Oral Presentation Annual Meetings of the Endocrine Society of Australia and Society for Reproductive Biology and Australia and New Zealand Bone and Mineral Society 2016

Mechanical control of human endometrium: Role of ECM-mediated TGFβ signalling in uterine epithelium (#241)

Subhransu Sahoo 1 , Pradeep Tanwar 1
  1. Gynaecology Oncology group, School of Biomedical Sciences and Pharmacy, University of Newcastle, Callaghan 2308, NSW, Australia

The extracellular matrix (ECM) is a major component of the cellular microenvironment and regulates normal tissue development and homeostasis. ECM of female reproductive tract undergoes extensive structural remodelling for decidualization, implantation and endometrium regeneration. In contrast, abnormal ECM dynamics contribute to pathological processes such as endometriosis, infertility and cancer.  The signalling alteration in uterine stroma or ECM that regulates remodelling of differentiated endometrium to a disease state is currently unclear.

To investigate this, we cultured 10 different endometrial cancer (EC) cell lines in 3D matrix, which revealed distinct glandular and non-glandular colonies.  We analysed differentially expressed genes in 3D vs monolayer-cultured (2D) cells using RNA seq and identified that the members of TGFβ (Transforming growth factor beta) signalling pathway were most upregulated in EC spheroids forming disintegrated colonies (MFE 296) than round morphology (Ishikawa). MFE 296 cells displayed overexpression of pSmad2 protein (>2-fold) in 3D matrix suggesting ECM regulates TGFβ signalling. In 3D culture, TGFβ1 cytokine treatment led to increase in pSmad2 protein expression in Ishikawa cells whereas treatment of TGFβ signalling inhibitor (SB431542) decreased basal level pSmad2 expression, proliferation and invasion of MFE 296 cells (p<0.01). 3D cultured Ishikawa and MFE 296 cells distinctly expressed epithelial and mesenchymal markers. Moreover, TGFβ1 treatment disrupted polarized and glandular architecture of Ishikawa cells and elevated actin disorganization. In contrast, SB431542 induced reverted glandular morphology of MFE 296 with decreased invasive propensity. Specifically, treatment of TGFβ1 stimulated slug protein (mesenchymal marker) expression in Ishikawa cells whereas SB431542 inhibited basal level snail, slug and zeb1 protein expression in MFE 296 cells. Collectively, our results depicted the role of ECM-derived TGFβ signalling in endometrial cell behaviour.