Possible Therapeutic Strategy Involving the Purine Synthesis Pathway Regulated by ITK in Tongue Squamous Cell Carcinoma
Abstract
:Simple Summary
Abstract
1. Introduction
2. Materials and Methods
2.1. Patients and Tissue Samples
2.2. Immunohistochemistry (IHC)
2.3. Cell Lines, Antibodies, and Drugs
2.4. Establishment of Stably Infected Cells
2.5. Western Blot Analysis
2.6. In Vitro Cell Growth Assay
2.7. Animal Experiments
2.8. Preparation and Digestion of Protein Lysate
2.9. Mass Spectrometry Analysis for Quantitative Phosphotyrosine Proteomics
2.10. Identification and Quantification of Phosphopeptides
2.11. Bioluminescence Resonance Energy Transfer (BRET) Assay
2.12. Immunofluorescence Cytochemistry
2.13. Metabolomic Analysis
2.14. Ion Chromatography-Tandem Mass Spectrometry for Anionic Metabolites
2.15. Growth Inhibition Analysis with ITK Inhibitor
2.16. Statistical Analysis
3. Results
3.1. ITK Protein Expression in Patients with TSCC
3.2. Hazard Ratios (HRs) for Death and Prognostic Significance of ITK Protein Expression in TSCC
3.3. Role of ITK in Cancer Cell Proliferation In Vitro and In Vivo
3.4. Proteomic Analysis of ITK Tyrosine Phosphorylation
3.5. Elucidation of the Association between ITK and GART
3.6. Assessment of De Novo Synthesis of Purines in ITK-Expressing Cells
3.7. Inhibition of CellG and Suppression of GART Phosphorylation and Purine Metabolism Using an ITK Inhibitor
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Number of Cases (%) | ITK-Negative | ITK-Positive | p-value a | |
---|---|---|---|---|
Total | 86 | 73 | 13 | |
Age (years) | 0.3655 | |||
<64 | 43 (50.0) | 38 | 5 | |
≥64 | 43 (50.0) | 35 | 8 | |
Sex | 0.1715 | |||
Male | 48 (55.8) | 43 | 5 | |
Female | 38 (44.2) | 30 | 8 | |
Stage b | 0.9776 | |||
I | 46 (53.5) | 39 | 7 | |
II | 40(46.5) | 34 | 6 | |
Histologic differentiation | 0.1760 | |||
Poor/Moderate | 25 (29.1) | 18 | 6 | |
Well | 61 (70.9) | 55 | 7 | |
Mode of invasion c | 0.0000 * | |||
1,2 | 58 (67.4) | 56 | 2 | |
3,4 | 28 (32.6) | 17 | 11 | |
Late metastasis of cervical lymph nodes | 0.0006 * | |||
Negative | 68 (79.1) | 63 | 5 | |
Positive | 18 (20.9) | 10 | 8 | |
Lymphovascular invasion | 0.0101 * | |||
Negative | 75 (87.2) | 67 | 8 | |
Positive | 11 (12.8) | 6 | 5 | |
Perineural invasion | ||||
Negative | 82 (95.3) | 71 | 11 | 0.1067 |
Positive | 4 (4.7) | 2 | 2 |
Covariate | Univariate Analysis (n = 86) | Multivariate Analysis (n = 86) | ||||||
---|---|---|---|---|---|---|---|---|
HR | 95% CI | p-Value | HR | 95% CI | p-Value | |||
Age (years) | ||||||||
<64 | Reference | Reference | ||||||
≥64 | 2.866 | 0.923 | 8.894 | 0.068 | 2.611 | 0.836 | 8.153 | 0.099 |
ITK protein expression | ||||||||
Negative | Reference | Reference | ||||||
Positive | 3.544 | 1.225 | 10.247 | 0.020 * | 3.167 | 1.089 | 9.215 | 0.034 * |
Sex | ||||||||
Male | Reference | |||||||
Female | 1.235 | 0.463 | 3.296 | 0.673 | ||||
Stage a | ||||||||
Stage I | Reference | |||||||
Stage II | 1.519 | 0.567 | 4.068 | 0.405 | ||||
Histologic differentiation | ||||||||
Well | Reference | |||||||
Poor and moderate | 3.401 | 1.264 | 9.149 | 0.015 * | ||||
Mode of invasion b | ||||||||
1, 2 | Reference | |||||||
3, 4 | 5.112 | 1.768 | 14.783 | 0.003 * | ||||
Late metastasis of cervical lymph nodes | ||||||||
Negative | Reference | |||||||
Positive | 4.497 | 1.684 | 12.010 | 0.003 * | ||||
Lymphovascular invasion | ||||||||
Negative | Reference | |||||||
Positive | 3.497 | 1.210 | 10.110 | 0.021 * | ||||
Perineural invasion | ||||||||
Negative | Reference | |||||||
Positive | N.A.c |
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Onidani, K.; Miura, N.; Sugiura, Y.; Abe, Y.; Watabe, Y.; Kakuya, T.; Mori, T.; Yoshimoto, S.; Adachi, J.; Kiyoi, T.; et al. Possible Therapeutic Strategy Involving the Purine Synthesis Pathway Regulated by ITK in Tongue Squamous Cell Carcinoma. Cancers 2021, 13, 3333. https://doi.org/10.3390/cancers13133333
Onidani K, Miura N, Sugiura Y, Abe Y, Watabe Y, Kakuya T, Mori T, Yoshimoto S, Adachi J, Kiyoi T, et al. Possible Therapeutic Strategy Involving the Purine Synthesis Pathway Regulated by ITK in Tongue Squamous Cell Carcinoma. Cancers. 2021; 13(13):3333. https://doi.org/10.3390/cancers13133333
Chicago/Turabian StyleOnidani, Kaoru, Nami Miura, Yuki Sugiura, Yuichi Abe, Yukio Watabe, Takanori Kakuya, Taisuke Mori, Seiichi Yoshimoto, Jun Adachi, Takao Kiyoi, and et al. 2021. "Possible Therapeutic Strategy Involving the Purine Synthesis Pathway Regulated by ITK in Tongue Squamous Cell Carcinoma" Cancers 13, no. 13: 3333. https://doi.org/10.3390/cancers13133333