Abstract B070: Global proteomics and phosphoproteomics profiling of prostate cancer patient derived xenografts tumors from the LuCaP series
Authors: Sychev Zoi E. , Larson Gabbrianne E. , Bergom Hannah E. , Day Abderrahman E. , Ludwig Megan E. , Ali Atef E. , Corey Eva , Plymate Stephen R. , Hwang Justin E. , Drake Justin M. 
Published: June 2023

Castration Resistant Prostate Cancer (CRPC) is a form of prostate cancer (PCa) that is resistant to androgen deprivation therapies. Resistance to these therapies leads to metastatic CRPC of adenocarcinoma (AD) origin and can transform to emergent aggressive variant prostate cancer (AVPC) which has genetic aberration features similar to neuroendocrine (NE) phenotypes. Understanding and identifying the underlying mechanisms of resistance and genetic transformations leading to protein alterations will enable us to develop novel therapeutic methods to counteract this resistance and improve outcomes for men with lethal metastatic prostate cancer. To this end, we used patient-derived xenografts (PDXs), an in-vivo clinically relevant model from advanced prostate cancer (PC) patients, to investigate the biology, identify novel protein targets and evaluate new treatment modalities. We aimed to identify known and novel protein kinase targets that might be phospho-regulated unique between CRPC, in AD and AVPC in NE. To pursue this, we applied a mass spectrometry base phosphoproteomics analysis. We processed 48 PDX samples, which included 15 non-castrated (NCR), 18 castration resistant (CR), which are the AD samples and 15 AVPC, the NE tumors. To enrich for global phosphorylated residues, we used sequential metal oxide affinity chromatography. We sequenced peptides from each sample using liquid chromatography in tandem with Orbitrap Eclipse Tribid MS and FAIMS technology for 2 hour gradients. To normalize and calibrate the LC method and to compare across all 48 runs, we spiked-in 400 femtomol of internal standards (iRT) to each sample. For peptide sequence and search analysis, we used Maxquant search algorithm. Using a &gt; 0.75 probability phospho-residue cut-off and 5% FDR, we identified more than 9,700 phospho-residues. We identified RET, ASCL1, CHGA and SYP in NEPC and Androgen Receptor (AR), FOXA1, HOXB13, NKX3.1 and TACSTD2 in the adenocarcinoma PDX LuCap tumor samples, which confirms that our approach detected specific proteins already previously established. We subsequently performed a kinase substrate enrichment analysis and identified GSK-3, ERK1, ERK2, CDK5 kinases to be significantly enriched in the NE tumors while ATM kinase and PKC alpha and beta were enriched in the AD PDX tumor samples. Hierarchical clustering analysis across NE versus AD tumor samples indicated that samples within each group clustered uniquely where we observed intra and inter sample variability. We generated pathway enrichment and GSEA analysis, identified novel targets for therapeutic treatments and novel surface proteins. In conclusion, we have performed a global mass spectrometry based proteomic analysis and created an important first global phosphoproteomics and proteomics database that provides insights into the differentially regulated protein and phosphoproteins between AD and NE PDX tumors and provide a protein database source to develop new hypothesis.

Citation Format: Zoi E. Sychev, Gabbrianne E. Larson, Hannah E. Bergom, Abderrahman E. Day, Megan E. Ludwig, Atef E. Ali, Eva Corey, Stephen R. Plymate, Justin E. Hwang, Justin M. Drake. Global proteomics and phosphoproteomics profiling of prostate cancer patient derived xenografts tumors from the LuCaP series [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr B070.