Barbara Guertl

Adipose Triglyceride Lipase Contributes to Cancer-Associated Cachexia

Ablation of a gene controlling fat breakdown can protect mice from cancer-associated uncontrolled loss of fat and muscle. 10.1126/science.1198973 Cachexia is a multifactorial wasting syndrome most common in patients with cancer that is characterized by the uncontrolled loss of adipose and muscle mass. We show that the inhibition of lipolysis through genetic ablation of adipose triglyceride lipase (Atgl) or hormone-sensitive lipase (Hsl) ameliorates certain features of cancer-associated cachexia (CAC). In wild-type C57BL/6 mice, the injection of Lewis lung carcinoma or B16 melanoma cells causes tumor growth, loss of white adipose tissue (WAT), and a marked reduction of gastrocnemius muscle. In contrast, Atgl-deficient mice with tumors resisted increased WAT lipolysis, myocyte apoptosis, and proteasomal muscle degradation and maintained normal adipose and gastrocnemius muscle mass. Hsl-deficient mice with tumors were also protected although to a lesser degree. Thus, functional lipolysis is essential in the pathogenesis of CAC. Pharmacological inhibition of metabolic lipases may help prevent cachexia.

miR-192, miR-194, miR-215, miR-200c and miR-141 are downregulated and their common target ACVR2B is strongly expressed in renal childhood neoplasms

Micro RNAs (miRNAs) play an important role during renal development and show a tissue-specific enrichment in the kidney. Nephroblastomas, embryonal renal neoplasms of childhood, are considered to develop from nephrogenic rests (NRs) and resemble morphologically and genetically developing kidney. We therefore investigated the role of kidney-enriched miRNAs in the pathogenesis of nephroblastomas. miR-192, miR-215 and miR-194 had a significantly lower expression in nephroblastomas regardless of the subtype compared with mature kidney measured by quantitative real-time-PCR. miR-141 and miR-200c showed a significantly lower expression in blastema-type and mixed-type tumors. In comparison with NRs, a significantly lower expression of miR-192, miR-194 and miR-215 was identified in blastema-type, mixed-type and stroma-type nephroblastomas and of miR-141 and miR-200c in blastema-type tumors. Kidney parenchyma had a significantly higher expression of miR-192, miR-194, miR-215 and miR-200c compared with NRs. In this study, the activin receptor type 2B (ACVR2B), a member of the transforming growth factor (TGF)-β pathway, was identified as single common target gene for miR-192, miR-215, miR-194, miR-141 and miR-200c in silico for the first time. The interaction between all five miRNAs and ACVR2B was also verified by an in vitro assay. Additionally, a distinct protein expression of ACVR2B was detected in 53 of 55 nephroblastomas paralleled by an upregulation of ACVR2B messenger RNA demonstrated in 25 nephroblastomas of all subtypes. A differential regulation of ACVR2B by miRNAs in NRs and nephroblastomas appears to be an important step in the pathogenesis of nephroblastomas implicating for the first time the TGF-β pathway in this process.

Metabolic cardiomyopathies: Metabolic cardiomyopathies

The energy needed by cardiac muscle to maintain proper function is supplied by adenosine Ariphosphate primarily (ATP) production through breakdown of fatty acids. Metabolic cardiomyopathies can be caused by disturbances in metabolism, for example diabetes mellitus, hypertrophy and heart failure or alcoholic cardiomyopathy. Deficiency in enzymes of the mitochondrial β‐oxidation show a varying degree of cardiac manifestation. Aberrations of mitochondrial DNA lead to a wide variety of cardiac disorders, without any obvious correlation between genotype and phenotype. A completely different pathogenetic model comprises cardiac manifestation of systemic metabolic diseases caused by deficiencies of various enzymes in a variety of metabolic pathways. Examples of these disorders are glycogen storage diseases (e.g. glycogenosis type II and III), lysosomal storage diseases (e.g. Niemann‐Pick disease, Gaucher disease, I‐cell disease, various types of mucopolysaccharidoses, GM1 gangliosidosis, galactosialidosis, carbohydrate–deficient glycoprotein syndromes and Sandhoffs disease). There are some systemic diseases which can also affect the heart, for example triosephosphate isomerase deficiency, hereditary haemochromatosis, CD 36 defect or propionic acidaemia.

The pluripotent renal stem cell regulator SIX2 is activated in renal neoplasms and influences cellular proliferation and migration

Embryonal renal mesenchyme contains pluripotent progenitor cells characterized by expression of SIX2, which suppresses cellular differentiation. Additionally hypomethylation of the promotor region in renal neoplasms indicates a role of SIX2 in tumorigenesis. This study focuses therefore on the investigation of SIX2 in different renal neoplasms and the mode and consequences of SIX2 activation. Expression of SIX2 was determined in renal cell carcinomas, nephroblastomas, and dysplastic kidneys using immunohistochemistry and quantitative real-time polymerase chain reaction. Its potential mode of activation was assessed by measuring upstream activators by quantitative real-time polymerase chain reaction and the level of methylation of the promoter region by quantitative DNA methylation analysis. Consequences of SIX2 activation were investigated by overexpressing SIX2 in a cell line. Forty-seven of 49 renal clear cell carcinomas showed nuclear staining of SIX2, whereas all papillary carcinomas were negative. In nephroblastomas of various subtypes blastema showed a significant up-regulation (P < .01) and a strong nuclear protein expression of SIX2 in contrast to negative epithelial and mesenchymal areas. 11 cases of dysplastic kidneys were entirely negative. Upstream activators of SIX2 indicated an activation of the signal transduction pathway in most samples. No difference of promoter methylation status was observed between blastema and epithelial structures. A significantly higher percentage of cells in the S-phase and an increased migration were detected in the cell-line overexpressing SIX2. Our study suggests that activation of SIX2 might contribute to the pathogenesis of renal clear cell carcinomas and nephroblastomas. SIX2 also appears to be a valuable marker for minimal residual blastema contributing to the prognosis of nephroblastomas.

Nephroblastomas show low expression of MicroR-204 and high expression of its target, the oncogenic transcription factor MEIS1

By comparing several studies we identified a possible deregulation of the transcription factors PBX2 (pre–B-cell leukemia homeobox 2) and one of its binding partners, MEIS1 (Meis homeobox 1) in nephroblastomas. The regulation of MEIS1 is complex, and its expression is known to be influenced by changes of promoter methylation and binding of microRNA-204 (miR-204). Therefore, in our study, we assessed the expression of MEIS1 and PBX2 and the factors regulating expression of MEIS1 in nephroblastomas. MEIS1 and PBX2 messenger RNA (mRNA) and protein levels were investigated by quantitative real-time-polymerase chain reaction (qRT-PCR) and immunohistochemistry. Promoter methylation of MEIS1 was evaluated using a methylation-specific PCR assay. Expression levels of miR-204 were examined by qRT-PCR. Eighteen of 21 nephroblastomas showed a high level of MEIS1 mRNA, and 22 of 26 samples had a specific nuclear protein expression. MicroRNA-204 had a statistically significantly lower expression in all nephroblastomas investigated compared with renal parenchyma, but no change of MEIS1 promoter methylation status was noted. Eleven of 23 nephroblastomas had a high expression of PBX2 mRNA, and 15 of 23 samples had a specific nuclear protein expression was noted. In our study, we demonstrated an expression of MEIS1 and its binding partner PBX2 in most nephroblastomas. The statistically significantly lower expression of miR-204 in all nephroblastomas investigated might point to an involvement of miR-204 in the regulation of MEIS1 in nephroblastomas.

Lim1, an Embryonal Transcription Factor, Is Absent in Multicystic Renal Dysplasia, but Reactivated in Nephroblastomas

Objective:Lim1 (Lim homeobox 1) plays an important role during rodent renal development; however, its rolein human kidney development and disease is still unclear. Methods: We investigated LIM1 expression during human renal development, in dysplastic kidneys and in renal neoplasms using immunohistochemistry. RNA levels in renal carcinomas were determined by quantitative RT-PCR, and the potential roles of LIM1 in mesenchymal-epithelial transition and cell cycle were investigated in a cell culture model. Results: LIM1 was detected in pretubular aggregates, S-shaped and comma-shaped bodies as well as immature glomeruli between 10 and 30 weeks of gestation. Eleven dysplastic kidneys showed no expression of LIM1. In contrast, 12 of 32 nephroblastomas showed nuclear positivity. One regressive nephroblastoma had diffuse expression of LIM1 in tubular structures, all others showed focal positivity in mesenchymal, blastemal and epithelial structures. Renal cell carcinomas revealed no expression of LIM1. Overexpression of LIM1 in a cell culture model led to an increase in KERATIN7 expression but no change in the cell cycle. Conclusion: Our study supports the concept of a causative role of LIM1 deficiency in the development of multicystic kidney. In a small subset of nephroblastomas with a more diffuse expression pattern LIM1 might also contribute to the pathogenesis of these lesions.

Genetic clonality is a feature unifying nephroblastomas regardless of the variety of morphological subtypes

Nephroblastomas are embryonal tumors exhibiting a wide variety of different morphological features and genetic changes. Some of the genetic aberrations were associated with a certain histological subtype. It is generally assumed that nephroblastomas develop as subclonal proliferations from nephrogenic rests. However, so far, a very limited amount of tumors from only part of the morphological spectrum of nephroblastomas was investigated. We therefore investigated the clonality of 45 tumors of all different histological subtypes. The number of each subtype was in accordance with the percentage of occurrence of the respective subtype. We analyzed a highly polymorphic locus of the human androgen receptor gene for nonrandom X-inactivation of genomic DNA using a methylation-sensitive restriction enzyme. Data were obtained for 39 tumors. Eighteen of the tumors included were noninformative in the genetic locus examined, the remaining 21 tumors were monoclonal regardless of the histological subtype. Our findings therefore support the hypothesis that Wilms’ tumors are monoclonal proliferations despite their large variety of morphological features.

Activation of beta-catenin is a late event in the pathogenesis of nephroblastomas and rarely correlated with genetic changes of the APC gene.

Aims: Activation of &bgr;-catenin has been identified as a possible mechanism for the development of nephroblastomas. In our study we investigated whether this activation occurs already in precursor lesions of nephroblastomas, called nephrogenic rests (NRs). Inactivation of the adenomatous polyposis coli (APC) protein is an important regulatory mechanism of activating &bgr;-catenin. We clarified the role of APC by assessing loss of heterozygosity (LOH) and possible mutations within the genomic region. Methods: Activation of &bgr;-catenin was examined by immunohistochemistry identifying nuclear translocation. Two polymorphic loci of the APC gene were investigated for LOH and sequence analysis was performed for the mutation cluster region of the APC gene on formalin fixed, paraffin embedded samples. Results: Four of the 18 nephroblastomas available for immunohistochemistry exhibited nuclear staining of &bgr;-catenin, but none of the NRs. Analysis of LOH revealed 14 homozygous samples, 10 heterozygous tumours and six tumours exhibiting LOH of the APC gene. One blastema-type nephroblastoma showed nuclear localisation of &bgr;-catenin in conjunction with LOH of the APC gene. Analysis of 12 nephroblastomas revealed no sequence aberration. Conclusion: Our results indicate that nuclear activation of &bgr;-catenin is a late event in the tumorigenesis of nephroblastomas coinciding in some tumours with LOH of the APC gene.

Is Predisposition for Nephroblastoma Linked to Polymorphisms of the WTX Gene

Inactivation of Wilms´ tumor X (WTX) gene has been linked to the pathogenesis of a varying percentage of nephroblastomas. In contrast, germline mutations of WTX were identified to cause bone dysplasia, but not to induce the development of nephroblastomas. In our study we investigated whether tumor promotion of nephroblastoma by inactivation of WTX gene is linked to certain single nucleotide polymorphisms (SNPs). Therefore 8 SNPs—distributed over the whole length of the WTX gene—were investigated by high resolution melting curve analysis (HRMA) and sequencing of genomic DNA from nephroblastoma patients (NB) and controls. No difference was detected in the 8 SNPs investigated, which were distributed over the whole length of the gene. Additionally, sequence analysis of the coding part of the WTX gene of the tumor samples revealed no chromosomal aberration. Our study indicates, that inactivation of WTX appears to be a late event in tumorigenesis of nephroblastoma in a subgroup of nephroblastomas.

Loss of PTEN/MMAC1 activity is a rare and late event in the pathogenesis of nephroblastomas

Recent genetic investigations of nephroblastomas point to an activation of the Wnt pathway. Data indicate however that activation might be partly due to cross talk of different signaling pathways including the tumor suppressor gene PTEN (phosphatase and tensin homolog on chromosome 10). Therefore, we examined expression and chromosomal aberrations of PTEN in nephroblastomas of different subtypes and the corresponding nephrogenic rests. Loss of heterozygosity was analyzed by high-resolution melting analysis of 4 different single nucleotide polymorphisms. Results were confirmed by sequence analysis of the polymerase chain reaction products. In addition, an intragenic insertion-deletion polymorphism of the PTEN gene was investigated. Protein expression was assessed by immunohistochemistry. Twenty-two nephroblastomas and their corresponding nephrogenic rests were included in the study. In the high-resolution melting analysis, 15 samples were homozygous, 6 were heterozygous, and for 1 sample results could not be obtained for technical reasons. None of the samples showed loss of heterozygosity. Nineteen of the tumors and corresponding nephrogenic rests were also examined immunohistochemically. All tumors showed cytoplasmic positivity, with the exception of 1 tumor that showed complete loss of staining. In 1 tumor, the epithelial component showed distinct cytoplasmic staining, whereas the immature muscle and hyaline cartilage were negative. All nephrogenic rests exhibited positive cytoplasmic staining of all components. Our results establish that inactivation of PTEN is a rare and late event in the pathogenesis of nephroblastomas.