PRC2/EED-EZH2 Complex Is Up-Regulated in Breast Cancer Lymph Node Metastasis Compared to Primary Tumor and Correlates with Tumor Proliferation In Situ

Background

Lymph node metastasis is a key event in the progression of breast cancer. Therefore it is important to understand the underlying mechanisms which facilitate regional lymph node metastatic progression.

Methodology/Principal Findings

We performed gene expression profiling of purified tumor cells from human breast tumor and lymph node metastasis. By microarray network analysis, we found an increased expression of polycomb repression complex 2 (PRC2) core subunits EED and EZH2 in lymph node metastatic tumor cells over primary tumor cells which were validated through real-time PCR. Additionally, immunohistochemical (IHC) staining and quantitative image analysis of whole tissue sections showed a significant increase of EZH2 expressing tumor cells in lymph nodes over paired primary breast tumors, which strongly correlated with tumor cell proliferation in situ. We further explored the mechanisms of PRC2 gene up-regulation in metastatic tumor cells and found up-regulation of E2F genes, MYC targets and down-regulation of tumor suppressor gene E-cadherin targets in lymph node metastasis through GSEA analyses. Using IHC, the expression of potential EZH2 target, E-cadherin was examined in paired primary/lymph node samples and was found to be significantly decreased in lymph node metastases over paired primary tumors.

Conclusions/Significance

This study identified an over expression of the epigenetic silencing complex PRC2/EED-EZH2 in breast cancer lymph node metastasis as compared to primary tumor and its positive association with tumor cell proliferation in situ. Concurrently, PRC2 target protein E-cadherin was significant decreased in lymph node metastases, suggesting PRC2 promotes epithelial mesenchymal transition (EMT) in lymph node metastatic process through repression of E-cadherin. These results indicate that epigenetic regulation mediated by PRC2 proteins may provide additional advantage for the outgrowth of metastatic tumor cells in lymph nodes. This opens up epigenetic drug development possibilities for the treatment and prevention of lymph node metastasis in breast cancer.     

Epigenetic silencing of genes enhanced by collective role of reactive oxygen species and MAPK signaling downstream ERK/Snail axis: Ectopic application of hydrogen peroxide repress CDH1 gene by enhanced DNA methyltransferase activity in human breast cancer

Loss of E-cadherin and epithelial to mesenchymal transition (EMT) are key steps in cancer progression. Reactive oxygen species (ROS) play significant roles in cellular physiology and homeostasis. Roles of E-cadherin (CDH1), EMT and ROS are intriguingly illustrated in many cancers without focusing their collective concert during cancer progression. We report that hydrogen peroxide (H₂O₂) treatment modulate CDH1 gene expression by epigenetic modification(s). Sublethal dosage of H₂O₂ treatment decrease E-cadherin, increase DNMT1, HDAC1, Snail, Slug and enrich H3K9me3 and H3K27me3 in the CDH1 promoter. The effect of H₂O₂ was attenuated by ROS scavengers; NAC, lupeol and beta-sitosterol. DNMT inhibitor, AZA prevented the H₂O₂ induced promoter-CpG-island methylation of CDH1. Treatment of cells with U0126 (inhibitor of ERK) reduced the expression of DNMT1, Snail and Slug, increased CDH1. This implicates that CDH1 is synergistically repressed by histone methylation, DNA methylation and histone deacetylation mediated chromatin remodelling and activation of Snail and Slug through ERK pathway. Increased ROS leads to activation of epigenetic machineries and EMT activators Snail/Slug which in their course of action inactivates CDH1 gene and lack of E-cadherin protein promotes EMT in breast cancer cells. ROS and ERK signaling facilitate epigenetic silencing and support the fact that subtle increase of ROS above basal level act as key cell signaling molecules. Free radical scavengers, lupeol and beta-sitosterol may be tested for therapeutic intervention of breast cancer. This work broadens the amplitude of epigenome and open avenues for investigations on conjoint effects of canonical and intrinsic metabolite signaling and epigenetic modulations in cancer.     

FOXO3a inhibits the EMT and metastasis of breast cancer by regulating TWIST-1 mediated miR-10b/CADM2 axis

BackgroundBreast cancer is the most common malignancy and has been considered as a leading cause of cancer death in women. Exploring the mechanism of breast cancer metastasis is extremely important for seeking novel therapeutic strategies and improving prognosis.MethodsClinical specimens and pathological characteristics were collected for evaluating the expression of forkhead box class O 3a (FOXO3a) and twist-related protein 1 (TWIST-1) in breast cancer tissues. CCK-8 assay was used to analyze cell proliferation. Cell invasion and migration were assessed by transwell assays. The expression of FOXO3a, TWIST-1, miR-10b, CADM2, FAK, phosphor-AKT and the epithelial-mesenchymal transition (EMT)-related protein (N-cadherin, E-cadherin and vimentin) were analyzed by RT-qPCR, immunohistochemical staining, immunofluorescence assay or western blot, respectively. Xenograft mouse models were used to analyze the role of the FOXO3a in breast cancer.ResultsFOXO3a was down-regulated and TWIST-1 was up-regulated in breast cancer tissues. Overexpression of FOXO3a or knockdown of TWIST-1 suppressed the proliferation, invasion, migration and EMT of breast cancer cells. Overexpression of TWIST-1 could reverse the effect of FOXO3a on the proliferation, invasion, migration and EMT of breast cancer. Moreover, FOXO3a suppressed the growth and metastasis of breast cancer by targeting TWIST1 in vivo.ConclusionFOXO3a inhibited the EMT and metastasis of breast cancer via TWIST-1/miR-10b/CADM2 axis.     

Histone Demethylase KDM6B Promotes Epithelial-Mesenchymal Transition

Epithelial-mesenchymal transition (EMT) is a critical event that occurs in embryonic development, tissue repair control, organ fibrosis, and carcinoma invasion and metastasis. Although significant progress has been made in understanding the molecular regulation of EMT, little is known about how chromatin is modified in EMT. Chromatin modifications through histone acetylation and methylation determine the precise control of gene expression. Recently, histone demethylases were found to play important roles in gene expression through demethylating mono-, di-, or trimethylated lysines. KDM6B (also known as JMJD3) is a histone demethylase that might activate gene expression by removing repressive histone H3 lysine 27 trimethylation marks from chromatin. Here we report that KDM6B played a permissive role in TGF-β-induced EMT in mammary epithelial cells by stimulating SNAI1 expression. KDM6B was induced by TGF-β, and the knockdown of KDM6B inhibited EMT induced by TGF-β. Conversely, overexpression of KDM6B induced the expression of mesenchymal genes and promoted EMT. Chromatin immunoprecipitation (ChIP) assays revealed that KDM6B promoted SNAI1 expression by removing histone H3 lysine trimethylation marks. Consistently, our analysis of the Oncomine database found that KDM6B expression was significantly increased in invasive breast carcinoma compared with normal breast tissues. The knockdown of KDM6B significantly inhibited breast cancer cell invasion. Collectively, our study uncovers a novel epigenetic mechanism regulating EMT and tumor cell invasion, and has important implication in targeting cancer metastasis.     

CRH suppressed TGFβ1-induced Epithelial–Mesenchymal Transition via induction of E-cadherin in breast cancer cells

Since its discovery in biopsies from breast cancer patients, the effect of corticotropin-releasing hormone (CRH) on carcinoma progression is still unclear. Transforming growth factorβ1 (TGFβ1) promotes Epithelial–Mesenchymal Transition (EMT) and induces Snail1 and Twist1 expressions. Loss of epithelial cadherin (E-cadherin) mainly repressed by Snail1 and Twist1, has been considered as hallmark of Epithelial–Mesenchymal Transition (EMT). Two breast cancer cell lines, MCF-7 and MDA-MB-231 were used to investigate the effect of CRH on TGFβ1-induced EMT by transwell chamber. And HEK293 cells were transiently transfected with CRHR1 or CRHR2 to explore the definite effects of CRH receptor. We reported that CRH inhibited migration of human breast cancer cells through downregulation of Snail1 and Twist1, and subsequent upregulation of E-cadherin. CRH inhibited TGFβ1-mediated migration of MCF-7 via both CRHR1 and CRHR2 while this inhibition in MDA-MB-231 was mainly via CRHR2. Ectopic re-expression of CRHR1 or CRHR2 respectively in HEK293 cells increased E-cadherin expression after CRH stimulation. Furthermore, CRH repressed expression of mesenchymal marker, N-cadherin and induced expression of Occludin, inhibiting EMT in MCF-7 & MDA-MB-231. Our results suggest that CRH may function as a tumor suppressor, at least partly by regulating TGFβ1-mediated EMT. These results may contribute to uncovering the effect of CRH in breast tumorigenesis and progression.     

HIF-1α Promotes Epithelial-Mesenchymal Transition and Metastasis through Direct Regulation of ZEB1 in Colorectal Cancer

It is well recognized that hypoxia-inducible factor 1 alpha (HIF-1α) is involved in cancer metastasis, chemotherapy and poor prognosis. We previously found that deferoxamine, a hypoxia-mimetic agent, induces epithelial-mesenchymal transition (EMT) in colorectal cancer. Therefore, here we explored a new molecular mechanism for HIF-1α contributing to EMT and cancer metastasis through binding to ZEB1. In this study, we showed that overexpression of HIF-1α with adenovirus infection promoted EMT, cell invasion and migration in vitro and in vivo. On a molecular level, HIF-1α directly binding to the proximal promoter of ZEB1 via hypoxia response element (HRE) sites thus increasing the transactivity and expression of ZEB1. In addition, inhibition of ZEB1 was able to abrogate the HIF-1α-induced EMT and cell invasion. HIF-1α expression was highly correlated with the expression of ZEB1 in normal colorectal epithelium, primary and metastatic CRC tissues. Interestingly, both HIF-1α and ZEB1 were positively associated with Vimentin, an important mesenchymal marker of EMT, whereas negatively associated with E-cadherin expression. These findings suggest that HIF-1α enhances EMT and cancer metastasis by binding to ZEB1 promoter in CRC. HIF-1α and ZEB1 are both widely considered as tumor-initiating factors, but our results demonstrate that ZEB1 is a direct downstream of HIF-1α, suggesting a novel molecular mechanism for HIF-1α-inducing EMT and cancer metastasis.     

Celastrol inhibits TGF-β1-induced epithelial–mesenchymal transition by inhibiting Snail and regulating E-cadherin expression

The epithelial–mesenchymal transition (EMT) is a pivotal event in the invasive and metastatic potentials of cancer progression. Celastrol inhibits the proliferation of a variety of tumor cells including leukemia, glioma, prostate, and breast cancer; however, the possible role of celastrol in the EMT is unclear. We investigated the effect of celastrol on the EMT. Transforming growth factor-beta 1 (TGF-β1) induced EMT-like morphologic changes and upregulation of Snail expression. The downregulation of E-cadherin expression and upregulation of Snail in Madin–Darby Canine Kidney (MDCK) and A549 cell lines show that TGF-β1-mediated the EMT in epithelial cells; however, celastrol markedly inhibited TGF-β1-induced morphologic changes, Snail upregulation, and E-cadherin expression. Migration and invasion assays revealed that celastrol completely inhibited TGF-β1-mediated cellular migration in both cell lines. These findings indicate that celastrol downregulates Snail expression, thereby inhibiting TGF-β1-induced EMT in MDCK and A549 cells. Thus, our findings provide new evidence that celastrol suppresses lung cancer invasion and migration by inhibiting TGF-β1-induced EMT.     

The calcium pump PMCA4 prevents epithelial-mesenchymal transition by inhibiting NFATc1-ZEB1 pathway in gastric cancer

Epithelial-mesenchymal transition (EMT) is considered as the key mechanism involved in cancer metastasis. Several studies showed that various cell membrane calcium channels play different roles in cancer metastasis. In the present study, the potential role of ATPase plasma membrane Ca²⁺ transporting 4 (PMCA4) in regulating EMT in gastric cancer (GC) was investigated. GC patients who underwent radical surgery were enrolled in this study. In vitro human GC cell lines MKN45 and NCI-N87 were used, and MKN45 cells were injected in nude mice to evaluate tumor development. Our results showed that low PMCA4 expression was associated with advanced TNM stage and poor prognosis in GC patients. Knockdown of PMCA4 suppressed E-cadherin, grainyhead like 2 (GRHL2) and ovo-like 1 (OVOL1) expression, up-regulated vimentin expression, increased migration and invasion ability, and promoted the resistance to cytotoxic drug. Furthermore, GC cells displayed an elongated fibroblastoid morphology when PMCA4 was knockdown. PMCA4 overexpression resulted in an up-regulated E-cadherin expression and decreased migration and invasion ability. In vivo metastasis assay showed that PMCA4 overexpression resulted in a decreased incidence of lung metastasis. PMCA4 inhibition increased ZEB1 expression and nuclear accumulation of nuclear factor of activated T-cell isoform c1 (NFATc1). EMT induced by PMCA4 inhibition could be prevented by the knockdown of NFATc1 or ZEB1. In addition, cyclosporine A prevented EMT induced by PMCA4 inhibition by suppressing the NFATc1-ZEB1 pathway. Our data identified a novel mechanism in the regulation of EMT in GC, and provided a novel target in the treatment of EMT subtype in GC.     

Epithelial-mesenchymal transition and cancer stemness: the Twist1-Bmi1 connection

EMT (epithelial-mesenchymal transition), a major mechanism of cancer metastasis, is a process that generates cells with stem-like properties. These stem-like cells in tumours are described as cancer stem cells. The link between EMT and cancer stemness is well documented without detailed mechanistic proof. Bmi1 belongs to the PRC1 (polycomb repressive complex 1) maintaining self-renewal and stemness together with EZH2 (enhancer of zeste homologue 2), which is a component of PRC2. Bmi1 is frequently overexpressed in different types of human cancers. Recent demonstration of an EMT regulator, Twist1, directly regulating the expression of Bmi1 provides a mechanistic explanation of the relationship between EMT and cancer stemness. The functional interdependence between Twist1 and Bmi1 provides a fresh insight into the common mechanism mediating EMT and cancer stemness. This observation is also confirmed using head and neck cancer patient samples. These results provide a critical mechanism of Twist1-induced EMT and cancer stemness in cancer cells through chromatin remodelling. The role of hypoxia and microRNAs in regulating EMT and cancer stemness is also discussed.     

Inhibition of mTORC1 induces loss of E-cadherin through AKT/GSK-3β signaling-mediated upregulation of E-cadherin repressor complexes in non-small cell lung cancer cells

Background

mTOR, which can form mTOR Complex 1 (mTORC1) or mTOR Complex 2 (mTORC2) depending on its binding partners, is frequently deregulated in the pulmonary neoplastic conditions and interstitial lung diseases of the patients treated with rapalogs. In this study, we investigated the relationship between mTOR signaling and epithelial mesenchymal transition (EMT) by dissecting mTOR pathways.

Methods

Components of mTOR signaling pathway were silenced by shRNA in a panel of non-small cell lung cancer cell lines and protein expression of epithelial and mesenchymal markers were evaluated by immunoblotting and immunocytochemistry. mRNA level of the E-cadherin repressor complexes were evaluated by qRT-PCR.

Results

IGF-1 treatment decreased expression of the E-cadherin and rapamycin increased its expression, suggesting hyperactivation of mTOR signaling relates to the loss of E-cadherin. Genetic ablation of rapamycin-insensitive companion of mTOR (Rictor), a component of mTORC2, did not influence E-cadherin expression, whereas genetic ablation of regulatory-associated protein of mTOR (Raptor), a component of mTORC1, led to a decrease in E-cadherin expression at the mRNA level. Increased phosphorylation of AKT at Ser473 and GSK-3β at Ser9 were observed in the Raptor-silenced NSCLC cells. Of the E-cadherin repressor complexes tested, Snail, Zeb2, and Twist1 mRNAs were elevated in raptor-silenced A549 cells, and Zeb2 and Twist1 mRNAs were elevated in Raptor-silenced H2009 cells. These findings were recapitulated by treatment with the GSK-3β inhibitor, LiCl. Raptor knockdown A549 cells showed increased expression of N-cadherin and vimentin with mesenchymal phenotypic changes.

Conclusions

In conclusion, selective inhibition of mTORC1 leads to hyperactivation of the AKT/GSK-3β pathway, inducing E-cadherin repressor complexes and EMT. These findings imply the existence of a feedback inhibition loop of mTORC1 onto mTORC2 that plays a role in the homeostasis of E-cadherin expression and EMT, requiring caution in the clinical use of rapalog and selective mTORC1 inhibitors.