DACH1 Research Articles

Abstract 5581: The DACH1 gene, frequently co-deleted with BRCA2 in prostate cancer, governs PARP inhibitor resistance

Abstract Background: Prostate cancer (PCa), the second leading cause of death in American men, includes distinct genetic subtypes with distinct therapeutic vulnerabilities (1,2). The DACH1 gene, in the 13q21.31-q21.33 region, encodes a winged helix/Forkhead DNA-binding protein that governs cell-fate determination that is deleted in ~ 18% of human PCa, where it is associated with poor prognosis (3). Prostate-specific deletion of the Dach1 gene enhanced prostatic intraepithelial neoplasia (PIN). Methods: Analysis of DACH1 gene deletion and expression was conducted from public data bases and human prostate cancer samples. Cells derived from Dach1 gene deletion mice and human prostate cancer cell lines with DACH1 overexpression were analyzed for PARP inhibitor responses and mechanisms of PARPi resistance. Transgenic prostate Oncomice were generated in which the Dach1 gene was deleted in the prostate. Results: Herein, DACH1 was shown to be co-deleted with BRCA2 in each of 8 separate cohorts (N=2,186 patients), in ~3-12% of patients. Dach1 gene deletion or knockdown conferred PARP inhibitor resistance (talazoparib > niraparib > olaparib > rucaparib > veliparib). DACH1 enhanced PARP sensitivity through several mechanisms affecting replication stress, PARP1 binding, inhibiting G9a (reducing H3K9me2), inhibiting CHK1p and CDK1p and inducing expression of snoRNA, specifically the snoRNA known to bind PARP. PARP1 has a BRCT (BRCA1 C-terminus 1) homology domain governing PARP1 dimerization and binding to intact DNA in a complex within a nucleosome. DACH1 binding to PARP1 requires the BRCT domain. Conclusions: There is a correlation between DACH1a-/- and PARPi resistance, correlating with PARPi trapping ability. As reduced Dach1a expression may define a subclass of PCa that warrants specific therapies, testing for DACH1a may be warranted.

The DACH1 gene is frequently deleted in prostate cancer, restrains prostatic intraepithelial neoplasia, decreases DNA damage repair, and predicts therapy responses

Prostate cancer (PCa), the second leading cause of death in American men, includes distinct genetic subtypes with distinct therapeutic vulnerabilities. The DACH1 gene encodes a winged helix/Forkhead DNA-binding protein that competes for binding to FOXM1 sites. Herein, DACH1 gene deletion within the 13q21.31-q21.33 region occurs in up to 18% of human PCa and was associated with increased AR activity and poor prognosis. In prostate OncoMice, prostate-specific deletion of the Dach1 gene enhanced prostatic intraepithelial neoplasia (PIN), and was associated with increased TGFβ activity and DNA damage. Reduced Dach1 increased DNA damage in response to genotoxic stresses. DACH1 was recruited to sites of DNA damage, augmenting recruitment of Ku70/Ku80. Reduced Dach1 expression was associated with increased homology directed repair and resistance to PARP inhibitors and TGFβ kinase inhibitors. Reduced Dach1 expression may define a subclass of PCa that warrants specific therapies.

Abstract 2598: The DACH1 gene is frequently deleted in prostate cancer, restrains prostatic intraepithelial neoplasia, augments DNA damage repair and predicts therapy responses

Abstract Background: Prostate cancer (PCa), the second leading cause of death in American men, includes distinct genetic subtypes with distinct therapeutic vulnerabilities. The DACH1 gene encodes a winged helix/Forkhead DNA-binding protein that competes for binding to FOXM1 sites. Methods: Analysis of DACH1 gene deletion and gene expression was conducted from public data bases and human prostate cancer samples. Transgenic mice were generated in which the Dach1 gene was deleted in the prostate of prostate Oncomice. Cells derived from Dach1 gene deletion mice and human prostate cancer cell lines with Dach1 knockdown were analyzed for DNA damage repair responses. Results: DACH1 gene deletion within the 13q21.31-q21.33 region, occurred in up to 18% of human PCa, and was associated with increased AR activity, and poor prognosis. DACH1 homozygous deletions more frequent in the metastatic site than in the primary tumors (Mich: 10% vs. 18%, N=59; FHCRC: 4% vs.11%, N= 54, SU2C: not profiled vs. 3.3, N=150). The prevalence of DACH1 heterozygous deletions was higher in the metastatic lesions than in primary tumors within a given cohort for three of six cohorts (Mich: 27.3% vs. 36%, N=59; FHCRC: 15% vs. 59%, N=54; SU2C: 0% vs. 26%, N=150, respectively). The patients with homozygous DACH1 deletions had reduced overall survival (medians of 84 vs. 120 months, N=667, log rank test P=9.3x10-3). Low DACH1 gene expression (expressed as a z-score with a z-score threshold of -1.25) was significantly correlated with earlier biochemical recurrence (BCR, log rank p value = 4.7x10-4, n=79). In prostate OncoMice, prostate-specific deletion of the Dach1 gene enhanced prostatic intraepithelial neoplasia (PIN) and was associated with increased DNA damage. Reduced Dach1 increased DNA damage in responses to genotoxic stresses. DACH1 was recruited to sites of DNA damage, augmenting recruitment of Ku70/Ku80. Reduced Dach1 expression was associated with resistance to TGFβ kinase and PARP inhibitors. Conclusions: Reduced Dach1 expression may define a subclass of PCa that warrants specific therapies. Citation Format: Zhiping Li, Xuanmao Jiao, Gordon Robertson, Gabriele Di Sante, Anthony W. Ashton, Agnese DiRocco, Min Wang, Jun Zhao, Sankar Addya, Chenguang Wang, Peter A. McCue, Andrew P. South, Carlos Cordon-Cardo, Runzhi Liu, Kishan Patel, Rasha Hamid, Jorim Parmar, James B. DuHadaway, Nikolaus Schultz, Andrew Kossenkov, Lai Yee Phoon, Hao Chen, Li Lan, Yunguang Sun, Kenneth A. Iczkowski, Hallgeir Rui, Richard G. Pestell. The DACH1 gene is frequently deleted in prostate cancer, restrains prostatic intraepithelial neoplasia, augments DNA damage repair and predicts therapy responses [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2598.

DACH1 mutation frequency in endometrial cancer is associated with high tumor mutation burden in a nationwide cohort.

e17634 Background: DACH1 is a novel transcriptional repressor and tumor suppressor gene. DACH1 mutations are associated with poor prognostic features and reduced overall survival in endometrial cancer, with an increased prevalence in the Appalachian region of Kentucky. Preliminary studies have suggested an association with an increase in tumor mutation burden. In this follow up study, we utilized the nationwide Oncology Research Information Exchange Network (ORIEN) to determine the frequency of DACH1 mutations in patients with endometrial cancer through a multi-institution analysis and evaluate its impact on RNA expression, clinical correlates, and outcomes. Methods: We obtained clinical and genomic data for 691 patients with endometrial cancer from nine U.S. institutions within the ORIEN collaborative. We examined the clinical attributes of the cancers with DACH1 status by comparing whole-exome sequencing (WES), RNA Sequencing (RNASeq), microsatellite instability (MSI), and tumor mutational burden (TMB). Results: Appalachian women with endometrial cancer had an increased frequency of DACH1 mutations (6/41 patients, 15%) compared to the non-Appalachian endometrial cancer population (24/581 patients, 4.1%) with p-value = 0.010, with the non-Appalachian DACH1 mutation frequency mirroring the rate of DACH1 gene mutation seen in TCGA at 3.8%. DACH1 mutated patients have a higher tumor mutation burden compared to DACH1 wild-type (32.2 vs. 4.62, p-value = 0.001) though no differences in microsatellite instability between DACH1 mutated and wild-type were present (p-value = 0.350). DACH1 mutations showed significant gene co-occurrence patterns with POLE, MLH1, MSH2, MSH6 and PMS2. Conclusions: DACH1 mutations are prevalent in Kentucky patients with endometrial cancer, particularly those from the Appalachian region. These results were again reflected in the TCGA PanCancer Atlas as well as the ORIEN multi-institution cohort. These mutations are associated with high tumor mutational burden and co-occur with genome destabilizing gene mutations. These findings suggest DACH1 as a candidate biomarker for future trials with immunotherapy, particularly in endometrial cancers.

3105 – IMPACT OF DACH1 DOWNREGULATION ON MLL-AF4 INFANT LEUKEMOGENESIS

Leukaemia in infants, caused by a rearrangement of the MLL gene, has a very bad prognosis. The most common type of infant leukaemia is caused by the t(4;11) translocation that fuses the MLL and AF4 genes. Current lack of knowledge of its developmental origins and early stages of pathogenesis is one of the reasons for the slow progress in developing more targeted therapies. RNA-Seq studies have recently been completed in the lab to gain a more comprehensive insight into the molecular determinants that make foetal haematopoietic stem and progenitor cells more susceptible to transformation by MLL-AF4. A lack of expression of the DACH1 gene, which has been described as a tumour suppressor in a range of different cancers, was noted. The aim of this work is to assess the impact of DACH1 on the initiation of MLL-AF4+ infant leukaemia and on its maintenance. To determine the role of DACH1 on the initiation of leukaemia, we used our pre-leukaemia mouse model with Mll-AF4 expression in foetal cells and then transduced the cells with lentiviruses carrying a DACH1 overexpression construct. Using methylcellulose assays under B lymphoid or myeloid conditions, we observed that DACH1 reduces the size and numbers of B cell colonies and has a general negative effect on cell proliferation and differentiation. To establish whether DACH1 repression is required for leukaemia maintenance, we have overexpressed DACH1 in the SEM leukaemia cell line, which was derived from an MLL-AF4+ paediatric B-ALL. Our work shows that DACH1 affects negatively the proliferation and survival of the cells. To conclude, our data give new insights into the role of DACH1 in MLL-AF4 leukaemia. In the future, we will identify downstream targets of this protein to determine which genes are directly repressed by DACH1 to counteract the leukaemic phenotype.

DACH1 protects podocytes and tubules from injury.

DACH1 protects podocytes and tubules from injury.

Biallelic variant in DACH1, encoding Dachshund Homolog 1, defines a novel candidate locus for recessive postaxial polydactyly type A

Polydactyly or hexadactyly is characterized by an extra digit/toe with or without a bone. Currently, variants in ten genes have been implicated in the non-syndromic form of polydactyly.DNA from a single affected individual having bilateral postaxial polydactyly was subjected to whole exome sequencing (WES), followed by Sanger sequencing. Homology modeling was performed for the identified variant and advance microscopy imaging approaches were used to reveal the localization of the DACH1 protein at the base of primary cilia.A disease-causing biallelic missense variant (c.563G > A; p.Cys188Tyr; NM_080760.5) was identified in the DACH1 gene segregating perfectly within the family. Structural analysis using homology modeling of the DACH1 protein revealed secondary structure change that might result in loss of function or influence downstream interactions. Moreover, siRNA-mediated depletion of DACH1 showed a key role of DACH1 in ciliogenesis and cilia function.This study provides the first evidence of involvement of the DACH1 gene in digits development in humans and its role in primary cilia. This signifies the importance and yet unexplored role of DACH1.

Investigating the role of dachshund b in the development of the pancreatic islet in zebrafish.

Aims/Introductionβ‐Cell dysfunction is a hallmark of type 2 diabetes. In a previous pilot study, we identified an association between genetic variants within the human DACH1 gene and young‐onset type 2 diabetes. Here, we characterized the function of dachb, the only dach homologue to be expressed in the pancreas, in developing zebrafish embryos.Materials and MethodsWe injected one‐cell stage embryos with a dachb‐morpholino (MO) or with the dachb‐MO and dachb messenger ribonucleic acid, and determined the effect on the development of the pancreatic islet. We also carried out quantitative polymerase chain reaction and ribonucleic acid sequencing on the dachb‐MO group to determine the effect of dachb knockdown on gene expression.ResultsMO‐mediated dachb knockdown resulted in impaired islet cell development, with a significant decrease in both the β‐cell and islet cell numbers. This islet developmental defect was rescued when embryos were co‐injected with dachb‐MO and dachb messenger ribonucleic acid. Knockdown of dachb was associated with a significant downregulation of the β‐cell specific marker gene, insa, and the somatostatin cell marker, sst2, as well as regulators of pancreas development, ptf1a, neuroD, pax6a and nkx6.1, and the cell cycle gene, insm1a. Furthermore, ribonucleic sequencing analysis showed an upregulation of genes enriched in the forkhead box O and mitogen‐activated protein kinase signaling pathways in the dachb‐MO group, when compared with the control groups.ConclusionsTogether, our results suggest the possible role of dachb in islet development in zebrafish.

Abstract 1730: DACH1 gene deletion extends portraits of human prostate cancer

Abstract Purpose of the study: This study was conducted to define the role of Dachshund in prostate cancer, through assessing human prostate cancer samples and through genetic deletion in the mouse. Prostate cancer (PCa), the second leading cause of death in American men, is a genetically heterogeneous disease, likely representing distinct genetic drivers, with terminal events caused primarily by metastasis. Substratification of PCa into genetic subtypes, forms the basis of rational therapy for PCa. A better molecular understanding of the disease is necessary in order to develop novel targeted therapies of metastatic PCa. Known genetic drivers to tumor initiation include PTEN and NKX3.1 deletions, rearrangements of the TMPRSS2 gene to the oncogenic ETS transcription factor, ERG, and genetic predisposing factors include germline DNA-repair gene mutations. The DACH1 gene, initially cloned as an inhibitor of Elipse, the hyperactive epidermal growth factor (EGFR) in Drosophila, was found to be reduced in abundance in several malignancies including breast and prostate cancer. Results: In order to determine whether the DACH1 gene is deleted or mutated in prostate cancer we interrogated the genomic sequencing analysis of over 490 patients from 5 population cohorts. Homozygous deletion of DACH1 was identified in 18% (N=61), 11% (N=136), 10% (N=492), 7% (N=103) and 3% (N=150) of prostate cancer in 5 distinct cohorts. The prevalence of DACH1 gene deep deletions was higher in the metastasis than in the primary tumors. The Transgenic Adenocarcinoma Mouse Prostate (TRAMP) transgenic, Dach1fl/fl, and Probasin-Cre, ROSA26mT/mG transgenic mice were used to generate a prostate epithelial cell specific Dach1 gene knockout mouse (Probasin-Cre-Dach1fl/fl ROSA26mT/mG-TRAMP) lines. Prostate specific deletion of the murine Dach1 gene enhanced progression of prostatic intraepithelial neoplasia (PIN), associated with increased prostate epithelial cell proliferation, epithelial mesenchymal transition (EMT), DNA damage and inflammation. Conclusions: DACH1 gene deletion may define a distinct subclass of prostate cancer that may benefit from PARP inhibitors, and platinum-based chemotherapy. Citation Format: Xuanmao Jiao, Gabriele Di Sante, Zhiping Li, Agnese Di Rocco, Min Wang, Adam Ertel, Peter A. McCue, Andrew P. South, Carlos Cordon-Cardo, Matthew P. Stokes, Marco Marra, Steven J. Jones, Andrew Kossenkov, Richard G. Pestell. DACH1 gene deletion extends portraits of human prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1730.

Role of Dach1 revealed using a novel inner ear-specific Dach1-knockdown mouse model.

ABSTRACTThe Dach1 gene is expressed in the inner ear of normal mouse embryos in the area that differentiates into the cochlear stria vascularis (SV). We hypothesised that Dach1 downregulation in the inner ear would lead to SV dysplasia. However, because Dach1 knockout is embryonic lethal in mice, the role of Dach1 in the inner ear is unclear. Here, we established inner ear-specific Dach1-knockdown mice and showed that Dach1 downregulation resulted in hearing loss, reduced endocochlear potential and secondary outer hair cell loss. There were no abnormalities in marginal cells and basal cells in the SV or spiral ligament in inner ear-specific Dach1-knockdown mature mice. However, intermediate cell dysplasia and thinning of the SV were observed. Moreover, dynamic changes in the expression of key genes related to the epithelial-mesenchymal transition were observed in the lateral wall of the cochlear epithelium, which differentiated into the SV in inner ear-specific Dach1-knockdown mice at embryonic stages. In summary, suppression of Dach1 expression in the inner ear caused the epithelial-mesenchymal transition in the lateral wall of cochlear epithelium, resulting in loss of intermediate cells in the SV and SV dysplasia.This article has an associated First Person interview with the first author of the paper.

Dachshund Depletion Disrupts Mammary Gland Development and Diverts the Composition of the Mammary Gland Progenitor Pool.

SummaryDACH1 abundance is reduced in human malignancies, including breast cancer. Herein DACH1 was detected among multipotent fetal mammary stem cells in the embryo, among mixed lineage precursors, and in adult basal cells and (ERα+) luminal progenitors. Dach1 gene deletion at 6 weeks in transgenic mice reduced ductal branching, reduced the proportion of mammary basal cells (Lin− CD24med CD29high) and reduced abundance of basal cytokeratin 5, whereas DACH1 overexpression induced ductal branching, increased Gata3 and Notch1, and expanded mammosphere formation in LA-7 breast cells. Mammary gland-transforming growth factor β (TGF-β) activity, known to reduce ductal branching and to reduce the basal cell population, increased upon Dach1 deletion, associated with increased SMAD phosphorylation. Association of the scaffold protein Smad anchor for receptor activation with Smad2/3, which facilitates TGF-β activation, was reduced by endogenous DACH1. DACH1 increases basal cells, enhances ductal formation and restrains TGF-β activity in vivo.

Genome-wide association studies for two exterior traits in Chinese Dongxiang spotted pigs.

Pigs show extensive variation in exterior appearance. This variation has been explored as one of the selection targets to form breeding features in the pig industry. In this study, a customized Affymetrix Axiom Pig1.4M array plate was used to conduct genome-wide association studies (GWAS) for two exterior traits-spotted coat color (SC) and facial type (FT)-in Chinese Dongxiang spotted pigs. Two and 12 single nucleotide polymorphisms (SNPs) were identified to be associated with SC and FT at the 5% genome-wide significant level, respectively. Of these SNPs, two associated with SC were located around the DACH1 gene on Sus Scrofa chromosome (SSC)11. Eleven SNPs associated with FT were located within a 7.68Mb region (29.03-36.71Mb) on SSC7. Based on the GWAS results and the biological functions of genes, we highlight EDNRB as a candidate gene for SC and HMGA1 and RPS10 as the potential genes affecting facial variation. The findings contribute to the final characterization of causative genes and mutations underlying the effects of the SSC7 and SSC11 loci and improve our understanding of the genetic basis of phenotypic variation in Chinese indigenous pigs.

Expression and Promoter Methylation Analysis of DACH1 Gene in Tumor and Normal Breast Tissue in Breast Cancer

Expression and Promoter Methylation Analysis of DACH1 Gene in Tumor and Normal Breast Tissue in Breast Cancer

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.

Focal cerebral ischemia induces the neurogenic potential of mouse Dach1-expressing cells in the dorsal part of the lateral ventricles

The mouse Dach1 gene, involved in the development of the neocortex and the hippocampus, is expressed by neural stem cells (NSCs) during early neurogenesis, and its expression also continues in a subpopulation of cells in the dorsal part of the lateral ventricles (LV) of the adult mouse brain. In this study we aimed to elucidate the role of Dach1-expressing cells in adult neurogenesis/gliogenesis under physiological as well as post-ischemic conditions, employing transgenic mice in which the expression of green fluorescent protein (GFP) is controlled by the D6 promotor of the mouse Dach1 gene. A neurosphere-forming assay of GFP+ cells isolated from the dorsal part of the LV was carried out with subsequent differentiation in vitro. To elucidate the neurogenic/gliogenic potential of GFP+ cells in the dorsal part of the LV, in situ immunohistochemical/electrophysiological analyses of GFP+ cells in adult sham-operated brains (controls) and those after middle cerebral artery occlusion (MCAo) were performed. The GFP+ cells isolated from the dorsal part of the LV of controls formed neurospheres and differentiated solely into a glial phenotype, while those isolated after MCAo also gave rise to cells with the properties of neuronal precursors. In situ analyses revealed that GFP+ cells express the phenotype of adult NSCs or neuroblasts in controls as well as following ischemia. Following MCAo we found a significantly increased number of GFP+ cells expressing doublecortin as well as a number of GFP+ cells migrating through the rostral migratory stream into the olfactory bulb, where they probably differentiated into calretinin+ interneurons. Collectively, our results suggest the involvement of the mouse Dach1 gene in adult neurogenesis; cells expressing this gene exhibit the properties of adult NSCs or neuroblasts and respond to MCAo by enhanced neurogenesis.

Abstract 4157: DACH1 Inhibited proliferation and tumor growth of lung adenocarcinoma cells

Abstract Lung cancer is the leading cause of cancer death worldwide. A key driver to the proliferation of lung cancer includes increased activity of the gene encoding the epidermal growth factor receptor (EGFR) and mutation of K-ras oncogene. Target therapies through small molecules to inhibit the tyrosine kinase domain of EGFR (Gefitinib) or monoclonal antibody to EGFR (Cetuximab) have demonstrated clinical benefit. Unfortunately, development of resistance to those treatments occurs in most patients within one year. Studies of the hyperactive EGFR in drosophila identified a genetic inhibitor of EGFR signaling, known as dachshund (dac). Activate ras inhibits DACH1 expression in malignant peripheral nerve sheath tumor. Dachshund can activate or represses transcription, and regulates cell proliferation and differentiation in multiple context-dependent manners. Our studies have proved that DACH1, human homolog of dac, functions as a potential tumor suppressor of breast and prostate cancer. Expression of DACH1 is lost during cancer progression and re-expression of DACH1 gene inhibited cellular proliferation, migration and tumor formation. The expression levels and the role of DACH1 in lung cancer are largely unknown. As both EGFR and Ras signaling interact with DACH1, we hypotheses that loss of endogenous DACH1 may contribute to the lung tumorigenesis. The mRNA expression of DACH1 was decreased in lung adenocarcinoma compared with normal lung tissues. Ectopic expression of DACH1 by stably transduction inhibited cellular proliferation of lung cancer cell lines A549 and SKLU, cell cycle progression and clone formation rate in contact-dependent and -independent growth. ELISA using conditional medium from A549 cells expressing vector control or DACH1 demonstrated remarkable inhibition of secreting factors, such as CXCL1, CXCL2, CXCL5 and CXCL8. Those chemokines have already been proved to positively regulating angiogenesis and proliferation. To test in vivo tumorigenesis, 2x106 A549 cells expressing DACH1 or vector control were injected subcutaneously into nude mouse and tumor size was monitored weekly. Tumor growth was dramatically blocked by DACH1. Tumor weight was 297.8 ± 38.6mg for vector control and 31 ± 6.1mg for cells expressing DACH1(p value <0.001). Importantly, over-expression of CXCL5 can partly rescue tumor growth inhibition by DACH1 in A549 cells. Together, our study demonstrated that DACH1 is a potentially tumor suppressor of lung cancer though repression of chemokines secretion. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4157. doi:1538-7445.AM2012-4157

Abstract LB-144: A p53-dependent G2/M checkpoint governed by the cell-fate factor dachshund in non-small cell lung cancer

Abstract Lung cancer is the leading cause of cancer mortality worldwide with more than 170,000 cases diagnosed each year in the United States. Genetic studies have identified several oncogenes activated in lung cancer including K-Ras and EGFR. Tumor suppressor inactivation occurs including p53 in which LOH is observed in - 60% and mutations arise in 50%. Initially cloned as a dominant inhibitor of the hyperactive EGFR, Ellipse in Drosophila, the mammalian DACH1 gene regulates gene expression of target genes in part through interacting with DNA-binding transcription factors and in part through intrinsic DNA-sequence specific binding properties. Clinical studies have demonstrated a reduced expression of the DACH1 in breast, prostate and endometrial cancer. Given the importance of the EGFR in human lung cancer, we examined the role of DACH1 in lung cancer cellular growth, migration and DNA damage response. Herein, endogenous DACH1 was reduced in human non-small cell lung cancer (NSCLS). Expression levels of DACH1 correlated inversely with clinical stage and pathological grade. Re-expression of DACH1 reduced lung cancer cell colony formation and cellular migration. Cell cycle analyses demonstrated G2/M block by ectopic expression of DACH1 occurs synergistically with p53. DACH1 band p53, requiring p53 R248 and R273, residues commonly mutated in lung cancer. Fluorescent microscopy demonstrated co-localization of DACH1 with p53. Immunoprecipitation and western blot assay showed DACH1 association with p53 required the C-terminus of DACH1. DACH1 enhanced p53 transcriptional induction of p21 promoter, p53 and DACH1 repressed Rad51 expression. DACH1 enhanced the cytotoxcity of cisplatin and doxorubicin, two commonly used drugs for NSCLS. Together, our studies demonstrate that p53 is DACH1 binding partner that inhibits NSCLS cellular proliferation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-144. doi:1538-7445.AM2012-LB-144

Analysis of the status of DACH1 gene promoter methylation in endometrial carcinoma and its clinical significance

To analyze the status of DACH1 gene promoter methylation and explore its association with the expression of DACH1 gene promoter methylation and clinical significance of endometrium carcinoma (EC). From February 2004 to August 2008, a total of 80 EC tissue samples with comprehensive surgical pathology staging were collected and used for this study. Twenty normal endometrium tissues in 2008 were abstained from the fractional curettage because of dysfunctional uterine bleeding as control. All samples were confirmed pathologically. Methylation specific PCR (MSP) was performed to detect the promoter methylation of DACH1 gene, and analyze its influence on the expression of DACH1 and the relationship between DACH1 promoter methylation and clinicopathological factors in EC. DACH1 protein expression was detected by western blot. Chi-square test and Pearson test were used for statistical analysis. The rate of promoter methylation of DACH1 gene in the EC tissues was significantly higher than that in the normal endometrium issues (30% vs. 5%, P < 0.05). There was an association between the expression of DACH1 and DACH1 gene promoter methylation (r = -0.30, P < 0.01). There was statistical difference between the methylation of DACH1 and the pathological grade (P < 0.05) or histological type (P < 0.05). But DACH1 gene methylation was not related with the age, stage, myometrial invasion depth and lymphnode metastasis (P > 0.05). DACH1 gene promoter methylaion could lead to a decrease or absence in the DACH1 expression in EC. The promoter methylation of DACH1 gene may induce the inhibition of DACH1 expression, which might be one of the mechanisms of DACH1 gene inactivation in human EC.

Dachshund homologues play a conserved role in islet cell development

All metazoans use insulin to control energy metabolism, but they secrete it from different cells: neurons in the central nervous system in invertebrates and endocrine cells in the gut or pancreas in vertebrates. Despite their origins in different germ layers, all of these insulin-producing cells share common functional features and gene expression patterns. In this study, we tested the role in insulin-producing cells of the vertebrate homologues of Dachshund, a transcriptional regulator that marks the earliest committed progenitors of the neural insulin-producing cells in Drosophila. Both zebrafish and mice expressed a single dominant Dachshund homologue in the pancreatic endocrine lineage, and in both species loss of this homologue reduced the numbers of all islet cell types including the insulin-producing β-cells. In mice, Dach1 gene deletion left the pancreatic progenitor cells unaltered, but blocked the perinatal burst of proliferation of differentiated β-cells that normally generates most of the β-cell mass. In β-cells, Dach1 bound to the promoter of the cell cycle inhibitor p27Kip1, which constrains β-cell proliferation. Taken together, these data demonstrate a conserved role for Dachshund homologues in the production of insulin-producing cells.

The Cell Fate Determination Factor Dachshund Inhibits Androgen Receptor Signaling and Prostate Cancer Cellular Growth

Initially isolated as the dominant suppressor of the mutant epidermal growth factor receptor (ellipse), the Dachshund gene plays a key role in metazoan development regulating the Retinal Determination Gene Network. Herein, the DACH1 gene was expressed in normal prostate epithelial cells with reduced expression in human prostate cancer. DACH1 inhibited prostate cancer cellular DNA synthesis, growth in colony forming assays, and blocked contact-independent growth in soft agar assays. DACH1 inhibited androgen receptor (AR) activity, requiring a conserved DS Domain (Dachshund domain conserved with Ski/Sno) that bound NCoR/HDAC and was recruited to an androgen-responsive gene promoter. DACH1 inhibited ligand-dependent activity of AR mutations identified in patients with androgen-insensitive prostate cancer. The DS domain was sufficient for repression of the AR wild-type but failed to repress an AR acetylation site point mutant. These studies show a role for the Retinal Determination Gene Network in regulating cellular growth and signaling in prostate cancer.