DACH1 Expression Research Articles

Dynamic and Cell-Specific DACH1 Expression in Human Neocortical and Striatal Development.

DACH1 is the human homolog of the Drosophila dachshund gene, which is involved in the development of the eye, nervous system, and limbs in the fly. Here, we systematically investigate DACH1 expression patterns during human neurodevelopment, from 5 to 21 postconceptional weeks. By immunodetection analysis, we found that DACH1 is highly expressed in the proliferating neuroprogenitors of the developing cortical ventricular and subventricular regions, while it is absent in the more differentiated cortical plate. Single-cell global transcriptional analysis revealed that DACH1 is specifically enriched in neuroepithelial and ventricular radial glia cells of the developing human neocortex. Moreover, we describe a previously unreported DACH1 expression in the human striatum, in particular in the striatal medium spiny neurons. This finding qualifies DACH1 as a new striatal projection neuron marker, together with PPP1R1B, BCL11B, and EBF1. We finally compared DACH1 expression profile in human and mouse forebrain, where we observed spatio-temporal similarities in its expression pattern thus providing a precise developmental description of DACH1 in the 2 mammalian species.

Metformin blocks myeloid-derived suppressor cell accumulation through AMPK-DACH1-CXCL1 axis

ABSTRACTPurpose: Tumor development has been closely linked to tumor microenvironment, particularly in terms of myeloid-derived suppressive cells (MDSCs), a heterogeneous population of immature myeloid cells that protect tumors from elimination by immune cells. Approaches aimed at blocking MDSC accumulation could improve cancer clinical outcome.Experimental Design: We investigated that metformin suppressed MDSC migration to inhibit cancer progression. Primary tumor tissues were incubated with metformin, and proinflammatory chemokine production was measured. To study MDSC chemotaxis in vivo, BALB/C nude mice were injected subcutaneously with TE7 cells and treated with metformin. Migration of adoptively transferred MDSCs was analyzed using flow cytometry and immunohistochemistry.Results: The frequency of tumor-infiltrated polymorphonuclear (PMN)-MDSCs was increased compared to their circulating counterparts. There was a significant correlation between PMN-MDSCs accumulation in tumors and ESCC prognosis. Moreover, PMN-MDSCs displayed immunosuppressive activity in vitro. Treatment with metformin reduced MDSC migration in patients. Metformin inhibited CXCL1 secretion in ESCC cells and tumor xenografts by enhancing AMPK phosphorylation and inducing DACH1 expression, leading to NF-κB inhibition and reducing MDSC migration. Knockdown of AMPK and DACH1 expression blocked the effect of metformin on MDSC chemotaxis.Conclusions: A novel anti-tumor effect of metformin, which is mediated by reducing PMN-MDSC accumulation in the tumor microenvironment via AMPK/DACH1/CXCL1 axis.

The transcription factor Dach1 is essential for podocyte function.

Dedifferentiation and loss of podocytes are the major cause of chronic kidney disease. Dach1, a transcription factor that is essential for cell fate, was found in genome‐wide association studies to be associated with the glomerular filtration rate. We found that podocytes express high levels of Dach1 in vivo and to a much lower extent in vitro. Parietal epithelial cells (PECs) that are still under debate to be a type of progenitor cell for podocytes expressed Dach1 only at low levels. The transfection of PECs with a plasmid encoding for Dach1 induced the expression of synaptopodin, a podocyte‐specific protein, demonstrated by immunocytochemistry and Western blot. Furthermore, synaptopodin was located along actin fibres in a punctate pattern in Dach1‐expressing PECs comparable with differentiated podocytes. Moreover, dedifferentiating podocytes of isolated glomeruli showed a significant reduction in the expression of Dach1 together with synaptopodin after 9 days in cell culture. To study the role of Dach1 in vivo, we used the zebrafish larva as an animal model. Knockdown of the zebrafish ortholog Dachd by morpholino injection into fertilized eggs resulted in a severe renal phenotype. The glomeruli of the zebrafish larvae showed morphological changes of the glomerulus accompanied by down‐regulation of nephrin and leakage of the filtration barrier. Interestingly, glomeruli of biopsies from patients suffering from diabetic nephropathy showed also a significant reduction of Dach1 and synaptopodin in contrast to control biopsies. Taken together, Dach1 is a transcription factor that is important for podocyte differentiation and proper kidney function.

SIX1 and DACH1 influence the proliferation and apoptosis of hepatocellular carcinoma through regulating p53

ABSTRACTSThis research aimed to explore effects of SIX1 and DACH1 on hepatocellular carcinoma (HCC) cell proliferation, apoptosis and cell cycle. Fifty paired hepatocellular carcinoma tissues were screened for differentially expressed genes. SIX1 and DACH1 expressions were subjected to qRT-PCR and western blot in tumor tissues and cells. The knockdown efficiency of siRNAs and transfection efficiency of cDNAs and siRNAs were validated by qRT-PCR and western blot as well. Then colony formation assay and flow cytometry were applied to observe cell proliferation, cell apoptosis and cell cycle changes. Immunofluorescence co-localization and immunoprecipitation were used to analyze the interaction between proteins which was quantified using western blot. Effects of SIX1 and DACH1 on tumor growth and their expressions in tumors were confirmed in vitro in nude mice model. Results of these experiments showed that SIX1 was overexpressed while DACH1 was suppressed in HCC tissues and cells. The suppression of SIX1 and overexpression of DACH1 not only inhibited cell proliferation, but also induced cell apoptosis and arrested cell cycle in G2/M phase compared with control group. Results of immunofluorescence co-localization suggested that SIX1, p53 and DACH1 were significantly overlapped. Immunoprecipitation showed that DACH1 (marked with Flag tag) could pull down p53 and SIX1, but SIX1 (marked with His tag) could only pull down DACH1, which indicated that an indirect regulation between SIX1 and p53. Validated with western blot afterwards, DACH1 overexpression suppressed tumorigenesis in vivo by up-regulating p53 expression while SIX1 overexpression accelerated tumor growth by down-regulating p53 expression. Therefore, the decrease of SIX1 facilitated the expression of DACH1, thus activated the expression of p53 and suppressed the progression of HCC both in vitro and in vivo.

DACH1 stimulates shear stress-guided endothelial cell migration and coronary artery growth through the CXCL12-CXCR4 signaling axis.

Sufficient blood flow to tissues relies on arterial blood vessels, but the mechanisms regulating their development are poorly understood. Many arteries, including coronary arteries of the heart, form through remodeling of an immature vascular plexus in a process triggered and shaped by blood flow. However, little is known about how cues from fluid shear stress are translated into responses that pattern artery development. Here, we show that mice lacking endothelial Dach1 had small coronary arteries, decreased endothelial cell polarization, and reduced expression of the chemokine Cxcl12 Under shear stress in culture, Dach1 overexpression stimulated endothelial cell polarization and migration against flow, which was reversed upon CXCL12/CXCR4 inhibition. In vivo, DACH1 was expressed during early arteriogenesis but was down in mature arteries. Mature artery-type shear stress (high, uniform laminar) specifically down-regulated DACH1, while the remodeling artery-type flow (low, variable) maintained DACH1 expression. Together, our data support a model in which DACH1 stimulates coronary artery growth by activating Cxcl12 expression and endothelial cell migration against blood flow into developing arteries. This activity is suppressed once arteries reach a mature morphology and acquire high, laminar flow that down-regulates DACH1. Thus, we identified a mechanism by which blood flow quality balances artery growth and maturation.

Deficiency of the c-JUN Inhibitor DACH1 in FA Macrophages Accounts for the Cytokine Overproduction Phenotype

▪The inflammatory cytokines TNFα and IL-1β contribute to the bone marrow failure phenotype in Fanconi anemia (FA) as well as to clonal evolution to MDS and AML. Mononuclear phagocytes deficient in FANCA or FANCC overproduce these cytokines in response to toll-like receptor agonists (TLRa), but the precise molecular mechanisms by which FA proteins suppress cytokine production remains enigmatic. We show here that pretreatment of control macrophages with interferon (IFN) α enhances (by 3-fold) TLRa-induced production of both TNFα and IL-1β. FANCC-deficient cells produce more of these proteins (by 3-fold) after exposure to TLRa alone and IFNα does not enhance production of these cytokines. We therefore hypothesized that TLR stimulation of FANCC-deficient cells activates an IFNα-like pathway, one constrained by FANCC in normal cells. To test this notion, we performed gene expression microarray analysis (Affymetrix HTA 2.0) using RNA from FANCC-deficient (T-shFC) and control (T-shNT) THP-1 human mononuclear phagocytes treated with IFNα, the TLR7/8 agonist R848, or a combination of IFNα plus R848. We found that treatment of T-shFC cells with R848 alone was sufficient to enhance expression of 49 genes that were activated by IFNα in control cells. Thirteen of these genes were not induced by R848 in control cells but were induced by R848 in T-shFC cells (one of which, IFNgamma-inducible protein 30 (IFI30), is known to be overexpressed in Fancd2 -/- progenitor cells). These results support our hypothesis that FANCC functions normally to constrain IFNα pathway activation in TLRa-activated macrophages. Of greater functional importance vis-à-vis cytokine production, DACH1 mRNA was suppressed by 11-fold and DACH1 protein was barely detectable in FANCC-deficient cells under all conditions when compared to control cells. DACH1 is known to bind to and suppress the activities of a variety of transcription factors, notably c-JUN in fibroblasts. We tested the hypothesis that DACH1 deficiency is sufficient to account for the TLRa hypersensitivity of FA macrophages using gain- and loss-of-function studies. Knockdown of DACH1 in control (T-shNT) macrophages resulted in a 7-fold enhancement of R848-induced TNFα production, increased c-JUN protein (1.6-fold) and c-JUN phosphorylation (2.6-fold). Reporter gene expression (secreted embryonic alkaline phosphatase [SEAP]) from constructs containing both AP-1 and NF-κB sites was activated by DACH1 knockdown but deletion of the AP-1 site completely abrogated activation. Results of gain-of-function experiments with wild-type and mutant DACH1 cDNA sequences now taking place will be reported. Taken together, these results suggest that FANCC directly or indirectly enhances ground-state expression of DACH1 and thereby suppresses the TLR pathway in normal cells specifically by inhibiting R848-dependent activation of c-JUN. Conversely, loss of DACH1 expression in FANCC-deficient cells leads to unconstrained c-JUN activity, resulting in overproduction inflammatory cytokines. In light of the contribution of such cytokines to both bone marrow failure and clonal evolution, activation of DACH1 gene expression is a rational therapeutic objective in the management of patients with Fanconi anemia and is worthy of investigating in preclinical (animal) models. DisclosuresNo relevant conflicts of interest to declare.

Abstract B34: Antitumor activity and sensitivity evaluation of novel BET inhibitors in neuroblastoma

Abstract Children with neuroblastoma exhibit marked variability in outcome based on age at diagnosis, disease stage and tumor biologic characteristics. Genomic amplification of MYCN is a potent oncogenic driver and negative prognostic marker in neuroblastoma patients. Therefore, there is a pressing need to identify therapeutic agents that target MYCN gene expression. The bromodomain and extra-terminal (BET) family of proteins are epigenetic regulators known to control expression of genes involved in cell growth and oncogenesis. Previous reports demonstrate that BET inhibition (BETi) significantly attenuates cellular proliferation in numerous cancer models, perhaps via modulation of the MYC and/or MYCN oncogenes. Here, we show that potent BET inhibition induces anti-tumor effects in preclinical neuroblastoma models. Specifically, GSK726 and GSK762 (GlaxoSmithKline) were used for in vitro cytotoxicity and in vivo therapeutic studies, respectively. Neuroblastoma cell lines (n=22) were treated with GSK726 to calculate IC50 values in a luminescence-based cell viability assay and differential sensitivity was observed with an IC50 range of 27 nM to 9.5 µM. In sensitive cell lines, cell cycle distribution and induction of apoptosis were also measured. In these cell lines, GSK726 treatment resulted in MYCN depletion, G1 arrest within 24 hours, and apoptosis as measured by cleaved-PARP. To assess in vivo efficacy, GSK762 was subcutaneously administered in xenograft models and in genetically engineered neuroblastoma mouse models overexpressing MYCN and MYCN/ALK F1174L in the neural crest. In both models, GSK762 treatment resulted in tumor growth delay. Further assessment of results seen in vitro and in vivo indicated that MYCN amplification status did not fully predict sensitivity to GSK726 or GSK762. Thus, to determine additional biomarkers of sensitivity, we examined baseline gene expression data at the extremes of IC50 values by comparing sensitive (n=6; IC50<128 nM) and resistant (n=4; IC50>940 nM) neuroblastoma cell lines. Gene expression data from the neuroblastoma cell lines generated on HuGene1.0ST expression microarrays (Affymetrix) were utilized, and data were analyzed using the Limma package in R/Bioconductor. Univariate analysis, with a false discovery rate less than 0.25, revealed 6 genes (PTER, PCDHB14, RFTN1, JAK2, MYCBP, and DACH1) that were differentially expressed between sensitive and resistant cell lines in the MYCN amplified setting. While all 6 genes predicted BETi sensitivity in the MYCN-amplified subset of cell lines, only DACH1 expression predicted sensitivity to BET inhibition irrespective of MYCN amplification status (p=7.92x10-7). In addition, high DACH1 expression was correlated with poor patient outcome (p=1.74x10-5). As a result, high DACH1 expression serves as a candidate biomarker for future studies with respect to sensitivity to BETi. Ultimately, these studies will help to optimize the clinical utility of BETi in neuroblastoma and perhaps other MYC-driven malignancies. Citation Format: Jason R. Healy, Robert W. Schnepp, Lori S. Hart, Pichai Raman, Laura Danielson, Michael Russell, Priya Khurana, Maria Gagliardi, Ryan M. Kinsey, Anastasia Wyce, Olena Barbash, Peter J. Tummino, Louis Chesler, John M. Maris. Antitumor activity and sensitivity evaluation of novel BET inhibitors in neuroblastoma. [abstract]. In: Proceedings of the AACR Special Conference on Myc: From Biology to Therapy; Jan 7-10, 2015; La Jolla, CA. Philadelphia (PA): AACR; Mol Cancer Res 2015;13(10 Suppl):Abstract nr B34.

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].

Abstract 1074: Epigenetic silencing of DACH1 in triple negative breast cancer contributes to the tumorigenesis

Abstract BACKGROUND: Increasing evidence indicates that cell fate determination factor DACH1 functions as a tumor suppressor. Specifically, in estrogen receptor positive (luminal), highly expression of DACH1 predicted longer cancer specific survival and disease free interval, as well as reduced metastasis formation. Our analysis of over 2,000 patients demonstrated increased nuclear DACH1 expression correlated inversely with cellular mitosis and predicted 40 months survival advantage in breast cancer patients. But, endogenous DACH1 was dramatically reduced in breast cancer cell lines with high expression of tumor initiation cell markers and in patient samples of triple negative breast cancer, the most aggressive subtype. We proposed that epigenetically change might contribute to the loss expression of DACH1 in breast cancer. MATERIALS AND METHODS: Clinical samples included 10cases of normal breast tissues, 26 cases of luminal breast cancer(ER+,PR+,HER1-2+), and 32 cases of triple negative breast cancer(ER-,PR-,HER-). Genomic DNA was extracted from formalin-fixed tissue and methylation-specific PCR of DACH1 promoter was performed. DACH1 protein expression was detected in human breast cancer tissue arrays by immunohistochemistry. Human triple negative breast cancer cell lines MDA-MB-231 and sum159 were cultured in DME medium supplied with 10% fetal bovine serum and growth factors. Stable cell lines expressing DACH1 were established in MDA-MB-231 and Sum159 though lentivirus transfection. In vitro cell growth was evaluated by MTT assay and colony formation. In vivo tumor growth was measured in xenograft mice model. RESULTS: There is no methylation of DACH1 promoter region was found in normal breast tissues, but methylation was found in 2 out of 26 (7.7%) in luminal breast cancer and 10 of 32(32%) in triple negative breast cancer. Consisting with previous report, immunohistochemical stain revealed nuclear stain of DACH1 in all normal and majority of luminal type breast cancer tissues, but lost or very weak expression of DACH1 in triple negative cancer tissues. Correspondingly, expression microarrays demonstrated that DACH1 mRNA levels in triple negative breast cancer cell lines were dramatically less than that of normal breast cell line and luminal breast cancer cell lines. Treatment of MDA-MB-231 and Sum159 cells with 5-Aza-2′-deoxyazacytidine induced the expression of DACH1 and decreased cellular proliferation. Ectopic expression of DACH1 inhibited in vitro cell growth and in vivo tumor formation. CONCLUSION: Promoter hypermethylation is a main factor leading to the inactivation of DACH1 in triple negative breast cancer; Re-expression of DACH1by epigenetic modification might provide a novel therapeutic strategy for this aggressive cancer. Citation Format: Qian Chu, Na Han, Yan Dong, Xun Yuan, Mingzhou Guo, Hanxiao Xu, Shiying Yu, Kongming Wu. Epigenetic silencing of DACH1 in triple negative breast cancer contributes to the tumorigenesis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1074. doi:10.1158/1538-7445.AM2015-1074

Abstract 515: Cell fate determination factor DACH1 inhibits lung adenocarcinoma invasion and tumor growth through repression of CXCL5 signaling

Abstract The onset of human lung cancer involves the loss of tumor suppressor genes and activation of tumor inducing genes. Current studies have proved EGFR, RAS, ALK as as key driver genes, however, in about 50% lung cancer samples there are no known genes to explain tumorigenesis, suggesting many novel pathways remain to be elucidated. Recent study on whole-genome and transcriptome sequencing NSCLC identified cell fate determinatiton gene DACH1 as novel alteration in genes involved in NSCLC. However, the role of DACH1 in NSCLC and its downstrem targets are largely unknown. We showed that expression of DACH1 was significantly decreased in human NSCLC tissues andits protein abundance was inversely correlated with the tumor stage and grade. Restoration of DACH1 function in NSCLC cells significantly reduced cellular proliferation, clone formation, migrasion and invasion in vitro, as well as tumor growth in vivo. By unbiased screen of secreting factors, we found out that CXCL5 was dramaticcaly repressed by DACH1. Furthermore, functional study suggested that CXCL5 was contribute to the DACH1-mediated repression of migrative in vitro and tumorigenesis in vivo. Consistingly, there was a inverse relationship between the mRNA level of DACH1 and CXCL5 in both lung cancer cell lines and huamn NSCLC tissues. Moreover, Kaplan-Meier analysis of human NSCLC samples demonstrated that high mRNA abundance of DACH1 was predicted favorate prognosis for both relaspse-free survivl and overall survival; in contrast, high CXCL5 expression predict worse prognosis for survival. To our understanding, this is the first study to demonstrate DACH1 regulated paracrine signaling in lung cancer and DACH1 could predict prognosis. In conclusion, DACH1 acts as a tumor suppressor gene through repression of paracrine signaling and is a potential biomarker for prognosis. Citation Format: Na Han, Xun Yuan, Hua Wu, Hanxiao Xu, Qian Chu, Mingzhou Guo, Shiying Yu, Yuan Chen, Kongming Wu. Cell fate determination factor DACH1 inhibits lung adenocarcinoma invasion and tumor growth through repression of CXCL5 signaling. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 515. doi:10.1158/1538-7445.AM2015-515

DACH1, a zona glomerulosa selective gene in the human adrenal, activates transforming growth factor-β signaling and suppresses aldosterone secretion.

Common somatic mutations in CACNAID and ATP1A1 may define a subgroup of smaller, zona glomerulosa (ZG)-like aldosterone-producing adenomas. We have therefore sought signature ZG genes, which may provide insight into the frequency and pathogenesis of ZG-like aldosterone-producing adenomas. Twenty-one pairs of zona fasciculata and ZG and 14 paired aldosterone-producing adenomas from 14 patients with Conn's syndrome and 7 patients with pheochromocytoma were assayed by the Affymetrix Human Genome U133 Plus 2.0 Array. Validation by quantitative real-time polymerase chain reaction was performed on genes >10-fold upregulated in ZG (compared with zona fasciculata) and >10-fold upregulated in aldosterone-producing adenomas (compared with ZG). DACH1, a gene associated with tumor progression, was further analyzed. The role of DACH1 on steroidogenesis, transforming growth factor-β, and Wnt signaling activity was assessed in the human adrenocortical cell line, H295R. Immunohistochemistry confirmed selective expression of DACH1 in human ZG. Silencing of DACH1 in H295R cells increased CYP11B2 mRNA levels and aldosterone production, whereas overexpression of DACH1 decreased aldosterone production. Overexpression of DACH1 in H295R cells activated the transforming growth factor-β and canonical Wnt signaling pathways but inhibited the noncanonical Wnt signaling pathway. Stimulation of primary human adrenal cells with angiotensin II decreased DACH1 mRNA expression. Interestingly, there was little overlap between our top ZG genes and those in rodent ZG. In conclusion, (1) the transcriptome profile of human ZG differs from rodent ZG, (2) DACH1 inhibits aldosterone secretion in human adrenals, and (3) transforming growth factor-β signaling pathway is activated in DACH1 overexpressed cells and may mediate inhibition of aldosterone secretion in human adrenals.

DACH1 is a novel predictive and prognostic biomarker in hepatocellular carcinoma as a negative regulator of Wnt/β-catenin signaling.

The cell fate determination factor Dachshund (DACH1) functions as a novel suppressor in the progression of various neoplasms. Previous study has suggested that hypermethylation of promoter region was responsible for the reduction of DACH1 expression in hepatocellular carcinoma (HCC), and associated with the progression of HCC, but the clinical significance and the exact molecular mechanisms of DACH1 in the progression of HCC remain unclear. In this study, we employed public microarray data analysis and tissue microarrays (TMAs) technologies and showed that DACH1 expression was reduced in HCC even at early stage and associated with the tumor progression. Notably, Kaplan-Meier analysis further indicated DACH1 could be an independent prognostic factor for the overall survival of HCC. Further, mechanistic studies revealed that overexpression of DACH1 inhibited the growth and migration of HCC cell line, which were dependent in part on the inactivation of Wnt pathway via phosphorylation of GSK3β to suppress β-catenin. In agreement, the abundance of DACH1 was inversely correlated with several Wnt target genes. Collectively, our study indicated β-catenin is a novel target of DACH1 in HCC.

DACH1 inhibits lung adenocarcinoma invasion and tumor growth by repressing CXCL5 signaling.

Whole-genome and transcriptome sequencing of non-small cell lung cancer (NSCLC) identified that DACH1, is a human homolog of drosophila gene dac, is involved in NSCLC. Here we showed that expression of DACH1 was significantly decreased in human NSCLC tissues and DACH1 abundance was inversely correlated with tumor stages and grades. Restoration of DACH1 expression in NSCLC cells significantly reduced cellular proliferation, clone formation, migration and invasion in vitro, as well as tumor growth in vivo. Unbiased screen and functional study suggested that DACH1 mediated effects were dependent in part on suppression of CXCL5. There was an inverse correlation between DACH1 mRNA levels and CXCL5 in both lung cancer cell lines and human NSCLC tissues. Kaplan-Mier analysis of human NSCLC samples demonstrated that high DACH1 mRNA levels predicted favorable prognosis for relapse-free and overall survival. In agreement, high CXCL5 expression predicted a worse prognosis for survival.

MiR-217 Promotes Tumor Proliferation in Breast Cancer via Targeting DACH1.

Objective: The expression of DACH1 was frequently lost in human breast cancer, which significantly correlated with poor prognosis. Herein, we aim to investigate its underlying mechanisms.Methods: The expression of miR-217 was detected by Taqman PCR. The mRNA and protein level of DACH1 were investigated by real time PCR and western blot. The dual-luciferase reporter system was used to determine the direct interaction between miR-217 and DACH1. A series of gain&loss of function assays were performed to measure the affects of miR-217 on tumor proliferation and cell cycle distribution.Results: Compared to that in normal breast samples, the expression of miR-217 was significantly upregulated in breast cancer tissues. High level of miR-217 was notably correlated with highly histological grade, the triple negative subtype and advanced tumor stage. Moreover, the expression of miR-217 was negatively correlated with the expression of DACH1. The results of dual-luciferase reporter assay demonstrated that miR-217 directly targets and inhibits the transcriptive activity of DACH1. In vitro, treatment with miR-217 mimics significantly suppressed the proliferation of MCF-7 cells, induced G1 phase arrest and inhibited the expression of cyclin D1; while these effects were significantly reversed by the restoration of DACH1. In MDA-MB-231 cells, treatment with miR-217 inhibitors enhanced the cellular proliferation, promoted cell cycle progression and upregulated the expression of cyclin D1, which were neutralized by the pre-treatment of siRNA-DACH1. In vivo, inhibition of miR-217 significantly suppressed the xenografts growth and downregulated the expression of cyclin D1.Conclusion: We found that miR-217 was commonly overexpressed in breast cancer, which could enhance tumor proliferation via promoting cell cycle progression. Moreover, the DACH1 (the cell fate determination factor) was identified as a novel target of miR-217. Our results proposed inhibiting miR-217 to be a potent therapeutic strategy for breast cancer.

Quantitative high-throughput gene expression profiling of human striatal development to screen stem cell–derived medium spiny neurons

A systematic characterization of the spatio-temporal gene expression during human neurodevelopment is essential to understand brain function in both physiological and pathological conditions. In recent years, stem cell technology has provided an in vitro tool to recapitulate human development, permitting also the generation of human models for many diseases. The correct differentiation of human pluripotent stem cell (hPSC) into specific cell types should be evaluated by comparison with specific cells/tissue profiles from the equivalent adult in vivo organ. Here, we define by a quantitative high-throughput gene expression analysis the subset of specific genes of the whole ganglionic eminence (WGE) and adult human striatum. Our results demonstrate that not only the number of specific genes is crucial but also their relative expression levels between brain areas. We next used these gene profiles to characterize the differentiation of hPSCs. Our findings demonstrate a temporal progression of gene expression during striatal differentiation of hPSCs from a WGE toward an adult striatum identity. Present results establish a gene expression profile to qualitatively and quantitatively evaluate the telencephalic hPSC-derived progenitors eventually used for transplantation and mature striatal neurons for disease modeling and drug-screening.

Androgen therapy reverses injuries caused by ethanol consumption in the prostate: Testosterone as a possible target to ethanol-related disorders

AimsChronic ethanol consumption leads to reproductive damages, since it can act directly in the tissues or indirectly, causing a hormonal imbalance. Prostate is a hormone-dependent gland and, consequently, susceptible to ethanol. The potential of testosterone therapy in the ethanol-related disorders was investigated in the prostate microenvironment. Main methodsUChB rats aged 90days were divided into 2 experimental groups (n=20): C: drinking water only and EtOH: drinking 10% (v/v) ethanol at >2g/kg body weight/day+water. At 150days old, 10 rats from each group received subcutaneous injections of testosterone cypionate (5mg/kg body weight) diluted in corn oil every other day for 4weeks, constituting T and EtOH+T, while the remaining animals received corn oil as vehicle. Animals were euthanized at 180days old, by decapitation. Blood was collected to obtain hormone concentrations and ventral prostate was dissected and processed for light microscope and molecular analyses. Key findingsVentral prostate weight, plasma testosterone and DHT and intraprostatic testosterone concentrations were increased after testosterone treatment. Plasma estradiol level was reduced in the EtOH+T. Inflammatory foci, metaplasia and epithelial atrophy were constantly found in the prostate of EtOH and were not observed after hormonal therapy. No differences were found in the expression of AR, ERβ and DACH-1. Additionally, testosterone treatment down-regulated ERα and increased the e-cadherin and α-actinin immunoreactivities. SignificanceTestosterone was able to reverse damages caused by ethanol consumption in the prostate microenvironment and becomes a possible target to be investigated to ethanol-related disorders.

DACH1 inhibits cyclin D1 expression, cellular proliferation and tumor growth of renal cancer cells.

BackgroundRenal cell carcinoma (RCC) is a complex with diverse biological characteristics and distinct molecular signature. New target therapies to molecules that drive RCC initiation and progression have achieved promising responses in some patients, but the total effective rate is still far from satisfaction. Dachshund (DACH1) network is a key signaling pathway for kidney development and has recently been identified as a tumor suppressor in several cancer types. However, its role in renal cell carcinoma has not been fully investigated.MethodsImmunohistochemical staining for DACH1, PCNA and cyclin D1 was performed on human renal tissue microaraays and correlation with clinic-pathological characteristics was analyzed. In vitro proliferation, apoptosis and in vivo tumor growth were evaluated on human renal cancer cell lines with decitabine treatment or ectopic expression of DACH1. Downstream targets and potential molecular mechanism were investigated through western blot, immunoprecipitation and reporter gene assays.ResultsExpression of DACH1 was significantly decreased in human renal carcinoma tissue. DACH1 protein abundance was inversely correlated with the expression of PCNA and cyclin D1, tumor grade, and TNM stage. Restoration of DACH1 function in renal clear cell cancer cells inhibited in vitro cellular proliferation, S phase progression, clone formation, and in vivo tumor growth. In mechanism, DACH1 repressed cyclin D1 transcription through association with AP-1 protein.ConclusionOur results indicated that DACH1 was a novel molecular marker of RCC and it attributed to the malignant behavior of renal cancer cells. Re-activation of DACH1 may represent a potential therapeutic strategy.

Abstract B75: Defining the antitumor activity and sensitivity profiles of BET inhibitors in neuroblastoma

Abstract Background: MYCN amplification is the most frequent somatically acquired genomic alteration in neuroblastoma and is a potent oncogenic driver. Targeting MYCN therapeutically has been complicated by the challenges inherent in targeting transcription factors. Recently, several groups have shown that inhibiting bromodomain and extra-terminal (BET) proteins (BRD2, BRD3, BRD4, and BRDT) and their ability to bind to acetylated lysine residues within histone tails resulted in silencing of MYC family protein expression and demonstrated therapeutic efficacy in preclinical cancer models. We hypothesized that potent inhibition of bromodomain-chromatin interactions would show anti-tumor activity in preclinical models of neuroblastoma and that reliable biomarkers of activity could be discovered. Methods: GlaxoSmithKline compounds GSK726 and GSK762 potently inhibit BET proteins (BETi) and were used for in vitro cytotoxicity and in vivo therapeutic studies, respectively. We exposed a panel of 15 cell lines to a 4-log dose range of GSK726 to determine IC50s in a luminescence-based cell viability assay. We also measured cell cycle changes by flow cytometry and apoptosis by immunoblotting for cleaved PARP. We tested the in vivo efficacy of the GSK762 in subcutaneous xenograft models (3 sensitive and 2 resistant cell lines) and in genetically engineered neuroblastoma mouse models (overexpressing MYCN and MYCN/ALK F1174L in the neural crest). For biomarker discovery, gene expression data from the neuroblastoma cell lines generated on HuGene1.0ST expression microarrays (Affymetrix) were utilized, and data were analyzed using the Limma package in R/Bioconductor. Results: Neuroblastoma cell lines (N=15) were differentially sensitive to GSK726, with a median IC50 of 128nM and a range of 17nM to >10uM. In sensitive cell lines, GSK726 treatment resulted in MYCN depletion, G1 arrest within 24 hours, and apoptosis. In subcutaneous xenograft models and genetically engineered neuroblastoma mouse models, GSK762 treatment resulted in tumor growth delay. Importantly, MYCN amplification status did not fully predict sensitivity to GSK726 or GSK762. Thus, to determine additional biomarkers of sensitivity, we examined baseline gene expression data at the extremes of IC50s by comparing sensitive (N=6; IC50<128 nM) and resistant (N=4; IC50>940 nM) neuroblastoma cell lines. Univariate analysis, with an FDR < 0.25, revealed 6 genes (PTER, PCDHB14, RFTN1, JAK2, MYCBP, and DACH1) differentially expressed between sensitive and resistant cell lines in the MYCN amplified setting. While all 6 genes predicted BETi sensitivity in the MYCN-amplified subset of cell lines, only DACH1 expression predicted sensitivity to BET inhibition irrespective of MYCN amplification status (7.92x10-7). Moreover, DACH1 levels were heterogeneously expressed in a panel of 88 primary neuroblastoma tumors (Versteeg-88) and high expression was correlated with poor patient outcome (1.74x10-5). Conclusions: BET inhibitors demonstrated significant anti-tumor activity in a subset of neuroblastoma preclinical models. Although MYCN protein levels decreased in neuroblastoma cell lines that were sensitive to BETi, MYCN amplification status alone did not fully explain BETi sensitivity. DACH1 expression serves as a candidate biomarker for sensitivity to BET inhibition. These studies will help to optimize the clinical utility of BETi in neuroblastoma and perhaps other MYC-driven malignancies. Citation Format: Robert Schnepp, Lori Hart, Pichai Raman, Laura Danielson, Maria Gagliardi, Ryan Kinsey, Anastasia Wyce, Olena Barbash, Peter Tummino, Louis Chesler, John Maris. Defining the antitumor activity and sensitivity profiles of BET inhibitors in neuroblastoma. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr B75.

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.

Epigenetic silencing of DACH1 induces the invasion and metastasis of gastric cancer by activating TGF-β signalling.

Gastric cancer (GC) is the fourth most common malignancy in males and the fifth most common malignancy in females worldwide. DACH1 is frequently methylated in hepatic and colorectal cancer. To further understand the regulation and mechanism of DACH1 in GC, eight GC cell lines, eight cases of normal gastric mucosa, 98 cases of primary GC and 50 cases of adjacent non-tumour tissues were examined. Methylation-specific PCR, western blot, transwell assay and xenograft mice were used in this study. Loss of DACH1 expression correlated with promoter region methylation in GC cells, and re-expression was induced by 5-Aza-2′-deoxyazacytidine. DACH1 is methylated in 63.3% (62/98) of primary GC and 38% (19/50) of adjacent non-tumour tissues, while no methylation was found in normal gastric mucosa. Methylation of DACH1 correlated with reduced expression of DACH1 (P < 0.01), late tumour stage (stage III/IV) (P < 0.01) and lymph node metastasis (P < 0.05). DACH1 expression inhibited epithelial–mesenchymal transition and metastasis by inhibiting transforming growth factor (TGF)-β signalling and suppressed GC cell proliferation through inducing G2/M phase arrest. The tumour size is smaller in DACH1-expressed BGC823 cell xenograft mice than in unexpressed group (P < 0.01). Restoration of DACH1 expression also sensitized GC cells to docetaxel. These studies suggest that DACH1 is frequently methylated in human GC and expression of DACH1 was controlled by promoter region methylation. DACH1 suppresses GC proliferation, invasion and metastasis by inhibiting TGF-β signalling pathways both in vitro and in vivo. Epigenetic silencing DACH1 may induce GC cells' resistance to docetaxel.