Cell Proliferation In Endometriosis Research Articles

Retracted] The AMPK‑mTOR axis requires increased MALAT1 expression for promoting granulosa cell proliferation in endometriosis.

[This retracts the article DOI: 10.3892/etm.2020.9453.].

Phosphorylation of PFKFB4 by PIM2 promotes anaerobic glycolysis and cell proliferation in endometriosis

Endometriosis (EM) is one of the vanquished wonted causes of chronic pelvic sting in women and is closely associated with infertility. The long-term, complex, systemic, and post-treatment recurrence of EM wreaks havoc on women’s quality of life. Extensive metabolic reprogramming (aerobic glycolysis, glucose overweening intake, and high lactate production) and cancer-like changes have been found in EM, which bears striking similarities to tumorigenesis. The key glycolysis regulator PFKFB4 is overexpressed in EM. However, the mechanism of PFKFB4 in EM remains unknown. We found that PFKFB4 was upregulated and was closely related to the progression of EM. We identified focus PIM2 as a new pioneering adjoin protein of PFKFB4. Vigorous biochemical methods were used to confirm that PIM2 phosphorylated site Thr140 of PFKFB4. PIM2 also could enhance PFKFB4 protein expression through the ubiquitin–proteasome pathway. Moreover, PIM2 expression was really corresponding prevalent with PFKFB4 in endometriosis in vivo. Importantly, phosphorylation of PFKFB4 on Thr140 by PIM2 promoted EM glycolysis and cell growth. Our study demonstrates that PIM2 mediates PFKFB4 Thr140 phosphorylation thus regulating glycolysis and EM progression. We illustrated a new mechanism that PIM2 simulated a central upstream partnership in the regulation of PFKFB4, and reveal a novel means of PIM2-PFKFB4 setting EM growth. Our research provided new theoretical support for further clarifying the reprogramming of EM glucose metabolism, and provided new clues for exploring non-contraceptive treatments for EM.

The AMPK-mTOR axis requires increased MALAT1 expression for promoting granulosa cell proliferation in endometriosis.

Endometriosis is a common reproductive disorder in women, with a global prevalence of 10-15%. Long noncoding RNAs (lncRNAs) are critical to gene transcription, cell cycle modulation and immune response. The lncRNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) reportedly mediates autophagy of endometrial stromal cells in endometriosis. The present study aimed to evaluate the role and mechanism of MALAT1 in granulosa cells (GCs) in endometriosis. Consequently, MALAT1 expression was upregulated in GCs obtained from patients with endometriosis and in the steroidogenic human granulosa-like tumor cell line KGN. However, MALAT1 knockdown consequently decreased the proliferation and viability of these cells, as determined by MTT and 5-ethynyl-2'-deoxyuridine staining assays. Both Annexin V-fluorescein isothiocyanate/propidium iodide flow cytometry and western blotting performed to detect proapoptotic factors indicated that MALAT1 depletion might promote KGN cell apoptosis. Furthermore, MALAT1 knockdown increased GC autophagy, as evidenced by microtubule-associated protein 1A/1B-light chain 3 (LC3) cleavage upregulation and p62 degradation. In addition, although 5'-AMP-activated protein kinase (AMPK) mRNA expression and protein levels decreased in GCs obtained from patients with endometriosis and KGN cells, MALAT1 knockdown restored AMPK levels. However, addition of BML-275 (MALAT1 inhibitor) to MALAT1-knockdown KGN cells recovered their viability and proliferative capacity and simultaneously reduced their apoptotic and autophagic capacity. Therefore, MALAT1 may regulate GC proliferation via AMPK-mTOR-mediated cell apoptosis and autophagy.

Oncogenic mutation in RAS-RAF axis leads to increased expression of GREB1, resulting in tumor proliferation in colorectal cancer.

BRAFV600E mutation accounts for up to 90% of all BRAF mutations in human colorectal cancer (CRC), and constitutively activates the MEK‐MAPK pathway. It is recognized that neutralizing mAbs for epidermal growth factor receptor alone are not effective for CRC with BRAFV600E mutation. Therefore, there is increasing interest in identification of the possible therapeutic targets in downstream of BRAF mutation in CRCs. To address this, we studied genome engineered mouse models for colonic neoplasia that has BrafV600E mutation on the basis of Apc inactivation, induced in 2 distinct Cre mouse models, CDX2P‐G22Cre and CDX2P‐CreERT2 mice. We carried out oligonucleotide microarray analysis for colonic neoplasia generated in these mouse models, and compared gene expression profiles among Kras/Braf WT, Kras‐mutated, and Braf‐mutated mouse colon tumors to seek new molecular targets corresponding to the KRAS‐BRAF‐MAPK axis. We found that the expression of the growth regulation by estrogen in breast cancer protein 1 (Greb1) was the most upregulated gene in Braf‐mutated mouse tumors compared to Kras/Braf WT counterparts. The silencing of GREB1 significantly reduced the proliferation and tumorigenesis of CRC cell lines, whereas the overexpression of GREB1 promoted cell proliferation. Although GREB1 was first identified as a hormone‐responsive gene mediating estrogen‐stimulated cell proliferation in endometriosis, breast, and ovarian cancers, these results suggest that RAS‐RAF‐MAPK signaling upregulates GREB1 expression in CRC, resulting in cellular proliferation. Thus, GREB1 is a possible therapeutic target for CRCs with BrafV600E mutation.

Endometriosis Cell Proliferation Induced by Bone Marrow Mesenchymal Stem Cells.

Endometriosis is an estrogen-dependent gynecological disorder that affects 10% of reproductive-aged women and causes pelvic pain and infertility. Bone marrow-derived stem cells (BMDCs) are known to engraft endometriosis in association with lesion growth; however, they do not undergo significant clonal expansion. The indirect effects of BMDCs on endometriosis growth and cell proliferation are not well characterized. Here, we demonstrate that BMDCs' co-culture increased endometrial stromal cell proliferation. In vitro studies using endometrial cells showed that BMDCs increased cell proliferation and activation of CDK1 in both an endometriosis cell line and primary endometrial stromal cells from women with endometriosis, however not in normal endometrial cells. In vivo studies using a mouse model of endometriosis showed increased CDK1+ expression associated with engrafted GFP + BMDCs. These results suggest that endometrial cell proliferation is induced by stem cell-derived trophic factors leading to the growth of endometriotic lesions. Targeting the specific signaling molecules secreted by BMDC may lead to novel therapeutic strategies for controlling cell proliferation in endometriosis.

Thalidomide Reduces Cell Proliferation in Endometriosis Experimentally Induced in Rats.

To analyze the effect of thalidomide on the progression of endometriotic lesions experimentally induced in rats and to characterize the pattern of cell proliferation by immunohistochemical Proliferating Cell Nuclear Antigen (PCNA) labeling of eutopic and ectopic endometrium. Fifteen female Wistar rats underwent laparotomy for endometriosis induction by resection of one uterine horn, isolation of the endometrium and fixation of a tissue segment to the pelvic peritoneum. Four weeks after, the animals were divided into 3 groups: control (I), 10mg/kg/day (II) and 1mg/kg/day (III) intraperitoneal thalidomide for 10 days. The lesion was excised together with the opposite uterine horn for endometrial gland and stroma analysis. Eutopic and ectopic endometrial tissue was submitted to immunohistochemistry for analysis of cell proliferation by PCNA labeling and the cell proliferation index (CPI) was calculated as the number of labeled cells per 1,000 cells. Group I showed a mean CPI of 0.248 ± 0.0513 in the gland and of 0.178 ± 0.046 in the stroma. In contrast, Groups II and III showed a significantly lower CPI, that is, 0.088 ± 0.009 and 0.080 ± 0.021 for the gland (p < 0.001) and 0.0945 ± 0.0066 and 0.075 ± 0.018 for the stroma (p < 0.001), respectively. Also, the mean lesion area of Group I was 69.2 mm2, a significantly higher value compared with Group II (49.4 mm2, p = 0.023) and Group III (48.6 mm2, p = 0.006). No significant difference was observed between Groups II and III. Thalidomide proved to be effective in reducing the lesion area and CPI of the experimental endometriosis implants both at the dose of 1 mg/kg/day and at the dose of 10 mg/kg/day.

17βE 2 promotes cell proliferation in endometriosis by decreasing PTEN via NFκB-dependent pathway

The objective of this study was to explore the mechanism of phosphatase and tensin homolog (PTEN) loss in endometriosis. We found that aberrant PTEN expression and mitogen-activated protein kinases (MAPK)/ERK, phosphoinositide 3-kinase (PI3K)/AKt, and nuclear factor-kappaB (NFκB) signaling overactivities coexisted in endometriosis. In vitro, 17β-estradiol rapidly activated the 3 pathways in endometriotic cells and specific inhibitions on the 3 pathways respectively blocked 17β-estradiol-induced cell proliferation. 17β-estradiol suppressed PTEN transcription and expression in endometriotic cells which was abolished by specific NFκB inhibition. Conclusion(s) Total/nuclear PTEN-loss and MAPK/ERK, PI3K/AKt, and NFκB signal overactivities coexist in endometriosis. In vitro, 17β-estradiol can promotes cell proliferation in endometriosis by activating PI3K/AKt pathway via an NFκB/PTEN-dependent pathway. For the first time we propose the possibility of the presence of a positive feedback-loop: 17β-estradiol → high NFκB → low PTEN → high PI3K → high NFκB, in endometriosis, which may finally promote the proliferation of ectopic endometrial epithelial cells and in turn contributes to the progression of the disease.

Endometriotic stromal cells lose the ability to regulate cell-survival signaling in endometrial epithelial cells in vitro

In normal endometrium, stromal factors regulate the growth of epithelial cells. However, epithelial cells in endometriotic lesions display increased proliferation and decreased apoptosis. This work tested the hypothesis that in endometriosis stromal cells lose the ability to regulate survival signaling and cell growth in epithelial cells. Primary normal, endometriotic eutopic and ectopic epithelial cells were cultured in the presence of medium conditioned by normal, eutopic and ectopic endometriotic endometrial stromal cells. Endometriotic epithelial cells showed higher Survivin expression than normal epithelial cells. Conditioned medium (CM) from normal or eutopic endometriotic stromal cells significantly inhibited the Survivin expression and AKt phosphorylation in normal or eutopic endometriotic epithelial cells. However, CM from ectopic endometriotic stromal cells did not have an inhibitory effect on normal or ectopic endometriotic epithelial cells. Inhibition of AKt phosphorylation and Survivin expression in normal or eutopic endometriotic epithelial cells in the presence of stromal factors from normal or eutopic endometriotic stromal cells was enhanced by progesterone, whereas progesterone had little effect in the presence of stromal factors from ectopic endometriotic stromal cells. The inability of ectopic endometriotic stromal cells to regulated PI3K/AKt/Survivin signaling and mediate the progesterone response in endometriotic epithelial cells may facilitate epithelial cell proliferation in endometriosis and promote the survival of endometriotic lesions.

The involvement of T lymphocytes in the pathogenesis of endometriotic tissues overgrowth in women with endometriosis.

BACKGROUND: Endometriosis, uncontrolled proliferation of ectopic and eutopic endometriotic tissues, is common in women at reproductive age, and may affect fertility. The role of macrophages in the pathogenesis is well proved, but engagement of T cells in the pathogenesis of endometriosis is a matter of controversy. AIMS: T-cell involvement in the pathogenesis of endometriosis was the objective of our study performed on women aged 24-46 years with diagnosed endometriosis. All the patients that were studied underwent diagnostic laparoscopy. METHODS: We evaluated the distribution of T-lymphocyte subpopulations in peripheral blood (PB), peritoneal fluid (PF) and in endometriotic tissues (ET), as well as cytokines [interleukin (IL)-2, IL-4, IL-10, IL-12, interferon (IFN)-gamma] production by peripheral blood lymphocytes. IFN-gamma, tumor necrosis factor (TNF)-alpha, IL-4 and IL-6 were investigated for their intracellular presence. The experiments were carried out before and after 6 months treatment with the GnRH-Analogous Goserelin (Zeneca Pharmaceuticals). The number of performed investigations is presented. Statistical analysis was performed using Statistica/Win 5.0 software and Student's t-test, the paired Student t-test and Fisher's exact test when appropriate. RESULTS: We have compared the lymphocyte subset re-distribution with regard to the American Fertility Society (AFS) stages and scores, but no differences were observed. The significant increase in CD4:CD8 ratio, the decrease in the number of natural killer (NK) cells in PB and the decrease in CD4:CD8 ratio in PF and ET of women with endometriosis was noted. The diminished IFN-gamma secretion by phytohemagglutinim (PHA)-stimulated lymphocytes in vitro derived from women with endometriosis and increased IL-4 production may be responsible for defective immunosurveillance against overgrowth of endometriotic tissues. The diminished NK cells number in PB of women with endometriosis argues for such a hypothesis. The increased deposits of proinflammatory IL-6 and TNF-alpha in the T lymphocytes of women with endometriosis may be related to T-regulatory lymphocyte function and their inability to suppress cell proliferation in endometriosis. GnRH-Analogous Goserelin treatment normalises cytokine production and induces patient recovery. CONCLUSIONS: The significant functional and phenotypic differences between the lymphocytes from healthy women and women with endometriosis were noted. The diminished IFN-gamma production in relation to decreased NK cells number and the increased IL-4 production before the treatment and normalisation after the treatment suggest the involvement of the deregulated T-cell system in the growth stimulation and recruitment of endometriotic cells. The increased CD4:CD8 ratio, IL-6, TNF-alpha deposits and diminished anti-inflammatory IL-10 production by lymphocytes may participate in the pathogenesis of endometriosis, and may secondarily affect the monocyte/macrophage function.

Cytokine regulation of cellular proliferation in endometriosis.

This study was designed (1) to characterize the resident leukocyte population in ectopic endometrium (EE), (2) to assess proliferative activity of cellular components in EE, (3) to assess whether resident leukocytes in EE express IFN gamma mRNA and (4) to demonstrate endometrial epithelial cell IFN gamma receptors in EE. Biopsies of EE and normal eutopic endometrium (UE) were studied immunocytochemically using monoclonal antibodies specific for CD45 leukocyte common antigen, CD3 (a T cell marker), CD11c (a macrophage marker), and Ki67 (proliferation marker). Leukocyte types were identified immunocytochemically, followed by in situ hybridization to assess expression of IFN gamma mRNA. IFN gamma receptor expression was assessed by immunocytochemistry. The percentage of scattered stromal cells staining for each CD marker was greater in EE than in UE. The proliferative activity of endometrial stromal cells and epithelial cells was significantly less in EE than in UE. The overall concentration of T cells and macrophages expressing IFN gamma mRNA was significantly greater in EE than in UE. The percentage of each leukocyte type expressing IFN gamma mRNA was also greater in EE than in UE, and IFN gamma receptors were present in glandular epithelium of EE. These findings support a possible paracrine role for resident leukocytes and IFN gamma in regulating cell proliferation in endometriosis.

Enhanced expression of resident leukocyte interferon gamma mRNA in endometriosis.

Previous studies have shown that the endometrial epithelial/stromal cell proliferative activity of endometriosis is significantly less than that of normal endometrium and that the concentration of resident stromal leukocytes is significantly greater in ectopic than in eutopic endometrium. Other work has shown that interferon gamma (IFN gamma), secreted by resident leukocytes, inhibits endometrial cell proliferation in vitro. Accordingly, we hypothesized that the lower proliferative activity of endometriosis may be related to enhanced resident leukocyte IFN gamma production. This study was designed to assess whether resident leukocytes in endometriosis express IFN gamma mRNA and to compare this expression to that of normal endometrium. Biopsies of ectopic endometrium (N = 16) from women in the follicular phase and normal proliferative (N = 9) and secretory (N = 8) endometria were examined for IFN gamma expression. Using monoclonal antibodies specific for CD45 (leukocyte common antigen), CD3 (a T-cell marker) and CD11c (a macrophage marker), leukocyte types were identified immunocytochemically, followed by in situ hybridization to examine expression of IFN gamma mRNA. Results demonstrated that (1) the overall concentration of T cells and macrophages expressing IFN gamma mRNA is significantly greater in endometriosis as compared to eutopic endometrium, and (2) the percent of each leukocyte type expressing IFN gamma mRNA is greater in endometriosis than in normal endometrium. These findings support a possible paracrine role for resident leukocytes in regulating cell proliferation in endometriosis.