SKP2 regulates ZEB1 expression and stimulates eutopic endometrial stromal cell invasion and proliferation of adenomyosis

Though endometriosis is benign, however, it shares certain characteristics with cancers, such as the ability to invade and metastasize. Previous studies have demonstrated that S-phase kinase associated protein2 (SKP2) promotes invasion, tumorigenesis, and metastasis. However, its correlation with adenomyosis is unclear. Herein, we aimed to look into SKP2 expression patterns and regulatory effects on endometrial stromal cell (ESC) proliferation and invasion, and its internal mechanism in adenomyosis. Western blot, qRT-PCR, and immunochemistry were carried out for detecting SKP2 and ZEB1 expression in ESC of adenomyosis and adenomyosis endometrial tissue. The primary ESCs were identified using immunofluorescence. SKP2 knockdown was accomplished in vitro by transfecting a particular lentivirus vector. The colony formation and CCK-8 assays were carried out for assessing cell proliferation, while cell invasion potential was assessed using the transwell assay. Both SKP2 and ZEB1 were found to be significantly upregulated in adenomyosis endometrial tissue. Knockdown of SKP2 inhibited adenomyotic ESC invasion and proliferation. Further experiments showed that knocking out SKP2 reduced ZEB1 expression in adenomyotic ESCs. Our results showed that SKP2 could regulate ZEB1 expression, and increased SKP2 may play a role in the pathogenesis of adenomyosis and stimulating ESC proliferation and invasion.     

Hypoxia induced‐disruption of lncRNA TUG1/PRC2 interaction impairs human trophoblast invasion through epigenetically activating Nodal/ALK7 signalling

Inadequate trophoblastic invasion is considered as one of hallmarks of preeclampsia (PE), which is characterized by newly onset of hypertension (>140/90 mmHg) and proteinuria (>300 mg in a 24‐h urine) after 20 weeks of gestation. Accumulating evidence has indicated that long noncoding RNAs are aberrantly expressed in PE, whereas detailed mechanisms are unknown. In the present study, we showed that lncRNA Taurine upregulated 1 (TUG1) were downregulated in preeclamptic placenta and in HTR8/SVneo cells under hypoxic conditions, together with reduced enhancer of zeste homolog2 (EZH2) and embryonic ectoderm development (EED) expression, major components of polycomb repressive complex 2 (PRC2), as well as activation of Nodal/ALK7 signalling pathway. Mechanistically, we found that TUG1 bound to PRC2 (EZH2/EED) in HTR8/SVneo cells and weakened TUG1/PRC2 interplay was correlated with upregulation of Nodal expression via decreasing H3K27me3 mark at the promoter region of Nodal gene under hypoxic conditions. And activation of Nodal signalling prohibited trophoblast invasion via reducing MMP2 levels. Overexpression of TUG1 or EZH2 significantly attenuated hypoxia‐induced reduction of trophoblastic invasiveness via negative modulating Nodal/ALK7 signalling and rescuing expression of its downstream target MMP2. These investigations might provide some evidence for novel mechanisms responsible for inadequate trophoblastic invasion and might shed some light on identifying future therapeutic targets for PE.     

Curcumin inhibits the growth of triple‐negative breast cancer cells by silencing EZH2 and restoring DLC1 expression

Enhancer of zeste homolog 2 (EZH2), an oncogene, is a commonly up‐regulated epigenetic factor in human cancer. Hepatocellular carcinoma deletion gene 1 (DLC1) is an antioncogene that is either expressed at low levels or not expressed in many malignant tumours. Curcumin is a promising anticancer drug that has antitumour effects in many tumours, but its mechanism of action is unclear. Our research demonstrated that EZH2 was up‐regulated in breast cancer (BC) tissues and cells, whereas DLC1 was down‐regulated, and the expression of EZH2 and DLC1 was negatively correlated in BC. By analysing the characteristics of clinical cases, we found that positive expression of EZH2 and negative expression of DLC1 may be predictors of poor prognosis in patients with triple‐negative breast cancer (TNBC). Moreover, knockdown of EZH2 expression restored the expression of DLC1 and inhibited the migration, invasion and proliferation, promoted the apoptosis, and blocked the cell cycle of MDA‐MB‐231 cells. Furthermore, we found that curcumin restored the expression of DLC1 by inhibiting EZH2; it also inhibited the migration, invasion and proliferation of MDA‐MB‐231 cells, promoted their apoptosis and blocked the cell cycle. Finally, xenograft tumour models were used to demonstrate that curcumin restored DLC1 expression by inhibiting EZH2 and also inhibited the growth and promoted the apoptosis of TNBC cells. In conclusion, our results suggest that curcumin can inhibit the migration, invasion and proliferation, promote the apoptosis, block the cycle of TNBC cells and restore the expression of DLC1 by inhibiting the expression of EZH2.     

Retracted: Upregulation of long noncoding RNA TUG1 contributes to the development of laryngocarcinoma by targeting miR-145-5p/ROCK1 axis

Laryngocarcinoma is the most common head and neck cancer and has a high incidence and mortality, causing about 83 000 deaths per year worldwide. Our research aimed to investigate the possible role of long noncoding RNA (lncRNA) taurine upregulated gene 1 (TUG1) in laryngocarcinoma development. The messenger RNA (mRNA) levels of TUG1 in tumor tissues and control (plasma) samples of laryngocarcinoma patients as well as in laryngocarcinoma cells were detected. The influences of TUG1 suppression on cell biological processes (viability, apoptosis, migration, and invasion) and cytoskeleton rearrangement in laryngocarcinoma cells were tested. Moreover, we investigated the regulatory interaction between TUG1 and miR-145-5p, and identified the target gene of miR-145-5p. The association between TUG1 and the protein expressions of RhoA/rho associated coiled-coil containing protein kinase (ROCK)/matrix metalloproteinases (MMPs) pathway-associated factors were detected. TUG1 was found to be highly expressed in tumor tissues and plasma samples of laryngocarcinoma patients as well as in laryngocarcinoma cells. Suppression of TUG1 decreased laryngocarcinoma cell viability, increased apoptosis, and suppression migration, invasion, and cytoskeleton rearrangement. Moreover, TUG1 negatively regulated miR-145-5p. TUG1 regulated tumor growth (viability and apoptosis) and metastasis through miR-145-5p. Furthermore, ROCK1 was targeted by miR-145-5p, and miR-145-5p/ROCK1 partner was involved in the process of tumor growth and metastasis. Finally, we found that TUG1 functioned on laryngocarcinoma by activating RhoA/ROCK/MMPs pathway. Our study reveals that lncRNA TUG1 is upregulated in laryngocarcinoma and may be involved in the process of laryngocarcinoma through miR-145-5p downregulation and activating the RhoA/ROCK/MMPs signals.     

Oestrogen‐induced angiogenesis promotes adenomyosis by activating the Slug‐VEGF axis in endometrial epithelial cells

Adenomyosis is an oestrogen‐dependent disease characterized by the invasion of endometrial epithelial cells into the myometrium of uterus, and angiogenesis is thought to be required for the implantation of endometrial glandular tissues during the adenomyotic pathogenesis. In this study, we demonstrate that compared with eutopic endometria, adenomyotic lesions exhibited increased vascularity as detected by sonography. Microscopically, the lesions also exhibited an oestrogen‐associated elevation of microvascular density and VEGF expression in endometrial epithelial cells. We previously reported that oestrogen‐induced Slug expression was critical for endometrial epithelial–mesenchymal transition and development of adenomyosis. Our present studies demonstrated that estradiol (E2) elicited a Slug‐VEGF axis in endometrial epithelial cells, and also induced pro‐angiogenic activity in vascular endothelial cells. The antagonizing agents against E2 or VEGF suppressed endothelial cells migration and tubal formation. Animal experiments furthermore confirmed that blockage of E2 or VEGF was efficient to attenuate the implantation of adenomyotic lesions. These results highlight the importance of oestrogen‐induced angiogenesis in adenomyosis development and provide a potential strategy for treating adenomyosis through intercepting the E2‐Slug‐VEGF pathway.     

Three-dimensional culture sensitizes epithelial ovarian cancer cells to EZH2 methyltransferase inhibition

Inhibitors of EZH2 methyltransferase activity have been demonstrated to selectively suppress the growth of diffused large B cell lymphoma (DLBCL) cells with gain-of-function mutations in EZH2, while exhibiting very limited effects on the growth of DLBCL cells with wild-type EZH2. Given that EZH2 is often overexpressed but not mutated in solid tumors, it is important to investigate the determinants of sensitivity of solid tumor cells to EZH2 inhibitors. In the current study, we show that three-dimensional (3D) culture of epithelial ovarian cancer (EOC) cells that overexpress EZH2 sensitizes these cells to EZH2 methyltransferase inhibition. Treatment of EOC cells with GSK343, a specific inhibitor of EZH2 methyltransferase, decreases the level of H3K27Me3, the product of EZH2’s enzymatic activity. However, GSK343 exhibited limited effects on the growth of EOC cells in conventional two-dimensional (2D) culture. In contrast, GSK343 significantly suppressed the growth of EOC cells cultured in 3D matrigel extracellular matrix (ECM), which more closely mimics the tumor microenvironment in vivo. Notably, GSK343 induces apoptosis of EOC cells in 3D but not 2D culture. In addition, GSK343 significantly inhibited the invasion of EOC cells. In summary, we show that the 3D ECM sensitizes EOC cells to EZH2 methyltransferase inhibition, which suppresses cell growth, induces apoptosis and inhibits invasion. Our findings imply that in EZH2 wild-type solid tumors, the ECM tumor microenvironment plays an important role in determining sensitivity to EZH2 inhibition and suggest that targeting the ECM represents a novel strategy for enhancing EZH2 inhibitor efficacy.     

Proteomics identification of annexin A2 as a key mediator in the metastasis and proangiogenesis of endometrial cells in human adenomyosis

Adenomyosis is a common estrogen-dependent disorder of females characterized by a downward extension of the endometrium into the uterine myometrium and neovascularization in ectopic lesions. It accounts for chronic pelvic pain, dysmenorrhea, menorrhagia, and infertility in 8.8-61.5% women worldwide. However, the molecular mechanisms for adenomyosis development remain poorly elucidated. Here, we utilized a two-dimensional polyacrylamide gel electrophoresis/MS-based proteomics analysis to compare and identify differentially expressed proteins in matched ectopic and eutopic endometrium of adenomyosis patients. A total of 93 significantly altered proteins were identified by tandem MS analysis. Further cluster analysis revealed a group of estrogen-responsive proteins as dysregulated in adenomyosis, among which annexin A2, a member of annexin family proteins, was found up-regulated most significantly in the ectopic endometrium of adenomyosis compared with its eutopic counterpart. Overexpression of ANXA2 was validated in ectopic lesions of human adenomyosis and was found to be tightly correlated with markers of epithelial to mesenchymal transition and dysmenorrhea severity of adenomyosis patients. Functional analysis demonstrated that estrogen could remarkably up-regulate ANXA2 and induce epithelial to mesenchymal transition in an in vitro adenomyosis model. Enforced expression of ANXA2 could mediate phenotypic mesenchymal-like cellular changes, with structural and functional alterations in a β-catenin/T-cell factor (Tcf) signaling-associated manner, which could be reversed by inhibition of ANXA2 expression. We also proved that enforced expression of ANXA2 enhanced the proangiogenic capacity of adenomyotic endometrial cells through HIF-1α/VEGF-A pathway. In vivo, we demonstrated that ANXA2 inhibition abrogated endometrial tissue growth, metastasis, and angiogenesis in an adenomyosis nude mice model and significantly alleviated hyperalgesia. Taken together, our data unraveled a dual role for ANXA2 in the pathogenesis of human adenomyosis through conferring endometrial cells both metastatic potential and proangiogenic capacity, which could serve as a potential therapeutic target for the treatment of adenomyosis patients.     

Overexpression of MYC and EZH2 cooperates to epigenetically silence MST1 expression

Hippo-like MST1 protein kinase regulates cell growth, organ size, and carcinogenesis. Reduction or loss of MST1 expression is implicated in poor cancer prognosis. However, the mechanism leading to MST1 silencing remains elusive. Here, we report that both MYC and EZH2 function as potent suppressors of MST1 expression in human prostate cancer cells. We demonstrated that concurrent overexpression of MYC and EZH2 correlated with the reduction or loss of MST1 expression, as shown by RT-qPCR and immunoblotting. Methylation sensitive PCR and bisulfite genomic DNA sequencing showed that DNA methylation caused MST1 silencing. Pharmacologic and RNAi experiments revealed that MYC and EZH2 silenced MST1 expression by inhibiting its promoter activity, and that EZH2 was a mediator of the MYC-induced silencing of MST1. In addition, MYC contributed to MST1 silencing by partly inhibiting the expression of microRNA-26a/b, a negative regulator of EZH2. As shown by ChIP assays, EZH2-induced DNA methylation and H3K27me3 modification, which was accompanied by a reduced H3K4me3 mark and RNA polymerase II occupancy on the MST1 promoter CpG region, were the underlying cause of MST1 silencing. Moreover, potent pharmacologic inhibitors of MYC or EZH2 suppressed prostate cancer cell growth in vitro, and the knockdown of MST1 caused cells’ resistance to MYC and EZH2 inhibitor-induced growth retardation. These findings indicate that MYC, in concert with EZH2, epigenetically attenuates MST1 expression and suggest that the loss of MST1/Hippo functions is critical for the MYC or EZH2 mediation of cancer cell survival.     

Overexpression of MYC and EZH2 cooperates to epigenetically silence MST1 expression

Hippo-like MST1 protein kinase regulates cell growth, organ size, and carcinogenesis. Reduction or loss of MST1 expression is implicated in poor cancer prognosis. However, the mechanism leading to MST1 silencing remains elusive. Here, we report that both MYC and EZH2 function as potent suppressors of MST1 expression in human prostate cancer cells. We demonstrated that concurrent overexpression of MYC and EZH2 correlated with the reduction or loss of MST1 expression, as shown by RT-qPCR and immunoblotting. Methylation sensitive PCR and bisulfite genomic DNA sequencing showed that DNA methylation caused MST1 silencing. Pharmacologic and RNAi experiments revealed that MYC and EZH2 silenced MST1 expression by inhibiting its promoter activity, and that EZH2 was a mediator of the MYC-induced silencing of MST1. In addition, MYC contributed to MST1 silencing by partly inhibiting the expression of microRNA-26a/b, a negative regulator of EZH2. As shown by ChIP assays, EZH2-induced DNA methylation and H3K27me3 modification, which was accompanied by a reduced H3K4me3 mark and RNA polymerase II occupancy on the MST1 promoter CpG region, were the underlying cause of MST1 silencing. Moreover, potent pharmacologic inhibitors of MYC or EZH2 suppressed prostate cancer cell growth in vitro, and the knockdown of MST1 caused cells’ resistance to MYC and EZH2 inhibitor-induced growth retardation. These findings indicate that MYC, in concert with EZH2, epigenetically attenuates MST1 expression and suggest that the loss of MST1/Hippo functions is critical for the MYC or EZH2 mediation of cancer cell survival.     

EZH2 promotes invasion and tumour glycolysis by regulating STAT3 and FoxO1 signalling in human OSCC cells

The enhancer of zeste homolog 2 (EZH2), known as a member of the polycomb group (PcG) proteins, is an oncogene overexpressed in a variety of human cancers. Here, we found that EZH2 correlated with poor survival of oral squamous cell carcinoma (OSCC) patients using immunohistochemistry staining. EZH2 overexpression led to a significant induction in tumour glycolysis, Epithelial‐mesenchymal transition (EMT), migration and invasion of OSCC cells. Conversely, silencing of EZH2 inhibited tumour glycolysis, EMT, migration and invasion in OSCC cells. Ectopic overexpression of EZH2 increased phosphorylation of STAT3 at pY705 and decreased FoxO1 expression, and FoxO1 expression was enhanced when inhibiting STAT3. In addition, EZH2 overexpression led to a significant decrease in FoxO1 mRNA levels in nude mice xenograft. These results indicated that regulation of EZH2 might have the potential to be targeted for OSCC treatment.     

Upregulated lncRNA UCA1 inhibits trophoblast cell invasion and proliferation by downregulating JAK2

Numerous studies have suggested that urothelial cancer-associated 1 (UCA1) acts as a suppressor gene affecting cell proliferation and migration. However, the biological role and the potential mechanism of UCA1 in the progression of pre-eclampsia (PE) remains unclear. The UCA1 level was markedly upregulated in PE pregnancies relative to non-PE ones in GSE75010 and tissues. A higher body mass index (BMI), maximum systolic blood pressure (BP), and maximum diastolic BP were observed in PE pregnancies, whereas the newborn weight z-score was lower compared with those of non-PE pregnancies. Knockdown of UCA1 accelerated the proliferative migratory abilities and cell cycle progression, but inhibited apoptosis of HTR-8/SVneo and JAR cells. Then, we found that Janus kinases 2 (JAK2) was negatively correlated with UCA1. In addition, JAK2 was downregulated in the placenta of PE pregnancies and was negatively regulated by UCA1. UCA1 was mainly enriched in the nucleus. Knockdown of UCA1 reduced the occupancies of the enhancer of zeste 2 polycomb repressive complex 2 subunit (EZH2) and H3K27me3 on the Janus kinase 2 (JAK2) promoter regions. Finally, rescue experiments found that transfection of short-hairpin JAK2 attenuated proliferative and migratory abilities of trophoblasts, which were partially reversed after UCA1 knockdown. In short, UCA1 is upregulated in the trophocytes of PE pregnancies and accelerates trophoblast cell invasion and proliferation by downregulating JAK2.     

Enhancer of zeste homolog 2 promotes the proliferation and invasion of epithelial ovarian cancer cells

Enhancer of zeste homolog 2 (EZH2) is the catalytic subunit of the polycomb repressive complex 2 (PRC2) that includes noncatalytic subunits suppressor of zeste 12 (SUZ12) and embryonic ectoderm development (EED). When present in PRC2, EZH2 catalyzes trimethylation on lysine 27 residue of histone H3 (H3K27Me3), resulting in epigenetic silencing of gene expression. Here, we investigated the expression and function of EZH2 in epithelial ovarian cancer (EOC). When compared with primary human ovarian surface epithelial (pHOSE) cells, EZH2, SUZ12, and EED were expressed at higher levels in all 8 human EOC cell lines tested. Consistently, H3K27Me3 was also overexpressed in human EOC cell lines compared with pHOSE cells. EZH2 was significantly overexpressed in primary human EOCs (n = 134) when compared with normal ovarian surface epithelium (n = 46; P < 0.001). EZH2 expression positively correlated with expression of Ki67 (P < 0.001; a marker of cell proliferation) and tumor grade (P = 0.034) but not tumor stage (P = 0.908) in EOC. There was no correlation of EZH2 expression with overall (P = 0.3) or disease-free survival (P = 0.2) in high-grade serous histotype EOC patients (n = 98). Knockdown of EZH2 expression reduced the level of H3K27Me3 and suppressed the growth of human EOC cells both in vitro and in vivo in xenograft models. EZH2 knockdown induced apoptosis of human EOC cells. Finally, we showed that EZH2 knockdown suppressed the invasion of human EOC cells. Together, these data demonstrate that EZH2 is frequently overexpressed in human EOC cells and its overexpression promotes the proliferation and invasion of human EOC cells, suggesting that EZH2 is a potential target for developing EOC therapeutics.     

Long non-coding RNA TUG1/microRNA-187-3p/TESC axis modulates progression of pituitary adenoma via regulating the NF-κB signaling pathway

The molecule mechanisms of long non-coding RNAs (lncRNAs) and microRNAs (miRNAs) in human diseases have been broadly studied recently, therefore, our research aimed to assess the effect of lncRNA taurine upregulated gene 1 (TUG1)/miR-187-3p/tescalcin (TESC) axis in pituitary adenoma (PA) by regulating the nuclear factor-kappa B (NF-κB) signaling pathway. We observed that TUG1 was upregulated in PA tissues and was associated with invasion, knosp grade and tumor size. TUG1 particularly bound to miR-187-3p. TUG1 knockdown inhibited cell proliferation, invasion, migration, and epithelial–mesenchymal transition, promoted apoptosis, and regulated the expression of NF-κB p65 and inhibitor of κB (IκB)-α in PA cells lines in vitro, and also inhibited tumor growth in vivo, and these effects were reversed by miR-187-3p reduction. Similarly, miR-187-3p elevation inhibited PA cell malignant behaviors and modulated the expression of NF-κB p65 and IκB-α in PA cells, and reduced in vivo tumor growth as well. TUG1 inhibition downregulated TESC, which was targeted by miR-187-3p. In conclusion, this study suggests that TUG1 sponges miR-187-3p to affect PA development by elevating TESC and regulating the NF-κB signaling pathway.Subject terms: Cell biology, Diseases     

NUSAP1 knockdown inhibits cell growth and metastasis of non-small-cell lung cancer through regulating BTG2/PI3K/Akt signaling

Non-small-cell lung cancer (NSCLC) is the most common malignancy along with high mortality rate worldwide. Recently, nucleolar and spindle-associated protein 1 (NUSAP1) has been reported to be involved in the malignant progression of several cancers. However, in NSCLC, the biological function of NUSAP1 and its molecular mechanism have not been reported. Here, our findings indicated that the NUSAP1 messenger RNA expression level was remarkably upregulated in NSCLC tissues compared with that of adjacent normal tissues. We also found that NUSAP1 gene expression was notably upregulated in NSCLC cell lines (A549, 95-D, H358, and H1299) compared with that of normal human bronchial epithelial cell line (16HBE). Subsequently, the biological function of NUSAP1 was investigated in A549 and H358 cells transfected with NUSAP1 small interfering RNA (siRNA), respectively. Results showed that NUSAP1 knockdown inhibited NSCLC cell proliferation, and promoted cell apoptosis. Furthermore, the number of cell migration and invasion was significantly suppressed by NUSAP1 knockdown. In addition, our results indicated that NUSAP1 knockdown increased the gene expression of B-cell translocation gene 2 (BTG2), but decreased the expression levels of phosphoinositide 3-kinase (PI3K) and phosphorylated serine/threonine kinase (p-AKT). BTG2 siRNA partly abrogates the effect of NUSAP1 knockdown on BTG2 gene expression. Fumonisin B1 (FB1), a AKT activator, reversed the effect of NUSAP1 knockdown on the biological function in NSCLC. Taken together, NUSAP1 knockdown promotes NSCLC cell apoptosis, and inhibits cell proliferation, cell migration, and invasion, which is associated with regulating BTG2/PI3K/Akt signal pathway. Our findings suggest that NUSAP1 is a promising molecular target for NSCLC treatment.