104 CHAPTER 4 set, we have not been able to validate our 78-phosphosite candidate signature that may predict treatment outcome of sunitinib. For most (uniquely identified) differential phosphosites no antibodies were available for (technical) Western blot validation of the phosphoproteomic data. An exploratory comparison of our findings from the (phospho)proteomics analysis to transcriptome data as a proxy for (phospho)protein expression, using a comparable (n = 53) RCC cohort44 showed limited overlap (36 of 173) between the differentially regulated proteins and transcripts. In addition to sample size as contributing factor, it is known that transcriptomic and (phospho)proteomic data provide different levels of biological information23,78,79. However, in resistant patients, three proteins/transcripts overlapped: PLAUR, SLC2A3 and EIF4A1. Interestingly, EIF4A1, a regulator of ERK signaling80, was differentially upregulated on protein and transcript level, while its nearly identical homolog EIF4A2 was exclusively phosphorylated in resistant patients and represented in the candidate signature, stressing its potential importance in sunitinib resistance. Several identified differential kinases and substrates in our analysis show overlap with previous findings23,58, while some, such as WEE1 and BAP1, did not surface in our study. Although these kinases/substrates are important in RCC pathogenesis, they may not differ between sunitinib sensitive or resistant patients. CONCLUSIONS This MS-based analysis of the RCC (tyrosine-phospho)proteome revealed disctinctive phosphosite and protein signatures and differential kinase and pathway activities that are associated with sunitinib sensitivity and resistance. One protein (EIF4A1 and its homolog EIF4A2) was confirmed to be differentially expressed on phosphosite, protein and RNA level. These findings warrant validation in an independent cohort and the clinical utility for treatment selection remains to be demonstrated. A targeted assay or immunohistochemistry analysis with a selection of differential phosphosites and/or proteins could facilitate the implementation of these signatures as a decision-making tool for treatment selection in clinical practice. Such an assay would prevent toxicity and enable alternative (combination) treatment in patients upfront predicted to be resistant to sunitinib.
RkJQdWJsaXNoZXIy MTk4NDMw