Interplay between post-translational cyclooxygenase-2 modifications and the metabolic and proteomic profile in a colorectal cancer cohort

Abstract

Background: Colorectal cancer (CRC) is the second most common cause of cancer death worldwide. It is broadly described that cyclooxygenase-2 (COX-2) is mainly overexpressed in CRC but less is known regarding post-translational modifications of this enzyme that may regulate its activity, intracellular localization and stability. Since metabolic and proteomic profile analysis is essential for cancer prognosis and diagnosis, our hypothesis is that the analysis of correlations between these specific parameters and COX-2 state in tumors of a high number of CRC patients could be useful for the understanding of the basis of this cancer in humans.

Aim: To analyze COX-2 regulation in colorectal cancer and to perform a detailed analysis of their metabolic and proteomic profile.

Methods: Biopsies from both healthy and pathological colorectal tissues were taken under informed consent from patients during standard colonoscopy procedure in the University Hospital of Bellvitge (Barcelona, Spain) and Germans Trias i Pujol University Hospital (Campus Can Ruti) (Barcelona, Spain). Western blot analysis was used to determine COX-2 levels. Deglycosylation assays were performed in both cells and tumor samples incubating each sample with peptide N-glycosidase F (PNGase F). Prostaglandin E₂ (PGE₂) levels were determined using a specific ELISA. ¹H high resolution magic angle spinning (HRMAS) analysis was performed using a Bruker AVIII 500 MHz spectrometer and proteomic analysis was performed in a nano-liquid chromatography-tandem mass spectrometer (nano LC-MS/MS) using a QExactive HF orbitrap MS.

Results: Our data show that COX-2 has a differential expression profile in tumor tissue of CRC patients vs the adjacent non-tumor area, which correspond to a glycosylated and less active state of the protein. This fact was associated to a lesser PGE₂ production in tumors. These results were corroborated in vitro performing deglycosylation assays in HT29 cell line where COX-2 protein profile was modified after PNGase F incubation, showing higher PGE₂ levels. Moreover, HRMAS analysis indicated that tumor tissue has altered metabolic features vs non-tumor counterparts, presenting increased levels of certain metabolites such as taurine and phosphocholine and lower levels of lactate. In proteomic experiments, we detected an enlarged number of proteins in tumors that are mainly implicated in basic biological functions like mitochondrial activity, DNA/RNA processing, vesicular trafficking, metabolism, cytoskeleton and splicing.

Conclusion: In our colorectal cancer cohort, tumor tissue presents a differential COX-2 expression pattern with lower enzymatic activity that can be related to an altered metabolic and proteomic profile.

Keywords: Carcinoma; Colon; Cyclooxygenase; High resolution magic angle spinning; Prostaglandin; Proteomics.

Figure 1

Analysis of cyclooxygenase-2 protein levels and prostaglandin E₂ measurement. A: Representative image of western blot analysis of cyclooxygenase-1 (COX-1), cyclooxygenase-2 (COX-2) and 15-hydroxyprostaglandin dehydrogenase (15-PGDH) normalized by glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of tumor (T) or non-tumor (N) samples. B and C: Graphs represent normalized COX-1 (B) or 15-PGDH (C) bands in western blot in T vs N tissue. D: Graph represent normalized COX-2 protein levels corresponding to upper (Up) or lower (Down) band in western blot in T vs N tissue. E: Graphs represent the percentage of samples expressing lower, upper, both or none bands in western blot for COX-2. F: Levels of PGE₂ in Tumor vs Non-tumor samples. G: Relative levels of prostaglandin E₂ only in samples from colorectal cancer patients showing the upper band of COX-2 in western blot. n = 45. Graphs show mean ± SD. ^aP ≤ 0.05, ^bP ≤ 0.01 and ^cP ≤ 0.001 vs the non-tumor condition. COX-1: Cyclooxygenase-1; COX-2: Cyclooxygenase-2; 15-PGDH: 15-hydroxyprostaglandin dehydrogenase; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; T: Tumor; N: Non-tumor; PGE₂: Prostaglandin E₂.

Figure 2

Deglycosylation assays of cyclooxygenase-2. A: Representative images of western blot of cyclooxygenase-2 (COX-2) protein levels corresponding to tumor tissue (T), negative control (NC) and deglycosylated tumor tissue (DT). B: Graphs correspond to upper or lower COX-2 bands quantifications. n = 4. C: Representative images of western blot of COX-2 protein levels in HT29 cells under basal conditions (C) or deglycosylated (DG). D: Graphs correspond to upper or lower COX-2 bands quantifications. n = 8. E. Relative levels of prostaglandin E₂ (PGE₂) normalized by total COX-2 band obtained in the corresponding western blot analysis. n = 11. Graphs show mean ± SD. ^aP ≤ 0.05, ^bP ≤ 0.01 and ^cP ≤ 0.001 vs the glycosylated condition. COX-2: Cyclooxygenase-2; T: Tumor; NC: Negative control; DT: Deglycosylated tumor; C: Cells under basal conditions; DG: Deglycosylated; PGE₂: Prostaglandin E₂.

Figure 3

High resolution magic angle spinning analysis of tumor tissue. A: Schematic diagram of high resolution magic angle spinning spectra of tumor tissue (red line) and non-tumor tissue (blue line). Black arrows indicate two peaks corresponding to taurine δ ppm 3.26 and 3.42. B: Graph represents the quantification of both taurines normalized by creatine of tumor (T) and non-tumor (N) tissue. C and D: Correlation graphs of taurine 3.26 (C) and taurine 3.42 (D) in T vs N subdivided depending on the stage of the Tumor: 1-2 vs 3-4. E and F: Graphs represent the quantification of phosphocholine (E) or lactate (F) in T vs N. n = 29. Graphs show mean ± SD. ^bP ≤ 0.01, ^cP ≤ 0.001 of T vs N and ^dP ≤ 0.05, ^fP ≤ 0.001 between stages in tumors. T: Tumor; N: Non-tumor.

Figure 4

Biostatistical analysis of the correlation between the presence of cyclooxygenase-2 bands in western blot and both taurine peaks. A-F: Statistical analysis was performed searching the correlation coefficient between the presence of upper or lower cyclooxygenase-2 band or 15-hydroxyprostaglandin dehydrogenase in western blot and the taurine peaks (3.26 and 3.42 δ ppm) levels of 29 Tumor/Non-tumor pairs of samples. The Pearson’s correlation coefficient is shown in each figure for non-tumor or tumor sample. ^aP ≤ 0.05. COX-2: Cyclooxygenase-2; 15-PGDH: 15-hydroxyprostaglandin dehydrogenase.

Figure 5

Proteomic analysis of tumor tissue of colorectal cancer patients and the corresponding non-tumor adjacent tissue. A: Venn diagram with specific and overlapped proteins. 2188 and 4091 proteins were identified (FDR < 1%) in non-tumor and tumor tissue, respectively. Among them, 1573 were identified in both tissues, and 615 and 2518 were unique in non-tumor and tumor tissue, respectively. B: Enrichment analysis of specific proteins identified in tumor tissue by mass spectrometry performed with proteins that were identified with 5 or more peptides (271 most abundant proteins). Most of these proteins had mitochondrial and metabolic function, or were involved in processes related to nucleic acids processing, cytoskeleton, vesicular trafficking, or splicing. C: Specific tumor tissue proteins identified with 15 or more peptides in liquid chromatography-mass spectrometry analyses. T: Tumor; N: Non-tumor; LC/MS: Liquid chromatography-mass spectrometry.