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

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.

Characterisation of Levonorgestrel-Resistant Endometrial Cancer Cells.

BACKGROUND: Endometrial cancer (EC) is the most common gynaecologic malignancy in the developed world, and incidence is increasing in premenopausal women. The levonorgestrel intrauterine system (LNG-IUS) is gaining traction as an alternative treatment for hyperplasia and early-stage EC for women who are unable to undergo surgery. Thirty to 60% of the women do not respond to this treatment, making the unknown mechanisms of levonorgestrel (LNG) resistance a critical obstacle for the conservative management of EC. This study aimed to characterise LNG-IUS treatment resistance in early-stage endometrial cancer in cell-line models. METHODS: LNG-resistant endometrial cancer cell lines (MFE296R and MFE319R) and cultures from three early stage endometrial cancer patients were developed. The behavioural profile of MFE296R and MFE319R was analysed using proliferation, adhesion, migration (wound healing and transwell) and invasion (spheroid) assays. LNG-sensitive cell lines (MFE296S and MFE319S) were compared to LNGR cell lines (MFE296R and MFE319R). A literature search was conducted to identify possible candidate biomarkers of LNG resistance. RT-qPCR was used to analyse the mRNA expression of 17 candidate biomarkers in MFE296R and MFE319R. mRNA expression of the top differentially expressed genes was measured using RT-qPCR in primary cultures. RESULTS: LNG resistance did not affect proliferation or invasion in immortalised endometrial cancer cells. Transwell migration was significantly increased in MFE319R cells (p=0.03). Cellular adhesion significantly decreased in both MFE296R cells (p=0.012) and MFE319R cells (p=0.04). mRNA expression of KLF4 and SATB2 was significantly amplified in MFE296R and MFE319R cells. mRNA expression of KLF4 was significantly upregulated LNG-resistant primary cell lines. CONCLUSION: LNG-resistant cells may have more oncogenic potential than their LNG-sensitive counterparts. Significant changes in the mRNA expression of KLF4 and SATB2 of LNG-resistant cells is a promising preliminary result in biomarker discovery for guiding LNG-IUS treatment of early stage endometrial cancer.

Efficacy of the LNG-IUS for treatment of endometrial hyperplasia and early stage endometrial cancer: Can biomarkers predict response?

Endometrial Cancer (EC) is the most common gynaecologic malignancy in the developed world, and is increasing in premenopausal women. The surgical standard of care for early-stage EC is not possible in women with concurrent comorbidities or women who desire a fertility sparing approach. The Levonorgestrel Intrauterine System (LNG-IUS) is gaining traction as an alternative treatment for endometrial hyperplasia and early stage EC in inoperable women. Whilst early evidence appears promising, predictive biomarkers need to be established to determine non-responders, which make up one in three women. This timely review discusses the current literature around the identification of clinical, molecular and novel biomarkers that show potential to predict response to progesterone treatment, including the LNG-IUS.