👤 Martin Götte

What is the physiological role of transforming growth factor-beta (TGF-β1) and syndecans (SDC1, SDC4) in endometriotic cells in women with endometriosis? We observed an abnormal, pro-invasive phenotype in a subgroup of samples with ovarian endometriosis, which was reversed by combining gene silencing of SDC1 with the TGF-β1 treatment. Women with endometriosis express high levels of TGF-β1 and the proteoglycan co-receptors SDC1 and SDC4 within endometriotic cysts. However, how SDC1 and SDC4 expression is regulated by TGF-β1 and the physiological significance of the high expression in endometriotic cysts remains unknown as does the potential role in disease severity. We utilized a pre-validated panel of stem- and cancer cell-associated markers on endometriotic tissue (n = 15) to stratify subgroups of women with endometriosis. Furthermore, CD90+CD73+CD105+ (SC+) endometriotic stromal cells from these patient subgroups were explored for their invasive behaviour in vitro by transient gene inhibition of SDC1 or SDC4, both in the presence or absence of TGF-β1 treatment. Endometriotic cyst biopsies (n = 15) were obtained from women diagnosed with ovarian endometriosis (ASRM Stage III-IV). Gene expression variability was assessed on tissue samples by applying gene clustering tools for the dataset generated from the pre-validated panel of markers. Three-dimensional (3D) spheroids from endometriotic SC+ were treated in vitro with increasing doses of TGF-β1 or the TGFBRI/II inhibitor Ly2109761 and assessed for SDC1, SDC4 expression and in vitro 3D-spheroid invasion. Transcriptomic signatures from the invaded 3D spheroids were evaluated upon combining transient gene silencing of SDC1 or SDC4, both in presence or absence of TGF-β1 treatment. Furthermore, nanoscale changes on the surface of endometriotic cells were analysed after treatment with TGF-β1 or TGFBRI/II inhibitor using atomic force microscopy. Gene clustering analysis revealed that endometriotic tissues displayed variability in their gene expression patterns; a small subgroup of samples (2/15, Endo-hi) exhibited high levels of SDC1, SDC4 and molecules involved in TGF-β signalling (TGF-β1, ESR1, CTNNB1, SNAI1, BMI1). The remaining endometriotic samples (Endo-lo) showed a uniform, low gene expression profile. Three-dimensional spheroids derived from Endo-hi SC+ but not Endo-lo SC+ samples showed an aberrant expression of SDC1 and exhibited enhanced 3D-spheroid invasion in vitro, upon rhTGF-β1 treatment. However, this abnormal, pro-invasive response of Endo-hi SC+ was reversed upon gene silencing of SDC1 with the TGF-β1 treatment. Interestingly, transcriptomic signatures of 3D spheroids silenced for SDC1 and consecutively treated with TGF-β1, showed a down-regulation of cancer-associated pathways such as WNT and GPCR signalling. Transcriptomic data were deposited in NCBI's Gene Expression Omnibus (GEO) and could be retrieved using GEO series accession number: GSE135122. It is estimated that about 2.5% of endometriosis patients have a potential risk for developing ovarian cancer later in life. It is possible that the pro-oncogenic molecular changes observed in this cohort of endometriotic samples may not correlate with clinical occurrence of ovarian cancer later in life, thus a validation will be required. This study emphasizes the importance of interactions between syndecans and TGF-β1 in the pathophysiology of endometriosis. We believe that this knowledge could be important in order to better understand endometriosis-associated complications such as ovarian cancer or infertility. This study was funded by Cancerfonden (CAN 2016/696), Radiumhemmets Forskningsfonder (Project no. 154143 and 184033), EU MSCA-RISE-2015 project MOMENDO (691058), Estonian Ministry of Education and Research (IUT34-16), Enterprise Estonia (EU48695) and Karolinska Institute. Authors do not have any conflict of interest. Show less

Mutations in MYBPC3 are the most common cause of hypertrophic cardiomyopathy (HCM). These mutations produce dysfunctional protein that is quickly degraded and not incorporated in the myofilaments. Most patients are heterozygous and allelic expression differs between cells. We hypothesized that this would lead to cell-to-cell variation in cardiac myosin binding protein-C (cMyBP-C, encoded by MYBPC3 gene) protein levels. Twelve HCM patients were included (six had no sarcomere mutations (HCM Protein and mRNA analysis revealed significantly reduced cMyBP-C levels in MYBPC3 This is the first study to demonstrate intercellular variation of myofilament cMyBP-C protein expression within the myocardium from HCM patients with heterozygous MYBPC3 mutations. Show less

Proteoglycans and glycosaminoglycans modulate numerous cellular processes relevant to tumour progression, including cell proliferation, cell-matrix interactions, cell motility and invasive growth. Among the glycosaminoglycans with a well-documented role in tumour progression are heparan sulphate, chondroitin/dermatan sulphate and hyaluronic acid/hyaluronan. While the mode of biosynthesis differs for sulphated glycosaminoglycans, which are synthesised in the ER and Golgi compartments, and hyaluronan, which is synthesized at the plasma membrane, these polysaccharides partially compete for common substrates. In this study, we employed a siRNA knockdown approach for heparan sulphate (EXT1) and heparan/chondroitin/dermatan sulphate-biosynthetic enzymes (β4GalT7) in the aggressive human breast cancer cell line MDA-MB-231 to study the impact on cell behaviour and hyaluronan biosynthesis. Knockdown of β4GalT7 expression resulted in a decrease in cell viability, motility and adhesion to fibronectin, while these parameters were unchanged in EXT1-silenced cells. Importantly, these changes were associated with a decreased expression of syndecan-1, decreased signalling response to HGF and an increase in the synthesis of hyaluronan, due to an upregulation of the hyaluronan synthases HAS2 and HAS3. Interestingly, EXT1-depleted cells showed a downregulation of the UDP-sugar transporter SLC35D1, whereas SLC35D2 was downregulated in β4GalT7-depleted cells, indicating an intricate regulatory network that connects all glycosaminoglycans synthesis. The results of our in vitro study suggest that a modulation of breast cancer cell behaviour via interference with heparan sulphate biosynthesis may result in a compensatory upregulation of hyaluronan biosynthesis. These findings have important implications for the development of glycosaminoglycan-targeted therapeutic approaches for malignant diseases. Show less