Identification of Reference Genes in Chordoma Cells Allows Cross-Comparison of Expression Studies Across Subtypes.

AIM: To present the best housekeeping genes including clival/sacral based chordoma, and the nucleus pulposus cells. MATERIAL AND METHODS: We investigated 13 candidate reference genes in public chordoma array transcriptome datasets, validated these genes by using RT-PCR, and evaluated their stability with NormFinder, geNorm, and Bestkeeper. RESULTS: YWHAZ, TBP and PGK1 genes were identified as the most stable reference genes as confirmed with three different approaches. Conversely, KRT8, KRT19 and GAPDH genes are less stable and not appropriate for use in chordoma research. CONCLUSION: For normalization of RT-PCR experiments in gene profiling of chordoma, we recommend the use of the stable genes YWHAZ, TBP and PGK1.

NANOG Dominates Interleukin-6-Induced Sphere Formation in Prostate Cancer.

OBJECTIVE: Identifying the dynamics of prostate tumor aggressiveness is essential to find new therapeutics for the treatment. Cancer stem cells contribute to cancer progression by promoting tumor growth and metastasis, resisting treatment, and evading the immune system. Interleukin 6 (IL-6) is a pleiotropic cytokine that functions in inflammation, immune response, etc. However, dysregulated expression of IL-6 plays a pathological role in such conditions as cancer. In this study, we aimed to elucidate the effect of IL-6 on cancer stemness genes in prostate cancer cells. METHODS: Enrichment of stem-like cells was achieved through the formation of tumor spheres using the DU-145 cell line. Sphere formation was conducted in a medium supplemented with IL-6 and compared to a control group. The number of spheres was quantified, and the resulting pellet was collected for quantitative reverse transcription polymerase chain reaction analysis to assess the impact of IL-6 induction on the expression of stemness-related genes. RESULTS: Tumor sphere numbers and sizes increased in IL-6-induced environment. NANOG expression elevated in an IL-6-enriched environment compared to the nontreated spheres. Our results demonstrated that IL-6 induction in prostate tumor spheres upregulates NANOG gene expression. CONCLUSION: Inducing IL-6 in prostate tumor spheres stimulates stemness biomarker NANOG genes. NANOG may be suggested as a therapeutic target for metastatic prostate cancer.

TRIM28 haploinsufficiency predisposes to Wilms tumor.

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.

Biallelic Expression of Mucin-1 in Autosomal Dominant Tubulointerstitial Kidney Disease: Implications for Nongenetic Disease Recognition.

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.

Investigation of the SLC22A23 gene in laryngeal squamous cell carcinoma.

BACKGROUND: Laryngeal squamous cell carcinoma (LSCC) is the second most common cancer of the head and neck. In order to identify differentially expressed genes which may have a role in LSCC carcinogenesis, we performed GeneFishing Assay. One of the differentially expressed genes was the SLC22A23 (solute carrier family 22, member 23) gene. SLC22A23 belongs to a family of organic ion transporters that are responsible for the absorption or excretion of many drugs, xenobiotics and endogenous compounds in a variety of tissues. SLC22A23 is expressed in a various tissues but no substrates or functions have been identified for it. Although the exact function is unknown, single nucleotide polymorphisms (SNPs) which are located in SLC22A23 gene were associated with inflammatory bowel disease (IBD), endometriosis-related infertility and the clearance of antipsychotic drugs. On the other hand SLC22A23 is identified as a prognostic gene to predict the recurrence of triple-negative breast cancer. METHODS: To understand the role of the SLC22A23 gene in laryngeal carcinogenesis, we investigated its mRNA expression level in laryngeal tumor tissue and adjacent non-cancerous tissue samples obtained from 83 patients by quantitative real-time PCR. To understand the association between SNPs in SLC22A23 and LSCC, selected genetic variations (rs4959235, rs6923667, rs9503518) were genotyped. RESULTS: We found that SLC22A23 expression was increased in 46 of 83 tumor tissues (55.4%) and was decreased in 30 of 83 (36.1%) tumor tissues compared to normal tissues. 77.2% of patients were homozygote for genotype rs9503518-AA and they most frequently had histological grade 2 and 3 tumors. We also found that rs9503518-AA genotype is associated with increased SLC22A23 expression. CONCLUSIONS: Our results indicate that SLC22A23 may play a role in the development of laryngeal cancer.

Downregulation of Rab25 activates Akt1 in head and neck squamous cell carcinoma.

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.

DLEC1 is not silenced solely by promoter methylation in head and neck squamous cell carcinoma.

Different types of genetic and epigenetic changes are associated with HNSCC. The molecular mechanisms of HNSCC carcinogenesis are still undergoing intensive investigation. The Deleted in lung and esophageal cancer 1 (DLEC1) gene is frequently silenced by methylation in various kinds of cancer. However, there is no data in the literature investigating the DLEC1 gene in the HNSCC. Tumor tissues from 97 patients were analyzed by real-time quantitative RT-PCR and DLEC1 expression levels were correlated with the methylation of the DLEC1 gene promoter. A statistically significant down-regulation was observed in tumors compared to non-cancerous tissue samples (p = 0.00). However, this down-regulation was not directly associated with hypermethylation of the promoter (p ≥ 0.05). Our results indicate that the DLEC1 gene may play an important role in the development of HNSCC. However, its down-regulation is not associated with the clinicopathological parameters and is not solely under the control of promoter methylation.

LKB1 mutations and their correlation with LKB1 and Rheb expression in bladder cancer.

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.