Recently, few studies have reported alterations in the metabolomic or proteomic phenotype of EC in serum²⁵, urine²⁶ or in other sample types^{27, 28}, underscoring the clinical translational relevance of these technologies in furthering the personalized medicine initiative. In this study, we used a global metabolomics profiling approach in order to understand the metabolic changes that take place in EC carcinogenesis and during tumor progression (Fig. 4).

Our study reveals an array of metabolites dysregulated in EC, some of which have been previously described in endometrial carcinogenesis. Glycerophospholipid class of metabolites was found to be upregulated in tumor tissues, including PCs, PEs, and PIs. Lipid biosynthesis and catabolism is known to be altered in several diseases, including cancer^28–32. Consistent with our findings, Trousil et al.²⁸ also observed an increase (up to 70%) in PC levels in EC tissues. We also observed a downregulation of the acylamido analogs of endocannabinoids such as palmitamide, stearamide and oleamide in EC. This downregulation has been previously reported³³. We also observed a decreased proportion of picolinic acid in tumor samples. Picolinic acid and quinolinic acid are the end products of the Kynurenine pathway and have been shown to have anti-tumoral and pro-tumoral activity respectively³⁴. Moreover, the activity of one of the main enzymes of the pathway, indoleamine 2,3-dioxygenase (IDO) has also been studied in EC³⁵. Decreased levels of Glu Phe Arg Trp and inosine as well as upregulation of 3-Deoxyvitamin D3 and UDP-N-acetyl-D-galactosamine were also found in our EC tumor sample set compared to control tissues. We also analyzed metabolomic changes that underscore tumor progression. Our data suggest significant changes in the lipidome including PC (16:0/20:5), PC (16:0/22:6), PE (16:0/22:6), PE (22:6/P-18:1) and PE (18:1/22:6) at different stages of cancer progression, as well as a significant increase in the levels of UDP-N-acetyl-D-galactosamine and arachidonic acid at advanced stages of EC. Further studies would be needed to fully understand the scope and impact of these alterations in cancer progression.

The dysregulation of inosine was further studied to dissect the functional implications of the RNA editing pathway in EC. The dysregulation of inosine is indicative of a possible imbalance in the I/A ratio, which was also reported by Trousil et al.²⁸ in studies with EC tissue compared to normal endometrium. The A-to-I conversion is the most common type of RNA editing found in mammals mediated by the ADAR enzymes. Although, to date, the RNA editing pathway and the expression and function of the ADAR gene family has not been interrogated in EC, it has been reported that the expression of ADAR enzymes is upregulated in many cancers^{18, 19}, including breast¹⁷ and esophageal squamous cell carcinoma^{36, 37}. The ADAR family is comprised of three members: ADAR1 and ADAR2, that are present in most human tissues; and ADAR3, that is brain specific²³. Changes in editing frequencies have been described in other diseases including prostate, lung, kidney and testis tumors while reduced RNA levels of ADAR1, ADAR2 and ADAR3 have been observed in brain tumors^{38, 39}. ADAR enzymes are also involved in physiological events such neuronal development, immune response, cell response to viruses and regulation of miRNA expression among others^{40, 41}.

Hence, we asked if the expression of ADAR enzymes had any correlation with EC initiation and progression. Our findings not only elucidate ADAR1 and ADAR2 enzymes to be significantly upregulated in EC tumor tissues compared to healthy endometrium, but also demonstrate a significant correlation between ADARs expression and the malignancy of the tumor. We found that the ADARs expression increased progressively with tumor grade. More importantly, our data showed a significant increase in the expression of ADAR1 and ADAR2 in the most aggressive subtype of EC, the NEEC, that have the worst predicted survival³.

The role of the RNA editing pathway in EC was further investigated by knocking-down the expression of ADAR1 and ADAR2 in HEC-1A, RL95-2 and Ishikawa EC lines. Our results demonstrate the impact of decreased ADAR2 expression on an array of cellular functions in EC cell lines including a significant decrease of cell proliferation and viability, increased apoptosis rate, and reduced migration capabilities in vitro. Similar to our observations, silencing of ADARs in breast cancer cell lines led to less cell proliferation and more apoptosis¹⁷ and overexpression of editing enzymes accelerated growth rate and colony formation in esophageal squamous cell carcinoma in vitro ³⁶. Taken together, our results strongly suggest, for the first time, that the RNA editing gene family, specifically ADAR2, may play an important role promoting EC carcinogenesis.

In conclusion, a global molecular profiling approach using high resolution mass spectrometry has been useful, not only to describe changes in the metabolome and lipidome in human endometrial carcinogenesis and EC progression but also led to the discovery of an important alteration of the RNA editing pathway in EC. We further demonstrated the role of this pathway in proliferation and viability, apoptosis, and migration of EC cells, leading us to conclude that the activation of the RNA editing pathway is an oncogenic process in EC. This study opens several avenues for further investigations of ADAR2 as possible target for the development of therapeutic approaches for the treatment of EC patients.