Functional Exploration of the Adult Ovarian Granulosa Cell Tumor-Associated Somatic FOXL2 Mutation p.Cys134Trp (c.402C>G)

Background

The somatic mutation in the FOXL2 gene c.402C>G (p.Cys134Trp) has recently been identified in the vast majority of adult ovarian granulosa cell tumors (OGCTs) studied. In addition, this mutation seems to be specific to adult OGCTs and is likely to be a driver of malignant transformation. However, its pathogenic mechanisms remain elusive.

Methodology/Principal Findings

We have sequenced the FOXL2 open reading frame in a panel of tumor cell lines (NCI-60, colorectal carcinoma cell lines, JEG-3, and KGN cells). We found the FOXL2 c.402C>G mutation in the adult OGCT-derived KGN cell line. All other cell lines analyzed were negative for the mutation. In order to gain insights into the pathogenic mechanism of the p.Cys134Trp mutation, the subcellular localization and mobility of the mutant protein were studied and found to be no different from those of the wild type (WT). Furthermore, its transactivation ability was in most cases similar to that of the WT protein, including in conditions of oxidative stress. A notable exception was an artificial promoter known to be coregulated by FOXL2 and Smad3, suggesting a potential modification of their interaction. We generated a 3D structural model of the p.Cys134Trp variant and our analysis suggests that homodimer formation might also be disturbed by the mutation.

Conclusions/Significance

Here, we confirm the specificity of the FOXL2 c.402C>G mutation in adult OGCTs and begin the exploration of its molecular significance. This is the first study demonstrating that the p.Cys134Trp mutant does not have a strong impact on FOXL2 localization, solubility, and transactivation abilities on a panel of proven target promoters, behaving neither as a dominant-negative nor as a loss-of-function mutation. Further studies are required to understand the specific molecular effects of this outstanding FOXL2 mutation.     

Aromatase is a direct target of FOXL2: C134W in granulosa cell tumors via a single highly conserved binding site in the ovarian specific promoter

Background

Granulosa cell tumors (GCT) of the ovary often express aromatase and synthesize estrogen, which in turn may influence their progression. Recently a specific point mutation (C134W) in the FOXL2 protein was identified in >94% of adult-type GCT and it is likely to contribute to their development. A number of genes are known to be regulated by FOXL2, including aromatase/CYP19A1, but it is unclear which are direct targets and whether the C134W mutation alters their regulation. Recently, it has been reported that FOXL2 forms a complex with steroidogenic factor 1 (SF-1) which is a known regulator of aromatase in granulosa cells.

Methodology/Principal Findings

In this work, the human GCT-derived cell lines KGN and COV434 were heterozygous and wildtype for the FOXL2:C134W mutation, respectively. KGN had abundant FOXL2 mRNA expression but it was not expressed in COV434. Expression of exogenous FOXL2:C134W in COV434 cells induced higher expression of a luciferase reporter for the ovarian specific aromatase promoter, promoter II (PII) (−516bp) than expression of wildtype FOXL2, but did not alter induction of a similar reporter for the steroidogenic acute regulatory protein (StAR) promoter (−1300bp). Co-immunoprecipitation confirmed that FOXL2 bound SF-1 and that it also bound its homologue, liver receptor homologue 1 (LRH-1), however, the C134W mutation did not alter these interactions or induce a selective binding of the proteins. A highly conserved putative binding site for FOXL2 was identified in PII. FOXL2 was demonstrated to bind the site by electrophoretic mobility shift assays (EMSA) and site-directed mutagenesis of this element blocked its differential induction by wildtype FOXL2 and FOXL2:C134W.

Conclusions/Significance

These findings suggest that aromatase is a direct target of FOXL2:C134W in adult-type GCT via a single distinctive and highly conserved binding site in PII and therefore provide insight into the pathogenic mechanism of this mutation.     

Variants of the PPARD Gene and Their Clinicopathological Significance in Colorectal Cancer

Background

Peroxisome proliferator-activated receptor delta (PPARD) is nuclear hormone receptor involved in colorectal cancer (CRC) differentiation and progression. The purpose of this study was to determine prevalence and spectrum of variants in the PPARD gene in CRC, and their contribution to clinicopathological endpoints.

Methods and Findings

Direct sequencing of the PPARD gene was performed in 303 primary tumors, in blood samples from 50 patients with ≥3 affected first-degree relatives, 50 patients with 2 affected first-degree relatives, 50 sporadic patients, 360 healthy controls, and in 6 colon cancer cell lines. Mutation analysis revealed 22 different transversions, 7 of them were novel. Three of all variants were somatic (c.548A>G, p.Y183C, c.425-9C>T, and c.628-16G>A). Two missense mutations (p.Y183C and p.R258Q) were pathogenic using in silico predictive program. Five recurrent variants were detected in/adjacent to the exons 4 (c.1-87T>C, c.1-67G>A, c.130+3G>A, and c.1-101-8C>T) and exon 7 (c.489T>C). Variant c.489C/C detected in tumors was correlated to worse differentiation (P = 0.0397).

Conclusions

We found 7 novel variants among 22 inherited or acquired PPARD variants. Somatic and/or missense variants detected in CRC patients are rare but indicate the clinical importance of the PPARD gene.     

Four Common Vascular Endothelial Growth Factor Polymorphisms (?2578C>A, ?460C>T, +936C>T,and +405G>C) in Susceptibility to Lung Cancer: A Meta-Analysis

Background and Objective

Vascular endothelial growth factor (VEGF) is one of the key initiators and regulators of angiogenesis and it plays a vital role in the onset and development of malignancy. The association between VEGF gene polymorphisms and lung cancer risk has been extensively studied in recent years, but currently available results remain controversial or ambiguous. The aim of this meta-analysis is to investigate the associations between four common VEGF polymorphisms (i.e., −2578C>A, −460C>T, +936C>T and +405C>G) and lung cancer risk.

Methods

A comprehensive search was conducted to identify all eligible studies to estimate the association between VEGF polymorphisms and lung cancer risk. Crude odds ratios (ORs) with 95% confidence intervals (CIs) were used to evaluate the strength of this association.

Results

A total of 14 published case-control studies with 4,664 cases and 4,571 control subjects were identified. Our meta-analysis provides strong evidence that VEGF −2578C>A polymorphism is capable of increasing lung cancer susceptibility, especially among smokers and lung squamous cell carcinoma (SCC) patients. Additionally, for +936C>T polymorphism, increased lung cancer susceptibility was only observed among lung adenocarcinoma patients. In contrast, VEGF −460C>T polymorphism may be a protective factor among nonsmokers and SCC patients. Nevertheless, we did not find any association between +405C>G polymorphism and lung cancer risk, even when the groups were stratified by ethnicity, smoking status or histological type.

Conclusion

This meta-analysis recommends more investigations into the relationship between −2578C>A and −460C>T lung cancer risks. More detailed and well-designed studies should be conducted to identify the causal variants and the underlying mechanisms of the possible associations.     

Forkhead Box L1 Is Frequently Downregulated in Gallbladder Cancer and Inhibits Cell Growth through Apoptosis Induction by Mitochondrial Dysfunction

Background

Forkhead box L1 (FOXL1), considered as a novel candidate tumor suppressor, suppresses proliferation and invasion in certain cancers. However, the regulation and function of FOXL1 in gallbladder cancer (GBC) remains unclear.

Methods

FOXL1 expression at mRNA and protein levels in GBC tissues and cell lines were examined by RT-PCR, immunohistochemistry and western blot assay. FOXL1 expression in GBC cell lines was up-regulated by transfection with pcDNA-FOXL1. The effects of FOXL1 overexpression on cell proliferation, apoptosis, migration and invasion were evaluated in vitro or in vivo. In addition, the status of mediators involved in migration, invasion and apoptosis was examined using western blot after transfection with pcDNA-FOXL1.

Results

FOXL1 was frequently downregulated in GBC tissues and cell lines. Its higher expression is associated with better prognosis, while its lower expression is correlated with advanced TNM stage and poor differentiation. FOXL1 overexpression in NOZ cells significantly suppresses cell proliferation, migration and invasion in vitro and tumorigenicity in nude mice. FOXL1 overexpression disrupted mitochondrial transmembrane potential and triggered mitochondria-mediated apoptosis in NOZ cells. In addition, FOXL1 overexpression suppressed ZEB1 expression and induced E-cadherin expression in NOZ cells.

Conclusion

Our findings suggested that dysregulated FOXL1 is involved in tumorigenesis and progression of GBC and may serve as a predictor of clinical outcome or even a therapeutic target for patients with GBC.     

Activin Signaling in Microsatellite Stable Colon Cancers Is Disrupted by a Combination of Genetic and Epigenetic Mechanisms

Background

Activin receptor 2 (ACVR2) is commonly mutated in microsatellite unstable (MSI) colon cancers, leading to protein loss, signaling disruption, and larger tumors. Here, we examined activin signaling disruption in microsatellite stable (MSS) colon cancers.

Methods

Fifty-one population-based MSS colon cancers were assessed for ACVR1, ACVR2 and pSMAD2 protein. Consensus mutation-prone portions of ACVR2 were sequenced in primary cancers and all exons in colon cancer cell lines. Loss of heterozygosity (LOH) was evaluated for ACVR2 and ACVR1, and ACVR2 promoter methylation by methylation-specific PCR and bisulfite sequencing and chromosomal instability (CIN) phenotype via fluorescent LOH analysis of 3 duplicate markers. ACVR2 promoter methylation and ACVR2 expression were assessed in colon cancer cell lines via qPCR and IP-Western blots. Re-expression of ACVR2 after demethylation with 5-aza-2′-deoxycytidine (5-Aza) was determined. An additional 26 MSS colon cancers were assessed for ACVR2 loss and its mechanism, and ACVR2 loss in all tested cancers correlated with clinicopathological criteria.

Results

Of 51 MSS colon tumors, 7(14%) lost ACVR2, 2 (4%) ACVR1, and 5(10%) pSMAD2 expression. No somatic ACVR2 mutations were detected. Loss of ACVR2 expression was associated with LOH at ACVR2 (p<0.001) and ACVR2 promoter hypermethylation (p<0.05). ACVR2 LOH, but not promoter hypermethylation, correlated with CIN status. In colon cancer cell lines with fully methylated ACVR2 promoter, loss of ACVR2 mRNA and protein expression was restored with 5-Aza treatment. Loss of ACVR2 was associated with an increase in primary colon cancer volume (p<0.05).

Conclusions

Only a small percentage of MSS colon cancers lose expression of activin signaling members. ACVR2 loss occurs through LOH and ACVR2 promoter hypermethylation, revealing distinct mechanisms for ACVR2 inactivation in both MSI and MSS subtypes of colon cancer.     

Mutation Screening of Retinal Dystrophy Patients by Targeted Capture from Tagged Pooled DNAs and Next Generation Sequencing

Purpose

Retinal dystrophies are genetically heterogeneous, resulting from mutations in over 200 genes. Prior to the development of massively parallel sequencing, comprehensive genetic screening was unobtainable for most patients. Identifying the causative genetic mutation facilitates genetic counselling, carrier testing and prenatal/pre-implantation diagnosis, and often leads to a clearer prognosis. In addition, in a proportion of cases, when the mutation is known treatment can be optimised and patients are eligible for enrolment into clinical trials for gene-specific therapies.

Methods

Patient genomic DNA was sheared, tagged and pooled in batches of four samples, prior to targeted capture and next generation sequencing. The enrichment reagent was designed against genes listed on the RetNet database (July 2010). Sequence data were aligned to the human genome and variants were filtered to identify potential pathogenic mutations. These were confirmed by Sanger sequencing.

Results

Molecular analysis of 20 DNAs from retinal dystrophy patients identified likely pathogenic mutations in 12 cases, many of them known and/or confirmed by segregation. These included previously described mutations in ABCA4 (c.6088C>T,p.R2030*; c.5882G>A,p.G1961E), BBS2 (c.1895G>C,p.R632P), GUCY2D (c.2512C>T,p.R838C), PROM1 (c.1117C>T,p.R373C), RDH12 (c.601T>C,p.C201R; c.506G>A,p.R169Q), RPGRIP1 (c.3565C>T,p.R1189*) and SPATA7 (c.253C>T,p.R85*) and new mutations in ABCA4 (c.3328+1G>C), CRB1 (c.2832_2842+23del), RP2 (c.884-1G>T) and USH2A (c.12874A>G,p.N4292D).

Conclusions

Tagging and pooling DNA prior to targeted capture of known retinal dystrophy genes identified mutations in 60% of cases. This relatively high success rate may reflect enrichment for consanguineous cases in the local Yorkshire population, and the use of multiplex families. Nevertheless this is a promising high throughput approach to retinal dystrophy diagnostics.     

Overexpression of LIM and SH3 Protein 1 Leading to Accelerated G2/M Phase Transition Contributes to Enhanced Tumourigenesis in Oral Cancer

Background

LIM and SH3 protein 1 (LASP-1) is a specific focal adhesion protein involved in several malignant tumors. However, its role in oral squamous cell carcinoma (OSCC) is unknown. The aim of this study was to characterize the role and molecular status/mechanism of LASP-1 in OSCC.

Methods

We evaluated LASP-1 mRNA and protein expressions in OSCC-derived cell lines and primary OSCCs. Using an shRNA system, we analyzed the effect of LASP-1 on the biology and function of the OSCC cell lines, HSC-3 and Ca9-22. The cells also were subcutaneously injected to evaluate tumor growth in vivo. Data were analyzed by the Fisher’s exact test or the Mann-Whitney U test. Bonferroni correction was used for multiple testing.

Results

Significant up-regulation of LASP-1 was detected in OSCC-derived cell lines (n = 7, P<0.007) and primary OSCCs (n = 50, P<0.001) compared to normal controls. LASP-1 knockdown cells significantly inhibited cellular proliferation compared with shMock-transfected cells (P<0.025) by arresting cell-cycle progression at the G2 phase. We observed dramatic reduction in the growth of shLASP-1 OSCC xenografts compared with shMock xenografts in vivo.

Conclusion

Our results suggested that overexpression of LASP-1 is linked closely to oral tumourigenicity and further provide novel evidence that LASP-1 plays an essential role in tumor cellular growth by mediating G2/M transition.     

Whole Exome Sequencing Identifies Recessive PKHD1 Mutations in a Chinese Twin Family with Caroli Disease

Background

Mutations in PKHD1 cause autosomal recessive Caroli disease, which is a rare congenital disorder involving cystic dilatation of the intrahepatic bile ducts. However, the mutational spectrum of PKHD1 and the phenotype-genotype correlations have not yet been fully established.

Methods

Whole exome sequencing (WES) was performed on one twin sample with Caroli disease from a Chinese family from Shandong province. Routine Sanger sequencing was used to validate the WES and to carry out segregation studies. We also described the PKHD1 mutation associated with the genotype-phenotype of this twin.

Results

A combination of WES and Sanger sequencing revealed the genetic defect to be a novel compound heterozygous genotype in PKHD1, including the missense mutation c.2507 T>C, predicted to cause a valine to alanine substitution at codon 836 (c.2507T>C, p.Val836Ala), and the nonsense mutation c.2341C>T, which is predicted to result in an arginine to stop codon at codon 781 (c.2341C>T, p.Arg781*). This compound heterozygous genotype co-segregates with the Caroli disease-affected pedigree members, but is absent in 200 normal chromosomes.

Conclusions

Our findings indicate exome sequencing can be useful in the diagnosis of Caroli disease patients and associate a compound heterozygous genotype in PKHD1 with Caroli disease, which further increases our understanding of the mutation spectrum of PKHD1 in association with Caroli disease.     

XPF-673C>T Polymorphism Effect on the Susceptibility to Esophageal Cancer in Chinese Population

Purpose

Xeroderma pigmentsum group F (XPF) plays a pivotal role in DNA nucleotide excision repair and has been linked to the development of various cancers. This study aims to assess the association of XPF genetic variants with the susceptibility to esophageal squamous cell carcinoma (ESCC) in Chinese population.

Methods

This two-stage case-control study was conducted in a total of 1524 patients with ESCC and 1524 controls. Genotype of XPF -673C>T and 11985A>G variants were determined by polymerase chain reaction-based restriction fragment length polymorphism (PCR-RFLP). Logistic regression analysis was performed to estimate odd ratios (ORs) and 95% confidence intervals (95% CI).

Results

Our case-control study showed that XPF -673TT genotype was associated with a decreased risk of ESCC compared with CC genotype in both case-control sets (Tangshan set: OR = 0.58; 95%CI = 0.34–0.99, P = 0.040; Beijing set: OR = 0.66; 95%CI = 0.46–0.95, P = 0.027). Stratified analyses revealed that a multiplicative interaction between -673C>T variant and age, sex or smoking status was evident (Gene-age: P_interaction = 0.002; Gene-sex: P_interaction = 0.002; Gene-smoking: P_interaction = 0.002). For XPF 11985A>G polymorphism, there was no significant difference of genotype distribution between ESCC cases and controls.

Conclusion

These findings indicated that genetic variants in XPF might contribute to the susceptibility to ESCC.     

Lymphotoxin Alpha (LTA) Polymorphism Is Associated with Prognosis of Non-Hodgkin’s Lymphoma in a Chinese Population

Background

Non-Hodgkin’s lymphoma (NHL) has been widely reported to be associated with autoimmune and pro-inflammatory response, and genetic polymorphisms of candidate genes involved in autoimmune and pro-inflammatory response may influence the survival and prognosis of NHL patients. To evaluate the role of such genetic variations in prognosis of NHL, we conducted this study in a Chinese population.

Methods

We used the TaqMan assay to genotype six single nucleotide polymorphisms (SNPs) (TNF rs1799964T>C, LTA rs1800683G>A, IL-10 rs1800872T>G, LEP rs2167270G>A, LEPR rs1327118C>G, TNFAIP8 rs1045241C>T) for 215 NHL cases. Kaplan-Meier analysis was performed to compare progression free survival among two common genotypes. Cox proportional hazard models were used to identify independent risk factors.

Results

We observed that LTA rs1800683G>A was significantly associated with risk of progression or relapse in NHL patients (HR = 1.63, 95%CI = 1.06–2.51; P = 0.028), particularly in Diffuse large B cell lymphoma (DLBCL) cases (HR = 1.50, 95%CI = 1.10–2.04, P = 0.01). Both univariate and multivariate Cox regression analysis showed that in DLBCL patients, Ann Arbor stage III/IV, elevated LDH level before treatment and LTA rs1800683 AA genotype carrier were independent risk factors for progression or relapse. While in NK/T cell lymphoma, Ann Arbor stage III/IV and elevated β₂-MG level before treatment indicated poorer prognosis.

Conclusions

The polymorphism of LTA rs1800683G>A contributes to NHL prognosis in a Chinese population. Further large-scale and well-designed studies are needed to confirm these results.     

Mutation analysis of BRAF, MEK1 and MEK2 in 15 ovarian cancer cell lines: implications for therapy

Background

Among gynecologic cancers, ovarian cancer is the second most common and has the highest death rate. Cancer is a genetic disorder and arises due to the accumulation of somatic mutations in critical genes. An understanding of the genetic basis of ovarian cancer has implications both for early detection and for therapeutic intervention in this population of patients.

Methodology/Principal Findings

Fifteen ovarian cancer cell lines, commonly used for in vitro experiments, were screened for mutations using bidirectional direct sequencing in all coding regions of BRAF, MEK1 and MEK2. BRAF mutations were identified in four of the fifteen ovarian cancer cell lines studied. Together, these four cell lines contained four different BRAF mutations, two of which were novel. ES-2 had the common B-Raf p.V600E mutation in exon 15 and Hey contained an exon 11 missense mutation, p.G464E. The two novel B-Raf mutants identified were a 5 amino acid heterozygous deletion p.N486-P490del in OV90, and an exon 4 missense substitution p.Q201H in OVCAR 10. One of the cell lines, ES-2, contained a mutation in MEK1, specifically, a novel heterozygous missense substitution, p.D67N which resulted from a nt 199 G→A transition. None of the cell lines contained coding region mutations in MEK2. Functional characterization of the MEK1 mutant p.D67N by transient transfection with subsequent Western blot analysis demonstrated increased ERK phosphorylation as compared to controls.

Conclusions/Significance

In this study, we report novel BRAF mutations in exon 4 and exon 12 and also report the first mutation in MEK1 associated with human cancer. Functional data indicate the MEK1 mutation may confer alteration of activation through the MAPK pathway. The significance of these findings is that BRAF and MEK1/2 mutations may be more common than anticipated in ovarian cancer which could have important implications for treatment of patients with this disease and suggests potential new therapeutic avenues.     

Loss of secreted frizzled-related protein 4 correlates with an aggressive phenotype and predicts poor outcome in ovarian cancer patients

Background

Activation of the Wnt signaling pathway is implicated in aberrant cellular proliferation in various cancers. In 40% of endometrioid ovarian cancers, constitutive activation of the pathway is due to oncogenic mutations in β-catenin or other inactivating mutations in key negative regulators. Secreted frizzled-related protein 4 (SFRP4) has been proposed to have inhibitory activity through binding and sequestering Wnt ligands.

Methodology/Principal Findings

We performed RT-qPCR and Western-blotting in primary cultures and ovarian cell lines for SFRP4 and its key downstream regulators activated β-catenin, β-catenin and GSK3β. SFRP4 was then examined by immunohistochemistry in a cohort of 721 patients and due to its proposed secretory function, in plasma, presenting the first ELISA for SFRP4. SFRP4 was most highly expressed in tubal epithelium and decreased with malignant transformation, both on RNA and on protein level, where it was even more profound in the membrane fraction (p<0.0001). SFRP4 was expressed on the protein level in all histotypes of ovarian cancer but was decreased from borderline tumors to cancers and with loss of cellular differentiation. Loss of membrane expression was an independent predictor of poor survival in ovarian cancer patients (p = 0.02 unadjusted; p = 0.089 adjusted), which increased the risk of a patient to die from this disease by the factor 1.8.

Conclusions/Significance

Our results support a role for SFRP4 as a tumor suppressor gene in ovarian cancers via inhibition of the Wnt signaling pathway. This has not only predictive implications but could also facilitate a therapeutic role using epigenetic targets.     

Systematic Study on Genetic and Epimutational Profile of a Cohort of Amsterdam Criteria-Defined Lynch Syndrome in Singapore

Background

Germline defects of mismatch repair (MMR) genes underlie Lynch Syndrome (LS). We aimed to gain comprehensive genetic and epigenetic profiles of LS families in Singapore, which will facilitate efficient molecular diagnosis of LS in Singapore and the region.

Methods

Fifty nine unrelated families were studied. Mutations in exons, splice-site junctions and promoters of five MMR genes were scanned by high resolution melting assay followed by DNA sequencing, large fragment deletions/duplications and promoter methylation in MLH1, MSH2, MSH6 and PMS2 were evaluated by multiplex ligation-dependent probe amplification. Tumor microsatellite instability (MSI) was assessed with five mononucleotide markers and immunohistochemical staining (IHC) was also performed.

Results

Pathogenic defects, all confined to MLH1 and MSH2, were identified in 17 out of 59 (28.8%) families. The mutational spectrum was highly heterogeneous and 28 novel variants were identified. One recurrent mutation in MLH1 (c.793C>T) was also observed. 92.9% sensitivity for indication of germline mutations conferred by IHC surpassed 64.3% sensitivity by MSI. Furthermore, 15.6% patients with MSS tumors harbored pathogenic mutations.

Conclusions

Among major ethnic groups in Singapore, all pathogenic germline defects were confined to MLH1 and MSH2. Caution should be applied when the Amsterdam criteria and consensus microsatellite marker panel recommended in the revised Bethesda guidelines are applied to the local context. We recommend a screening strategy for the local LS by starting with tumor IHC and the hotspot mutation testing at MLH1 c.793C>T followed by comprehensive mutation scanning in MLH1 and MSH2 prior to proceeding to other MMR genes.