Prognostic value of MYBL2 and its potential implications for immunotherapy: a study of bioinformatics and experimental validation.

PurposeThis study aimed to investigate the effect of MYO3B on endometrial cancer (EC) proliferation and invasion.MethodsThe expression of MYO3B in EC tissues and cells was analyzed using TCGA database, immunohistochemical staining, real-time PCR, and western blot (WB). Cell proliferation was detected by CCK8, Annexin V-APC/PI flow cytometry was used to detect apoptosis, intracellular calcium ion (Ca2+) was detected by flow cytometry with Fluo-4 AM fluorescent probe, cell migration by scratch assay, and cell invasion by Transwell assay, and the expression of proteins related to Ca2+ homeostasis and RhoA/ROCK1 signaling pathway was detected by WB and immunofluorescence staining.ResultsThe expression of MYO3B was an influential factor in EC recurrence, and the expression of MYO3B was significantly up-regulated in EC tissues and cells, but down-regulated in KLE cells, and MYO3B knockdown inhibited the proliferation, migration, and invasion ability of EC cells and promoted apoptosis, suggesting that MYO3B plays a tumor-promoting role in EC. Furthermore, MYO3B knockdown decreased Ca2+ concentration in EC cells and the RhoA/ROCK1 signaling pathway was inhibited, and the effect of MYO3B knockdown on RhoA/ROCK1 signaling was reversed by treatment with the Calmodulin agonist CALP-2, and the effects of MYO3B knockdown on cell proliferation, migration, and invasion were reversed after treatment with the RhoA agonist U-46,619.ConclusionMYO3B promotes the proliferation and migration of endometrial cancer cells via Ca2+-RhoA/ROCK1 signaling pathway. High expression of MYO3B may be a biomarker for EC metastasis.

Loss of exosomal miR‐26a‐5p contributes to endometrial cancer lymphangiogenesis and lymphatic metastasis

This study intended to investigate the role of lncRNA NR2F1-AS1 in endometrial cancer (EC). The expression level of NR2F1-AS1 in tumor tissues and EC cells was measured. After sh-NR2F1-AS1 transfection, the cell viability, apoptosis, migration and invasion of EC cells were analyzed. Luciferase reporter assay was conducted to investigate the target gene of miR-363. The expression levels of PI3K/AKT/GSK-3β pathway-associated factors were assayed using western blot. NR2F1-AS1 was significantly overexpressed in EC tissues and cells. NR2F1-AS1 inhibition decreased EC cell viability, migration and invasion, while promoted cell apoptosis. miR-363 was negatively regulated by NR2F1-AS1. SOX4 was a target of miR-363. NR2F1-AS1 functioned on EC progression via PI3K/AKT/GSK-3β pathway. The results demonstrated that NR2F1-AS1 was highly expressed in EC, which involved in the proliferation and migration of EC cells through downregulation of miR-363 to target SOX4 and regulating PI3K/AKT/GSK-3β pathway.

CCL18, originating from M2-polarized tumor-associated macrophages (M2-TAMs) is recognized for its vital role in endometrial cancer (EC) development. Nonetheless, its precise mechanisms remain largely undefined. The primary objective of this research was to elucidate the underlying mechanism of M2-TAM-isolated CCL18 in EC progression. TWIST1 and HMGA1 expressions were assessed in EC tissues and cells by qRT-PCR or western blotting. M2 macrophages were differentiated from human monocyte THP-1 cells, and characterized via flow cytometry and western blotting. CCL18 levels were evaluated using western blotting and ELISA assay. CCK8, Transwell, and wound healing assays were employed to assess EC cell vitality, invasion, and migration, respectively, while western blotting was utilized to measure related protein markers. The binding relationship between TWIST1 and HMGA1 was validated via ChIP and dual-luciferase reporter assays. TWIST1 and HMGA1 were increased in EC tissues and cells. After being treated with M2-TAM-isolated CCL18, EC cell vitality, migration, and invasion were enhanced. Additionally, CCL18 derived from M2-TAM upregulated TWIST1 levels in EC cells. Further mechanistic analyses unveiled that TWIST could positively regulate HMGA1 in EC cells. Notably, HMGA1 knockdown restrained the malignancy of EC cells, which was reversed by TWIST1 overexpression. M2-TAM-isolated CCL18 facilitated EC progression by activating the TWIST1/HMGA1 axis. These observations might offer new directions for developing targeted curative interventions for EC.

The long noncoding RNA (lncRNA) THOR is highly conserved and expressed in various human cancer tissues, although its potential role and underlying mechanism in endometrial cancer (EC) remain unknown. This study aims to explore THOR's biological function and molecular mechanism in EC progression. THOR expression in EC tissues and cell lines was detected by quantitative reverse transcription PCR (qRT-PCR) and in situ hybridization (ISH). THOR expression based on The Cancer Genome Atlas (TCGA) and clinical sample analyses was significantly higher in EC tissues than normal tissues, and higher THOR levels were closely associated with poor overall survival in EC. Additionally, a positive correlation between ISH-detected THOR expression and pathological grade was observed. CCK-8, colony formation, and transwell migration and invasion assays revealed that THOR significantly enhances the proliferation, migration, and invasion abilities of EC cells. Moreover, IGF2BP1 protein expression and ERK and AKT protein phosphorylation levels in EC cells increased significantly with THOR overexpression in EC cells. In conclusion, our findings suggest that THOR promotes EC cell growth and invasion, and IGF2BP1-mediated AKT and ERK signaling pathways activation might be involved. Clinically, THOR is significantly expressed in EC, and high THOR expression correlates with poor prognosis, making it a potential prognostic marker for EC.

Crosstalk between estrogen receptor and mitogen-activated protein kinase signaling in the development and progression of endometrial cancer.

ObjectiveThe aim of this study was to examine the effect of TRIB3 on proliferation, apoptosis, and migration of endometrial cancer (EC) cells and explore the relationship between TRIB3 and AKT signaling pathway in EC progression.MethodsImmunohistochemical analysis was performed to measure the expression level of TRIB3 in normal endometrium tissues and EC tissues. Overexpression and shRNA knockdown techniques were applied by transfecting EC cells (ISK and AN3CA), and the effect of TRIB3 on EC cell biological behaviors was evaluated. Cell Counting Kit-8 and colony formation assays were utilized to investigate EC cell proliferation ability, and flow cytometry was performed to assess the apoptosis of EC cells. Moreover, the migration and invasion of EC cells were detected by transwell assay, and the levels of MMP-2 and MMP-9 were measured by ELISA. Additionally, Western blot analysis was carried out to determine the levels of AKT and p-AKT.ResultsThe expression level of TRIB3 was higher in EC than normal endometrium tissues, and its overexpression promoted apoptosis and suppressed proliferation of EC cells. Furthermore, TRIB3 retarded the migration and invasion of EC cells and decreased the levels of MMP-2 and MMP-9. Conversely, TRIB3 inhibition enhanced the expression levels of MMP-2 and MMP-9, and proliferation and migration of EC cells but suppressed their apoptosis. Similarly, TRIB3 overexpression reduced while its knockdown increased the level of p-AKT.ConclusionTRIB3 inhibited proliferation and migration and promoted apoptosis of EC cells probably through regulating AKT signaling pathway.

To investigate the role of Trophinin-associated protein (TROAP) in endometrial cancer (EC) progression and elucidate how the transcription factor E2F transcription factor 1 (E2F1) modulates EC by upregulating TROAP expression. TROAP expression in EC tissues and cell lines was analyzed using bioinformatics databases, quantitative real-time polymerase chain reaction (qRT-PCR), western blot, and immunohistochemistry. TROAP was knocked down in EC cells to assess its effects on proliferation, migration, invasion, and glycolysis. Potential transcription factors regulating TROAP were identified, and the relationship between E2F1 and TROAP gene regulation was examined using dual luciferase assay. In vivo tumor growth was evaluated using a mouse xenograft model. TROAP was overexpressed in EC tissues and cell lines compared with normal controls. High TROAP expression correlated with poor differentiation, advanced stage, lymph node metastasis, and worse overall survival in EC patients. Knockdown of TROAP inhibited the proliferation, migration, invasion, and glycolytic capacity of EC cells. E2F1 was identified as a transcriptional activator of TROAP. E2F1 overexpression enhanced TROAP expression and promoted EC cell proliferation, migration, and glycolysis in a TROAP-dependent manner. TROAP knockdown suppressed tumor growth in vivo. TROAP is transcriptionally activated by E2F1 and promotes EC progression by enhancing cell proliferation, metastasis, and glycolysis. The E2F1-TROAP axis may serve as a potential therapeutic target for EC treatment.

Although the medical science has been developed for decades, the molecular mechanism of endometrial cancer (EC) is not yet completely clear. Previous studies have shown that the tripartite motif containing 28 (TRIM28) plays a crucial role in tumor development. However, TRIM28 is rarely studied in EC, and its role and mechanism need to be further determined. This study was aimed to delve into the related molecular mechanism underling the role of TRIM28 in EC cell growth and migration. qPCR assays and Western blot assays revealed that the expression level of TRIM28 was higher in EC tissues or cell lines (HEC1B, AN3CA, and Ishikawa) than normal tissue or human endometrial epithelial cells (hEEC), respectively. Then, CCK-8 cell viability assay and clone formation assay were performed in HEC1B and AN3CA cell lines after overexpression or knockdown of TRIM28. The results verified that suppression of TRIM28 expression inhibited the proliferation of EC cells. The wound scratch healing assay and transwell assay were performed in HEC1B and AN3CA cell lines after overexpression or knockdown of TRIM28. The results showed that suppression of TRIM28 expression inhibited the invasion and migration of EC cells. Finally, the Western blot assays hinted that overexpression or knockdown of TRIM28 in HEC1B and AN3CA cell lines would promote or inhibit the phosphorylation of AKT and mTOR protein. These findings indicated that TRIM28 promoted the growth and migration of EC cells via regulating the AKT/mTOR pathway.

Sirtuin-7 is an evolutionarily conserved NAD-dependent deacetylase, which serves an important role in carcinogenesis. However, the potential mechanism of sirtuin-7 in endometrial cancer has not yet been investigated. The purpose of the present study was to investigate whether sirtuin-7 exhibits inhibitory effects on endometrial cancer cells. The potential mechanisms mediated by sirtuin-7 in endometrial cancer cells were also investigated. The expression levels of sirtuin-7 in endometrial cancer cells were compared with normal endometrial cells using western blotting. The results demonstrated that sirtuin-7 is overexpressed in endometrial cancer cells compared with normal endometrial cells. The downregulation of sirtuin-7 inhibited the growth and invasiveness of endometrial cancer cells. The knockdown of sirtuin-7 was observed to increase the sensitivity of the endometrial cancer cells to cisplatin treatment in vitro. An investigation into the potential molecular mechanism demonstrated that sirtuin-7 knockdown promoted the apoptosis of endometrial cancer cells by regulating the nuclear factor (NF)-κB signaling pathway. The knockdown of sirtuin-7 inhibited NF-κB expression and resulted in a decrease in the expression of NF-κB target proteins that are anti-apoptotic: Bcl-xl, Bcl-2 and Mcl-1. Sirtuin-7 knockdown also resulted in an increase of the NF-κB target proteins that are pro-apoptotic: Caspase-3, Bad and Bax. In conclusion, the present study demonstrated that sirtuin-7 knockdown was able to markedly inhibit the growth of endometrial cancer cells, suggesting that sirtuin-7 may be a potential therapeutic target for endometrial cancer therapy.

Common risk factors for the development of endometrial and breast cancer include early menarche, late menopause, null parity, and later age at first birth, indicating that “lifetime” exposure to estrogens increases the incidence of both tumors. In contrast, smoking protects against the development of endometrial cancer, whereas the role of smoking in breast cancer incidence is equivocal and may be dependent on the timing and duration of smoking. Constituents of cigarette smoke bind and activate the aryl hydrocarbon receptor (AhR), and research in this laboratory has focused on characterizing the inhibitory AhR-estrogen receptor (ER) a crosstalk in endometrial and breast cancer cell lines. Both Ishikawa and ECC1 endometrial cancer cells express the AhR and ERoc proteins by Western blot analysis. Moreover, AhR ligands such as benzo[a]pyrene (BaP) and/or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induce CYP1A1-dependent activity or reporter gene activity in cells transfected with constructs containing dioxin-responsive elements as promoters. Estrogen responsiveness was also confirmed in these cells, as evidenced by gene/reporter gene assay and the induction of cell proliferation by 17β-estradiol (E2). Inhibitory AhR-ERα crosstalk studies have shown that TCDD and/or BaP inhibit E2-induced growth of endometrial cancer cells and also block hormone-activated reporter gene/gene responses. Although there are several possible mechanisms for the interaction between AhR and ERα signaling pathways, the role of AhR-mediated downregulation of ERα will be discussed as one possible mechanism. In addition, selective AhR modulators have been developed for the treatment of breast and endometrial cancer and the potential use of these compounds alone or in combination with tamoxifen will be outlined.

Endometriosis-associated carcinomas (EACs) and endometrial malignancies have many commonalities yet their pathophysiology, particularly those with high-risk histology such as clear cell, is understudied. EACs are histologically and clinically distinct from other gynecological cancers due to their unique origin within endometriosis. The endometriotic microenvironment is uniquely hypoxic with high levels of iron due to recurrent menstrual cycling, and this iron-rich microenvironment has been linked to promoting carcinogenesis through iron-induced oxidative stress. However, the mechanism by which the endometriotic microenvironment contributes to EAC tumorigenesis and how this compares with endometrial cancer is unknown. Most of our current knowledge of EACs is built on either epidemiologic analyses or correlative studies. Additionally, most studies of the endometriotic microenvironment primarily focus on the epithelial component while the role of the stroma has been largely unexplored. Mesenchymal stem cells (MSC) are a critical component of the stromal microenvironment. Recently, we identified a key role of endometriosis derived MSCs (enMSCs) in altering ovarian clear cell carcinoma iron regulation. Interestingly, enMSCs resemble the primary endometrium derived MSCs, which suggested their role in endometrial cancer. This stromal subset is characterized by the loss of surface CD10 expression. It is known that cancer cells, particularly clear cell carcinoma (CCC), have a unique requirement for high levels of iron. We hypothesize that CD10 negative enMSCs promote growth of EAC and endometrial cancer through iron regulation. MSCs were isolated from primary human benign endometriosis, benign endometrium, or endometrial cancer. Flow cytometry was used to measure surface CD10 expression. We investigated the role of CD10 negative enMSCs on CCC iron regulation. Our results showed that CD10 negative enMSCs promote CCC growth by exporting iron through the upregulation of Hephaestin (HEPH) and Ferroportin (FPO) which then led to increase labile iron pool (LIP) in CCC cells. However, increasing the LIP does not occur with iron supplementation alone, this indicates the critical role of CD10 negative enMSCs in regulating iron handling in CCC cells. Additionally, our data showed that CD10 negative enMSCs alter the balance of Ferritin L and Ferritin H- the main iron storage proteins- in CCC cells. Mechanistically, our preliminary data showed that CD10 negative enMSCs directly donate Ferritin L into CCC cells. Further, CD10 negative enMSCs enhance Ferritin H degradation through the upregulation of ferritinophagy in CCC cells. In conclusion, our results indicate there is a sub-population of enMSCs, marked by the loss of CD10 expression, that enhances EAC and endometrial cancer cell growth through iron regulation. This highlights the existence of a tumor-promoting stromal cell within both the endometrium and endometriosis which can be capitalized on as an “Achilles' heel” to uncover novel treatment targets for EAC and endometrial cancer. Citation Format: Huda I. Atiya, Lan G. Coffman. Role of stromal CD10 expression in progression of endometrial and endometriosis-associated cancers [abstract]. In: Proceedings of the AACR Special Conference on Endometrial Cancer: Transforming Care through Science; 2023 Nov 16-18; Boston, Massachusetts. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(5_Suppl):Abstract nr A014.

Background: LncRNA DLX6-AS1 has been uncovered to exert effects on various cancers. Nevertheless, the impacts of DLX6-AS1 on endometrial cancer (EC) development remained obscure. The study explored the influence of DLX6-AS1 on EC progression via the microRNA (miR)-374a-3p/zinc-finger protein (ZFX) axis.Methods: EC cell lines were collected and DLX6-AS1, miR-374a-3p, and ZFX levels in EC cell lines were detected. The EC cells were transfected with DLX6-AS1, miR-374a-3p, and ZFX constructs to examine the biological functions of EC cells. The xenograft model was established for detecting tumor growth. Rescue experiments were conducted to verify the interaction of DLX6-AS1, miR-374a-3p, and ZFX in EC cells.Results: DLX6-AS1 and ZFX levels were elevated, while miR-374a-3p exhibited a reduced level in EC cells. Silencing DLX6-AS1 and elevated miR-374a-3p expressions repressed the biological activities of EC cells. Reduced DLX6-AS1 repressed tumor development. MiR-374a-3p silencing reversed the impacts of DLX6-AS1 silencing, while ZFX overexpression abrogated the impacts of miR-374a-3p elevation on EC cell growth. Mechanically, DLX6-AS1 was found to bind to miR-374a-3p, and miR-374a-3p targeted ZFX.Conclusion: DLX6-AS1 depletion restricts the malignant phenotype of EC cells. The study might provide novel therapeutic biomarkers for EC treatment.

Endometrial cancer is the most common malignancy of the female genital tract. However, in spite of a huge advance in our understanding of endometrial cancer biology, therapeutic modalities haven't significantly changed over the past 40 years. Recent studies have indicated that endoplasmic reticulum stress, the unfolded protein response activation and altered GRP78 expression can play an important role in a variety of tumors development and progression. Our previous studies reported for the first time that GRP78 is increased in endometrial tumors. In this study, we further analyzed the role of UPR and GRP78 in endometrial cancer progression. We found that GRP78 plays a role in endometrial cancer progression since its silencing attenuate both the growth and invasion of endometrial cancer cells. Interestingly, we also show that metformin, an antidiabetic drug with anticancer properties, is able to inhibit endometrial cancer cells growth and this is accompanied by the inhibition of GRP78 expression and upregulation of proapoptotic UPR genes such as ATF4 and CHOP. Finally, we describe that metformin affects β-catenin signaling, a frequently activated signaling pathway in endometrial cancer. These observations highlight the possibility that GRP78 might represent an intriguing therapeutic target of metformin action in the treatment of endometrial cancer.

An accumulation of evidence has demonstrated that abnormal microRNA (miRNA or miR) expression is associated with different types of cancer, including endometrial cancer (EC). The dysregulation of miRNAs may serve important roles in the development and progression of EC by regulating multiple aggressive biological behaviors, including cell proliferation, apoptosis, metastasis and angiogenesis. An in-depth understanding of the miRNAs associated with EC initiation and progression may be crucial for identifying successful therapeutic techniques. miR-873 has been demonstrated to be dysregulated in different types of cancer. However, the expression status and regulatory roles of miR-873 are yet to be elucidated in EC. In the present study, reverse transcription-quantitative PCR was carried out to detect miR-873 expression in EC tissues and cell lines. Cell Counting Kit-8 and in vitro invasion assays were utilized to determine the influence of miR-873 on the proliferation and invasion of EC cells. miR-873 expression was revealed to be downregulated in EC tissues and cell lines. Decreased miR-873 expression was significantly associated with International Federation of Gynecology and Obstetrics stage and lymph node metastasis of patients with EC. Functional assays revealed that resumed miR-873 expression suppressed the proliferation and invasion of EC cells. Additionally, hepatoma-derived growth factor (HDGF) was indicated to be a direct target gene of miR-873 in EC cells. HDGF was highly expressed in EC tissues and inversely correlated with miR-873 expression. HDGF silencing also imitated the tumor-suppressor activity of miR-873 overexpression in EC cells. A series of rescue experiments identified that recovered HDGF expression hindered the anti-proliferative and anti-invasive roles of miR-873 upregulation in EC cells. In conclusion, the present study indicated that miR-873 serves an important role as a tumor suppressor in EC development by directly targeting HDGF. The results may provide a novel insight into clinical treatments, which can be used to prevent EC aggression.