Grainyhead-Like 2 Expression Is Associated with Disease Progression, Chemoresistance and Poor Survival in Tubo-Ovarian High-Grade Serous Carcinoma

Grainyhead-like 2 (GRHL2) is a transcription factor that suppresses epithelial-to-mesenchymal transition (EMT). It has been previously shown that GRHL2 can confer both oncogenic and tumor-suppressive roles in human cancers, including breast, pancreatic and colorectal cancers. However, its role in lung cancer remains elusive. In the present study, a meta-analysis of multiple gene expression datasets with clinical data revealed that GRHL2 expression was increased in lung cancer compared with that in the normal tissues. Copy number analysis of GRHL2, performed using datasets of whole exome sequencing involving 151 lung cancer cell lines, revealed frequent amplifications, suggesting that the increased GRHL2 expression may have resulted from gene amplification. A survival meta-analysis of GRHL2 using The Cancer Genome Atlas (TCGA) dataset showed no association of GRHL2 expression with overall survival. GRHL2 expression was found to be associated with EMT status in lung cancer in TCGA dataset and lung cancer cell lines. GRHL2 knockdown induced partial EMT in the hTERT/Cdk4-immortalized normal lung epithelial cell line HBEC4KT without affecting proliferation measured by CCK-8 assays. In addition, GRHL2 silencing caused three lung cancer cell lines, H1975, H2009 and H441, to undergo partial EMT. However, the proliferative effects differed significantly. GRHL2 silencing promoted proliferation but not colony formation in H1975 cells whilst suppressing colony formation without affecting proliferation in H2009 cells, but it did not affect proliferation in H441 cells. These results suggest cell type-dependent effects of GRHL2 knockdown. Downstream, GRHL2 silencing enhanced the phosphorylation of AKT and ERK, assessed by western blotting with phospho-specific antibodies, in HBEC4KT, H1975 and H2009 cell lines but not in the H441 cell line. By contrast, transient GRHL2 overexpression did not affect A549 cell proliferation, which lack detectable endogenous expression of the GRHL2 protein. However, GRHL2 overexpression did suppress E-cadherin expression in A549 cells. These results suggested that GRHL2 does not only function as a tumor suppressor of EMT but can also behave as an oncogene depending on the lung cancer cell-type context.

Using a retrovirus-mediated cDNA expression cloning approach, we identified the grainyhead-like 2 (GRHL2) transcription factor as novel protooncogene. Overexpression of GRHL2 in NIH3T3 cells induced striking morphological changes, an increase in cell proliferation, anchorage-independent growth, and tumor growth in vivo. By combining a microarray analysis and a phylogenetic footprinting analysis with various biochemical assays, we identified the epidermal growth factor receptor family member Erbb3 as a novel GRHL2 target gene. In breast cancer cell lines, shRNA-mediated knockdown of GRHL2 expression or functional inactivation of GRHL2 using dominant negative GRHL2 proteins induces down-regulation of ERBB3 gene expression, a striking reduction in cell proliferation, and morphological and phenotypical alterations characteristic of an epithelial-to-mesenchymal transition (EMT), thus implying contradictory roles of GRHL2 in breast carcinogenesis. Interestingly, we could further demonstrate that expression of GRHL2 is directly suppressed by the transcription factor zinc finger enhancer-binding protein 1 (ZEB1), which in turn is a direct target for repression by GRHL2, suggesting that the EMT transcription factors GRHL2 and ZEB1 form a double negative regulatory feedback loop in breast cancer cells. Finally, a comprehensive immunohistochemical analysis of GRHL2 expression in primary breast cancers showed loss of GRHL2 expression at the invasive front of primary tumors. A pathophysiological relevance of GRHL2 in breast cancer metastasis is further demonstrated by our finding of a statistically significant association between loss of GRHL2 expression in primary breast cancers and lymph node metastasis. We thus demonstrate a crucial role of GRHL2 in breast carcinogenesis.

GRHL2-Dependent Enhancer Switching Maintains a Pluripotent Stem Cell Transcriptional Subnetwork after Exit from Naive Pluripotency

Purpose: Grainyhead-like 2 (GRHL2) is one of the three mammalian homologues of Drosophila Grainyhead involved in epithelial morphogenesis. GRHL2 also controls epithelial cell proliferation and differentiation. Our objective was to evaluate the role of GRHL2 in oral carcinogenesis and the underlying mechanism. Experimental Design: GRHL2 expression was evaluated in multiple oral cancer cell lines and tissues of oral squamous cell carcinomas (OSCCs) by qRT-PCR, western blot assay, immunohistochemistry and laser-captured microdissection assay. The effects of shRNA-mediated GRHL2 knockdown were evaluated by tumor spheroid assay, colony formation assay, gene promoter luciferase assay and immunofluorescent staining and chromatin immunoprecipitation assay. In vivo the effect of GRHL2 loss on cancer cell tumorigenicity was evaluated by tumor xenograft assay. Results: GRHL2 expression was elevated in cells and tissues of oral squamous cell carcinomas (OSCCs) compared with normal counterparts. Knockdown of GRHL2 resulted in the loss of in vivo tumorigenicity, cancer stemness, and epithelial phenotype of oral cancer cells. GRHL2 inhibition decreased oral cancer cell proliferation and colony formation. GRHL2 regulated the expression of miR-200 family and Octamer-binding transcription factor 4 (Oct-4) genes through direct promoter DNA binding. Overexpression of miR-200 genes in the oral cancer cells depleted of GRHL2 partially restored the epithelial phenotype, proliferative rate and cancer stemness. Conclusion: GRHL2 plays pivotal role in the maintenance of the transformed phenotype in OSCCs. Downregulation of GRHL2 leads to the decrease of tumorigenicity, cancer stemness of oral cancer cell, in which miR-200 and Oct-4 genes partially mediate the tumorigenic effects of GRHL2 in OSCC. This study was supported in part by the grants R56DE024593 and Jack Weichman Endowed Fund. Citation Format: Wei Chen, Shebli Mehrazarin, Ki-Hyuk S Shin, Yi-Ling Lin, Reuben H. Kim, No-Hee Park, Mo K. Kang. Grainyhead-like 2 (GRHL2) regulates epithelial plasticity and stemness in oral cancers. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2097. doi:10.1158/1538-7445.AM2015-2097

Epithelial ovarian cancer (EOC) is clinically heterogeneous, comprising different histological and biological subtypes. Despite tumor heterogeneity, patient stratification is lacking and targeted therapeutic options for EOC are limited. Epithelial-mesenchymal transition (EMT), an important developmental process that allows the conversion of epithelial cells to mesenchymal phenotype, has been implicated in contributing to the molecular heterogeneity in EOC. Emerging evidence suggests that EMT in EOC may be associated with tumor progression and metastasis. We aim to decipher the underlying EMT mechanisms by using a panel of 42 EMT-phenotyped EOC cell lines. We found that Grainyhead-like 2 (GRHL2), a transcription factor, was expressed exclusively in EOC cells with epithelial phenotype by using qPCR, ELISA and western blots. EOC tumors with Mesenchymal molecular subtype also showed a decrease in GRHL2 expression. These data suggest that GRHL2 is required for maintaining cell polarity or cell-cell adhesion. Therefore, manipulating the expression of GRHL2 might switch the EMT-related molecular subtypes in EOC. Here, we showed that knocking down of GRHL2 resulted in EMT changes in EOC cells with Epithelial molecular subtype, accompanied by an increase in cell motility and directional migration. GRHL2-deficient cells also showed a tendency to migrate individually instead of collectively, thus suggesting that GRHL2 might be required for collective cell migration. Knock-down of GRHL2 not only downregulates E-cadherin expression, but also interferes with the stability of E-cadherin at cell-cell junctions. Chromatin-immunoprecipitation sequencing (ChIP-seq) identified GRHL2 transcriptional targets that might be the downstream mediators for this molecular subtype switch. In conclusion, GRHL2 is an important regulator in maintaining the epithelial phenotype and crucial player in molecular heterogeneity in EOC. Citation Format: Vin Yee Chung, Meng Kang Wong, Kuee Theng Kuay, Tuan Zea Tan, Ernesto Guccione, Jean Paul Thiery, Ruby Yun-Ju Huang. Grainyhead-like 2 regulates molecular subtype switching in epithelial ovarian cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1058. doi:10.1158/1538-7445.AM2014-1058

The transcription factor grainyhead-like 2 (GRHL2) plays a crucial role in various developmental processes. Although GRHL2 recently has attracted considerable interest in that it could be identified as a novel suppressor of the epithelial-to-mesenchymal transition, evidence is emerging that GRHL2 also exhibits tumour-promoting activities. Aim of the present study therefore was to help defining the relevance of GRHL2 for human cancers by performing a comprehensive immunohistochemical analysis of GRHL2 expression in normal (n = 608) and (n = 3,143) tumour tissues using tissue microarrays. Consistent with its accepted role in epithelial morphogenesis, GRHL2 expression preferentially but not exclusively was observed in epithelial cells. Regenerative and proliferating epithelial cells with stem cell features showed a strong GRHL2 expression. Highly complex GRHL2 expression patterns indicative of both reduced and elevated GRHL2 expression in tumours, possibly reflecting potential tumour-suppressing as well as oncogenic functions of GRHL2 in distinct human tumours, were observed. A dysregulation of GRHL2 expression for the first time was found in tumours of non-epithelial origin (e.g., astrocytomas, melanomas). We also report GRHL2 copy number gains which, however, did not necessarily translate into increased GRHL2 expression levels in cancer cells. Results obtained by meta-analysis of gene expression microarray data in conjunction with functional assays demonstrating a direct regulation of HER3 expression further point to a potential therapeutic relevance of GRHL2 in ovarian cancer. Hopefully, the results presented in this study may pave the way for a better understanding of the yet largely unknown function of GRHL2 in the initiation, progression and also therapy of cancers.

Esophageal cancer (EC) is one of the most common causes of cancer-related mortality in the world. Although much effort has been made to improve the 5-year survival rate of patients with EC, it still remains low due to diagnosis at an advanced stage, aggressive local invasion, early metastasis, and resistance to chemotherapy. Although grainyhead-like 2 (GRHL2) has attracted interest since it has been recently identified as a novel suppressor of the epithelial–mesenchymal transition, clinical values of GRHL2 and its relationship with the metastasis-related factors, such as hypoxia-inducible factor 1α (HIF-1α) and vascular endothelial growth factor (VEGF), remain unclear. In order to investigate the expression of GRHL2, HIF-1α, and VEGF, and their correlation with angiogenesis in EC, 63 patients with EC were examined. The expression of GRHL2, HIF-1α, and VEGF in tumor tissues was higher than that in adjacent tissues and was associated with tumor differentiation. GRHL2 expression was significantly correlated with lymph node metastasis and invasion depth, whereas VEGF expression was associated with tumor (TNM) stage. A significant correlation was found between the expression of GRHL2 and HIF-1α. The patients expressing low GRHL2 and high HIF-1α showed significant reduction in both overall survival rate and disease-free survival rate. The results demonstrated that abnormal expression of GRHL2 is common in EC, and low expression of GRHL2 accompanied by a high expression of HIF-1α indicates poor prognosis.

Knockdown of GRHL2 inhibited proliferation and induced apoptosis of colorectal cancer by suppressing the PI3K/Akt pathway

GRHL2 is a proto-oncogene that regulates epithelial proliferation and differentiation through transcriptional regulation of large number of target genes. The current study was performed to determine its role in oral carcinogenesis through regulation of forkhead box 1 (FoxM1), which is a known oncogene for many solid tumors. GRHL2 expression was highly elevated in oral squamous cell carcinoma (OSCC) tissues compared with normal oral epithelium (NHOM), and its protein expression is localized in the proliferative cells at the outer border of invasive islands of OSCC. In cultured OSCCs, GRHL2 and FoxM1 levels were both highly upregulated compared with normal human oral keratinocytes (NHOK), supporting their roles in oral carcinogenesis. When GRHL2 was knocked down in SCC4 cells by RNA interference, the cells have lost their proliferative capacity and colony formation when plated at low density. GRHL2 knockdown also resulted in marked loss of tumor spheroid formation, which is hallmark of cancer self-renewal. GRHL2 knockdown also led to loss of tumorigenic ability of cells upon xenograft transplantation in immunocompromised mice. GRHL2 appeared to determine the epithelial phenotype in OSCC cells. GRHL2 knockdown led to loss of E-cadherin and miR-200 family genes, which are epithelial markers. These data indicate that GRHL2 is required to maintain the transformed phenotype in OSCC. We also noted that GRHL2 knockdown led to drastic loss of FoxM1 expression, suggesting that GRHL2 may regulate FoxM1. To test this possibility, we performed in vivo binding assay by chromatin immunoprecipitation (ChIP), which showed GRHL2 binding at the proximal promoter of FoxM1. Luciferase reporter plasmid under FoxM1 promoter showed that GRHL2 directly regulates the gene promoter activity. FoxM1 knockdown resulted in loss of OSCC proliferation and tumor spheroid formation, while its overexpression conferred transformed phenotype in non-tumorigenic human oral keratinocytes (HOKs). Therefore, our data indicate that GRHL2 is required for the maintenance of transformed phenotype and that it plays a role in oral carcinogenesis through regulation of FoxM1. This study was supported in part by the grants from NIDCR/NIH (DE18295 and DE18959) and Jack Weichman Endowed Fund. Note: This abstract was not presented at the meeting. Citation Format: Wei Chen, Jin-Kyu Yi, Ki-Hyuk Shin, Reuben Kim, Shebli Mehrazarin, No-Hee Park, Mo K. Kang. Grainyhead-like 2 (GRHL2) regulates the expression of forkhead box transcription factor M1 (FoxM1) in human oral cancer cells and determines tumorigenicity. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1598. doi:10.1158/1538-7445.AM2014-1598

ABSTRACTOur 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, β-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, β-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.

Grainyhead like 2 (GRHL2) is an essential transcription factor for development and function of epithelial tissues. It has dual roles in cancer by supporting tumor growth while suppressing epithelial to mesenchymal transitions (EMT). GRHL2 cooperates with androgen and estrogen receptors (ER) to regulate gene expression. We explore genome wide GRHL2 binding sites conserved in three ER⍺/GRHL2 positive luminal breast cancer cell lines by ChIP-Seq. Interaction with the ER⍺/FOXA1/GATA3 complex is observed, however, only for a minor fraction of conserved GRHL2 peaks. We determine genome wide transcriptional dynamics in response to loss of GRHL2 by nascent RNA Bru-seq using an MCF7 conditional knockout model. Integration of ChIP- and Bru-seq pinpoints candidate direct GRHL2 target genes in luminal breast cancer. Multiple connections between GRHL2 and proliferation are uncovered, including transcriptional activation of ETS and E2F transcription factors. Among EMT-related genes, direct regulation of CLDN4 is corroborated but several targets identified in other cells (including CDH1 and ZEB1) are ruled out by both ChIP- and Bru-seq as being directly controlled by GRHL2 in luminal breast cancer cells. Gene clusters correlating positively (including known GRHL2 targets such as ErbB3, CLDN4/7) or negatively (including TGFB1 and TGFBR2) with GRHL2 in the MCF7 knockout model, display similar correlation with GRHL2 in ER positive as well as ER negative breast cancer patients. Altogether, this study uncovers gene sets regulated directly or indirectly by GRHL2 in luminal breast cancer, identifies novel GRHL2-regulated genes, and points to distinct GRHL2 regulation of EMT in luminal breast cancer cells.6_BaCm7NaLGTK4K5qg2XWLVideo

The oncogenic Epithelial-Mesenchymal Transition (EMT) enhances malignant progression by promoting invasion and survival of tumor cells. EMT is induced by microenvironmental factors including TGF-β and Wnt agonists, and transcription factors including the E-box binding factors Twist, Snail and ZEB. Previously, we reported that a member of the mammalian Grainyhead family of wound healing-regulatory transcription factors, Grainyhead-like-2 (GRHL2), suppresses EMT and restores sensitivity to anoikis by repressing ZEB1 expression and inhibiting TGF-β signaling. Here, 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; for Six1, this occurred 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 CD44highCD24low cells in a cell line where this marker set correlates with cancer stem cell phenotype. GRHL2 was down-regulated in recurrent mouse tumors that had evolved to an oncogene-independent, EMT-like state, supporting a role for GRHL2 down-regulation in this tumor phenotype transition modeling disease recurrence. The combination of TGF-β and Wnt activation repressed GRHL2 expression through direct interaction of ZEB1 with the GRHL2 promoter, inducing EMT. These observations indicate that a reciprocal feedback loop between GRHL2 and ZEB1 controls epithelial vs. mesenchymal phenotypes and EMT-driven tumor progression. Citation Format: Benjamin Cieply, Heide L. Ford, Joshua Farris, Steven Frisch. A reciprocal feedback loop between grainyhead-like-2 and ZEB1 controls EMT and tumor suppression. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr A15.

The transcription factor Grainyhead-like 2 (GRHL2) is a critical transcription factor for epithelial tissues that has been reported to promote cancer growth in some and suppress aspects of cancer progression in other studies. We investigated its role in different breast cancer subtypes. In breast cancer patients, GRHL2 expression was increased in all subtypes and inversely correlated with overall survival in basal-like breast cancer patients. In a large cell line panel, GRHL2 was expressed in luminal and basal A cells, but low or absent in basal B cells. The intersection of ChIP-Seq analysis in 3 luminal and 3 basal A cell lines identified conserved GRHL2 binding sites for both subtypes. A pathway analysis of ChIP-seq data revealed cell-cell junction regulation and epithelial migration as well as epithelial proliferation, as candidate GRHL2-regulated processes and further analysis of hub genes in these pathways showed similar regulatory networks in both subtypes. However, GRHL2 deletion in a luminal cell line caused cell cycle arrest while this was less prominent in a basal A cell line. Conversely, GRHL2 loss triggered enhanced migration in the basal A cells but failed to do so in the luminal cell line. ChIP-Seq and ChIP-qPCR demonstrated GRHL2 binding to CLDN4 and OVOL2 in both subtypes but not to other GRHL2 targets controlling cell-cell adhesion that were previously identified in other cell types, including CDH1 and ZEB1. Nevertheless, E-cadherin protein expression was decreased upon GRHL2 deletion especially in the luminal line and, in agreement with its selectively enhanced migration, only the basal A cell line showed concomitant induction of vimentin and N-cadherin. To address how the balance between growth reduction and aspects of EMT upon loss of GRHL2 affected in vivo behavior, we used a mouse basal A orthotopic transplantation model in which the GRHL2 gene was silenced. This resulted in reduced primary tumor growth and a reduction in number and size of lung colonies, indicating that growth suppression was the predominant consequence of GRHL2 loss. Altogether, these findings point to largely common but also distinct roles for GRHL2 in luminal and basal breast cancers with respect to growth and motility and indicate that, in agreement with its negative association with patient survival, growth suppression is the dominant response to GRHL2 loss.

Purpose Abnormal methylation of Grainyhead-like 2 (GRHL2) is associated with a substantial role in the malignant phenotype of tumor patients. Our present research is aimed at studying the abnormal expression of GRHL2 and the association of methylation in patients with acute leukemia and its relationship with prognosis. Materials and Methods We used quantitative real-time polymerase chain reaction (qRT-PCR) for detecting the aberrant expression level of GRHL2 in 60 patients with acute leukemia and 60 normal controls. We analyzed the significant correlation between the expression level of GRHL2 with clinicopathological features and patients' prognosis in acute leukemia using the corresponding statistical methods. Secondly, we employed qRT-PCR and Western blotting to detect the mRNA and protein levels of GRHL2 in leukemia cell lines. Next, we used methylation-specific polymerase chain reaction (MSP) technology for detecting the methylation of GRHL2 in clinical samples with acute leukemia and cell lines. Then we investigated the demethylating effect of arsenic trioxide and 5-azacitidine on the mRNA and protein expression levels of GRHL2 in cell lines of acute leukemia. Finally, we studied the effects of arsenide trioxide and 5-azacitidine on the proliferation of leukemia cells and the TGF-β signaling pathway. Results We found a lower level of GRHL2 expression not only in acute leukemia patients but also in cell lines when compared with normal controls. At the same time, the expression level of GRHL2 in patients with acute leukemia was significantly correlated with leukocyte count, platelet count, and cytogenetic risk grouping. In addition, the lower GRHL2 expression group showed a significantly lower overall survival rate in acute leukemia patients than that of patients with a higher GRHL2 expression group. Univariate and multivariate analyses revealed that the expression of GRHL2 is an independent risk factor in acute leukemia patients. The methylation level of the GRHL2 promoter region in acute leukemia patients and cell lines was significantly higher than the normal control group, and we found the elevated mRNA and protein levels of GRHL2 in acute leukemia cell lines after the use of the demethylation drug arsenic trioxide and 5-azacitidine. At the same time, arsenide trioxide and 5-azacitidine are associated with the inhibition of cellular proliferation of acute leukemia cells and also promote the elevated expression of TGF-β signaling pathway-linked proteins, including TGF-β, Smad2, Smad3, and Smad4. Conclusion Increased expression and methylation level of GRHL2 are closely associated with the prognosis and malignant phenotype of acute leukemia patients and play an irreplaceable role in the occurrence and development of patients with acute leukemia.

<div>Abstract<p>Epithelial–mesenchymal transition (EMT) in carcinoma cells enhances malignant progression by promoting invasion and survival. EMT is induced by microenvironmental factors, including TGF-β 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-β 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<sup>high</sup>CD24<sup>low</sup> 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-β 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. <i>Cancer Res; 73(20); 6299–309. ©2013 AACR</i>.</p></div>