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Background The adhesion molecule, FAT4, has a tumor suppressor function with a critical role in the epithelial-to-mesenchymal-transition (EMT) and anti-malignant growth in several cancers. No study has investigated yet its role in epithelial ovarian cancer (EOC) progression. In the present study, we examined the role of FAT4 in proliferation and metastasis, and its mechanisms of interaction in these processes. Methods We have performed cell viability, colony formation, and invasion assays in ovarian cancer cells treated with siRNA to knockdown FAT4 gene expression. The regulatory effects of FAT4 on proteins involved in apoptotic, Wnt, Hippo, and retinoblastoma signaling pathways were evaluated by Western blotting following FAT4 repression. Also, 426 ovarian tumor samples and 88 non-tumor samples from the Gene Expression Profiling Interactive Analysis (GEPIA) database were analyzed for the expression of FAT4. Pearson’s correlation was performed to determine the correlation between FAT4 and the E2F5, cyclin D1, cdk4, and caspase 9 expressions. Results Lower expression of FAT4 was observed in ovarian cancer cell lines and human samples as compared to non-malignant tissues. This down-regulation seems to enhance cell viability, invasion, and colony formation. Silencing FAT4 resulted in the upregulation of E2F5, vimentin, YAP, β-catenin, cyclin D1, cdk4, and Bcl2, and in the downregulation of GSK-3-β, and caspase 9 when compared to control. Furthermore, regulatory effects of FAT4 on the EMT and aggressive phenotype seem to occur through Hippo, Wnt, and cell cycle pathways. Conclusion FAT4 downregulation promotes increased growth and invasion through the activation of Hippo and Wnt-β-catenin pathways.

Breast and ovarian cancer are heterogeneous diseases. While breast cancer accounts for 25% of cancers worldwide, ovarian cancer accounts for 3.5% of all cancers and it is considered to be the most lethal type of cancer among women. In Oman, breast cancer accounts for 25% and ovarian cancer for 4.5% of all cancer cases. Various risk factors, including variable biological and clinical traits, are involved in the onset of breast and ovarian cancer. Although highly developed diagnostic and therapeutic methods have paved the way for better management, targeted therapy against specific biomarkers has not yet shown any significant improvement, particularly in triple-negative breast cancer and epithelial ovarian cancer, which are associated with high mortality rates. Thus, elucidating the mechanisms underlying the pathology of these diseases is expected to improve their prevention, prognosis and management. The aim of the present study was to provide a comprehensive review and updated information on genomics and proteomics alterations associated with cancer pathogenesis, as reported by several research groups worldwide. Furthermore, molecular research in our laboratory, aimed at identifying new pathways involved in the pathogenesis of breast and ovarian cancer using microarray and chromatin immunoprecipitation (ChIP), is discussed. Relevant candidate genes were found to be either up- or downregulated in a cohort of breast cancer cases. Similarly, ChIP analysis revealed that relevant candidate genes were regulated by the E2F5 transcription factor in ovarian cancer tissue. An ongoing study aims to validate these genes with a putative role as biological markers that may contribute to the development of targeted therapies for breast and ovarian cancer.

This paper reports in vitro anticancer activity of hexane, chloroform, ethyl acetate, butanol and water extracts obtained from twelve (12) medicinal plants including Aloe dhufarensis, Calotropis procera, Juniperus servaschanica, Lawsonia inermis, Maytenus dhofarensis, Moringa peregrina, Polygala senensis, Punica granatum, Rhazya stricta, Solanum incanum, Teucrium mascatense and Zataria multiflora collected from Oman. Crude ethanol extracts prepared by maceration of plant materials in ethanol were Kupchan partitioned to give hexane, chloroform, ethyl acetate, butanol and aqueous fractions. Cytotoxicities of the prepared fractions were measured by Alamar blue assay against ovarian cancer cell line (MCAS) and breast cancer cell line (MDA MB231). Thirteen extracts from five plants C. procera, J. servaschanica, M. dhofarensis, S. incanum and T. mascatense were found active against MCAS; hexane extract from J. servaschanica was the most active followed by chloroform extract from leaves of S. incanum (IC50 = 8.50 and 10.90 µg/ml, respectively). Furthermore, nine extracts from these plants except C. procera inhibited the growth of MDA MB321; hexane extract from J. servaschanica was again the most active followed by butanol extract of S. incanum. (IC50 = 11.4 and 19.44 µg/ml, respectively). Ethnobotanical and ethnopharmacological information on flora growing in this intemperate, hot climate region could provide new chemical entities for development of new and more potent cancer chemotherapeutics.

Introduction/Background FBXW7, a tumor suppressor gene plays a critical role in cell cycle progression under the regulation of the transcription factor E2F5. While FBXW7 is silenced in several cancers; its role in ovarian cancer pathogenesis is still unclear. This study explored FBXW7 promotor methylation status in ovarian cancer and its functional significance. Methodology FBXW7 expression at mRNA and protein levels were assessed in OVSAHO and MCAS cells using RT-qPCR and western blotting respectively. Expression of FBXW7 was high in MCAS and low in OVSAHO cells. FBXW7 gene methylation status was analyzed in cell lines (MCAS & OVSAHO) as well as in 23 EOC patients (5 normal, 8 benign, 4 borderline, 5 high grade tumors) by both methylation-specific PCR and MS-HRM analysis. Results In addition to OVSAHO cells, more than 60% of patients analyzed showed methylation. To evaluate its functional importance, FBXW7 expression was restored in OVSAHO cells by treatment with the demethylation agent, 5’-aza-2’deoxycitidine. Western blotting in FBXW7-restored OVSAHO cells and FBXW7-positive cell line (MCAS) showed an upregulation in caspases-2, 3, 7, BAX, P53, BAD, Cyclin D1, pNFkB and pGSK3-beta, whereas Bcl-2, Bcl-x, pBad, STAT3, GSK3-beta and NFkB were downregulated; indicating cells to undergo apoptosis via STAT3 pathway. Conclusion This study described an alternative mechanism for FBXW7 inactivation, namely promoter specific methylation in ovarian cancer and identified FBXW7 as a potential target for guiding the development of therapeutic strategies against ovarian cancer. Disclosure Nothing to disclose.

Introduction/Background Epithelial ovarian cancer (EOC) accounts for about 70% of all types of ovarian cancer.It is a heterogeneous disease and has variable prognosis. In a previous study, chromatin immunoprecipitation (ChIP) was used to study expression of E2F5, a transcription factor, and revealed that FAT4, is regulated by E2F5. FAT4, a tumor suppressor gene, is an upstream regulator of the Hippo pathway and its expression in EOC remains unclear. Objective We aimed to investigate the involvement of FAT4 gene in EOC by quantifying its expression in MCAS, OVSAHO, A2780cp and A2780s ovarian cancer cell lines and compare to HOSE, a normal ovarian cell line. Methodology We evaluated the differential expression of FAT4 and E2F5 in five ovarian cancer cell lines using qRT-PCR and western blotting. We knocked down FAT4 and E2F5 in MCAS and OVSAHO using siRNA and monitored it at the mRNA and protein levels. The effect on viability and invasion following knockdown were assessed by alamar blue and invasion assay. The mechanism linking FAT4 and E2F5 was evaluated by assessing proteins involved in Hippo, Retinoblastoma and apoptotic pathways. Results MCAS and OVSAHO showed higher FAT4 expression compared to A2780s and A2780cp. FAT4 and E2F5 were knocked down significantly using siRNA in MCAS (FAT4: p Conclusion Down regulation of FAT4 in ovarian cancer cell lines promotes growth and invasion via the modulation of apoptosis, Rb and Hippo pathways. Disclosure Nothing to disclose.

Introduction/Background Epithelial Ovarian Cancer (EOC) is the most lethal disease among gynecologic cancers. It is usually diagnosed at late stages with a poor outcome due to its asymptomatic nature. In Oman, the incidence rate for 2013 was estimated at 2.9 per 100,000. There is an urgent need to identify biomarkers for an early detection of EOC. we used chromatin immune precipitation (ChIP) to identify downstream genes regulated by the E2F5 transcription factor (TF) in ovarian cancer cell lines (MCAS and OVSAHO). Results revealed that SOCS-2 was regulated by the TF E2F5. SOCS (1–8) family have a role in JAK/STAT transcription pathway. Any disruption in this pathway may lead to many diseases such as cancers. Some of these SOCS genes found to act as biomarker for some cancers. SOCS2 was recently shown to act as an early biomarker for prostate and colorectal cancer. The aim of this study is to explore the role of SOCS2 gene in EOC by monitoring the expression of the gene in stages variety of EOC tissues and cell lines. Methodology We examined the expression of SOCS2 in tissues and cell lines using the quantitative reverse transcription-polymerase chain reaction (qRT-PCR) to monitor the differential expression of SOCS2 in cancerous and normal tissues. Results Significant down-regulation of SOCS2 was found in borderline tissues but it was not significant in other cancer stages such as benign and malignant tissues. Although all examined cell lines (MCAS, OVSAHO and 2008OV) showed a significant downregulation of the gene. SOCS2 methylation was investigated using MSP technique. It was revealed that SOCS2 is methylated in OVSAHO cell line but not in MCAS cells while cancerous tissues showed partially methylation. Conclusion The down regulation of SOCS2 gene in EOC is not caused by methylation; other mechanisms might be involved. Disclosure Nothing to disclose.

Objectives The SOCS3 gene is a key regulator for JAK/STAT pathway, responsible for inflammation and proliferation response, was found to be regulated by E2F5 and its down regulation seems to have a role in the pathogenesis of different tumors including gastric and pancreatic cancers. Our aim to investigate the involvement of SOCS3 gene in EOC by monitoring its expression in ovarian cancer cell lines and EOC tissue samples, then to compare results to normal ovarian tissue samples. We also examine methylation status of both, ovarian cell lines and ovarian tissue specimen. Methods Real time qPCR was used to assess gene expression using Taq Man gene expression assay. Five cell lines (MCAS, OVSAHO, OV 2008, A2780s and A2780cp) and 19 tissue samples with different histopathology types (6 normal, 4 benign, 5 borderlines and 4 high grade serous) were examined. Methylation status analysis was performed by methylation specific PCR Results OVSAHO, OV2008 and A2780s cell lines showed down regulation of SOCS3 expression when compared to normal. Benign, borderline and high grades samples, displayed also significant down regulation of SOCS3 expression. Analysis of methylation pattern showed no hyper-methylation in both cell lines and tissues. Conclusions Down regulation of SOCS3 gene expression was detected in ovarian cancer cell lines and EOC tissue samples, suggesting a putative role of SOCS3 in the pathogenesis of EOC. Other epigenetics mechanisms such as micro-RNAs are involved in the regulation of SOCS3 expression in addition to hyper-methylation and therefore, further study is needed to uncover these mechanisms.

Objectives: Marine organisms are a rich source of bioactive molecules with potential applications in medicine, biotechnology and industry; however, few bioactive compounds have been isolated from organisms inhabiting the Arabian Gulf and the Gulf of Oman. This study aimed to isolate and screen the anti-cancer activity of compounds and extracts from 40 natural products of marine organisms collected from the Gulf of Oman. Methods: This study was carried out between January 2012 and December 2014 at the Sultan Qaboos University, Muscat, Oman. Fungi, bacteria, sponges, algae, soft corals, tunicates, bryozoans, mangrove tree samples and sea cucumbers were collected from seawater at Marina Bandar Al-Rowdha and Bandar Al-Khayran in Oman. Bacteria and fungi were isolated using a marine broth and organisms were extracted with methanol and ethyl acetate. Compounds were identified from spectroscopic data. The anti-cancer activity of the compounds and extracts was tested in a Michigan Cancer Foundation (MCF)-7 cell line breast adenocarcinoma model. Results: Eight pure compounds and 32 extracts were investigated. Of these, 22.5% showed strong or medium anti-cancer activity, with malformin A, kuanoniamine D, hymenialdisine and gallic acid showing the greatest activity, as well as the soft coral Sarcophyton sp. extract. Treatment of MCF-7 cells at different concentrations of Sarcophyton sp. extracts indicated the induction of concentration-dependent cell death. Ultrastructural analysis highlighted the presence of nuclear fragmentation, membrane protrusion, blebbing and chromatic segregation at the nuclear membrane, which are typical characteristics of cell death by apoptosis induction. Conclusion: Some Omani marine organisms showed high anti-cancer potential. The efficacy, specificity and molecular mechanisms of anti-cancer compounds from Omani marine organisms on various cancer models should be investigated in future in vitro and in vivo studies. Keywords: Anticancer Agents; Cancer Screening; Breast Cancer; Marine Organisms; Biological Products; Apoptosis; Oman.

HOXB13, a member of the homeobox proteins family, is a key regulator of the epithelial differentiation in the prostate gland. HOXB13 is overexpressed during malignant progression of the prostatic tissue and suspected to contribute in the pathogenesis of the prostate gland. In androgen deprived conditions, HOXB13 is thought to act through inhibition of the tumour suppressor protein p21. Since HOXB13 has a multifaceted role in ventral prostate development, its critical partners in the cascade need to be elucidated for a further understanding of its role in prostate malignancy. In this report, we review the functions attributed to HOXB13, by highlighting the most recent findings supporting the hypothesis that HOXB13 might serve as a novel biomarker for the prognosis of prostate cancer.

Micrometastasis is a health burden affecting a large population worldwide, where early stage circulating tumor cells are clinically below the detection limit of the currently used techniques in diagnosis. These cells are considered one of the sources related to the disease spread, usually associated with poor prognosis and resistant to conventional therapies. With the recent advances in technology, various molecular and biological techniques including cytological examination, RT-PCR immunocytochemistry, immuno-magnetic separation and cell-enrichment techniques have emerged to improve the early detection of circulating tumor cells in different carcinomas. However, the sensitivity and specificity of these techniques along with their prognostic influence are still contested. This review aims to discuss the role of key player molecules including cell adhesion molecules, integrins and proteases in promoting micrometastasis and the current techniques used for an early detection of these malignant cells. Understanding mechanisms underlying this invasive process, will pave the way for designing new tools to unravel difficulties associated with early detection of CTCs and will improve therapies.

To determine whether PAX-2 and PAX-8 are involved in Wilms tumor (WT) pathogenesis, we sought mutations in these two genes in 99 Wilms tumors of favorable histology. We screened the entire protein coding sequences as well as the intronic regions adjacent to exons, using denaturing HPLC followed by sequencing of samples displaying abnormal chromatograms. In PAX-2, a silent polymorphism was found within exon 2 and exon 8 in 1% and 21% of cases, respectively. Three apparently silent polymorphisms were also found in PAX-8, two in exon 5 (2 of 99 cases or 2%) and one in exon 6 (22 of 99 cases or 22%), all of which were located 3' to the exons. In conclusion, no evidence for disease causing mutation was found using this technique, and so the direct involvement of either of these two genes in WT is unlikely.

The forkhead C1 (FOXC1) transcription factor is involved in the development and regulation of several organs, including the eye, where FOXC1 alterations cause iris, trabecular meshwork and corneal anomalies. Using nickel agarose chromatin enrichment with human anterior segment cells, we previously identified the fibroblast growth factor 19 (FGF19) locus as a gene potentially regulated by FOXC1. Here, we demonstrate that FGF19 is a direct target of FOXC1 in the eye. FOXC1 positively regulates FGF19 expression in corneal and periocular mesenchymal cells in cell culture and in zebrafish embryos. Through the FGFR4 tyrosine kinase, FGF19 promotes MAPK phosphorylation in the developing and mature cornea. During development, loss of either FOXC1 or FGF19 results in complementary, but distinct, anterior segment dysgeneses. This study reveals an important role for FOXC1 in the direct regulation of the FGF19-FGFR4-MAPK pathway to promote both the development and maintenance of anterior segment structures within the eye.

The FOX family of transcription factor genes is an evolutionary conserved, yet functionally diverse class of transcription factors that are important for regulation of energy homeostasis, development and oncogenesis. The proteins encoded by FOX genes are characterized by a conserved DNA-binding domain known as the forkhead domain (FHD). To date, disease-causing mutations have been identified in eight human FOX genes. Many of these mutations result in single amino acid substitutions in the FHD. We analyzed the molecular consequences of two disease-causing missense mutations (R121H and S125L) occurring in the FHD of the FOXC2 gene that were identified in patients with hereditary lymphedema with distichiasis (LD) to test the predictive capacity of a FHD structure/function model. On the basis of the FOXC2 solution structure, both FOXC2 missense mutations are located on the DNA-recognition helix of the FHD. A mutation model based on the parologous FOXC1 protein predicts that these FOXC2 missense mutations will impair the DNA-binding and transcriptional activation ability of the FOXC2 protein. When these mutations were analyzed biochemically, we found that both mutations did indeed reduce the DNA binding and transcriptional capacity. In addition, the R121H mutation affected nuclear localization of FOXC2. Together, these data indicate that these FOXC2 missense mutations are functional nulls and that FOXC2 haploinsufficiency underlies hereditary LD and validates the predictive ability of the FOXC1-based FHD mutational model.

We have examined the cytotoxic effect of gemcitabine in intravesical therapy using an in vitro co-cultured spheroid model composed of transitional cell carcinoma (TCC) and fibroblasts from both human and rat species. Immunohistochemistry analysis of the co-cultured spheroids, using cytokeratin-13 and vimentin antibodies against TCC and fibroblasts, respectively, showed the central location of fibroblasts within the spheroid, whereas TCC formed the peripheral layers. Spheroids composed of human TCC and fibroblasts (MGH-U3/CRL-1120 or RT-112/CRL-1120) as well as rat TCC and their corresponding fibroblasts (AY-27/RF-Ed1) displayed the same drug tolerance profile after an exposure of 0, 1, 3, 5, 7 and 14 days. As confirmed by time-lapse photography, MTT essay and vital dye staining, gemcitabine selectively killed the human and rat bladder cancer cell lines, but did not affect un-transformed human and rat fibroblast lines.

We have studied p16INK4 mutation (by PCR-SSCP) and deletion (by Southern blotting and/or multiplex PCR) in a series of 47 bilharziasis-associated tumors from Egypt and compared the results with those obtained on a series of 17 established bladder cell lines and non-bilharziasis-associated bladder cancers from the Netherlands. In the cell lines we found 9 homozygous deletions and 1 mutation (59% of p16INK4 alterations in cell lines), whereas in cases from the Netherlands deletions were found in 4 of 22 samples. No mutations were detected in the 46 samples screened. Interestingly, in bilharziasis-associated bladder cancer, deletions were present in 23 samples and mutations in a further 2 cases (53% of p16INK4 alteration in bilharziasis-associated bladder cancer). No correlation was found between p16INK4 alteration and histopathological data. Likewise, the same frequency of alteration was found in tumors with different differentiation patterns (squamous, transitional or adenocarcinoma). Three conclusions can be drawn from our findings: (i) p16INK4 alterations are more frequent in cell lines than in primary tumors; (ii) in primary bladder tumors (bilharziasis-associated or not), p16INK4 deletions are much more frequent than p16INK4 mutations; (iii) p16INK4 alterations are more frequent in bilharziasis-associated bladder tumors than in other bladder tumors. This high frequency of deletion is not related to a specific histological type but to the specific etiology of these tumors. © 1996 Wiley-Liss, Inc.

We have analyzed the short arm of chromosome 1 using loss of heterozygosity (LOH) analysis in Wilms tumors (WT) to identify a minimal region of loss. 1909 WT, 22 malignant rhabdoid tumors of the kidney and 90 clear cell carcinomas of the kidney (CCSK) were subjected to LOH analysis using five markers flanked by D1S243 and D1S244. 225 WT and 4 CCSK displayed LOH for this region. A group of 16 cases which had lost heterozygosity for at least one locus but also retained heterozygosity for at least one locus within this region were more finely analyzed using a panel of 24 microsatellite markers. A minimum region of loss located between D1S2694 and D1S244 spanning an area of 3.23 Mb was found in 15/16 of these tumors. No evidence for a second locus within this region was identified. This region of loss overlaps that found in neuroblastoma and harbors candidate genes highly expressed in fetal kidney i.e., LZIC, ICAT, and DNB5. Denaturing HPLC and quantitative RT-PCR analysis of these three genes, however, revealed no aberrations in WT samples retaining heterozygosity (8 cases) or displaying LOH 1p (8 cases). Further studies are required to identify the presumed tumor suppressor gene located within this region of 1p.

Epithelial ovarian cancer (EOC) is a deadly q disease and the related statistics are alarming with 15000 lives claimed in USA each year. EOC is a subtle disease since the majority of patients at presentation are diagnosed with a higher stage disease characterized by an aggressive potential. Early diagnosis seems to be of importance since complete cure can reach up to 90% if the disease is diagnosed at an early stage. Therefore, biological markers to detect patients at an early stage of the disease progression are urgently needed. In this study, the focus was centered on the transcription factor E2F5 used as biomarker and highly expressed at early stages of EOC pathogenesis. Therefore, we hypothesize that downstream genes regulated by this transcription factor might be also involved in the early events leading to ovarian cancer and can therefore potentially serve as useful biomarkers. Chromatin Immuno-Precipitation (ChIP) was performed using two ovarian cancer cell lines SK-OV-3 and OVCAR-3 and an antibody against E2F5 transcription factor. The enriched chromatin was cloned and sequenced before using available databases for BLAST and identification of downstream regulated genes. A short list of relevant genes, all involved in cancer, was obtained and analysis for their putative role in early detection of EOC is ongoing. The selected genes will be also validated using frozen human ovarian tissues available in our institution. ChIP is a robust technology to identify genes regulated by E2F5 with a putative potential to play a crucial role in the early steps of ovarian cancer pathogenesis. Citation Format: Yahya Tamimi, Ikram Burney, Moza Al-Kalbani, Ritu Lakhtari, Roseline Godbout, Mansour Al-Moundhri. Identification of putative genes involved in early steps of epithelial ovarian cancer pathogenesis [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(16 Suppl):Abstract nr POSTER-TECH-1130.

Cyclin-dependent kinase-4 inhibitor gene (p16INK4) has recently been mapped to chromosome 9p21. Homozygous deletions of this gene have been found at high frequency in cell lines derived from different types of tumours. These findings suggested therefore, that p16INK4 is a tumour-suppressor gene involved in a wide variety of human cancers. To investigate the frequency of p16INK mutations/deletions in prostate cancer, we screened 20 primary prostate tumours and four established cell lines by polymerase chain reaction (PCR) and single-strand conformation polymorphism (SSCP) analysis for exon 1 and exon 2. In contrast to most previous reports, no homozygous deletions were found in prostate cancer cell lines, but one cell line (DU145) has revealed to a mutation at codon 76. Only two SSCP shifts were detected in primary tumours: one of them corresponds to a mutation at codon 55 and the other one probably corresponds to a polymorphism. These data suggest that mutation of the p16INK4 gene is not a frequent genetic alteration implicated in prostate cancer development. Images Figure 1 Figure 2

Up to 50% of the transitional cell carcinomas (TCC) express an activated EGF pathway involving MAP/MEK and RAF kinase thus providing a novel means to selectively eliminate transformed cells expressing such proteins. This EGF pathway expression phenotype was also confirmed in our MGH-U3 and room temperature-112 human TCC cell lines, which makes them a suitable model target for the reovirus oncolysis. We report here on an in vitro assay of co-culture spheroids using either human or rat TCC cells with their corresponding fibroblasts to examine the potential of viral selective lysis for TCC. Reovirus, a respiratory enteric orphan virus, which mammals are exposed to early in life, was used in this study. Selective killing of transformed versus normal cells was assayed by time-lapse photography, vital dye staining, immunohistochemistry, and MTT assay. In this in vitro bladder cancer model, reovirus selectively destroyed the transformed cells by lysis or induction of apoptosis. Based on these findings we have initiated an in vivo pre-clinical study on intravesical administration of reovirus in an animal model to further explore the effect of reovirus-mediated oncolysis of TCC.

PURPOSE. To overcome the problem of antibody availability, often encountered during chromatin immunoprecipitation (ChIP) assays, nickel agarose– based chromatin enrichment (NACE) was developed. Based on the affinity of (His)-6-tagged proteins for the nickel ion, this modified form of ChIP allows the isolation of chromatin in the absence of specific antibodies. METHODS. Nonpigmented ciliary epithelium cells were transfected with (His)-6-tagged FOXC1. FOXC1-enriched chromatin complexes were isolated by using the tight electrostatic interaction between histidine residues of the recombinant FOXC1 protein and nickel. One hundred fifty NACE-enriched clones were sequenced and subjected to in silico and biochemical analyses. RESULTS. Twenty-six clones were detected near known genes: Eight were near predicted but uncharacterized genes, eight were within areas where neither known nor predicted genes have yet been mapped, four were chimeric, and the rest were either repetitive (n 81) or poor-quality (n 23) sequences. Twenty of the 26 known genes were expressed in the eye. Five of the NACE-enriched clones (BMP2K, DACH, FVT-1, SIX-1, and PGE-2 receptor), as well as nine clones selected from the literature, were validated by PCR amplification in two independent lots of NACE-enriched chromatin. All five NACE-selected genes were detected in two independent assays, as well as four (BMP7, SMAD2, TGF-B1, and WNT6) of the nine genes selected from the literature, consistent with these genes’ being regulated by FOXC1. CONCLUSIONS. NACE is a useful technique allowing specific chromatin enrichment in cases where antibodies are unavailable. Specific recovery of PTGER, DACH1, WNT6, and FVT-1 implicates FOXC1 in a variety of cellular events including modulation of intraocular pressure, cell cycle, ocular development, and oncogenesis. (Invest Ophthalmol Vis Sci. 2004;45: 3904 –3913) DOI:10.1167/iovs.04-0628

The PAX2 gene encodes a transcription factor expressed during development. In humans, PAX2 mutations cause the renal-coloboma syndrome, whereas homozygous mutations are lethal, causing severe organ malformation, notably in the brain and kidney. Wilms tumor (WT) of the kidney results from a failure in the mesenchymal-epithelial transition, a crucial step partly controlled by PAX2 . Downstream target genes regulated by PAX2 are still undefined. We therefore hypothesized that identification and characterization of the genes regulated by PAX2 may improve our understanding of developmentally related malignancies including WT. We used nickel agarose chromatin enrichment, chromatin immunoprecipitation, and the human embryonic kidney-derived cell line HEK293 to identify regulatory elements responding to PAX2 . Among others, we identified WNT5A as a gene potentially regulated by PAX2 . Here, we demonstrate that WNT5A is a direct target of PAX2 in HEK293 cells, using both transactivation and electrophoretic mobility shift assays. We were unable to find any WNT5A disease-associated mutations after screening a panel of 99 WT samples. However, quantitative reverse transcription-polymerase chain reaction in human favorable-histology WT revealed that ∼66% of the cases expressed significantly less WNT5A than human fetal kidney. Moreover, the WiT9 WT cell line revealed a weak expression of the WNT5A gene. A correlation of decreased WNT5A expression with predominant blastemal histology tumors suggests a possible inhibitory role in WT pathogenesis. This study underlines the importance of PAX2 in the regulation of WNT5A . Further in vivo study is necessary to determine whether the PAX2 and WNT5A are truly involved in WT pathogenesis.