Characterisation of Levonorgestrel-Resistant Endometrial Cancer Cells

Abstract

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 (MFE296 R and MFE319 R ) and cultures from three early stage endometrial cancer patients were developed. The behavioural profile of MFE296 R and MFE319 R was analysed using proliferation, adhesion, migration (wound healing and transwell) and invasion (spheroid) assays. LNG-sensitive cell lines (MFE296 S and MFE319 S ) were compared to LNG R cell lines (MFE296 R and MFE319 R ). 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 MFE296 R and MFE319 R . 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 MFE319 R cells (p=0.03). Cellular adhesion significantly decreased in both MFE296 R cells (p=0.012) and MFE319 R cells (p=0.04). mRNA expression of KLF4 and SATB2 was significantly amplified in MFE296 R and MFE319 R cells. mRNA expression of KLF4 was significantly upregulated LNG-resistant primary cell lines. Conclusion: LNG-resistant cells may have more oncogenic potential than their LNGsensitive 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.

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

“…We previously characterized the LNG resistant cell lines used in the current study, and investigated potential predictive biomarkers of LNG resistance found in the literature. KLF4 and SATB2 were identified as significantly differentially expressed between LNG resistant and non‐resistant EC cell lines 14 . It is important to note that these genes did not appear in the top significant DEGs in the current study.…”

“…Yang et al, conducted a study using the GSE121367 data set from Li et al 26 and SATB2 were identified as significantly differentially expressed between LNG resistant and non-resistant EC cell lines. 14 It is important to note that these genes did not appear in the top significant DEGs in the current study. We did however, observe a nonsignificant increased FC in KLF4 in both cell lines.…”

“…LNG resistant Endometrioid EC cell lines MFE296 R and MFE319 R were developed as previously described 14 . MFE296 and MFE319 cell lines were gifted by the Gynecological Cancer Research Group at the University of New South Wales, Sydney, Australia.…”

“…LNG resistant Endometrioid EC cell lines MFE296 R and MFE319 R were developed as previously described. 14 Thermo Fisher Scientific #11875-093) containing 20% FBS. All media was supplemented with 100U/ml penicillin/streptomycin (Gibco; Thermo Fisher Scientific, #15070-063).…”

Serum HE4 predicts progestin treatment response in endometrial cancer and atypical hyperplasia: A prognostic study

“…This may be in part explained by the fact that women over the age of 50 years had a higher rate of endometrial cancer (25/41, 61%) compared with those younger than 50 years (19/35, 54%). Endometrial cancer is less likely to respond than AEH, 29 and histopathological features that indicate more aggressive disease are predictive of treatment failure 28,30 . Serum HE4 is associated with clinical markers of disease severity including myometrial invasion of 50% or more, FIGO stage, lymphovascular space invasion and tumour size, 13,15,31–36 offering a potential explanation for its association with treatment failure and highlighting the importance of identifying non‐responders before treatment.…”