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Molecular Mechanisms of Endometriosis

Deliverables

Impact of HA-CD44 inhibition on an endometriotic stem cell phenotype

Extending the analyses described in Deliverable 19, KI will perform sphere formation assays, as well as lineage-specific differentiation assays in the experimental systems subjected to 4MU inhibition. Results of TaqMan low density array analysis on identified targets will be confirmed by these functional analyses.

Identification and functional analysis of pain-related microRNAs

In cooperation with partner CHARITE, WWU and CONICET, we will investigate microRNAs known to be dysregulated in endometriosis (34-38) which are predicted to target molecular compounds known to be involved in pain generation in endometriosis (see WP1,WP2). Backed up by in silico analysis of predicted targets, we will focus on miRNAs targeting the confirmed candidate genes of nocireceptive neuronal markers (see WP2) as well as compounds of the NGF signaling pathways. Target regulation of miRNA targets identified in deliverable 36 will be confirmed at the mRNA, protein and 3’UTR level as described above, whereas we will employ the neurite outgrowth assays available at CHARITE, as well as assays in endometrial stroma cells (ST-T1b cell line and primary cells) mimicking the inflammatory stroma as a functional readout for alterations in pain-generating molecular mechanisms.

CSC marker analysis and Nanostic analysis in patient tissues

To further investigate CSCs/ESCs, KI will explore CSC markers in endometrial /endometriotic samples in a large number (n=50) of endometriosis patients to identify endometriosis patients with high risk for EAOEC and develop a prophylactic strategy. This study will be assisted by partners CONICET and WWU. Proliferative phase endometrial and endometriotic samples will be collected at the time of surgery from women undergoing surgery for endometriosis. The expression levels of CSC markers will be quantified by real time PCR and protein quantification setup Wes.This work will be complemented by a studying the whole transcriptome (mRNA, miRNA) and by epigenetic profiling of endometrial /endometriotic samples from women with endometriosis expressing cancer stem cells to understand the molecular mechanism and find new markers to develop prophylactic measures. For this, next generation RNA sequencing with illumina platform available at KI will be used. Differential expression between the groups will be compared using bioinformatics platform, Ingenuity Pathway Analysis (IPA) at KI. This study will give knowledge on functional and non-functional genes and its association with signalling pathways, which have carcinogenic linkage. Genome wide scanning for epigenetic profile in endometrium and endometrioma will be studied using high throughput meDI . These results will be analysed together with transcriptome data using IPA. Nanostic atomic force microscopy to characterize enSC with the expression of CSC to screen endometrium of high risk patients for EAOEC by partner KI will be done with partner Serend-IP.

Role of NFkB-dependent microRNAs in endometriotic cell behaviour

To identify novel downstream regulators of this pathway in endometriosis, partners WWU and UNIVERSIDAD DE CHILE will investigate if activation of this pathway in endometriotic stroma cells leads to an upregulation of miR-9/9* and miR146a, reported to be regulated by NFkB in other systems (73-75). The functional consequences of NFkB-dependent miRNA regulation on invasive behaviour and a stem cell phenotype of these cells will be investigated analogously to WP3 and WP6, and potential targets responsible for the phenotype will be identified following bioinformatic targtet prediction analysis and functional characterisation after transfection with miRNA precursor molecules (see WP6).

Pathogenetic role of the NFkB pathway following iron overload

We will study study endometrial epithelial and stromal cells NF-B-dependent responses to iron overload as a mechanism promoting endometriosis origin, development and maintenance: assessing endometrial stromal and epithelial cell cultures in response to iron overload, evaluating p65-NF-B activation, inflammatory NF-B-dependent cytokines expression (IL-1, IL-8), cell proliferation and apoptosis. Furthermore, by using intracellular iron chelators, this project aims to suggest new drugs for the prevention and treatment of endometriosis: to determine if the iron chelators deferoxamine and deferasirox are able to prevent iron overload-induced changes in endometrial stromal and epithelial cell cultures. In cooperation with partner WWU, UNIVERSIDAD DE CHILE will aim to discover the main function(s) of the alternative NF-kB pathway in human endometrial cells. This will be achieved by silencing RelB expression by using small interfering RNA (siRNA) in endometrial stromal and epithelial cell cultures, by assessing the protein expression of proinflammatory cytokines in response to RelB silencing in endometrial cells using ELISA-based methodology, and by assessing cell proliferation and apoptosis in response to RelB silencing in endometrial cells.

Atomic force microscopy

The impact of SARM treatment on the invasive phenotype of endometriotic cells will be evaluated by quantitative atomic force microscopy by partner SERENDIP.

Nocireceptive neuronal marker study

CHARITE will characterize tissues of more than 120 endometriosis patients and controls for the appearance of nociceptive innervation with the classical markers for nociceptive neurons substance P and TRPV1. The cellular / tissue phenotype will be correlated to a pain score with patients’ pain questionnaires. Using a screening assay allowing to quantify neuronal outgrowth of rodent cultured neurons, sensory neurons will be incubated with with patients’ peritoneal fluid and neurite outgrowth will be quantified. Furthermore, by fluorescence microscopy, the cellular outgrowth phenotype in vitro will be determined as a readout for cells potentially generating nociceptive neurons.The RNA and protein expression of 22 gene candidates, specifically expressed in nociceptive neurons, for neuronal outgrowth after treatment with patients’ peritoneal fluid in rat dorsal root ganglia neurons will be studied to characterise the molecular phenotype.Candidate gene expression will be independelty confirmed in already available patient tissue and/or modulated by specific cellular and animal experiments (65) leading to the development of novel therapeutic opportunities for the treatment, preventions, and understanding of endometriosis related pelvic pain (WP2).

Impact of miRNA expression on an invasive phenotype

Endometriotic cells (12Z cell line or primary eutopic and ectopic endometrial stroma stroma cells from at least 5 endometriosis patients) will be transfected with 20nM miRNA precursors or a negative control miRNA and analyzed for changes in invasive cell behaviour by matrigel invasion chamber assays or video microscopy.Endometriotic cells (12Z cell line or primary eutopic and ectopic endometrial stroma stroma cells from at least 5 endometriosis patients) will be transfected with 20nM miRNA precursors or a negative control miRNA. Changes in predicted target gene expression potentially linked to the invasive phenotype will be monitored by qPCR, Western Blotting and confocal immunofluorescence microscopy (WWU, CONICET). Regulation of selected targets will be confirmed by 3’UTR luciferase reporter assays and plasmid-based complementation analysis using target constructs devoid of the endogenous 3’UTR (WWU).Endometriotic cells (12Z cell line or primary eutopic and ectopic endometrial stroma stroma cells from at least 5 endometriosis patients) will be transfected with 20nM miRNA precursors or a negative control miRNA.Altered signal transduction processes will be analyzed by phosphokinase array screening (R&D systems) and phosphokinase blotting (WWU, CONICET). Finally, we will employ proprietary nanoscale topography analysis (nanostic TM) provided by SEREND-IP to determine cell surface properties (filopodia formation, cell shape, etc.) in cooperation with partners WWU and CONICET. This technique has the potential to develop a marker-free diagnostic tool for invasive endometriotic cell behavious based on quantitative atomic force microscopy.

Effect of herbal medicine on inflammation, angiogenesis and apoptotic resistance

CONICET has previously demondtrated a beneficial therapeutic effect of the natural compounds Wogonin (WG), an active constituent of Chinese Herbal Medicine, and two of the main antioxidant compounds found in rosemary extract: CA and RA, on the development of experimental endometriosis both in vitro and in vivo. CA, RA and WG inhibited significantly cell proliferation in human endometrial stromal cell line T-HESC, and in primary cultures. In a Balb/c-based murine model, these compounds diminished significantly the development of endometriotic-like lesions, reducing considerably its size. Moreover, all compounds decreased endometriotic epithelial and stromal cell proliferation evaluated by immunohistochemistry for PCNA (71,72). Based on these promising results, the following objectives will be addressed in this WP, in collaboration with CHARITE, and UEDIN: (i) To evaluate the effect of these natural compounds on apoptosis in T-HESC cell line and primary cultures. (ii) To evaluate the effect of these compounds on expression and activity of COX-2 and the NFkB pathway in an in vitro model of endometriosis, with particular relevance to WP1 (inflammatory microenvironment), (iii) regarding antioxidant effects in vitro and in vivo, on experimental endometriosis and (iv) on angiogenesis

Evaluation of anti-endocrine therapy in vitro and in vivo

To evaluate the contribution of endometriotic-derived estrogens to ovarian epithelial carcinomas onset and development in vivo an experimental mouse model harbouring the two pathologic entities will be developed by inducing ovarian epithelial carcinomas in immunosuppressed mice with experimental endometriosis.The specific effects exerted by the estrogens secreted by patients-derived endometriotic tissue explants culture on cellular proliferation, migration, matrix invasion, survival and differential gene expression will be determined in human endometrioid and clear cell-derived carcinoma cell lines which express both estrogen receptors, one of them, or none. Finally, in all experimental models, the blockage of estrogens will be carried out to elucidate if this strategy has a potential therapeutic value.

Project meetings and workshops

A kickoff meeting of MOMENDO will by organized by WWU and will take place in Muenster, Germany.Workshops will ensure further communication between participants and dissemination and results: A workshop on microRNAs in endometriosis in Münster organized by WWU in the first project year, a Young Scientist Workshop on the Pathobiology of Endometriosis organizet by UEDIN in Edinburgh in the second project year, a workshop on Endometrial Stem Cells in Stockholm organized by KI and WWU in the third year, and a workshop on Endometriosis and Pain organized by Charite and UEDIN in Berlin in the fourth project year. Finally, WWU will organize Annual meetings of the general assembly and regular Skype meetings, which will be help in combination with the kickoff meeting and workshops.

Molecular role and therapeutic potential of HA in endometriosis

CONICET has demonstrated that inhibition of HA synthesis using 4-methylumbelliferone (4-MU) at low doses, lead to a significant reduction of the vascularized area and functional microvessel density as revealed by in vivo imaging in a mouse model of endometriosis, which was supported by a diminished CD31 staining (Olivares et al, manuscript under revision). We will induce peritoneal endometriosis in BALB/c mice treated with 4-MU and evaluate its effect on endometriotic lesions regarding attachment, proliferation and apoptosis, as well as on angiogenesis and inflammation markers.We will study the role of CD44 and HA on endometriosis by performing the following experiments: 1- cell proliferation and apoptosis evaluation of endometrial stromal and epithelial cells obtained from endometriosis patients and controls treated with 4-MU in vitro; 2- validation of the results obtained on primary cell cultures on two endometrial epithelial and stromal cell lines treated with 4-MU in order to continue with objectives 3 and 4; 3- study of the pathways implicated in the results obtained on the endometrial cell lines treated with 4-MU in vitro, in particular the CD44, AKT/pAKT, ErbB2/pErbB2, beta-catenin and COX-2 pathway; 4- use siRNA approach at different levels of the pathway of interest evaluating how it affects cell survival; 5- evaluation of inflammation and angiogenesis related molecules modulation after treatment of primary cell cultures with 4-MU in vitro or after siRNA introduction in endometrial cell lines.

Therapeutic effect of SARMS

Lead participant UEDIN has identified androgen target genes in primary endometrial stromal cells and obtained evidence that AR agonists can oppose oestrogen-dependent proliferation in this cell type (55). (i) In vitro cell systems developed in Edinburgh using 3D culture scaffolds will be used to evaluate the impact of a range of selective androgen receptor modulators (SARMs) alone, and in combination with E2, on cell behavior (e.g. proliferation, apoptosis, migration) and gene expression. A range of analysis methods will be used to stratify the response to different SARMs including analysis using pathway focused RT² Profiler PCR Arrays (e.g. AR signaling targets, EMT, cell cycle), qRTPCR, reporter assays, Westerns, immunohistochemistry). To test whether SARMs are capable of activating the mouse Ar we will quantify their ability to induce expression of a luciferase reporter gene in female ‘androgen reporter mice’ (68). Uterine tissues will be retrieved from the mice following an injection of BrDu and both Ar expression and cell proliferation determined. We will assess the impact of SARMs identified as having the most potent impact on uterine cells using our in house model of mouse endometriosis (66): endpoints will include lesion size and pain scores. In addition, UEDIN will identify DCA-responsive proteins/functional pathways as future candidate targets for therapeutic development.

Effect and molecular mechanism of Syndecan-1 inhibition on endometriosis

The impact of shRNA-mediated knockdown of Syndecan-1 will be assessed in the in vivo endometriosis models available to partner CONICET. WWU and KI in cooperation with CONICET will employ several in vitro assays of endometriotic stem cell phenotypic analysis, including flow cytometric side population analysis, measurement of ALDH activity, qPCR and FACS analysis of stemness-associated cell surface markers, colony formation and sphere formation assays. The impact of Syndecan-1 and the HA-CD44 assays will be studied in the endometriotic cell line 12Z, and in patient-derived primary endometriotic stroma cells by siRNA knockdown and 4-MU inhibition. TaqMan low density array analysis will be used by WWU to screen for affected pathways, and targets will be confirmed by qPCR and Western blotting.Sphere formation assays, as well as lineage-specific differentiation assays will be performed in the experimental systems subjected to Syndecan-1 shRNA treatment. Results of TaqMan low density array analysis on identified targets will be confirmed by these functional analyses.

NFkB pathway analysis

UNIVERSIDAD DE CHILE will study NfKB-dependent signaling both in rodent primary neurons exposed to peritoneal fluid of endometriosis patients with CHARITE, and in primary endometrial stroma cells of endometriosis patients and controls. Methods include p65-NF-B activation assays (TransAM ® kit (Active Motif Europe)) ELISAs for inflammatory NF-B-dependent cytokine expression (IL-1, IL-8), and proliferation and apoptosis assays following siRNA-mediated knockdown of components of the NFkB, pathway, including RelB.

Therapeutic effects of DCA

We will determine whether dichloroacetate (DCA) can alter the peritoneal environment and halt the growth of endometriosis lesions in women with endometriosis. We will evaluate the impact of DCA in an in-vivo model of endometriosis on (a) glycolysis/lactate production in the peritoneal mesothelium (b) endometriosis lesion growth (c) endometriosis-associated pain behaviour. The effect of DCA will also be tested in vitro, on CSC spheroids generated from endometrium/endometrioma, in collaboration with our KI partner (see WP5). In addition, UEDIN and AQUILA will identify DCA-responsive proteins/functional pathways as future candidate targets for therapeutic development. The effect of Dichloroacetate on a putative stem cell phenotype will be tested in vitro, on CSC spheroids generated from endometrium/endometrioma (see WP5).

In vivo and in vitro models of inflammation-associated pain

UEDIN and partner AQUILA will explore how the peripheral inflammatory environment in endometriosis causes pain. The team has previously used a mouse model of endometriosis using syngeneic menstrual endometrial tissue introduced into the peritoneum of immunocompetent mice to understand the origin of macrophages present in endometriosis lesions (66) and to demonstrate a role for estradiol in regulating both neuroangiogenesis (67) and neuroinflammation (Greaves et al, in press) in endometriosis. UEDIN will use these mice to explore the dynamics of macrophages present in endometriosis lesions and how these cells may contribute to the inflammatory milieu. Analysis techniques will include confocal and multi-photon intra-vital imaging, gene expression analysis and behavioural assessments. We will use in vitro systems to explore how the peripheral inflammatory environment observed in endometriosis influences specific cell types (macrophages and sensory neurons). Analysis techniques will include fluorescent activated cell sorting (FACS), gene expression analysis, enzyme linked immunosorbent assay (ELISA) and calcium assays.

Induced differentiation of ESCs and CSCs as a therapeutic approach

The notch and CD44 pathways will be targeted using in vitro cell culture systems after inhibition with gamma secretase inhibitors, Musashi-1 and -2 siRNA, or 4MU (see WP3) respectively, in cooperation with partners KI, WWU and CONICET. A phenotypic stem cell analysis (see WP3), apoptosis and cell viability assays, as well as sphereformation assays will be utilized as readouts.

Webpage and Social Media

WWU will furthermore be the lead organizer of Results Dissemination via a Website and Facebook site of the project.

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Publications

The endometrial stem cell markers notch-1 and numb are associated with endometriosis

Author(s): Andreas N. Schüring, Beatrice Dahlhues, Anna Korte, Ludwig Kiesel, Ulf Titze, Birthe Heitkötter, Christian Ruckert, Martin Götte
Published in: Reproductive BioMedicine Online, Issue 36, 2017, Page(s) 294-301, ISSN 1472-6483
DOI: 10.1016/j.rbmo.2017.11.010

DNA methylation alterations - potential cause of endometriosis pathogenesis or a reflection of tissue heterogeneity?

Author(s): Merli Saare, Kertu Liis Krigul, Triin Laisk-Podar, Sakthi Vignesh-Srinivasan, Nilufer Rahmioglu, Parameswaran Grace Lalit Kumar, Krina Zondervan, Andres Salumets, Maire Peters
Published in: Biology of Reproduction, 2018, ISSN 0006-3363
DOI: 10.1093/biolre/ioy067

Whole exome sequencing of benign pulmonary metastasizing leiomyoma reveals mutation in the BMP8B gene

Author(s): Deniss Sõritsa, Hindrek Teder, Retlav Roosipuu, Hannes Tamm, Triin Laisk-Podar, Pille Soplepmann, Alan Altraja, Andres Salumets, Maire Peters
Published in: BMC Medical Genetics, Issue 19/1, 2018, ISSN 1471-2350
DOI: 10.1186/s12881-018-0537-5

Differentially-Expressed miRNAs in Ectopic Stromal Cells Contribute to Endometriosis Development: The Plausible Role of miR-139-5p and miR-375

Author(s): Kadri Rekker, Tõnis Tasa, Merli Saare, Külli Samuel, Ülle Kadastik, Helle Karro, Martin Götte, Andres Salumets, Maire Peters
Published in: International Journal of Molecular Sciences, Issue 19/12, 2018, Page(s) 3789, ISSN 1422-0067
DOI: 10.3390/ijms19123789

Hyperglycosylated hCG activates LH/hCG-receptor with lower activity than hCG

Author(s): Hannu Koistinen, Mariann Koel, Maire Peters, Ago Rinken, Karolina Lundin, Timo Tuuri, Juha S. Tapanainen, Henrik Alfthan, Andres Salumets, Ulf-Håkan Stenman, Darja Lavogina
Published in: Molecular and Cellular Endocrinology, Issue 479, 2019, Page(s) 103-109, ISSN 0303-7207
DOI: 10.1016/j.mce.2018.09.006

Endometrial receptivity revisited: endometrial transcriptome adjusted for tissue cellular heterogeneity

Author(s): Marina Suhorutshenko, Viktorija Kukushkina, Agne Velthut-Meikas, Signe Altmäe, Maire Peters, Reedik Mägi, Kaarel Krjutškov, Mariann Koel, Francisco M Codoñer, Juan Fco Martinez-Blanch, Felipe Vilella, Carlos Simón, Andres Salumets, Triin Laisk
Published in: Human Reproduction, Issue 33/11, 2018, Page(s) 2074-2086, ISSN 0268-1161
DOI: 10.1093/humrep/dey301

TAC-seq: targeted DNA and RNA sequencing for precise biomarker molecule counting

Author(s): Hindrek Teder, Mariann Koel, Priit Paluoja, Tatjana Jatsenko, Kadri Rekker, Triin Laisk-Podar, Viktorija Kukuškina, Agne Velthut-Meikas, Olga Fjodorova, Maire Peters, Juha Kere, Andres Salumets, Priit Palta, Kaarel Krjutškov
Published in: npj Genomic Medicine, Issue 3/1, 2018, ISSN 2056-7944
DOI: 10.1038/s41525-018-0072-5

Aberrant expression of genes associated with stemness and cancer in endometria and endometrioma in a subset of women with endometriosis

Author(s): Sakthivignesh Ponandai-Srinivasan, Karin L Andersson, Monica Nister, Merli Saare, Halima A Hassan, Suby J Varghese, Maire Peters, Andres Salumets, Kristina Gemzell-Danielsson, Parameswaran Grace Luther Lalitkumar
Published in: Human Reproduction, Issue 33/10, 2018, Page(s) 1924-1938, ISSN 0268-1161
DOI: 10.1093/humrep/dey241

Gamma‐Secretase inhibition affects viability, apoptosis and the stem cell phenotype of endometriotic cells

Author(s): Laura Ramirez Williams, Kathrin Brüggemann, Marina Hubert, Nurjannah Achmad, Ludwig Kiesel, Sebastian D. Schäfer, Burkhard Greve, Martin Götte
Published in: Acta Obstetricia et Gynecologica Scandinavica, 2019, ISSN 0001-6349
DOI: 10.1111/aogs.13707

rs10732516 polymorphism at the IGF2/H19 locus associates with genotype-specific effects on placental DNA methylation and birth weight of newborns conceived by assisted reproductive technology

Author(s): Heidi Marjonen, Pauliina Auvinen, Hanna Kahila, Olga Tšuiko, Sulev Kõks, Airi Tiirats, Triin Viltrop, Timo Tuuri, Viveca Söderström-Anttila, Anne-Maria Suikkari, Andres Salumets, Aila Tiitinen, Nina Kaminen-Ahola
Published in: Clinical Epigenetics, Issue 10/1, 2018, ISSN 1868-7075
DOI: 10.1186/s13148-018-0511-2

MUC20 expression marks the receptive phase of the human endometrium

Author(s): Artjom Stepanjuk, Mariann Koel, Martin Pook, Merli Saare, Kersti Jääger, Maire Peters, Kaarel Krjutškov, Sulev Ingerpuu, Andres Salumets
Published in: Reproductive BioMedicine Online, Issue 39/5, 2019, Page(s) 725-736, ISSN 1472-6483
DOI: 10.1016/j.rbmo.2019.05.004

Chemosensitivity and chemoresistance in endometriosis – differences for ectopic versus eutopic cells

Author(s): Darja Lavogina, Külli Samuel, Arina Lavrits, Alvin Meltsov, Deniss Sõritsa, Ülle Kadastik, Maire Peters, Ago Rinken, Andres Salumets
Published in: Reproductive BioMedicine Online, Issue 39/4, 2019, Page(s) 556-568, ISSN 1472-6483
DOI: 10.1016/j.rbmo.2019.05.019

Challenges in endometriosis miRNA studies - from tissue heterogeneity to disease specific miRNAs

Author(s): Merli Saare, Kadri Rekker, Triin Laisk-Podar, Nilufer Rahmioglu, Krina Zondervan, Andres Salumets, Martin G?tte, Maire Peters
Published in: Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease, Issue 1863, 2017, Page(s) 2282-2292, ISSN 0925-4439
DOI: 10.1016/j.bbadis.2017.06.018