Methylation of PITX2, HOXD3, RASSF1 and TDRD1 predicts biochemical recurrence in high-risk prostate cancer

PURPOSE To explore differential methylation of HAAO, HOXD3, LGALS3, PITX2, RASSF1 and TDRD1 as a molecular tool to predict biochemical recurrence (BCR) in patients with high-risk prostate cancer (PCa). METHODS A multiplexed nested methylation-specific PCR was applied to quantify promoter methylation of the selected markers in five cell lines, 42 benign prostatic hyperplasia (BPH) and 71 high-risk PCa tumor samples. Uni- and multivariate Cox regression models were used to assess the importance of the methylation level in predicting BCR. RESULTS A PCa-specific methylation marker HAAO in combination with HOXD3 and a hypomethylation marker TDRD1 distinguished PCa samples (>90 % of tumor cells each) from BPH with a sensitivity of 0.99 and a specificity of 0.95. High methylation of PITX2, HOXD3 and RASSF1, as well as low methylation of TDRD1, appeared to be significantly associated with a higher risk for BCR (HR 3.96, 3.44, 2.80 and 2.85, correspondingly) after correcting for established risk factors. When DNA methylation was treated as a continuous variable, a two-gene model PITX2 × 0.020677 + HOXD3 × 0.0043132 proved to be the best predictor of BCR (HR 4.85) compared with the individual markers. This finding was confirmed in an independent set of 52 high-risk PCa tumor samples (HR 11.89). CONCLUSIONS Differential promoter methylation of HOXD3, PITX2, RASSF1 and TDRD1 emerges as an independent predictor of BCR in high-risk PCa patients. A two-gene continuous DNA methylation model "PITX2 × 0.020677 + HOXD3 × 0.0043132" is a better predictor of BCR compared with individual markers.

The role of LMX1B and PITX2 in anterior segment development and adult ocular function

Several congenital syndromes associated with anterior segment (AS) anomalies can lead to impaired vision and glaucoma, such as nail-patella syndrome (NPS), caused by mutations in the LIM homeodomain transcription factor LMX1B and Axenfeld-Rieger's syndrome (ARS), caused by mutations in the bicoid-related homeodomain transcription factor PITX2. Targeted mutations in lmx1b and pitx2 and RNA in situ analysis reveal that both genes are required for AS development and are co-expressed within the periocular mesenchyme, suggesting they participate in a shared genetic pathway. Lmx1b homozygous mutants display iris and corneal stroma hypoplasia, and defects in ciliary body formation. In contrast, pitx2 homozygous mutants exhibit a more severe phenotype: the AS chamber, corneal endothelium, and extraocular muscles (EOM) fail to develop. The absence of EOM in pitx2 mutants suggests pitx2 acts upstream of lmx1b, or that other lmx1b family members, such as lmx1a, can compensate for lmx1b function. Lmxla/lmx1b double homozygous mutants have a reduced capacity to generate EOM, implying that lmx1 gene products have a redundant function in EOM development and that lmx1 family members may act downstream of pitx2. However, analysis of pitx2 expression in the AS tissues of lmx1b mutants and reciprocal studies of lmx1b expression in pitx2 mutants indicate that these genes do not function in a simple linear pathway. Instead, lmx1b and pitx2 may regulate a shared set of downstream targets or both genes may work in parallel transcribing unique targets required for a common biological process. Ultrastructural analysis of lmx1b and pitx2 mutant corneas indicates that collagen fibrillogenesis is perturbed, revealing a common role for both genes in the deposition of extracellular matrix. Furthermore, lmx1b/pitx2 double heterozygotes develop corneal opacities not observed in single heterozygotes demonstrating that lmx1b and pitx2 genetically interact. Data suggests that defects in the basement membrane of the corneal endothelium underlie the opacities observed in double heterozygotes. Additionally, double heterozygotes develop anterior synechias that occlude the trabecular meshwork, potentially blocking aqueous humor drainage. These data suggest that lmx1b and pitx2 are responsible for ECM deposition in multiple cell types and imply that such defects may contribute to the glaucomas observed in NPS and ARS patients. ^

The functions of pitx2 and Bmp signaling in craniofacial development

The formation of the vertebrate face is an extremely complex developmental process, which needs to coordinate the outgrowth of several facial primordia. Facial primordia are small buds made up of mesenchymal masses enclosed by an epithelial layer that surrounds the primitive mouth. The upper jaw is formed by the maxillary process, the lateral nasal process, and the frontonasal process while the mandibular process forms the lower jaw. Recent experiments using genetics in mice and bead implantation approaches have shown that the pitx2 homeobox gene and Bmp signaling play important roles in this complex developmental process. However, the molecular mechanisms underlying the function of pitx2 and Bmp in these events are still unclear. Here, we show that pitx2 is required for oral epithelium maintenance, and branchial arch signaling is pitx2 dosage sensitive by using pitx2 allelic combinations that encode varying levels of pitx2. Maintenance of fgf8 signaling requires only low pitx2 dosage while repression of Bmp signaling requires high pitx2 levels. Different incisor and molar phenotypes in low level pitx2 mutant embryos suggest a distinct requirement for pitx2 in tooth-type development. The results show that pitx2 is required for craniofacial muscle formation and expanded Bmp signaling results in excess bone formation in pitx2 mutant embryos. Fate-mapping studies show that ectopic bone results from excessive bone growth, instead of muscle transformation. Moreover, by using cre/loxp system we show that partial loss of Bmpr-IA in the facial primordia results in cleft lip/palate, abnormal teeth, ectopic teeth and tooth transformation. These phenotypes suggest that Bmp signaling has multiple functions during craniofacial development. The mutant palate shelves can fuse with each other when cultured in vitro, suggesting that cleft palate is secondary to the partial loss of Bmpr-IA. Furthermore, we prove that Bmp4, one of the ligands of Bmpr-IA, plays a role during lip fusion developmental process and partial loss of Bmp4 in the facial primordia results in the lip fusion delay. These results have provided insight to understand the complex signaling cascades that regulate craniofacial development. ^

The roles of Smads and Pitx2 in cardiac development

The heart is the first organ to form in vertebrates during embryogenesis, and its circulatory function is essential to embryonic survival. Cardiac morphogenesis comprises a complex series of interactions involving cells from several embryonic origins. These cell-cell interactions are regulated temporally and spatially by programs of inductive signaling events, including BMP signaling transduced by Smads and left-right asymmetry signaling mediated by Pitx2. Disruptions of BMP signaling and left-right asymmetry signaling result in abnormal cardiac morphogenesis that causes congenital heart disease in humans. In this study, conventional and conditional gene targeting approaches were employed to dissect the functions of Smad8 and Smad1, intracellular BMP signaling transducers, and Pitx2, a direct target of left-right signaling, in cardiac development. We generated the Smad8mt mutant allele and the Smad8lacZ knock-in allele. Smad8 homozygous mutant mice were viable and fertile without obvious abnormalities. The Smad8lacZ knock-in allele showed that Smad8 was expressed in the myocardium of cardiac outflow tract and atrioventricular cushions. We did not find defects in these Smad8-expressing cardiac regions in Smad8mt/mt and Smad8lacZ/lacZ mutants, indicating that Smad8 is dispensable for cardiac development. Conditional knockout of Smad1 using the Nkx2.5Cre allele in cardiac mesoderm resulted in partial inactivation of Smad1 in the myocardium and complete deletion of Smad1 in the epicardium, and caused ventricular hypoplasia featured with a thinner compact zone, suggesting that Smad1 signaling in the epicardium is required for myocardial morphogenesis in ventricles. Previous data have shown that Pitx2 null mutants exhibit defects in the cardiac outflow tract, a region populated with cells from the cardiac mesoderm and the cardiac neural crest. We found that the cardiac neural crest normally populated into the outflow tract in Pitx2 null mutant. Moreover, specific deletion of Pitx2 in the neural crest resulted in normal heart formation. Deletion of Pitx2 in the cardiac mesoderm caused defective outflow tract, revealing that the function of Pitx2 in the cardiac outflow tract resides in splanchnic and branchial arch mesoderm, and is independent of cardiac neural crest cells. ^

Genetic analysis of Pitx2 in left -right asymmetry and cardiovascular development

Pitx2, a paired-related homeobox gene that is mutated in human Rieger Syndrome, plays a key role in transferring the early asymmetric signals to individual organs. Pitx2 encodes three isoforms, Pitx2a, Pitx2b and Pitx2c. I found that Pitx2c was the Pitx2 isoform for regulating left-right asymmetry in heart, lung and the predominant isoform in guts. Previous studies suggested that the generation of left-right asymmetry within individual organs is an all or none, random event. Phenotypic analysis of various Pitx2 allelic combinations, that encode graded levels of Pitx2c, reveals an organ-intrinsic mechanism for regulating left-right asymmetric morphogenesis based on differential response to Pitx2c levels. The heart needs low Pitx2c levels, while the lungs and duodenum require higher doses of Pitx2c. In addition, the duodenal rotation is under strict control of Pitx2c activity. Left-right asymmetry development for aortic arch arteries involves complex vascular remodeling. Left-sided expression of Pitx2c in these developing vessels implied its potential function in this process. In order to determine if Pitx2c also can regulate the left-right asymmetry of the aortic arch arteries, a Pitx2c-specific loss of function mutation is generated. Although in wild type mice, the direction of the aortic arch is always oriented toward the left side, the directions of the aortic arches in the mutants were randomized, showing that Pitx2c also determined the left-right asymmetry of these vessels. I have further showed that the cardiac neural crest wasn't involved in this vascular remodeling process. In addition, all mutant embryos had Double Outlet Right Ventricle (DORV), a common congenital heart disease. This study provided insight into the mechanism of Pitx2c-mediated late stages of left-right asymmetry development and identified the roles of Pitx2c in regulation of aortic arch remodeling and heart development. ^

Cited2 is required both for heart morphogenesis and establishment of the left-right axis in mouse development

Establishment of the left-right axis is a fundamental process of vertebrate embryogenesis. Failure to develop left-right asymmetry leads to incorrect positioning and morphogenesis of numerous internal organs, and is proposed to underlie the etiology of several common cardiac malformations. The transcriptional modulator Cited2 is essential for embryonic development: Cited2-null embryos die during gestation with profound developmental abnormalities, including cardiac malformations, exencephaly and adrenal agenesis. Cited2 is also required for normal establishment of the left-right axis; we demonstrate that abnormal heart looping and right atrial and pulmonary isomerism are consistent features of the left-right-patterning defect. We show by gene expression analysis that Cited2 acts upstream of Nodal, Lefty2 and Pitx2 in the lateral mesoderm, and of Lefty1 in the presumptive floor plate. Although abnormal left-right patterning has a major impact on the cardiac phenotype in Cited2-null embryos, laterality defects are only observed in a proportion of these embryos. We have therefore used a combination of high-resolution imaging and three-dimensional (3D) modeling to systematically document the full spectrum of Cited2-associated cardiac defects. Previous studies have focused on the role of Cited2 in cardiac neural crest cell development, as Cited2 can bind the transcription factor Tfap2, and thus affect the expression of Erbb3 in neural crest cells. However, we have identified Cited2-associated cardiac defects that cannot be explained by laterality or neural crest abnormalities. In particular, muscular ventricular septal defects and reduced cell density in the atrioventricular (AV) endocardial cushions are evident in Cited2-null embryos. As we found that Cited2 expression tightly correlated with these sites, we believe that Cited2 plays a direct role in development of the AV canal and cardiac septa. We therefore propose that, in addition to the previously described reduction of cardiac neural crest cells, two other distinct mechanisms contribute to the spectrum of complex cardiac defects in Cited2-null mice; disruption of normal left-right patterning and direct loss of Cited2 expression in cardiac tissues.

A gene expression signature that distinguishes desmoid tumours from nodular fasciitis.

Nodular fasciitis (NF) is a rapidly growing cellular mass composed of fibroblasts/myofibroblasts, usually localized in subcutaneous tissues, that typically undergoes fibrosis and almost never recurs. Desmoid tumours (DTs) are rare forms of fibroblastic/myofibroblastic growth that arise in deep soft tissues, display a propensity for local infiltration and recurrence, but fail to metastasize. Given that both entities are primarily fibroblastic/myofibroblastic lesions with overlapping histological features, their gene expression profiles were compared to identify differentially expressed genes that may provide not only potential diagnostic markers, but also clues as to the pathogenesis of each disorder. Differentially expressed transcripts (89 clones displaying increased expression in DTs and 246 clones displaying increased expression in NF) included genes encoding several receptor and non-receptor tyrosine kinases (EPHB3, PTPRF, GNAZ, SYK, LYN, EPHA4, BIRC3), transcription factors (TWIST1, PITX2, EYA2, OAS1, MITF, TCF20), and members of the Wnt signalling pathway (AXIN2, WISP1, SFRP). Remarkably, almost one-quarter of the differentially expressed genes encode proteins associated with inflammation and tissue remodelling, including members of the interferon (IFN), tumour necrosis factor (TNF), and transforming growth factor beta (TGF-beta) signalling pathways as well as metalloproteinases (MMP1, 9, 13, 23), urokinase plasminogen activator (PLAU), and cathepsins. The observations provide the first comparative molecular characterization of desmoid tumours and nodular fasciitis and suggest that selected tyrosine kinases, transcription factors, and members of the Wnt, TGF-beta, IFN, and TNF signalling pathways may be implicated in influencing and distinguishing their fate.

Formation and differentiation of multiple mesenchymal lineages during lung development is regulated by {beta}-catenin signaling.

BACKGROUND: The role of ss-catenin signaling in mesodermal lineage formation and differentiation has been elusive. METHODOLOGY: To define the role of ss-catenin signaling in these processes, we used a Dermo1(Twist2)(Cre/+) line to target a floxed beta-catenin allele, throughout the embryonic mesenchyme. Strikingly, the Dermo1(Cre/+); beta-catenin(f/-) conditional Knock Out embryos largely phenocopy Pitx1(-/-)/Pitx2(-/-) double knockout embryos, suggesting that ss-catenin signaling in the mesenchyme depends mostly on the PITX family of transcription factors. We have dissected this relationship further in the developing lungs and find that mesenchymal deletion of beta-catenin differentially affects two major mesenchymal lineages. The amplification but not differentiation of Fgf10-expressing parabronchial smooth muscle progenitor cells is drastically reduced. In the angioblast-endothelial lineage, however, only differentiation into mature endothelial cells is impaired. CONCLUSION: Taken together these findings reveal a hierarchy of gene activity involving ss-catenin and PITX, as important regulators of mesenchymal cell proliferation and differentiation.

Les facteurs à homéodomaine Pitx et Irx au cours du développement des membres postérieurs

Les facteurs de transcription Pitx ont été impliqués dans la croissance et la détermination de l’identité des membres postérieurs. D’abord, l’inactivation de Pitx1 chez la souris résulte en la transformation partielle des membres postérieurs en membres antérieurs. Ensuite, la double mutation de Pitx1 et de Pitx2 a montré l’activité redondante de ces facteurs pour la croissance des membres postérieurs. Ainsi, les souris mutantes Pitx1-/-;Pitx2néo/néo montrent une perte des éléments squelettiques proximaux et antérieurs. Des travaux récents ont impliqué les gènes de la famille des Iroquois dans le développement des membres. Tout particulièrement, les souris Irx3-/-;Irx5-/- montrent la perte des éléments squelettiques proximaux et antérieurs, exclusivement au niveau des membres postérieurs. Cette phénocopie entre les souris mutantes pour Pitx1/2 et Irx3/5 nous a amenés à poser trois hypothèses : (1) les Pitx sont responsables de l’expression de Irx dans les bourgeons postérieurs ; (2) à l’inverse, les Irx dirigent l’expression des Pitx ; (3) les Pitx et les Irx participent ensemble au programme génétique de croissance des bourgeons postérieurs. Nous avons pu conclure que les Pitx et les Irx font partie de cascades de régulation indépendantes l’une de l’autre et qu’ils sont capables d’interaction transcriptionnelle autant sur un promoteur générique que sur des régions conservées du locus de Tbx4. Enfin, autant l’inactivation Pitx que celle des Irx mène à un retard d’expression de Pax9 exclusivement dans les bourgeons postérieurs. Ainsi, les Pitx et les Irx semblent agir sur des programmes génétiques parallèles impliqués dans la croissance et le patterning des membres postérieurs.

Limiting glutamate transmission in a Vglut2-expressing subpopulation of the subthalamic nucleus is sufficient to cause hyperlocomotion

The subthalamic nucleus (STN) is a key area of the basal ganglia circuitry regulating movement. We identified a subpopulation of neurons within this structure that coexpresses Vglut2 and Pitx2, and by conditional targeting of this subpopulation we reduced Vglut2 expression levels in the STN by 40%, leaving Pitx2 expression intact. This reduction diminished, yet did not eliminate, glutamatergic transmission in the substantia nigra pars reticulata and entopeduncular nucleus, two major targets of the STN. The knock-out mice displayed hyperlocomotion and decreased latency in the initiation of movement while preserving normal gait and balance. Spatial cognition, social function, and level of impulsive choice also remained undisturbed. Furthermore, these mice showed reduced dopamine transporter binding and slower dopamine clearance in vivo, suggesting that Vglut2-expressing cells in the STN regulate dopaminergic transmission. Our results demonstrate that altering the contribution of a limited population within the STN is sufficient to achieve results similar to STN lesions and high-frequency stimulation, but with fewer side effects.

Alteraciones genéticas y epigenéticas en el cáncer de endometrio

Programa de doctorado: Cáncer: biología y clínica.

Novel genetic markers associate with atrial fibrillation risk in Europeans and Japanese

OBJECTIVES This study sought to identify nonredundant atrial fibrillation (AF) genetic susceptibility signals and examine their cumulative relations with AF risk. BACKGROUND AF-associated loci span broad genomic regions that may contain multiple susceptibility signals. Whether multiple signals exist at AF loci has not been systematically explored. METHODS We performed association testing conditioned on the most significant, independently associated genetic markers at 9 established AF loci using 2 complementary techniques in 64,683 individuals of European ancestry (3,869 incident and 3,302 prevalent AF cases). Genetic risk scores were created and tested for association with AF in Europeans and an independent sample of 11,309 individuals of Japanese ancestry (7,916 prevalent AF cases). RESULTS We observed at least 4 distinct AF susceptibility signals on chromosome 4q25 upstream of PITX2, but not at the remaining 8 AF loci. A multilocus score comprised 12 genetic markers demonstrated an estimated 5-fold gradient in AF risk. We observed a similar spectrum of risk associated with these markers in Japanese. Regions containing AF signals on chromosome 4q25 displayed a greater degree of evolutionary conservation than the remainder of the locus, suggesting that they may tag regulatory elements. CONCLUSIONS The chromosome 4q25 AF locus is architecturally complex and harbors at least 4 AF susceptibility signals in individuals of European ancestry. Similar polygenic AF susceptibility exists between Europeans and Japanese. Future work is necessary to identify causal variants, determine mechanisms by which associated loci predispose to AF, and explore whether AF susceptibility signals classify individuals at risk for AF and related morbidity.

Conservation of early odontogenic signaling pathways in Aves

Teeth have been missing from birds (Aves) for at least 60 million years. However, in the chick oral cavity a rudiment forms that resembles the lamina stage of the mammalian molar tooth germ. We have addressed the molecular basis for this secondary loss of tooth formation in Aves by analyzing in chick embryos the status of molecular pathways known to regulate mouse tooth development. Similar to the mouse dental lamina, expression of Fgf8, Pitx2, Barx1, and Pax9 defines a potential chick odontogenic region. However, the expression of three molecules involved in tooth initiation, Bmp4, Msx1, and Msx2, are absent from the presumptive chick dental lamina. In chick mandibles, exogenous bone morphogenetic protein (BMP) induces Msx expression and together with fibroblast growth factor promotes the development of Sonic hedgehog expressing epithelial structures. Distinct epithelial appendages also were induced when chick mandibular epithelium was recombined with a tissue source of BMPs and fibroblast growth factors, chick skin mesenchyme. These results show that, although latent, the early signaling pathways involved in odontogenesis remain inducible in Aves and suggest that loss of odontogenic Bmp4 expression may be responsible for the early arrest of tooth development in living birds.

Salivary Gland and Tooth Abnormalities Massively Increase Caries Susceptibility in A Mouse Model of Cleft Lip/Palate

Thesis (Ph.D.)--University of Washington, 2016-04

On the use of transgenic mice and optogenetics to characterize genetically defined subpopulations of neurons

Neuroscientists have a variety of perspectives with which to classify different parts of the brain. With the rise of genetic-based techniques such as optogenetics, it is increasingly important to identify whether a group of cells, defined by morphology, function or anatomical location possesses a distinct pattern of expression of one or more genetic promoters. This would allow for better ways to study of these genetically defined subpopulations of neurons. In this work, I present a theoretical discussion and threeexperimental studies in which this was the main question being addressed. Paper I discusses the issues involved in selecting a promoter to study structures and subpopulations in the Ventral Tegmental Area. Paper II characterizes a subpopulation of cells in the Ventral Tegmental Area that shares the expression of a promoter and is anatomically very restricted, and induces aversion when stimulated. Paper III utilizes a similar strategy to investigate a subpopulation in the subthalamic nucleus that expresses PITX2 and VGLUT2 which, when inactivated, causes hyperlocomotion. Paper IV exploits the fact that a previously identified group of cells in the ventral hippocampus expresses CHRNA2, and indicates that this population may be necessary and sufficient for the establishment of the theta rhythm (2-8 Hz) in the Local Field Potential of anesthetized mice. All of these studies were guided by the same strategy of characterizing and studying the role of a genetically defined subpopulation of cells, and they demonstrate the different ways in which this approach can generate new discoveries.