Gene Summary

Gene:OTX2; orthodenticle homeobox 2
Aliases: CPHD6, MCOPS5
Summary:This gene encodes a member of the bicoid subfamily of homeodomain-containing transcription factors. The encoded protein acts as a transcription factor and plays a role in brain, craniofacial, and sensory organ development. The encoded protein also influences the proliferation and differentiation of dopaminergic neuronal progenitor cells during mitosis. Mutations in this gene cause syndromic microphthalmia 5 (MCOPS5) and combined pituitary hormone deficiency 6 (CPHD6). This gene is also suspected of having an oncogenic role in medulloblastoma. Alternative splicing results in multiple transcript variants encoding distinct isoforms. Pseudogenes of this gene are known to exist on chromosomes two and nine. [provided by RefSeq, Jul 2012]
Databases:OMIM, HGNC, Ensembl, GeneCard, Gene
Protein:homeobox protein OTX2
Source:NCBIAccessed: 31 August, 2019


What does this gene/protein do?
Show (29)

Cancer Overview

Research Indicators

Publications Per Year (1994-2019)
Graph generated 31 August 2019 using data from PubMed using criteria.

Literature Analysis

Mouse over the terms for more detail; many indicate links which you can click for dedicated pages about the topic.

  • Teratoma
  • DNA Methylation
  • Cell Movement
  • Oligonucleotide Array Sequence Analysis
  • Retinoblastoma
  • Chromosome 14
  • Hedgehog Proteins
  • Basic Helix-Loop-Helix Transcription Factors
  • World Health Organization
  • Otx Transcription Factors
  • Wnt Proteins
  • Visual Perception
  • Cell Proliferation
  • Medulloblastoma
  • Homeodomain Proteins
  • Adolescents
  • Nerve Tissue Proteins
  • Childhood Cancer
  • Gene Expression Profiling
  • siRNA
  • Wnt Signaling Pathway
  • Cell Differentiation
  • p53 Protein
  • Infant
  • Molecular Sequence Data
  • Brain Tumours
  • Retinoic Acid
  • Transcription Factors
  • Promoter Regions
  • Neurons
  • Repressor Proteins
  • Transcriptome
  • Base Sequence
  • Cerebellar Neoplasms
  • Messenger RNA
  • Single Nucleotide Polymorphism
  • Biomarkers, Tumor
  • Cancer Gene Expression Regulation
  • Proto-Oncogene Proteins c-myc
  • Brain Tumours
  • Trans-Activators
Tag cloud generated 31 August, 2019 using data from PubMed, MeSH and CancerIndex

Specific Cancers (4)

Data table showing topics related to specific cancers and associated disorders. Scope includes mutations and abnormal protein expression.

Note: list is not exhaustive. Number of papers are based on searches of PubMed (click on topic title for arbitrary criteria used).

Latest Publications: OTX2 (cancer-related)

Ferrucci V, de Antonellis P, Pennino FP, et al.
Metastatic group 3 medulloblastoma is driven by PRUNE1 targeting NME1-TGF-β-OTX2-SNAIL via PTEN inhibition.
Brain. 2018; 141(5):1300-1319 [PubMed] Related Publications
Genetic modifications during development of paediatric groups 3 and 4 medulloblastoma are responsible for their highly metastatic properties and poor patient survival rates. PRUNE1 is highly expressed in metastatic medulloblastoma group 3, which is characterized by TGF-β signalling activation, c-MYC amplification, and OTX2 expression. We describe the process of activation of the PRUNE1 signalling pathway that includes its binding to NME1, TGF-β activation, OTX2 upregulation, SNAIL (SNAI1) upregulation, and PTEN inhibition. The newly identified small molecule pyrimido-pyrimidine derivative AA7.1 enhances PRUNE1 degradation, inhibits this activation network, and augments PTEN expression. Both AA7.1 and a competitive permeable peptide that impairs PRUNE1/NME1 complex formation, impair tumour growth and metastatic dissemination in orthotopic xenograft models with a metastatic medulloblastoma group 3 cell line (D425-Med cells). Using whole exome sequencing technology in metastatic medulloblastoma primary tumour cells, we also define 23 common 'non-synonymous homozygous' deleterious gene variants as part of the protein molecular network of relevance for metastatic processes. This PRUNE1/TGF-β/OTX2/PTEN axis, together with the medulloblastoma-driver mutations, is of relevance for future rational and targeted therapies for metastatic medulloblastoma group 3.10.1093/brain/awy039_video1awy039media15742053534001.

Stromecki M, Tatari N, Morrison LC, et al.
Characterization of a novel OTX2-driven stem cell program in Group 3 and Group 4 medulloblastoma.
Mol Oncol. 2018; 12(4):495-513 [PubMed] Free Access to Full Article Related Publications
Medulloblastoma (MB) is the most common malignant primary pediatric brain cancer. Among the most aggressive subtypes, Group 3 and Group 4 originate from stem/progenitor cells, frequently metastasize, and often display the worst prognosis, yet we know the least about the molecular mechanisms driving their progression. Here, we show that the transcription factor orthodenticle homeobox 2 (OTX2) promotes self-renewal while inhibiting differentiation in vitro and increases tumor initiation from MB stem/progenitor cells in vivo. To determine how OTX2 contributes to these processes, we employed complementary bioinformatic approaches to characterize the OTX2 regulatory network and identified novel relationships between OTX2 and genes associated with neuronal differentiation and axon guidance signaling in Group 3 and Group 4 MB stem/progenitor cells. In particular, OTX2 levels were negatively correlated with semaphorin (SEMA) signaling, as expression of 9 SEMA pathway genes is upregulated following OTX2 knockdown with some being potential direct OTX2 targets. Importantly, this negative correlation was also observed in patient samples, with lower expression of SEMA4D associated with poor outcome specifically in Group 4 tumors. Functional proof-of-principle studies demonstrated that increased levels of select SEMA pathway genes are associated with decreased self-renewal and growth in vitro and in vivo and that RHO signaling, known to mediate the effects of SEMA genes, is contributing to the OTX2 KD phenotype. Our study provides mechanistic insight into the networks controlled by OTX2 in MB stem/progenitor cells and reveals novel roles for axon guidance genes and their downstream effectors as putative tumor suppressors in MB.

El Nagar S, Zindy F, Moens C, et al.
A new genetically engineered mouse model of choroid plexus carcinoma.
Biochem Biophys Res Commun. 2018; 496(2):568-574 [PubMed] Free Access to Full Article Related Publications
Choroid plexus carcinomas (CPCs) are highly malignant brain tumours predominantly found in children and associated to poor prognosis. Improved therapy for these cancers would benefit from the generation of animal models. Here we have created a novel mouse CPC model by expressing a stabilised form of c-Myc (MycT58A) and inactivating Trp53 in the choroid plexus of newborn mice. This induced aberrant proliferation of choroid plexus epithelial cells, leading to aggressive tumour development and death within 150 days. Choroid plexus tumours occurred with a complete penetrance in all brain ventricles, with prevalence in the lateral and fourth ventricles. Histological and cellular analysis indicated that these tumours were CPCs resembling their human counterparts. Comparison of gene expression profiles of CPCs and non-neoplastic tissues revealed profound alterations in cell cycle regulation and DNA damage responses, suggesting that dysregulation of cell division and DNA checkpoint pathways may represent key vulnerabilities. This novel animal model of CPC provides an invaluable tool to elucidate the mechanism of CPC formation and to develop successful therapies against this devastating paediatric cancer.

Sivagurunathan S, Arunachalam JP, Chidambaram S
PIWI-like protein, HIWI2 is aberrantly expressed in retinoblastoma cells and affects cell-cycle potentially through OTX2.
Cell Mol Biol Lett. 2017; 22:17 [PubMed] Free Access to Full Article Related Publications
Retinoblastoma (RB), a childhood cancer, is caused by biallelic mutation of the

Ke C, Wang J, Xi S, et al.
An Unusual Combination of Mirror-Image Dextrocardia with Familial Medulloblastoma: Is There a Histogenetic Relationship?
World Neurosurg. 2017; 107:860-867 [PubMed] Related Publications
BACKGROUND: The occurrence of medulloblastoma in the absence of hereditary syndromes is rare. Dextrocardia with situs inversus is also called mirror-image dextrocardia. A combination of mirror-image dextrocardia with medulloblastoma has not been reported previously. To the best of our knowledge, this is the first report of this rare combination in a family with medulloblastoma.
METHODS: The clinical manifestation, radiographic characteristics, treatment, and outcomes of 3 medulloblastoma cases in 2 cousins and their maternal uncle was described. Tumor samples of the 2 cousins were first examined for histologic subtypes. Total RNA of their tumors was extracted from formalin-fixed and paraffin-embedded samples. Then, expression of 22 subgroup-specific genes and 3 housekeeping genes was analyzed by the NanoString nCounter Analysis System. The posttest data were normalized by NanoStringNorm package for molecular subgroup prediction.
RESULTS: The proband remains tumor free and alive up to the latest follow-up. His cousin, who had combined mirror-image dextrocardia with situs inversus, died of anoxia after surgery and his uncle died of tumor 2.5 years after surgery. Medulloblastoma of the 2 cousins was classified as classic and molecular group 4 subtype.
CONCLUSIONS: The same classic and molecular group 4 subtype of the 2 cousins may suggest a similar genetic predisposition. Involvement of the Otx2 gene dysfunction in both group 4 subtype medulloblastoma and mirror-image dextrocardia with situs inversus points to a possible mechanism that dysfunction of a shared signaling pathway such as Otx2 might be the underlying cause of these 2 conditions in this family.

Pirrone C, Chiaravalli AM, Marando A, et al.
OTX1 and OTX2 as possible molecular markers of sinonasal carcinomas and olfactory neuroblastomas.
Eur J Histochem. 2017; 61(1):2730 [PubMed] Free Access to Full Article Related Publications
OTX Homeobox genes are involved in embryonic morphogenesis and in the development of olfactory epithelium in adult. Mutations occurring in the OTX genes are reported to be associated to tumorigenisis in human. No reports correlate the expression of OTX genes and neoplasms of the nasal cavity. Thus, through immunohistochemical and Real-time PCR analysis we investigated OTX1 and OTX2 expression in the more frequent types of nasal and sinonasal tumours. Variable expression of both genes were found in normal sinonasal mucosa and in tumours. Interestingly, no expression of both OTX genes were detected in sinonasal intestinal-type adenocarcinomas; only OTX1 was found in non-intestinal-type adenocarcinomas and OTX2 was selectively expressed in olfactory neuroblastomas. In conclusion, OTX1 and OTX2 genes might have a role in the pathogenesis of different types of sinonasal neoplasms.

Boulay G, Awad ME, Riggi N, et al.
OTX2 Activity at Distal Regulatory Elements Shapes the Chromatin Landscape of Group 3 Medulloblastoma.
Cancer Discov. 2017; 7(3):288-301 [PubMed] Free Access to Full Article Related Publications
Medulloblastoma is the most frequent malignant pediatric brain tumor and is divided into at least four subgroups known as WNT, SHH, Group 3, and Group 4. Here, we characterized gene regulation mechanisms in the most aggressive subtype, Group 3 tumors, through genome-wide chromatin and expression profiling. Our results show that most active distal sites in these tumors are occupied by the transcription factor OTX2. Highly active OTX2-bound enhancers are often arranged as clusters of adjacent peaks and are also bound by the transcription factor NEUROD1. These sites are responsive to

Japp AS, Klein-Hitpass L, Denkhaus D, Pietsch T
OTX2 Defines a Subgroup of Atypical Teratoid Rhabdoid Tumors With Close Relationship to Choroid Plexus Tumors.
J Neuropathol Exp Neurol. 2017; 76(1):32-38 [PubMed] Related Publications
Atypical teratoid rhabdoid tumors (ATRT) are highly malignant brain tumors of early childhood that have been regarded as a homogenous entity characterized by inactivation of the SMARCB1/INI1 or SMARCA4/BRG1 genes as the only characteristic alteration. Recent studies suggest that similar to other embryonal tumors ATRT can also be divided into subgroups based on their mRNA or methylation profiles. Using microarray-based expression analysis of 12 patient ATRT specimens we demonstrated the existence of 2 subgroups of ATRT. One subgroup is characterized by high expression of OTX2, encoding a transcription factor involved in brain development. OTX2 expression was verified by immunohistochemistry and might function as a novel therapeutic target for this fatal tumor. High expression of OTX2 as well as expression of Kir7.1/KCNJ13, TRPM3 and ENPP2, which have all previously been linked to either choroid plexus epithelium or choroid plexus tumors (CPTs), suggests a close histogenetic relation of this subgroup to CPTs.

Daugaard I, Dominguez D, Kjeldsen TE, et al.
Identification and validation of candidate epigenetic biomarkers in lung adenocarcinoma.
Sci Rep. 2016; 6:35807 [PubMed] Free Access to Full Article Related Publications
Lung cancer is the number one cause of cancer-related deaths worldwide. DNA methylation is an epigenetic mechanism that regulates gene expression, and disease-specific methylation changes can be targeted as biomarkers. We have compared the genome-wide methylation pattern in tumor and tumor-adjacent normal lung tissue from four lung adenocarcinoma (LAC) patients using DNA methylation microarrays and identified 74 differentially methylated regions (DMRs). Eighteen DMRs were selected for validation in a cohort comprising primary tumors from 52 LAC patients and tumor-adjacent normal lung tissue from 32 patients by methylation-sensitive high resolution melting (MS-HRM) analysis. Significant increases in methylation were confirmed for 15 DMRs associated with the genes and genomic regions: OSR1, SIM1, GHSR, OTX2, LOC648987, HIST1H3E, HIST1H3G/HIST1H2BI, HIST1H2AJ/HIST1H2BM, HOXD10, HOXD3, HOXB3/HOXB4, HOXA3, HOXA5, Chr1(q21.1).A, and Chr6(p22.1). In particular the OSR1, SIM1 and HOXB3/HOXB4 regions demonstrated high potential as biomarkers in LAC. For OSR1, hypermethylation was detected in 47/48 LAC cases compared to 1/31 tumor-adjacent normal lung samples. Similarly, 45/49 and 36/48 LAC cases compared to 3/31 and 0/31 tumor-adjacent normal lung samples showed hypermethylation of the SIM1 and HOXB3/HOXB4 regions, respectively. In conclusion, this study has identified and validated 15 DMRs that can be targeted as biomarkers in LAC.

Pietsch T, Haberler C
Update on the integrated histopathological and genetic classification of medulloblastoma - a practical diagnostic guideline.
Clin Neuropathol. 2016 Nov/Dec; 35(6):344-352 [PubMed] Free Access to Full Article Related Publications
The revised WHO classification of tumors of the CNS 2016 has introduced the concept of the integrated diagnosis. The definition of medulloblastoma entities now requires a combination of the traditional histological information with additional molecular/genetic features. For definition of the histopathological component of the medulloblastoma diagnosis, the tumors should be assigned to one of the four entities classic, desmoplastic/nodular (DNMB), extensive nodular (MBEN), or large cell/anaplastic (LC/A) medulloblastoma. The genetically defined component comprises the four entities WNT-activated, SHH-activated and TP53 wildtype, SHH-activated and TP53 mutant, or non-WNT/non-SHH medulloblastoma. Robust and validated methods are available to allow a precise diagnosis of these medulloblastoma entities according to the updated WHO classification, and for differential diagnostic purposes. A combination of immunohistochemical markers including β-catenin, Yap1, p75-NGFR, Otx2, and p53, in combination with targeted sequencing and copy number assessment such as FISH analysis for MYC genes allows a precise assignment of patients for risk-adapted stratification. It also allows comparison to results of study cohorts in the past and provides a robust basis for further treatment refinement.

Nagel S, Meyer C, Kaufmann M, et al.
Aberrant expression of homeobox gene SIX1 in Hodgkin lymphoma.
Oncotarget. 2015; 6(37):40112-26 [PubMed] Free Access to Full Article Related Publications
In Hodgkin lymphoma (HL) we recently identified deregulated expression of homeobox genes MSX1 and OTX2 which are physiologically involved in development of the embryonal neural plate border region. Here, we examined in HL homeobox gene SIX1 an additional regulator of this embryonal region mediating differentiation of placodal precursors. SIX1 was aberrantly activated in 12 % of HL patient samples in silico, indicating a pathological role in a subset of this B-cell malignancy. In addition, SIX1 expression was detected in HL cell lines which were used as models to reveal upstream factors and target genes of this basic developmental regulator. We detected increased copy numbers of the SIX1 locus at chromosome 14q23 correlating with enhanced expression while chromosomal translocations were absent. Moreover, comparative expression profiling data and pertinent gene modulation experiments indicated that the WNT-signalling pathway and transcription factor MEF2C regulate SIX1 expression. Genes encoding the transcription factors GATA2, GATA3, MSX1 and SPIB - all basic lymphoid regulators - were identified as targets of SIX1 in HL. In addition, cofactors EYA1 and TLE4, respectively, contrastingly mediated activation and suppression of SIX1 target gene expression. Thus, the protein domain interfaces may represent therapeutic targets in SIX1-positive HL subsets. Collectively, our data reveal a gene regulatory network with SIX1 centrally deregulating lymphoid differentiation and support concordance of lymphopoiesis/lymphomagenesis and developmental processes in the neural plate border region.

Nagel S, Ehrentraut S, Meyer C, et al.
Aberrantly Expressed OTX Homeobox Genes Deregulate B-Cell Differentiation in Hodgkin Lymphoma.
PLoS One. 2015; 10(9):e0138416 [PubMed] Free Access to Full Article Related Publications
In Hodgkin lymphoma (HL) we recently reported that deregulated homeobox gene MSX1 mediates repression of the B-cell specific transcription factor ZHX2. In this study we investigated regulation of MSX1 in this B-cell malignancy. Accordingly, we analyzed expression and function of OTX homeobox genes which activate MSX1 transcription during embryonal development in the neural plate border region. Our data demonstrate that OTX1 and OTX2 are aberrantly expressed in both HL patients and cell lines. Moreover, both OTX loci are targeted by genomic gains in overexpressing cell lines. Comparative expression profiling and subsequent pathway modulations in HL cell lines indicated that aberrantly enhanced FGF2-signalling activates the expression of OTX2. Downstream analyses of OTX2 demonstrated transcriptional activation of genes encoding transcription factors MSX1, FOXC1 and ZHX1. Interestingly, examination of the physiological expression profile of ZHX1 in normal hematopoietic cells revealed elevated levels in T-cells and reduced expression in B-cells, indicating a discriminatory role in lymphopoiesis. Furthermore, two OTX-negative HL cell lines overexpressed ZHX1 in correlation with genomic amplification of its locus at chromosomal band 8q24, supporting the oncogenic potential of this gene in HL. Taken together, our data demonstrate that deregulated homeobox genes MSX1 and OTX2 respectively impact transcriptional inhibition of (B-cell specific) ZHX2 and activation of (T-cell specific) ZHX1. Thus, we show how reactivation of a specific embryonal gene regulatory network promotes disturbed B-cell differentiation in HL.

Kaur R, Aiken C, Morrison LC, et al.
OTX2 exhibits cell-context-dependent effects on cellular and molecular properties of human embryonic neural precursors and medulloblastoma cells.
Dis Model Mech. 2015; 8(10):1295-309 [PubMed] Free Access to Full Article Related Publications
Medulloblastoma (MB) is the most common malignant primary pediatric brain tumor and is currently divided into four subtypes based on different genomic alterations, gene expression profiles and response to treatment: WNT, Sonic Hedgehog (SHH), Group 3 and Group 4. This extensive heterogeneity has made it difficult to assess the functional relevance of genes to malignant progression. For example, expression of the transcription factor Orthodenticle homeobox2 (OTX2) is frequently dysregulated in multiple MB variants; however, its role may be subtype specific. We recently demonstrated that neural precursors derived from transformed human embryonic stem cells (trans-hENs), but not their normal counterparts (hENs), resemble Groups 3 and 4 MB in vitro and in vivo. Here, we tested the utility of this model system as a means of dissecting the role of OTX2 in MB using gain- and loss-of-function studies in hENs and trans-hENs, respectively. Parallel experiments with MB cells revealed that OTX2 exerts inhibitory effects on hEN and SHH MB cells by regulating growth, self-renewal and migration in vitro and tumor growth in vivo. This was accompanied by decreased expression of pluripotent genes, such as SOX2, and was supported by overexpression of SOX2 in OTX2+ SHH MB and hENs that resulted in significant rescue of self-renewal and cell migration. By contrast, OTX2 is oncogenic and promotes self-renewal of trans-hENs and Groups 3 and 4 MB independent of pluripotent gene expression. Our results demonstrate a novel role for OTX2 in self-renewal and migration of hENs and MB cells and reveal a cell-context-dependent link between OTX2 and pluripotent genes. Our study underscores the value of human embryonic stem cell derivatives as alternatives to cell lines and heterogeneous patient samples for investigating the contribution of key developmental regulators to MB progression.

Li J, Di C, Jing J, et al.
OTX2 is a therapeutic target for retinoblastoma and may function as a common factor between C-MYC, CRX, and phosphorylated RB pathways.
Int J Oncol. 2015; 47(5):1703-10 [PubMed] Related Publications
The homeobox transcription factor orthodenticle homeobox 2 (OTX2) plays a critical role in very early neurogenesis, but can become oncogenic when aberrantly expressed later in life. We previously discovered its novel oncogenic role in the malignant childhood brain tumor medulloblastoma and hypothesize an oncogenic role in retinoblastoma. Primary retinoblastoma tumors and cell lines were analyzed by quantitative-PCR, immunoblotting and immunohistochemistry for OTX2. The effect of modulating OTX2 expression on tumorigenesis was tested pharmacologically and by siRNA. A lentiviral shRNA-engineered vector was used for conditional knockdown studies on tumor growth in vivo. A luciferase reporter assay was used to analyze ATRA's effect on OTX2's promoter. In this study on retinoblastoma, OTX2 was frequently amplified and/or overexpressed in primary tumors and cell lines. Knockdown of OTX2 expression by siRNA or pharmacologic inhibition by all-trans retinoic acid (ATRA) repressed OTX2 expression and cell proliferation and significantly decreased tumor growth in vivo. Loss of OTX2 expression also resulted in decreased expression of C-MYC and CRX, genes previously implicated in retinoblastoma tumorigenesis. Loss of OTX2 expression increased the phosphorylation of RB, a potential mechanism of modulating cell proliferation. Aberrant expression of OTX2 may contribute to the development of retinoblastoma. OTX2 may serve as a common transcription factor that interlinks multiple tumor-driving pathways. These results also show that OTX2 can be genetically and pharmacologically targeted, providing an exciting new therapeutic option that may be less toxic and more efficacious than current treatments.

Japp AS, Gessi M, Messing-Jünger M, et al.
High-resolution genomic analysis does not qualify atypical plexus papilloma as a separate entity among choroid plexus tumors.
J Neuropathol Exp Neurol. 2015; 74(2):110-20 [PubMed] Related Publications
Choroid plexus tumors are rare neoplasms that mainly affect children. They include papillomas, atypical papillomas, and carcinomas. Detailed genetic studies are rare, and information about their molecular pathogenesis is limited. Molecular inversion probe analysis is a hybridization-based method that represents a reliable tool for the analysis of highly fragmented formalin-fixed paraffin-embedded tissue-derived DNA. Here, analysis of 62 cases showed frequent hyperdiploidy in papillomas and atypical papillomas that appeared very similar in their cytogenetic profiles. In contrast, carcinomas showed mainly losses of chromosomes. Besides recurrent focal chromosomal gains common to all choroid plexus tumors, including chromosome 14q21-q22 (harboring OTX2), chromosome 7q22 (LAMB1), and chromosome 9q21.12 (TRPM3), Genomic Identification of Significant Targets in Cancer analysis uncovered focal alterations specific for papillomas and atypical papillomas (e.g. 7p21.3 [ARL4A]) and for carcinomas (16p13.3 [RBFOX1] and 6p21 [POLH, GTPBP2, RSPH9, and VEGFA]). Additional RNA expression profiling and gene set enrichment analysis revealed greater expression of cell cycle-related genes in atypical papillomas in comparison with that in papillomas. These findings suggest that atypical papillomas represent an immature variant of papillomas characterized by increased proliferative activity, whereas carcinomas seem to represent a genetically distinct tumor group.

Shou Y, Robinson DM, Amakye DD, et al.
A five-gene hedgehog signature developed as a patient preselection tool for hedgehog inhibitor therapy in medulloblastoma.
Clin Cancer Res. 2015; 21(3):585-93 [PubMed] Related Publications
PURPOSE: Distinct molecular subgroups of medulloblastoma, including hedgehog (Hh) pathway-activated disease, have been reported. We identified and clinically validated a five-gene Hh signature assay that can be used to preselect patients with Hh pathway-activated medulloblastoma.
EXPERIMENTAL DESIGN: Gene characteristics of the Hh medulloblastoma subgroup were identified through published bioinformatic analyses. Thirty-two genes shown to be differentially expressed in fresh-frozen and formalin-fixed paraffin-embedded tumor samples and reproducibly analyzed by RT-PCR were measured in matched samples. These data formed the basis for building a multi-gene logistic regression model derived through elastic net methods from which the five-gene Hh signature emerged after multiple iterations. On the basis of signature gene expression levels, the model computed a propensity score to determine Hh activation using a threshold set a priori. The association between Hh activation status and tumor response to the Hh pathway inhibitor sonidegib (LDE225) was analyzed.
RESULTS: Five differentially expressed genes in medulloblastoma (GLI1, SPHK1, SHROOM2, PDLIM3, and OTX2) were found to associate with Hh pathway activation status. In an independent validation study, Hh activation status of 25 medulloblastoma samples showed 100% concordance between the five-gene signature and Affymetrix profiling. Further, in medulloblastoma samples from 50 patients treated with sonidegib, all 6 patients who responded were found to have Hh-activated tumors. Three patients with Hh-activated tumors had stable or progressive disease. No patients with Hh-nonactivated tumors responded.
CONCLUSIONS: This five-gene Hh signature can robustly identify Hh-activated medulloblastoma and may be used to preselect patients who might benefit from sonidegib treatment.

Xu J, Margol A, Asgharzadeh S, Erdreich-Epstein A
Pediatric brain tumor cell lines.
J Cell Biochem. 2015; 116(2):218-24 [PubMed] Related Publications
Pediatric brain tumors as a group, including medulloblastomas, gliomas, and atypical teratoid rhabdoid tumors (ATRT) are the most common solid tumors in children and the leading cause of death from childhood cancer. Brain tumor-derived cell lines are critical for studying the biology of pediatric brain tumors and can be useful for initial screening of new therapies. Use of appropriate brain tumor cell lines for experiments is important, as results may differ depending on tumor properties, and can thus affect the conclusions and applicability of the model. Despite reports in the literature of over 60 pediatric brain tumor cell lines, the majority of published papers utilize only a small number of these cell lines. Here we list the approximately 60 currently-published pediatric brain tumor cell lines and summarize some of their central features as a resource for scientists seeking pediatric brain tumor cell lines for their research.

Wortham M, Guo C, Zhang M, et al.
Chromatin accessibility mapping identifies mediators of basal transcription and retinoid-induced repression of OTX2 in medulloblastoma.
PLoS One. 2014; 9(9):e107156 [PubMed] Free Access to Full Article Related Publications
Despite an emerging understanding of the genetic alterations giving rise to various tumors, the mechanisms whereby most oncogenes are overexpressed remain unclear. Here we have utilized an integrated approach of genomewide regulatory element mapping via DNase-seq followed by conventional reporter assays and transcription factor binding site discovery to characterize the transcriptional regulation of the medulloblastoma oncogene Orthodenticle Homeobox 2 (OTX2). Through these studies we have revealed that OTX2 is differentially regulated in medulloblastoma at the level of chromatin accessibility, which is in part mediated by DNA methylation. In cell lines exhibiting chromatin accessibility of OTX2 regulatory regions, we found that autoregulation maintains OTX2 expression. Comparison of medulloblastoma regulatory elements with those of the developing brain reveals that these tumors engage a developmental regulatory program to drive OTX2 transcription. Finally, we have identified a transcriptional regulatory element mediating retinoid-induced OTX2 repression in these tumors. This work characterizes for the first time the mechanisms of OTX2 overexpression in medulloblastoma. Furthermore, this study establishes proof of principle for applying ENCODE datasets towards the characterization of upstream trans-acting factors mediating expression of individual genes.

Shi JA, Lu DL, Huang X, Tan W
miR-219 inhibits the proliferation, migration and invasion of medulloblastoma cells by targeting CD164.
Int J Mol Med. 2014; 34(1):237-43 [PubMed] Related Publications
It is known that microRNA-219 (miR-219) expression is downregulated in medulloblastoma. In the present study, we investigated the expression, targets and functional effects of miR-219 in D283-MED medulloblastoma cells. We first demonstrated that miR-219 not only inhibits proliferation, but also suppresses the invasion and migration of D283-MED cells. Moreover, the knockdown of miR-219 promoted the proliferation, migration and invasion of the D283-MED cells. Secondly, we predicted that miR-219 targets the 3' untranslated region (3'UTR) of CD164 and orthodenticle homeobox 2 (OTX2) and then confirmed that it significantly downregulated the protein expression of CD164 and OTX2 in D283-MED cells. Finally, we demonstrated that the proliferation, invasion and migration of D283-MED cells were promoted by theectopic expression of CD164. These results indicate that miR-219 suppresses the proliferation, migration and invasion of medulloblastoma cells by targeting CD164. The results also suggest that miR-219 may serve as a potential therapeutic agent for medulloblastoma.

McEvoy J, Nagahawatte P, Finkelstein D, et al.
RB1 gene inactivation by chromothripsis in human retinoblastoma.
Oncotarget. 2014; 5(2):438-50 [PubMed] Free Access to Full Article Related Publications
Retinoblastoma is a rare childhood cancer of the developing retina. Most retinoblastomas initiate with biallelic inactivation of the RB1 gene through diverse mechanisms including point mutations, nucleotide insertions, deletions, loss of heterozygosity and promoter hypermethylation. Recently, a novel mechanism of retinoblastoma initiation was proposed. Gallie and colleagues discovered that a small proportion of retinoblastomas lack RB1 mutations and had MYCN amplification [1]. In this study, we identified recurrent chromosomal, regional and focal genomic lesions in 94 primary retinoblastomas with their matched normal DNA using SNP 6.0 chips. We also analyzed the RB1 gene mutations and compared the mechanism of RB1 inactivation to the recurrent copy number variations in the retinoblastoma genome. In addition to the previously described focal amplification of MYCN and deletions in RB1 and BCOR, we also identified recurrent focal amplification of OTX2, a transcription factor required for retinal photoreceptor development. We identified 10 retinoblastomas in our cohort that lacked RB1 point mutations or indels. We performed whole genome sequencing on those 10 tumors and their corresponding germline DNA. In one of the tumors, the RB1 gene was unaltered, the MYCN gene was amplified and RB1 protein was expressed in the nuclei of the tumor cells. In addition, several tumors had complex patterns of structural variations and we identified 3 tumors with chromothripsis at the RB1 locus. This is the first report of chromothripsis as a mechanism for RB1 gene inactivation in cancer.

Mol BM, Massink MP, van der Hout AH, et al.
High resolution SNP array profiling identifies variability in retinoblastoma genome stability.
Genes Chromosomes Cancer. 2014; 53(1):1-14 [PubMed] Related Publications
Both hereditary and nonhereditary retinoblastoma (Rb) are commonly initiated by loss of both copies of the retinoblastoma tumor suppressor gene (RB1), while additional genomic changes are required for tumor initiation and progression. Our aim was to determine whether there is genomic heterogeneity between different clinical Rb subtypes. Therefore, 21 Rb tumors from 11 hereditary patients and 10 nonhereditary Rb patients were analyzed using high-resolution single nucleotide polymorphism (SNP) arrays and gene losses and gains were validated with Multiplex Ligation-dependent Probe Amplification. In these tumors only a few focal aberrations were detected. The most frequent was a focal gain on chromosome 2p24.3, the minimal region of gain encompassing the oncogene MYCN. The genes BAZ1A, OTX2, FUT8, and AKT1 were detected in four focal regions on chromosome 14 in one nonhereditary Rb. There was a large difference in number of copy number aberrations between tumors. A subset of nonhereditary Rbs turned out to be the most genomic unstable, while especially very young patients with hereditary Rb display stable genomes. Established Rb copy number aberrations, including gain of chromosome arm 1q and loss of chromosome arm 16q, turned out to be preferentially associated with the nonhereditary Rbs with later age of diagnosis. In contrast, copy number neutral loss of heterozygosity was detected mainly on chromosome 13, where RB1 resides, irrespective of hereditary status or age. Focal amplifications and deletions and copy number neutral loss of heterozygosity besides chromosome 13 appear to be rare events in retinoblastoma.

Zakrzewska M, Grešner SM, Zakrzewski K, et al.
Novel gene expression model for outcome prediction in paediatric medulloblastoma.
J Mol Neurosci. 2013; 51(2):371-9 [PubMed] Related Publications
Medulloblastoma is the most frequent type of embryonal tumour in the paediatric population. The disease progression in patients with this tumour may be connected with the presence of stem/tumour-initiating cells, but the precise source and characteristics of such cells is still a subject of debate. Thus, we tried to analyse biomarkers for which a connection with the presence of stem/tumour-initiating cells was suggested. We evaluated the transcriptional level of the ATOH1, FUT4, NGFR, OTX1, OTX2, PROM1 and SOX1 genes in 48 samples of medulloblastoma and analysed their usefulness in the prediction of disease outcome. The analyses showed a strong correlation of PROM1, ATOH1 and OTX1 gene expression levels with the outcome (p ≤ 0.2). On the basis of the multivariate Cox regression analysis, we propose a three-gene model predicting risk of the disease, calculated as follows: RS(risk score) =( 0:81 x PROM1) + (0:18 x OTX1) + (0:02 x ATOH1). Survival analysis revealed a better outcome among standard-risk patients, with a 5-year survival rate of 65 %, compared to the 40 % rate observed among high-risk patients. The most promising advantage of such molecular analysis consists in the identification of molecular markers influencing clinical behaviour, which may in turn be useful in therapy optimization.

Bunt J, Hasselt NA, Zwijnenburg DA, et al.
OTX2 sustains a bivalent-like state of OTX2-bound promoters in medulloblastoma by maintaining their H3K27me3 levels.
Acta Neuropathol. 2013; 125(3):385-94 [PubMed] Related Publications
Recent studies showed frequent mutations in histone H3 lysine 27 (H3K27) demethylases in medulloblastomas of Group 3 and Group 4, suggesting a role for H3K27 methylation in these tumors. Indeed, trimethylated H3K27 (H3K27me3) levels were shown to be higher in Group 3 and 4 tumors compared to WNT and SHH medulloblastomas, also in tumors without detectable mutations in demethylases. Here, we report that polycomb genes, required for H3K27 methylation, are consistently upregulated in Group 3 and 4 tumors. These tumors show high expression of the homeobox transcription factor OTX2. Silencing of OTX2 in D425 medulloblastoma cells resulted in downregulation of polycomb genes such as EZH2, EED, SUZ12 and RBBP4 and upregulation of H3K27 demethylases KDM6A, KDM6B, JARID2 and KDM7A. This was accompanied by decreased H3K27me3 and increased H3K27me1 levels in promoter regions. Strikingly, the decrease of H3K27me3 was most prominent in promoters that bind OTX2. OTX2-bound promoters showed high levels of the H3K4me3 and H3K9ac activation marks and intermediate levels of the H3K27me3 inactivation mark, reminiscent of a bivalent modification. After silencing of OTX2, H3K27me3 levels strongly dropped, but H3K4me3 and H3K9ac levels remained high. OTX2-bound bivalent genes showed high expression levels in D425, but the expression of most of these genes did not change after OTX2 silencing and loss of the H3K27me3 mark. Maintaining promoters in a bivalent state by sustaining H3K27 trimethylation therefore seems to be an important function of OTX2 in medulloblastoma, while other transcription factors might regulate the actual expression levels of these genes.

Bai RY, Staedtke V, Lidov HG, et al.
OTX2 represses myogenic and neuronal differentiation in medulloblastoma cells.
Cancer Res. 2012; 72(22):5988-6001 [PubMed] Free Access to Full Article Related Publications
The brain development transcription factor OTX2 is overexpressed and/or genomically amplified in most medulloblastomas, but the mechanistic basis for its contributions in this setting are not understood. In this study, we identified OTX2 as a transcriptional repressor and a gatekeeper of myogenic and neuronal differentiation in medulloblastoma cells. OTX2 binds to the MyoD1 core enhancer through its homeobox domain, and the remarkable repressor activity exhibited by the homeobox domain renders OTX2 transcriptionally repressive. RNA interference-mediated attenuation of OTX2 expression triggered myogenic and neuronal differentiation in vitro and prolonged the survival in an orthotopic medulloblastoma mouse model. Conversely, inducing myogenic conversion of medulloblastoma cells led to the loss of OTX2 expression. In medullomyoblastoma, a medulloblastoma subtype containing muscle elements, myogenic cells share cytogenetic signatures with the primitive tumor cells and OTX2 expression was lost in the differentiated myogenic cells. Thus, OTX2 functions via its homeobox domain as a suppressor of differentiation, and the loss of OTX2 expression is linked to the myogenesis in medullomyoblastoma. Together, our findings illustrate the origin of muscle cells in medullomyoblastomas and the oncogenic mechanism of OTX2 as a repressor of diverse differentiating potential.

Lian ZQ, Wang Q, Li WP, et al.
Screening of significantly hypermethylated genes in breast cancer using microarray-based methylated-CpG island recovery assay and identification of their expression levels.
Int J Oncol. 2012; 41(2):629-38 [PubMed] Related Publications
To screen candidate methylation markers for early detection of breast cancer and to explore the relationship between methylation and gene expression, we performed methylated-CpG island recovery assay (MIRA) combined with CpG island array on 61982 CpG sites across 4162 genes in 10 cancerous and 10 non-cancerous breast tissues. Direct bisulfite sequencing and combined bisulfite restriction analysis (COBRA) were carried out in independent cancerous and non-cancerous samples. Gene expression was analyzed by microarrays and validated using RT-PCR. We detected 70 significantly hypermethylated genes in breast cancer tissues, including many novel hypermethylated genes such as ITGA4, NFIX, OTX2 and FGF12. Direct bisulfite sequencing showed widespread methylation occurring in intragenic regions of the WT1, PAX6 and ITGA4 genes and in the promoter region of the OTX2 gene in breast cancer tissues. COBRA assay confirmed that the WT1, OTX2 and PAX6 genes were hypermethylated in breast cancer tissues. Clustering analysis of the gene expression of 70 significantly hypermethylated genes revealed that most hypermethylated genes in breast cancer were not expressed in breast tissues. RT-PCR assay confirmed that WT1 and PITX2 were only weakly expressed in the breast cancer tissues and were not expressed in most non-cancerous breast tissues. OTX2 and PAX6 were not expressed in either breast cancer or non-cancerous tissues. In conclusion, these results will expand our knowledge of hypermethylated genes and methylation sites for early detection of breast cancer and deepen our understanding of the relationship between methylation and gene expression. The MIRA approach can screen candidate methylated genes for further clinical validation more effectively than gene expression microarray-based strategy.

Wortham M, Jin G, Sun JL, et al.
Aberrant Otx2 expression enhances migration and induces ectopic proliferation of hindbrain neuronal progenitor cells.
PLoS One. 2012; 7(4):e36211 [PubMed] Free Access to Full Article Related Publications
Dysregulation of Otx2 is a hallmark of the pediatric brain tumor medulloblastoma, yet its functional significance in the establishment of these tumors is unknown. Here we have sought to determine the functional consequences of Otx2 overexpression in the mouse hindbrain to characterize its potential role in medulloblastoma tumorigenesis and identify the cell types responsive to this lineage-specific oncogene. Expression of Otx2 broadly in the mouse hindbrain resulted in the accumulation of proliferative clusters of cells in the cerebellar white matter and dorsal brainstem of postnatal mice. We found that brainstem ectopia were derived from neuronal progenitors of the rhombic lip and that cerebellar ectopia were derived from granule neuron precursors (GNPs) that had migrated inwards from the external granule layer (EGL). These hyperplasias exhibited various characteristics of medulloblastoma precursor cells identified in animal models of Shh or Wnt group tumors, including aberrant localization and altered spatiotemporal control of proliferation. However, ectopia induced by Otx2 differentiated and dispersed as the animals reached adulthood, indicating that factors restricting proliferative lifespan were a limiting factor to full transformation of these cells. These studies implicate a role for Otx2 in altering the dynamics of neuronal progenitor cell proliferation.

Wang R, Hu Y, Song G, et al.
MiR-206 regulates neural cells proliferation and apoptosis via Otx2.
Cell Physiol Biochem. 2012; 29(3-4):381-90 [PubMed] Related Publications
MiR-206 was involved in a series of cellular activities, such as the growth and development of skeletal muscle and the tumorigenesis. MiR-206 was characterized previously as a differentially expressed gene in sodium arsenite (SA)-induced neural tube defects (NTDs) in chick embryos via miRNA microarray analysis. However, the role of miR-206 in the pathological process of nerve cells remained elusive. In this study we found differential expression of miR-206 in SA-treated chick embryos by Northern blot analysis. Ectopic expression of miR-206 inhibited cell proliferation, and promoted cell apoptosis in U343 and SK-N-SH cell by using MTT, Edu Apollo assay and Flow cytometry analysis. Further investigation revealed that miR-206 can interact with 3'-untranslated region (UTR) of Otx2. MiR-206 mimics down-regulated the endogeneous Otx2 expression, whereas the miR-206 inhibitor obviously up-regulated the expression of Otx2. These findings indicate that overexpression of miR-206 promotes cell apoptosis and low expression of miR-206 inhibits cell apoptosis. Otx2 may play an important role in the process of miR-206-mediated cell apoptosis.

Rauch TA, Wang Z, Wu X, et al.
DNA methylation biomarkers for lung cancer.
Tumour Biol. 2012; 33(2):287-96 [PubMed] Related Publications
Changes in DNA methylation patterns are an important characteristic of human cancer including lung cancer. In particular, hypermethylation of CpG islands is a signature of malignant progression. Methylated CpG islands are promising diagnostic markers for the early detection of cancer. However, the full extent and sequence context of DNA hypermethylation in lung cancer has remained unknown. We have used the methylated CpG island recovery assay and high-resolution microarray analysis to find hypermethylated CpG islands in squamous cell carcinomas (SCC) and adenocarcinomas of the lung. Each tumor contained several hundred hypermethylated CpG islands. In an initial microarray screen, 36 CpG islands were methylated in five of five (=100%) of the SCC tumors tested and 52 CpG islands were methylated in at least 75% of the adenocarcinomas tested (n=8). Using sodium-bisulfite-based approaches, 12 CpG islands (associated with the BARHL2, EVX2, IRX2, MEIS1, MSX1, NR2E1, OC2, OSR1, OTX1, PAX6, TFAP2A, and ZNF577 genes) were confirmed to be methylated in 85% to 100% of the squamous cell carcinomas and 11 CpG islands (associated with the CHAD, DLX4, GRIK2, KCNG3, NR2E1, OSR1, OTX1, OTX2, PROX1, RUNX1, and VAX1 genes) were methylated in >80% of the adenocarcinomas. From the list of genes that were methylated in lung adenocarcinomas, we identified the gene FAT4 and found that this gene was methylated in 39% of the tumors. FAT4 is the closest mammalian homologue of the Drosophila tumor suppressor Fat which is an important component of the Hippo growth control pathway. Many of these newly discovered methylated CpG islands hold promise for becoming biomarkers for the early detection of lung cancer.

Bunt J, Hasselt NE, Zwijnenburg DA, et al.
Joint binding of OTX2 and MYC in promotor regions is associated with high gene expression in medulloblastoma.
PLoS One. 2011; 6(10):e26058 [PubMed] Free Access to Full Article Related Publications
Both OTX2 and MYC are important oncogenes in medulloblastoma, the most common malignant brain tumor in childhood. Much is known about MYC binding to promoter regions, but OTX2 binding is hardly investigated. We used ChIP-on-chip data to analyze the binding patterns of both transcription factors in D425 medulloblastoma cells. When combining the data for all promoter regions in the genome, OTX2 binding showed a remarkable bi-modal distribution pattern with peaks around -250 bp upstream and +650 bp downstream of the transcription start sites (TSSs). Indeed, 40.2% of all OTX2-bound TSSs had more than one significant OTX2-binding peak. This OTX2-binding pattern was very different from the TSS-centered single peak binding pattern observed for MYC and other known transcription factors. However, in individual promoter regions, OTX2 and MYC have a strong tendency to bind in proximity of each other. OTX2-binding sequences are depleted near TSSs in the genome, providing an explanation for the observed bi-modal distribution of OTX2 binding. This contrasts to the enrichment of E-box sequences at TSSs. Both OTX2 and MYC binding independently correlated with higher gene expression. Interestingly, genes of promoter regions with multiple OTX2 binding as well as MYC binding showed the highest expression levels in D425 cells and in primary medulloblastomas. Genes within this class of promoter regions were enriched for medulloblastoma and stem cell specific genes. Our data suggest an important functional interaction between OTX2 and MYC in regulating gene expression in medulloblastoma.

Bunt J, Hasselt NE, Zwijnenburg DA, et al.
OTX2 directly activates cell cycle genes and inhibits differentiation in medulloblastoma cells.
Int J Cancer. 2012; 131(2):E21-32 [PubMed] Related Publications
The transcription factor OTX2 has been implicated as an oncogene in medulloblastoma, which is the most common malignant brain tumor in children. It is highly expressed in most medulloblastomas and amplified in a subset of them. To study the role OTX2 has in medulloblastoma we investigated the downstream pathway of OTX2. We generated D425 medulloblastoma cells in which endogenous OTX2 can be silenced by inducible shRNA. Silencing of OTX2 strongly inhibited cell proliferation and resulted in a neuronal-like differentiation. Expression profiling of time courses after silencing showed a progressive change in gene expression for many cellular processes. Downregulated genes were highly enriched for cell cycle and visual perception genes, while upregulated genes were enriched for genes involved in development and differentiation. This shift is reminiscent of expression changes described during normal cerebellum development where proliferating granule progenitor cells have high OTX2 expression, which diminishes when these cells exit the cell cycle and start to differentiate. ChIP-on-chip analyses of OTX2 in D425 cells identified cell cycle and perception genes as direct OTX2 targets, while regulation of most differentiation genes appeared to be indirect. The expression of many directly regulated genes correlated to OTX2 expression in primary tumors, suggesting the in vivo relevance of these genes and their potential as targets for therapeutic intervention. These analyses provide more insight in the molecular network of OTX2, demonstrating that OTX2 is essential in medulloblastoma and directly drives proliferation by regulation of cell cycle genes.

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