Adjacent Myometrium Tissue Research Articles

SAT632 The Functional Role And Regulatory Mechanism Of FTO M6A RNA Demethylase In Human Uterine Leiomyosarcoma

Abstract Disclosure: Q. Yang: None. A. Al-Hendy: None. Introduction: Uterine leiomyosarcoma (uLMS) is the most frequent subtype of uterine sarcoma that presents a poor prognosis, high rates of recurrence, and metastasis. However, the origin and molecular mechanism underlying driving its clinical and biological behavior remain to be determined. Recently, we and others have revealed the role of microRNAs, DNA methylation, and histone modifications contributing to the pathogenesis of uLMS. However, the connectivity between reversible RNA m6A methylation and uLMS remains unclear. In this study, we assessed the role and mechanism of the m6A demethylase fat mass- and obesity-associated protein (FTO) in the pathogenesis of uLMS. Materials and Methods: We performed immunohistochemistry (IHC) and immunoblot analysis to measure the m6A erasers FTO and ALKBH5 protein levels in LMS and adjacent myometrium tissues (n=8) and cell lines. We performed LMS cell proliferation and cell cycle phase distribution analysis. RNA-seq was performed to determine the molecular mechanism underlying m6A eraser inhibition in LMS cells (n=4 for each group). The R package limma+voom was used for this analysis. Hallmark pathway analysis was used to identify enriched pathways in response to FTO inhibition. Real-time PCR was performed to validate the differentially expressed genes. Ingenuity pathway analysis (IPA) was performed to determine the upstream regulators targeting cell cycle-related genes. We used a cutoff −1.5 > fold-change > 1.5 and an adjusted p-value of 0.05. In addition, data were analyzed by a two-tailed unpaired Student’s t-test between any two groups. Results: IHC analysis revealed that m6A demethylases FTO and ALKBH5 are aberrantly upregulated in uLMS tissues compared to adjacent myometrium (p<0.01, H-score). Furthermore, inhibition of FTO with its small, potent inhibitor (Dac51) decreased the uLMS proliferation dose-dependently via cell cycle arrest at G1. Notably, RNA-seq analysis revealed that inhibition of FTO with Dac51 exhibited a significant decrease in the expression of multiple CDK members and a significant increase in the expression of CDKN1A. Furthermore, IPA analysis revealed that transcription factors, including SP1 and E2F members are the upstream regulators targeting CDK members in response to Dac51 treatment. Moreover, Dac51 treatment altered the expression of several epigenetic regulators, including Sirt1 and 2, HDAC10 and SUV39H1, and rewiring of several critical pathways, including inflammation response, G2M checkpoint, and C-Myc signaling, among others, leading to suppression of uLMS phenotype. Conclusion: Our findings uncover the anti-uLMS function of a potent FTO inhibitor via reprogramming the oncogenic epitranscriptome. Therefore, targeted inhibition of FTO may provide an additional regulatory layer for a promising and novel strategy for treating patients with this aggressive uterine cancer. Presentation: Saturday, June 17, 2023

P-526 Targeting histone modifications: H3K27 acetylation regulates the expression of genes involved in key processes of uterine leiomyoma pathogenesis

Abstract Study question Does histone mark H3K27ac regulate the expression of genes involved in uterine leiomyoma (UL) pathogenesis, and can histone deacetylation inhibition be a new therapeutic approach? Summary answer H3K27ac regulates genes implicated in UL pathogenesis through angiogenesis and extracellular matrix (ECM), and histone deacetylation reversion could offer a therapeutic approach to treat UL. What is known already UL is a benign tumor arising from myometrium (MM). Women present symptoms such as pelvic pain and infertility. There is no fully effective treatment with minimal side effects. Available medical options focus on the relief of symptoms and not in mechanisms implicated in UL development. Histone modifications are altered in tumors, particularly via histone acetylation which is correlated with gene activation. Herein, we aimed to identify if the modification H3K27ac is involved in UL pathogenesis, determining the relationship between H3K27ac and gene expression in UL versus MM and if its reversion may be a therapeutic option to treat UL. Study design, size, duration Prospective study comparing transcriptome (GEO:GSE192354 and GSE142332) by RNA sequencing (RNA-seq) (n = 48) and H3K27ac profile (GEO:GSE142332) by Chromatin Immunoprecipitation Sequencing (CHIP-seq) (n = 19) in UL versus adjacent MM tissue. Human uterine leiomyoma primary (HULP) cells obtained from individual patients (n = 10) were treated with 0.01% DMSO (control) and 10 µM SAHA, a histone deacetylases inhibitor, for 48 hours. Participants/materials, setting, methods Samples were collected from 48 women aged 31-54 years. Bioinformatics analysis were performed within the R/Bioconductor (4.1.1.). Differential expressed genes (DEGs) were analysed using three methods: DESeq2, edgeR and limma. Common DEGs (FDR<0.01,log2FC>1 or <-1) were considered for further analysis. Differential H2K27ac peak enrichment analysis of selected genes was performed with limma and functional enrichment analysis (FDR<0.05) with Shiny Go (version 0.741). SAHA effect on hypoacetylated/downregulated genes was assessed in HULP cells by qRT-PCR. Main results and the role of chance CHIP-seq analysis showed a lower amount of global H3K27ac peak enrichment level in UL versus MM (p-value<2.2e-16). RNA-seq showed 922 common DEGs in UL versus MM, being 559 up-regulated and 363 down-regulated (FDR<0.01, log2FC>1 or <-1). Integration of CHIP-seq and RNA-seq data showed that among 922 selected genes, 482 also presented the histone mark H3K27ac. Differential peak enrichment analysis demonstrated that 82 of them presented differential acetylation (FDR<0.05) in UL versus MM, being 29 hyper-acetylated/up-regulated and 53 hypo-acetylated/down-regulated. Functional enrichment analysis of 82 DEGs regulated by H3K27ac showed biological processes deregulated in UL that were related to angiogenesis. Additionally, we found cellular components enriched in UL, which were related to an alteration of ECM, one of the key features of UL. We studied further these 82 genes controlled by H3K27ac and found hyperacetylation/upregulation of oncogenes (NDP, TFAP2C, HOXA13, COL24A1 and IGFL3) and hypoacetylation/downregulation of tumor suppressor genes (CD40, GIMAP8, IL15, GPX3 and DPT) in UL, which are related to immune system, angiogenesis, invasion, metabolism, ECM, TGFβ3 and Wnt/β-catenin pathway dysregulation. Finally, SAHA treatment in HULP cells significantly increased expression of the tumor suppressor genes that were hypoacetylated/downregulated in UL versus MM (CD40, GIMAP8, IL15, GPX3 and DPT) (p < 0.05). Limitations, reasons for caution This is a preliminary study including only 19 participants in ChIP-seq analysis, thereby we should be cautious extrapolating our results to the general population. Further studies are necessary to determine the effectiveness of histone deacetylases inhibition, SAHA dose and adverse effects on UL in vivo. Wider implications of the findings H3K27 acetylation regulates the expression of oncogenes and tumor suppressor genes involved in key processes of UL pathogenesis, such as angiogenesis and ECM formation. Histone deacetylation reversion by SAHA upregulated the expression of tumor suppressor genes in HULP cells, suggesting histone deacetylation as a potential therapeutic approach for UL patients. Trial registration number Not applicable

INHIBITION OF BRD PROTEINS SUPPRESSES THE PHENOTYPE OF UTERINE FIBROIDS VIA REGULATION OF N6-METHYLADENOSINE REGULATORS

Bromodomain (BRD)-containing proteins are involved in many biological processes, most notably epigenetic regulation of transcription, and BRD protein dysfunction has been linked to many diseases including tumorigenesis. However, the role of BRD proteins in the pathogenesis of uterine fibroids (UFs), the most common pelvic tumors in women of reproductive age, is unknown. The present study aimed to determine the expression pattern of BRD proteins in UFs and matching myometrium and assess the effect of BRD inhibitors on UF phenotype and epitranscriptomic changes. Human UFs (n=22) and adjacent myometrium tissues (MyoF, n=7) were collected at the time of hysterectomy at the University of Chicago. Western blot (WB) was performed to determine the levels of BRD proteins in UFs and myometrium. Fourteen BRD inhibitors from the Epigenetic Chemical Probe Library provided by the University of Illinois at Chicago were used to determine the cell proliferation by MTT assay. Two BRD inhibitors (I-BRD9 and TP-472) were further used to explore their mechanistic actions. The student’s t-test was used to determine the significant difference. Our studies demonstrated that among 22 UFs analyzed, 96% (21/22, p<0.01), 55% (12/22), 82% (18/22, p<0.01) exhibited the upregulation of BRD2, BRD3, and BRD9 respectively, suggesting the aberrant BRD protein expression may contribute to the pathogenesis of UFs. To examine the biological impact of BRD inhibition on the UFs, 14 BRD inhibitors were screened to identify specific and potent BRD inhibitors in UF cells. 43% (6/14) of these BRD inhibitors selectively inhibited UF, but not myometrial cell proliferation. Furthermore, targeting BRD proteins with BRD inhibitors (I-BRD9 and TP-472) decreased the protein levels of extracellular matrix proteins (Type I Collagen, fibronectin), PCNA (cell proliferation marker), and Bcl-2 (anti-apoptotic marker) in a dose- and time-dependent manner in UF cells. Notably, inhibition of BRD proteins with I-BRD9 and TP-472 downregulated the expression of METTL3, YTHDC2, and YTHDF2, which are the key regulators for N6-methyladenosine (m6A) modification, indicating the tight link between BRD proteins and epitranscriptomic landscape. These results demonstrate for the first time that BRD proteins are aberrantly expressed in UFs. BRD inhibitors suppress the UF phenotype while concomitantly altering the expression of the central m6A regulators.

5-aza-2\u2032-deoxycitidine inhibits cell proliferation, extracellular matrix formation and Wnt/\u03b2-catenin pathway in human uterine leiomyomas

BackgroundUterine leiomyoma is a benign tumor with unclear pathogenesis and inaccurate treatment. This tumor exhibits altered DNA methylation related to disease progression. DNMT inhibitors as 5-aza-2′-deoxycytidine (5-aza-CdR), have been suggested to treat tumors in which DNA methylation is altered. We aimed to evaluate whether DNA methylation reversion with 5-aza-CdR reduces cell proliferation and extracellular matrix (ECM) formation in uterine leiomyoma cells to provide a potential treatment option.MethodsProspective study using uterine leiomyoma and adjacent myometrium tissues and human uterine leiomyoma primary (HULP) cells (n = 16). In tissues, gene expression was analyzed by qRT-PCR and DNMT activity by ELISA. Effects of 5-aza-CdR treatment on HULP cells were assessed by CellTiter, western blot, and qRT-PCR.ResultsDNMT1 gene expression was higher in uterine leiomyoma vs myometrium. Similarly, DNMT activity was greater in uterine leiomyoma and HULP cells (6.5 vs 3.8 OD/h/mg; 211.3 vs 63.7 OD/h/mg, respectively). After 5-aza-CdR treatment on HULP cells, cell viability was reduced, significantly so at 10 μM (85.3%). Treatment with 10 μM 5-aza-CdR on HULP cells significantly decreased expression of proliferation marker PCNA (FC = 0.695) and of ECM proteins (COLLAGEN I FC = 0.654; PAI-1, FC = 0.654; FIBRONECTIN FC = 0.733). 5-aza-CdR treatment also decreased expression of Wnt/β-catenin pathway final targets, including WISP1 protein expression (10 μM, FC = 0.699), c-MYC gene expression (2 μM, FC = 0.745 and 10 μM, FC = 0.728), and MMP7 gene expression (5 μM, FC = 0.520 and 10 μM, FC = 0.577).Conclusions5-aza-CdR treatment inhibits cell proliferation, ECM formation, and Wnt/β-catenin signaling pathway targets in HULP cells, suggesting that DNA methylation inhibition is a viable therapeutic target in uterine leiomyoma.

SWATH-MS Analysis of FFPE Tissues Identifies Stathmin as a Potential Marker of Endometrial Cancer in Patients Exposed to Tamoxifen

A specific form of endometrial cancer (EC) can develop in breast cancer patients previously treated with tamoxifen (ET), an antagonist of estrogen receptor (ER) that inhibits proliferation of ER positive breast cancer. ET tumors have a different phenotype than endometrial tumors, which typically develop de novo without previous exposure to tamoxifen (EN). Here we aimed to identify specific protein markers that could serve as specific molecular targets in either phenotype. A set of total 45 formalin-fixed paraffin-embedded (FFPE) endometrial tumor tissues and adjacent myometrium tissue samples were analyzed using LC-MS/MS in SWATH-MS mode. We found that calcyphosin (CAPS) levels were elevated in EN tumors compared to ET tumors. The higher CAPS level in EC tissue invading to myometrium supports its relationship to EC aggressiveness. Further, stathmin (STMN1) levels were found significantly elevated in ET versus EN tumors and significantly associated with patient survival. This finding connects elevated levels of this cell cycle regulating, proliferation-associated protein with tamoxifen exposure. In summary, using SWATH-MS we show that CAPS and STMN1 should be recognized as clinicopathologically different EC markers of which STMN1 is specifically connected with a previous tamoxifen exposition.

ERα inhibited myocardin-induced differentiation in uterine fibroids

Uterine fibroids, also known as uterine leiomyomas, are a benign tumor of the human uterus and the commonest estrogen-dependent benign tumor found in women. Myocardin is an important transcriptional regulator in smooth and cardiac muscle development. The role of myocardin and its relationship with ERα in uterine fibroids have barely been addressed. We noticed that the expression of myocardin was markedly reduced in human uterine fibroid tissue compared with corresponding normal or adjacent myometrium tissue. Here we reported that myocardin induced the transcription and expression of differentiation markers SM22α and alpha smooth muscle actin (α-SMA) in rat primary uterine smooth muscle cells (USMCs) and this effect was inhibited by ERα. Notably, we showed that, ERα induced expression of proliferation markers PCNA and ki-67 in rat primary USMCs. We also found ERα interacted with myocardin and formed complex to bind to CArG box and inhibit the SM22α promoter activity. Furthermore, ERα inhibited the transcription and expression of myocardin, and reduced the levels of transcription and expression of downstream target SM22α, a SMC differentiation marker. Our data thus provided important and novel insights into how ERα and myocardin interact to control the cell differentiation and proliferation of USMCs. Thus, it may provide potential therapeutic target for uterine fibroids.

MiR-139-5p regulates proliferation, apoptosis, and cell cycle of uterine leiomyoma cells by targeting TPD52.

BackgroundUterine leiomyoma is one of the most common benign tumors in women. It dramatically decreases the quality of life in the affected women. However, there is a lack of effective treatment paradigms. Micro-RNAs are small noncoding RNA molecules that are extensively expressed in organisms, and they are interrelated with the occurrence and development of the tumor. miR-139-5p was found to be downregulated in various cancers, but its function and mechanism in uterine leiomyoma remain unknown. The aim of this study was to investigate the role of miR-139-5p and its target gene in uterine leiomyoma.MethodsBy using a bioinformatic assay, it was found that TPD52 was a potential target gene of miR-139-5p. Then, expressions of miR-139-5p and TPD52 in uterine leiomyoma and adjacent myometrium tissues were evaluated by quantitative real-time polymerase chain reaction and Western blot. Proliferation, apoptosis, and cell cycle of uterine leiomyoma cells transfected by miR-139-5p mimics or TPD52 siRNA were determined.ResultsIt was observed that the expression of miR-139-5p in uterine leiomyoma tissues was significantly lower (P<0.001) than that in the adjacent myometrium tissues. Overexpression of miR-139-5p inhibited the growth of uterine leiomyoma cells and induced apoptosis and G1 phase arrest. Dual-luciferase reporter assay and Western blot validated that TPD52 is the target gene of miR-139-5p. Furthermore, downregulation of TPD52 by siRNA in uterine leiomyoma cells inhibited cell proliferation and induced cell apoptosis and G1 phase arrest.ConclusionData suggested that miR-139-5p inhibited the proliferation of uterine leiomyoma cells and induced cell apoptosis and G1 phase arrest by targeting TPD52.

Stro-1/CD44 as putative human myometrial and fibroid stem cell markers

To identify and characterize myometrial/fibroid stem cells by specific stem cell markers in human myometrium, and to better understand the stem cell contribution in the development of uterine fibroids. Prospective, experimental human and animal study. University research laboratory. Women undergoing hysterectomy for treatment of symptomatic uterine fibroids and female NOD/SCID/IL-2Rγ(null) mice. Identification and isolation of stem cells from human fibroids and adjacent myometrium tissues using Stro-1/CD44-specific surface markers. Flow cytometry, semiquantitative polymerase chain reaction, clonogenicity assays, cell culture, molecular analysis, immunocyto-histochemistry, invitro differentiation, and xenotransplantation assays. Using Stro-1/CD44 surface markers, we were able to isolate stem cells from adjacent myometrium and human fibroid tissues. The undifferentiated status of isolated cells was confirmed by the expression of ABCG2 transporter, as well as additional stem cell markers OCT4, NANOG, and GDB3, and the low expression of steroid receptors ERα and PR-A/PR-B. Mesodermal cell origin was established by the presence of typical mesenchymal markers (CD90, CD105, and CD73) and absence of hematopoietic stem cell markers (CD34, CD45), and confirmed by the ability of these cells to differentiate invitro into adipocytes, osteocytes, and chondrocytes. Finally, their functional capability to form fibroid-like lesions was established in a xenotransplantation mouse model. The injected cells labeled with superparamagnetic iron oxide were tracked by both magnetic resonance imaging and fluorescence imaging, thus demonstrating the regenerative potential of putative fibroid stem cells invivo. We have demonstrated that Stro-1/CD44 can be used as specific surface markers to enrich a subpopulation of myometrial/fibroids cells, exhibiting key features of stem/progenitor cells. These findings offer a useful tool to better understand the initiation of uterine fibroids, and may lead to the establishment of effective therapeutic options.

Expression and functional analysis of platelet-derived growth factor in uterine leiomyomata

Objective: To examine the expression of PDGF and its receptors in leiomyoma tissue and to investigate the regulation of PDGF on leiomyoma cell proliferation.Materials and Methods: The expression of PDGF and its receptors was examined in 21 pairs of uterine leiomyoma and the adjacent myometrium tissues using an immunostaining method. Paired cultures of leiomyoma and normal myometrium cells were established and treated with PDGF. Total RNA was extracted from leiomy yoma and myometrial cells for detection of the expression of proliferating cell nuclear antigen (PCNA) and collagen _1 (I, III) by semi-quantitative PCR.Results: The expression of PDGF-AA and PDGF-BB in leiomyoma tissue is obviously higher than that in the matched myometrial tissue (P

Breast cancer, basic and clinical aspects

To identify and characterize myometrial/fibroid stem cells by specific stem cell markers in human myometrium, and to better understand the stem cell contribution in the development of uterine fibroids.Prospective, experimental human and animal study.University research laboratory.Women undergoing hysterectomy for treatment of symptomatic uterine fibroids and female NOD/SCID/IL-2Rγnull mice.Identification and isolation of stem cells from human fibroids and adjacent myometrium tissues using Stro-1/CD44–specific surface markers.Flow cytometry, semiquantitative polymerase chain reaction, clonogenicity assays, cell culture, molecular analysis, immunocyto-histochemistry, in vitro differentiation, and xenotransplantation assays.Using Stro-1/CD44 surface markers, we were able to isolate stem cells from adjacent myometrium and human fibroid tissues. The undifferentiated status of isolated cells was confirmed by the expression of ABCG2 transporter, as well as additional stem cell markers OCT4, NANOG, and GDB3, and the low expression of steroid receptors ERα and PR-A/PR-B. Mesodermal cell origin was established by the presence of typical mesenchymal markers (CD90, CD105, and CD73) and absence of hematopoietic stem cell markers (CD34, CD45), and confirmed by the ability of these cells to differentiate in vitro into adipocytes, osteocytes, and chondrocytes. Finally, their functional capability to form fibroid-like lesions was established in a xenotransplantation mouse model. The injected cells labeled with superparamagnetic iron oxide were tracked by both magnetic resonance imaging and fluorescence imaging, thus demonstrating the regenerative potential of putative fibroid stem cells in vivo.We have demonstrated that Stro-1/CD44 can be used as specific surface markers to enrich a subpopulation of myometrial/fibroids cells, exhibiting key features of stem/progenitor cells. These findings offer a useful tool to better understand the initiation of uterine fibroids, and may lead to the establishment of effective therapeutic options.

Creatine kinase isoenzyme patterns in normal smooth muscle and smooth muscle neoplasms

The CK isoenzyme composition of leiomyoma tissue is predominantly CK-BB and similar to adjacent myometrium tissue, while the leiomyosarcoma revealed a lesser quantity of CK-BB, but a greater quantity of CK-MM. The reasons for the discrepancy between the two types of neoplasms is not clear, but may reflect the changes which occur when smooth muscle becomes malignant.