ABSTRACT: Grainyhead genes are involved in wound healing and developmental neural tube closure. In light of the high degree of similarity between the epithelial-mesenchymal transitions (EMTs) occurring in wound healing processes and the cancer stem cell-like compartment of tumors, including TGF-β-dependence, we investigated the role of a Grainyhead gene (GRHL2) in oncogenic EMT. Grainyhead was specifically down-regulated in the claudin-low subclass of mammary tumors and in the basal-B subclass of breast cancer cell lines. Functionally, GRHL2 suppressed TGF-β-induced, Twist-induced or spontaneous EMT, enhanced anoikis-sensitivity, and suppressed mammosphere generation in mammary epithelial cells. These effects were mediated, in part, by its suppression of ZEB1 expression, through direct repression of the ZEB1 promoter. GRHL2 also inhibited Smad-mediated transcription, and up-regulated mir200b/c as well as the TGF-β receptor antagonist, BMP2. The expression of GRHL2 in the breast cancer cell line MDA-MB-231 triggered a mesenchymal-to-epithelial transition and sensitized the cells to anoikis. These results indicate that GRHL2 is a suppressor of the oncogenic EMT. 3 biologic replicates for each cell line. Comparison of HMLE+Twist-ER cells expressing GRHL2/pMIG vs. HMLE+Twist-ER cells expressing empty pMIG.
SUBMITTER: Phillip M Pifer Philip RileySteven M FrischJoseph WidmeyerJoseph B AddisonAlexey V IvanovBenjamin CieplyJames Denvir
Project description:Grainyhead genes are involved in wound healing and developmental neural tube closure. In light of the high degree of similarity between the epithelial-mesenchymal transitions (EMT) occurring in wound-healing processes and the cancer stem cell-like compartment of tumors, including TGF-? dependence, we investigated the role of the Grainyhead gene, Grainyhead-like-2 (GRHL2) in oncogenic EMT. GRHL2 was downregulated specifically in the claudin-low subclass breast tumors and in basal-B subclass breast cancer cell lines. GRHL2 suppressed TGF-?-induced, Twist-induced or spontaneous EMT, enhanced anoikis sensitivity, and suppressed mammosphere generation in mammary epithelial cells. These effects were mediated in part by suppression of ZEB1 expression via direct repression of the ZEB1 promoter. GRHL2 also inhibited Smad-mediated transcription and it upregulated mir-200b/c as well as the TGF-? receptor antagonist, BMP2. Finally, ectopic expression of GRHL2 in MDA-MB-231 breast cancer cells triggered an MET and restored sensitivity to anoikis. Taken together, our findings define a major role for GRHL2 in the suppression of oncogenic EMT in breast cancer cells.
Project description:Epithelial-mesenchymal transition (EMT) in carcinoma cells enhances malignant progression by promoting invasion and survival. EMT is induced by microenvironmental factors, including TGF-? and Wnt agonists, and by the E-box-binding transcription factors Twist, Snail, and ZEB. Grainyhead-like-2 (GRHL2), a member of the mammalian Grainyhead family of wound-healing regulatory transcription factors, suppresses EMT and restores sensitivity to anoikis by repressing ZEB1 expression and inhibiting TGF-? signaling. In this study, we elucidate the functional relationship between GRHL2 and ZEB1 in EMT/MET and tumor biology. At least three homeodomain proteins, Six1, LBX1, and HoxA5, transactivated the ZEB1 promoter, in the case of Six1, through direct protein-promoter interaction. GRHL2 altered the Six1-DNA complex, inhibiting this transactivation. Correspondingly, GRHL2 expression prevented tumor initiation in xenograft assays, sensitized breast cancer cells to paclitaxel, and suppressed the emergence of CD44(high)CD24(low) cells (defining the cancer stem cell phenotype in the cell type studied). GRHL2 was downregulated in recurrent mouse tumors that had evolved to an oncogene-independent, EMT-like state, supporting a role for GRHL2 downregulation in this phenotypic transition, modeling disease recurrence. The combination of TGF-? and Wnt activation repressed GRHL2 expression by direct interaction of ZEB1 with the GRHL2 promoter, inducing EMT. Together, our observations indicate that a reciprocal feedback loop between GRHL2 and ZEB1 controls epithelial versus mesenchymal phenotypes and EMT-driven tumor progression.
Project description:Developmental morphogenesis and tumor progression require a transient or stable breakdown of epithelial junctional complexes to permit programmed migration, invasion, and anoikis resistance, characteristics endowed by the epithelial-mesenchymal transition (EMT). The epithelial master-regulatory transcription factor Grainyhead-like 2 (GRHL2) suppresses and reverses EMT, causing a mesenchymal-epithelial transition to the default epithelial phenotype. Here we investigated the role of GRHL2 in tubulogenesis of Madin-Darby canine kidney cells, a process requiring transient, partial EMT. GRHL2 was required for cystogenesis, but it suppressed tubulogenesis in response to hepatocyte growth factor. Surprisingly, GRHL2 suppressed this process by inhibiting the histone acetyltransferase coactivator p300, preventing the induction of matrix metalloproteases and other p300-dependent genes required for tubulogenesis. A 13-amino acid region of GRHL2 was necessary for inhibition of p300, suppression of tubulogenesis, and interference with EMT. The results demonstrate that p300 is required for partial or complete EMT occurring in tubulogenesis or tumor progression and that GRHL2 suppresses EMT in both contexts through inhibition of p300.
Project description:The epithelial-mesenchymal transition (EMT) and mesenchymal-epithelial transition (MET) contribute to cancer metastasis of pancreatic ductal adenocarcinoma (PDAC). We explored the role of grainyhead-like 2 (GRHL2), a suppressor of EMT, in the progression of PDAC. Expressions of GRHL2 were assessed using surgically resected PDAC tissues by immunohistochemistry analysis, and in vitro using human and mouse PDAC cells. Effects on epithelial plasticity and stemness of GRHL2 were examined in vitro using liver metastatic PDAC cells (CFPAC-1) with GRHL2 knockdown by specific siRNAs. GRHL2 has a significantly positive correlation with E-cadherin and CD133 in 155 resected human primary PDAC tissues. GRHL2 is highly expressed in liver metastatic cells than in primary invasive cells of both human and mouse PDAC, accompanied by a positive correlation with E-cadherin expression. GRHL2 knockdown CFPAC-1 cells demonstrated morphological changes into mesenchymal appearances and reduced proliferation through EMT. Notably, knockdown studies followed by flow cytometry analysis for a subpopulation of CD133+ showed that GRHL2 facilitates CFPAC-1 cells to maintain stem-like characters including self-renewal capacity and anoikis resistance. GRHL2 regulates epithelial plasticity along with stemness in PDAC, both of which are crucial for metastasis, implicating the possibility of GRHL2 as a therapeutic target for PDAC liver metastasis.
Project description:Twist is a key EMT inducer, expression of Twist will induce EMT in HMLE and breast tumor T47D cells By expressing Twist in HMLE and T47D cells, which lack the expression of Twist, will identify the genes regulated by Twist Expressing Twist in HMLE and T47D cells, stable clones were selected and treated with BET inhibitor JQ1 and RNA were prepared for microarray analysis
Project description:Cancer cells exhibit phenotypic plasticity during epithelial-mesenchymal transition (EMT) and mesenchymal-epithelial transition (MET) involving intermediate states. To study genome-wide epigenetic remodeling associated with EMT plasticity, we integrate the analyses of DNA methylation, ChIP-sequencing of five histone marks (H3K4me1, H3K4me3, H3K27Ac, H3K27me3 and H3K9me3) and transcriptome profiling performed on ovarian cancer cells with different epithelial/mesenchymal states and on a knockdown model of EMT suppressor Grainyhead-like 2 (GRHL2). We have identified differentially methylated CpG sites associated with EMT, found at promoters of epithelial genes and GRHL2 binding sites. GRHL2 knockdown results in CpG methylation gain and nucleosomal remodeling (reduction in permissive marks H3K4me3 and H3K27ac; elevated repressive mark H3K27me3), resembling the changes observed across progressive EMT states. Epigenetic-modifying agents such as 5-azacitidine, GSK126 and mocetinostat further reveal cell state-dependent plasticity upon GRHL2 overexpression. Overall, we demonstrate that epithelial genes are subject to epigenetic control during intermediate phases of EMT/MET involving GRHL2.
Project description:Epithelial-mesenchymal transition (EMT), a biological process by which polarized epithelial cells convert into a mesenchymal phenotype, has been implicated to contribute to the molecular heterogeneity of epithelial ovarian cancer (EOC). Here we report that a transcription factor--Grainyhead-like 2 (GRHL2) maintains the epithelial phenotype. EOC tumours with lower GRHL2 levels are associated with the Mes/Mesenchymal molecular subtype and a poorer overall survival. shRNA-mediated knockdown of GRHL2 in EOC cells with an epithelial phenotype results in EMT changes, with increased cell migration, invasion and motility. By ChIP-sequencing and gene expression microarray, microRNA-200b/a is identified as the direct transcriptional target of GRHL2 and regulates the epithelial status of EOC through ZEB1 and E-cadherin. Our study demonstrates that loss of GRHL2 increases the levels of histone mark H3K27me3 on promoters and GRHL2-binding sites at miR-200b/a and E-cadherin genes. These findings support GRHL2 as a pivotal gatekeeper of EMT in EOC via miR-200-ZEB1.
Project description:Until now the essential transcription factor that determines the epithelial phenotype of breast cancer has not been identified and its role in epithelial-to-mesenchymal transition (EMT) and tumor progression remain unclear. Here, by analyzing large expression profiles of human breast cancer cells, we found an extraordinary correlation between the expression of Grainyhead transcription factor Grhl2 and epithelial marker E-cadherin. Knockdown of Grhl2 expression by shRNA in human mammary epithelial cell MCF10A leads to down-regulation of E-cadherin and EMT. Grhl2 is down-regulated in disseminated cancer cells that have undergone EMT, and over-expression of Grhl2 is sufficient to induce epithelial gene expression. Large clinical datasets reveal that expression of Grhl2 is significantly associated with poor relapse free survival and increased risk of metastasis in breast cancer patients. In mouse models, over-expression of Grhl2 significantly promotes tumor growth and metastasis. Further testing of several Grhl2 regulated genes leads to the same conclusions that the tumorigenic and metastatic potentials of tumor cells are linked to epithelial phenotype but not mesenchymal phenotype. In conclusion, our findings indicate that Grhl2 plays an essential role in the determination of epithelial phenotype of breast cancers, EMT and tumor progression.
Project description:Our previous study has demonstrated that knockdown of Grainyhead-like 2(GRHL2) in colorectal cancer (CRC) cells inhibited cell proliferation by targeting ZEB1. This study aimed at researching whether knockdown of GRHL2 promoted CRC progression and metastasis via inducing epithelial-mesenchymal transition (EMT). GRHL2-upregulated SW-620/GRHL2+ and GRHL2-knockdown HCT116/GRHL2-KD, HT29/GRHL2-KD cells and their control cells were generated. The morphological changes after overexpression and knockdown GRHL2 were observed. qRT-PCR, Western blotting, and Immunofluorescence were used to detect EMT markers: E-cadherin, Vimentin, p-catein, ZO-1 and ZEB1 expression. Then, sh-ZEB1 was transfected to GRHL2 knockdown cells to research the relationship between GRHL2 and ZEB1. Transwell and wound healing assays were further performed to detect the impact of GRHL2 on invasion and migration in vitro. CRC cells were injected into mice tail vein to verify the impact of GRHL2 on CRC metastasis. Morphological change of mesenchymal-epithelial transition (MET) could be observed in SW620/GRHL2+ cell. The expression of epithelial markers: E-cadherin, ?-catenin, ZO-1 were up-regulated, while mesenchymal markers: Vimentin was decreased. Meanwhile, opposite EMT morphological change could be observed in HCT116/GRHL2-KD cell, accompanied by reverse change of E-cadherin, ?-catenin, ZO-1, and Vimentin. The expression level of GRHL2 and ZEB1 was found negative in both SW620/GRHL2+ and HCT116/GRHL2-KD cells. Knockdown of ZEB1 by siRNA in HCT116/GRHL2-KD and HT29/GRHL2-KD could upregulate expression of E-cadherin and GRHL2. GRHL2 knockdown also promoted migration, invasion in vitro and CRC metastasis in mice model. In conclusion, GRHL2/ZEB1 axis inhibits CRC progression and metastasis via oppressing EMT.