Quantitative Chemical Proteomics Reveals New Potential Drug Targets in Head and Neck Cancer

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 labelfree 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.

Systematic Identification of the HSP90 Regulated Proteome

“…VSN is able to stabilize the variance across the entire intensity range and addresses the error structure in the data. The application of VSN has previously been shown to be beneficial for MS-based quantification (27,28). To investigate the data distribution and ensure the appropriate application of statistical tools, normal quantile-quantile plots were created for all protein intensities in each cell line.…”

“…Kinobead Affinity Purification-Kinobead pulldowns were performed as described previously (16,22). Briefly, cell lysates were diluted with equal volumes of 1ϫ compound pulldown buffer (50 mM Tris/HCl pH 7.5, 5% glycerol, 1.5 mM MgCl 2 , 150 mM NaCl, 20 mM NaF, 1 mM sodium orthovanadate, 1 mM dithiothreitol, 5 mM calyculin A, and protease inhibitors).…”

“…S1) and "heavy" cells were left untreated. Cell lysates were mixed 1:1 and subjected in parallel to protein separation by 1D-SDS-PAGE and to the enrichment of kinases using an affinity reagent called kinobeads (a set of unselective kinase inhibitors immobilized on Sepharose beads, (16,22)). Following digestion into tryptic peptides, protein identification and differential quantification was performed by LC-MS/MS on an LTQ-Orbitrap XL mass spectrometer and using the software package MaxQuant.…”

Preclinical Evaluation of the WEE1 Inhibitor MK-1775 as Single-Agent Anticancer Therapy

“…High expression of WEE1 was found in malignant melanoma and correlated with poor disease-free survival in this population (11). Aberrant WEE1 expression has been implicated in additional tumor types such as hepatocellular carcinoma (12), breast cancer (13), colon carcinoma (14), lung carcinoma (15), and head and neck squamous cell carcinoma (16). Advanced tumors with an increased level of genomic instability may require functional checkpoints to allow the repair of DNA perturbations that accompany genomic instability.…”

Inhibition of SRC family kinases reduces myeloid‐derived suppressor cells in head and neck cancer

“…10 Several high-throughput analyses in HNSCC cell lines and specimens indicated that LYN is a novel candidate target for individualized therapy. 11,12 However, the relationship between SFKs and their clinical implications in HNSCC patients remains unclear, and direct evidence to confirm the biological function of SFKs in tumor immunosuppression remains insufficient. Accumulating evidence suggests that the high infiltration of immature myeloid cells, including myeloid-derived suppressor cells (MDSCs) and tumor associated macrophages (TAMs), fosters potent immunosuppressive tumor microenvironment activity by dampening T-cell activation.…”