Funktionelle Charakterisierung des putativen Tumorsuppressors "Epithelial Membrane Protein 3"

EMP3 has been proposed as a potential tumor suppressor gene in neuroblastomas, gliomas and other solid tumors, due to its differential methylation and expression pattern. In these tumors EMP3 is often transcriptionally silenced by promoter hypermethylation. The biological function of EMP3 itself is largely unknown. Based on homologies to other members of the protein family, it was presumed that EMP3 is involved in the regulation of proliferation, apoptosis and cell-cell-interactions. The aim of this work is the functional characterization of EMP3 and its role in glioma formation and progression. To this end we analyzed the methylation and expression pattern of EMP3 in gliomas, non-neoplastic tissues and cell lines. In addition we studied the effects of RNA-interference-mediated knockdown of EMP3 on proliferation, migration and apoptosis in an in vitro cancer cell line model. Furthermore we utilized different interaction assays to identify novel protein-protein interaction partners of EMP3. EMP3 is expressed in almost all analyzed normal tissues, with the highest levels in leukocytes, neurons and astrocytic cells. In gliomas, the EMP3 protein levels are highest in glioblastomas, of which over 80% show very strong EMP3 expression, while in low-grade gliomas only 20% show elevated levels of EMP3. High levels of EMP3 also correlate with shorter progression-free and overall patient survival. RNA-interference-mediated repression of EMP3 significantly reduces the proliferation and migration of cancer cells in vitro and increased their susceptibility to induced cell death. This is in part caused by decreased phosphorylation and activation of the EGFR, AKT and ERK signaling kinases. EMP3 is part of a complex interaction and signaling network, of which we could identify 10 novel interacting proteins. Through this network EMP3 is possibly involved in the regulation of several important signaling and trafficking pathways. EMP3 is therefore likely to be a mediator and regulator of intracellular trafficking and signal transduction. The data support a role for EMP3 in the progression of cancer, but, at least in glioma, not as a tumor suppressor. Nevertheless EMP3 could be a valid prognostic and possibly even predictive biomarker in the diagnosis of glioma as well as a potential novel therapeutic target in different tumors and other diseases

Prognostic Value of Three Different Methods of MGMT Promoter Methylation Analysis in a Prospective Trial on Newly Diagnosed Glioblastoma

Hypermethylation in the promoter region of the MGMT gene encoding the DNA repair protein O6-methylguanine-DNA methyltransferase is among the most important prognostic factors for patients with glioblastoma and predicts response to treatment with alkylating agents like temozolomide. Hence, the MGMT status is widely determined in most clinical trials and frequently requested in routine diagnostics of glioblastoma. Since various different techniques are available for MGMT promoter methylation analysis, a generally accepted consensus as to the most suitable diagnostic method remains an unmet need. Here, we assessed methylation-specific polymerase chain reaction (MSP) as a qualitative and semi-quantitative method, pyrosequencing (PSQ) as a quantitative method, and methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) as a semi-quantitative method in a series of 35 formalin-fixed, paraffin-embedded glioblastoma tissues derived from patients treated in a prospective clinical phase II trial that tested up-front chemoradiotherapy with dose-intensified temozolomide (UKT-05). Our goal was to determine which of these three diagnostic methods provides the most accurate prediction of progression-free survival (PFS). The MGMT promoter methylation status was assessable by each method in almost all cases (n = 33/35 for MSP; n = 35/35 for PSQ; n = 34/35 for MS-MLPA). We were able to calculate significant cut-points for the continuous methylation signals at each CpG site analysed by PSQ (range, 11.5 to 44.9%) and at one CpG site assessed by MS-MLPA (3.6%) indicating that a dichotomisation of continuous methylation data as a prerequisite for comparative survival analyses is feasible. Our results show that, unlike MS-MLPA, MSP and PSQ provide a significant improvement of predicting PFS compared with established clinical prognostic factors alone (likelihood ratio tests: p<0.001). Conclusively, taking into consideration prognostic value, cost effectiveness and ease of use, we recommend pyrosequencing for analyses of MGMT promoter methylation in high-throughput settings and MSP for clinical routine diagnostics with low sample numbers

Rare heterozygous GDF6 variants in patients with renal anomalies

Although over 50 genes are known to cause renal malformation if mutated, the underlying genetic basis, most easily identified in syndromic cases, remains unsolved in most patients. In search of novel causative genes, whole-exome sequencing in a patient with renal, i.e., crossed fused renal ectopia, and extrarenal, i.e., skeletal, eye, and ear, malformations yielded a rare heterozygous variant in the GDF6 gene encoding growth differentiation factor 6, a member of the BMP family of ligands. Previously, GDF6 variants were reported to cause pleiotropic defects including skeletal, e.g., vertebral, carpal, tarsal fusions, and ocular, e.g., microphthalmia and coloboma, phenotypes. To assess the role of GDF6 in the pathogenesis of renal malformation, we performed targeted sequencing in 193 further patients identifying rare GDF6 variants in two cases with kidney hypodysplasia and extrarenal manifestations. During development, gdf6 was expressed in the pronephric tubule of Xenopus laevis, and Gdf6 expression was observed in the ureteric tree of the murine kidney by RNA in situ hybridization. CRISPR/Cas9-derived knockout of Gdf6 attenuated migration of murine IMCD3 cells, an effect rescued by expression of wild-type but not mutant GDF6, indicating affected variant function regarding a fundamental developmental process. Knockdown of gdf6 in Xenopus laevis resulted in impaired pronephros development. Altogether, we identified rare heterozygous GDF6 variants in 1.6% of all renal anomaly patients and 5.4% of renal anomaly patients additionally manifesting skeletal, ocular, or auricular abnormalities, adding renal hypodysplasia and fusion to the phenotype spectrum of GDF6 variant carriers and suggesting an involvement of GDF6 in nephrogenesis

Rare heterozygous GDF6 variants in patients with renal anomalies.

Although over 50 genes are known to cause renal malformation if mutated, the underlying genetic basis, most easily identified in syndromic cases, remains unsolved in most patients. In search of novel causative genes, whole-exome sequencing in a patient with renal, i.e., crossed fused renal ectopia, and extrarenal, i.e., skeletal, eye, and ear, malformations yielded a rare heterozygous variant in the GDF6 gene encoding growth differentiation factor 6, a member of the BMP family of ligands. Previously, GDF6 variants were reported to cause pleiotropic defects including skeletal, e.g., vertebral, carpal, tarsal fusions, and ocular, e.g., microphthalmia and coloboma, phenotypes. To assess the role of GDF6 in the pathogenesis of renal malformation, we performed targeted sequencing in 193 further patients identifying rare GDF6 variants in two cases with kidney hypodysplasia and extrarenal manifestations. During development, gdf6 was expressed in the pronephric tubule of Xenopus laevis, and Gdf6 expression was observed in the ureteric tree of the murine kidney by RNA in situ hybridization. CRISPR/Cas9-derived knockout of Gdf6 attenuated migration of murine IMCD3 cells, an effect rescued by expression of wild-type but not mutant GDF6, indicating affected variant function regarding a fundamental developmental process. Knockdown of gdf6 in Xenopus laevis resulted in impaired pronephros development. Altogether, we identified rare heterozygous GDF6 variants in 1.6% of all renal anomaly patients and 5.4% of renal anomaly patients additionally manifesting skeletal, ocular, or auricular abnormalities, adding renal hypodysplasia and fusion to the phenotype spectrum of GDF6 variant carriers and suggesting an involvement of GDF6 in nephrogenesis

Estimated cut-points for PSQ and MS-MLPA.

<p>Estimated cut-points for PSQ and MS-MLPA.</p

Schematic overview of the <i>MGMT</i> promoter region including CpG sites interrogated by each diagnostic method.

<p>Grey arrow, <i>MGMT</i> promoter region; filled yellow box, complete CpG island; red boxes, single CpG sites; understriked red labelled sequence, <i>MGMT</i> exon 1 with start codon marked in bold type; purple bars, methylation-specific (M) primers for MSP according to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033449#pone.0033449-Esteller1" target="_blank">[2]</a>; mint bars, unmethylation-specific (U) primers for MSP according to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033449#pone.0033449-Esteller1" target="_blank">[2]</a>; dark blue box, PSQ region comprising five CpG sites; light blue boxes, three GCGC <i>Hha</i>l sites for MS-MLPA.</p

Prediction error curves for each diagnostic method with respect to (A) PFS and (B) OS.

<p>To assess the predictive accuracy of models including methylation data, the cumulating prediction error curves over 18 months follow-up time and a time-dependent R²-like measure were computed for the marginal Kaplan-Meier estimates (Kaplan-Meier), the Cox model using clinical data only (i.e., age, gender, Karnofsky performance status, extent of resection [Clinical]), and the Cox model using combined clinical plus methylation data as determined by MSP (Clinical + MSP), PSQ CpG sites 1 to 5 (Clinical + PSQ), dichotomised PSQ CpG sites 1 to 5 (Clinical + dPSQ), and MS-MLPA sites 1 to 3 (Clinical + MS-MLPA). Prediction error curves for dichotomised methylation data at MS-MLPA sites 1 to 3 (PFS and OS) and pyrosequencing CpG sites 1 to 5 (OS) were not feasible due to insignificant cut-points (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033449#pone-0033449-t002" target="_blank">Table 2</a>).</p

Results of <i>MGMT</i> promoter methylation assessed by MSP (qualitative, +/−, and semi-quantitative, M/U ratio), PSQ and MS-MLPA.

<p>For PSQ and MS-MLPA, the mean methylation signal averaged over all CpGs addressed in each method is indicated. N/A, not applicable.</p

Method-dependent Kaplan-Meier estimates of PFS.

<p>PFS data for binary methylation covariates were obtained from (<b>A</b>) MSP, (<b>B</b>) PSQ CpG sites 1 to 5 and (<b>C</b>) MS-MLPA sites 1 to 3 based on missing data imputation (<i>n</i> = 35) for each diagnostic method applied. Respective cut-points allowing the conversion from continuous to binary methylation data in (B) and (C) are indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033449#pone-0033449-t002" target="_blank">Table 2</a>. Of note, Kaplan-Meier curves for MS-MLPA sites 2 and 3 were computed on grounds of insignificant cut-points (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033449#pone-0033449-t002" target="_blank">Table 2</a>). Respective <i>p</i> values of the maximally selected log-rank test and respective numbers (percentages) of tumours assigned to each group, methylated or unmethylated, are given in each plot. Black curves, unmethylated; red curves, methylated.</p