Leiomyoma Stem Cells Research Articles

From Elegance to Complexity: The Intricate Dance of Myometrial Stem Cells Transforming into Leiomyoma Stem Cells: Unraveling the Symphony of Genetic and Epigenetic Refinement

Uterine leiomyomas (LM), commonly referred to as uterine fibroids (UFs), pelvic tumor in women of reproductive age globally and are main indicators of hysterectomies if become symptomatic. The progression of uterine LM involves a complex interplay of hormones, stem cells, growth factors, and genetic/epigenetic irregularities. Both endometrial and myometrial stem/progenitor cells play pivotal roles in the uterus’ response to hormonal stimuli and return to its basal state. LM, arising from a single stem cell of smooth muscle cells with acquired mutations, exhibit diverse cellular transcriptomic patterns influenced by genotype. Affecting over 70% of women at some point in their lives, with significant clinical symptoms, such as pelvic pain, infertility, and heavy uterine bleeding. Recent literature underscores the potential importance of LM stem cells and their paracrine interactions with specialized cells, connecting the gap between medications targeting leiomyoma expansion and potential eradication strategies. Keeping these points in view, this review discusses the current understanding of the involvement of myometrial stem/progenitor cells and the genetic and epigenetic changes occurring in these cells during the pathogenesis of uterine LM.

Simvastatin inhibits stem cell proliferation in human leiomyoma via TGF‐β3 and Wnt/β‐Catenin pathways

Uterine leiomyoma (UL) is the most common gynaecologic tumour, affecting an estimated 70 to 80% of women. Leiomyomas develop from the transformation of myometrial stem cells into leiomyoma stem (or tumour‐initiating) cells. These cells undergo self‐renewal and differentiation to mature cells, both are necessary for the maintenance of tumour stem cell niche and tumour growth, respectively. Wnt/β‐catenin and TGF‐β/SMAD pathways, both overactive in UL, promote stem cell self‐renewal, crosstalk between stem and mature cells, cellular proliferation, extracellular matrix (ECM) accumulation and drive overall UL growth. Recent evidence suggests that simvastatin, an antihyperlipidemic drug, may have anti‐leiomyoma properties. Herein, we investigated the effects of simvastatin on UL stem cells. We isolated leiomyoma stem cells by flow cytometry using DyeCycle Violet staining and Stro‐1/CD44 surface markers. We found that simvastatin inhibits proliferation and induces apoptosis in UL stem cells. In addition, it also suppressed the expression of the stemness markers Nanog, Oct4 and Sox2. Simvastatin significantly decreased the production of the key ECM proteins, collagen 1 and fibronectin. Finally, it inhibited genes and/or proteins expression of TGF‐β1, 2 and 3, SMAD2, SMAD4, Wnt4, β‐Catenin, LRP6, AXIN2 and Cyclin D1 in UL stem cells, all are key drivers of the TGF‐β3/SMAD2 and Wnt4/β‐Catenin pathways. Thus, we have identified a novel stem cell‐targeting anti‐leiomyoma simvastatin effect. Further studies are needed to replicate these findings in vivo.

Progesterone receptor-DNA methylation crosstalk regulates depletion of uterine leiomyoma stem cells: A potential therapeutic target.

SummaryUterine leiomyoma (LM) is the most common tumor in women. Via its receptor (PGR) expressed in differentiated LM cells, progesterone stimulates paracrine signaling that induces proliferation of PGR-deficient LM stem cells (LSCs). Antiprogestins shrink LM but tumors regrow after treatment cessation possibly due to persisting LSCs. Using sorted primary LM cell populations, we found that the PGR gene locus and its target cistrome are hypermethylated in LSCs, inhibiting the expression of genes critical for progesterone-induced LSC differentiation. PGR knockdown shifted the transcriptome of total LM cells toward LSCs and increased global DNA methylation by regulating TET methylcytosine dioxygenases. DNA methylation inhibitor 5′-Aza activated PGR signaling, stimulated LSC differentiation, and synergized with antiprogestin to reduce tumor size in vivo. Taken together, targeting the feedback loop between DNA methylation and progesterone signaling may accelerate the depletion of LSCs through rapid differentiation and sensitize LM to antiprogestin therapy, thus preventing tumor regrowth.

Wnt/β-catenin signaling pathway in uterine leiomyoma: role in tumor biology and targeting opportunities.

Uterine leiomyoma is the most common tumor of the female reproductive system and originates from a single transformed myometrial smooth muscle cell. Despite the immense medical, psychosocial, and financial impact, the exact underlying mechanisms of leiomyoma pathobiology are poorly understood. Alterations of signaling pathways are thought to be instrumental in leiomyoma biology. Wnt/β-catenin pathway appears to be involved in several aspects of the genesis of leiomyomas. For example, Wnt5b is overexpressed in leiomyoma, and the Wnt/β-catenin pathway appears to mediate the role of MED12 mutations, the most common mutations in leiomyoma, in tumorigenesis. Moreover, Wnt/β-catenin pathway plays a paracrine role where estrogen/progesterone treatment of mature myometrial or leiomyoma cells leads to increased expression of Wnt11 and Wnt16, which induces proliferation of leiomyoma stem cells and tumor growth. Constitutive activation of β-catenin leads to myometrial hyperplasia and leiomyoma-like lesions in animal models. Wnt/β-catenin signaling is also closely involved in mechanotransduction and extracellular matrix regulation and relevant alterations in leiomyoma, and crosstalk is noted between Wnt/β-catenin signaling and other pathways known to regulate leiomyoma development and growth such as estrogen, progesterone, TGFβ, PI3K/Akt/mTOR, Ras/Raf/MEK/ERK, IGF, Hippo, and Notch signaling. Finally, evidence suggests that inhibition of the canonical Wnt pathway using β-catenin inhibitors inhibits leiomyoma cell proliferation. Understanding the molecular mechanisms of leiomyoma development is essential for effective treatment. The specific Wnt/β-catenin pathway molecules discussed in this review constitute compelling candidates for therapeutic targeting.

Activation of protein kinase B by WNT4 as a regulator of uterine leiomyoma stem cell function

To investigate the functional interaction between the Wnt/β-catenin and protein kinase B (Akt) pathways in leiomyoma stem cells (LSC). Laboratory study. Research laboratory. Premenopausal women (n = 36; age range: 28 to 49 years) undergoing hysterectomy or myomectomy for leiomyoma. None. Gene expression, protein phosphorylation, and cell proliferation. Cells from human leiomyoma tissues were sorted by fluorescence-activated cell sorting (FACS) into three populations: LSC, intermediate cells (LIC), and differentiated cells (LDC) with the function of the Wnt/β-catenin and Akt signaling pathways in leiomyoma cells evaluated using real-time quantitative polymerase chain reaction and immunoblot analyses. The Wnt/β-catenin signaling pathway components were differentially expressed in each leiomyoma cell population. WNT4 was distinctly overexpressed in LIC, and its receptor FZD6 was primarily expressed in LSC. WNT4 stimulated Akt phosphorylation, activated β-catenin, and increased primary leiomyoma cell proliferation. These stimulatory effects were abolished by cotreatment with the Akt inhibitor, MK-2206. WNT4 up-regulated the expression of pro-proliferative genes, c-Myc and cyclin D1, specifically in LSC; this was also abrogated by Akt inhibition. Our data suggest that WNT4 regulates LSC proliferation via Akt-dependent β-catenin activation, representing a key step toward a better understanding of LSC regulation and potentially novel therapeutic targets.

Targeting DNA Methylation Depletes Uterine Leiomyoma Stem Cell-enriched Population by Stimulating Their Differentiation.

Uterine leiomyoma (LM) is the most common tumor in women and can cause severe morbidity. Leiomyoma growth requires the maintenance and proliferation of a stem cell population. Dysregulated deoxyribonucleic acid (DNA) methylation has been reported in LM, but its role in LM stem cell regulation remains unclear. Here, we fluorescence-activated cell sorting (FACS)-sorted cells from human LM tissues into 3 populations: LM stem cell–like cells (LSC, 5%), LM intermediate cells (LIC, 7%), and differentiated LM cells (LDC, 88%), and we analyzed the transcriptome and epigenetic landscape of LM cells at different differentiation stages. Leiomyoma stem cell–like cells harbored a unique methylome, with 8862 differentially methylated regions compared to LIC and 9444 compared to LDC, most of which were hypermethylated. Consistent with global hypermethylation, transcript levels of TET1 and TET3 methylcytosine dioxygenases were lower in LSC. Integrative analyses revealed an inverse relationship between methylation and gene expression changes during LSC differentiation. In LSC, hypermethylation suppressed the genes important for myometrium- and LM-associated functions, including muscle contraction and hormone action, to maintain stemness. The hypomethylating drug, 5′-Aza, stimulated LSC differentiation, depleting the stem cell population and inhibiting tumor initiation. Our data suggest that DNA methylation maintains the pool of LSC, which is critical for the regeneration of LM tumors.

Pathogenomics of Uterine Fibroids Development.

We review recent studies dealing with the molecular genetics and basic results of omics analysis of uterine leiomyoma (LM)—a common benign muscle tumor of the uterus. Whole genome studies of LM resulted in the discovery of many new gene nets and biological pathways, including its origin, transcriptomic, and epigenetic profiles, as well as the impact of the inter-cell matrix in LM growth and involvement of microRNA in its regulation. New data on somatic cell mutations ultimately involved in the origin, distribution and growth of LM are reviewed. Putative identification of LM progenitor SC (stem cells) giving rise to maternal fibroid nodes and junctional zones provide a new clue for hypotheses on the pathogenomics of LM. The reviewed data are consistent with at least two different but probably intimately interacted molecular mechanisms of LM. One of them (the genetic hypothesis) is focused primarily on the MED12 gene mutations and suggests its onset in the side population of embryonic myoblasts of the female reproductive system, which later gave rise to multiple small and medium fibroids. The single and usually large-size fibroids are induced by predominantly epigenetic disorders in LM SC, provoked by enhanced expression of the HMGA2 gene caused by its hypomethylation and epigenetic deregulation enhanced by hypoxia, muscle tension, or chromosome instability/aberrations. The pathogenomics of both genetic and epigenetic programs of LM with many peculiarities at the beginning later became rather similar and partly overlapped due to the proximity of their gene nets and epigenetic landscape. Pathogenomic studies of LM open ways for elaboration of novel strategies of prevention and treatment of this common disease.

Endometriosis and nuclear receptors.

Endometriosis is recognized as a steroid-dependent disorder; however, the precise roles of nuclear receptors (NRs) in steroid responsiveness and other signaling pathways are not well understood. Over the past several years, a number of paradigm-shifting breakthroughs have occurred in the area of NRs in endometriosis. We review and clarify new information regarding the mechanisms responsible for: (i) excessive estrogen biosynthesis, (ii) estrogen-dependent inflammation, (iii) defective differentiation due to progesterone resistance and (iv) enhanced survival due to deficient retinoid production and action in endometriosis. We emphasize the roles of the relevant NRs critical for these pathological processes in endometriosis. We conducted a comprehensive search using PubMed for human, animal and cellular studies published until 2018 in the following areas: endometriosis; the steroid and orphan NRs, estrogen receptors alpha (ESR1) and beta (ESR2), progesterone receptor (PGR), steroidogenic factor-1 (NR5A1) and chicken ovalbumin upstream promoter-transcription factor II (NR2F2); and retinoids. Four distinct abnormalities in the intracavitary endometrium and extra-uterine endometriotic tissue underlie endometriosis progression: dysregulated differentiation of endometrial mesenchymal cells, abnormal epigenetic marks, inflammation activated by excess estrogen and the development of progesterone resistance. Endometriotic stromal cells compose the bulk of the lesions and demonstrate widespread epigenetic abnormalities. Endometriotic stromal cells also display a wide range of abnormal NR expression. The orphan NRs NR5A1 and NR2F2 compete to regulate steroid-synthesizing genes in endometriotic stromal cells; NR5A1 dominance gives rise to excessive estrogen formation. Endometriotic stromal cells show an abnormally low ESR1:ESR2 ratio due to excessive levels of ESR2, which mediates an estrogen-driven inflammatory process and prostaglandin formation. These cells are also deficient in PGR, leading to progesterone resistance and defective retinoid synthesis. The pattern of NR expression, involving low ESR1 and PGR and high ESR2, is reminiscent of uterine leiomyoma stem cells. This led us to speculate that endometriotic stromal cells may display stem cell characteristics found in other uterine tissues. The biologic consequences of these abnormalities in endometriotic tissue include intense inflammation, defective differentiation and enhanced survival. Steroid- and other NR-related abnormalities exert genome-wide biologic effects via interaction with defective epigenetic programming and enhance inflammation in endometriotic stromal cells. New synthetic ligands, targeting PGR, retinoic acid receptors and ESR2, may offer novel treatment options.

RANKL/RANK Pathway and Its Inhibitor RANK-Fc in Uterine Leiomyoma Growth.

Uterine leiomyomas are the most common type of gynecologic tumor in women. To determine the role of the cytokine receptor activator of nuclear factor κ-Β ligand (RANKL); its receptor, receptor activator of nuclear factor κ-Β (RANK); and the RANKL/RANK pathway inhibitor RANK-Fc in leiomyoma growth. Messenger RNA (mRNA) or protein levels of RANKL, RANK, and proliferation markers cyclin D1 and Ki67 were assessed in various leiomyoma tissues and cell populations. Human xenograft experiments were performed to determine the effects of RANK-Fc on leiomyoma growth in vivo. Research laboratory. Twenty-four regularly cycling premenopausal women (age 28 to 49 years) who were not receiving hormone therapy. None. Tumor growth in a murine xenograft model following targeting of the RANKL/RANK pathway with RANK-Fc. RANKL mRNA levels in leiomyoma were significantly higher than those in myometrial tissues. The highest RANK levels were found in the leiomyoma stem cell population, which is deficient in progesterone receptor (PR). Conversely, the highest RANKL levels were found in the PR-rich leiomyoma intermediate cell (LIC) population. R5020, a PR agonist, specifically increased RANKL expression in LICs. RANK-Fc blocked RANKL-induced expression of the proliferative gene cyclin D1. Treatment with RANK-Fc also significantly decreased tumor growth in vivo and diminished the expression of proliferation marker Ki67 in tumors (P < 0.01; n = 4). Treatment with the RANKL/RANK pathway inhibitor RANK-Fc significantly decreased human leiomyoma cell proliferation and tumor growth. This suggests that the RANKL/RANK pathway could serve as a potential target for the prevention and treatment of uterine leiomyoma.

Synergistic effects of simvastatin and ulipristal acetate on uterine leiomyoma

Human fibroids are highly prevalent uterine tumors composed of proliferating transformed myometrial cells and excessive disordered extracellular matrix (ECM). The gonadal hormone progesterone is known to induce and maintain leiomyoma growth. Simvastatin, primarily used to treat hyperlipidemia, was recently shown to have anti-proliferative and pro-apoptotic effect on leiomyoma cells and xenograft model. Moreover, simvastatin is thought to modulate progesterone levels. Our objectives are to examine effect of simvastatin on ECM production and leiomyoma stem cells, and evaluate the combined effect of simvastatin and ulipristal acetate (UPA), a progesterone receptor modulator.

Paracrine Pathways in Uterine Leiomyoma Stem Cells Involve Insulinlike Growth Factor 2 and Insulin Receptor A.

Uterine leiomyomas (fibroids) are the most common benign tumors in women. Recently, three populations of leiomyoma cells were discovered on the basis of CD34 and CD49b expression, but molecular differences between these populations remain unknown. To define differential gene expression and signaling pathways in leiomyoma cell populations. Cells from human leiomyoma tissue were sorted by flow cytometry into three populations: CD34+/CD49b+, CD34+/CD49b-, and CD34-/CD49b-. Microarray gene expression profiling and pathway analysis were performed. To investigate the insulinlike growth factor (IGF) pathway, real-time quantitative polymerase chain reaction, immunoblotting, and 5-ethynyl-2'-deoxyuridine incorporation studies were performed in cells isolated from fresh leiomyoma. Research laboratory. Eight African American women. None. Gene expression patterns, cell proliferation, and differentiation. A total of 1164 genes were differentially expressed in the three leiomyoma cell populations, suggesting a hierarchical differentiation order whereby CD34+/CD49b+ stem cells differentiate to CD34+/CD49b- intermediary cells, which then terminally differentiate to CD34-/CD49b- cells. Pathway analysis revealed differential expression of several IGF signaling pathway genes. IGF2 was overexpressed in CD34+/CD49b- vs CD34-/CD49b- cells (83-fold; P < 0.05). Insulin receptor A (IR-A) expression was higher and IGF1 receptor lower in CD34+/CD49b+ vs CD34-/CD49b- cells (15-fold and 0.35-fold, respectively; P < 0.05). IGF2 significantly increased cell number (1.4-fold; P < 0.001), proliferation indices, and extracellular signal-regulated kinase (ERK) phosphorylation. ERK inhibition decreased IGF2-stimulated cell proliferation. IGF2 and IR-A are important for leiomyoma stem cell proliferation and may represent paracrine signaling between leiomyoma cell types. Therapies targeting the IGF pathway should be investigated for both treatment and prevention of leiomyomas.

Stem cells and uterine leiomyomas: What is the evidence?

Uterine leiomyomas, also known as uterine fibroids or uterine myomas, are the most common benign gynecologic tumors found in women of reproductive age. In spite of the numerous published studies evaluating the hormonal dependency, epidemiology, molecular biology, pathology, and genetics of leiomyomas, many questions remain unanswered. The remodeling of the uterus in response to hormonal stimuli and its return to a basal state may be related to adult stem/progenitor cells residing in the endometrial and myometrial layers. Recent published papers on stem cells and their paracrine interactions with more specialized cell populations within leiomyomas may help establish the missing link between the development of treatments designed to stop the growth of leiomyomas and therapies devised to eliminate them. Therefore, this study aimed to address the current paradigm regarding the evidence available on the role of stem/progenitor cells in the pathogenesis of uterine leiomyoma. Only a handful of studies involving humans have been published to date describing the presence of somatic stem cells (SSCs) in the myometrium and leiomyomas. No solid conclusion has been established thus far. Despite the fact that these studies strongly pointed to the vital role human leiomyoma stem cells might play in initiating the development of myomas, huge gaps still persist in the literature. Studies to identify putative myometrial and leiomyoma-specific markers might offer new possibilities for understanding the origin of these tumors and perhaps help develop new nonsurgical noninvasive treatments.

Uterine Leiomyoma Stem Cells: Linking Progesterone to Growth.

Uterine leiomyomas (fibroids) represent the most common class of benign tumors in women. Multiple leiomyomas usually arise from the uterus of a symptomatic woman. These tumors cause a variety of symptoms, including abnormal uterine bleeding, pelvic pain, bladder or bowel dysfunction, and recurrent pregnancy loss, and are responsible for more than 200,000 hysterectomies in the United States annually. Each leiomyoma seems to arise from the clonal expansion of a single myometrial smooth muscle cell transformed by a mutation. Tumor expansion is sustained by cell proliferation together with the production of large amounts of extracellular matrix. Estrogen and progesterone stimulate the growth of leiomyomas. Estrogen, together with its receptor ERα, enables progesterone action via induction of progesterone receptor (PR) expression. Progesterone induces the growth of leiomyoma by regulation of a set of key genes that control proliferation and apoptosis. A distinct cell population with stem-progenitor properties is indispensable for progesterone-dependent growth of leiomyomas. This stem-progenitor cell population is deficient in ERα and PR and dependent on the much higher levels of these steroid receptors in surrounding mature leiomyoma or myometrial cells. Progesterone sends paracrine signals from these mature cells to stem cells. The WNT/β-catenin pathway comprises a key component of this paracrine signaling system. The majority of medical treatments currently available for leiomyoma works by inhibiting estrogen or progesterone production or action, but tumors tend to regrow once treatment is stopped. Targeting stem cells and their paracrine interactions with more differentiated cell populations within leiomyoma may lead to the development of more effective therapeutics.

Paracrine activation of WNT/β-catenin pathway in uterine leiomyoma stem cells promotes tumor growth

Uterine leiomyomas are extremely common estrogen and progesterone-dependent tumors of the myometrium and cause irregular uterine bleeding, severe anemia, and recurrent pregnancy loss in 15-30% of reproductive-age women. Each leiomyoma is thought to arise from a single mutated myometrial smooth muscle stem cell. Leiomyoma side-population (LMSP) cells comprising 1% of all tumor cells and displaying tumor-initiating stem cell characteristics are essential for estrogen- and progesterone-dependent in vivo growth of tumors, although they have remarkably lower estrogen/progesterone receptor levels than mature myometrial or leiomyoma cells. However, how estrogen/progesterone regulates the growth of LMSP cells via mature neighboring cells is unknown. Here, we demonstrate a critical paracrine role of the wingless-type (WNT)/β-catenin pathway in estrogen/progesterone-dependent tumorigenesis, involving LMSP and differentiated myometrial or leiomyoma cells. Estrogen/progesterone treatment of mature myometrial cells induced expression of WNT11 and WNT16, which remained constitutively elevated in leiomyoma tissues. In LMSP cells cocultured with mature myometrial cells, estrogen-progesterone selectively induced nuclear translocation of β-catenin and induced transcriptional activity of its heterodimeric partner T-cell factor and their target gene AXIN2, leading to the proliferation of LMSP cells. This effect could be blocked by a WNT antagonist. Ectopic expression of inhibitor of β-catenin and T-cell factor 4 in LMSP cells, but not in mature leiomyoma cells, blocked the estrogen/progesterone-dependent growth of human tumors in vivo. We uncovered a paracrine role of the WNT/β-catenin pathway that enables mature myometrial or leiomyoma cells to send mitogenic signals to neighboring tissue stem cells in response to estrogen and progesterone, leading to the growth of uterine leiomyomas.

Identification and characterization of the human leiomyoma side population as putative tumor-initiating cells

To isolate and characterize human leiomyoma stem cells by the side population (SP) method. Prospective experimental human and animal study. University research laboratory-affiliated infertility clinic. Women undergoing laparoscopic myomectomy. Female non-obese diabetic severe combined immune deficiency (NOD-SCID) mutation mice. Obtainment of human leiomyoma SP cells as candidate tumor-initiating cells and establishment of two leiomyoma SP lines. Flow cytometry, semiquantitative polymerase chain reaction, clonogenicity assays, cDNA microarrays hybridization, cell culture, karyotype, molecular analysis, immunocytochemistry, invitro differentiation, xenotransplantation assays, immunohistochemistry. SP cells from human leiomyomas were isolated, identified, and characterized. Two leiomyoma's SP cell lines with a normal karyotype were thus established. Undifferentiated status was confirmed by the expression of OCT-4, NANOG, DNMT3B, GDF3. Presence of typical mesenchymal markers (CD90, CD105, CD73) and absence of hematopoietic stem cell markers (CD34, CD45) supported their mesodermal origin. Mesenchymal lineage commitment was also demonstrated by their ability to differentiate invitro into adipogenic and osteogenic lineages. Finally, their functional capability was established in an animal model by leiomyoma tissue reconstruction. SP cells from human leiomyoma exhibit key features of tumor-initiating cells, opening up new possibilities of understanding the origin and developing new nonsurgical approaches for leiomyomas.