Characterization of a splice-site mutation in the tumor suppressor gene FLCN associated with renal cancer.

BACKGROUND: Renal cell carcinoma is among the most prevalent malignancies. It is generally sporadic. However, genetic studies of rare familial forms have led to the identification of mutations in causative genes such as VHL and FLCN. Mutations in the FLCN gene are the cause of Birt-Hogg-Dubé syndrome, a rare tumor syndrome which is characterized by the combination of renal cell carcinoma, pneumothorax and skin tumors. METHODS: Using Sanger sequencing we identify a heterozygous splice-site mutation in FLCN in lymphocyte DNA of a patient suffering from renal cell carcinoma. Furthermore, both tumor DNA and DNA from a metastasis are analyzed regarding this mutation. The pathogenic effect of the sequence alteration is confirmed by minigene assays and the biochemical consequences on the protein are examined using TALEN-mediated transgenesis in cultured cells. RESULTS: Here we describe an FLCN mutation in a 55-year-old patient who presented himself with progressive weight loss, bilateral kidney cysts and renal tumors. He and members of his family had a history of recurrent pneumothorax during the last few decades. Histology after tumor nephrectomy showed a mixed kidney cancer consisting of elements of a chromophobe renal cell carcinoma and dedifferentiated small cell carcinoma component. Subsequent FLCN sequencing identified an intronic c.1177-5_-3delCTC alteration that most likely affected the correct splicing of exon 11 of the FLCN gene. We demonstrate skipping of exon 11 to be the consequence of this mutation leading to a shift in the reading frame and the insertion of a premature stop codon. Interestingly, the truncated protein was still expressed both in cell culture and in tumor tissue, though it was strongly destabilized and its subcellular localization differed from wild-type FLCN. Both, altered protein stability and subcellular localization could be partly reversed by blocking proteasomal and lysosomal degradation. CONCLUSIONS: Identification of disease-causing mutations in BHD syndrome requires the analysis of intronic sequences. However, biochemical validation of the consecutive alterations of the resulting protein is especially important in these cases. Functional characterization of the disease-causing mutations in BHD syndrome may guide further research for the development of novel diagnostic and therapeutic strategies.

High glucose uptake unexpectedly is accompanied by high levels of the mitochondrial ß-F1-ATPase subunit in head and neck squamous cell carcinoma.

A hallmark of solid tumors is the consumption of large amounts of glucose and production of lactate, also known as Warburg-like metabolism. This metabolic phenotype is typical for aggressive tumor growth, and can be visualized by 18F-fluorodeoxyglucose (18F-FDG) uptake detected by positron emission tomography (PET). High 18F-FDG uptake inversely correlates with survival and goes along with reduced expression of the catalytic beta-subunit of the H+-ATP synthase (ß-F1-ATPase) in several tumor entities analyzed so far.For this study we characterized a series of 15 head and neck squamous cell carcinoma (HNSCC) by (i) determining 18F-FDG-uptake; (ii) quantitative expression analysis of ß-F1-ATPase (Complex V), NDUF-S1 (Complex I) and COX1 (Complex IV) of the mitochondrial electron transport chain (ETC), as well as Hsp60 (mitochondrial mass) and GAPDH (glycolysis) in tumor cells; (iii) sequencing of the mtDNA of representative tumor samples.Whereas high 18F-FDG-uptake also correlates with poor prognosis in HNSCC, it surprisingly is accompanied by high levels of ß-F1-ATPase, but not by any of the other analyzed proteins.In conclusion, we here describe a completely new phenotype of metabolic adaptation possibly enabling those tumors with highest levels of ß-F1-ATPase to rapidly proliferate even in hypoxic zones, which are typical for HNSCC.

Immunohistochemical analysis of cytochrome C oxidase facilitates differentiation between oncocytoma and chromophobe renal cell carcinoma.

In this study, immunohistochemical staining pattern of cytochrome c oxidase subunit 1 (CCO1) was investigated in the differentiation of renal oncocytoma (RO) from eosinophilic (EoC) and classic chromophobe renal cell carcinoma (ChRCC). A feature found in ChRCC/EoC but not in RO is the predominance of a perinuclear halo when stained for CCO1. In a cohort of 103 mixed cases including 44 RO, 37 classic ChRCC and 22 EoC, the diagnosis based on this immunohistochemical feature alone was consistent with the previous routine diagnosis in 95.7%. We reached 100% specificity and 81.4% sensitivity of this pattern in ChRCC. Specificity for RO was 93.2% and sensitivity correspondingly 95.5%. We propose a novel and easily interpretable immunohistochemical method for the discrimination of benign RO from certain subtypes of malignant ChRCC. Because of strong similarity in morphology of the 2 entities the diagnosis often cannot be made based on standard histopathology alone. The study describes for the first time the formation of a perinuclear halo in CCO1 immunohistochemistry as a highly specific marker for the diagnosis of ChRCC. We think this method can be a strong amendment for routine diagnostics in renal cell carcinoma.

Deficiency in four and one half LIM domain protein 2 (FHL2) aggravates liver fibrosis in mice.

BACKGROUND: Four and one half LIM domain protein 2 (FHL2) has been reported to be a key regulator in many cellular processes being associated with fibrogenesis such as cell migration and contraction. Moreover, hepatic FHL2 is involved in regulation pathways mediating proliferation and cell death machineries. We here investigated the role of FHL2 in the setting of experimental and clinical liver fibrosis. METHODS: FHL2(-/-) and wild type (wt) mice were challenged with CCl(4). Fibrotic response was assessed by quantitative real time PCR (qRT-PCR) of fibrotic marker genes, measurement of hydroxyproline content and histological methods. Murine FHL2(-/-) and hepatic stellate cells (HSC) were isolated and investigated via immunofluorescence. Human fibrotic and normal liver samples were analysed immunohistochemically using antibodies directed against FHL2. RESULTS: FHL2(-/-) mice displayed aggravated liver fibrosis compared to wt mice. However, immunofluorescence revealed no significant morphological changes in cultured FHL2(-/-) and wt myofibroblasts (MFB). In human liver samples, FHL2 was strongly expressed both in the nucleus and cytoplasm in MFB of fibrotic livers. In contrast, FHL2 expression was absent in normal liver tissue. CONCLUSIONS: Deficiency of FHL2 results in aggravation of murine liver fibrosis. In human liver samples, FHL2 is expressed in activated HSCs and portal fibroblasts in human fibrotic livers, pointing to a central role of FHL2 for human hepatic fibrogenesis as well.

NLRP10 enhances Shigella-induced pro-inflammatory responses.

Members of the NLR family evolved as intracellular sensors for bacterial and viral infection. However, our knowledge on the implication of most of the human NLR proteins in innate immune responses still remains fragmentary. Here we characterized the role of human NLRP10 in bacterial infection. Our data revealed that NLRP10 is a cytoplasmic localized protein that positively contributes to innate immune responses induced by the invasive bacterial pathogen Shigella flexneri. SiRNA-mediated knock-down studies showed that NLRP10 contributes to pro-inflammatory cytokine release triggered by Shigella in epithelial cells and primary dermal fibroblasts, by influencing p38 and NF-κB activation. This effect is dependent on the ATPase activity of NLRP10 and its PYD domain. Mechanistically, NLRP10 interacts with NOD1, a NLR that is pivotally involved in sensing of invasive microbes, and both proteins are recruited to the bacterial entry point at the plasma membrane. Moreover, NLRP10 physically interacts with downstream components of the NOD1 signalling pathway, such as RIP2, TAK1 and NEMO. Taken together, our data revealed a novel role of NLRP10 in innate immune responses towards bacterial infection and suggest that NLRP10 functions as a scaffold for the formation of the NOD1-Nodosome.

Hepatocarcinogenesis in non-cirrhotic liver is associated with a reduced number of clonal hepatocellular patches in non-tumorous liver parenchyma.

We investigated circumscribed cell proliferations in healthy livers in comparison to non-cirrhotic livers bearing hepatocellular carcinoma. Using histochemical staining for cytochrome c oxidase, the fourth complex of the respiratory chain, we visualized patch-forming descendents of regeneratively active liver cells. The clonal nature of these patches was verified by laser-capture microdissection and Sanger sequencing of the enzyme's core subunits in patches carrying marker mutations on the mtDNA. We demonstrate a highly significant increase of the patch size and also a highly significant increase in the number of patches carrying marker mutations between hepatocellular carcinoma-free and -bearing livers. Thus, the carcinoma-bearing livers accumulated more genetic damage on mtDNA than the control group. Furthermore, for the first time, we present evidence in hepatocellular carcinoma-bearing non-cirrhotic livers of a significantly reduced pool of regeneratively active liver cells that are genetically and functionally altered. The analogy to ageing-related changes is suggestive of premature ageing of stem cells in non-cirrhotic hepatocellular carcinoma-bearing liver as an early step to hepatocarcinogenesis.

The pattern-recognition molecule Nod1 is localized at the plasma membrane at sites of bacterial interaction.

The pattern-recognition molecule Nod1 is a critical sensor for bacterial derived diaminopimelic acid-containing peptidoglycan fragments which induces innate immune responses in epithelial cells. Here we report the subcellular localization of this protein in human epithelial cells. Nod1 is localized in the cytosol and at the plasma membrane in human cells. This membrane association is dependent on the integrity of the protein, on its signalling capacity and on an intact actin cytoskeleton. Signalling-inactive mutants of Nod1 or disruption of the actin cytoskeleton interferes with this localization pattern and impacts on downstream NF-kappaB activation. Moreover, the invasive bacterium Shigella flexneri was used as a model for physiological activation of Nod1. Imaging revealed that Nod1 is recruited to the site of bacterial entry, where it colocalizes with NEMO. Our data provide evidence that membrane association is linked to Nod1 function and, in view of recent findings on Nod2, that this may be a common feature of NLR family members.

Hodgkin's lymphoma in a patient with Jo-1 syndrome.

Jo-1 syndrome is an autoimmune disease with autoantibodies against the histidyl tRNA synthetase. Characteristic clinical findings include inflammatory myopathy and interstitial lung disease. We present the first case of a patient with Jo-1 syndrome (positive Jo-1 autoantibodies, myositis, interstitial alveolitis) who developed Hodgkin's lymphoma of nodular-sclerosing type. Thus, patients with Jo-1 syndrome and immunosuppressive therapy similar to other patients with autoimmune disease are at risk to develop lymphomas and should therefore be monitored carefully.

The Nfs1 interacting protein Isd11 has an essential role in Fe/S cluster biogenesis in mitochondria.

Formation of iron/sulfur (Fe/S) clusters, protein translocation and protein folding are essential processes in the mitochondria of Saccharomyces cerevisiae. In a systematic approach to characterize essential proteins involved in these processes, we identified a novel essential protein of the mitochondrial matrix, which is highly conserved from yeast to human and which we termed Isd11. Depletion of Isd11 caused a strong reduction in the levels of the Fe/S proteins aconitase and the Rieske protein, and a massive decrease in the enzymatic activities of aconitase and succinate dehydrogenase. Incorporation of iron into the Fe/S protein Leu1 and formation of the Fe/S cluster containing holoform of the mitochondrial ferredoxin Yah1 were inhibited in the absence of Isd11. This strongly suggests that Isd11 is required for the assembly of Fe/S proteins. We show that Isd11 forms a stable complex with Nfs1, the cysteine desulfurase of the mitochondrial machinery for Fe/S cluster assembly. In the absence of Isd11, Nfs1 is prone to aggregation. We propose that Isd11 acts together with Nfs1 in an early step in the biogenesis of Fe/S proteins.