Expression Of GRHL2 Research Articles

Expression Patterns of Grainyhead-Like 2 and Ovo-Like 2 in Mouse Mammary Gland Development During Pregnancy, Lactation, and Weaning.

Grainyhead-like 2 (Grhl2) is a transcription factor that regulates cell adhesion genes in mammary ductal development and serves as a repressor of the epithelial-mesenchymal transition. Conversely, Ovo-like2 (Ovol2) is a target gene of Grhl2 but functions as a substitute in Grhl2-deficient mice, facilitating successful epithelial barrier formation and lumen expansion in kidney-collecting ductal epithelial cells. Our objective was to examine the expression patterns of Grhl2, Ovol2, and their associated genes during the intricate phases of mouse mammary gland development. The mRNA expression of Grhl2 and Ovol2 increased after pregnancy. We observed Grhl2 protein presence in the epithelial cell's region, coinciding with acini formation, and its signal significantly correlated with E-cadherin (Cdh1) expression. However, Ovol2 was present in the epithelial region without a correlation with Cdh1. Similarly, Zeb1, a mesenchymal transcription factor, showed Cdh1-independent expression. Subsequently, we explored the interaction between Rab25, a small G protein, and Grhl2/Ovol2. The expressions of Grhl2 and Ovol2 exhibited a strong correlation with Rab25 and claudin-4, a tight junction protein. These findings suggest that Grhl2 and Ovol2 may collaborate to regulate genes associated with cell adhesion and are crucial for maintaining epithelial integrity during the different phases of mammary gland development.

Inhibition of retinoic acid signaling impairs cranial and spinal neural tube closure in mice lacking the Grainyhead-like 3 transcription factor

Neural tube closure is a dynamic morphogenic event in early embryonic development. Perturbations of this process through either environmental or genetic factors induce the severe congenital malformations known collectively as neural tube defects (NTDs). Deficiencies in maternal folate intake have long been associated with NTDs, as have mutations in critical neurulation genes that include the Grainyhead-like 3 (Grhl3) gene. Mice lacking this gene exhibit fully penetrant thoraco-lumbo-sacral spina bifida and a low incidence of exencephaly. Previous studies have shown that exposure of pregnant mice carrying hypomorphic Grhl3 alleles to exogenous retinoic acid (RA) increases the incidence and severity of NTDs in their offspring. Here, we demonstrate that inhibition of RA signaling using a high affinity pan-RA receptor antagonist administered to pregnant mice at E7.5 induces fully penetrant exencephaly and more severe spina bifida in Grhl3-null mice. Later administration, although prior to neural tube closure has no effect. Similarly, blockade of RA in the context of reduced expression of Grhl2, a related gene known to induce NTDs, has no effect. Taken together, these findings provide new insights into the complexities of the interplay between RA signaling and Grhl3-induced neurulation.

Hypermethylation of the GRHL2 promoter region is associated with ovarian endometriosis.

Abnormal gene expression caused by epigenetic changes, including DNA methylation, is associated with the development and progression of endometriosis. Grainyhead-like 2 gene (GRHL2), a suppressor of epithelial-mesenchymal transition, has been suggested to be associated with the occurrence, progression and poor survival of a variety of cancers. Although endometriosis is a benign disease, it has the biological behaviour of migration and invasion as malignant tumor. This study aims to determine whether the abnormal expression of the GRHL2 caused by aberrant methylation of its promoter is associated with the pathogenesis of ovarian endometriosis. Our results demonstrated that GRHL2 promoter region was significantly hypermethylated in the ectopic endometrium of patients with ovarian endometriosis compared with the normal endometrium of control patients. In contrast, the levels of GRHL2 mRNA and protein were significantly lower in the ectopic endometrium than in the control endometrium. Correlation analysis showed the methylation levels of GRHL2 were significantly negatively correlated with the mRNA expression of GRHL2. Moreover, the in vitro results suggested that the knockdown of GRHL2 could significantly increase the invasion and migration ability of EECs and may promote ZEB1 and vimentin expression while decreasing the expression of E-cadherin in EECs. Taken together, these results suggest that the low expression of GRHL2 caused by hypermethylation of the GRHL2 promoter is associated with ovarian endometriosis. The knockdown of GRHL2 may be involved in the occurrence of endometriosis by increasing EEC migration and invasion. This study provides more evidence for the hypothesis that endometriosis may be an epigenetic regulatory disorder.

Correlation analysis between CD133, Klk3 and grhl2 expression and tumor characteristics in prostate cancer.

Prostate cancer is a common clinical disease in men. It is known that prostate cancer ranks 3rd in the incidence of malignant tumors of the male genitourinary system in China, which is able to evaluate the riskiness of life expectancy of male patients. Therefore, we investigated the expression of CD133, recombinant human kallikrein 3 (Klk3), grainy head like 2 (grhl2) in prostate cancer, and correlation with tumor characteristics in the present study. A total of 167 prostate cancer patients who underwent surgical treatment in our hospital from February 2017 to April 2021 were selected. Their cancer and adjacent tissues were resected, and CD133 was detected by double staining using immunohistochemistry, Klk3 and grhl2 were detected by RT-PCR analysis, and CD133, Klk3 were analyzed by Pearson's method in different clinical stages, Gleason grade Correlation of grhl2 with tumor characteristics. The expression of CD133, KLK3, and GRHL2 in cancer tissue was increased compared with adjacent tissue (P < 0.05). The expression of CD133, KLK3, and GRHL2 increased with the aggravation of the clinical stage and Gleason grade (P < 0.05). CD133, KLK3, and GRHL2 showed a positive correlation in prostate cancer. The Pearson method found a positive correlation between CD133, KLK3, GRHL2 and clinical stage, Gleason grade, and lymph node metastasis. In general, high CD133, Klk3, and grhl2 expression was observed in prostate cancer and increased with the disease. They presented a positive correlation in prostate cancer presence, and these three gene products correlated with tumor characteristics.

Impact of Epithelial-Mesenchymal Transition on the Immune Landscape in Breast Cancer.

Simple SummaryThe process of epithelial–mesenchymal transition (EMT) is thought to influence breast cancer tumor progression by affecting both tumor cells and the tumor microenvironment (TME). We aimed to study the impact of EMT-related markers on a breast cancer cohort and specifically analyze the involvement of Snail, Twist, ZEB1, N-cadherin, Vimentin, GRHL2, E-cadherin, and EpCAM and their respective outcome on both immune infiltration of the TME and clinicopathological features. The inflammatory infiltrate was more often associated with poorly differentiated carcinomas including triple-negative breast cancer (TNBC). The altered pattern of protein expression of epithelial markers markedly influenced the magnitude of the inflammatory infiltrate found in the TME. Overall, our data highlight the potential beneficial association of the EMT signature with the immune inflammatory response. This may open new avenues for rational decision making in the clinical use of immunotherapy in subsets of breast cancer patients, specifically TNBC.The impact of epithelial–mesenchymal transition (EMT) signature on the immune infiltrate present in the breast cancer tumor microenvironment (TME) is still poorly understood. Since there is mounting interest in the use of immunotherapy for the treatment of subsets of breast cancer patients, it is of major importance to understand the fundamental tumor characteristics which dictate the inter-tumor heterogeneity in immune landscapes. We aimed to assess the impact of EMT-related markers on the nature and magnitude of the inflammatory infiltrate present in breast cancer TME and their association with the clinicopathological parameters. Tissue microarrays were constructed from 144 formalin-fixed paraffin-embedded invasive breast cancer tumor samples. The protein expression patterns of Snail, Twist, ZEB1, N-cadherin, Vimentin, GRHL2, E-cadherin, and EpCAM were examined by immunohistochemistry (IHC). The inflammatory infiltrate in the TME was assessed semi-quantitatively on hematoxylin and eosin (H&E)-stained whole sections and was characterized using IHC. The inflammatory infiltrate was more intense in poorly differentiated carcinomas and triple-negative carcinomas in which the expression of E-cadherin and GRHL2 was reduced, while EpCAM was overexpressed. Most EMT-related markers correlated with plasma cell infiltration of the TME. Taken together, our findings reveal that the EMT signature might impact the immune response in the TME.

Medication for Acromegaly Reduces Expression of MUC16, MACC1 and GRHL2 in Pituitary Neuroendocrine Tumour Tissue.

Acromegaly is a disease mainly caused by pituitary neuroendocrine tumor (PitNET) overproducing growth hormone. First-line medication for this condition is the use of somatostatin analogs (SSAs), that decrease tumor mass and induce antiproliferative effects on PitNET cells. Dopamine agonists (DAs) can also be used if SSA treatment is not effective. This study aimed to determine differences in transcriptome signatures induced by SSA/DA therapy in PitNET tissue. We selected tumor tissue from twelve patients with somatotropinomas, with half of the patients receiving SSA/DA treatment before surgery and the other half treatment naive. Transcriptome sequencing was then carried out to identify differentially expressed genes (DEGs) and their protein–protein interactions, using pathway analyses. We found 34 upregulated and six downregulated DEGs in patients with SSA/DA treatment. Three tumor development promoting factors MUC16, MACC1, and GRHL2, were significantly downregulated in therapy administered PitNET tissue; this finding was supported by functional studies in GH3 cells. Protein–protein interactions and pathway analyses revealed extracellular matrix involvement in the antiproliferative effects of this type of the drug treatment, with pronounced alterations in collagen regulation. Here, we have demonstrated that somatotropinomas can be distinguished based on their transcriptional profiles following SSA/DA therapy, and SSA/DA treatment does indeed cause changes in gene expression. Treatment with SSA/DA significantly downregulated several factors involved in tumorigenesis, including MUC16, MACC1, and GRHL2. Genes that were upregulated, however, did not have a direct influence on antiproliferative function in the PitNET cells. These findings suggested that SSA/DA treatment acted in a tumor suppressive manner and furthermore, collagen related interactions and pathways were enriched, implicating extracellular matrix involvement in this anti-tumor effect of drug treatment.

GRHL2 Upregulation Predicts a Poor Prognosis and Promotes the Resistance of Serous Ovarian Cancer to Cisplatin.

BackgroundGRHL2 has been shown to function in ovarian carcinogenesis. However, the relationship between GRHL2 and cisplatin (DDP) resistance in serous ovarian cancer (SOC) is not clear. The purpose of this study was to elucidate the function and mechanism of GRHL2 in DDP resistance of SOC.Materials and MethodsImmunohistochemistry (IHC) was utilized to identify GRHL2 protein expression in DDP resistant and sensitive SOC tissues. GRHL2 mRNA and protein levels were identified using quantitative real-time PCR (qRT-PCR) and Western blotting in SKOV3/DDP and SKOV3 cell lines. We conducted loss- and gain-of-function experiments to uncover the consequence of GRHL2 knockdown or overexpression on the sensitivity of ovarian cancer cells to DDP in vitro and in vivo and the underlying mechanism.ResultsIt was observed that expression of GRHL2 was higher in DDP resistant SOC tissues relative to DDP sensitive SOC tissues. In addition, the increased expression of GRHL2 led to shorter progression-free survival (PFS) and overall survival (OS). Meanwhile, the GRHL2 transcript and protein levels in SKOV3/DDP were also higher than SKOV3. Small hairpin RNA (shRNA)-facilitated GRHL2 gene knockdown considerably heightened the sensitivity of SKOV3/DDP cells to DDP by inhibiting proliferation and promoting apoptosis, while up-regulation of GRHL2 significantly reduced the sensitivity of SKOV3 cells to DDP by promoting proliferation and decreasing apoptosis. In addition, GRHL2 promotes DDP resistance of SOC through activation of ERK/MAPK signaling pathways.ConclusionOur results suggest that GRHL2 up-regulation predicts a poor prognosis and promotes the resistance of SOC to DDP. Therefore, GRHL2 may be a possible treatment target for cisplatin-resistant serous ovarian cancer.

GRHL2 Acts as an Anti-Oncogene in Bladder Cancer by Regulating ZEB1 in Epithelial-Mesenchymal Transition (EMT) Process.

PurposeGRHL2 played important roles in different cancers. In this study, we aimed to investigate the roles of GRHL2 in bladder cancer.MethodsThe immunohistochemistry assay was performed to detect the expression of GRHL2 in bladder cancer tissues and adjacent noncancerous tissues and the expression levels of GRHL2 and zinc finger E-box binding homeobox (ZEB1) mRNA in tissues were determined by qRT-PCR. In addition, qRT-PCR and Western blotting were applied to detect the expression levels of GRHL2 and ZEB1 in bladder cancer cell lines (RT4, BIU-87, 5637, T24) and immortalized human bladder epithelial cell line (SV-HUC-1). The cell models with up-regulated and down-regulated expression of GRHL2 were constructed using bladder cancer cell lines T24 and 5637 to investigate the underlying roles of GRHL2 on the proliferation, migration, invasion and EMT process of bladder cancer cells. After that, cell proliferation was evaluated by CCK8 assay, cell cycle assay and colony formation assay. Transwell assay and wound healing assay were performed to determine the invasion and migration ability of the bladder cancer cells. The expressions of epithelial-mesenchymal transition (EMT) related proteins (E-cadherin, Vimentin, Slug and Snail) were assessed by Western blot analysis. Moreover, ZEB1 and GRHL2 were co-transfected into T24 and 5637 cells and their effects on EMT process and invasive capacity of cells were examined.ResultsThe expression of GRHL2 was down-regulated in bladder cancer tissues and human bladder cancer cell lines compared with the normal bladder tissues and immortalized human bladder epithelial cell line. Besides, down-regulation of GRHL2 improved the proliferation ability of bladder cancer cells and promoted the EMT process through up-regulation of ZEB1. The overexpression of ZEB1 partially reversed the inhibitory effect of GRHL2 on EMT.ConclusionGRHL2 acts as an anti-oncogene to regulate bladder cancer cell proliferation and inhibit EMT by targeting ZEB1. This study may provide a theoretical basis for further research.

Abstract A49: Modeling “decathlon winner” cancer cells that drive therapy resistance and metastasis in triple-negative breast cancer

Abstract Introduction: There is a pressing need for reliable models for testing potential therapeutic drugs that accurately predict how drugs will act in cancer patients. Cancer resembles an evolution-like process in the body involving epigenetic and genetic alterations in tumor cells accompanied by a selection for their fitness to survive multiple challenges in the body. Therefore, we are developing an approach designed to test whether a therapy would affect most adaptable/evolvable cancer cells. We hypothesize that there is a two-way linkage between the regulatory state and the metabolic state, which can be exploited for selecting highly adaptable “decathlon winner” cancer cells. Methods: To model the intrinsic resistance in triple-negative breast cancer that often overwhelms currently offered therapies, we selected rare cancer cells (0.01% in population) based on their ability to survive a severe metabolic challenge, i.e., a prolonged lack of glutamine in culture medium of SUM149 and FC-IBC02 triple-negative Inflammatory Breast Cancer (IBC) cell lines. The rationale is that if a cancer cell can survive such a severe challenge, it can survive all other challenges encountered in the body. Results: The rare metabolically adaptable (MA) cancer cells, which survive and grow without glutamine indefinitely, are resistant to chemotherapeutic drugs, and highly metastatic to multiple organs—lungs, liver, brain, and skin—from fat pad xenografts in nude mice (Singh et al., PLoS ONE, 2012). The MA cells are resistant to most drugs tested thus far as single agent, supporting the validity of our cell-based model for testing new therapies (Singh et al., PLoS ONE, 2014). We have strong evidence of epithelial-to-mesenchymal transition (EMT) in MA cells, as indicated by reduced expression of GRHL2, increased expression of ZEB1, and reduced expression of ESRP1 (epithelial splicing regulatory protein 1) and a consequently increased level of CD44s, to name a few critical alterations. There is a strong correlation between EMT and a progenitor-like cell state. Investigating the molecular characteristics of SUM149-MA cells, we found that MA cells produced a very low level of TET2 methylcytosine dioxygenase (5- to 10-fold reduction) compared with the parental SUM149 cell line. TET2 could represent a link between the metabolic state and the epigenetic state in progenitor-like MA cells; its activity could be regulated both in terms of expression levels and by allosteric regulation with metabolites. We have recently reported that an RNA demethylase, FTO, which is important in organismal survival under food shortages and also controls obesity, plays an important role in the survival of MA cells (Singh et al., PLoS ONE, 2016). We also observed that SUM149-MA cells overexpress ADARB2, an RNA-editing adenosine deaminase converting adenosine to inosine, which is interestingly associated with extreme old age in humans. ADARB2 can also influence lifespan in Caenorhabditis elegans. These investigations of MA cells revealed how resistant cancer cells can exploit the regulatory molecular networks that evolution has selected for organismal fitness. We are currently evaluating therapeutic strategies that will eradicate or disable MA cells. Conclusions: A highly abnormal and highly adaptable subpopulation of cancer cells modeled through our approach is ideally suited for evaluating combination therapies that are required for combating a heterogeneous and evolving disease. Such studies are likely to improve outcomes for breast cancer patients with high-risk of relapse, e.g., those with triple-negative breast cancer and IBC. Supported by a State of Texas Grant for Rare and Aggressive Cancers. Citation Format: Balraj Singh, Vanessa N. Sarli, Anthony Lucci. Modeling “decathlon winner” cancer cells that drive therapy resistance and metastasis in triple-negative breast cancer [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr A49.

Expression of Grhl2 and Its Target Gene Products in Developing Mouse Submandibular Gland

Expression of Grhl2 and Its Target Gene Products in Developing Mouse Submandibular Gland

Mesenchymal-Epithelial Transition in Sarcomas Is Controlled by the Combinatorial Expression of MicroRNA 200s and GRHL2.

Phenotypic plasticity involves a process in which cells transiently acquire phenotypic traits of another lineage. Two commonly studied types of phenotypic plasticity are epithelial-mesenchymal transition (EMT) and mesenchymal-epithelial transition (MET). In carcinomas, EMT drives invasion and metastatic dissemination, while MET is proposed to play a role in metastatic colonization. Phenotypic plasticity in sarcomas is not well studied; however, there is evidence that a subset of sarcomas undergo an MET-like phenomenon. While the exact mechanisms by which these transitions occur remain largely unknown, it is likely that some of the same master regulators that drive EMT and MET in carcinomas also act in sarcomas. In this study, we combined mathematical models with bench experiments to identify a core regulatory circuit that controls MET in sarcomas. This circuit comprises the microRNA 200 (miR-200) family, ZEB1, and GRHL2. Interestingly, combined expression of miR-200s and GRHL2 further upregulates epithelial genes to induce MET. This effect is phenocopied by downregulation of either ZEB1 or the ZEB1 cofactor, BRG1. In addition, an MET gene expression signature is prognostic for improved overall survival in sarcoma patients. Together, our results suggest that a miR-200, ZEB1, GRHL2 gene regulatory network may drive sarcoma cells to a more epithelial-like state and that this likely has prognostic relevance.

AR-V7 Transcripts in Whole Blood RNA of Patients with Metastatic Castration Resistant Prostate Cancer Correlate with Response to Abiraterone Acetate

The expression of AR-V7 (androgen receptor splice variant) 7 in circulating tumor cells has been associated with resistance to abiraterone and enzalutamide in patients with metastatic castration resistant prostate cancer. We used a sensitive, whole blood reverse transcriptase-polymerase chain reaction assay that does not require circulating tumor cell enrichment to correlate outcomes of abiraterone with whole blood expression of AR-V7 and other prostate cancer associated transcripts. We assessed the expression of AR-V7, FOXA1, GRHL2, HOXB13, KLK2, KLK3 and TMPRSS2:ERG mRNA in 2.5 ml whole blood from each of 27 patients with metastatic castration resistant prostate cancer and 33 controls without cancer as the discovery cohort. Cycle threshold values of controls with the highest gene expression were set as the threshold for a positive test. Thresholds were then applied to a validation cohort of 37 patients with metastatic castration resistant prostate cancer who were commencing abiraterone. Gene expression was correlated with the prostate specific antigen response rate using the chi-square test, and with time to prostate specific antigen progression and overall survival using the log rank test. In the discovery cohort 3 of 27 patients (11.1%) with metastatic castration resistant prostate cancer were AR-V7 positive vs 4 of 37 (10.8%) in the validation cohort. In the validation cohort patients with a positive AR-V7 test had a lower prostate specific antigen response rate (0% vs 42%, p = 0.27) together with shorter median prostate specific antigen progression (0.7 vs 4.0months, p <0.001) and median overall survival (5.5 vs 22.1 months, p<0.001). Reverse transcriptase-polymerase chain reaction detection of AR-V7 transcripts in whole blood was associated with inferior outcomes in patients treated with abiraterone. These results reinforce the potential usefulness of AR-V7 as a prognostic and predictive biomarker for metastatic castration resistant prostate cancer.

A mutation in the tuft mouse disrupts TET1 activity and alters the expression of genes that are crucial for neural tube closure.

ABSTRACTGenetic variations affecting neural tube closure along the head result in malformations of the face and brain. Neural tube defects (NTDs) are among the most common birth defects in humans. We previously reported a mouse mutant called tuft that arose spontaneously in our wild-type 3H1 colony. Adult tuft mice present midline craniofacial malformations with or without an anterior cephalocele. In addition, affected embryos presented neural tube closure defects resulting in insufficient closure of the anterior neuropore or exencephaly. Here, through whole-genome sequencing, we identified a nonsense mutation in the Tet1 gene, which encodes a methylcytosine dioxygenase (TET1), co-segregating with the tuft phenotype. This mutation resulted in premature termination that disrupts the catalytic domain that is involved in the demethylation of cytosine. We detected a significant loss of TET enzyme activity in the heads of tuft embryos that were homozygous for the mutation and had NTDs. RNA-Seq transcriptome analysis indicated that multiple gene pathways associated with neural tube closure were dysregulated in tuft embryo heads. Among them, the expressions of Cecr2, Epha7 and Grhl2 were significantly reduced in some embryos presenting neural tube closure defects, whereas one or more components of the non-canonical WNT signaling pathway mediating planar cell polarity and convergent extension were affected in others. We further show that the recombinant mutant TET1 protein was capable of entering the nucleus and affected the expression of endogenous Grhl2 in IMCD-3 (inner medullary collecting duct) cells. These results indicate that TET1 is an epigenetic determinant for regulating genes that are crucial to closure of the anterior neural tube and its mutation has implications to craniofacial development, as presented by the tuft mouse.

Expression and clinical significance of GRHL2 in colorectal cancer

The purpose of this study was to detect the expression of GRHL2 in colorectal cancer (CRC) tissues, and to assess the relationship between GRHL2 expression and clinicopathological features. Immunohistochemistry was used to examine GRHL2 in 75 CRC tissues. GRHL2 mRNA and protein levels in the CRC tissues were also analyzed by qRT-PCR and Western blot. The relationship between GRHL2 and clinicopathological features was assessed by Pearson's chi-square (χ(2)) test. Positive immunoreactivity for GRHL2 was detected in the nuclei of CRC cells. GRHL2 expression was increased in CRC tissues compared withthat in the paired non-tumor tissues (61.3% vs. 44.0%, P<0.01). Moreover, qRT-PCR results showed that the relative expression level of GRHL2 mRNA in the colorectal cancer tissue was (2.64±0.35), significantly higher than that of normal mucosa tissue (1.19±0.23, P<0.001). The expression level of GRHL2 mRNA was higher in stage III-IV patients (2.84±0.36) than that of stage I-II cases (2.31±0.32, P<0.05). Western blot results also showed that the expression level of GRHL2 protein in the colorectal carcinoma tissue was significantly higher than that in the normal mucosa (P<0.05). GRHL2 expression was positively correlated with tumor size, TNM stage and Ki-67 (P<0.05, respectively). Taking together, our findings demonstrate that GRHL2 is overexpressed in CRC, and plays an important role in the progression of CRC.

Downregulation of GRHL2 inhibits the proliferation of colorectal cancer cells by targeting ZEB1

Previous reports have associated GRHL2 with tumor progression. However, the biological role of GRHL2 in human colorectal cancer (CRC) has not been explored. We examined the expression of GRHL2 in 75 CRC samples, as well as the paired non-tumor tissues, by immunohistochemistry, qRT-PCR, and western blot analysis. The association between GRHL2 expression and various clinicopathological parameters including Ki-67, a marker of proliferative activity, was also evaluated. We performed lentivirus-mediated shRNA transfection to knock down GRHL2 gene expression in HT29 and HCT116 CRC cells. Cell proliferation was examined by the CCK-8 (Cell Counting Kit-8) assay, colony formation, and cell cycle assay in vitro. Tumorigenesis in vivo was assessed using a mouse xenograft model. Moreover, we transiently silenced ZEB1 expression in GRHL2-knockdown CRC cells using specific shRNA, and then examined the effects on GRHL2 and E-cadherin expression, as well as cell proliferation. Herein, we demonstrated that enhanced GRHL2 expression was detected in CRC, and correlated with higher levels of Ki-67 staining, larger tumor size, and advanced clinical stage. Knocking down GRHL2 in HT29 and HCT116 CRC cells significantly inhibited cell proliferation by decreasing the number of cells in S phase and increasing that in the G0/G1 phaseof the cell cycle. This resulted in inhibition of tumorigenesis in vivo, as well as increased expression of ZEB1. Furthermore, transient ZEB1 knockdown dramatically enhanced cell proliferation and increased GRHL2 and E-cadherin expression. Collectively, our study has identified ZEB1 as a target of GRHL2 and suggested a reciprocal GRHL2-ZEB1 repressive relationship, providing a novel mechanism through which proliferation may be modulated in CRC cells.

Expression and role of grainyhead-like 2 in gastric cancer

The aim of this study was to evaluate the expression and role of Grhl2 in gastric cancer. Immunohistochemistry was performed to explore the expression of Grhl2 in gastric cancer and surrounding non-tumor tissues. Moreover, the mRNA and protein expression level of Grhl2 in human immortalized gastric epithelial cell line GES-1 and four gastric cancer cell lines (MGC803, SGC7901, MKN45, HGC27) were detected by qRT-PCR and Western blotting, respectively. To further investigate the role of Grhl2 in gastric cancer as well as the potential mechanisms, SGC7901 cells were transfected with lentiviral constructs expressing Grhl2 or empty vector, and then proliferation and apoptosis of SGC7901 cells were evaluated by MTT assay and flow cytometry, respectively. Finally, the protein expression level of c-Myc and Bcl-2 was detected by Western blotting. Both mRNA and protein expression level of Grhl2 were significantly downregulated in gastric cancer. Exogenous Grhl2 transduced into SGC7901 cells significantly inhibited the proliferation and promoted apoptosis. Meanwhile, over-expression of Grhl2 decreased c-Myc and Bcl-2 protein expression level. Taken together, our results demonstrated that Grhl2 downregulated in gastric cancer and may function as a tumor suppressor and play an important role in the development and progression of gastric cancer. These results may provide a new clue for treatment for gastric cancer.

Correction: Grhl2 Determines the Epithelial Phenotype of Breast Cancers and Promotes Tumor Progression

[This corrects the article on p. e50781 in vol. 7.].

Grhl2 Determines the Epithelial Phenotype of Breast Cancers and Promotes Tumor Progression

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.

Abstract 3436: Suppression of the epithelial-mesenchymal transition (EMT) by a wound-healing gene, Grainyhead-like-2

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 (EMT) 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, by 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, and provide a novel molecular focus for the wound-healing/oncogenic EMT relationship. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3436. doi:1538-7445.AM2012-3436

The unique and cooperative roles of the Grainy head-like transcription factors in epidermal development reflect unexpected target gene specificity

The Grainy head-like 3 (Grhl3) gene encodes a transcription factor that plays essential roles in epidermal morphogenesis during embryonic development, with deficient mice exhibiting failed skin barrier formation, defective wound repair, and loss of eyelid fusion. Despite sharing significant sequence homology, overlapping expression patterns, and an identical core consensus DNA binding site, the other members of the Grhl family (Grhl1 and -2) fail to compensate for the loss of Grhl3 in these processes. Here, we have employed diverse genetic models, coupled with biochemical studies, to define the inter-relationships of the Grhl factors in epidermal development. We show that Grhl1 and Grhl3 have evolved complete functional independence, as evidenced by a lack of genetic interactions in embryos carrying combinations of targeted alleles of these genes. In contrast, compound heterozygous Grhl2/Grhl3 embryos displayed failed wound repair, and loss of a single Grhl2 allele in Grhl3-null embryos results in fully penetrant eyes open at birth. Expression of Grhl2 from the Grhl3 locus in homozygous knock-in mice corrects the wound repair defect, but these embryos still display a complete failure of skin barrier formation. This functional dissociation is due to unexpected differences in target gene specificity, as both GRHL2 and GRHL3 bind to and regulate expression of the wound repair gene Rho GEF 19, but regulation of the barrier forming gene, Transglutaminase 1 (TGase1), is unique to GRHL3. Our findings define the mechanisms underpinning the unique and cooperative roles of the Grhl genes in epidermal development.