Identification of differentially expressed proteins in direct expressed prostatic secretions of men with organ-confined versus extracapsular prostate cancer

Abstract

Current protocols for the screening of prostate cancer cannot accurately discriminate clinically indolent tumors from more aggressive ones. One reliable indicator of outcome has been the determination of organ-confined versus nonorgan-confined disease but even this determination is often only made following prostatectomy. This underscores the need to explore alternate avenues to enhance outcome prediction of prostate cancer patients. Fluids that are proximal to the prostate, such as expressed prostatic secretions (EPS), are attractive sources of potential prostate cancer biomarkers as these fluids likely bathe the tumor. Direct-EPS samples from 16 individuals with extracapsular (n = 8) or organ-confined (n = 8) prostate cancer were used as a discovery cohort, and were analyzed in duplicate by a nine-step MudPIT on a LTQ-Orbitrap XL mass spectrometer. A total of 624 unique proteins were identified by at least two unique peptides with a 0.2% false discovery rate. A semiquantitative spectral counting algorithm identified 133 significantly differentially expressed proteins in the discovery cohort. Integrative data mining prioritized 14 candidates, including two known prostate cancer biomarkers: prostate-specific antigen and prostatic acid phosphatase, which were significantly elevated in the direct-EPS from the organ-confined cancer group. These and five other candidates (SFN, MME, PARK7, TIMP1, and TGM4) were verified by Western blotting in an independent set of direct-EPS from patients with biochemically recurrent disease (n = 5) versus patients with no evidence of recurrence upon follow-up (n = 10). Lastly, we performed proof-of-concept SRM-MS-based relative quantification of the five candidates using unpurified heavy isotope-labeled synthetic peptides spiked into pools of EPS-urines from men with extracapsular and organ-confined prostate tumors. This study represents the first efforts to define the direct-EPS proteome from two major subclasses of prostate cancer using shotgun proteomics and verification in EPS-urine by SRM-MS.

Fig. 1.

Workflow of study design from the discovery of differentially expressed proteins to verification of candidates in prostatic fluids.

Fig. 2.

A, Average number of unique proteins identified in the duplicate MudPIT runs for each discovery direct-EPS sample. B, Venn diagram depicting the total number of proteins exclusively expressed in the extracapsular or organ-confined groups. C, Gene Ontology enrichment of the extracapsular (EC) and organ-confined (OC) direct-EPS.

Fig. 3.

A, Distribution of all 624 proteins identified in the current direct-EPS data set. Blue and red dots represent the 133 differentially expressed proteins based on a fold change of at least 2 and a FDR of ≤0.05 by QSpec analysis (red dots are proteins up-regulated in the extracapsular tumor group, blue dots are significantly down-regulated in the extracapsular tumor group, with the 14 short-listed candidates' indicated by their names). EC denotes extracapsular, OC denotes organ-confined. B, Two significantly enriched pathways in the 133 differentially expressed proteins involved in hemostatic and immunological processes. Pink circles represent proteins up-regulated in the extracapsular tumor group; blue circles represent proteins down-regulated in the extracapsular group; orange triangles represent linkers.

Fig. 4.

Protein prioritization scheme for the selection of candidates for verification.

Fig. 5.

A, Measurement of PSA and PAP in direct-EPS from patients with extracapsular (EC) and organ-confined (OC) prostate tumors in the discovery cohort by mass spectrometry (reported as normalized spectral abundance factors, NSAF) and in a new set of samples by ELISA (reported as μg/ml; ** p value ≤0.01; * p value ≤0.05). B, Differential expression of verified candidates by mass spectrometry in extracapsular and organ-confined direct-EPS and Western blotting in biochemically recurrent (R) and nonrecurrent (NR) patient direct-EPS (reported as densitometry values; ** p value ≤0.01; * p value ≤0.05; x, p value ≤0.1; NS denotes an insignificant p value).

Fig. 6.

Verification of differential expression of candidates in EPS-urines pooled from patients with extracapsular (EC) or organ-confined (OC) prostate tumors. A, Five candidates were measured by Western blot and SRM-MS using heavy isotope-labeled peptide standards. Relative quantitative values are shown for each technical replicate and as the average ratio of light/heavy peptide in the organ-confined tumor group divided by the light/heavy peptide in the extracapsular tumor group ± standard deviation for each peptide. B, PSA measurements in individual EPS-urines from patients with extracapsular and organ-confined prostate tumors by ELISA (NS denotes insignificant p value). The same patient samples were pooled and assayed by Western blot for PSA as well as SRM-MS.