Abstract
Simple SummaryWe identified overexpression of interleukin-2–inducible T-cell kinase (ITK) as a novel biomarker for predicting the prognosis of tongue squamous cell carcinoma patients with poor outcomes. Oral cancer cell lines overexpressing ITK exhibited significantly increased proliferation in three-dimensional culture assays and murine inoculation models as compared with mock control cells. Phosphorylation proteomics analysis revealed that ITK expression induces the phosphorylation of a novel tyrosine residue of the trifunctional purine biosynthetic protein adenosine-3 (GART), an enzyme in the purine biosynthesis pathway. A significant increase in de novo purine biosynthesis was observed in cells expressing ITK. These results suggest that ITK is involved in enhancing the proliferation of cancer cells in the malignant phenotype by activating de novo purine biosynthesis through phosphorylation of GART. In this study, we report a possible therapeutic strategy involving the purine synthesis pathway regulated by ITK in tongue squamous cell carcinoma.The epidermal growth factor receptor is the only available tyrosine kinase molecular target for treating oral cancer. To improve the prognosis of tongue squamous cell carcinoma (TSCC) patients, a novel molecular target for tyrosine kinases is thus needed. We examined the expression of interleukin-2–inducible T-cell kinase (ITK) using immunohistochemistry, and the biological function of ITK was investigated using biochemical, phosphoproteomic, and metabolomic analyses. We found that ITK is overexpressed in TSCC patients with poor outcomes. The proliferation of oral cancer cell lines expressing ITK via transfection exhibited significant increases in three-dimensional culture assays and murine inoculation models with athymic male nude mice as compared with mock control cells. Suppressing the kinase activity using chemical inhibitors significantly reduced the increase in cell growth induced by ITK expression. Phosphoproteomic analyses revealed that ITK expression triggered phosphorylation of a novel tyrosine residue in trifunctional purine biosynthetic protein adenosine-3, an enzyme in the purine biosynthesis pathway. A significant increase in de novo biosynthesis of purines was observed in cells expressing ITK, which was abolished by the ITK inhibitor. ITK thus represents a potentially useful target for treating TSCC through modulation of purine biosynthesis.
Highlights
A number of molecular-targeted drugs, including kinase-targeted drugs, are used clinically in the treatment of many types of cancer to improve the survival time of patients
Expression of inducible T-cell kinase (ITK) protein was not observed in normal tongue mucosa adjacent to the tumor (Figure 1A,B), and lymphocytes were stained by the ITK antibody in all 86 patient tongue squamous cell carcinoma (TSCC) tissues on the same pathology sections as the internal controls (Figure 1E,H)
Elucidation of the Association between ITK and GART We investigated the direct association between ITK and GART, as GART is endogenously expressed in oral cancer cell lines and human embryonic kidney 293 (HEK 293) cells (Figures 1K and 2H)
Summary
A number of molecular-targeted drugs, including kinase-targeted drugs, are used clinically in the treatment of many types of cancer to improve the survival time of patients. Cetuximab, a monoclonal antibody that blockades the epidermal growth factor receptor (EGFR), is the only kinase-targeted drug available for treating tongue squamous cell carcinoma (TSCC) [1]. It was recently reported that ITK gene expression is higher in tumor tissues than in normal tissues and associated with poor prognosis in head and neck cancer [3]. ITK protein expression reportedly increases with nevus to metastatic melanoma progression and is associated with tumor development and progression in melanoma [4]. Little is known about ITK protein expression and function in almost all types of solid tumors. Trifunctional purine biosynthetic protein adenosine-3 (GART) is an enzyme in the de novo purine biosynthesis pathway. The mechanism of the de novo purine biosynthesis pathway through GART in TSCC has not been elucidated in detail
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