Tumor-associated macrophages (TAMs) depend on ZEB1 for their cancer-promoting roles.

Accumulation of tumor-associated macrophages (TAMs) associates with malignant progression in cancer. However, the mechanisms that drive the pro-tumor functions of TAMs are not fully understood. ZEB1 is best known for driving an epithelial-to-mesenchymal transition (EMT) in cancer cells to promote tumor progression. However, a role for ZEB1 in macrophages and TAMs has not been studied. Here we describe that TAMs require ZEB1 for their tumor-promoting and chemotherapy resistance functions in a mouse model of ovarian cancer. Only TAMs that expressed full levels of Zeb1 accelerated tumor growth. Mechanistically, ZEB1 expression in TAMs induced their polarization toward an F4/80low pro-tumor phenotype, including direct activation of Ccr2 In turn, expression of ZEB1 by TAMs induced Ccl2, Cd74, and a mesenchymal/stem-like phenotype in cancer cells. In human ovarian carcinomas, TAM infiltration and CCR2 expression correlated with ZEB1 in tumor cells, where along with CCL2 and CD74 determined poorer prognosis. Importantly, ZEB1 in TAMs was a factor of poorer survival in human ovarian carcinomas. These data establish ZEB1 as a key factor in the tumor microenvironment and for maintaining TAMs' tumor-promoting functions.

Regulation of muscle atrophy-related genes by the opposing transcriptional activities of ZEB1/CtBP and FOXO3.

Multiple physiopathological and clinical conditions trigger skeletal muscle atrophy through the induction of a group of proteins (atrogenes) that includes components of the ubiquitin-proteasome and autophagy-lysosomal systems. Atrogenes are induced by FOXO transcription factors, but their regulation is still not fully understood. Here, we showed that the transcription factor ZEB1, best known for promoting tumor progression, inhibits muscle atrophy and atrogene expression by antagonizing FOXO3-mediated induction of atrogenes. Compared to wild-type counterparts, hindlimb immobilization in Zeb1-deficient mice resulted in enhanced muscle atrophy and higher expression of a number of atrogenes, including Atrogin-1/Fbxo32, MuRF1/Trim63, Ctsl, 4ebp1, Gabarapl1, Psma1 and Nrf2. Likewise, in the C2C12 myogenic cell model, ZEB1 knockdown augmented both myotube diameter reduction and atrogene upregulation in response to nutrient deprivation. Mechanistically, ZEB1 directly represses in vitro and in vivo Fbxo32 and Trim63 promoter transcription in a stage-dependent manner and in a reverse pattern with MYOD1. ZEB1 bound to the Fbxo32 promoter in undifferentiated myoblasts and atrophic myotubes, but not in non-atrophic myotubes, where it is displaced by MYOD1. ZEB1 repressed both promoters through CtBP-mediated inhibition of FOXO3 transcriptional activity. These results set ZEB1 as a new target in therapeutic approaches to clinical conditions causing muscle mass loss.

ZEB1 promotes inflammation and progression towards inflammation-driven carcinoma through repression of the DNA repair glycosylase MPG in epithelial cells.

OBJECTIVE: Chronic inflammation is a risk factor in colorectal cancer (CRC) and reactive oxygen species (ROS) released by the inflamed stroma elicit DNA damage in epithelial cells. We sought to identify new drivers of ulcerative colitis (UC) and inflammatory CRC. DESIGN: The study uses samples from patients with UC, mouse models of colitis and CRC and mice deficient for the epithelial-to-mesenchymal transition factor ZEB1 and the DNA repair glycosylase N-methyl-purine glycosylase (MPG). Samples were analysed by immunostaining, qRT-PCR, chromatin immunoprecipitation assays, microbiota next-generation sequencing and ROS determination. RESULTS: ZEB1 was induced in the colonic epithelium of UC and of mouse models of colitis. Compared with wild-type counterparts, Zeb1-deficient mice were partially protected from experimental colitis and, in a model of inflammatory CRC, they developed fewer tumours and exhibited lower levels of DNA damage (8-oxo-dG) and higher expression of MPG. Knockdown of ZEB1 in CRC cells inhibited 8-oxo-dG induction by oxidative stress (H2O2) and inflammatory cytokines (interleukin (IL)1ß). ZEB1 bound directly to the MPG promoter whose expression inhibited. This molecular mechanism was validated at the genetic level and the crossing of Zeb1-deficient and Mpg-deficient mice reverted the reduced inflammation and tumourigenesis in the former. ZEB1 expression in CRC cells induced ROS and IL1ß production by macrophages that, in turn, lowered MPG in CRC cells thus amplifying a positive loop between both cells to promote DNA damage and inhibit DNA repair. CONCLUSIONS: ZEB1 promotes colitis and inflammatory CRC through the inhibition of MPG in epithelial cells, thus offering new therapeutic strategies to modulate inflammation and inflammatory cancer.

ZEB1-induced tumourigenesis requires senescence inhibition via activation of DKK1/mutant p53/Mdm2/CtBP and repression of macroH2A1.

OBJECTIVE: Understand the role of ZEB1 in the tumour initiation and progression beyond inducing an epithelial-to-mesenchymal transition. DESIGN: Expression of the transcription factor ZEB1 associates with a worse prognosis in most cancers, including colorectal carcinomas (CRCs). The study uses survival analysis, in vivo mouse transgenic and xenograft models, gene expression arrays, immunostaining and gene and protein regulation assays. RESULTS: The poorer survival determined by ZEB1 in CRCs depended on simultaneous high levels of the Wnt antagonist DKK1, whose expression was transcriptionally activated by ZEB1. In cancer cells with mutant TP53, ZEB1 blocked the formation of senescence-associated heterochromatin foci at the onset of senescence by triggering a new regulatory cascade that involves the subsequent activation of DKK1, mutant p53, Mdm2 and CtBP to ultimately repress macroH2A1 (H2AFY). In a transgenic mouse model of colon cancer, partial downregulation of Zeb1 was sufficient to induce H2afy and to trigger in vivo tumour senescence, thus resulting in reduced tumour load and improved survival. The capacity of ZEB1 to induce tumourigenesis in a xenograft mouse model requires the repression of H2AFY by ZEB1. Lastly, the worst survival effect of ZEB1 in patients with CRC ultimately depends on low expression of H2AFY and of senescence-associated genes. CONCLUSIONS: The tumourigenic capacity of ZEB1 depends on its inhibition of cancer cell senescence through the activation of a herein identified new molecular pathway. These results set ZEB1 as a potential target in therapeutic strategies aimed at inducing senescence.

Porphyromonas gingivalis initiates a mesenchymal-like transition through ZEB1 in gingival epithelial cells.

The oral anaerobe Porphyromonas gingivalis is associated with the development of cancers including oral squamous cell carcinoma (OSCC). Here, we show that infection of gingival epithelial cells with P. gingivalis induces expression and nuclear localization of the ZEB1 transcription factor, which controls epithelial-mesenchymal transition. P. gingivalis also caused an increase in ZEB1 expression as a dual species community with Fusobacterium nucleatum or Streptococcus gordonii. Increased ZEB1 expression was associated with elevated ZEB1 promoter activity and did not require suppression of the miR-200 family of microRNAs. P. gingivalis strains lacking the FimA fimbrial protein were attenuated in their ability to induce ZEB1 expression. ZEB1 levels correlated with an increase in expression of mesenchymal markers, including vimentin and MMP-9, and with enhanced migration of epithelial cells into matrigel. Knockdown of ZEB1 with siRNA prevented the P. gingivalis-induced increase in mesenchymal markers and epithelial cell migration. Oral infection of mice by P. gingivalis increased ZEB1 levels in gingival tissues, and intracellular P. gingivalis were detected by antibody staining in biopsy samples from OSCC. These findings indicate that FimA-driven ZEB1 expression could provide a mechanistic basis for a P. gingivalis contribution to OSCC.

Phosphorylation Regulates Functions of ZEB1 Transcription Factor.

ZEB1 transcription factor is important in both development and disease, including many TGFß-induced responses, and the epithelial-to-mesenchymal transition (EMT) by which many tumors undergo metastasis. ZEB1 is differentially phosphorylated in different cell types; however the role of phosphorylation in ZEB1 activity is unknown. Luciferase reporter studies and electrophoresis mobility shift assays (EMSA) show that a decrease in phosphorylation of ZEB1 increases both DNA-binding and transcriptional repression of ZEB1 target genes. Functional analysis of ZEB1 phosphorylation site mutants near the second zinc finger domain (termed ZD2) show that increased phosphorylation (due to either PMA plus ionomycin, or IGF-1) can inhibit transcriptional repression by either a ZEB1-ZD2 domain clone, or full-length ZEB1. This approach identifies phosphosites that have a substantial effect regulating the transcriptional and DNA-binding activity of ZEB1. Immunoprecipitation with anti-ZEB1 antibodies followed by western analysis with a phospho-Threonine-Proline-specific antibody indicates that the ERK consensus site at Thr-867 is phosphorylated in ZEB1. In addition to disrupting in vitro DNA-binding measured by EMSA, IGF-1-induced MEK/ERK phosphorylation is sufficient to disrupt nuclear localization of GFP-ZEB1 fusion clones. These data suggest that phosphorylation of ZEB1 integrates TGFß signaling with other signaling pathways such as IGF-1. J. Cell. Physiol. 231: 2205-2217, 2016. © 2016 Wiley Periodicals, Inc.

Cell type-specific transcriptome analysis reveals a major role for Zeb1 and miR-200b in mouse inner ear morphogenesis.

Cellular heterogeneity hinders the extraction of functionally significant results and inference of regulatory networks from wide-scale expression profiles of complex mammalian organs. The mammalian inner ear consists of the auditory and vestibular systems that are each composed of hair cells, supporting cells, neurons, mesenchymal cells, other epithelial cells, and blood vessels. We developed a novel protocol to sort auditory and vestibular tissues of newborn mouse inner ears into their major cellular components. Transcriptome profiling of the sorted cells identified cell type-specific expression clusters. Computational analysis detected transcription factors and microRNAs that play key roles in determining cell identity in the inner ear. Specifically, our analysis revealed the role of the Zeb1/miR-200b pathway in establishing epithelial and mesenchymal identity in the inner ear. Furthermore, we detected a misregulation of the ZEB1 pathway in the inner ear of Twirler mice, which manifest, among other phenotypes, malformations of the auditory and vestibular labyrinth. The association of misregulation of the ZEB1/miR-200b pathway with auditory and vestibular defects in the Twirler mutant mice uncovers a novel mechanism underlying deafness and balance disorders. Our approach can be employed to decipher additional complex regulatory networks underlying other hearing and balance mouse mutants.

EMT-activating transcription factors in cancer: beyond EMT and tumor invasiveness.

Cancer is a complex multistep process involving genetic and epigenetic changes that eventually result in the activation of oncogenic pathways and/or inactivation of tumor suppressor signals. During cancer progression, cancer cells acquire a number of hallmarks that promote tumor growth and invasion. A crucial mechanism by which carcinoma cells enhance their invasive capacity is the dissolution of intercellular adhesions and the acquisition of a more motile mesenchymal phenotype as part of an epithelial-to-mesenchymal transition (EMT). Although many transcription factors can trigger it, the full molecular reprogramming occurring during an EMT is mainly orchestrated by three major groups of transcription factors: the ZEB, Snail and Twist families. Upregulated expression of these EMT-activating transcription factors (EMT-ATFs) promotes tumor invasiveness in cell lines and xenograft mice models and has been associated with poor clinical prognosis in human cancers. Evidence accumulated in the last few years indicates that EMT-ATFs also regulate an expanding set of cancer cell capabilities beyond tumor invasion. Thus, EMT-ATFs have been shown to cooperate in oncogenic transformation, regulate cancer cell stemness, override safeguard programs against cancer like apoptosis and senescence, determine resistance to chemotherapy and promote tumor angiogenesis. This article reviews the expanding portfolio of functions played by EMT-ATFs in cancer progression.

The EMT factor ZEB1 paradoxically inhibits EMT in BRAF-mutant carcinomas.

Despite being in the same pathway, mutations of KRAS and BRAF in colorectal carcinomas (CRCs) determine distinct progression courses. ZEB1 induces an epithelial-to-mesenchymal transition (EMT) and is associated with worse progression in most carcinomas. Using samples from patients with CRC, mouse models of KrasG12D and BrafV600E CRC, and a Zeb1-deficient mouse, we show that ZEB1 had opposite functions in KRAS- and BRAF-mutant CRCs. In KrasG12D CRCs, ZEB1 was correlated with a worse prognosis and a higher number of larger and undifferentiated (mesenchymal or EMT-like) tumors. Surprisingly, in BrafV600E CRC, ZEB1 was associated with better prognosis; fewer, smaller, and more differentiated (reduced EMT) primary tumors; and fewer metastases. ZEB1 was positively correlated in KRAS-mutant CRC cells and negatively in BRAF-mutant CRC cells with gene signatures for EMT, cell proliferation and survival, and ERK signaling. On a mechanistic level, ZEB1 knockdown in KRAS-mutant CRC cells increased apoptosis and reduced clonogenicity and anchorage-independent growth; the reverse occurred in BRAFV600E CRC cells. ZEB1 is associated with better prognosis and reduced EMT signature in patients harboring BRAF CRCs. These data suggest that ZEB1 can function as a tumor suppressor in BRAF-mutant CRCs, highlighting the importance of considering the KRAS/BRAF mutational background of CRCs in therapeutic strategies targeting ZEB1/EMT.

Metastatic progression of prostate cancer and e-cadherin regulation by zeb1 and SRC family kinases.

Expression of E-cadherin is used to monitor the epithelial phenotype, and its loss is suggestive of epithelial-mesenchymal transition (EMT). EMT triggers tumor metastasis. Exit from EMT is marked by increased E-cadherin expression and is considered necessary for tumor growth at sites of metastasis; however, the mechanisms associated with exit from EMT are poorly understood. Herein are analyzed 185 prostate cancer metastases, with significantly higher E-cadherin expression in bone than in lymph node and soft tissue metastases. To determine the molecular mechanisms of regulation of E-cadherin expression, three stable isogenic cell lines from DU145 were derived that differ in structure, migration, and colony formation on soft agar and Matrigel. When injected into mouse tibia, the epithelial subline grows most aggressively, whereas the mesenchymal subline does not grow. In cultured cells, ZEB1 and Src family kinases decrease E-cadherin expression. In contrast, in tibial xenografts, E-cadherin RNA levels increase eight- to 10-fold despite persistent ZEB1 expression, and in all ZEB1-positive metastases (10 of 120), ZEB1 and E-cadherin proteins were co-expressed. These data suggest that transcriptional regulation of E-cadherin differs in cultured cells versus xenografts, which more faithfully reflect E-cadherin regulation in cancers in human beings. Furthermore, the aggressive nature of xenografts positive for E-cadherin and the frequency of metastases positive for E-cadherin suggest that high E-cadherin expression in metastatic prostate cancer is associated with aggressive tumor growth.

Novel ZEB1 expression in bladder tumorigenesis.

UNLABELLED: What's known on the subject? and What does the study add? Epithelial-mesenchymal transition (EMT) is involved in tumor progression where the underlying cellular changes associated with EMT have been identified in in vitro models and confirmed in a limited number of in vivo studies. ZEB1, which targets E-cadherin repression, is a transcriptional regulator that has been implicated in EMT, and is associated with uterine and colorectal cancers. Regulation of ZEB1 expression has been shown to involve different microRNAs (miRNAs), identifying a potential role for miRNA in EMT. In the present study we have identified novel expression of ZEB1 in bladder tumours and shown a role for ZEB1 in enhanced migration and invasion potential in in vitro assays. Confirmation of ZEB1 expression in bladder tumours was shown in tissue microarrays (TMAs). OBJECTIVE: To evaluate ZEB1 expression in bladder tumorigenesis and define a possible role for this transcription factor in urothelial carcinomas of the bladder (UCBs). MATERIALS AND METHODS: Five hundred and fifty-eight samples were assembled in 10 tissue microarrays (TMAs; 263 non-muscle-invasive Ta/T1/Tis, 295 muscle-invasive T2-T4). All tumours were transitional cell carcinomas (TCCs) and processed for immunohistochemistry to assess nuclear ZEB1 expression. Expression levels of ZEB1 were modulated in bladder carcinoma cell lines CUBIII or UM-UC-3 after forced expression or shRNA knockdown, respectively. Protein expression levels were determined using western blot analysis and transfectants were assessed for migration and invasion potential in standard in vitro assays. RESULTS: Nuclear ZEB1 expression was recorded in 22.8% of non-muscle-invasive UCBs and 21.7% of muscle-invasive UCBs, including 24.1% grade I/II and 21.1% grade III tumours, and absent in normal bladder mucosa. No significant correlation was observed for tumour stage and grade, nodal involvement, vascular invasion, metastasis and overall or cancer-specific survival. The introduction or knockdown of ZEB1 expression in bladder carcinoma cell lines showed enhanced or reduced migration and invasive potential, respectively. Changes in ZEB1 expression were accompanied by altered microRNA (miRNA) expression underlying events linked to epithelial-mesenchymal transition (EMT). CONCLUSION: The results in the present study showed novel expression of ZEB1 in bladder cancer in the absence of a link to clinical variables of change, including metastasis and survival. However, in vitro assays showed enhanced or reduced migration and invasion after the introduction or reduction of ZEB1, respectively, in transfected bladder cell lines. Modulation in expression of ZEB1 was closely linked to changes in the miR-200 family along with alternative known prognostic indicators of bladder tumour progression.

Mesenchymal cell remodeling during mouse secondary palate reorientation.

The formation of mammalian secondary palate requires a series of developmental events such as growth, elevation, and fusion. Despite recent advances in the field of palate development, the process of palate elevation remains poorly understood. The current consensus on palate elevation is that the distal end of the vertical palatal shelf corresponds to the medial edge of the elevated horizontal palatal shelf. We provide evidence suggesting that the prospective medial edge of the vertical palate is located toward the interior side (the side adjacent to the tongue), instead of the distal end, of the vertical palatal shelf and that the horizontal palatal axis is generated through palatal outgrowth from the side of the vertical palatal shelf rather than rotating the pre-existing vertical axis orthogonally. Because palate elevation represents a classic example of embryonic tissue re-orientation, our findings here may also shed light on the process of tissue re-orientation in general.

The EMT Transcription Factor ZEB2 Promotes Proliferation of Primary and Metastatic Melanoma While Suppressing an Invasive, Mesenchymal-Like Phenotype.

Epithelial-to-mesenchymal transition (EMT)-inducing transcription factors (TF) are well known for their ability to induce mesenchymal states associated with increased migratory and invasive properties. Unexpectedly, nuclear expression of the EMT-TF ZEB2 in human primary melanoma has been shown to correlate with reduced invasion. We report here that ZEB2 is required for outgrowth for primary melanomas and metastases at secondary sites. Ablation of Zeb2 hampered outgrowth of primary melanomas in vivo, whereas ectopic expression enhanced proliferation and growth at both primary and secondary sites. Gain of Zeb2 expression in pulmonary-residing melanoma cells promoted the development of macroscopic lesions. In vivo fate mapping made clear that melanoma cells undergo a conversion in state where ZEB2 expression is replaced by ZEB1 expression associated with gain of an invasive phenotype. These findings suggest that reversible switching of the ZEB2/ZEB1 ratio enhances melanoma metastatic dissemination. SIGNIFICANCE: ZEB2 function exerts opposing behaviors in melanoma by promoting proliferation and expansion and conversely inhibiting invasiveness, which could be of future clinical relevance. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/14/2983/F1.large.jpg.

ZEB1 protects skeletal muscle from damage and is required for its regeneration.

The mechanisms linking muscle injury and regeneration are not fully understood. Here we report an unexpected role for ZEB1 regulating inflammatory and repair responses in dystrophic and acutely injured muscles. ZEB1 is upregulated in the undamaged and regenerating myofibers of injured muscles. Compared to wild-type counterparts, Zeb1-deficient injured muscles exhibit enhanced damage that corresponds with a retarded p38-MAPK-dependent transition of their macrophages towards an anti-inflammatory phenotype. Zeb1-deficient injured muscles also display a delayed and poorer regeneration that is accounted by the retarded anti-inflammatory macrophage transition and their intrinsically deficient muscle satellite cells (MuSCs). Macrophages in Zeb1-deficient injured muscles show lower phosphorylation of p38 and its forced activation reverts the enhanced muscle damage and poorer regeneration. MuSCs require ZEB1 to maintain their quiescence, prevent their premature activation following injury, and drive efficient regeneration in dystrophic muscles. These data indicate that ZEB1 protects muscle from damage and is required for its regeneration.

ZEB1 expression in type I vs type II endometrial cancers: a marker of aggressive disease.

Zinc-finger E-box-binding homeobox 1 (ZEB1) is a transcription factor containing two clusters of Kruppel-type zinc-fingers, by which it binds E-box-like sequences on target DNAs. A role for ZEB1 in tumor progression, specifically, epithelial to mesenchymal transitions, has recently been revealed. ZEB1 acts as a master repressor of E-cadherin and other epithelial markers. We previously demonstrated that ZEB1 is confined to the stromal compartment in normal endometrium and low-grade endometrial cancers. Here, we quantify ZEB1 protein expression in endometrial samples from 88 patients and confirm that it is expressed at significantly higher levels in the tumor-associated stroma of low-grade endometrioid adenocarcinomas (type I endometrial cancers) compared to hyperplastic or normal endometrium. In addition, as we previously reported, ZEB1 is aberrantly expressed in the epithelial-derived tumor cells of highly aggressive endometrial cancers, such as FIGO grade 3 endometrioid adenocarcinomas, uterine serous carcinomas, and malignant mixed Müllerian tumors (classified as type II endometrial cancers). We now demonstrate, in both human endometrial cancer specimens and cell lines, that when ZEB1 is inappropriately expressed in epithelial-derived tumor cells, E-cadherin expression is repressed, and that this inverse relationship correlates with increased migratory and invasive potential. Forced expression of ZEB1 in the nonmigratory, low-grade, relatively differentiated Ishikawa cell line renders them migratory. Conversely, reduction of ZEB1 in a highly migratory and aggressive type II cell line, Hec50co, results in reduced migratory capacity. Thus, ZEB1 may be a biomarker of aggressive endometrial cancers at high risk of recurrence. It may help identify women who would most benefit from chemotherapy. Furthermore, if expression of ZEB1 in type II endometrial cancers could be reversed, it might be exploited as therapy for these highly aggressive tumors.

Zeb1 mutant mice as a model of posterior corneal dystrophy.

PURPOSE: The zinc finger transcription factor Zeb1 binds to E-box-like sequences and is important for maintaining repression of epithelial specification genes in vivo. Overexpression of Zeb1 in cancer triggers epithelial-mesenchymal transition, which facilitates metastasis. The mutation of ZEB1 in humans is linked to posterior polymorphous corneal dystrophy (PPCD), in which an epithelial transition of the corneal endothelium is associated with abnormal endothelial proliferation. The purpose of this study is to determine whether Zeb1 null or heterozygous mice may provide an animal model for PPCD. METHODS: Corneal morphology, protein and mRNA expression, and cell proliferation were compared in wild-type and Zeb1 gene knockout mice by immunostaining, real-time PCR, and BrdU incorporation. mRNA expression in isolated embryo fibroblasts derived from wild-type, Zeb1 heterozygous, and null mice was analyzed by real-time PCR RESULTS: Zeb1 null mice late in gestation show ectopic expression of epithelial genes in the corneal endothelium and keratocytes, including the basement membrane component COL4A3, which is ectopically expressed by the corneal endothelium in PPCD. These embryos also show abnormal corneal endothelial and keratocyte proliferation, corneal thickening, and corneolenticular and iridocorneal adhesions. Adult Zeb1 heterozygous mice exhibit these same corneal defects. The ectopic expression of epithelial genes extended to embryonic fibroblasts derived from Zeb1 heterozygous and null mice, suggesting that Zeb1 may have a more general role in the suppression of an epithelial phenotype. CONCLUSIONS: The authors conclude that Zeb1 heterozygous and null mice show features of PPCD and thus should provide an animal model for genetic dissection of pathways contributing to the disease.

The zinc finger transcription factor ZFHX1A is linked to cell proliferation by Rb-E2F1.

ZFHX1A is expressed in proliferating cells in the developing embryo, and in the present study we provide evidence that its expression is confined to proliferating cells through dependence on the Rb (retinoblastoma protein) family/E2F cell cycle pathway. Mutation of the Rb or E2F1 genes lead to induction of ZFHX1A mRNA, implying that the Rb-E2F1 repressor complex is important for repression of ZFHX1A. This repression is associated with recruitment of an E2F-Rb-histone deacetylase repressor complex to the promoter. A dominant-negative form of E2F1 inhibited ZFHX1A expression in p16INK4a- cells where Rb is constitutively hyperphosphorylated and inactive, suggesting that E2F can contribute to ZFHX1A transactivation in the absence of functional Rb. ZFHX1A is an E-box-binding transcription factor whose binding sites overlap with those bound by Snail1 and 2, and ZFHX1B/SIP1 (leading to at least partially overlapping function; for example, each of the proteins can repress E-cadherin expression). We found that expression of Snail1 and ZFHX1B/SIP1 is also regulated by E2Fs, but in contrast with ZFHX1A this regulation is Rb-family-independent. Snail2 expression was unaffected by either E2F or the Rb family. We propose that the differential effects of the Rb family/E2F pathway on expression of these E-box-binding proteins are important in maintaining their distinct patterns (and thus distinct functions) during embryogenesis.

Estrogen enhances gonadotropin-releasing hormone-stimulated transcription of the luteinizing hormone subunit promoters via altered expression of stimulatory and suppressive transcription factors.

Transcription of the LH subunit genes is stimulated by GnRH and may be modulated physiologically by steroids such as 17beta-estradiol (E). We found that E treatment amplified GnRH stimulation of the rat LHbeta and alpha-subunit promoters, and expression of the endogenous mRNA, in LbetaT2 gonadotrope cells 2- to 5-fold above GnRH alone. We examined gene expression in LbetaT2 cells after E and/or GnRH treatment, and found that E suppressed expression of transcription factor Zfhx1a, and enhanced GnRH stimulation of Egr-1 mRNA and protein. E effects were abolished in the presence of antiestrogen. Egr-1 is critical for LHbeta expression; however, the role of Zfhx1a, which binds to E-box sequences, was untested. We found E-box motifs in both the rat LHbeta (-381, -182, and -15 bp) and alpha-subunit (-292, -64, -58 bp) promoters. Zfhx1a overexpression suppressed basal and GnRH-stimulated activity of both promoters. Mutation of the alpha-subunit promoter E boxes at either -64 or -58 bp eliminated Zfhx1a suppression, whereas mutation of the -292 bp E box had no effect. Gel shift assays demonstrated that Zfhx1a bound to the -64 and -58, but not -292, bp E-box DNA. Similarly, mutation of LHbeta promoter E boxes at either -381 or -182, but not -15, bp reduced Zfhx1a suppression, correlating with binding of Zfhx1a. The -381 bp LHbeta E box overlaps with an Sp1 binding site in the distal GnRH-stimulatory region, and increased Sp1 expression overcame Zfhx1a suppression. Thus, one mechanism by which E may enhance GnRH-stimulated LH subunit promoter activity is through regulation of both activators and suppressors of transcription.

ZEB1 Regulates Multiple Oncogenic Components Involved in Uveal Melanoma Progression.

Human uveal melanoma (UM) is a major ocular malignant tumor with high risk of metastasis and requires multiple oncogenic factors for progression. ZEB1 is a zinc finger E-box binding transcription factor known for participating epithelial-mesenchymal transition (EMT), a critical cellular event for metastasis of malignant tumors of epithelium origin. ZEB1 is also expressed in UM and high expression of ZEB1 correlates with UM advancement, but has little effect on cell morphology. We show that spindle UM cells can become epithelioid but not vice versa; and ZEB1 exerts its tumorigenic effects by promoting cell dedifferentiation, proliferation, invasiveness, and dissemination. We provide evidence that ZEB1 binds not only to repress critical genes involving in pigment synthesis, mitosis, adherent junctions, but also to transactivate genes involving in matrix degradation and cellular locomotion to propel UM progression towards metastasis. We conclude that ZEB1 is a major oncogenic factor required for UM progression and could be a potential therapeutic target for treating UM in the clinic.

Interplay between Notch1 and Notch3 promotes EMT and tumor initiation in squamous cell carcinoma.

Notch1 transactivates Notch3 to drive terminal differentiation in stratified squamous epithelia. Notch1 and other Notch receptor paralogs cooperate to act as a tumor suppressor in squamous cell carcinomas (SCCs). However, Notch1 can be stochastically activated to promote carcinogenesis in murine models of SCC. Activated form of Notch1 promotes xenograft tumor growth when expressed ectopically. Here, we demonstrate that Notch1 activation and epithelial-mesenchymal transition (EMT) are coupled to promote SCC tumor initiation in concert with transforming growth factor (TGF)-ß present in the tumor microenvironment. We find that TGFß activates the transcription factor ZEB1 to repress Notch3, thereby limiting terminal differentiation. Concurrently, TGFß drives Notch1-mediated EMT to generate tumor initiating cells characterized by high CD44 expression. Moreover, Notch1 is activated in a small subset of SCC cells at the invasive tumor front and predicts for poor prognosis of esophageal SCC, shedding light upon the tumor promoting oncogenic aspect of Notch1 in SCC.