Abstract 5305: Phosphoproteomics analysis reveals differential signaling pathways among clear cell and papillary renal cell carcinoma tumors

Abstract

Abstract Introduction: The use of kinase inhibitors with targets such as VEGFR has heralded a new era of treatment for metastatic renal cell carcinoma (RCC) patients. However, patients develop resistance with time. Therefore, novel targets must be identified and a systems level description of RCC produced. To this end, we used mass spectrometry based phosphoproteomics to nominate targets as well as compare RCC subtypes. Methods: We developed an integrative strategy combining RCC phosphoproteomics and functional interrogation using drug screening. Tyrosine phosphorylated peptide immunoprecipitation and purification was performed in clear cell RCC (ccRCC) and normal renal cortex cell lines and analyzed via nano-liquid chromatography/tandem mass spectrometry interfaced with an electrospray hybrid ion-trap mass spectrometer. Eight ccRCC and five papillary RCC (pRCC) tumor tissues were also analyzed. We assembled a library of 180 small molecules, including 60 tyrosine kinase inhibitors (TKIs), and screened them at multiple concentrations in 10 clear cell RCC (ccRCC) cell lines. Results: Among the ccRCC cell lines, 153 phosphosites (114 proteins) were identified. Receptor tyrosine kinases included EGFR, MET, ERBB4, and EPHB2 and nonreceptor tyrosine kinases included ABL2, JAK2, KALRN, and PTK2. Serine/threonine kinases were the most abundant kinases and included, among others, several CDK and MAPK members. Other frequently observed phosphosites corresponded to cytoskeleton (10%) and cell adhesion (6%) proteins. Among the ccRCC tumors, 119 phosphosites (99 proteins) were identified. In contrast to the cell lines, the ccRCC tumors demonstrated increased Src family kinase signaling but less RTK signaling. Among the pRCC tumors, 113 phosphosites (98 proteins) were identified, including 37 not identified in the ccRCC tumors. This data, coupled with the TKI screen, offered some interesting observations. MET phosphotyrosine peptide intensity was relatively low in ccRCC compared to pRCC and among the eight MET inhibitors tested, seven failed to significantly inhibit cell viability. These results suggest that MET signaling may be more active in pRCC than ccRCC. Other divergent patterns of phosphotyrosine signaling emerged. For example, certain tyrosine phosphatases that could impact signaling displayed higher phosphorylation patterns in ccRCC. Other kinases currently not targeted by RCC therapeutics were more highly phosphorylated in pRCC relative to ccRCC tumors. The functional impact of these findings is currently undergoing evaluation. Conclusions: Integrating global phosphoproteomics of cell lines and tumors with functional analyses can shed light on key survival and growth pathways in clear cell and papillary RCC. Further studies in human tissues are ongoing and will be presented. Citation Format: Scott M. Haake, Jiannong Li, Yun Bai, Bin Fang, Julio Pow-Sang, John Koomen, Mayer Fishman, Eric B. Haura. Phosphoproteomics analysis reveals differential signaling pathways among clear cell and papillary renal cell carcinoma tumors. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5305. doi:10.1158/1538-7445.AM2014-5305