Tryptophan 2,3-Dioxygenase-2 in Uterine Leiomyoma: Dysregulation by MED12 Mutation Status.

Uterine leiomyomas (fibroids) are common benign tumors in women. The tryptophan metabolism through the kynurenine pathway plays important roles in tumorigenesis in general. Leiomyomas expressing mutated mediator complex subunit 12 (mut-MED12) were reported to contain significantly decreased tryptophan levels; the underlying mechanism and the role of the tryptophan metabolism-kynurenine pathway in leiomyoma tumorigenesis, however, remain unknown. We here assessed the expression and regulation of the key enzymes that metabolize tryptophan. Among these, the tissue mRNA levels of tryptophan 2,3-dioxygenase (TDO2), the rate limiting enzyme of tryptophan metabolism through the kynurenine pathway, was 36-fold higher in mut-MED12 compared to adjacent myometrium (P < 0.0001), and 14-fold higher compared to wild type (wt)-MED12 leiomyoma (P < 0.05). The mRNA levels of other tryptophan metabolizing enzymes, IDO1 and IDO2, were low and not significantly different, suggesting that TDO2 is the key enzyme responsible for reduced tryptophan levels in mut-MED12 leiomyoma. R5020 and medroxyprogesterone acetate (MPA), two progesterone agonists, regulated TDO2 gene expression in primary myometrial and leiomyoma cells expressing wt-MED12; however, this effect was absent or blunted in leiomyoma cells expressing G44D mut-MED12. These data suggest that MED12 mutation may alter progesterone-mediated TDO2 expression in leiomyoma, leading to lower levels of tryptophan in mut-MED12 leiomyoma. This highlights that fibroids can vary widely in their response to progesterone as a result of mutation status and provides some insight for understanding the effect of tryptophan-kynurenine pathway on leiomyoma tumorigenesis and identifying targeted interventions for fibroids based on their distinct molecular signatures.

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

Uterine 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.

Identification of core gene in obese type 2 diabetes patients using bioinformatics analysis.

Objectives Adipocytes and adipocyte lipid metabolism are closely related with obesity and type 2 diabetes, but the molecular mechanism still needs further investigation. The aim of this study is to discover the adipocyte genes and pathways involved in obesity and type 2 diabetes using bioinformatics analysis.Methods The GSE27951 gene expression profile was obtained. Software and online tools (STRING, Cytoscape, BioGPS, CTD, and FunRich) were used to identify core genes.21 human subcutaneous adipose samples, with 10 from type 2 diabetic patients and 11 from normal controls, were included in these analyses.Results 184 differentially expressed genes (DEGs) including 42 up-regulated genes and 142 down-regulated genes were found to be enriched in metabolism, receptor activity, collagen type IV and glutamine biosynthesis I pathway by using the enrichment analysis. Seven hub genes were identified from the PPI network using various software (Cytoscape, STRING, BioGPS, and CTD). Four core genes (COL4A2, ACACB, GLUL, and CD36) were found to be highly expressed in subcutaneous adipose tissue of obese patients accompanying type 2 diabetes.Conclusion COL4A2, ACACB, GLUL and CD36 might be the core molecular biomarkers of obesity in patients with or without type 2 diabetes.

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

OBJECTIVE: To investigate the functional interaction between the Wnt/ß-catenin and protein kinase B (Akt) pathways in leiomyoma stem cells (LSC). DESIGN: Laboratory study. SETTING: Research laboratory. PATIENT(S): Premenopausal women (n = 36; age range: 28 to 49 years) undergoing hysterectomy or myomectomy for leiomyoma. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Gene expression, protein phosphorylation, and cell proliferation. RESULT(S): 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/ß-catenin and Akt signaling pathways in leiomyoma cells evaluated using real-time quantitative polymerase chain reaction and immunoblot analyses. The Wnt/ß-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 ß-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. CONCLUSION(S): Our data suggest that WNT4 regulates LSC proliferation via Akt-dependent ß-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.

Predicting MicroRNA Target Genes and Identifying Hub Genes in IIA Stage Colon Cancer Patients Using Bioinformatics Analysis.

BACKGROUND: Colon cancer is a heterogeneous disease, differing in clinical symptoms, epigenetics, and prognosis for each individual patient. Identifying the core genes is important for early diagnoses and it provides a more precise method for treating colon cancer. MATERIALS AND METHODS: In this study, we wanted to pinpoint these core genes so we obtained GSE101502 microRNA profiles from the GEO database, which resulted in 17 differential expressed microRNAs that were identified by GEO2R analysis. Then, 875 upregulated and 2920 downregulated target genes were predicted by FunRich. GO and KEGG pathway were used to do enrich analysis. RESULTS: GO analysis indicated that upregulated genes were significantly enriched in the regulation of cell communication and signaling and in nervous system development, while the downregulated genes were significantly enriched in nervous system development and regulation of transcription from the RNA polymerase II promoter. KEGG pathway analysis suggested that the upregulated genes were enriched in axon guidance, MAPK signaling pathway, and endocytosis, while the downregulated genes existed in pathways in cancer, focal adhesion, and PI3K-Akt signaling pathway. The top four molecules including 82 hub genes were identified from the PPI network and involved in endocytosis, spliceosome, TGF-beta signaling pathway, and lysosome. Finally, NUDT21, GNB1, CLINT1, and COL1A2 core gene were selected due to their correlation with the prognosis of IIA stage colon cancer. CONCLUSION: this study suggested that NUDT21, GNB1, CLINT1, and COL1A2 might be the core genes for colon cancer that play an important role in the development and prognosis of IIA stage colon cancer.

Progesterone receptor integrates the effects of mutated MED12 and altered DNA methylation to stimulate RANKL expression and stem cell proliferation in uterine leiomyoma.

Progesterone and its receptor, PR, are essential for uterine leiomyoma (LM, a.k.a., fibroid) tumorigenesis, but the underlying cellular and molecular mechanisms remain unclear. The receptor activator of NF-κB (RANKL) was recently identified as a novel progesterone/PR-responsive gene that plays an important role in promoting LM growth. Here, we used RANKL as a representative gene to investigate how steroid hormone, genetic, and epigenetic signals are integrated to regulate LM stem cell (LSC) function. We demonstrated that RANKL specifically upregulates LSC proliferation through activation of Cyclin D1. RANKL gene transcription was robustly induced by the progesterone agonist R5020, leading to a dramatically higher RANKL expression in LM compared to adjacent myometrial (MM) tissue. MethylCap-Seq revealed a differentially methylated region (DMR) adjacent to the distal PR-binding site (PRBS) 87 kb upstream of the RANKL transcription start site. Hypermethylation of the DMR inhibited recruitment of PR to the adjacent PRBS. Luciferase assays indicated that the DMR and distal PRBS constitute a novel RANKL distal regulatory element that actively regulates RANKL expression. Furthermore, MED12 physically interacts with PR in LM tissue. The interaction between MED12 and PR, binding of PR and MED12 to PRBS, and RANKL gene expression are significantly higher in LM containing a distinct MED12 mutation (G44D) than in LM with wild-type MED12. In summary, our findings suggest that DNA methylation and MED12 mutation together constitute a complex regulatory network that affects progesterone/PR-mediated RANKL gene expression, with an important role in activating stem cell proliferation and fibroid tumor development.