102 CHAPTER 4 resistant patients to 43 sensitive patients in this independent cohort, 815 out of 17,561 transcripts were differentially expressed (p < 0.05) between the two groups. Thirty-six of the 173 differentially upregulated proteins in our analysis were also differentially upregulated at the RNA level in the independent cohort (3 in resistant (PLAUR, SLC2A3 and EIF4A1) and 33 in sensitive patients). DISCUSSION To our knowledge, this is the first combined mass spectrometry-based tyrosine-phosphoproteomics and expression proteomics analysis on tumor tissue from patients with advanced RCC in order to identify candidate predictive molecular biomarkers for treatment benefit of sunitinib. We report distinctive phosphosite and protein signatures and differential kinase and pathway activities that are associated with sensitive and resistant tumors. Exploring the differences in biology between sensitive and resistant tumors, we first focused on the characteristics of primary resistant patients. In this group, 22 phosphosites were differentially upregulated, of which 4 phosphosites were uniquely identified in this group (BCAR3_ Y117, EIF4A2_Y251, NOP58_Y272, GDI1_Y93) (Table 2). BCAR3 and GDI1 have a role in tumor development and progression and are correlated with resistance to systemic therapy in other tumor types, including breast cancer28,49-53. EIF4A2 mutations are found in 0.7% of ccRCC54, when found in other types of cancer, these mutations are associated with unfavorable prognosis and resistance to therapy55,56. EIF4A2 is a highly homologous paralog of, and functionally indistinguishable from EIF4A157, which was also differentially expressed in our cohort on the protein level and, in an independent study44, on the RNA level. Interestingly, comparing tumor and normal adjacent ccRCC tissue samples, Li et al report EIF4EBP1, another member of the translation initiation complex, as a downstream substrate of mTOR, and EIF4EBP1 phosphorylation was decreased in vitro by mTOR inhibition58. These four in resistant patients uniquely identified phosphosites have not previously been implied in RCC prognosis or prediction of sunitinib treatment outcome. Other differential phosphosites, yet non-uniquely upregulated in one of the groups, included STAT4_Y693 which is regulated upstream by TYK2, and ALOX5_Y95 which has a role in inflammatory processes59,60. Looking further into the biology of primary resistant tumors by analyzing enriched phosphosite-centric signatures (PTM-SEA), we found that Fibroblast Growth Factor (FGF) 1 and PROLACTIN pathways and EPHA2 substrates were significantly enriched signatures (Figure 1e). FGF is known to play a critical role in driving VEGF-independent tumor angiogenesis and FGFR signaling is an established resistance mechanism of VEGFR inhibition61,62. Prolactin has been reported to be elevated in 45% of ccRCC patients63, acting in a cytokine-like manner and as an important stimulatory regulator of the immune system. EPHA2 is overexpressed in renal cell carcinoma, associated with more advanced disease and angiogenesis64 and has been implied as a mediator of sunitinib resistance in RCC65.
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