The pivotal role of long non-coding RNAs as potential biomarkers and modulators of chemoresistance in ovarian cancer (OC).

Ovarian cancer (OC) is a fatal gynecological disease that is often diagnosed at later stages due to its asymptomatic nature and the absence of efficient early-stage biomarkers. Previous studies have identified genes with abnormal expression in OC that couldn't be explained by methylation or mutation, indicating alternative mechanisms of gene regulation. Recent advances in human transcriptome studies have led to research on non-coding RNAs (ncRNAs) as regulators of cancer gene expression. Long non-coding RNAs (lncRNAs), a class of ncRNAs with a length greater than 200 nucleotides, have been identified as crucial regulators of physiological processes and human diseases, including cancer. Dysregulated lncRNA expression has also been found to play a crucial role in ovarian carcinogenesis, indicating their potential as novel and non-invasive biomarkers for improving OC management. However, despite the discovery of several thousand lncRNAs, only one has been approved for clinical use as a biomarker in cancer, highlighting the importance of further research in this field. In addition to their potential as biomarkers, lncRNAs have been implicated in modulating chemoresistance, a major problem in OC. Several studies have identified altered lncRNA expression upon drug treatment, further emphasizing their potential to modulate chemoresistance. In this review, we highlight the characteristics of lncRNAs, their function, and their potential to serve as tumor markers in OC. We also discuss a few databases providing detailed information on lncRNAs in various cancer types. Despite the promising potential of lncRNAs, further research is necessary to fully understand their role in cancer and develop effective strategies to combat this devastating disease.

Epigenetic status of FBXW7 gene and its role in Ovarian cancer pathogenesis.

BACKGROUND: Chromatin immunoprecipitation (ChIP) analysis revealed that the FBXW7 gene and the long non-coding RNA (LINC01588) are potential candidates in epithelial ovarian cancer (EOC) pathogenesis. However, their exact role in EOC is not yet known. Thus, the present study sheds light on the impact of the mutations/ methylation status of the FBXW7 gene. MATERIALS AND METHODS: We used public databases to assess the correlation between mutations/ methylation status and the FBXW7 expression. Furthermore, we performed Pearson's correlation analysis between the FBXW7 gene and LINC01588. We performed gene panel exome sequencing and Methylation-specific PCR (MSP) in HOSE 6-3, MCAS, OVSAHO, and eight EOC patients' samples to validate the bioinformatics results. RESULTS: The FBXW7 gene was less expressed in EOC, particularly in stages III and IV, compared to healthy tissues. Furthermore, bioinformatics analysis, gene panel exome sequencing, and MSP revealed that the FBXW7 gene is neither mutated nor methylated in EOC cell lines and tissues, suggesting alternative mechanisms for FBXW7 gene regulation. Interestingly, Pearson's correlation analysis showed an inverse, significant correlation between the FBXW7 gene and LINC01588  expression, suggesting a potential regulatory role of LINC01588. CONCLUSION: Neither mutations nor methylation is the causative mechanism for the FBXW7 downregulation in EOC, suggesting alternative means involving the lncRNA LINC01588.

Exosomes as crucial emerging tools for intercellular communication with therapeutic potential in ovarian cancer.

More than two-thirds of epithelial ovarian cancer (EOC) patients are diagnosed at advanced stages due to the lack of sensitive biomarkers. Currently, exosomes are intensively investigated as non-invasive cancer diagnostic markers. Exosomes are nanovesicles released in the extracellular milieu with the potential to modulate recipient cells' behavior. EOC cells release many altered exosomal cargoes that exhibit clinical relevance to tumor progression. Exosomes represent powerful therapeutic tools (drug carriers or vaccines), posing a promising option in clinical practice for curing EOC in the near future. In this review, we highlight the importance of exosomes in cell-cell communication, epithelial-mesenchymal transition (EMT), and their potential to serve as diagnostic and prognostic factors, particularly in EOC.

Exosomes are nanovesicles released in the extracellular milieu by diverse cell types and serve as carriers of proteins, and nucleic acids, with the potential to modulate the recipient cells. Understanding the functions of exosomes as messengers between cancer and healthy cells unfolds new avenues in intercellular signaling mechanisms. In this review, we have highlighted the importance of exosomes in cell­cell communication and their potential to serve as diagnostic and prognostic factors in EOC. Furthermore, we believe that exosomes could represent a powerful therapeutic tool due to their ability to function as drug carriers or vaccines posing a promising option in curing EOC.

Identification and validation of a novel long non-coding RNA (LINC01465) in ovarian cancer.

Epithelial Ovarian Cancer (EOC) is a heterogeneous disease usually diagnosed at advanced stages. Therefore, early detection is crucial for better survival. Despite the advances in ovarian research, mechanisms underlying EOC carcinogenesis are not elucidated. We performed chromatin immunoprecipitation sequencing to identify genes regulated by E2F5, a transcription factor involved in ovarian carcinogenesis. Results revealed several putative candidate genes (115 protein-coding genes, 20 lncRNAs, 6 pseudogenes, and 4 miRNAs). A literature review and bioinformatics analysis of these genes revealed a novel lncRNA candidate (LINC01465) in EOC. We validated LINC01465 by quantifying its expression in EOC cell lines and selected OVSAHO and SKOV3 as a model with high LINC01465 levels. We silenced LINC01465 and performed proliferation, wound healing, invasion, and drug resistance assays. Knocking-down LINC01465 resulted in reduced migration, suggesting potential involvement in EOC. Furthermore, to identify the significance of LINC01465 in chemoresistance, we assessed the LINC01465 levels in A2780 S cells treated with malformin, which revealed higher LINC01465 expression as compared to untreated A2780S cells implying the involvement of LINC01465 in cell death. Thus, this study unraveled the repertoire of E2F5 regulated candidate genes and suggested a putative role of LINC01465 in malformin-induced cell death in EOC.

Addition of Gallic Acid Overcomes Resistance to Cisplatin in Ovarian Cancer Cell Lines.

OBJECTIVE: Ovarian cancer is one of the leading causes of cancer-related mortality in women, and is often associated with drug resistance. Therefore, finding effective drugs, including naturally derived compounds, is urgently needed. Herein, we aimed to test the anti-cancer potential of gallic acid monohydrate (GA) and its congeners on cisplatin-sensitive (A2780S), and resistant (A2780CP) ovarian cancer and normal ovarian (HOSE6-3) cell lines. METHODS: Cytotoxicity was assessed by AlamarBlue and CCK08 assays by exposing cells to different concentrations of cisplatin (0-21µg/mL), GA and its congeners (0-100µg/mL), and a combination of GA and cisplatin. Apoptosis was estimated by Hoechst stain and monitoring the relative RNA expression of the apoptotic effector caspase-3 using qRT-PCR. RESULTS: GA decreased cell viability in a concentration-dependent manner in all cell lines, with an IC50 of 19.39µg/mL (A2780S), 35.59 µg/mL (A2780CP), and 49.32µg/mL (HOSE6-3). GA displayed higher cytotoxicity than its congeners. An apoptotic rate estimation of approximately 20% and 30% was obtained in A2780S and A2780CP. While the cytotoxicity observed with cisplatin and GA was comparable, combining the two enhanced the cytotoxicity significantly, especially in the A2780CP cell line (p<0.05). CONCLUSION: These data suggest that GA may help overcome the resistance. Hence, the cytotoxic effects of GA, especially on chemo-resistant ovarian cancer cells merit further investigation.
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SOCS3 gene silencing does not occur through methylation and mutations in gastric cancer.

Gastric cancer (GC) is ranked the third leading cause of cancer-related deaths worldwide. Mutations and epigenetic alterations in several essential genes, including p53, KRAS, PIK3CA, FAT4 and ARID1A, are often reported. Furthermore, loss of SOCS3 expression was reported in GC, suggesting its tumor suppressor role. To assess the mutational and methylation status of SOCS3, we performed gene panel exome sequencing on 47 human GC samples. The SOCS3 gene was rarely mutated, suggesting alternative regulation mechanisms, such as promoter hypermethylation and/or long non-coding RNAs (lncRNAs). We first explored SOCS3 promoter methylation status in 44 human GC samples by methylation-specific PCR (MS-PCR). Thirteen out of forty-four patients (29.5%) displayed a methylation pattern. Then, to see whether SOCS3 expression is silenced by CpG methylation, we examined publicly available databases (cbioportal and The Cancer Genome Atlas (TCGA)). The analysis revealed ß values lower than 0.1, indicating hypo-methylation in healthy and GC samples. Moreover, moderate methylation (ß < 0.4) and high methylation (ß > 0.4) did not affect the free survival, suggesting that methylation is unlikely to be the mechanism ruling SOCS3 silencing in GC. Next, to assess the regulatory effects of lncRNAs on SOCS3, we silenced the AC125807.2-lncRNA and quantified the SOCS3 gene expression in AGS and NCI-N87 gastric cancer cell line. SOCS3 was found to be downregulated following AC125807.2-lncRNA silencing in AGS cells, suggesting the potential implication of lncRNA AC125807.2 in SOCS3 regulation. However, in NCI-N87 cells, there was no significant change in SOCS3 expression. In conclusion, neither mutations nor hypermethylation was associated with the SOCS3 downregulation in GC, and alternative mechanisms, including non-coding RNAs-mediated gene silencing, may be proposed.

Hymenialdisine is Cytotoxic Against Cisplatin-Sensitive but Not Against Cisplatin-Resistant Cell Lines.

OBJECTIVES: New compounds are needed to overcome the resistance to commonly used cytotoxic chemotherapy for epithelial ovarian cancer. Marine sponges are a rich source of diverse chemical compounds and hymenialdisine has been found to have antiproliferative effects. This study aimed to investigate the cytotoxic effect of hymenialdisine in cisplatin-sensitive and cisplatin resistant ovarian cancer cell lines. METHODS: This study took place at Sultan Qaboos University, Muscat, Oman between August and November, 2019. The anti-cancer effects of hymenialdisine or cisplatin were assessed using treating cells with different concentrations of hymenialdisine and cisplatin. Cell viability was determined using the AlamarBlue® Assay. RESULTS: The half-maximal inhibitory concentration (IC50) of cisplatin was estimated at 31.4 µM for A2780S and 76.9 µM for A2780CP, whereas the IC50 of hymenialdisine was evaluated at 146.8 µM for A2780S cells. Despite the higher concentrations of hymenialdisine (up to 300 µM), IC50 could not be determined for the A2780CP cell line. CONCLUSION: When compared to cisplatin, hymenialdisine was less toxic against both A2780S and A2780CP ovarian cancer cell lines.

Malformin-A1 (MA1) Sensitizes Chemoresistant Ovarian Cancer Cells to Cisplatin-Induced Apoptosis.

High-grade epithelial ovarian cancer is a fatal disease in women frequently associated with drug resistance and poor outcomes. We previously demonstrated that a marine-derived compound MalforminA1 (MA1) was cytotoxic for the breast cancer cell line MCF-7. In this study, we aimed to examine the effect of MA1 on human ovarian cancer cells. The potential cytotoxicity of MA1was tested on cisplatin-sensitive (A2780S) and cisplatin-resistant (A2780CP) ovarian cancer cell lines using AlamarBlue assay, Hoechst dye, flow cytometry, Western blot, and RT-qPCR. MA1 had higher cytotoxic activity on A2780S (IC50 = 0.23 µM) and A2780CP (IC50 = 0.34 µM) cell lines when compared to cisplatin (IC50 = 31.4 µM and 76.9 µM, respectively). Flow cytometry analysis confirmed the cytotoxic effect of MA1. The synergistic effect of the two drugs was obvious, since only 13% of A2780S and 7% of A2780CP cells remained alive after 24 h of treatment with both MA1 and cisplatin. Moreover, we examined the expression of bcl2, p53, caspase3/9 genes at RNA and protein levels using RT-qPCR and Western blot, respectively, to figure out the cell death mechanism induced by MA1. A significant down-regulation in bcl2 and p53 genes was observed in treated cells compared to non-treated cells (p < 0.05), suggesting that MA1 may not follow the canonical pathway to induce apoptosis in ovarian cancer cell lines. MalforminA1 showed promising anticancer activity by inducing cytotoxicity in cisplatin-sensitive and cisplatin-resistant cancer cell lines. Interestingly, a synergistic effect was observed when MA1 was combined with cisplatin, leading to it overcoming its resistance to cisplatin.

E2F5 Promotes the Malignancy of Ovarian Cancer Via the Regulation of Hippo and Wnt Pathways.

Background: E2F5 is a transcription factor that is overexpressed in the early stages of ovarian cancer and has been suggested as a potential biomarker for early detection. In this study, we aimed to examine the role of E2F5 in invasion and proliferation of ovarian cancer cells. Materials and Methods: We performed cell viability, colony formation, and invasion assays using ovarian cancer cells treated with siRNA to knock down the E2F5 gene. The regulatory effects of E2F5 on proteins involved in the apoptotic, Wnt, Hippo, and retinoblastoma signaling pathways were evaluated by western blotting following E2F5 repression. In addition, we analyzed data available on Gene Expression Profiling Interactive Analysis for correlations between E2F5 and YAP, ß-catenin, cyclin D1, cdk4, and caspase-9. Results: E2F5 was highly expressed in ovarian cancer cell lines and samples when compared to the nonmalignant tissues. Downregulation of E2F5 inhibited cell viability and invasion and promoted the phosphorylation of YAP, GSK-3-ß, ß-catenin, and retinoblastoma. However, cyclin D1, cdk4, and caspase-9 were downregulated when compared to control. Conclusion: Overall, E2F5 promotes ovarian carcinogenesis via the regulation of Hippo and Wnt pathways.

FAT4 silencing promotes epithelial-to-mesenchymal transition and invasion via regulation of YAP and ß-catenin activity in ovarian cancer.

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, ß-catenin, cyclin D1, cdk4, and Bcl2, and in the downregulation of GSK-3-ß, 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-ß-catenin pathways.

BRIP1 overexpression is correlated with clinical features and survival outcome of luminal breast cancer subtypes.

In Oman, breast cancer is most common, representing approximately more than 25% of all cancers in women. Relatively younger populations of patients (25-40 years) present surprisingly with an aggressive phenotype and advanced tumor stages. In this study, we investigated differential gene expressions in Luminal A, Luminal B, triple-negative and Her2+ breast cancer subtypes and compared data to benign tumor samples. We identified a potential candidate gene BRIP1, showing differential expression in the four breast cancer subtypes examined, suggesting that BRIP1 has the profile of a useful diagnostic marker, suitable for targeted therapeutic intervention. RT-qPCR and Western blotting analysis showed higher BRIP1 expression in luminal samples as compared to triple-negative subtype patient's samples. We further screened BRIP1 for eventual mutations/SNPs/deletions by sequencing the entire coding region. Four previously identified polymorphisms were detected, one within the 5'-UTR region (c.141-64G > A) and three in the BRCA-binding domain (c.2755T > C, c.2647G > A and c.3411T > C). Kaplan-Meier analysis revealed that patients with overexpression of BRIP1 displayed a poor survival rate (P < 0.05). BRIP1 has a dual function of an oncogene and a tumor suppressor gene in addition to its role as a potential biomarker to predict survival and prognosis. Data obtained in this study suggest that BRIP1 can plausibly have an oncogenic role in sporadic cancers.

Molecular genetics complexity impeding research progress in breast and ovarian cancers.

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.

A novel missense mutation in the C2C domain of otoferlin causes profound hearing impairment in an Omani family with auditory neuropathy.

OBJECTIVES: To identify genetic defects in an Omani family diagnosed with deafness.  METHODS: A cross-sectional association study was conducted at the Department of Biochemistry, College of Medicine and Health Sciences, Sultan Qaboos University, Al-Khoud, Oman and the Centre of Medical Genetics, University of Antwerp, Antwerp, Belgium between August 2010 and September 2014. Microsatellites markers for nine non-syndromic genes were used to genotype the defective locus using the extracted DNA from family members. Sanger sequencing method was used to identify the disease causative mutation. Eazy linkage 5.05 was used to calculate the logarithm of odds score. Lasergene suite was used to detect the mutation position, and Phyre2, SMART, Rasmol, and GOR IV were used to predict the effects of the defect on protein structure and function.  RESULTS: The disease was linked to markers located on chromosome-2 and covering the OTOF (DFNB9) gene. A novel missense mutation that changed nucleotide C to G at position c.1469 and consequently the amino acid Proline to Arginine (P490R) on exon 15 was detected. Protein modeling analysis revealed the impact of the mutation on protein structure and the relevant C2C domain. The mutation seems to create a new protein isoform homologous to the complement component C1q.  CONCLUSION: These findings suggest that the mutation found in C2C domain of the OTOF gene is likely to cause deafness in the studied family reflecting the importance of C2 domains of otoferlin in hearing loss.

Screening for Anti-Cancer Compounds in Marine Organisms in Oman.

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.

Hoxb13, a potential prognostic biomarker for prostate cancer.

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.

Understanding the functional discrepancy of Pim-1 in cancer.

The Pim-1 gene encodes for a proto-oncogenic serine/threonine protein kinase and is generally involved in cytokine signaling as well as in various signaling pathways regulating cell cycle and apoptosis. Pim-1 kinase plays a role in the development of various tumors mainly, prostate cancer, Burkitt's lymphoma, oral cancer and various other hematopoietic lymphomas. This review will focus on the importance and mechanisms of Pim-1 in prostate cancer and the potential clinical relevance of its various inhibitors.

The p53 Mutation/Deletion Profile in a Small Cohort of the Omani Population with Diffuse Large B-Cell Lymphoma.

OBJECTIVES: Mutations/deletions affecting the TP53 gene are considered an independent marker predicting a poor prognosis for patients with diffuse large B-cell lymphoma (DLBCL). A cohort within a genetically isolated population was investigated for p53 mutation/deletion status. METHODS: Deoxyribonucleic acid (DNA) samples were extracted from 23 paraffin-embedded blocks obtained from DLBCL patients, and subjected to polymerase chain reaction (PCR) amplification and sequencing of exons 4-9 of the p53 gene. RESULTS: While 35% of patients analysed displayed allelic deletions (P <0.01), immunohistochemical analysis revealed a mutation rate of 69.5%. It is noteworthy that the rate of p53 mutations/deletions in this small cohort was found to be higher than that previously reported in the literature. Interestingly, patients with p53 mutations displayed a better overall survival when compared to those without. The survival of patients treated with rituximab-containing combination chemotherapy was significantly better than those who did not receive rituximab (P <0.05). Furthermore, a modelling analysis of the deleted form of p53 revealed a huge structural change affecting the DNA-binding domain. CONCLUSION: The TP53 mutation/deletion status plays a role in mechanism(s) ruling the pathogenesis of DLBCL and may be useful for stratifying patients into distinct prognostic subsets.

WNT5A is regulated by PAX2 and may be involved in blastemal predominant Wilms tumorigenesis.

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 approximately 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.

Identification of a minimal region of loss on the short arm of chromosome 1 in Wilms tumor.

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.

FGF19 is a target for FOXC1 regulation in ciliary body-derived cells.

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.