Project PXD006561

PRIDE Assigned Tags:
Biomedical Dataset



CDK4/6 inhibitor resistance in prostate cancer


CDK4/6 kinase inhibitors have shown great promise in clinical trials in various cancer types and have recently entered clinical trial for advanced prostate cancer. Although patients are expected to respond well to this class of drugs, development of resistance in some patients is anticipated. To pre-empt this and study how prostate cancer may evade CDK4/6 inhibition, new resistance models were generated from LNCaP and LAPC4 prostate cancer cells cells by prolonged culturing in presence of 0.5uM palbociclib. A shotgun phosphoproteomics approach was utilized and integrated with RNA sequencing data to unravel the molecular underpinnings of acquired resistance to palbociclib and resultant broad CDK4/6 inhibitor resistance.

Sample Processing Protocol

Protein digestion and phosphopeptide enrichment were performed as previously described(Humphrey, Azimifar, and Mann 2015; Drake et al. 2012; Zimman et al. 2010) with minor modifications. Briefly, LNCaP or LAPC4 prostate cancer cell lines that were sensitive or resistant to palbociclib were lysed in 6M guanidium hydrochloride buffer (6M Guanidinium chloride, 100mM Tris pH8.5, 10mM Tris (2-carboxyethyl) phosphine, 40mM 2-chloroacetamide, 2mM Vanadate, 2.5mM NaPyrophosphate, 1mM Beta-glycerophosphate, 10mg/ml N-octyl-glycoside). The lysate was sonicated, cleared, and protein was measured. 5 mg of protein was digested with trypsin and the resulting phosphopeptides were subjected to phosphotyrosine antibody-based enrichment via immunoprecipitation. The immunoprecipitate was washed and pY peptides were eluted. The supernatant from the pY immunoprecipitations was kept for pST peptide enrichment. 2.5 mg of pST peptides were de-salted using C18 columns and then separated using strong cation exchange chromatography. In separate reactions the pY and pST peptides were then further enriched using titanium dioxide columns to remove existing non-phosphorylated peptides. The pY and pST peptides were then de-salted using C18 tips prior to submission on the mass spectrometer. Samples were analyzed by LC-MS/MS using a dual pump nanoRSLC system (Dionex, Sunnyvale CA) interfaced with a Q Exactive HF (ThermoFisher, San Jose, CA)(Kelstrup et al. 2012; Scheltema et al. 2014). Samples were run in technical duplicates, and data were searched using MaxQuant Andromeda version and Mann 2008) against the Uniprot human reference proteome database with canonical and isoform sequences (downloaded September 2016 from MaxQuant Andromeda parameters were set as previously described(Drake et al. 2016).

Data Processing Protocol

MS Data Analysis MS data analysis was performed as previously described(Drake et al. 2016). For clustering, pY data were filtered using an FDR-corrected ANOVA p-value of 0.2, pS/pT data were filtered using an FDR-corrected ANOVA p-value of 0.05. Hierarchical clustering was performed using the Cluster version 3.0 with the Pearson correlation and pairwise complete linkage analysis(Eisen et al. 1998). Java TreeView version 1.1.6r4 was used to visualize clustering results(Saldanha 2004). Kinase Substrate Enrichment Analysis (KSEA) KSEA was performed as previously described(Drake et al. 2012). Briefly, the phosphopeptides were rank-ordered by average fold change between palbociclib resistant LNCaP cell lines and sensitive cells. The enrichment score was calculated using the Kolmogorov-Smirnov statistic. Statistical significance was calculated via permutation analysis. The normalized enrichment score (NES) was calculated by taking the enrichment score and dividing by the mean of the absolute values of all enrichment scores from the permutation analysis. We used the Benjamini-Hochberg procedure to calculate false discovery rate for each kinase.


Zhen Li, Rutgers Cancer Institute of New Jersey
Justin M. Drake, Rutgers Cancer Institute of New Jersey, New Brunswick, NJ, 08901, USA Graduate Program in Cellular and Molecular Pharmacology, Graduate School of Biomedical Sciences, Rutgers ( lab head )

Submission Date


Publication Date



Q Exactive HF


Not available


phosphorylated residue


Not available

Experiment Type

Shotgun proteomics


    de Leeuw R, McNair C, Schiewer MJ, Neupane NP, Brand LJ, Augello MA, Li Z, Cheng LC, Yoshida A, Courtney SM, Hazard S, Hardiman G, Hussain M, Diehl JA, Drake JM, Kelly WK, Knudsen KE. MAPK reliance via acquired CDK4/6 inhibitor resistance in cancer. Clin Cancer Res. 2018 PubMed: 29739788