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1.
Head Neck ; 45(1): 147-155, 2023 01.
Artigo em Inglês | MEDLINE | ID: mdl-36285353

RESUMO

BACKGROUND: We have recently shown a frequent upregulation of Src-family kinases (SFK) in head and neck squamous cell carcinoma (HNSCC). Here we tested, if SFK targeting is effective especially in HNSCC cells with upregulated SFK signaling. METHODS: The impact of SFK inhibitors SU6656, PP2 and dasatinib on three HNSCC cell lines with different SFK activity levels was analyzed using proliferation and colony formation assays, Western blot and functional kinomics. RESULTS: Proliferation was blocked by all inhibitors in a micro-molar range. With respect to cell kill, dasatinib was most effective, while SU6656 showed moderate and PP2 minor effects. Cellular signaling was affected differently, with PP2 having no effect on SFK signaling while dasatinib probably has non-SFK specific effects. Only SU6656 showed clear SFK specific effects on signaling. CONCLUSION: The results demonstrate potential benefit of SFK inhibition in HNSCC but they also highlight challenges due to non-specificities of the different drugs.


Assuntos
Neoplasias de Cabeça e Pescoço , Quinases da Família src , Humanos , Dasatinibe/farmacologia , Quinases da Família src/metabolismo , Pirimidinas/farmacologia , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Carcinoma de Células Escamosas de Cabeça e Pescoço/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Linhagem Celular Tumoral
2.
Mol Ther Methods Clin Dev ; 22: 249-262, 2021 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-34485609

RESUMO

Since the introduction of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9), genome editing has been broadly applied in basic research and applied biotechnology, whereas translation into clinical testing has raised safety concerns. Indeed, although frequencies and locations of off-target events have been widely addressed, little is known about their potential biological consequences in large-scale long-term settings. We have developed a long-term adverse treatment effect (LATE) in vitro assay that addresses potential toxicity of designer nucleases by assessing cell transformation events. In small-scale proof-of-principle experiments we reproducibly detected low-frequency (<0.5%) growth-promoting events in primary human newborn foreskin fibroblasts (NUFF cells) resulting from off-target cleavage in the TP53 gene. Importantly, the LATE assay detected not only off-target effects in TP53 not predicted by popular online tools but also growth-promoting mutations in other tumor suppressor genes, such as p21 and PLZF. It convincingly verified strongly reduced off-target activities of high fidelity compared with first-generation Cas9. Finally, the LATE assay was readily adapted to other cell types, namely clinically relevant human mesenchymal stromal cells (hMSCs) and retinal pigmented epithelial (RPE-1) cells. In conclusion, the LATE assay allows assessment of physiological adverse effects of the CRISPR/Cas system and might therefore be useful for preclinical safety studies.

3.
Int J Cancer ; 149(5): 1166-1180, 2021 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-33890294

RESUMO

Signal transduction via protein kinases is of central importance in cancer biology and treatment. However, the clinical success of kinase inhibitors is often hampered by a lack of robust predictive biomarkers, which is also caused by the discrepancy between kinase expression and activity. Therefore, there is a need for functional tests to identify aberrantly activated kinases in individual patients. Here we present a systematic analysis of the tyrosine kinases in head and neck cancer using such a test-functional kinome profiling. We detected increased tyrosine kinase activity in tumors compared with their corresponding normal tissue. Moreover, we identified members of the family of Src kinases (Src family kinases [SFK]) to be aberrantly activated in the majority of the tumors, which was confirmed by additional methods. We could also show that SFK hyperphosphorylation is associated with poor prognosis, while inhibition of SFK impaired cell proliferation, especially in cells with hyperactive SFK. In summary, functional kinome profiling identified SFK to be frequently hyperactivated in head and neck squamous cell carcinoma. SFK may therefore be potential therapeutic targets. These results furthermore demonstrate how functional tests help to increase our understanding of cancer biology and support the expansion of precision oncology.


Assuntos
Biomarcadores Tumorais/metabolismo , Neoplasias de Cabeça e Pescoço/patologia , Quinases da Família src/metabolismo , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Neoplasias de Cabeça e Pescoço/metabolismo , Humanos , Fosforilação , Prognóstico , Inibidores de Proteínas Quinases/farmacologia , Estudos Retrospectivos , Taxa de Sobrevida , Análise Serial de Tecidos , Células Tumorais Cultivadas , Quinases da Família src/antagonistas & inibidores
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