• 2019-10
  • 2019-11
  • 2020-03
  • 2020-07
  • 2020-08
  • 2021-03
  • br Fig A interacts with ER A Ectopically expressed


    Fig. 5. A20 interacts with ERα. A. Ectopically expressed A20 and ERα showed a mutual interaction. HEK-293T cells were co-transfected with Myc-A20 and Flag-HA (FH)-ERα constructs (A1) or Flag-A20 and Myc-ERα (A2) for 30 h. After being treated with 20 μM MG132 for 8 h, cell lysates were prepared for co-IP with anti-FLAG beads and western blotting analysis. B. Endogenous A20 and ERα proteins interacted with each other in HEC-1A cells. After being treated with 20 μM MG132 for 8 h, cell lysates were prepared for co-IP with anti-ERα (B1) or anti-A20 (B2) antibody and western blotting analysis. C. ERα and A20 co-localized in EC cell nuclei. EC cells were immunostained with anti-ERα (red) and anti-A20 (green) Methylpiperidino pyrazole and visualized by confocal microscopy. DAPI (blue) was used to indicate cell nuclei. Scale bars.
    stabilize ERα protein and maintain estrogen signaling by inhibiting ERα ubiquitination degradation through interactions with ERα protein. This sustained estrogen signaling renders patients more susceptible to es-trogen-dependent EC.
    4. Discussion
    In this study, we demonstrated that CD163+ macrophage-associated chronic inflammation could stabilize ERα protein through A20-medi-ated deubiquitination modifications to sensitize EC cells to estrogen. Therefore, our study revealed that post-translational regulation was also an important mechanism involved in infiltrating macrophage-in-duced upregulation of estrogen sensitivity in endometrium.
    Macrophage infiltration is one of the main characteristics of chronic inflammation that is closely related to endometrial carcinogenesis [10,12]. In this study, we found that CD163+ macrophage infiltration is the early event of endometrial tumorigenesis. Although endometrial atypical hyperplasia is distinct from endometrial cancer, it is a pre-cancerous stage which develops gradually into EEC G1 [34,35]. The findings that CD163+ macrophages were abundantly infiltrated in EAH and EEC G1 suggested that CD163+ macrophages might be involved in the progress from EAH to EEC. In this study, continuous section slides of endometrial lesions were used to stain A20, ERα and CD163 by im-munohistochemistry, which indirectly proved that Methylpiperidino pyrazole A20 and ERα co-localized in endometrial gland cells in CD163+ macrophage-rich me-senchyme around endometrial glands. The expression of both A20 and ERα increased in EEC G1 compared with normal endometrium. How-ever, when the lesions progressed from EEC G1 to G3, ERα presented with more pronounced decline than A20. These results suggest that when cells gained cancerous property under the stimulation of  Cancer Letters 442 (2019) 137–147
    estrogen, estrogen is no longer the primary engine for cancer progres-sion [36]. Therefore, we see low or even loss expression of ERα but moderate expression of A20 in EEC G3. In other words, EC no longer relies on ERα signaling or A20-mediated ERα protein stability when EEC G1 progressed to G3.
    CD163+ macrophage-associated chronic inflammation can provide a carcinogenic microenvironment [37,38]. In EC, the aromatase activity of stromal cells is upregulated by IL6, suggesting increased 17β-es-trogen bio-synthesis [5]. Moreover, EC patients with insulin resistance showed increased GPER expression in EC tissues compared with those without insulin resistance [8,9,39]. Consistent with these reports, our findings also suggested that upregulation of ERα protein by CD163+ macrophage-associated chronic inflammation is an important me-chanism for estrogen-driven endometrial carcinogenesis.
    The mechanisms by which CD163+ macrophages increased ERα protein abundance are intriguing. In the present study, we demon-strated that A20 could positively regulate ERα protein stability through its N-terminal deubiquitinase activity. In conjunction with our recent report [6], we surmised that an inflammatory microenvironment with CD163+ macrophages could upregulate ERα expression by both upre-gulating ERα expression through TET1-mediated epigenetic modulation of ESR1 and stabilizing ERα protein through A20-mediated deubiqui-tination modification. Furthermore, the synergistic effects of these two mechanisms would be helpful for cascade amplification of ERα sig-naling, which ultimately promote the development of EC.