Retinal Determination Gene Network Research Articles

Vestigial organs alter fossil placements in an ancient group of terrestrial chelicerates

Vestigial organs provide a link between ancient and modern traits and therefore have great potential to resolve the phylogeny of contentious fossils that bear features not seen in extant species. Here we show that extant daddy-longlegs (Arachnida, Opiliones), a group once thought to possess only one pair of eyes, in fact additionally retain a pair of vestigial median eyes and a pair of vestigial lateral eyes. Neuroanatomical gene expression surveys of eye-patterning transcription factors, opsins, and other structural proteins in the daddy-longlegs Phalangium opilio show that the vestigial median and lateral eyes innervate regions of the brain positionally homologous to the median and lateral eye neuropils, respectively, of chelicerate groups like spiders and horseshoe crabs. Gene silencing of eyes absent shows that the vestigial eyes are under the control of the retinal determination gene network. Gene silencing of dachshund disrupts the lateral eyes, but not the median eyes, paralleling loss-of-function phenotypes in insect models. The existence of lateral eyes in extant daddy-longlegs bears upon the placement of the oldest harvestmen fossils, a putative stem group that possessed both a pair of median eyes and a pair of lateral eyes. Phylogenetic analysis of harvestman relationships with an updated understanding of lateral eye incidence resolved the four-eyed fossil group as a member of the extant daddy-longlegs suborder, which in turn resulted in older estimated ages of harvestman diversification. This work underscores that developmental vestiges in extant taxa can influence our understanding of character evolution, placement of fossils, and inference of divergence times.

Systemic paralogy and function of retinal determination network homologs in arachnids

BackgroundArachnids are important components of cave ecosystems and display many examples of troglomorphisms, such as blindness, depigmentation, and elongate appendages. Little is known about how the eyes of arachnids are specified genetically, let alone the mechanisms for eye reduction and loss in troglomorphic arachnids. Additionally, duplication of Retinal Determination Gene Network (RDGN) homologs in spiders has convoluted functional inferences extrapolated from single-copy homologs in pancrustacean models.ResultsWe investigated a sister species pair of Israeli cave whip spiders, Charinus ioanniticus and C. israelensis (Arachnopulmonata, Amblypygi), of which one species has reduced eyes. We generated embryonic transcriptomes for both Amblypygi species, and discovered that several RDGN homologs exhibit duplications. We show that duplication of RDGN homologs is systemic across arachnopulmonates (arachnid orders that bear book lungs), rather than being a spider-specific phenomenon. A differential gene expression (DGE) analysis comparing the expression of RDGN genes in field-collected embryos of both species identified candidate RDGN genes involved in the formation and reduction of eyes in whip spiders. To ground bioinformatic inference of expression patterns with functional experiments, we interrogated the function of three candidate RDGN genes identified from DGE using RNAi in the spider Parasteatoda tepidariorum. We provide functional evidence that one of these paralogs, sine oculis/Six1 A (soA), is necessary for the development of all arachnid eye types.ConclusionsOur work establishes a foundation to investigate the genetics of troglomorphic adaptations in cave arachnids, and links differential gene expression to an arthropod eye phenotype for the first time outside of Pancrustacea. Our results support the conservation of at least one RDGN component across Arthropoda and provide a framework for identifying the role of gene duplications in generating arachnid eye diversity.

RDGN-based predictive model for the prognosis of breast cancer

BackgroundBreast cancer is the most diagnosed malignancy in females in the United States. The members of retinal determination gene network (RDGN) including DACH, EYA, as well as SIX families participate in the proliferation, apoptosis, and metastasis of multiple tumors including breast cancer. A comprehensive predictive model of RDGN might be helpful to herald the prognosis of breast cancer patients.MethodsIn this study, the Gene Expression Ominibus (GEO) and Gene Set Expression Analysis (GSEA) algorithm were used to investigate the effect of RDGN members on downstream signaling pathways. Besides, based on The Cancer Genome Atlas (TCGA) database, we explored the expression patterns of RDGN members in tumors, normal tissues, and different breast cancer subtypes. Moreover, we estimated the relationship between RDGN members and the outcomes of breast cancer patients. Lastly, we constructed a RDGN-based predictive model by Cox proportional hazard regression and verified the model in two separate GEO datasets.ResultsThe results of GSEA showed that the expression of DACH1 was negatively correlated with cell cycle and DNA replication pathways. On the contrary, the levels of EYA2 and SIX1 were significantly positively correlated with DNA replication, mTOR, and Wnt pathways. Further investigation in TCGA database indicated that DACH1 expression was lower in breast cancers especially basal-like subtype. In the meanwhile, SIX1 was remarkably upregulated in breast cancers while EYA2 level was increased in Basal-like and Her-2 enriched subtypes. Survival analyses demonstrated that DACH1 was a favorable factor while EYA2 and SIX1 were risk factors for breast cancer patients. Given the results of Cox proportional hazard regression analysis, two members of RDGN were involved in the present predictive model and patients with high model index had poorer outcomes.ConclusionThis study showed that aberrant RDGN expression was an unfavorable factor for breast cancer. This RDGN-based comprehensively framework was meaningful for predicting the prognosis of breast cancer patients.

Dach1 regulates neural crest migration during embryonic development

Dachshund 1(Dach1) is a key component of the retinal determination gene network that plays significant roles in cell fate regulation. The vertebrate homolog of Drosophila dachshund has gained considerable importance as an essential regulator of development, but its functions during embryonic development remain elusive. We investigated the functional significance of dach1 during Xenopus embryogenesis using loss-of-function studies. Reverse transcription-polymerase chain reaction demonstrated the maternal nature of dach1, showing enhanced expression at the neurula stage of development, and morpholino oligonucleotide injection of dach1 induced phenotypic anomalies of microcephaly and reduced body length. Animal cap assays followed by whole-mount in-situ hybridization indicated the perturbed expression of neural and neural crest (NC) markers. Our data suggest the prerequisite functions of dach1 in NC migration during Xenopus embryogenesis. However, the developmental pathways regulated by dach1 during embryogenesis require further elucidation.

SIX1 Activates STAT3 Signaling to Promote the Proliferation of Thyroid Carcinoma via EYA1.

As a critical member of the Retinal Determination Gene Network (RDGN), SIX1 has been regarded as a tumor promoter in various types of cancer. However, its role in papillary thyroid carcinoma (PTC) has never been investigated. In this study, thyroid carcinoma tissue microarray staining was employed to identify the expression patterns of SIX1 and its co-activator EYA1. Papillary thyroid cancer cell lines, BCPAP, and TPC-1 cells were used to investigate the potential mechanism of SIX1 in vitro and in vivo. Flow cytometry analysis, MTT assay, the growth curve assay, colony formation assay, EdU incorporation and xenograft assay were performed to demonstrate the role of SIX1 in the malignant change of PTC cells. Western blot and Real-time PCR were used to detect the interaction among the SIX1, EYA1, and STAT3 signaling. In comparison with normal tissue, high expressions of SIX1 and EYA1 were associated with a malignant tumor. Importantly, SIX1 strongly correlated with EYA1 in thyroid carcinoma tissue microarray. Functional assays indicated SIX1 increased EYA1 expression by stabilizing EYA1 at the post-transcriptional level. Besides, SIX1 promoted the proliferation and invasion of thyroid carcinoma via activation of STAT3 signaling and its downstream targets in an EYA1-dependent manner. SIX1 can integrate with EYA1 to contribute to PTC development via activation of the classical STAT3 signaling. These data suggested targeting the abnormal activation of the SIX1/EYA1 complex may represent a novel therapeutic strategy for advanced PTC patients.

The regulation of cytokine signaling by retinal determination gene network pathway in cancer.

Tumor environment plays a pivotal role in determining cancer biology characteristics. Cytokine factors, as a critical component in tumor milieu, execute distinct functions in the process of tumorigenesis and progression via the autocrine or paracrine manner. The retinal determination gene network (RDGN), which mainly comprised DACH, SIX, and EYA family members, is required for the organ development in mammalian species. While the aberrant expression of RDGN is involved in the proliferation, apoptosis, angiogenesis, and metastasis of tumors via interacting with different cytokine-related signals, such as CXCL8, IL-6, TGF-β, FGF, and VEGF, in a cell- or tissue-dependent manner. Thus, joint detection of this pathway might be used as a potential biomarker for the stratification of target therapy and for the precision prediction of the prognosis of cancer patients.

Homeodomain-interacting protein kinase phosphorylates the Drosophila Paired box protein 6 (Pax6) homologues Twin of eyeless and Eyeless.

Homeodomain-interacting protein kinase (Hipk), the Drosophila homologue of mammalian HIPK2, plays several important roles in regulating differentiation, proliferation, apoptosis, and stress responses and acts as a mediator for signals of diverse pathways, such as Notch or Wingless signalling. The Paired box protein 6 (Pax6) has two Drosophila homologues, Twin of eyeless (Toy) and Eyeless (Ey). Both stand atop the retinal determination gene network (RDGN), which is essential for proper eye development in Drosophila. Here, we set Hipk and the master regulators Toy and Ey in an enzyme-substrate relationship. Furthermore, we prove a physical interaction between Toy and Hipk in vivo using bimolecular fluorescence complementation. Using in vitro kinase assays with different truncated Toy constructs and mutational analyses, we mapped four Hipk phosphorylation sites of Toy, one in the paired domain (Ser121 ) and three in the C-terminal transactivation domain of Toy (Thr395 , Ser410 and Thr452 ). The interaction and phosphorylation of the master regulator Toy by Hipk may be important for precise tuning of signalling within the RDGN and therefore for Drosophila eye development.

Mutations that impair Eyes absent tyrosine phosphatase activity in vitro reduce robustness of retinal determination gene network output in Drosophila.

A limited collection of signaling networks and transcriptional effectors directs the full spectrum of cellular behaviors that comprise development. One mechanism to diversify regulatory potential is to combine multiple biochemical activities into the same protein. Exemplifying this principle of modularity, Eyes absent (Eya), originally identified as a transcriptional co-activator within the retinal determination gene network (RDGN), also harbors tyrosine and threonine phosphatase activities. Although mounting evidence argues for the importance of Eya’s phosphatase activities to mammalian biology, genetic rescue experiments in Drosophila have shown that the tyrosine phosphatase function is dispensable for normal development. In this study, we repeated these rescue experiments in genetically sensitized backgrounds in which the dose of one or more RDGN factor was reduced. Heterozygosity for sine oculis or dachshund, both core RDGN members, compromised the ability of phosphatase-dead eya, but not of the control wild type eya transgene, to rescue the retinal defects and reduced viability associated with eya loss. We speculate that Eya’s tyrosine phosphatase activity, although non-essential, confers robustness to RDGN output.

Eye Development in Sepia officinalis Embryo: What the Uncommon Gene Expression Profiles Tell Us about Eye Evolution.

In metazoans, there is a remarkable diversity of photosensitive structures; their shapes, physiology, optical properties, and development are different. To approach the evolution of photosensitive structures and visual function, cephalopods are particularly interesting organisms due to their most highly centralized nervous system and their camerular eyes which constitute a convergence with those of vertebrates. The eye morphogenesis in numerous metazoans is controlled mainly by a conserved Retinal Determination Gene Network (RDGN) including pax, six, eya, and dac playing also key developmental roles in non-retinal structures and tissues of vertebrates and Drosophila. Here we have identified and explored the role of Sof-dac, Sof-six1/2, Sof-eya in eye morphogenesis, and nervous structures controlling the visual function in Sepia officinalis. We compare that with the already shown expressions in eye development of Sof-otx and Sof-pax genes. Rhodopsin is the pigment responsible for light sensitivity in metazoan, which correlate to correlate visual function and eye development. We studied Sof-rhodopsin expression during retina differentiation. By in situ hybridization, we show that (1) all of the RDGN genes, including Sof-pax6, are expressed in the eye area during the early developmental stages but they are not expressed in the retina, unlike Sof-otx, which could have a role in retina differentiation; (2) Sof-rhodopsin is expressed in the retina just before vision gets functional, from stage 23 to hatching. Our results evidence a role of Sof-six1/2, Sof-eya, and Sof-dac in eye development. However, the gene network involved in the retinal photoreceptor differentiation remains to be determined. Moreover, for the first time, Sof-rhodopsin expression is shown in the embryonic retina of cuttlefish suggesting the evolutionary conservation of the role of rhodopsin in visual phototransduction within metazoans. These findings are correlated with the physiological and behavioral observations suggesting that S. officinalis is able to react to light stimuli from stage 25 of organogenesis on, as soon as the first retinal pigments appear.

Retinal determination genes coordinate neuroepithelial specification and neurogenesis modes in the Drosophila optic lobe.

Differences in neuroepithelial patterning and neurogenesis modes contribute to area-specific diversifications of neural circuits. In the Drosophila visual system, two neuroepithelia, the outer (OPC) and inner (IPC) proliferation centers, generate neuron subtypes for four ganglia in several ways. Whereas neuroepithelial cells in the medial OPC directly convert into neuroblasts, in an IPC subdomain they generate migratory progenitors by epithelial-mesenchymal transition that mature into neuroblasts in a second proliferative zone. The molecular mechanisms that regulate the identity of these neuroepithelia, including their neurogenesis modes, remain poorly understood. Analysis of Polycomblike revealed that loss of Polycomb group-mediated repression of the Hox gene Abdominal-B (Abd-B) caused the transformation of OPC to IPC neuroepithelial identity. This suggests that the neuroepithelial default state is IPC-like, whereas OPC identity is derived. Ectopic Abd-B blocks expression of the highly conserved retinal determination gene network members Eyes absent (Eya), Sine oculis (So) and Homothorax (Hth). These factors are essential for OPC specification and neurogenesis control. Finally, eya and so are also sufficient to confer OPC-like identity, and, in parallel with hth, the OPC-specific neurogenesis mode on the IPC.

The retinal determination gene network: from developmental regulator to cancer therapeutic target.

Although originally identified for its function in Drosophila melanogaster eye specification, the Retinal Determination Gene Network (RDGN) is essential for the development of multiple organs in mammals. The RDGN regulates proliferation, differentiation and autocrine signaling, and interacts with other key signaling pathways. Aberrant expression of RDGN members such as DACH, EYA and SIX contributes to tumor initiation and progression; indeed, the levels of RDGN members are clinically prognostic factors in various cancer types. Stimulation or suppression of the activities of these crucial components can block cancer cell proliferation, prevent cancer stem cell expansion and even reverse the EMT process, thereby attenuating malignant phenotypes. Thus, cancer therapeutic interventions targeting RDGN members should be pursued in future studies.

Deployment of a retinal determination gene network drives directed cell migration in the sea urchin embryo.

Gene regulatory networks (GRNs) provide a systems-level orchestration of an organism's genome encoded anatomy. As biological networks are revealed, they continue to answer many questions including knowledge of how GRNs control morphogenetic movements and how GRNs evolve. The migration of the small micromeres to the coelomic pouches in the sea urchin embryo provides an exceptional model for understanding the genomic regulatory control of morphogenesis. An assay using the robust homing potential of these cells reveals a 'coherent feed-forward' transcriptional subcircuit composed of Pax6, Six3, Six1/2, Eya, and Dach1 that is responsible for the directed homing mechanism of these multipotent progenitors. The linkages of that circuit are strikingly similar to a circuit involved in retinal specification in Drosophila suggesting that systems-level tasks can be highly conserved even though the tasks drive unrelated processes in different animals.

Endogenous Dach1 in cancer.

The Dachshund gene is a key component of the retinal determination gene network in Drosophila eye development. Recent studies have demonstrated an important role for the human Dachshund homologue 1 (DACH1) in tumorigenesis [1]. Mechanistic studies demonstrated that DACH1 regulates its target genes via either directly binding to specific DNA sequences within chromatin or indirectly through forming protein complex with other transcriptional factors, including c-Jun and Smad4. DACH1 restrained proliferation, migration in vitro and repressed tumor growth and metastasis in vivo. There prior observations were based on cultured cells engineering DACH1 expression. The physiological role of the endogenous DACH1 had not been determined in part because genetic deletion in the mice was periembryonic lethal. A recent study report in the May 15 issue of Cancer Research determined the role of endogenous Dach1 in the prostate using tissue specific deletion mice [2]. Using prostate cancer cell lines of distinct genetic background, DACH1 was shown to inhibit proliferation in vitro and tumor growth in vivo of both AR negative cancer cell (PC-3) and castration resistant AR positive cell (C4-2 and 22RV-1). Consistent with the prior finding that DACH1 inhibited cell cycle progression though inhibiting Cyclin D1 expression, conditional Dach1 knock out in the prostate by using Dach1 fl/fl/Probasin-Cre bi-transgenic increased the expression of cyclin D1, E and A, accompanied by enhanced DNA synthesis and reduced apoptosis [2]. Whole genomic expression profiling with functional pathway analysis identified the cytokine-cytokine receptor interaction as a key target of endogenous Dach1. CXCL family member CXCL-1, 2, 5, 6 and IL-6, 8 expressions were inhibited by DACH1 in PC-3 cell. DACH1 mRNA expression was reduced in metastatic human prostate cancer [3] and DACH1 abundance was inversely correlated with IL-6 and IL-8 (Fig. ​(Fig.1).1). In agreement, there was 1000 fold activation of IL-6 and IL-8 secretion when endogenous Dach1 was deleted in vivo from prostate epithelial cells (PEC). Functional assays, using immune neutralizing antibodies or purified recombinant cytokines to conditioned medium from wt or Dach1 KO PEC, proved that IL-6 and KC (homolog of IL-8 in mice) were the key downstream targets of Dach1 in governing cell migration. In addition, various single oncogene (c-Myc, NeuT, H-Ras or v-Src) transformed prostate epithelial cells (PEC) reduced Dach1 expression both in cultured cells and in extirpated tumor tissues [2]. Combined with previous finding that DACH1 represses ligand induced transcriptional activation of the androgen receptor and prostate cancer cellular proliferation in tissue culture [3], it is likely that DACH1 conveys distinct functions at different stages of prostate cancer onset and progression. Figure 1 Reduced expression of DACH1 activates heterotypic signaling in prostate cancer The transition from hormone-dependent to castrate-resistant prostate cancer (CRPC) is the major cause of therapeutic failure. Clinical studies have shown that increased IL-6 and IL-8 signaling correlates with CRPC and predicts poor prognosis [4]. IL-6 and IL-8 produced by prostate cancer cell promote cancer cell proliferation and invasion. Moreover, IL-8 signaling from tumor cells initiates cancer cell-stromal interaction to induce treatment resistance and angiogenesis [5]. The finding that DACH1 is a key endogenous gene that restrains cytokine signaling may have therapeutic relevance. The finding that DNA demethylation agents could restore DACH1 expression in PC-3 cells [2] suggests targeting DACH1 may be practical. DACH1 also directly suppressed IL-8 in breast cancer cells and inhibited KC-mediated lung metastasis [6]. A subsequent study in lung cancer has also demonstrated that DACH1 can suppress the secretion of CXCL5, thereby reducing CXCL5-mediated proliferation, migration and invasion. Moreover, as with prostate cancer, a reverse relationship between DACH1 and CXCL5 was reported in tumor samples and low DACH1 correlated with reduced survival in lung cancer patients [7]. Thus, DACH1 governs cell fate through intracellular transcriptional regulation and also through heterotypic signaling that determines cancer-stromal interaction, a key hallmark of cancer [8].

The DACH/EYA/SIX gene network and its role in tumor initiation and progression.

The functional abnormality of developmental genes is a common phenomenon in cancer initiation and progression. The retinal determination gene network (RDGN) is a key signal in Drosophila eye specification, and this conservative pathway is also required for the development of multiple organs in mammalian species. Recent studies demonstrated that aberrant expressions of RDGN components in vertebrates, mainly Dach, Six, and Eya, represent a novel tumor signal. RDGN regulates proliferation, apoptosis, tumor growth and metastasis through interactions with multiple signaling pathways in a co-ordinated fashion; Dach acts as a tumor suppressor, whereas Six and Eya function as oncogenes. Clinical analyses demonstrated that the expression levels of RDGN correlate with tumor stage, metastasis and survival, suggesting that combinational detection of this pathway might be used as a promising biomarker for the stratification of therapy and for the prediction of the prognosis of cancer patients.

Interplay of retinal determination gene network with TGF-β signaling pathway in epithelial-mesenchymal transition.

As a fundamental event in the generation of tissues and organs during embryogenesis, the epithelial-mesenchymal transition (EMT) has also been implicated in cancer progression by its ability to alter the plasticity of epithelial cells to acquire invasive properties. Evidence is mounting that ectopic activation of transforming growth factors β (TGF-β)/bone morphogenetic protein (BMP) superfamily members to enhance tumorigenesis and metastasis. In this respect, the Retinal Determination Gene Network (RDGN), which was identified to govern the normal initiation of the morphogenetic furrow in Drosophila, has now been found to be de-regulated in various types of cancers, and the key members of this network, DACH, SIX, and EYA, have emerged as novel co-regulators of TGF- signaling during EMT. Understanding the molecular mechanism by which RDGN regulates TGF-β/BMP signaling to influence EMT may lead to novel strategies for targeted therapies.

733P - Expression Analyses of the Key Members of Rdgn in the Tissues of Benign Hepatic Diseases and Human Liver Cancers

ABSTRACT Aim: To analyze the differences of the expression of DACH1 in different groups of liver diseases (liver cirrhosis, fatty liver and chronic hepatitis), study the relationship between clinical pathological features (stage, pathologic grade and type, age and gender) of liver cancer and the expression of DACH1 and SIX1 which are the main members of The Retinal Determination Gene Network (RDGN) and explore the correlation among DACH1, SIX1 and cyclinD1 in the tissues of liver cancers. Methods: The expression of DACH1 in one tissue microarray including 120 cases of different hepatic diseases and the expression of DACH1, SIX1 and cyclinD1 in another tissue microarray including 120 cases of liver cancers and normal liver were detected by immunohistochemical methods. One-way ANOVA, Chi-square test, orderly logistic regression were used for data analysis. Results: In the liver cirrhosis, fatty liver and chronic hepatitis groups, DACH1 expression was lower than in the normal liver group. The amount of DACH1 in liver cancer tissues was lower than the three liver benign disease groups and normal group (P Conclusions: Several hepatic diseases were related to the low expression of DACH1 which has a negative correlation with pathologic grade and shows it may participate in regulating the occurrence and development of these diseases, especially in liver cancer tissues, and may play a protective role in these liver diseases. The high expression of SIX1 is associated with the low differentiation and advanced stage of the liver cancer, therefore, it may participate in relevant mechanisms of the progression of liver cancers. Disclosure: All authors have declared no conflicts of interest.

Silencing DACH1 promotes esophageal cancer growth by inhibiting TGF-β signaling.

Human Dachshund homologue 1 (DACH1) is a major component of the Retinal Determination Gene Network. Loss of DACH1 expression was found in breast, prostate, lung, endometrial, colorectal and hepatocellular carcinoma. To explore the expression, regulation and function of DACH1 in human esophageal cancer, 11 esophageal cancer cell lines, 10 cases of normal esophageal mucosa, 51 cases of different grades of dysplasia and 104 cases of primary esophageal squamous cancer were employed. Methylation specific PCR, immunohistochemistry, western blot, flow cytometry, small interfering RNAs, colony formation techniques and xenograft mice model were used. We found that DACH1 expression was regulated by promoter region hypermethylation in esophageal cancer cell lines. 18.8% (6 of 32) of grade 1, 42.1% (8 of 19) of grade 2 and grade 3 dysplasia (ED2,3), and 61.5% (64 of 104) of esophageal cancer were methylated, but no methylation was found in 10 cases of normal esophageal mucosa. The methylation was increased in progression tendency during esophageal carcinogenesis (P<0.01). DACH1 methylation was associated with poor differentiation (P<0.05) and late tumor stage (P<0.05). Restoration of DACH1 expression inhibited cell growth and activated TGF-β signaling in KYSE150 and KYSE510 cells. DACH1 suppressed human esophageal cancer cell tumor growth in xenograft mice. In conclusion, DACH1 is frequently methylated in human esophageal cancer and methylation of DACH1 is involved in the early stage of esophageal carcinogenesis. DACH1 expression is regulated by promoter region hypermethylation. DACH1 suppresses esophageal cancer growth by activating TGF-β signaling.

Hipk promotes photoreceptor differentiation through the repression of Twin of eyeless and Eyeless expression

Organogenesis is a complex developmental process, which requires tight regulation of selector gene expression to specify individual organ types. The Pax6 homolog Eyeless (Ey) is an example of such a factor and its expression pattern reveals it is dynamically controlled during development. Ey׳s paralog Twin of eyeless (Toy) induces its expression during embryogenesis, and the two genes are expressed in nearly identical patterns during the larval stages of development. While Ey must be expressed to initiate retinal specification, it must subsequently be repressed behind the morphogenetic furrow to allow for neuronal differentiation. Thus far, a few factors have been implicated in this repression including the signaling pathways Hedgehog (Hh) and Decapentaplegic (Dpp), and more recently downstream components of the retinal determination gene network (RDGN) Sine oculis (So), Eyes absent (Eya), and Dachshund (Dac). Homeodomain-interacting protein kinase (Hipk), a conserved serine–threonine kinase, regulates numerous factors during tissue patterning and development, including the Hh pathway. Using genetic analyses we identify Hipk as a repressor of both Toy and Ey and show that it may do so, in part, through Hh signaling. We also provide evidence that Ey repression is a critical step in ectopic eye development and that Hipk plays an important role in this process. Because Ey repression within the retinal field is a critical step in eye development, we propose that Hipk is a key link between eye specification and patterning.

Abstract P3-02-03: The phosphatase function of the eyes absent (EYA) homolog is required for the induction of breast cancer cellular proliferation via cyclin D1

Abstract The Drosophila Eyes Absent Homologue 1 (EYA1) is a component of the retinal determination gene network (RDGN) and serves as an H2AX phosphatase. The cell fate determination gene network includes the dachshund (dac), twin-of-eyeless (toy), eye absent (eya), teashirt (tsh) and sine oculis (So). In Drosophila, mutations of the RDGN leads to failure of eye formation, whereas, forced expression induces ectopic eye formation. EYA functions as a transcriptional co-activator being recruited in the context of local chromatin, but lacking intrinsic DNA binding activity. EYA family members EYA 1-4 are defined by a 275 amino acid carboxyl-terminal motif that is conserved between species, referred to as the EYA domain (ED). The human homologs EYA 1-4 are highly conserved in their EYA domain and amino termini, with the exception of a small tyrosine rich residue region named EYA domain II. Altered expression or functional activity of the RDGN has been documented in a variety of malignancies. DACH1 expression is reduced in breast, prostate, endometrial and brain cancer. EYA2 is up regulated in ovarian cancer, promoting tumor growth. EYA1 and EYA2 enhanced survival in response to DNA damage producing agents in HEK293 cells. Eya2 was required for Six1/TGFb signals that govern a prometastatic phenotype and epithelial mesenchymal transition (EMT). Although EYA proteins are expressed in human breast cancer, the relationship to molecular genetic subtype, prognosis and the molecular mechanisms governing contact-independent growth are not known. The cyclin D1 gene encodes the regulatory subunits of a holoenzyme that phosphorylates and inactivates the pRb protein. Herein, comparison with normal breast demonstrated EYA1 is overexpressed with cyclin D1 in luminal B breast cancer subtype. EYA1 enhanced breast tumor growth in mice in vivo requiring the phosphatase domain. EYA1 enhanced cellular proliferation, inhibited apoptosis, and induced contact-independent growth and cyclin D1 abundance. The induction of cellular proliferation and cyclin D1 abundance, but not apoptosis, was dependent upon the EYA1 phosphatase domain. The EYA1-mediated transcriptional induction of cyclin D1 occurred via the AP-1 binding site at -953 and required the EYA1 phosphatase function. The AP-1 mutation did not affect SIX1-dependent activation of cyclin D1. EYA1 was recruited in the context of local chromatin to the cyclin D1 AP-1 site. The EYA1 phosphatase function determined the recruitment of CBP, RNA polymerase II and acetylation of H3K9 at the cyclin D1 gene AP-1 site regulatory region in the context of local chromatin. The EYA1 phosphatase regulates cell cycle control via transcriptional complex formation at the cyclin D1 promoter. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P3-02-03.

Abstract P5-11-04: Post-translational modification of the cell-fate factor Dachshund determines p53 binding and signaling modules in breast cancer

Abstract Breast cancer is a leading form of cancer in the world. Initially cloned as a dominant inhibitor of the hyperactive EGFR, Ellipse, in Drosophila, the mammalian DACH1 regulates expression of target genes in part through interacting with DNA-binding transcription factors (c-Jun, Smads, Six, ERα), and in part through intrinsic DNA-sequence specific binding to Forkhead binding sites. The Drosophila dac gene is a key member of the retinal determination gene network (RDGN), which also includes eyes absent (eya), ey, twin of eyeless (toy), teashirt (tsh) and sin oculis (so), that specifies eye tissue identity. Several lines of evidence suggest DACH1 may function as a tumor suppressor. Clinical studies have demonstrated a correlation between poor prognosis and reduced expression of the cell-fate determination factor DACH1 in breast cancer, and loss of DACH1 expression has been observed in prostate and endometrial cancer. DACH1 inhibits breast cancer tumor metastasis and reduces breast cancer stem cell expansion via Sox2/Nanog. Although these studies suggest DACH1 may function as a tumor suppressor, the molecular mechanisms remain poorly defined. Herein, endogenous DACH1 co-localized with p53 in a nuclear, extranucleolar compartment and bound to p53 in human breast cancer cell lines, p53 and DACH1 bound common genes in ChIP-Seq. Full inhibition of breast cancer contact-independent growth by DACH1 required p53. The p53 breast cancer mutants R248Q and R273H, evaded DACH1 binding. DACH1 phosphorylation at serine residue (S439) inhibited p53 binding and phosphorylation at p53 amino-terminal sites (S15, S20) enhanced DACH1 binding. DACH1 binding to p53 was inhibited by NAD-dependent deacetylation via DACH1 K628. DACH1 repressed p21CIP1 and induced RAD51, an association found in basal breast cancer. DACH1 inhibits breast cancer cellular growth in an NAD and p53 dependent manner through direct protein-protein association. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-11-04.