Transcriptomic identification of differentially expressed genes in Levonorgestrel resistant endometrial cancer cell lines

Abstract

Endometrial cancer (EC) is the most common gynecologic malignancy in the world and incidence is steadily increasing. The Levonorgestrel Intrauterine System (LNG‐IUS) is an alternative conservative treatment for early‐stage EC, however, Levonorgestrel (LNG) resistance occurs for 1 in 3 people. This study aimed to present potential LNG resistance mechanisms and identify differentially expressed genes (DEGs) in EC cell lines. Two LNG resistant cell lines were developed through long term culture in LNG (MFE296R and MFE319R). Whole transcriptome sequencing was carried out on triplicate RNA samples. EdgeR v3.32.1 was used to identify differentially DEGs. Blast2go V6.0 (BioBam software) was used for functional annotation and analysis of genomic datasets. Protein interactions were investigated using the STRING database, including the identification of genes with high levels of interaction (HUB genes). Select DEGs and HUB genes were validated by quantitative real‐time polymerase chain reaction (qRT‐PCR) and Western blot. Fifteen DEGs were identified according to FDR < 0.05 and logFC < 2. Protein analysis identified six HUB genes with a degree of connectivity > 10. Relative mRNA expression of MAOA, MAOB, THRSP, CD80, NDP, LINC01474, DUSP2 and CXCL8 was significantly upregulated in both LNGR cell lines. Relative protein expression of GNAO1 and MAOA were significantly upregulated in both LNGR cell lines. This research identified novel markers of resistance in LNGR cell lines. We discussed potential mechanisms of LNG resistance including dedifferentiation and immunostimulation. The next step for this research is to validate these findings further in both translational and clinical settings.

Development of a long term, ex vivo, patient-derived explant model of endometrial cancer

“…On Day 21, the most viable explants from quasi-resistant tumours were fixed and stained immunohistochemically for their expression of five candidate proteins previously identified in a 2D model of EEC as biomarkers of resistance to LNG treatment in our laboratory [ 21 , 22 ]. The candidate biomarkers were examined in the current study to demonstrate the feasibility of using this PDE model for biomarker validation.…”

“…Previous cell line work in our laboratory generated EEC cells resistant to LNG [ 21 ]. Ensuing whole genome RNA sequencing and bioinformatic analysis revealed a number of differentially expressed genes in LNG resistant compared to LNG sensitive cell lines [ 22 ]. Many of these proteins had not been previously investigated in the context of EEC.…”

“…Five proteins were selected from previous biomarker discovery work in our laboratory which were demonstrated to be consistently upregulated on or within EEC cells that were resistant to LNG [ 21 , 22 ]. The following proteins were investigated for their expression pattern in explants by IHC: Special AT-rich sequence-binding protein 2 (SATB2), kruppel like factor 4 (KLF4), monoamine oxidase A (MAO-A), aldehyde dehydrogenase 1 family member A1 (ALDH1A1) and cluster of differentiation 80 (CD80) ( S2 Table ).…”