCC) is considered as an immunosuppressive disease, with impaired tumor-infiltrating T lymphocytes and increased suppressive immune cells. The efficacy of CD47 antibodies in immune checkpoint therapy is not clearly understood in HNSCC. In this study, human tissue microarrays and immunocompetent transgenic mouse models were used to explore the expression of CD47 and the use of CD47 antibodies in HNSCC. We identified overexpression of CD47 in HNSCC as compared with the control normal human tissue and also in HNSCC mouse models. The expression of CD47 also correlated with clinicopathological parameters as well as outcome. Furthermore, inhibition of CD47 delayed tumor growth and improved tumor microenvironment by stimulating effector T cells and decreasing suppressive immune cells and regulating the function of CD11b(+) Ly6G(+) MDSC. Our data suggest that CD47 blockade may be a potential immunotherapeutic target in human HNSCC.

Autophagy is considered as a double-edged sword. It can prolong the survival of cancer cells and enhance its resistance to apoptosis, and paradoxically, defective autophagy has been linked to increased tumorigenesis, but the mechanism behind this phenomenon is unclear. In this study, we demonstrated that decreased phosphorylation of signal transducer and activator of transcription 3 (p-STAT3) was correlated with increased autophagy through the Akt/mTOR and Erk signaling pathways in human head and neck squamous cell carcinoma (HNSCC). We also showed that blockage of STAT3 by NSC74859 could markedly induce apoptotic cell death and autophagy. Meanwhile, increased autophagy inhibited apoptosis. The pharmacological or genetic inhibition of autophagy and STAT3 further sensitized HNSCC cells to apoptosis. Furthermore, evidence from xenograft model proved that suppressed STAT3 activity combined with inhibition of autophagy promoted tumor regression better than either treatment alone. Taken together, this present study demonstrated that autophagy alleviates apoptotic cell death in HNSCC, and combination of inhibition of STAT3 by NSC74859 and autophagy might be a promising new therapeutic strategy for HNSCC.

// Si-Rui Ma 1 , Wei-Ming Wang 1 , Cong-Fa Huang 1 , Wen-Feng Zhang 1,2 and Zhi-Jun Sun 1,2 1 The State Key Laboratory Breeding Base of Basic Science of Stomatology & Key Laboratory of Oral Biomedicine Ministry of Education, Wuhan University, Wuhan, China 2 Department of Oral Maxillofacial-Head Neck Oncology, School and Hospital of Stomatology, Wuhan University, Wuhan, China Correspondence to: Zhi-Jun Sun, email: // Keywords : AGR2, HNSCC, cancer stemloid cell, epithelial mesenchymal transition Received : December 11, 2014 Accepted : February 10, 2015 Published : March 12, 2015 Abstract Anterior gradient protein 2 (AGR2) is a novel biomarker with potential oncogenic role. We sought to investigate the diagnostic and prognostic role of AGR2 on head and neck squamous cell carcinoma (HNSCC) with an emphasis on its correlation of cancer stemloid cells (CSC) and epithelial mesenchymal transition (EMT). We found that AGR2 protein levels were higher in HNSCC than in normal oral mucosa. High levels of AGR2 were associated with the T category, pathological grade and lymph node metastasis of HNSCC. Expression of AGR2 increased in recurring HNSCC after radiotherapy and in post cisplatin-based chemotherapeutic tissues. In HNSCC cell lines, knock-down of AGR2 induced apoptosis, reduced sphere formation, and down-regulated Survivin, Cyclin D1, Bcl2, Bcl2l1, Slug, Snail, Nanog and Oct4. In addition, over-expressed AGR2 in transgenic mice with spontaneous HNSCC was associated with lost function of Tgfbr1 and/ or lost function of Pten . In vitro knockdown TGFBR1 in HNSCC cell lines increased AGR2 expression. These results suggest that AGR2 is involved in EMT and self-renewal of CSC and may present a potential therapeutic target (oncotarget) for HNSCC.

The adenosine-induced immunosuppression hampers the immune response toward tumor cells and facilitates the tumor cells to evade immunosurveillance. CD73, an ecto-5-nucleotidase, is the ectoenzyme dephosphorylating extracellular AMP to adenosine. Here, using immunocompetent transgenic head and neck squamous cell carcinoma (HNSCC) mouse model, immune profiling showed high expression of CD73 on CD4+ and CD8+ T cells was associated with an "exhausted" phenotype. Further, treatment with anti-CD73 monoclonal antibody (mAb) significantly blunted the tumor growth in the mouse model, and the blockade of CD73 reversed the "exhausted" phenotype of CD4+ and CD8+ T cells through downregulation of total expression of PD-1 and CTLA-4 on T cells. Whereas the population of CD4+ CD73hi /CD8+ CD73hi T cells expressed higher CTLA-4 and PD-1 as compared to untreated controls. In addition, the human tissue microarrays showed the expression of CD73 is upregulated on tumor infiltrating immune cells in patients with primary HNSCC. Moreover, CD73 expression is an independent prognostic factor for poor outcome in our cohort of HNSCC patients. Altogether, these findings highlight the immunoregulatory role of CD73 in the development of HNSCC and we propose that CD73 may prove to be a promising immunotherapeutic target for the treatment of HNSCC.

Angiogenesis is an essential event in tumor growth and metastasis, and immune system also contributes to the tumor evasion. Emerging evidences have suggested the bidirectional link between angiogenesis and immunosuppression. Myeloid-derived suppressor cell (MDSC) is a kind of immunosuppressive cells and plays an important role in this process. However, the actual regulatory mechanisms of angiogenesis and MDSCs in head and neck squamous cell carcinoma (HNSCC) were unclear. In this study, through analyzing the immunohistochemistry staining of human HNSCC tissue microarray, we found that the microvascular density (MVD) was significantly increased in HNSCC patients. We also characterized angiogenic factors p-STAT3, VEGFA, CK2, and MDSCs marker CD11b in HNSCC tissue array, and found the close expression correlation among these markers. To determine the role of JAK2/STAT3 pathway in tumor microenvironment of HNSCC, we utilized AG490 (an inhibitor of JAK2/STAT3) for further research. Results showed that inhibition of JAK2/STAT3 suppressed angiogenesis by decreasing VEGFA and HIF1-α both in vitro and vivo. Moreover, in HNSCC transgenic mouse model, inhibiting JAK2/STAT3 not only suppressed angiogenesis but also reduced MDSCs in the tumor microenvironment through suppressing VEGFA and CK2. Our findings demonstrated the close relationship between angiogenesis and MDSCs in HNSCC, and inhibition of JAK2/STAT3 could reduce tumor-induced angiogenesis and decrease MDSCs.

Tumor necrosis factor receptor–associated factor 1, an adaptor protein of tumor necrosis factor 2, is involved in classical nuclear factor (NF)-κB activation and lymphocyte recruitment. However, less is known about the expression and association of tumor necrosis factor receptor–associated factor 1 with cancer stem cell markers in oral squamous cell carcinoma. This study aimed to investigate the expression of tumor necrosis factor receptor–associated factor 1 and stem cell characteristic markers (lin28 homolog B, B cell-specific Moloney murine leukemia virus integration site 1, and aldehyde dehydrogenase 1) in oral squamous cell carcinoma and analyze their relations. Paraffin-embedded tissues of 78 oral squamous cell carcinomas, 39 normal oral mucosa, and 12 oral dysplasia tissues were employed in tissue microarrays, and the expression of tumor necrosis factor receptor–associated factor 1, B cell-specific Moloney murine leukemia virus integration site 1, aldehyde dehydrogenase 1, and lin28 homolog B was measured by immunohistostaining and digital pathological analysis. The expression of tumor necrosis factor receptor–associated factor 1 was higher in the oral squamous cell carcinoma group as compared with the expression in the oral mucosa (p 2 = 0.109; p 2 = 0.64; and p 2 = 0.16) in oral squamous cell carcinoma. The patient survival rate was lower in the highly expressed tumor necrosis factor receptor–associated factor 1 group, although the difference was not significant. The clustering analysis showed that tumor necrosis factor receptor–associated factor 1 was most related to aldehyde dehydrogenase 1. These findings suggest that tumor necrosis factor receptor–associated factor 1 has potential direct/indirect regulations with the cancer stem cell markers in oral squamous cell carcinoma, which may help in further analysis of the cancer stem cell characteristics.

Immunotherapy with immune checkpoint molecule-specific monoclonal antibody have obtained encouraging results from preclinical studies and clinical trials, which promoted us to explore whether this kind of immunotherapy could be applicable to head and neck squamous cell carcinoma (HNSCC). Lymphocyte activation gene-3 (LAG-3) is an immune checkpoint control protein that negatively regulates T cells and immune response. Here, using the human tissue samples, we report these findings that LAG-3 is overexpressed on tumor-infiltrating lymphocytes (TILs; p < 0.001) and its overexpression correlates with the high pathological grades, lager tumor size and positive lymph node status in human primary HNSCC. Survival analysis identifies LAG-3 as a prognostic factor independent of tumor size and pathological grades for primary HNSCC patients with negative lymph node status (p = 0.014). Study in immunocompetent genetically defined HNSCC mouse model reports that LAG-3 is upregulated on CD4+ T cells, CD8+ T cells and CD4+Foxp3+ regulatory T cells (Tregs). In vivo study, administration of LAG-3-specific antibody retards tumor growth in a way associated with enhanced systemic antitumor response by potentiating the antitumor response of CD8+ T cells and decreasing the population of immunosuppressive cells. Taken together, our results offer a preclinical proof supporting the immunomodulatory effects of LAG-3 and suggest a potential therapeutic target of immunotherapy for HNSCC.

Objective Our aim is to evaluate the expression of SATB1 in human oral squamous cell carcinomas (OSCC) and its role in the invasiveness and metastasis of OSCC. Subjects and methods A human OSCC tissue microarray was used to evaluate the expression pattern of SATB1. SATB1 mRNA knockdown was performed in human OSCC cell lines SCC25 and Cal27 to assess the function of SATB1 in the invasiveness and metastasis of OSCC. Results SATB1 is highly expressed in human OSCC determined by immunohistochemistry, and its nuclear/cytoplasmic ratio of histoscore is significantly correlated with patients’ prognosis. Reduced cell motility, invasiveness, expression of epithelial to mesenchymal transition (EMT) markers (N-cadherin and β-catenin), and elevated expression of epithelial markers were observed in SATB1-knockdown cells in in vitro studies. Depletion of SATB1 also restored a cobblestone-like morphology in TGF-β1-treated cells. Conclusions These findings suggest SATB1 may play an important role in OSCC invasiveness and metastasis.

SRC family kinases (SFKs), a group of nonreceptor tyrosine kinases, modulate multiple cellular functions, such as cell proliferation, differentiation and metabolism. SFKs display aberrant activity in progressive stages of human cancers. However, the precise role of SFKs in the head and neck squamous cell carcinoma (HNSCC) signaling network is far from clear. In this study, we found that the inhibition of SFKs activity by dasatinib effectively reduced the tumor size and population of MDSCs in the HNSCC mouse model. Molecular analysis indicates that phosphorylation of LYN, rather than SRC, was inhibited by dasatinib treatment. Next, we analyzed LYN expression by immunostaining and found that it was overexpressed in the human HNSCC specimens. Moreover, LYN expression in stromal cells positively correlated with myeloid-derived suppressor cells (MDSCs) makers CD11b and CD33 in human HNSCC. The dual positive expression of LYN in epithelial and stromal cells (EPI

P21 activated kinase 2 (PAK2) is a member of Group I PAKs family and highly expressed in various cancers. Current studies have demonstrated that PAK2 played a pivotal role in tumor progression. However, the role of PAK2 in salivary adenoid cystic carcinoma is still unclear. This study aims to explore the expression and the function of PAK2 in AdCC. Human salivary gland tissue microarray, including 18 normal salivary glands (NSG), 12 pleomorphic adenoma (PMA) and 72 AdCC, and immunohistochemistry were used to evaluate the expression of PAK2. The result showed that PAK2 was significantly increased in AdCC compared with NSG and PMA. Then the Pearson correlation analysis using serial tissue sections showed a close correlation of PAK2 with Cyclin D1, Phospho-STAT3 at Tyrosine 705 (p-STAT3) and Ki-67. Further in vitro study utilizing PAK2 knockdown via siRNA transfection revealed significantly reduced migration and proliferation of AdCC cell lines compared with control group. Knockdown of PAK2 decreased the expression of Cyclin D1 in AdCC cell lines. In addition, the inhibition of STAT3 reduced the expression of PAK2 in AdCC cell lines. These findings suggested that PAK2 promotes AdCC cell migration and proliferation and may be a potential therapeutic target.

Cancer stem cells (CSCs) are considered responsible for tumor initiation and chemoresistance. This study was aimed to investigate the possibility of targeting head neck squamous cell carcinoma (HNSCC) by NOTCH1 pathway inhibition and explore the synergistic effect of combining NOTCH inhibition with conventional chemotherapy. NOTCH1/HES1 elevation was found in human HNSCC, especially in tissue post chemotherapy and lymph node metastasis, which is correlated with CSCs markers. NOTCH1 inhibitor DAPT (GSI-IX) significantly reduces CSCs population and tumor self-renewal ability in vitro and in vivo. Flow cytometry analysis showed that NOTCH1 inhibition reduces CSCs frequency either alone or in combination with chemotherapeutic agents, namely, cisplatin, docetaxel, and 5-fluorouracil. The combined strategy of NOTCH1 blockade and chemotherapy synergistically attenuated chemotherapy-enriched CSC population, promising a potential therapeutic exploitation in future clinical trial.

Checkpoint blockade therapy utilizing monoclonal antibodies to reactivate T cells and recover their antitumor activity makes an epoch in cancer immunotherapy. The role of B7-H4, a novel negative immune checkpoint, in oral squamous cell carcinoma (OSCC) has still not been elucidated. In this study, tissue samples from human OSCC, which contains 165 primary OSCC, 48 oral epithelial dysplasia and 43 normal oral mucosa specimens, and Tgfbr1/Pten 2cKO mice OSCC model were stained with B7-H4 antibody to analyze the correlations between B7-H4 expression and clinicopathological characteristics. Kaplan–Meier analysis was used to compare the survival of patients with high B7-H4 expression and patients with low B7-H4 expression. We found B7-H4 is highly expressed in human OSCC tissue, and the B7-H4 expression level was associated with the clinicopathological parameters containing pathological grade and lymph node status. Moreover, we confirmed that B7-H4 was overexpressed in Tgfbr1/Pten 2cKO mice OSCC model. Our data also indicated that patients with high B7-H4 expression had poor overall survival compared with those with low B7-H4 expression. Furthermore, this study demonstrated that B7-H4 was positively associated with PD-L1, CD11b, CD33, PI3Kα p110, and p-S6 (S235/236). Taken together, these findings suggest B7-H4 is a potential target in the treatment of OSCC.

C4.4A, a member of the Ly6/uPAR family of membrane proteins, has been identified as a metastasis-associated molecule, but little is known about its actual expression and possible function in head and neck squamous cell carcinoma (HNSCC). To explore diagnostic and prognostic roles of C4.4A in HNSCC, we investigated the expression of C4.4A in human HNSCC tissue array which contains 43 HNSCC, 6 epithelial dysplasia and 16 normal oral mucosa. Expression of C4.4A was significantly increased in epithelial dysplasia and HNSCC when compared with normal oral mucosa. Moreover, high C4.4A expression indicated a rather poor prognosis of HNSCC patients. To better understand the function of C4.4A in HNSCC progression, we investigated epithelial to mesenchymal transition (EMT) associated proteins including transforming growth factor (TGF-β1), Slug and CD147 in HNSCC. The expression of TGF-β1, Slug, and CD147 was significantly increased in HNSCC when compared with normal oral mucosa. Meanwhile, the expression of C4.4A was significantly correlated with TGF-β1, Slug, and CD147 in HNSCC tissue array. Furthermore, knockdown of C4.4A decreased the cell invasion and migration in CAL27 cell line and suppressed the EMT with increased E-cadherin and decreased N-cadherin and Slug. Our study demonstrated that C4.4A was a potential marker for prognosis of HNSCC, and C4.4A participated in EMT program in HNSCC progression.

Tumor-associated macrophages (TAMs) play an important role in the progression and prognostication of numerous cancers. However, the role and clinical significance of TAM markers in oral squamous cell carcinoma (OSCC) has not been elucidated. The present study was designed to investigate the correlation between the expression of TAM markers and pathological features in OSCC by tissue microarray. Tissue microarrays containing 16 normal oral mucosa, 6 oral epithelial dysplasia, and 43 OSCC specimens were studied by immunohistochemistry. We observed that the protein expression of the TAM markers CD68 and CD163 as well as the cancer stem cell (CSC) markers ALDH1, CD44, and SOX2 increased successively from the normal oral mucosa to OSCC. The expressions of CD68 and CD163 were significantly associated with lymph node status, and SOX2 was significantly correlated with pathological grade and lymph node status, whereas ALDH1 was correlated with tumor stage. Furthermore, CD68 was significantly correlated with CD163, SOX2, and ALDH1 (P<0.05). Kaplan-Meier analysis revealed that OSCC patients overexpressing CD163 had significantly worse overall survival (P<0.05). TAM markers are associated with cancer stem cell marker and OSCC overall survival, suggesting their potential prognostic value in OSCC.

Oxidative stress has been reported to play an important role in progression and prognostication in various kinds of cancers. However, the role and clinical significance of oxidative stress markers Keap1 and Nrf2 in oral squamous cell carcinoma (OSCC) has not been elucidated. This study aimed to investigate the correlation of oxidative stress markers Keap1 and Nrf2 expression and pathological features in OSCC by using tissue microarray. Tissue microarrays containing 17 normal oral mucosa, 7 oral epithelial dysplasia and 43 OSCC specimens were studied by immunohistochemistry. The association among these proteins and pathological features were analyzed. Expression of oxidative stress markers Keap1, Nrf2, and antioxidants PPIA, Prdx6, as well as CD147 was found to increase consecutively from normal oral mucosa to OSCC, and the Keap1, Nrf2, PPIA, Prdx6, CD147 expression in OSCC were significantly higher when compared to normal oral mucosa. Expression of Keap1, Nrf2 in tumors was not found to be significantly associated with T category, lymph node metastases, and pathological grade. Furthermore, we checked the relationship among these oxidative stress markers and found that Keap1 was significantly correlated with Nrf2, Prdx6 and CD147. Significant relationship between Nrf2 and Prdx6 was also detected. Finally, we found patients with overexpression of Keap1 and Nrf2 had not significantly worse overall survival by Kaplan-Meier analysis. These findings suggest that ROS markers are associated with carcinogenesis and progression of OSCC, which may have prognostic value and could be regarded as potential therapeutic targets in OSCC.

Cumulative evidence suggests that constitutively activated signal transducer and activator of transcription (STAT3) may contribute to sustaining immunosuppressive status, and that inhibiting STAT3 signaling represents a potential strategy to improve antitumor immunity. In the present study, we observed that high levels phosphorylated of STAT3 are significantly associated with the markers for both myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs) in human head and neck squamous cell carcinoma (HNSCC). Additionally, we showed that targeting STAT3 signaling with a tolerable selective inhibitor S3I-201 significantly decreased immature myeloid cells such as MDSCs, TAMs and iDCs in genetically defined mice HNSCC model. These findings highlight that targeting STAT3 signaling may be effective to enhance antitumor immunity via myeloid suppressor cells in HNSCC.

In the present study, we explored the expression and correlation of survivin with HIF-1α, TGF-β1 and TFE3 in adenoid cystic carcinoma (AdCC). The expression of survivin, HIF-1α, TGF-β1 and TFE3 was assessed by immunohistochemical staining of a tissue microarray containing tissue samples of normal salivary gland (NSG), pleomorphic adenoma (PA) and AdCC. Correlation analysis of these proteins revealed that increased survivin expression was associated with the overexpression of HIF-1α (P0.05). Selective inhibition of survivin by YM155 and siRNA significantly reduced human SACC-83 cell proliferation, with the corresponding decrease in expression of HIF-1α, TGF-β1 and TFE3. The data indicate that the overexpression of survivin in AdCC is related to HIF-1α, TGF-β1 and TFE3. We hypothesize from these findings that the inhibition of survivin may be a novel strategy for neoadjuvant chemotherapeutic and radiosensitive treatment of AdCC.