Abstract
Tumors of the head and neck represent a molecularly diverse set of human cancers, but relatively few proteins have actually been shown to drive the disease at the molecular level. To identify new targets for individualized diagnosis or therapeutic intervention, we performed a kinase centric chemical proteomics screen and quantified 146 kinases across 34 head and neck squamous cell carcinoma (HNSCC) cell lines using intensity-based label-free mass spectrometry. Statistical analysis of the profiles revealed significant intercell line differences for 42 kinases (p < 0.05), and loss of function experiments using siRNA in high and low expressing cell lines identified kinases including EGFR, NEK9, LYN, JAK1, WEE1, and EPHA2 involved in cell survival and proliferation. EGFR inhibition by the small molecule inhibitors lapatinib, gefitinib, and erlotinib as well as siRNA led to strong reduction of viability in high but not low expressing lines, confirming EGFR as a drug target in 10-20% of HNSCC cell lines. Similarly, high, but not low EPHA2-expressing cells showed strongly reduced viability concomitant with down-regulation of AKT and ERK signaling following EPHA2 siRNA treatment or EPHA1-Fc ligand exposure, suggesting that EPHA2 is a novel drug target in HNSCC. This notion is underscored by immunohistochemical analyses showing that high EPHA2 expression is detected in a subset of HNSCC tissues and is associated with poor prognosis. Given that the approved pan-SRC family kinase inhibitor dasatinib is also a very potent inhibitor of EPHA2, our findings may lead to new therapeutic options for HNSCC patients. Importantly, the strategy employed in this study is generic and therefore also of more general utility for the identification of novel drug targets and molecular pathway markers in tumors. This may ultimately lead to a more rational approach to individualized cancer diagnosis and therapy.
Highlights
From ‡Chair of Proteomics and Bioanalytics, Technische Universitat Munchen, 85354 Freising, Germany, the §Cell Signalling Group, Department of Oncology, Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, OX3 7BN United Kingdom, the ¶Cancer Research UK Cancer Therapeutics Unit, McElwain Laboratories, The Institute of Cancer Research, Belmont, Sutton, Surrey, SM2 5NG United Kingdom, the University of Pittsburgh Cancer Institute and the Department of Human Genetics, University of Pittsburgh, Pittsburgh, PA 15261, Pennsylvania, the **Department of Otorhinolaryngology, Head and Neck Surgery and Department of Medical Biochemistry and Genetics, Turku University Hospital and University of Turku, FIN20520 Turku, Finland, the ‡‡Institute for Pathology, Helmholtz Zentrum Munchen, 85764 Neuherberg, Germany, and the ¶¶Center for Integrated Protein Science Munich, Munich, Germany
Loss of function experiments using Small interfering RNA (siRNA) and small molecule kinase inhibitors showed that EGFR, EPHA2, NEK9, RIPK2, WEE1, and JAK1 are involved in cell survival, and the validation data assembled far suggest EPHA2 as a novel target for Head and neck squamous cell carcinoma (HNSCC) therapy
The analysis revealed a great molecular heterogeneity within this group of cell lines and identified a number of proteins previously implicated in the disease (EGFR, c-MET, etc.) as well as novel candidates indicative of individual tumor biology that open new avenues for the development of cancer drugs and companion diagnostics (EPHA2, NEK9, etc.)
Summary
Several other kinase-centric molecular mechanisms have been investigated These include aurora kinase A (AURKA) [17], polo-like kinase 1 (PLK1) [18], and c-MET [19], indicating that the observed molecular heterogeneity of the disease may be rooted in multiple kinase signaling pathways and underscoring the need for potential biomarkers and/or therapeutic targets for an individualized approach to the management of HNSCC. The first element is an affinity purification matrix termed Kinobeads, which consists of seven immobilized nonselective kinase inhibitors It allows the purification and identification of several hundred kinases and other ATP-binding proteins from cell lines or tissues [22, 23]. Quantitative profiling and statistical analysis of 146 protein kinases across 34 HNSCC cell lines revealed that 42 kinases showed highly significant differential protein expression These included disease associated kinases such as EGFR, c-MET, and AURKA and novel candidates. Loss of function experiments using siRNA and small molecule kinase inhibitors showed that EGFR, EPHA2, NEK9, RIPK2, WEE1, and JAK1 are involved in cell survival, and the validation data assembled far suggest EPHA2 as a novel target for HNSCC therapy
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