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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.

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.

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. [ABSTRACT FROM AUTHOR]

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+ SRT+) was associated with unfavorable overall survival of HNSCC patients. These findings indicate that SFKs may be a potential target for an effective immunotherapy of HNSCC by decreasing MDSCs and moreover, LYN will have an impact on such therapeutic strategy. [ABSTRACT FROM AUTHOR]

A 32-year-old male presented with a painless swelling on his nasal dorsum, persisting for over 3 months. He reported a gradual increase in the size of the mass, with no identifiable triggers except occasional skin redness. He denied nosebleeds, rhinorrhea, nasal obstruction, trauma, prior surgery, or spontaneous pain. His medical history revealed gout, managed with colchicine and diclofenac. Despite dietary and pharmaceutical interventions, he continued to have bouts of hyperuricemia, with blood uric acid levels measuring 739 μmol/L. Multiple tophi were evident, especially on the left first metatarsophalangeal joint (Figure 1A). Examination revealed an irregularly shaped, immobile, hard swelling at the nasal radix, measuring 3 cm×2 cm. Computerized tomography (CT) imaging of the nose showed bilateral nasal bone destruction from the lesion. Given its impact on the patient's appearance and his history of gout, the mass was initially diagnosed as unusual gouty tophus. The patient requested surgical removal of the lesion, and the dissection revealed a mass partly encased by a capsule-like connective tissue adherent to the nasal bone. As the lesion damaged the nasal bone, removal of the lesion led to defect of nasal bone. After an extensive rinse of the surgery site, the incision was sutured., Competing Interests: The authors report no conflicts of interest., (Copyright © 2024 by Mutaz B. Habal, MD.)

Background: Cancer immunotherapy offers a promising approach in cancer treatment. The adenosine A2A receptor (A2AR) could protect cancerous tissues from immune clearance via inhibiting T cells response. To date, the role of A2AR in head and neck squamous cell carcinoma (HNSCC) has not been investigated. Here, we sought to explore the expression and immunotherapeutic value of A2AR blockade in HNSCC., Methods: The expression of A2AR was evaluated by immunostaining in 43 normal mucosae, 48 dysplasia and 165 primary HNSCC tissues. The immunotherapeutic value of A2AR blockade was assessed in vivo in genetically defined immunocompetent HNSCC mouse model., Results: Immunostaining of HNSCC tissue samples revealed that increased expression of A2AR on tumor infiltrating immune cells correlated with advanced pathological grade, larger tumor size and positive lymph node status. Elevated A2AR expression was also detected in recurrent HNSCC and HNSCC tissues with induction chemotherapy. The expression of A2AR was found to be significantly correlated with HIF-1α, CD73, CD8 and Foxp3. Furthermore, the increased population of CD4 + Foxp3 + regulatory T cells (Tregs), which partially expressed A2AR, was observed in an immunocompetent mouse model that spontaneously develops HNSCC. Pharmacological blockade of A2AR by SCH58261 delayed the tumor growth in the HNSCC mouse model. Meanwhile, A2AR blockade significantly reduced the population of CD4 + Foxp3 + Tregs and enhanced the anti-tumor response of CD8 + T cells., Conclusions: These results offer a preclinical proof for the administration of A2AR inhibitor on prophylactic experimental therapy of HNSCC and suggest that A2AR blockade can be a potential novel strategy for HNSCC immunotherapy.

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 < 0.01) and oral dysplasia (p < 0.001) groups. In addition, the expression of tumor necrosis factor receptor-associated factor 1 was associated with those of B cell-specific Moloney murine leukemia virus integration site 1, aldehyde dehydrogenase 1, and lin28 homolog B (p = 0.032, r 2  = 0.109; p < 0.0001, r 2  = 0.64; and p < 0.001, r 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.

Salivary adenoid cystic carcinoma (AdCC) is a common head and neck cancer with the propensity for local spread and distant metastasis. In our previous study, elevated expression of Anterior gradient 2 (AGR2) was detected in head and neck squamous cell carcinoma (HNSCC), associated with epithelial-mesenchymal transition (EMT) and cancer stemness. However, to date, the expression and function of AGR2 in AdCC has yet to be elucidated. In the present study, human AdCC tissue microarrays including 18 cases of normal salivary gland (NSG), 12 cases of pleomorphic adenoma (PMA) and 72 cases of AdCC were employed for immunohistochemical staining analysis. Results indicated that AGR2, which was remarkably correlated with Ki-67, transforming growth factor beta-1 (TGF-β1) and CD147, was significantly elevated in human salivary AdCC tissues. Knockdown of AGR2 significantly repressed the proliferation and migration of human SACC-83 and SACC-LM cell lines. Additionally, AGR2 silencing obviously reversed the EMT phenomena induced by TGF-β1. Taken together, our present study revealed the potential pro-metastasis role of AGR2 in AdCC, indicating that AGR2 might be a novel therapeutic target of AdCC with distant metastasis.

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.

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.

Epithelial-mesenchymal transition (EMT) is considered to have pivotal roles in the invasive and metastatic of Adenoid cystic carcinoma (AdCC) which is marked by local infiltration and distant metastasis. Notch signaling abnormity has been implicated as important molecular events in recent next generation sequencing studies of AdCC, but the detail is still unclear. This study was designed to investigate the expression of Notch signaling pathway and its relation with EMT program in AdCC. We constructed custom-made Tissue microarray (TMA) to evaluate the immunoreactivity of Notch signaling and EMT program and found that Notch signaling increase consecutively from NSG, PMA to AdCC, suggesting Notch signaling pathway may be associated with human AdCC progression. Then, we carried out Pearson correlation analysis and showed a close correlation of Notch signaling and EMT progression. When blocking Notch signaling pathway with γ-secretase inhibitor DAPT, EMT progression was decreased and migration and invasion ability were declined. Collectively, these findings suggest the vital roles of Notch signaling pathway in AdCC progression through their relationship with EMT progress. Targeting Notch signaling may provide further understanding of the mechanism of invasion and metastasis of AdCC as well as potential clinical therapeutics.