Syndecan-1 modulates the invasive potential of endometrioma via TGF-ß signalling in a subgroup of women with endometriosis.

STUDY QUESTION: What is the physiological role of transforming growth factor-beta (TGF-ß1) and syndecans (SDC1, SDC4) in endometriotic cells in women with endometriosis? SUMMARY ANSWER: 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-ß1 treatment. WHAT IS KNOWN ALREADY: Women with endometriosis express high levels of TGF-ß1 and the proteoglycan co-receptors SDC1 and SDC4 within endometriotic cysts. However, how SDC1 and SDC4 expression is regulated by TGF-ß1 and the physiological significance of the high expression in endometriotic cysts remains unknown as does the potential role in disease severity. STUDY DESIGN, SIZE, DURATION: 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-ß1 treatment. PARTICIPANTS/MATERIALS, SETTING, METHODS: 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-ß1 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-ß1 treatment. Furthermore, nanoscale changes on the surface of endometriotic cells were analysed after treatment with TGF-ß1 or TGFBRI/II inhibitor using atomic force microscopy. MAIN RESULTS AND THE ROLE OF CHANCE: 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-ß signalling (TGF-ß1, 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-ß1 treatment. However, this abnormal, pro-invasive response of Endo-hi SC+ was reversed upon gene silencing of SDC1 with the TGF-ß1 treatment. Interestingly, transcriptomic signatures of 3D spheroids silenced for SDC1 and consecutively treated with TGF-ß1, showed a down-regulation of cancer-associated pathways such as WNT and GPCR signalling. LARGE SCALE DATA: Transcriptomic data were deposited in NCBI's Gene Expression Omnibus (GEO) and could be retrieved using GEO series accession number: GSE135122. LIMITATIONS, REASONS FOR CAUTION: 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. WIDER IMPLICATIONS OF THE FINDINGS: This study emphasizes the importance of interactions between syndecans and TGF-ß1 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. STUDY FUNDING/COMPETING INTEREST(S): 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.

Identification of CK2 as the kinase that phosphorylates Pax3 at Ser209 in early myogenic differentiation.

The myogenic transcription factor Pax3, a member of the paired class homeodomain family of transcription factors, plays an essential role in early skeletal muscle development. We previously demonstrated that Pax3 is phosphorylated at three specific residues (Ser201, Ser205, and Ser209) and that the pattern of phosphorylation at these sites changes throughout early myogenesis. Further, we demonstrated that the protein kinase CK2 phosphorylates Pax3 at Ser205 and that this phosphorylation event is required for the subsequent phosphorylation of Ser201 by GSK3ß. However, the kinase that phosphorylates Pax3 at Ser209 has yet to be identified. In the present work we use standard purification methods and in vitro biochemical analyses to provide solid evidence identifying the protein kinase CK2 as phosphorylating Pax3 at Ser209. Further, we qualitatively demonstrate that the phosphorylation of Pax3 at Ser209 by CK2 is enhanced when Ser205 is previously phosphorylated. Taken together, our results allow us to propose a mechanism to describe the ordered phosphorylation of Pax3 throughout early myogenesis.

Identification of serines 201 and 209 as sites of Pax3 phosphorylation and the altered phosphorylation status of Pax3-FOXO1 during early myogenic differentiation.

Pax3, a member of the paired class homeodomain family of transcription factors, is essential for early skeletal muscle development and is key in the development of the childhood solid muscle tumor alveolar rhabdomyosarcoma (ARMS). ARMS is primarily characterized by a t(2;13)(q35;q14) chromosomal translocation, which fuses the 5'-coding sequences of Pax3 with the 3'-coding sequence of the forkhead transcription factor FOXO1 generating the oncogenic fusion protein Pax3-FOXO1. We previously demonstrated that Pax3 and Pax3-FOXO1 are phosphorylated by the protein kinase CK2 at serine 205 in proliferating primary myoblasts and that this phosphorylation event is rapidly lost from Pax3, but not Pax3-FOXO1 upon the induction of differentiation. However, reports suggested that additional sites of phosphorylation might be present on Pax3. In this report we use in vitro and in vivo analyses to identify serines 201 and 209 as additional sites of phosphorylation and along with serine 205 are the only sites of phosphorylation on Pax3. We provide solid evidence supporting the role of the protein kinase GSK3ß as phosphorylating Pax3 at serine 201. Using phospho-specific antibodies we demonstrate a changing pattern of phosphorylation at serines 201, 205, and 209 throughout early myogenic differentiation and that this pattern of phosphorylation is different for Pax3-FOXO1 in primary myoblasts and in several ARMS cell lines. Taken together, our results allow us to propose a molecular model to describe the changing pattern of phosphorylation for Pax3 and the altered phosphorylation for Pax3-FOXO1 during early myogenic differentiation.