Homeobox regulator Wilms Tumour 1 is displaced by androgen receptor at cis-regulatory elements in the endometrium of PCOS patients.

Decidualisation, the process whereby endometrial stromal cells undergo morphological and functional transformation in preparation for trophoblast invasion, is often disrupted in women with polycystic ovary syndrome (PCOS) resulting in complications with pregnancy and/or infertility. The transcription factor Wilms tumour suppressor 1 (WT1) is a key regulator of the decidualization process, which is reduced in patients with PCOS, a complex condition characterized by increased expression of androgen receptor in endometrial cells and high presence of circulating androgens. Using genome-wide chromatin immunoprecipitation approaches on primary human endometrial stromal cells, we identify key genes regulated by WT1 during decidualization, including homeobox transcription factors which are important for regulating cell differentiation. Furthermore, we found that AR in PCOS patients binds to the same DNA regions as WT1 in samples from healthy endometrium, suggesting dysregulation of genes important to decidualisation pathways in PCOS endometrium due to competitive binding between WT1 and AR. Integrating RNA-seq and H3K4me3 and H3K27ac ChIP-seq metadata with our WT1/AR data, we identified a number of key genes involved in immune response and angiogenesis pathways that are dysregulated in PCOS patients. This is likely due to epigenetic alterations at distal enhancer regions allowing AR to recruit cofactors such as MAGEA11, and demonstrates the consequences of AR disruption of WT1 in PCOS endometrium.

Transcriptomic analysis reveals the anti-cancer effect of gestational mesenchymal stem cell secretome.

The environment created during embryogenesis contributes to reducing aberrations that drive structural malformations and tumorigenesis. In this study, we investigate the anti-cancer effect of mesenchymal stem cells (MSCs) derived from 2 different gestational tissues, the amniotic fluid (AF) and the chorionic villi (CV), with emphasis on their secretome. Transcriptomic analysis was performed on patient-derived AF- and CV-MSCs collected during prenatal diagnosis and identified both mRNAs and lncRNAs, involved in tissue homeostasis and inhibiting biological processes associated with the etiology of aggressive cancers while regulating immune pathways shown to be important in chronic disorders. Secretome enrichment analysis also identified soluble moieties involved in target cell regulation, tissue homeostasis, and cancer cell inhibition through the highlighted Wnt, TNF, and TGF-ß signaling pathways. Transcriptomic data were experimentally confirmed through in vitro assays, by evaluating the anti-cancer effect of the media conditioned by AF- and CV-MSCs and the exosomes derived from them on ovarian cancer cells, revealing inhibitory effects in 2D (by reducing cell viability and inducing apoptosis) and in 3D conditions (by negatively interfering with spheroid formation). These data provide molecular insights into the potential role of gestational tissues-derived MSCs as source of anti-cancer factors, paving the way for the development of therapeutics to create a pro-regenerative environment for tissue restoration following injury, disease, or against degenerative disorders.

Development and Characterisation of a New Patient-Derived Xenograft Model of AR-Negative Metastatic Castration-Resistant Prostate Cancer.

As the treatment landscape for prostate cancer gradually evolves, the frequency of treatment-induced neuroendocrine prostate cancer (NEPC) and double-negative prostate cancer (DNPC) that is deficient for androgen receptor (AR) and neuroendocrine (NE) markers has increased. These prostate cancer subtypes are typically refractory to AR-directed therapies and exhibit poor clinical outcomes. Only a small range of NEPC/DNPC models exist, limiting our molecular understanding of this disease and hindering our ability to perform preclinical trials exploring novel therapies to treat NEPC/DNPC that are urgently needed in the clinic. Here, we report the development of the CU-PC01 PDX model that represents AR-negative mCRPC with PTEN/RB/PSMA loss and CTNN1B/TP53/BRCA2 genetic variants. The CU-PC01 model lacks classic NE markers, with only focal and/or weak expression of chromogranin A, INSM1 and CD56. Collectively, these findings are most consistent with a DNPC phenotype. Ex vivo and in vivo preclinical studies revealed that CU-PC01 PDX tumours are resistant to mCRPC standard-of-care treatments enzalutamide and docetaxel, mirroring the donor patient's treatment response. Furthermore, short-term CU-PC01 tumour explant cultures indicate this model is initially sensitive to PARP inhibition with olaparib. Thus, the CU-PC01 PDX model provides a valuable opportunity to study AR-negative mCRPC biology and to discover new treatment avenues for this hard-to-treat disease.

Transcriptomic analysis of stem cells from chorionic villi uncovers the impact of chromosomes 2, 6 and 22 in the clinical manifestations of Down syndrome.

BACKGROUND: Down syndrome (DS) clinical multisystem condition is generally considered the result of a genetic imbalance generated by the extra copy of chromosome 21. Recent discoveries, however, demonstrate that the molecular mechanisms activated in DS compared to euploid individuals are more complex than previously thought. Here, we utilize mesenchymal stem cells from chorionic villi (CV) to uncover the role of comprehensive functional genomics-based understanding of DS complexity. METHODS: Next-generation sequencing coupled with bioinformatic analysis was performed on CV obtained from women carrying fetuses with DS (DS-CV) to reveal specific genome-wide transcriptional changes compared to their euploid counterparts. Functional assays were carried out to confirm the biological processes identified as enriched in DS-CV compared to CV (i.e., cell cycle, proliferation features, immunosuppression and ROS production). RESULTS: Genes located on chromosomes other than the canonical 21 (Ch. 2, 6 and 22) are responsible for the impairment of life-essential pathways, including cell cycle regulation, innate immune response and reaction to external stimuli were found to be differentially expressed in DS-CV. Experimental validation confirmed the key role of the biological pathways regulated by those genes in the etiology of such a multisystem condition. CONCLUSIONS: NGS dataset generated in this study highlights the compromised functionality in the proliferative rate and in the innate response of DS-associated clinical conditions and identifies DS-CV as suitable tools for the development of specifically tailored, personalized intervention modalities.

In silico enhancer mining reveals SNS-032 and EHMT2 inhibitors as therapeutic candidates in high-grade serous ovarian cancer.

BACKGROUND: Epigenomic dysregulation has been linked to solid tumour malignancies, including ovarian cancers. Profiling of re-programmed enhancer locations associated with disease has the potential to improve stratification and thus therapeutic choices. Ovarian cancers are subdivided into histological subtypes that have significant molecular and clinical differences, with high-grade serous carcinoma representing the most common and aggressive subtype. METHODS: We interrogated the enhancer landscape(s) of normal ovary and subtype-specific ovarian cancer states using publicly available data. With an initial focus on H3K27ac histone mark, we developed a computational pipeline to predict drug compound activity based on epigenomic stratification. Lastly, we substantiated our predictions in vitro using patient-derived clinical samples and cell lines. RESULTS: Using our in silico approach, we highlighted recurrent and privative enhancer landscapes and identified the differential enrichment of a total of 164 transcription factors involved in 201 protein complexes across the subtypes. We pinpointed SNS-032 and EHMT2 inhibitors BIX-01294 and UNC0646 as therapeutic candidates in high-grade serous carcinoma, as well as probed the efficacy of specific inhibitors in vitro. CONCLUSION: Here, we report the first attempt to exploit ovarian cancer epigenomic landscapes for drug discovery. This computational pipeline holds enormous potential for translating epigenomic profiling into therapeutic leads.

Bromodomain inhibitor i-BET858 triggers a unique transcriptional response coupled to enhanced DNA damage, cell cycle arrest and apoptosis in high-grade ovarian carcinoma cells.

BACKGROUND: Ovarian cancer has a specific unmet clinical need, with a persistently poor 5-year survival rate observed in women with advanced stage disease warranting continued efforts to develop new treatment options. The amplification of BRD4 in a significant subset of high-grade serous ovarian carcinomas (HGSC) has led to the development of BET inhibitors (BETi) as promising antitumour agents that have subsequently been evaluated in phase I/II clinical trials. Here, we describe the molecular effects and ex vivo preclinical activities of i-BET858, a bivalent pan-BET inhibitor with proven in vivo BRD inhibitory activity. RESULTS: i-BET858 demonstrates enhanced cytotoxic activity compared with earlier generation BETis both in cell lines and primary cells derived from clinical samples of HGSC. At molecular level, i-BET858 triggered a bipartite transcriptional response, comprised of a 'core' network of genes commonly associated with BET inhibition in solid tumours, together with a unique i-BET858 gene signature. Mechanistically, i-BET858 elicited enhanced DNA damage, cell cycle arrest and apoptotic cell death compared to its predecessor i-BET151. CONCLUSIONS: Overall, our ex vivo and in vitro studies indicate that i-BET858 represents an optimal candidate to pursue further clinical validation for the treatment of HGSC.

Selenium nanoparticles modulate histone methylation via lysine methyltransferase activity and S-adenosylhomocysteine depletion.

At physiological levels, the trace element selenium plays a key role in redox reactions through the incorporation of selenocysteine in antioxidant enzymes. Selenium has also been evaluated as a potential anti-cancer agent, where selenium nanoparticles have proven effective, and are well tolerated in vivo at doses that are toxic as soluble Se. The use of such nanoparticles, coated with either serum albumin or the naturally occurring alkaline polysaccharide chitosan, also serves to enhance biocompatibility and bioavailability. Here we demonstrate a novel role for selenium in regulating histone methylation in ovarian cancer cell models treated with inorganic selenium nanoparticles coated with serum albumin or chitosan. As well as inducing thioredoxin reductase expression, ROS activity and cancer cell cytotoxicity, coated nanoparticles caused significant increases in histone methylation. Specifically, selenium nanoparticles triggered an increase in the methylation of histone 3 at lysines K9 and K27, histone marks involved in both the activation and repression of gene expression, thus suggesting a fundamental role for selenium in these epigenetic processes. This direct function was confirmed using chemical inhibitors of the histone lysine methyltransferases EZH2 (H3K27) and G9a/EHMT2 (H3K9), both of which blocked the effect of selenium on histone methylation. This novel role for selenium supports a distinct function in histone methylation that occurs due to a decrease in S-adenosylhomocysteine, an endogenous inhibitor of lysine methyltransferases, the metabolic product of methyl-group transfer from S-adenosylmethionine in the one-carbon metabolism pathway. These observations provide important new insights into the action of selenium nanoparticles. It is now important to consider both the classic antioxidant and novel histone methylation effects of this key redox element in its development in cancer therapy and other applications.

Studying Activated Fibroblast Phenotypes and Fibrosis-Linked Mechanosensing Using 3D Biomimetic Models.

Fibrosis and solid tumor progression are closely related, with both involving pathways associated with chronic wound dysregulation. Fibroblasts contribute to extracellular matrix (ECM) remodeling in these processes, a crucial step in scarring, organ failure, and tumor growth, but little is known about the biophysical evolution of remodeling regulation during the development and progression of matrix-related diseases including fibrosis and cancer. A 3D collagen-based scaffold model is employed here to mimic mechanical changes in normal (2 kPa, soft) versus advanced pathological (12 kPa, stiff) tissues. Activated fibroblasts grown on stiff scaffolds show lower migration and increased cell circularity compared to those on soft scaffolds. This is reflected in gene expression profiles, with cells cultured on stiff scaffolds showing upregulated DNA replication, DNA repair, and chromosome organization gene clusters, and a concomitant loss of ability to remodel and deposit ECM. Soft scaffolds can reproduce biophysically meaningful microenvironments to investigate early stage processes in wound healing and tumor niche formation, while stiff scaffolds can mimic advanced fibrotic and cancer stages. These results establish the need for tunable, affordable 3D scaffolds as platforms for aberrant stroma research and reveal the contribution of physiological and pathological microenvironment biomechanics to gene expression changes in the stromal compartment.

Biomimetic Scaffolds Modulate the Posttraumatic Inflammatory Response in Articular Cartilage Contributing to Enhanced Neoformation of Cartilaginous Tissue In Vivo.

Focal chondral lesions of the knee are the most frequent type of trauma in younger patients and are associated with a high risk of developing early posttraumatic osteoarthritis. The only current clinical solutions include microfracture, osteochondral grafting, and autologous chondrocyte implantation. Cartilage tissue engineering based on biomimetic scaffolds has become an appealing strategy to repair cartilage defects. Here, a chondrogenic collagen-chondroitin sulfate scaffold is tested in an orthotopic Lapine in vivo model to understand the beneficial effects of the immunomodulatory biomaterial on the full chondral defect. Using a combination of noninvasive imaging techniques, histological and whole transcriptome analysis, the scaffolds are shown to enhance the formation of cartilaginous tissue and suppression of host cartilage degeneration, while also supporting tissue integration and increased tissue regeneration over a 12 weeks recovery period. The results presented suggest that biomimetic materials could be a clinical solution for cartilage tissue repair, due to their ability to modulate the immune environment in favor of regenerative processes and suppression of cartilage degeneration.

Novel Silicon Titanium Diboride Micropatterned Substrates for Cellular Patterning.

Both hard material photolithography and soft lithography are widely used for patterned cell culture. Soft lithography techniques enable bioactive molecule incorporation, however complex surface modifications are required to introduce specific ligands or proteins in conventional photolithography. In this study, we demonstrate human umbilical vein cell (HUVEC) and adult bone marrow derived mesenchymal stem cell (MSC) patterning on titanium diboride (TiB2) layers deposited on silicon (Si) substrates by electron-beam evaporation and micropatterned using photolithography. Micropatterned cell growth specificity on geometric shapes of circle and/or lines is achieved via differential growth factors adsorption in the presence of heparin. Specifically, the deposited films of TiB2 showed increased stiffness, hardness, hydrophilicity and surface charge when compared to background Si. These substrates were found to be compatible with HUVEC and MSC viability, based on biomarker expression and RNA-sequence transcriptome analysis. Cell-type dependent, micropattern selective cell growth, such as contact guidance, alignment, and durotaxis, were observed. In addition, MSC clustering was achieved, enabling a three-dimensional (3D) aggregate based microenvironment during culture. This study clearly demonstrates the potential of microfabricated Si and TiB2 biomaterials for patterned cell culture in vitro, independent of any additional surface modification.

Delayed endometrial decidualisation in polycystic ovary syndrome; the role of AR-MAGEA11.

Polycystic ovary syndrome (PCOS) is a common gynaecological disorder, with a prevalence of up to 12% of women of reproductive age, and is in part characterised by elevated circulating androgens and aberrant expression of androgen receptor (AR) in the endometrium. A high percentage of PCOS patients suffer from infertility, a condition that appears to be linked to mistimed and incomplete decidualisation critically affecting events surrounding embryo implantation. The aim of this study was to examine the involvement of MAGEA11, and the genome-wide role of AR in PCOS. We determined that elevated androgen levels on PCOS cells had an impact on the delayed and incomplete decidual transformation of endometrial cells. The AR co-regulator MAGEA11, a known enhancer of AR function, was constitutively overexpressed throughout the menstrual cycle of PCOS patients, co-localised in the nucleus of PCOS stromal tissue and cells and formed a molecular complex with AR. Genome-wide AR analysis in PCOS stromal cells revealed that AR targets included genes involved in cell death and apoptosis, as well as genes commonly dysregulated in endometrial cancer. Enhanced MAGEA11 and AR-mediated transcriptional regulation may impact on a correct endometrial decidualisation response, subsequently affecting endometrial receptivity in these infertile women. KEY MESSAGES: MAGEA11 and AR are overexpressed in hyperandrogenic PCOS patients. MAGEA11-AR overexpression in PCOS correlates with delayed decidualisation. AR and MAGEA11 associate in a molecular complex. AR directly regulates a unique set of genes controlling gene differentiation.

HBO1 directs histone H4 specific acetylation, potentiating mechano-transduction pathways and membrane elasticity in ovarian cancer cells.

New approaches to treat ovarian cancer, the fifth leading cause of cancer mortality among women, are being sought, with the targeting of epigenetic modulators now receiving much attention. The histone acetyltransferase HBO1 functions in regulating diverse molecular processes, including DNA repair, transcription and replication, and is highly expressed in primary ovarian cancer. Here we define both the molecular function and a role in cell biomechanics for HBO1 in ovarian cancer. HBO1 preferentially acetylates histone H4 through the concomitant overexpression of co-regulator JADE2, and is required for the expression of YAP1, an ovarian cancer oncogene and mechano-transductor signaling factor. HBO1 appears therefore to have a role in determining the mechano-phenotype in ovarian cancer cells, through both signal transduction processes, and the modulation of cell elasticity as observed using direct measurements on live cells via atomic force microscopy.