Quantitation of potential predictive biomarkers for hypoxia-activated prodrugs in head and neck squamous cell carcinomas by targeted proteomics

Abstract

Background: Hypoxia, a prevalent feature of head and neck squamous cell carcinoma (HNSCC) shown to contribute to adverse prognosis and treatment resistance, presents a compelling pharmacological target in clinical cancer drug development. The most notable approach to drugging tumour hypoxia involves the design of bioreductive (or hypoxia-activated) prodrugs, which can be reductively activated to release cytotoxic effectors in low-oxygen tumour compartments. The activity of these bioreductive agents likely impinges on the coincidence of three important determinants; tumour hypoxia, intrinsic sensitivity of tumour cells to the cytotoxic effectors and expression of reductases capable of prodrug activation. This thesis aimed to profile the expression of candidate prodrug-activating reductases and endogenous markers of hypoxic gene expression in HNSCC. Methods: We employed a targeted proteomic strategy based on selected reaction monitoring (SRM) by mass spectrometry to develop an assay for 14 candidate prodrug-activating reductases, 9 endogenous markers of hypoxia and 7 housekeeping proteins. The developed method was utilised to estimate absolute and relative abundances of proteotypic peptides unique to the proteins in HNSCC cell lines and xenografts. Relative transcript abundances of reductases and hypoxia markers were estimated from RNAseq analysis on the 27 HNSCC cell lines as well as on two clinical cohorts extracted from public databases. Results: The 14 reductases studied demonstrated variable protein expression across the 27 HNSCC cell lines and the HNSCC mouse xenograft tumours studied, with CYB5R3 and NQO1 exhibiting high expression, and POR, AKR1C3, TXNRD1 and FDXR exhibiting moderate expression. AKR1C3 was the only detectable reductase which demonstrated strong correlations between peptide abundance and transcript abundance in HNSCC in vitro. CYB5R3, NQO1 and TXNRD1 amongst the most abundant prodrug-activating reductases across two HNSCC clinical cohorts. 7 of the 9 endogenous hypoxia markers were upregulated at the protein level in FaDu and SiHa cell lines. Conclusions: SRM targeted proteomics has potential for evaluating determinants of sensitivity to bioreductive agents at the protein level. The prodrug-activating reductases, CYB5R3, NQO1, POR, TXNRD1, AKR1C3 and FDXR are well expressed at the protein level. The variation in expression of these reductases in HNSCC suggests that targeted proteomics could be used to help define subgroups for development of hypoxia-targeted bioreductive drugs.