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Graf, Norbert/0000-0002-2248-323X; Reis, Andre/0000-0002-6301-6363; Diets, Illja/0000-0001-7603-8898; Popp, Bernt/0000-0002-3679-1081; Thiel, Christian T/0000-0003-3817-7277; Roeleveld, Nel/0000-0002-3390-4466; Reis, AlessanRSS/0000-0001-8486-7469; Vasileiou, Georgia/0000-0002-1993-1134; Gessler, Manfred/0000-0002-7915-6045 WOS:000472571300008 PubMed ID: 30694527 Two percent of patients with Wilms tumors have a positive family history. In many of these cases the genetic cause remains unresolved. By applying germline exome sequencing in two families with two affected individuals with Wilms tumors, we identified truncating mutations in TRIM28. Subsequent mutational screening of germline and tumor DNA of 269 children affected by Wilms tumor was performed, and revealed seven additional individuals with germline truncating mutations, and one individual with a somatic truncating mutation in TRIM28. TRIM28 encodes a complex scaffold protein involved in many different processes, including gene silencing, DNA repair and maintenance of genomic integrity. Expression studies on mRNA and protein level showed reduction of TRIM28, confirming a loss-of-function effect of the mutations identified. The tumors showed an epithelial-type histology that stained negative for TRIM28 by immunohistochemistry. The tumors were bilateral in six patients, and 10/11 tumors are accompanied by perilobar nephrogenic rests. Exome sequencing on eight tumor DNA samples from six individuals showed loss-of-heterozygosity (LOH) of the TRIM28-locus by mitotic recombination in seven tumors, suggesting that TRIM28 functions as a tumor suppressor gene in Wilms tumor development. Additionally, the tumors showed very few mutations in known Wilms tumor driver genes, suggesting that loss of TRIM28 is the main driver of tumorigenesis. In conclusion, we identified heterozygous germline truncating mutations in TRIM28 in 11 children with mainly epithelial-type Wilms tumors, which become homozygous in tumor tissue. These data establish TRIM28 as a novel Wilms tumor predisposition gene, acting as a tumor suppressor gene by LOH. Dutch Cancer SocietyKWF Kankerbestrijding [KUN2012-5366]; KiKa Foundation [127] Grant sponsor: Dutch Cancer Society; Grant number: KUN2012-5366; Grant sponsor: The KiKa Foundation (project 127)

Background Providing the correct diagnosis for patients with tubulointerstitial kidney disease and secondary degenerative disorders, such as hypertension, remains a challenge. The autosomal dominant tubulointerstitial kidney disease (ADTKD) subtype caused by MUC1 mutations (ADTKD- MUC1 ) is particularly difficult to diagnose, because the mutational hotspot is a complex repeat domain, inaccessible with routine sequencing techniques. Here, we further evaluated SNaPshot minisequencing as a technique for diagnosing ADTKD- MUC1 and assessed immunodetection of the disease-associated mucin 1 frameshift protein (MUC1-fs) as a nongenetic technique. Methods We re-evaluated detection of MUC1 mutations by targeted repeat enrichment and SNaPshot minisequencing by haplotype reconstruction via microsatellite analysis in three independent ADTKD- MUC1 families. Additionally, we generated rabbit polyclonal antibodies against MUC1-fs and evaluated immunodetection of wild-type and mutated allele products in human kidney biopsy specimens. Results The detection of MUC1 mutations by SNaPshot minisequencing was robust. Immunostaining with our MUC1-fs antibodies and an MUC1 antibody showed that both proteins are readily detectable in human ADTKD- MUC1 kidneys, with mucin 1 localized to the apical membrane and MUC1-fs abundantly distributed throughout the cytoplasm. Notably, immunohistochemical analysis of MUC1-fs expression in clinical kidney samples facilitated reliable prediction of the disease status of individual patients. Conclusions Diagnosing ADTKD- MUC1 by molecular genetics is possible, but it is technically demanding and labor intensive. However, immunohistochemistry on kidney biopsy specimens is feasible for nongenetic diagnosis of ADTKD- MUC1 and therefore, a valid method to select families for further diagnostics. Our data are compatible with the hypothesis that specific molecular effects of MUC1-fs underlie the pathogenesis of this disease.

Although there are extensive studies on the genetics of bladder cancer, several questions remain unanswered. One of the pathways which are altered in bladder cancer is the mTOR signaling pathway. In the present study, we analyzed the expression of Rheb gene and genetic alterations in the LKB1 gene which are the key components of mTOR pathway. Nine exons of the LKB1 gene were analyzed by direct sequencing in 51 bladder cancer patients. To investigate the expression of Rheb and LKB1, real-time quantitative RT-PCR was performed in bladder tumor and normal bladder tissue samples. We did not observed a statistically significant difference in Rheb or LKB1 expression between the tumor and normal tissue samples. We detected a novel missense mutation creating stop codon in a high percent of the tumor samples. Five different single nucleotide substitutions were also observed in the introns. Our results indicate that LKB1 gene may play a role in the progression of bladder cancer. © 2012 Wiley Periodicals, Inc.

Several studies have suggested that Ras-associated binding 25 protein (Rab25) is involved in the pathogenesis of human cancer. Although it has been demonstrated that the development of head and neck squamous cell carcinoma (HNSCC) is the result of an accumulation of multiple sequential genetic and epigenetic alterations in key genes with important functions in cell growth and the cell cycle, recent studies have indicated that HNSCC is a complex and heterogenous disease. To the best of our knowledge, there is no data regarding the regulation of the Rab25 gene at the mRNA or protein level in HNSCC. Furthermore, available data on Rab25 expression in other types of cancer are conflicting. The aim of the present study was to investigate whether Rab25 is involved in the development and/or progression of HNSCC, and to analyze the mechanisms underlying its effects in this type of cancer. The expression of Rab25 mRNA in HNSCC tissues and adjacent non-tumor tissue samples was measured using reverse transcription-quantitative polymerase chain reaction, while the level of the Rab25, Akt1 and phosphorylated-Akt1 proteins was measured using western blotting. Expression of Rab25 mRNA and protein was downregulated in 69.1% and 56.1% of tumor tissue samples, respectively. This downregulation was associated with an increase in p-Akt1 expression, in the absence of a change in total Akt1 protein levels, in tumor tissues compared with normal tissues. The current findings suggest that Rab25 acts as a tumor suppressor in HNSCC.

Objective PI3K/Akt signalling pathway is frequently activated in several types of cancer. However, activator molecules have not been analysed systematically in HNSCC. The aim of this study was to investigate upstream activators and inhibitors of this pathway. Design Prospective study. Setting University hospital. Participants 108 patients with HNC who were operated at the Istanbul University, Cerrahpasa Medical Faculty, Department of Otorhinolaryngology. Methods Mutations in the coding exons and the flanking intronic sequences of the PIK3CA, PIK3R1 and AKT1 genes were analysed by direct sequencing. Expression levels in the tumours and non-cancerous tissue samples were analysed by quantitative RT-PCR, and Western blotting was performed to determine the phosphorylation levels of the Akt1 protein. Results Two synonymous alterations in exon 20 of the PIK3CA gene, a 6-bp duplication in the coding region of the PIK3R1 and two different alterations in the non-coding regions of the AKT1 and PIK3R1 genes were observed. Significant downregulation of LKB1 and PTEN mRNA expression levels were detected in tumour tissues compared to non-cancerous tissues. However, we did not observe any difference between the PIK3CA and AKT1 mRNA expression levels in the tumours and non-cancerous tissue samples. Akt1 phosphorylation was increased in 53.57% of the patients. Conclusions Our results indicate that the PI3K pathway has an important function in HNSCC progression with the contribution of more than one gene of this pathway. Our data suggest that in a high number of HNSCC tumours, PI3K/Akt signalling is activated by different molecules or by the combination of these molecules.

Head and neck squamous cell carcinoma (HNSCC) is a heterogeneous disease and despite therapeutic improvements and reconstructive modalities, the overall survival rate remains relatively poor. Although, tobacco and alcohol consumption are recognized as critical risk factors, genetic and epigenetics factors and viral infections also contribute to the progression of HNSCC. Several studies have assessed the status of known oncogenes and tumor suppressor genes to identify novel genomic events in HNSCC. Rab25 belongs to the Rab family of small GTPases. Rab25 has been described in conflicting previous studies as a promoter or suppressor of cancer. More recently, Zhang et al. reported that overexpression of Rab25 contributes to metastasis through activation of the Akt/GSK-3β/Snail pathway signaling in bladder cancer, which act as molecular switches that belong to the Ras protein superfamily and regulate vesicle trafficking in eucaryotic cells. The phosphotidylinositol 3-Kinase (PI3K) signaling pathway plays a critical role in cellular growth, proliferation and survival. Signaling through this pathway is commonly upregulated during tumorigenesis. Akt/PKB is a Ser/Thr kinase and the central member of the PI3K/Akt signaling pathway. It is activated by phosphorylation and subsequently phosphorylates diverse growth –controlling effectors and regulates the synthesis, stability and subcellular localization of cell cycle regulators. In this study, we investigated the phosphorylation of Akt1 and whether it occurs under the effect of Rab25 expression in HNSCC. For this purpose we analyzed the Rab25 protein by Western Blot in 57 HNSCC tumors and corresponding non-cancerous tissue samples. To detect the effect of the Rab25 protein on Akt1 activation we also analyzed the amount of total Akt1 and phosphorylated Akt1 in the same tissue samples. Our results indicate that Rab25 is down-regulated in 56.2% of the HNSCC tumor samples compared to their normal counterparts. On the other hand, the change in Akt1 phosphorylation was in the same direction with Rab25 protein expression (p: 0.04) Citation Format: Nur Buyru, Didem Seven, Soydan Dogan, Hikmet Koseoglu, Emin Karaman. Effect of Rab25 on Akt1 phosphorylation. [abstract]. In: Proceedings of the AACR Special Conference: Targeting the PI3K-mTOR Network in Cancer; Sep 14-17, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(7 Suppl):Abstract nr A25.

Bladder cancer is the most common cancer of the urinary tract and is the ninth the most common cancer among men. Its incidence directly increases with age, it rarely occurs before the age of 50 and it is three to four times more common in men compared to women. Although there is now exclusive information on bladder cancer genetics, epigenetics and gene expression, several questions remain unanswered. Global profiling via expression and genomic microarray technologies may provide answers to some of the outstanding questions. Many studies have assessed the status of known oncogenes and tumor suppressor genes and have investigated common chromosomal alterations to identify novel genomic events in bladder cancer. The phosphatidylinositol 3-kinase (PI3K) signaling pathway plays critical role in cell growth, proliferation, and apoptosis. As a result of genomic alterations and loss of some tumor suppressor genes signaling through this pathway is commonly altered in many types of cancer including bladder. In the present study, our interest was focused on the expression analysis of some critical genes in this pathway. For this purpose, expression of seven genes (mTOR, KRAS, PIK3CA, TSC1, STK11, Rheb, PTEN and AKT1) acting in this pathway was analyzed in 35 matched bladder tumor and non-cancerous tissue samples by quantitative RT-PCR. Expression of all genes except PIK3CA and PTEN were similar for the normal bladder and tumor tissue. We observed downregulated expression of PTEN and PIK3CA genes in bladder tumors. The median PTEN mRNA levels in non-cancerous and tumor tissue samples were 2.11 and 0.35, respectively and the difference between the two groups was statistically significant (p=0.00). PIK3CA was also downregulated in the tumor tissues. Tumor tissue samples displayed 6.62 fold decrease in PIK3CA expression compared to normal tissue samples (p=0.002). On the other hand, slightly decreased Rheb mRNA expression was also observed in tumor tissue compared to the non-cancerous tissue. However, the difference expression levels of Rheb between two groups was not statistically significant. Our result indicate that, PI3K pathway has an important function in bladder cancer progression with the contribution of more than one gene in this pathway. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2631. doi:1538-7445.AM2012-2631