Summary of the proceedings of the Basic Science of Uterine Fibroids meeting: new developments (February 28, 2020)
2020; Elsevier BV; Volume: 2; Issue: 1 Linguagem: Inglês
10.1016/j.xfss.2020.11.001
ISSN2666-335X
AutoresPhyllis C. Leppert, Ayman Al‐Hendy, Donna D. Baird, Serdar E. Bulun, William H. Catherino, Darlene Dixon, Merrick T. Ducharme, Quaker E. Harmon, Friederike L. Jayes, Emmanuel N. Paul, Aymara Mas Perucho, James H. Segars, Carlos Simón, Elizabeth A. Stewart, Jose M. Teixeira, Andrea Tinelli, Daniel J. Tschumperlin, Ami R. Zota,
Tópico(s)Gynecological conditions and treatments
ResumoScientists from multiple basic disciplines and an international group of physician-scientists from the fields of obstetrics and gynecology presented recent studies and discussed new and evolving theories of uterine fibroid etiology, growth, and development at the Basic Science of Uterine Fibroids meeting, sponsored by the Campion Fund and the National Institute of Environmental Health Sciences. The purpose was to share up-to-date knowledge and stimulate new concepts regarding the basic molecular biology and pathophysiology of uterine fibroids and promote future collaborations. The meeting was held at the National Institutes of Environmental Health Sciences in North Carolina on February 28, 2020. The speakers reviewed recent advances in cellular and molecular processes that contribute to fibroid growth and new opportunities for treatment. At the conclusion of the conference, attendees identified important new directions for future research. Scientists from multiple basic disciplines and an international group of physician-scientists from the fields of obstetrics and gynecology presented recent studies and discussed new and evolving theories of uterine fibroid etiology, growth, and development at the Basic Science of Uterine Fibroids meeting, sponsored by the Campion Fund and the National Institute of Environmental Health Sciences. The purpose was to share up-to-date knowledge and stimulate new concepts regarding the basic molecular biology and pathophysiology of uterine fibroids and promote future collaborations. The meeting was held at the National Institutes of Environmental Health Sciences in North Carolina on February 28, 2020. The speakers reviewed recent advances in cellular and molecular processes that contribute to fibroid growth and new opportunities for treatment. At the conclusion of the conference, attendees identified important new directions for future research. Discuss: You can discuss this article with its authors and other readers at https://www.fertstertdialog.com/posts/xfss-d-20-00042 Discuss: You can discuss this article with its authors and other readers at https://www.fertstertdialog.com/posts/xfss-d-20-00042 The Campion Fund and the National Institute of Environmental Health Sciences sponsored a meeting on the Basic Science of Uterine Fibroids on February 28, 2020. The purpose of the meeting was to present current research and stimulate further basic studies through collaboration. Presentations included recent discoveries and theories integrating current and evolving concepts, which are summarized in this article. The presentations demonstrated the complexity of fibroid biology. The final session was a discussion that led to a number of future research questions, which are included herein. Because this article presents material discussed at a meeting, it does not represent all the basic scientific concepts pertaining to uterine fibroids (UFs) and should not be considered as a review of the topic. Uterine leiomyomas or fibroids, which comprise abnormal smooth muscle cells and abundant extracellular matrix (ECM), are dependent on ovarian steroids for growth and can cause excessive uterine bleeding, anemia, and recurrent pregnancy loss. Each leiomyoma originates from clonal expansion of a single smooth muscle progenitor or stem cell, which bears a single distinct mutation (e.g., MED12 or HMGA2). These mutations initiate chromosomal instability or genome-wide epigenetic perturbations. Estrogen, together with estrogen receptor-α (ERα), renders leiomyoma responsive to progesterone by inducing progesterone receptor (PR) expression. PR binds to tens of thousands of DNA sites in leiomyoma smooth muscle cells to regulate multiple genes and promote proliferation, survival, and abnormal production of ECM. Treatment of patients using antiprogestins strikingly diminishes the tumor size and associated symptoms. A distinct leiomyoma stem cell population with self-renewing capacity, comprising 5% of the tumor, is indispensable for strong tumor formation in response to estrogen and progesterone in vivo. These stem cells are ERα- or PR-deficient but rich in cell surface receptors for cytokines and growth factors originating from the surrounding ERα/PR-rich differentiated cells in response to estrogen and progesterone (Fig. 1). Dysregulation of DNA methylation in tumor stem cells plays a crucial role in disease progression. Leiomyoma stem cells are uniquely DNA hypermethylated at the PR gene locus and its target regions, suppressing stem cell response to progesterone. The DNA methylation inhibitor 5'-Aza depletes stem cells by increasing PR expression and its target genes. 5'-Aza treatment significantly reduces the number of stem cells and their tumor initiation ability (1Liu S. Yin P. Kujawa S.A. Coon JS 5th Okeigwe I. Bulun S.E. Progesterone receptor integrates the effects of mutated MED12 and altered DNA methylation to stimulate RANKL expression and stem cell proliferation in uterine leiomyoma.Oncogene. 2019; 38: 2722-2735Crossref PubMed Scopus (12) Google Scholar, 2Ikhena D. Liu S. Kujawa S. Esencan E. Coon J.S. Robins J. et al.RANKL/RANK pathway and its inhibitor RANK-Fc in uterine leiomyoma growth.J Clin Endocrinol Metab. 2018; 103: 1842-1849Crossref PubMed Scopus (9) Google Scholar, 3Bulun S.E. Mechanisms of disease: uterine fibroids.N Engl J Med. 2013; 369: 1344-1355Crossref PubMed Scopus (312) Google Scholar, 4Ono M. Yin P. Navarro A. Moravek M.B. Coon J.S. Druschitz S.A. et al.Paracrine activation of WNT/β-catenin pathway in uterine leiomyoma stem cells promotes tumor growth.Proc Natl Acad Sci USA. 2013; 110: 17053-17058Crossref PubMed Scopus (100) Google Scholar). Mechanosensing is the ability of cells to respond to changes in their mechanical environment. This occurs through cellular interactions with other cells or ECM. ECM is deposited during development and maintained throughout life to serve as a scaffold and mechanical support for tissue function. Mesenchymal cells, including fibroblasts and smooth muscle cells, play a leading role in depositing the ECM and determining its architecture and mechanical properties (5Tschumperlin D.J. Ligresti G. Hilscher M.B. Shah V.H. Mechanosensing and fibrosis.J Clin Invest. 2018; 128: 74-84Crossref PubMed Scopus (89) Google Scholar). These cells are also responsive to changes in ECM composition and the mechanical environment, generating feedback loops between ECM remodeling and cellular mechanosensing. Such feedback loops can be important in maintaining tissue homeostasis but can also become corrupted in pathologic conditions, as is the case in fibrotic tissue remodeling (6Zhou Y. Horowitz J.C. Naba A. Ambalavanan N. Atabai K. Balestrini J. et al.Extracellular matrix in lung development, homeostasis and disease.Matrix Biol. 2018; 73: 77-104Crossref PubMed Scopus (79) Google Scholar). In such settings, matrices of pathologic stiffness promote profibrotic signaling and enhanced deposition and remodeling of ECM, leading to feedback loops that promote progressive fibrosis (6Zhou Y. Horowitz J.C. Naba A. Ambalavanan N. Atabai K. Balestrini J. et al.Extracellular matrix in lung development, homeostasis and disease.Matrix Biol. 2018; 73: 77-104Crossref PubMed Scopus (79) Google Scholar). There is growing evidence of mechanosensing being involved in fibroid growth (7Purdy M.P. Ducharme M. Haak A.J. Ravix J. Tan Q. Sicard D. et al.YAP/TAZ are activated by mechanical and hormonal stimuli in myometrium and exhibit increased baseline activation in uterine fibroids.Reprod Sci. 2020; 27: 1074-1085Crossref PubMed Scopus (3) Google Scholar). Work to identify the mechanically activated pathways that promote ECM deposition and disease-related changes in ECM composition and mechanics that regulate cell function in mesenchymal cells is ongoing. Uncovering control systems by which cells respond to and regulate the matrix and failures in these homeostatic mechanisms that lead to pathologic tissue remodeling remain major challenges. Ultimately, a detailed understanding of the molecular mechanisms linking the altered mechanical environment to cellular activation in UFs may enable new approaches to arrest or reverse fibroid growth and pathologic ECM remodeling. An unsupervised clustering of results from ribonucleic acid (RNA) sequencing (RNA-seq) and DNA methylation analyses distinguishes normal myometria from fibroids and further classifies the fibroids into subtypes based on the MED12 mutation or activation of either HMGA2 or HMGA1 expression. Hypomethylation of the HMGA2 gene body is observed in HMGA2-overexpressing fibroids compared with that observed in normal myometria and other fibroid subtypes. Chromatin compartmentalization of the HMGA2 locus is inferred from the DNA methylation results and shows open areas only in HMGA2-overexpressing fibroids (8George J.W. Fan H. Johnson B. Carpenter T.J. Foy K.K. Chatterjee A. et al.Integrated epigenome, exome, and transcriptome analyses reveal molecular subtypes and homeotic transformation in uterine fibroids.Cell Rep. 2019; 29: 4069-4085Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar). Additionally, homeobox cluster A; regulates the pattern of embryonic anatomical development (HOXA13). a critical homeobox gene for proper posterior Müllerian duct differentiation, is not normally expressed in the uterus but was expressed in fibroids (Fig. 2, left). The RNA-seq results showed a switch to a more posterior expression in the HOXA gene cluster in all fibroids. An overexpression of HOXA13 in a myometrial cell line correlates with an altered expression of typical UF genes, suggesting that the activation of HOXA13 expression could be a driving agent of the disease (8George J.W. Fan H. Johnson B. Carpenter T.J. Foy K.K. Chatterjee A. et al.Integrated epigenome, exome, and transcriptome analyses reveal molecular subtypes and homeotic transformation in uterine fibroids.Cell Rep. 2019; 29: 4069-4085Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar). HOXA13 is normally expressed in the cervix, and a significant overlap of differentially expressed genes between cervical stroma and UFs compared with those expressed in normal myometria was also observed in the RNA-seq results. These observations suggest that a homeotic transformation by chromatin modification in myometrial cells induces the cells to develop a more cervical stroma phenotype (Fig. 2, right). Exploring this epigenetically driven phenotypic change in more granular detail will be important for understanding the etiology and pathobiology of the disease (8George J.W. Fan H. Johnson B. Carpenter T.J. Foy K.K. Chatterjee A. et al.Integrated epigenome, exome, and transcriptome analyses reveal molecular subtypes and homeotic transformation in uterine fibroids.Cell Rep. 2019; 29: 4069-4085Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar). Observations based on light and electron microscopic, immunohistochemical, and morphometric studies have led to the hypothesis that fibroid changes over time may relate to an excessive production of collagen by phenotypically transformed myocytes. This accumulation of collagen results in a decreased microvessel density, followed by myocyte injury and atrophy, with eventual senescence and involution due to ischemic cellular degeneration and inanition. Uterine leiomyomas or fibroids are characterized by 2 histologic features: proliferation of myocytes and production of a collagenous ECM. In large tumors, a collagenous ECM is often abundant. In regions with the excessive accumulated collagen, the myocytes are progressively separated from their blood supply, resulting in myocyte atrophy and cell death eventually. It is within these hypocellular, hyalinized areas that the complete lifecycle of the fibroid myocyte occurs (9Flake G.P. Moore A.B. Sutton D. Flagler N. Clayton N. Kissling G.E. et al.The life cycle of the uterine fibroid myocyte.Curr Obstet Gynecol Rep. 2018; 7: 97-105Crossref PubMed Google Scholar, 10Flake G.P. Moore A.B. Flagler N. Wicker B. Clayton N. Kissling G.E. et al.The natural history of uterine leiomyomas: morphometric concordance with concepts of interstitial ischemia and inanosis.Obstet Gynecol Int. 2013; 285103PubMed Google Scholar, 11Flake G.P. Moore A.B. Sutton D. Kissling G.E. Horton J. Wicker B. et al.The natural history of uterine leiomyomas: light and electron microscopic studies of fibroid phases, interstitial ischemia, inanosis, and reclamation.Obstet Gynecol Int. 2013; 528376PubMed Google Scholar). It begins with the phenotypic transformation of a contractile cell to one characterized by proliferation and collagen synthesis, progresses through an intermediate stage of interstitial ischemia-related atrophy, and ends in cell death due to inanition. Finally, resorption of the inanotic cells seems to occur by a nonphagocytic, presumably enzymatic process of degradation and recycling, which is referred to as reclamation (Fig. 3). Phthalates are a class of multifunctional chemicals commonly used in personal care and consumer products (12Varshavsky J.R. Zota A.R. Woodruff T. A novel method for calculating potency-weighted cumulative phthalates exposure with implications for identifying racial/ethnic disparities among US reproductive-aged women in NHANES 2001–2012.Environ Sci Tech. 2016; 50: 10616-10624Crossref PubMed Scopus (29) Google Scholar). Exposure is widespread among reproductive-aged women and can vary by race or ethnicity (12Varshavsky J.R. Zota A.R. Woodruff T. A novel method for calculating potency-weighted cumulative phthalates exposure with implications for identifying racial/ethnic disparities among US reproductive-aged women in NHANES 2001–2012.Environ Sci Tech. 2016; 50: 10616-10624Crossref PubMed Scopus (29) Google Scholar). Evidence from both animal and human studies suggests that exposure to certain phthalates are associated with adverse reproductive outcomes (13CPSC Chronic Hazard Advisory Panel on Phthalates and Phthalate Alternatives Final ReportUS Consumer Product Safety Commission.2014Google Scholar). A preliminary, cross-sectional study of 57 premenopausal women undergoing hysterectomy or myomectomy was performed to evaluate the associations between phthalate exposure and measures of fibroid burden (i.e., diameter of the largest fibroid and uterine volume) and microRNA (miRNA) expression in fibroid tumors and myometrium (14Zota A.R. Geller R.J. Calafat A.M. Marfori C.Q. Baccarelli A.A. Moawad G.N. Phthalates exposure and uterine fibroid burden among women undergoing surgical treatment for fibroids: a preliminary study.Fertil Steril. 2019; 111: 112-121Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar, 15Zota A.R. Geller R.J. VanNoy B. Marfori C.Q. Tabbara S. Hu L.Y. et al.Phthalate exposures and microRNA expression in uterine fibroids: The FORGE Study.Epigenet Insights. 2020; 132516865720904057Crossref Scopus (7) Google Scholar). Most women were black, overweight or obese, and college-educated. The geometric mean of 3 phthalate metabolites was >30% higher in black women compared with that in white or Latina women. In multivariable models, a doubling in the sum of di(2-ethylhexyl) phthalate metabolites and the weighted sum of antiandrogenic phthalate metabolites was associated with 26.81% (95% confidence interval, 2.19, 57.37) and 33.19% (95% confidence interval, 6.59, 66.43) increases in the uterine volume, respectively (14Zota A.R. Geller R.J. Calafat A.M. Marfori C.Q. Baccarelli A.A. Moawad G.N. Phthalates exposure and uterine fibroid burden among women undergoing surgical treatment for fibroids: a preliminary study.Fertil Steril. 2019; 111: 112-121Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar). In epigenetic analyses of fibroid tumors, mono-hydroxybutyl phthalate and mono-hydroxyhexyl phthalate were positively associated with the miRNAs miR-10a-5p and miR-577, respectively. Eight phthalate-miRNA associations significantly varied by race or ethnicity. The messenger RNA gene targets of the phthalate-associated miRNAs were significantly associated with multiple fibroid-related processes, including angiogenesis, apoptosis, and proliferation of connective tissues (15Zota A.R. Geller R.J. VanNoy B. Marfori C.Q. Tabbara S. Hu L.Y. et al.Phthalate exposures and microRNA expression in uterine fibroids: The FORGE Study.Epigenet Insights. 2020; 132516865720904057Crossref Scopus (7) Google Scholar). Thus, phthalate exposure may be associated with fibroid outcomes, and miRNA regulation may be involved in biologic pathways linking phthalates to fibroid pathogenesis. A validation of these preliminary findings may provide an insight into modifiable risk factors and contribute to novel hypotheses regarding racial or ethnic disparities in fibroids. Uterine leiomyoma growth is directly related to an aberrant and excessive deposition of ECM consisting of collagen, proteoglycans, and fibronectin, among other proteins. Many indirect ECM regulators (vitamins A and D, targretin, fucoidan, celecoxib, simvastatin, curcumin, epigallocatechin gallate [EGCG], resveratrol, fasudil, locostatin, berberine, and isoliquiritigenin) (16Catherino W.H. Malik M. Uterine leiomyomas express a molecular pattern that lowers retinoic acid exposure.Fertil Steril. 2007; 87: 1388-1398Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 17Al-Hendy A. Diamond M.P. Boyer T.G. Halder S.K. 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The TGF-β response element has an overlapping site for AP-1 and cis-SMAD (serving as an AP-1 binding site), involved in the transactivation of COL1A2. The expression of multiple AP-1 members is altered in fibroids. The concentrations of pC-Jun, pC-Fos, JunB, JunD, C-Fos, FRA-1, and FRA-2 are lower in leiomyomas than in patient-matched myometrial cultures, and FosB proto-oncogene, an AP-1 transcripton factor subunit is at an increased concentration (38Pilgrim J, Arismendi J, DeAngelis A, Lewis T, Britten J, Malik M, et al. Characterization of the role of activator protein 1 (AP 1) signaling pathway on extracellular matrix deposition in uterine leiomyoma. Fertil Steril Sci. In press.Google Scholar). TGF-β3 stimulates the production of these ECM components, but this stimulation initially requires AP-1 activation. With AP-1 inhibition, the concentrations of COL1A, fibronectin, and versican are decreased. Although TGF-β3 treatment alone augments the expression of all the evaluated proteins, the cotreatment with the AP-1 inhibitor blocks TGF-β3 augmentation. Multiple signaling pathways are activated in leiomyomas, which converge in AP-1 signaling, to induce the phenotype with the aberrant and excessive ECM (Fig. 4). Despite the high prevalence and major negative impact of UFs on women's health, knowledge about tumor-initiating cells is scarce (39Walker C.L. Stewart E.A. Uterine fibroids: the elephant in the room.Science. 2005; 308: 1589-1592Crossref PubMed Scopus (404) Google Scholar). Whole-tissue studies have provided information on chromosomal and/or genetic alterations (40Mas A. Cervelló I. Fernández-Álvarez A. Faus A. Díaz A. 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The exponential growth of single-cell transcriptomics makes it possible to narrow down anatomy at the single-cell level, as demonstrated in the Human Cell Atlas initiative, to better understand the cellular hierarchy of UFs and myometrium. Based on this approach, single-cell RNA-seq analyses were performed by profiling a total of 5,432 single cells from UFs (F), fibroid-free matched myometrium (M), and healthy myometrium (hM) from 7 patients. Preliminary results based on dimensional reduction revealed the cellular heterogeneity of these tissues (F, M, and hM), consisting of 15 cell types and states. Specifically, 3 major lineages of smooth muscle cells, fibroblasts, and lymphatic endothelium were identified, which drastically differed from the tumor (F) and nontumor (M/hM) tissues. An overall inflammatory state in F was also observed, which manifested as a transcriptomic signature of immune cells, an abundance of lymphatic vessels, and an interplay between CCL21 and CCR7. This reveals a molecular and cellular cartography of the human myometrium and UFs through specific single-cell transcriptomes, which may provide molecular targets for a less invasive treatment approach for these benign tumors. In summary, novel human myometrial and UF-specific cell types and states were identified to advance the understanding of myometrial tumorigenesis. Despite multiple effective alternatives, hysterectomy with ovarian preservation is still the leading treatment for UFs. Moreover, a significant long-term risk of hysterectomy makes this treatment less than ideal (43Stewart AE, Missmer SA, Rocca, WA. Moving beyond reflexive and prophylactic gynecologic surgery. Mayo Clin Proc. In Press.Google Scholar) (Fig. 5). Hysterectomy remains important because both clinical trials and in vitro experiments have shown that there is always an unexplained heterogeneity of response among fibroids. Although the National Institute of Environmental Health Sciences Fibroid Growth Study has demonstrated that clinical characteristics, including age and race, are associated with an increased growth, we have no growth-directed or regression-triggering therapies. Clinically, there are unmeasured variables, such as adenomyosis and endometriosis, and individual and culturally influenced responses to specific symptoms. Nonetheless, biologic heterogeneity also likely plays a role. There are several barriers to fibroid research. First, although fibroids are clonal, multiple independent clones do arise within the same uterus. Moreover, although we commonly map the symptoms to specific fibroids, this has limited precision. Even genetic studies have demonstrated that a genotype does not predict the phenotype but only shows correlations and, thus, is not clinically useful for most women (44Hayden M.A. Ordulu Z. Gallagher C.S. Quade B.J. Anchan R.M. Middleton N.R. et al.Clinical, pathologic, cytogenetic, and molecular profiling in self-identified black women with uterine leiomyomata.Cancer Genet. 2018; 222-223: 1-8Abstract Full Text Full Text PDF PubMed Scopus (5) Google Scholar). Finally, a lack of understanding of the natural history of UFs handicaps our current clinical care. A longitudinal "Framingham Fibroid Project" would be useful. The Framingham Heart Study was started in 1948; it was almost a decade later that hypertension and hypercholesterolemia were found to increase heart disease and nearly 2 decades before exercise and obesity were linked to heart disease. Prospective registries, such as COMPARE-UF and CAPTURE, have been established in the past decade but have not
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