Influence of Bruton's Tyrosine Kinase (BTK) on Epithelial-Mesenchymal Transition (EMT) Processes and Cancer Stem Cell (CSC) Enrichment in Head and Neck Squamous Cell Carcinoma (HNSCC).

Constitutively active kinases play a crucial role in carcinogenesis, and their inhibition is a common target for molecular tumor therapy. We recently discovered the expression of two oncogenic isoforms of Bruton's Tyrosine Kinase (BTK) in head and neck squamous cell carcinoma (HNSCC), Btk-p80 and BTK-p65. However, the precise role of BTK in HNSCC remains unclear. Analyses of a tissue microarray containing benign and malignant as well as inflammatory tissue samples of the head and neck region revealed the preferential expression of BTK-p80 in malignant tissue, whereas BTK-p65 expression was confirmed in over 80% of analyzed metastatic head and neck tumor cases. Therefore, processes associated with metastasis, like cancer stem cell (CSC) enrichment and the epithelial-mesenchymal transition (EMT), which in turn depend on an appropriate cytokine milieu, were analyzed. Treatment of HNSCC-derived cell lines cultured under 3D conditions with the BTK inhibitor AVL-292 caused reduced sphere formation, which was accompanied by reduced numbers of ALDH1A1+ CSCs as well as biological changes associated with the EMT. Moreover, we observed reduced NF-κB expression as well as altered NF-κB dependent pro-tumorigenic and EMT-associated cytokine release of IL-6, IFNγ, and TNFα when BTK activity was dampened. Therefore, an autocrine regulation of the oncogenic BTK-dependent process in HNSCC can be suggested, with BTK inhibition expected to be an effective treatment option for HNSCC.

Adenosine receptor 2B activity promotes autonomous growth, migration as well as vascularization of head and neck squamous cell carcinoma cells.

Adenosine is a signaling molecule that exerts dual effects on tumor growth: while it inhibits immune cell function and thereby prevents surveillance by the immune system, it influences tumorigenesis directly via activation of adenosine receptors on tumor cells at the same time. However, the adenosine-mediated mechanisms affecting oncogenic processes particularly in head and neck squamous cell carcinomas (HNSCC) are not fully understood. Here, we investigated the role of adenosine receptor activity on HNSCC-derived cell lines. Targeting the adenosine receptor A2B (ADORA2B) on these cells with the inverse agonist/antagonist PSB-603 leads to inhibition of cell proliferation, transmigration as well as VEGFA secretion in vitro. At the molecular level, these effects were associated with cell cycle arrest as well as the induction of the apoptotic pathway. In addition, shRNA-mediated downmodulation of ADORA2B expression caused decreased proliferation. Moreover, in in vivo xenograft experiments, chemical and genetic abrogation of ADORA2B activity impaired tumor growth associated with decreased tumor vascularization. Together, our findings characterize ADORA2B as a crucial player in the maintenance of HNSCC and, therefore, as a potential therapeutic target for HNSCC treatment.

BTK Isoforms p80 and p65 Are Expressed in Head and Neck Squamous Cell Carcinoma (HNSCC) and Involved in Tumor Progression.

Here, we describe the expression of Bruton's Tyrosine Kinase (BTK) in head and neck squamous cell carcinoma (HNSCC) cell lines as well as in primary HNSCC samples. BTK is a kinase initially thought to be expressed exclusively in cells of hematopoietic origin. Apart from the 77 kDa BTK isoform expressed in immune cells, particularly in B cells, we identified the 80 kDa and 65 kDa BTK isoforms in HNSCC, recently described as oncogenic. Importantly, we revealed that both isoforms are products of the same mRNA. By investigating the mechanism regulating oncogenic BTK-p80/p65 expression in HNSSC versus healthy or benign tissues, our data suggests that the epigenetic process of methylation might be responsible for the initiation of BTK-p80/p65 expression in HNSCC. Our findings demonstrate that chemical or genetic abrogation of BTK activity leads to inhibition of tumor progression in terms of proliferation and vascularization in vitro and in vivo. These observations were associated with cell cycle arrest and increased apoptosis and autophagy. Together, these data indicate BTK-p80 and BTK-p65 as novel HNSCC-associated oncogenes. Owing to the fact that abundant BTK expression is a characteristic feature of primary and metastatic HNSCC, targeting BTK activity appears as a promising therapeutic option for HNSCC patients.