Impact of Phosphoproteomics in the Era of Precision Medicine for Prostate Cancer

Abstract

Prostate cancer is the most common malignancy in men in the United States. While androgen deprivation therapy results in tumor responses initially, there is relapse and progression to metastatic castration-resistant prostate cancer. Currently, all prostate cancer patients receive essentially the same treatment, and there is a need for clinically applicable technologies to provide predictive biomarkers toward personalized therapies. Genomic analyses of tumors are used for clinical applications, but with a paucity of obvious driver mutations in metastatic castration-resistant prostate cancer, other applications, such as phosphoproteomics, may complement this approach. Immunohistochemistry and reverse phase protein arrays are limited by the availability of reliable antibodies and evaluates a preselected number of targets. Mass spectrometry-based phosphoproteomics has been used to profile tumors consisting of thousands of phosphopeptides from individual patients after surgical resection or at autopsy. However, this approach is time consuming, and while a large number of candidate phosphopeptides are obtained for evaluation, limitations are reduced reproducibility, sensitivity, and precision. Targeted mass spectrometry can help eliminate these limitations and is more cost effective and less time consuming making it a practical platform for future clinical testing. In this review, we discuss the use of phosphoproteomics in prostate cancer and other clinical cancer tissues for target identification, hypothesis testing, and possible patient stratification. We highlight the majority of studies that have used phosphoproteomics in prostate cancer tissues and cell lines and propose ways forward to apply this approach in basic and clinical research. Overall, the implementation of phosphoproteomics via targeted mass spectrometry has tremendous potential to aid in the development of more rational, personalized therapies that will result in increased survival and quality of life enhancement in patients suffering from metastatic castration-resistant prostate cancer.

Keywords: clinical trials; kinase inhibitors; kinases; mass spectrometry; phosphoproteomics; prostate cancer; signaling pathways; targeted mass spectrometry.

Figure 1

PrCa progression and the current treatment landscape. Despite the availability of effective treatment for PrCa in its early stages, there are constant cycles of regression and recurrence due to therapeutic resistance via bypass mechanisms. Utilizing phosphoproteomics approaches to identify activated kinases in late-stage aggressive disease and precisely targeting these kinases with FDA-approved kinase inhibitors, in combination with other standard of care treatment, will lead to increased overall survival. ADT, androgen deprivation therapy; AVPC, aggressive variant prostate cancer; FDA, Food and Drug Administration; PrCa, prostate cancer; PSA, prostate-specific antigen.

Figure 2

General workflow for shotgun phosphoproteomics analysis. Tissue samples may include cultured cell lines, mouse xenografts, or clinical biopsy specimens. Tissue samples are lysed, homogenized, reduced, alkylated, and digested with the appropriate protease(s) (A). Phosphopeptide purification by immunoprecipitation (IP) and centrifugation will yield two fractions: pellet containing phosphotyrosine peptides (pY) and supernatant containing phosphoserine/phosphothreonine (pS/pT) peptides (B). Strong cation exchange is performed for the pS/pT peptides fraction before phosphopeptide enrichment step for both fractions [immobilized metal affinity chromatography (IMAC) or Titanium oxide (TiO₂)] (C) and analysis by LC-MS/MS (D).

Figure 3

Mutations in select kinases in metastatic castration-resistant prostate cancer (mCRPC). Columns represent individual patients, and rows represent genetic alterations detected in tyrosine (A) or serine/threonine (B) kinases. For the 6 studies mentioned (, , –70), samples from a total of 900 patients were sequenced revealing mutations in 59 patients (~7%) for tyrosine kinases and in 82 patients (~9%) for serine/threonine kinases. Importantly, driver mutations were only observed in 4 patients (~0.4%) for tyrosine kinases and 19 patients (~2%) for serine/threonine kinases, suggesting that a very small fraction of the mCRPC population have genomic identifiers of kinase activity. The proportion of patients with alterations in each kinase is listed on the left. Only patients with alterations are represented. Data were extracted from cBioPortal (71, 72).

Figure 4

General workflow for targeted phosphoproteomics analysis. Tissue samples may include cultures cell lines, mouse xenografts, or clinical biopsy specimens such as blood, urine, or tumor biopsies. Samples are processed as described in the shotgun workflow up to proteolytic digestion. Custom designed heavy-labeled peptide standards to specific targets of interest are spiked in with the tryptic peptides followed by enrichment and analysis by LC-MS/MS.

Figure 5

Overview of data integration. Data from a combination of phosphoproteomics, genomics, transcriptomics epigenomics, and metabolomics studies investigating the mutational landscape, phosphoproteomic signature, gene expression changes, and regulation in prostate cancer tumors of individual patients can be used clinically to determine disease drivers (mutations and/or activated kinases and aberrantly regulated signaling pathways) as diagnostic tools, to predict patient outcome, to design personalized therapeutic options, and to aid in better clinical trials design.