Metabolomic and Lipidomic Profiling Identifies The Role of the RNA Editing Pathway in Endometrial Carcinogenesis

Abstract

Endometrial cancer (EC) remains the most common malignancy of the genital tract among women in developed countries. Although much research has been performed at genomic, transcriptomic and proteomic level, there is still a significant gap in the metabolomic studies of EC. In order to gain insights into altered metabolic pathways in the onset and progression of EC carcinogenesis, we used high resolution mass spectrometry to characterize the metabolomic and lipidomic profile of 39 human EC and 17 healthy endometrial tissue samples. Several pathways including lipids, Kynurenine pathway, endocannabinoids signaling pathway and the RNA editing pathway were found to be dysregulated in EC. The dysregulation of the RNA editing pathway was further investigated in an independent set of 183 human EC tissues and matched controls, using orthogonal approaches. We found that ADAR2 is overexpressed in EC and that the increase in expression positively correlates with the aggressiveness of the tumor. Furthermore, silencing of ADAR2 in three EC cell lines resulted in a decreased proliferation rate, increased apoptosis, and reduced migration capabilities in vitro. Taken together, our results suggest that ADAR2 functions as an oncogene in endometrial carcinogenesis and could be a potential target for improving EC treatment strategies.

Figure 1

Multivariate analysis showing metabolic profiles in EC. Principal Component Analysis (PCA) plots showing separation between EC tissue samples (T, in green) and control tissue samples (N, in red) (Panel A) and separation between different EC FIGO stages (stage 1: TI, stage 2: TII, stage 3: TIII) (Panel B) for the positive MS ionization mode. X-axis shows interclass separation and Y-axis illustrates the intra-class variability. Panel C. Heat map of ion rankings, corresponding to their relative concentrations (intensity) for positive MS ionization mode, in the same cohort of subjects. Each row represents a unique feature with a specific m/z and RT while each column represents a unique subject. (m/z: mass to charge ratio; RT: retention time).

Figure 2

ADAR1 and ADAR2 proteins are overexpressed in EC. Panel A. ADAR1 and ADAR2 expression in EC tumors (T) compared to the paired control (C) tissues. Relative protein expression of each enzyme is plotted in the bar graphs. Panel B. Example of ADAR1 and ADAR2 staining levels in a matched EC tissue with the corresponding paired control. Panel C. ADAR1 and ADAR2 expression in EC tissues significantly correlate with the tumor grade. Panel D. Example of ADAR1 and ADAR2 staining levels in a set of 3 different EC tumor grades slides (grade 1, 2 and 3). Panel E. ADAR1 and ADAR2 levels are significantly increased in NEEC compared to EEC tumors. Panel F. Example of ADARs staining in EEC and NEEC tumors.

Figure 3

Functional assays revealing that ADAR2 presents oncogenic functions in vitro. HEC-1A, Ishikawa and RL95-2 EC cell lines were used for the functional assays. Panel A. Proliferation assay showing a significant decrease in cell viability (OD 590 nm) in the 3 cell lines when inhibiting ADAR2 expression. Panel B. Apoptosis assay showing a significant increase in apoptosis rate when silencing ADAR2. Panel C. Wound healing assay indicating a significant decrease in HEC-1A and RL95-2 migration capabilities (% of wound healing) when treating cells with siRNA-ADAR2. No significant changes were seen when inhibiting ADAR1.

Figure 4

Project workflow summary.