Sequencing of TGF-beta pathway genes in familial cases of intracranial aneurysm.

BACKGROUND AND PURPOSE: Familial aggregation of intracranial aneurysms (IA) strongly suggests a genetic contribution to pathogenesis. However, genetic risk factors have yet to be defined. For families affected by aortic aneurysms, specific gene variants have been identified, many affecting the receptors to transforming growth factor-beta (TGF-beta). In recent work, we found that aortic and intracranial aneurysms may share a common genetic basis in some families. We hypothesized, therefore, that mutations in TGF-beta receptors might also play a role in IA pathogenesis. METHODS: To identify genetic variants in TGF-beta and its receptors, TGFB1, TGFBR1, TGFBR2, ACVR1, TGFBR3, and ENG were directly sequenced in 44 unrelated patients with familial IA. Novel variants were confirmed by restriction digestion analyses, and allele frequencies were analyzed in cases versus individuals without known intracranial disease. Similarly, allele frequencies of a subset of known SNPs in each gene were also analyzed for association with IA. RESULTS: No mutations were found in TGFB1, TGFBR1, TGFBR2, or ACVR1. Novel variants identified in ENG (p.A60E) and TGFBR3 (p.W112R) were not detected in at least 892 reference chromosomes. ENG p.A60E showed significant association with familial IA in case-control studies (P=0.0080). No association with IA could be found for any of the known polymorphisms tested. CONCLUSIONS: Mutations in TGF-beta receptor genes are not a major cause of IA. However, we identified rare variants in ENG and TGFBR3 that may be important for IA pathogenesis in a subset of families.

Association of folate metabolic pathway genes with human spina bifida meningomyelocele

Neural tube defects including spina bifida meningomyelocele (SBMM) are common malformations of the brain and spinal cord, and include all abnormalities resulting from lack of closure of the developing neural tube during embryological development.^ The specific aims of this study were to determine if single nucleotide polymorphic variants (SNPs) in the folate/homocysteine metabolic pathway genes confer a risk for NTD susceptibility within this SBMM population.^ In completion of the first specific aim, two novel SNPs were identified in the FOLR1 gene in Chromosome 11of patients including one in non-coding exon 1 with a C → T transition at nucleotide position 71578317 and another in non-coding exon 3 with a T → G transversion at nucleotide position 71579123. It will be important to determine if these variants are present in the respective parents of these individuals. If they are in fact de novo variants, then these SNPs may be more likely to contribute to the birth defect.^ The second project aim was to analyze genotypes associated with SBMM risk by transmission disequilibrium tests (TDT) and association was detected on several SNPs across the folate metabolic pathway genes in this population. SNPs with significant RC-TDT values were found within the DHFR gene (rs1650723), the MTRR gene (rs327592), the FOLR2 gene (rs13908), four tightly linked variants in the FOLR3 gene (rs7925545, rs7926875, rs7926987, rs7926360) and a variant in the SLC19A1 gene (rs1888530). The product of each of these genes performs a vital function in the folate metabolic pathway. It is conceivable, therefore, that if the individual SNP or SNPs can be proven to perturb the function in some way that they may be involved in the disruption of folate metabolism and in the resulting birth defect. Validating the results of this study in other independent populations will further strengthen the evidence that dysfunction of folate enzymes and receptors may confer SBMM risk in humans. ^

Genetic variants within the Wnt/ B-catenin signaling pathway as indicators of bladder cancer clinical outcomes

The genetic factors that influence bladder cancer clinical outcomes are largely unknown. In this clinical outcomes study, I assessed genetic variations in the Wnt/β-catenin stem-cell pathway genes for association with recurrence and progression. A total of 230 SNPS in 40 genes from the Wnt/β-catenin pathway were genotyped in 419 histologically confirmed non-muscle invasive bladder cancer cases. Several significant associations were observed in the clinical outcomes analysis. Under the dominant model WNT8B: rs4919464 (HR: 1.55, 95% CI: 1.17-2.06, P=2.2x10-3) and WNT8B: rs3793771 (HR: 1.54, 95% CI: 1.09-1.62, P=4.6x10-3 ) were statistically significantly associated with an increase risk of recurrence while two other variants, APC2: rs11668593 (HR: 2.50, 95% CI: 1.43-4.35, P=1.2x10-3) and LRP5 : rs312778 (HR: 1.81, 95% CI: 1.23-2.65, P=2.7x10-3), were significantly associated with recurrence risk under the recessive model of inheritance. Four SNPs in the recessive model were associated with an increased risk of progression (AXIN2: rs1544427, LRP5: rs312778, AXIN1: rs370681, AXIN1: rs2301522). LRP5: rs312778 had the most significant increased risk of progression with a 2.68 (95% CI: 1.52-4.72, P=6.4x10-4)-fold increased risk. Stratification analysis based on treatment regimen (transurethral resection (TUR) and Bacillus Calmette-Guérin (BCG)) was also performed. Individuals with at least one variant in AXIN2: rs2007085 were found to have a 2.09 (95% CI: 1.24-3.52, P=5.4x10-3) -fold increased risk of recurrence in those that received TUR only, and no statistically significant effect was seen in those that received BCG. Individuals who received TUR with at least one variant in LEF1: rs10516550 were found to have a 2.26 (95% CI: 1.22-4.18, P=9.7x10-3)-fold increase risk of recurrence and no statistically significant effect was found in individuals who received BCG. Also, the recessive model of LRP6: rs2302684 in TUR only treatment was shown to have a 1.95 (95%CI: 1.18-3.21, P=8.8x10 -3)-fold increased risk of recurrence, and a suggested protective effect associated with a (HR: 0.83, 95% CI: 0.51-1.37, P=0.468) decreased risk of recurrence. Together, these findings implicate the Wnt/β-catenin stem-cell pathway as playing a role in bladder cancer clinical outcomes and have important implications for personalization of future treatment regimens. ^

Mechanisms regulating the p120-catenin/Kaiso pathway

The Wnt pathways contribute to many processes in cancer and developmental biology, with β-catenin being a key canonical component. P120-catenin, which is structurally similar to β-catenin, regulates the expression of certain Wnt target genes, relieving repression conferred by the POZ/ zinc-finger transcription factor Kaiso. In my first project, employing Xenopus embryos and mammalian cell lines, I found that the degradation machinery of the canonical Wnt pathway modulates p120-catenin protein stability, especially p120 isoform-1, through mechanisms shared with b-catenin. Exogenous expression of destruction-complex components such as GSK3b or Axin promotes p120-catenin degradation, and consequently, is able to rescue developmental phenotypes resulting from p120 over-expression during early Xenopus embryonic development. Conversely, as predicted, the in vivo depletion of either Axin or GSK3b coordinately increased p120 and b-catenin levels, while p120 levels decreased upon LRP5/6 depletion, which are positive modulators in the canonical Wnt pathway. At the primary sequence level, I resolved conserved GSK3b phosphorylation sites in p120’s (isoform 1) amino-terminal region. Point-mutagenesis of these residues inhibited the association of destruction complex proteins including those involved in ubiquitination, resulting in p120-catenin stabilization. Importantly, we found that two additional p120-catenin family members, ARVCF-catenin and d-catenin, in common with b-catenin and p120, associate with Axin, and are degraded in Axin’s presence. Thus, by similar means, it appears that canonical Wnt signals coordinately modulate multiple catenin proteins having roles in development and conceivably disease states. In my second project, I found that the Dyrk1A kinase exhibits a positive effect upon p120-catenin levels. That is, unlike the negative regulator GSK3b kinase, a candidate screen revealed that Dyrk1A kinase enhances p120-catenin protein levels via increased half-life. Dyrk1A is encoded by a gene located within the trisomy of chromosome 21, which contributes to mental retardation in Down Syndrome patients. I found that Dyrk1A expression results in increased p120 protein levels, and that Dyrk1A specifically associates with p120 as opposed to other p120-catenin family members or b-catenin. Consistently, Dyrk1A depletion in mammalian cell lines and Xenopus embryos decreased p120-catenin levels. I further confirmed that Dyrk overexpression and knock-down modulates both Siamois and Wnt11 gene expression in the expected manner based upon the resulting latered levels of p120-catenin. I determined that Dyrk expression rescues Kaiso depletion effects (gastrulation failure; increased endogenous Wnt11 expression), and vice versa. I then identified a putative Dyrk phosphorylation region within the N-terminus of p120-catenin, which may also be responsible for Dyrk1A association. I went on to make a phosphomimic mutant, which when over-expressed, had the predicted enhanced capacity to positively modulate endogenous Wnt11 and Siamois expression, and thereby generate gastrulation defects. Given that Dyrk1A modulates Siamois expression through stabilization of p120-catenin, I further observed that ectopic expression of Dyrk can positively influence b-catenin’s capacity to generate ectopic dorsal axes when ventrally expressed in early Xenopus embryos. Future work will investigate how Dyrk1A modulates the Wnt signaling pathway through p120-catenin, and possibly begin to address how dysfunction of Dyrk1A with respect to p120-catenin might relate to aspects of Down syndrome. In summary, the second phase of my graduate work appears to have revealed a novel aspect of Dyrk1A/p120-catenin action in embryonic development, with a functional linkage to canonical Wnt signaling. What I have identified as a “Dyrk1A/p120-catenin/Kaiso pathway” may conceivably assist in our larger understanding of the impact of Dyrk1A dosage imbalance in Down syndrome.

The p120-catenin/Kaiso signaling pathway

The canonical and non-canonical Wnt signaling pathways appear to interact with one another as a network in development, or when hyper-activated, in the progression of disease. A much studied key mediator of the canonical Wnt pathway, β-catenin, is characterized by a central armadillo-repeat domain that engages in multiple protein-protein interactions, such as those with cadherins functioning at cell-cell contact regions. In the nucleus, β-catenin forms a complex with the repressor TCF/LEF, promoting the activation of genes participating in processes such as proliferation, differentiation and stem cell survival. Somewhat similarly, the p120-catenin binds the distinct transcriptional repressor Kaiso, relieving Kaiso-mediated repression to promote gene activation. Here, employing Xenopus laevis, I report upon both downstream and upstream aspects of the p120-catenin/Kaiso pathway which was previously poorly understood. I first show that Kaiso, a BTB/POZ zinc-finger family member, directly represses canonical Wnt gene targets (Siamois, c-Fos, Cyclin-D1 and c-Myc) in conjunction with TCF. Depletion or dominant-negative inhibition of xKaiso results in Siamois de-repression, while xKaiso over-expression induces additional Siamois repression through recruitment of N-CoR co-repressor and chromatin modifications. Functional interdependencies are further corroborated by the capacity of Kaiso to suppress β-catenin-induced axis duplication. Thus, my work inter-relates the p120-catenin/Kaiso and β-catenin/TCF pathways at the level of specific gene promoters important in development and cancer progression. Regarding upstream aspects of the p120-catenin/Kaiso pathway, I collaboratively identified p120 in association with Frodo, a protein previously identified as a component of the canonical (β-catenin dependent) Wnt pathway. I determined that canonical Wnt signals result in Frodo-mediated stabilization of p120-catenin, resulting in the sequestration of Kaiso to the cytoplasm and thereby the activation (relief of repression) of gene targets. Developmental evidence supporting this view included findings that Frodo has the capacity to partially rescue Kaiso over-expression phenotypes in early Xenopus embryos. Taken together, my studies point to the convergence of p120-catenin/Kaiso and β-catenin/TCF signaling pathways at the level of gene transcription as well as at more upstream points during vertebrate development. ^

Analysis of Wnt/beta-catenin signaling in kidney tubulogenesis

The β-catenin/Lef/Tcf-mediated Wnt pathway is central to the developmental of all animals, stem cell renewal, and cancer progression. Prior studies in frogs and mice have indicated that the ligand Wnt-4 is essential for the mesenchyme to epithelial transition that generates tubules in the context of kidney organogenesis. More recently, Wnt-9b in mice, was likewise found to be required. Yet despite the importance of Wnt signals in renal development, the corresponding Frizzled receptor(s) and downstream signaling mechanim(s) are unclear. My work addresses these knowledge gaps using in vitro (Madin-Darby Canine Kidney cells) and in vivo (Xenopus laevis and zebrafish pronephros) tubulogenic kidney model systems. Employing established reporter constructs of Wnt/β-catenin pathway activity, I have determined that MDCK cells are highly responsive to Wnt-4, -1, and -3A, but not to Wnt-5A and control conditions. I have confirmed that Wnt-4's canonical signaling activity in MDCK cells is mediated by downstream effectors of the Wnt/β-catenin pathway using β-Engrailed and dnTCF-4, constructs that suppress this pathway. I have further found that MDCK cells express the Frizzled-6 receptor, and that Wnt-4 forms a biochemical complex with Frizzled-6, yet does not appear to transduce Wnt-4's canonical signal. Additionally, I demonstrate that standard Hepatocyte Growth Factor (HGF)-mediated (non-physiologic) induction of MDCK tubulogenesis in collagen matrices is not altered by activation or suppression of β-catenin signaling activity; however, β-catenin signaling maintains cell survival in this in vitro system. Using a Wnt/β-catenin signaling reporter in Xenopus laevis, I detect β-catenin signaling activity in the early pronephric epithelial kidney tubules. By inhibiting the Wnt/β-catenin signaling pathway in both zebrafish and Xenopus , a significant loss of kidney tubulogenesis is observed with little or no effect on adjoining axis or somite development. This inhibition also leads to the appearance of severe edema that phenocopies embryos depleted for Wnt-4. Tubulogenic loss does not appear to be caused by increased cell death in the Xenopus pronephric field, but rather by lessened expression of tubule epithelium genes associated with cellular differentiation. Together, my results show that Wnt/β-catenin signaling is required for renal tubule development and that Wnt-4 is a strong candidate for activating this pathway. ^

FZD6, MATN2 AND SLC25A32, POSSIBLE CANDIDATE GENES IN NONSYNDROMIC CLEFT LIP AND PALATE

Nonsyndromic cleft lip with or without cleft palate (NSCLP) is a common birth defect with a multifactorial etiology. Despite decades of research, the genetic underpinnings of NSCLP still remain largely unexplained. A genome wide association study (GWAS) of a large NSCLP African American family with seven affected individuals across three generations found evidence for linkage at 8q21.3-24.12 (LOD = 2.98). This region contained three biologically relevant candidate genes: Frizzled-6 (FZD6) (LOD = 2.8), Matrilin-2 (MATN2) (LOD = 2.3), and Solute Carrier Family 25, Member 32 (SLC26A32) (LOD = 1.6). Sequencing of the coding regions and the 5’ and 3’ UTRs of these genes in two affected family members identified a rare intronic variant, rs138557689 (c.-153+432A>C), in FZD6. The rs138557689/C allele segregated with the NSCLP phenotype; in silico analysis predicted and EMSA analysis showed that the 138557689/C allele creates new DNA binding sites. FZD6 is part of the WNT pathway, which is involved in craniofacial development, including midface development and upper lip fusion. Our novel findings suggest that an alteration in FZD6 gene regulation may perturb this tightly controlled biological pathway and in turn contribute to the development of NSCLP in this family. Studies are underway to further define how the rs138557689/C variant affects expression of FZD6.

Folate pathway polymorphisms and neuropsychological impairment after leukemia therapy

Neuropsychological impairment occurs in 20%-40% of childhood acute lymphoblastic leukemia (ALL) survivors, possibly mediated by folate depletion following methotrexate chemotherapy. We evaluated the relationship between two folate pathway polymorphisms and neuropsychological impairment after childhood ALL chemotherapy. Eighty-six childhood ALL survivors were recruited between 2004-2007 at Texas Children's Hospital after exclusion for central nervous system leukemia, cranial irradiation, and age<1 year at diagnosis. Neuropsychological evaluation at a median of 5.3 years off therapy included a parental questionnaire and the following child performance measures: Trail Making Tests A and B, Grooved Pegboard Test Dominant-Hand and Nondominant-Hand, and Digit Span subtest. We performed genotyping for polymorphisms in two folate pathway genes: reduced folate carrier (RFC1 80G>A, rs1051266) and dihydrofolate reductase (DHFR Intron-1 19bp deletion). Fisher exact test, logistic regression, Student's t-test, and ANOVA were used to compare neuropsychological test scores by genotype, using a dominant model to group genotypes. In univariate analysis, survivors with cumulative methotrexate exposure ≥9000 mg/m2 had an increased risk of attention disorder (OR=6.2, 95% CI 1.2 – 31.3), compared to survivors with methotrexate exposure <9000 mg/m2. On average, female survivors scored 8.5 points higher than males on the Digit Span subtest, a test of working memory (p=0.02). The RFC1 80G>A and DHFR Intron-1 deletion polymorphisms were not related to attention disorder or impairment on tests of attention, processing speed, fine motor speed, or memory. These data imply a strong relationship between methotrexate dose intensity and impairment in attention after childhood ALL therapy. We did not find an association between the RFC1 80G>A or DHFR Intron-1 deletion polymorphisms and long-term neuropsychological impairment in childhood ALL survivors.^

Genetic variants in the mammalian target of rapamycin (mTOR) signaling pathway as predictors of survival and clinical response in women with ovarian cancer

Background. The mTOR pathway is commonly altered in human tumors and promotes cell survival and proliferation. Preliminary evidence suggests this pathway's involvement in chemoresistance to platinum and taxanes, first line therapy for epithelial ovarian cancer. A pathway-based approach was used to identify individual germline single nucleotide polymorphisms (SNPs) and cumulative effects of multiple genetic variants in mTOR pathway genes and their association with clinical outcome in women with ovarian cancer. ^ Methods. The case-series was restricted to 319 non-Hispanic white women with high grade ovarian cancer treated with surgery and platinum-based chemotherapy. 135 SNPs in 20 representative genes in the mTOR pathway were genotyped. Hazard ratios (HRs) for death and Odds ratios (ORs) for failure to respond to primary therapy were estimated for each SNP using the multivariate Cox proportional hazards model and multivariate logistic regression model, respectively, while adjusting for age, stage, histology and treatment sequence. A survival tree analysis of SNPs with a statistically significant association (p<0.05) was performed to identify higher order gene-gene interactions and their association with overall survival. ^ Results. There was no statistically significant difference in survival by tumor histology or treatment regimen. The median survival for the cohort was 48.3 months. Seven SNPs were significantly associated with decreased survival. Compared to those with no unfavorable genotypes, the HR for death increased significantly with the increasing number of unfavorable genotypes and women in the highest risk category had HR of 4.06 (95% CI 2.29–7.21). The survival tree analysis also identified patients with different survival patterns based on their genetic profiles. 13 SNPs on five different genes were found to be significantly associated with a treatment response, defined as no evidence of disease after completion of primary therapy. Rare homozygous genotype of SNP rs6973428 showed a 5.5-fold increased risk compared to the wild type carrying genotypes. In the cumulative effect analysis, the highest risk group (individuals with ≥8 unfavorable genotypes) was significantly less likely to respond to chemotherapy (OR=8.40, 95% CI 3.10–22.75) compared to the low risk group (≤4 unfavorable genotypes). ^ Conclusions. A pathway-based approach can demonstrate cumulative effects of multiple genetic variants on clinical response to chemotherapy and survival. Therapy targeting the mTOR pathway may modify outcome in select patients.^

Mouse retinal development: Role of cell adhesion and mechanism of gene regulation

Cell differentiation and pattern formation are fundamental processes in animal development that are under intense investigation. The mouse retina is a good model to study these processes because it has seven distinct cell types, and three well-laminated nuclear layers that form during embryonic and postnatal life. β-catenin functions as both the nuclear effector for the canonical Wnt pathway and a cell adhesion molecule, and is required for the development of various organs. To study the function of β-catenin in retinal development, I used a Cre-loxP system to conditionally ablate β-catenin in the developing retina. Deletion of β-catenin led to disrupted laminar structure but did not affect the differentiation of any of the seven cell types. Eliminating β-catenin did not reduce progenitor cell proliferation, although enhanced apoptosis was observed. Further analysis showed that disruption of cell adhesion was the major cause of the observed patterning defects. Overexpression of β-catenin during retinal development also disrupted the normal retinal lamination and caused a transdifferentiation of neurons into pigmented cells. The results indicate that β-catenin functions as a cell adhesion molecule but not as a Wnt pathway component during retinal neurogenesis, and is essential for lamination but not cell differentiation. The results further imply that retinal lamination and cell differentiation are genetically separable processes. ^ Sonic hedgehog (shh) is expressed in retinal ganglion cells under the control of transcription factor Pou4f2 during retinal development. Previous studies identified a phylogenetically conserved region in the first intron of shh containing a Pou4f2 binding site. Transgenic reporter mice in which reporter gene expression was driven by this region showed that this element can direct gene expression specifically in the retina, but expression was not limited to the ganglion cells. From these data I hypothesized that this element is required for shh expression in the retina but is not sufficient for specific ganglion cell expression. To further test this hypothesis, I created a conditional allele by flanking this region with two loxP sites. Lines carrying this allele will be crossed with retinal-specific Cre lines to remove this element in the retina. My hypothesis predicts that alteration in shh expression and subsequent retinal defects will occur in the retinas of these mice. ^

Understanding the roles of Non-homologous end joining and p53 after DNA damage

The inability to maintain genomic stability and control proliferation are hallmarks of many cancers, which become exacerbated in the presence of unrepaired DNA damage. Such genotoxic stresses trigger the p53 tumor suppressor network to activate transient cell cycle arrest allowing for DNA repair; if the damage is excessive or irreparable, apoptosis or cellular senescence is triggered. One of the major DNA repair pathway that mends DNA double strand breaks is non-homologous end joining (NHEJ). Abrogating the NHEJ pathway leads to an accumulation of DNA damage in the lymphoid system that triggers p53-mediated apoptosis; complete deletion of p53 in this system leads to aggressive lymphomagenesis. Therefore, to study the effect of p53-dependent cell cycle arrest, we utilized a hypomorphic, separation-of-function mutant, p53p/p, which completely abrogates apoptosis yet retains partial cell cycle arrest ability. We crossed DNA ligase IV deficiency, a downstream ligase crucial in mending breaks during NHEJ, into the p53p/p background (Lig4-/-p53p/p). The accumulation of DNA damage activated the p53/p21 axis to trigger cellular senescence in developing lymphoid cells, which absolutely suppressed tumorigenesis. Interestingly, these mice progressively succumb to severe diabetes. Mechanistic analysis revealed that spontaneous DNA damage accumulated in the pancreatic b-cells, a unique subset of endocrine cells solely responsible for insulin production to regulate glucose homeostasis. The genesis of adult b-cells predominantly occurs through self-replication, therefore modulating cellular proliferation is an essential component for renewal. The progressive accumulation of DNA damage, caused by Lig4-/-, activated p53/p21-dependent cellular senescence in mutant pancreatic b-cells that lead to islet involution. Insulin levels subsequently decreased, deregulating glucose homeostasis driving overt diabetes. Our Lig4-/-p53p/p model aptly depicts the dichotomous role of cellular senescence—in the lymphoid system prevents tumorigenesis yet in the endocrine system leads to the decrease of insulin-producing cells causing diabetes. To further delineate the function of NHEJ in pancreatic b-cells, we analyzed mice deficient in another component of the NHEJ pathway, Ku70. Although most notable for its role in DNA damage recognition and repair within the NHEJ pathway, Ku70 has NHEJ-independent functions in telomere maintenance, apoptosis, and transcriptional regulation/repression. To our surprise, Ku70-/-p53p/p mutant mice displayed a stark increase in b-cell proliferation, resulting in islet expansion, heightened insulin levels and hypoglycemia. Augmented b-cell proliferation was accompanied with the stabilization of the canonical Wnt pathway, responsible for this phenotype. Interestingly, the progressive onset of cellular senescence prevented islet tumorigenesis. This study highlights Ku70 as an important modulator in not only maintaining genomic stability through NHEJ-dependent functions, but also reveals a novel NHEJ-independent function through regulation of pancreatic b-cell proliferation. Taken in aggregate, these studies underscore the importance for NHEJ to maintain genomic stability in b-cells as well as introduces a novel regulator for pancreatic b-cell proliferation.

The identification and regulation of a novel interaction occurring between $\beta$-catenin and the actin -bundling protein fascin

Catenins were first characterized as linking the cytoplasmic domains of cadherin cell-cell adhesion molecules to the cortical actin cytoskeleton. In addition to their essential role in modulating cadherin adhesion, catenins have more recently been indicated to participate in cell and developmental signaling pathways. $\beta$-catenin, for example, associates directly with receptor tyrosine kinases and transcription factors such as LEF-1/TCF, and tranduces developmental signals within the Wnt pathway. $\beta$-catenin also appear to a role in regulating cell proliferation via its interaction with the tumor supressor protein APC. I have employed the yeast two-hybrid method to reveal that fascin, a bundler of actin filaments, binds to $\beta$-catenin's central Armadillo-repeat domain. The $\beta$-catenin-fascin interaction exists in cell lines as well as in animal brain tissues as revealed by immunoprecipitation analysis, and substantiated in vitro with purified proteins. Fascin additionally binds to plakoglobin, which contains a more divergent Armadillo-repeat domain. Fascin and E-cadherin utilize a similar binding-site within $\beta$-catenin, such that they form mutually exclusive complexes with $\beta$-catenin. Fascin and $\beta$-catenin co-localize at cell-cell borders and dynamic cell leading edges of epithelial and endothelial cells. Total immunoprecipitable b-catein has several isoforms, only the hyperphosphorylated isoform 1 associated with fascin. An increased $\beta$-catenin-fascin interaction was observed in HGF stimulated cells, and in Xenopus embryos injected with src kinase RNAs. The increased $\beta$-catenin association with fascin is correlated with increased levels of $\beta$-catenin phosphorylation. $\beta$-catenin, but not fascin, can be readily phosphorylated on tyrosine in vivo following src injection of embryos, or in vitro following v-src addition to purified protein components. These observations suggest a role of $\beta$-catenin phosphorylation in regulating its interaction with fascin, and src kinase may be an important regulator of the $\beta$-catenin-fascin association in vivo. The $\beta$-catenin-fascin interaction represents a novel catenin complex, that may conceivably regulate actin cytoskeletal structures, cell adhesion, and cellular motility, perhaps in a coordinate manner with its functions in cadherin and APC complexes. ^

Identification of micro-RNAs targeting genes of PI3K/AKT pathway in ovarian cancer

Ovarian cancer is the leading cause of cancer-related death for females due to lack of specific early detection method. It is of great interest to find molecular-based biomarkers which are sensitive and specific to ovarian cancer for early diagnosis, prognosis and therapeutics. miRNAs have been proposed to be potential biomarkers that could be used in cancer prevention and therapeutics. The current study analyzed the miRNA and mRNA expression data extracted from the Cancer Genome Atlas (TCGA) database. Using simple linear regression and multiple regression models, we found 71 miRNA-mRNA pairs which were negatively associated between 56 miRNAs and 24 genes of PI3K/AKT pathway. Among these miRNA and mRNA target pairs, 9 of them were in agreement with the predictions from the most commonly used target prediction programs including miRGen, miRDB, miRTarbase and miR2Disease. These shared miRNA-mRNA pairs were considered to be the most potential genes that were involved in ovarian cancer. Furthermore, 4 of the 9 target genes encode cell cycle or apoptosis related proteins including Cyclin D1, p21, FOXO1 and Bcl2, suggesting that their regulator miRNAs including miR-16, miR-96 and miR-21 most likely played important roles in promoting tumor growth through dysregulated cell cycle or apoptosis. miR-96 was also found to directly target IRS-1. In addition, the results showed that miR-17 and miR-9 may be involved in ovarian cancer through targeting JAK1. This study might provide evidence for using miRNA or miRNA profile as biomarker.^

Analysis of Variation in Clubfoot Candidate Genes

Isolated clubfoot, a common birth defect occurring in more than 135,000 livebirths worldwide each year, is associated with significant health care and financial burdens. Clubfoot is defined by forefoot adduction, hindfoot varus, midfoot cavus and hindfoot equinus. Isolated clubfoot, which is the focus of these studies, is distinct from syndromic clubfoot because there are no other associated malformations. Population, family, twin and segregation analysis studies provide evidence that genetic and environmental factors play an etiologic role in isolated clubfoot. The studies described in this thesis were performed to define the role of genetic variation in isolated clubfoot. Interrogation of a deletion region associated with syndromic clubfoot, suggested that CASP8 and CASP10, two apoptotic genes, play a role in isolated clubfoot. To explore the role of apoptotic genes in clubfoot, SNPs spanning genes involved in the apoptotic pathway in the six chromosomal deletion regions, and limb patterning genes, HOXD and HOXA, were interrogated. SNPs in mitochondrial mediated apoptotic genes and several SNPs in HOXA and HOXD genes were modestly associated with clubfoot with the most significant SNP, rs3801776, located in the basal promoter of HOXA9. Several significant associations were found with SNPs in NFAT2 and TNIP2. Significant gene interactions were detected between SNPs in HOX and apoptotic genes. These findings suggest a model for clubfoot in which variation in one gene is not sufficient to cause the malformation but requires variation several genes to perturb protein expression sufficiently to alter muscle and foot development. These results significantly impact our knowledge base by delineating underlying mechanisms causing clubfoot.

CELL POLARITY REGULATES ORGAN GROWTH THROUGH THE HIPPO PATHWAY

Defects in apical-basal cell polarity and abnormal expression of cell polarity determinants are linked to human cancer. Loss of polarity is highly correlated with malignancy. In Drosophila, perturbation of apical-basal polarity, including overexpressing the apical determinant Crumbs, can lead to uncontrolled tissue growth. Cells mutant for the basolateral determinant scribble overproliferate and can form neoplastic tumors. Interestingly, scribble mutant clones that arise in wild-type tissues are eliminated and therefore do not manifest their tumorigenic potential. However, the mechanisms by which cell polarity coordinates with growth control pathways in developing organs to achieve appropriate organ size remain obscure. To investigate the function of apical determinants in growth regulation, I investigated the mechanism by which the apical determinant Crumbs affects growth in Drosophila imaginal discs. I found that crumbs gain and loss of function cause overgrowth and induction of Hippo target genes. In addition, Crumbs is required for the proper localization of Expanded, an upstream component of the Hippo pathway. Furthermore, we uncoupled the cell polarity and growth control function of Crb through structure-functional analysis. Taken together, our data identify a role of Crb in growth regulation specifically through modulation of the Hippo pathway. To further explore the role of polarity in growth control, I investigated how cells mutant for basolateral determinants are eliminated by using patches of cells mutant for scribble (scribble mutant clones) as a model system. We found that competitive cell-cell interactions eliminate tumorigenic scribble cells by modulation of the Hippo pathway. The regulation of Hippo signaling is required and sufficient to restrain the tumorous growth of scribble mutant cells. Artificially increasing the relative fitness of scribble mutant cells unleashes their tumorigenic potential. Therefore, we have identified a novel tumor-suppression mechanism that depends on signaling between normal and tumorigenic cells. These data identify evasion of cell competition as a critical step toward malignancy and illustrate a role for wild-type tissue in eliminating abnormal cells and preventing the formation of tumors.