Proteomic Characterization of Prostate Cancer to Distinguish Nonmetastasizing and Metastasizing Primary Tumors and Lymph Node Metastases

Abstract

Patients with metastatic prostate cancer (PCa) have a poorer prognosis than patients with organ-confined tumors. We strove to uncover the proteome signature of primary PCa and associated lymph node metastases (LNMs) in order to identify proteins that may indicate or potentially promote metastases formation. We performed a proteomic comparative profiling of PCa tissue from radical prostatectomy (RPE) of patients without nodal metastases or relapse at the time of surgical resection (n=5) to PCa tissue from RPE of patients who suffered from nodal relapse (n=5). For the latter group, we also included patient-matched tissue of the nodal metastases. All samples were formalin fixed and paraffin embedded. We identified and quantified more than 1200 proteins by liquid chromatography tandem mass spectrometry with subsequent label-free quantification. An increase of ribosomal or proteasomal proteins in LNM (compared to corresponding PCa) became apparent, while extracellular matrix components rather decreased. Immunohistochemistry (IHC) corroborated accumulation of poly-(ADP-ribose)-polymerase 1 and N-myc-downstream-regulated-gene 3, alpha/beta hydrolase domain-containing protein 11, and protein phosphatase slingshot homolog 3 in LNM. These findings strengthen the present interest in examining PARP inhibitors for the treatment of aggressive PCa. IHC also corroborated increased abundance of retinol dehydrogenase 11 in metastasized primary PCa compared to organ-confined PCa. Generally, metastasizing primary tumors were characterized by an enrichment of proteins involved in cellular lipid metabolic processes with concomitant decrease of cell adhesion proteins. This study highlights the usefulness of a combined proteomic-IHC approach to explore novel aspects in tumor biology. Our initial results open novel opportunities for follow-up studies.

Figure 1

Workflow of the study. After surgery (A), PCa and LNM tissues were formalin-fixed and paraffin-embedded (B). On HE-stained sections, experienced pathologists marked tissue areas for proteomic analysis. These were used as templates for the macrodissection of tumor tissue on the deparaffinized sections to analyze (C). Proteins were extracted from the tissue pieces, digested into peptides, and analyzed with LC-MS/MS (D). MaxQuant was used for peptide/protein identification and MaxLFQ for label-free quantification. During data analysis, we compared the sample groups TU with LNM, TU without LNM, and recurrent LNM against each other (E). IHC was performed to corroborate interesting protein candidates from proteomics data (F).

Figure 2

Overview of proteomic results. (A) The Venn diagram shows that 1294 proteins were shared among all three groups. This number and the other numbers given refer to proteins which were present in at least four of five replicates per group. (B) The average FCs were visualized as boxplots. (C) Histograms showing the average log2-transformed LFQ protein intensities of the three sample groups. (D and E) Volcano plots showing average FC values and limma-moderated P values. Comparing recurrent LNM to TU with LNM, 87 proteins met significance criteria (P value < .01). Comparing TU with LNM to TU without LNM, 35 proteins displayed a P value < .01.

Figure 3

Functional annotation of differentially regulated proteins. (A/B) GO term enrichment in TU with LNM compared to TU without LNM. Predominantly enriched biological process (BP) was “cellular metabolic process,” while “muscle contraction” was reported the most depleted BP. (C) STRING functional annotation clustering revealed several clusters, which are marked in green if clustering proteins (moderated P value < .05; limma) were enriched or in red if they were decreased in recurrent LNM compared to TU with LNM. PARP1 laid at the interface of several clusters.

Figure 4

IHC stainings for (A) PARP1, (B) NDRG3 (C) ABHD11, (D) SSH3, and (E) RDH11 in recurrent LNM, TU with LNM, and TU without LNM, respectively. Staining intensity was evaluated manually and scored as follows: 0 = negative, 1 = weak, 2 = moderate, or 3 = strong. The average intensity scores for each group are plotted in the bar charts on the right side. Statistically significant differences were marked with an asterisk (*) and the respective P value (Student's t test).

Figure 4

IHC stainings for (A) PARP1, (B) NDRG3 (C) ABHD11, (D) SSH3, and (E) RDH11 in recurrent LNM, TU with LNM, and TU without LNM, respectively. Staining intensity was evaluated manually and scored as follows: 0 = negative, 1 = weak, 2 = moderate, or 3 = strong. The average intensity scores for each group are plotted in the bar charts on the right side. Statistically significant differences were marked with an asterisk (*) and the respective P value (Student's t test).