Phospho-DIGE Identified Phosphoproteins Involved in Pathways Related to Tumour Growth in Endometrial Cancer

Abstract

Endometrial cancer (EC) is the most common gynecologic malignancy of the endometrium. This study focuses on EC and normal endometrium phosphoproteome to identify differentially phosphorylated proteins involved in tumorigenic signalling pathways which induce cancer growth. We obtained tissue samples from 8 types I EC at tumour stage 1 and 8 normal endometria. We analyzed the phosphoproteome by two-dimensional differential gel electrophoresis (2D-DIGE), combined with immobilized metal affinity chromatography (IMAC) and mass spectrometry for protein and phosphopeptide identification. Quantities of 34 phosphoproteins enriched by the IMAC approach were significantly different in the EC compared to the endometrium. Validation using Western blotting analysis on 13 patients with type I EC at tumour stage 1 and 13 endometria samples confirmed the altered abundance of HBB, CKB, LDHB, and HSPB1. Three EC samples were used for in-depth identification of phosphoproteins by LC-MS/MS analysis. Bioinformatic analysis revealed several tumorigenic signalling pathways. Our study highlights the involvement of the phosphoproteome in EC tumour growth. Further studies are needed to understand the role of phosphorylation in EC. Our data shed light on mechanisms that still need to be ascertained but could open the path to a new class of drugs that could hinder EC growth.

Keywords: 2D-DIGE; endometrial cancer; mass spectrometry; phosphoproteins.

Figure 1

A 2D-DIGE map of normal endometrium (C) and endometrial cancer (EC) phosphoproteome enriched by IMAC columns. Immobilized pH gradient 4–7 strips were used for the first dimension, and 12% polyacrylamide gel for the second dimension. The numbered circles indicate the differentially phosphorylated spots identified in Table 1.

Figure 2

Western blot analysis was utilized to confirm the alteration of phosphorylation of proteins HBB, HSPB1, LDHB and CKB in normal endometrium (C) and endometrial cancer (EC). The intensity of immunostained bands was normalized against the total protein intensities measured from the same blot stained with Red Ponceau. Results are displayed as a histogram (p < 0.05), and each bar represents mean ± standard deviation.

Figure 3

gProfiler classification of the EC phosphoproteins according to their molecular function (MF), biological processes (BP), and cellular component (CC).

Figure 4

Network build-up from one of the most significant bio-functions: (A) Protein folding (Endoplasmic reticulum resident protein 29 (ERP), Endoplasmic reticulum chaperone BiP (HSPA5), Thioredoxin (TNX), Heat shock protein beta-1 (HSPB1), Prefoldin subunit 2 (PFDN2), Protein disulfide-isomerase A6 (PDIA6); (B) Cellular infiltration (Annexin A3 (ANXA3), HSPB1, HSPA5, 2-phospho-D-glycerate hydro-lyase (ENO1), Cathepsin B (CTSB), Protein disulfide-isomerase (P4HB); (C) Catabolism of hydrogen peroxide Thioredoxin (TNX), Hemoglobin subunit beta (HBB); (D) Cellular viability. ENO1, HBB, HSPA5, HSPB1, Heat shock protein beta-6 (HSPB6), Protein mago nashi homolog (MAGOH), P4HB, PFDN2, Proteasome subunit alpha type-3 (PSMA3), Stress-induced-phosphoprotein 1 (STIP), TXN, CTSB. * Isoform indication.