Investigation into the Mechanisms of Prostate Cancer Androgen Independence Using Label-Free Data-Independent Quantitative LC-HDMSE and Pathway Analysis

Many patients receiving androgen deprivation treatment for prostate cancer [1] demonstrate progression to androgen-insensitive tumours that frequently progress to uncontrolled metastatic disease [2]. LNCaP cells are a prostate cancer cell line that shows androgen dependent growth. Sustained maintenance of LNCaP cells in steroid depleted medium resulted in the development of the LNCaP-Abl and Hof sub-lines, both of which grow independently of androgens [3]. To determine the molecular changes that accompany the development of androgen-independence, LNCaP, Abl and Hof cells were prepared (in triplicate) for label-free LC-MS analysis using both unfractionated and fractionated (SDS-PAGE) protein lysates. Quantitative LC-MS analysis was performed using a nanoAcquity UPLC and ion-mobility enabled Synapt G2 mass spectrometer (Waters) operating in data-independent HDMSE mode. Boinformatic and statistical analysis of the MS data was undertaken with TransOmics Informatics software and ProteinLynx Global Server (PLGS V2.5.2). TransOmics supported relative quantification between samples using precursor ion intensity data, as well as absolute quantification by use of a spiked internal standard of 150 fmol yeast ADH protein that was included in each sample. PLGS revealed a total of 1111 proteins with a mean protein sequence coverage of 15.00%. Numbers of peptides identified per protein was on average 6.29 (Max 49; Min 1). Comparing LNCaP and Abl cells, 428 proteins were seen in at least 2 replicates of ABL, and in 2 replicates of LNCaP. A two-tailed students t-test, assuming equal variance, revealed that there were 188 significantly regulated proteins (p<0.05). Initial pathway analysis revealed changes in the gonadotropin releasing hormone receptor pathway and glycolysis, both of which have been previously implicated in prostate cancer. We anticipate that further analysis of this data will reveal novel pathways altered in the androgen-independent cells and may provide rational targets for intervention in androgen-insensitive metastatic prostate cancer.