Identification of CTNNB1 mutations, CTNNB1 amplifications, and an Axin2 splice variant in juvenile angiofibromas.

Juvenile angiofibromas (JAs) are benign fibro-vascular tumors occurring nearly exclusively in adolescent males. Even less is known about this rare tumor entity, alterations affecting the Wnt-pathway seem to play a pivotal role in tumor biology as activating CTNNB1 mutations have been detected. However, the knowledge of Wnt-pathway changes is still limited. Therefore, we aimed to determine in JAs further insight into Wnt/ß-catenin pathway components. In our present study, genetic alterations of the Wnt-pathway members CTNNB1, APC, GSK3ß, and Axin2 detected by metaphase comparative genomic hybridization (CGH) were shown to result in elevated transcript levels in the majority of JA samples compared to nasal mucosa stroma (p < 0.001, p = 0.001, p = 0.046, and p = 0.006, respectively). Additionally, amplifications of CTNNB1 were validated by fluorescence in situ hybridization (FISH) and genomic qPCR. Moreover, our mutation analysis detected already known mutations as well as, to the best of our knowledge, mutations and an interstitial deletion of CTNNB1 not described in JAs before. Additionally, a so far unknown transcribed Axin2 splice variant was found, but no further Axin2 mutations. Taken together, our current study supports the importance of aberrant Wnt-signaling as a common event in JAs, most likely by the observed genetic alterations driven by mutations, interstitial deletions but also amplifications of CTNNB1 contributing to the stabilization of ß-catenin.

Mesenchymal-endothelial transition in juvenile angiofibroma?

CONCLUSION: Mesenchymal-endothelial transition is proposed for juvenile angiofibromas (JAs). Propranolol might be an interesting new medical option in JA treatment, as it reduces mesenchymal cell growth and decreases the number of CD31-positive cells in vitro. OBJECTIVE: Juvenile angiofibromas (JAs) are rare fibro-vascular tumors affecting almost exclusively adolescent males. Based on morphological aspects of irregularly configured vascular spaces and clinical features, JAs have been proposed to represent a vascular malformation. In general, mesenchymal-endothelial transition has been noted as an important process in tumorigenesis as well as in embryonal development. METHODS: The study analyzed effects of vascular endothelial growth factor (VEGF)/basic fibroblast growth factor (bFGF) and propranolol on endothelial differentiation (CD31+) of cultured JA cells and their expression of angiogenic growth factors using aortic ring assay and enzyme-linked immunosorbent assay. RESULTS: Following VEGF/bFGF supplement to cultured mesenchymal cells, an average of 4.47% (± 2.35%) CD31-positive cells were found (p < 0.001). Propranolol addition reduced the number of CD31-positive cells and inhibited mesenchymal cell growth. The aortic ring assay and ELISA investigation indicated no increased angiogenic growth factor secretion from cultured JA mesenchymal cells.

Genomic alterations in Warthin tumors of the parotid gland.

Despite the fact that Warthin tumors are the second most common type of benign salivary gland tumors, information regarding genetic alterations is extremely limited, and the tumorigenesis of these tumors has not been elucidated. The present results of the largest series of 30 tumors analyzed by comparative genomic hybridization (CGH) to date confirmed previous genetic findings and identified significant new candidate regions. The most commonly observed alterations were deletions of the short arm of chromosome 8, followed by deletions on 9p. Further representative changes were deletions on 16p and 22q with the minimal overlapping region at 16p12p13.1 and 22q12.1q12.3. Moreover, we indicated two different patterns of chromosomal aberrations. One group harbors deletions on 8p partly apparent with deletions on 9q, 11q 15q, 16p and 22. The second group shows gains on 22, partly apparent with gains on 1p and 20q and deletions on 9p. This leads to the assumption that Warthin tumors, in particular those with a high number of alterations, can be divided into two different genetic groups based on the pattern of numerical chromosomal aberrations. Further studies should address whether these subgroups also reflect a different clinical presentation.

New genetic findings in parotid gland pleomorphic adenomas.

BACKGROUND: Despite numerous studies, the tumor biology of pleomorphic adenomas, the most common salivary gland tumors, is still not completely defined. In order to identify further candidate genes important for tumor biology of pleomorphic adenomas, extended cytogenetic and molecular analysis are mandatory. METHODS: We performed a detailed molecular cytogenetic analysis using comparative genomic hybridization (CGH) followed by fluorescence in situ hybridization (FISH) with probes for chromosome X, 16p, 17, and 20 on a large cohort of pleomorphic adenomas (n = 29). RESULTS: We could confirm previously described deletions in pleomorphic adenomas affecting 16p, 17, 20q, and 22 by FISH and/or CGH analysis. Moreover, our CGH study revealed novel candidate regions on 8p23.1pter, 9p, 10q25.1q25.3, and 11q24qter in the series of analyzed pleomorphic adenomas. CONCLUSION: Our present study reveals new insights in novel candidate regions implicated in pleomorphic adenoma tumorigenesis which should be considered in further molecular studies.

Vitalization of porous polyethylene (Medpor®) with chondrocytes promotes early implant vascularization and incorporation into the host tissue.

Porous polyethylene (Medpor(®)) is frequently used in craniofacial reconstructive surgery. The successful incorporation of this alloplastic biomaterial depends on adequate vascularization. Here, we analyzed whether the early vascularization of porous polyethylene can be accelerated by vitalization with human chondrocytes. For this purpose, small polyethylene samples were coated with platelet-rich plasma (PRP) or a suspension of PRP and human chondrocytes. Uncoated polyethylene samples served as controls. Subsequently, the samples were implanted into the dorsal skinfold chamber of CD-1 nude mice to repetitively analyze their vascularization and biocompatibility by means of intravital fluorescence microscopy. PRP-chondrocyte-coated polyethylene exhibited an accelerated and improved vascularization when compared with the other two groups. This was indicated by a significantly higher functional capillary density of the microvascular network developing around the implants. Moreover, a leukocyte-endothelial cell interaction was found in a physiological range at the implantation site of all three groups, demonstrating that the vitalization with PRP and chondrocytes did not affect the good biocompatibility of the alloplastic material. Additional histological, immunohistochemical, and in situ hybridization analyses revealed that the chondrocytes formed a bioprotective tissue layer, which prevented the accumulation of macrophages and foreign body giant cells on the polyethylene surface. These findings clearly indicate that vitalization of polyethylene with chondrocytes promotes early implant vascularization and incorporation into the host tissue and, thus, may be a promising approach that prevents postoperative complications such as implant extrusion, migration, and infection.

Genome-wide copy number profiling using a 100K SNP array reveals novel disease-related genes BORIS and TSHZ1 in juvenile angiofibroma.

Juvenile angiofibroma (JA) is a unique fibrovascular tumor, which is almost exclusively found in the posterior nasal cavity of adolescent males. Although histologically classified as benign, the tumor often shows an aggressive growth pattern and has been associated with chromosomal imbalances, amplification of oncogenes and epigenetic dysregulation. We present the first genome-wide profiling of JAs (n=14) with a 100K single nucleotide polymorphism (SNP) microarray. Among the 30 novel JA-specific amplifications detected on autosomal chromosomes with this technique, the genes encoding the cancer-testis antigen BORIS (brother of the regulator of imprinted sites) and the developmental regulator protein TSHZ1 (teashirt zinc finger homeobox 1) were selected for further analysis. Gains for both BORIS (20q13.3) and TSHZ1 (18q22.3) were confirmed by quantitative genomic PCR. Furthermore, quantitative RT-PCR revealed a significant up-regulation of BORIS (p<0.001) and TSHZ1 transcripts (p<0.05) for JAs compared to nasal mucosa. Following detection of BORIS and TSHZ1 proteins in western blots of JAs, subcellular localization was determined for both proteins in immunostaining of JA cryosections. In conclusion, genomic copy number profiling using an SNP microarray has been proven to be a suitable and reliable tool for identifying novel disease-related genes in JAs and newly implicates BORIS and TSHZ1 overexpression in the pathogenesis of JAs. Detection of BORIS in JAs is described with special regard to tumor proliferation and epigenetic dysregulation, and the finding of TSHZ1 amplifications is discussed with special respect to the hypothesis of JAs as malformations of the first branchial arch artery.

The NP9 protein encoded by the human endogenous retrovirus HERV-K(HML-2) negatively regulates gene activation of the Epstein-Barr virus nuclear antigen 2 (EBNA2).

Epstein-Barr virus (EBV) is a human tumour virus that efficiently growth-transforms primary human B-lymphocytes in vitro. The viral nuclear antigen 2 (EBNA2) is essential for immortalisation of B-cells and stimulates viral and cellular gene expression through interaction with DNA-bound transcription factors. Like its cellular homologue Notch, it associates with the DNA-bound repressor RBPJκ (CSL/CBF1) thereby converting RBPJκ into the active state. For instance, both EBNA2 and Notch activate the cellular HES1 promoter. In EBV-transformed lymphocytes, the RNA of the NP9 protein encoded by human endogenous retrovirus HERV-K(HML-2) Type 1 is strongly up-regulated. The NP9 protein is detectable both in EBV-positive Raji cells, a Burkitt's lymphoma cell line, and in IB4, an EBV-transformed human lymphoblastoid cell line. NP9 binds to LNX that forms a complex with the Notch regulator Numb. Therefore, the function of NP9 vis-à-vis Notch and EBNA2 was analysed. Here, we show that NP9 binds to EBNA2 and negatively affects the EBNA2-mediated activation of the viral C- and LMP2A promoters. In contrast, NP9 did neither interfere in the activation of the HES1 promoter by Notch nor the induction of the viral LMP1 promoter by EBNA2. In an electrophoretic mobility shift analysis, NP9 reduced the binding of EBNA2 to DNA-bound RBPJκ by about 50%. The down-regulation of EBNA2-activity by NP9 might represent a cellular defence mechanism against viral infection or could, alternatively, represent an adaptation of the virus to prevent excessive viral protein production that might otherwise be harmful for the infected cell.

Loss-of-function mutations in sodium channel Nav1.7 cause anosmia.

Loss of function of the gene SCN9A, encoding the voltage-gated sodium channel Na(v)1.7, causes a congenital inability to experience pain in humans. Here we show that Na(v)1.7 is not only necessary for pain sensation but is also an essential requirement for odour perception in both mice and humans. We examined human patients with loss-of-function mutations in SCN9A and show that they are unable to sense odours. To establish the essential role of Na(v)1.7 in odour perception, we generated conditional null mice in which Na(v)1.7 was removed from all olfactory sensory neurons. In the absence of Na(v)1.7, these neurons still produce odour-evoked action potentials but fail to initiate synaptic signalling from their axon terminals at the first synapse in the olfactory system. The mutant mice no longer display vital, odour-guided behaviours such as innate odour recognition and avoidance, short-term odour learning, and maternal pup retrieval. Our study creates a mouse model of congenital general anosmia and provides new strategies to explore the genetic basis of the human sense of smell.

NIR, an inhibitor of histone acetyltransferases, regulates transcription factor TAp63 and is controlled by the cell cycle.

p63 Is a sequence-specific transcription factor that regulates epithelial stem cell maintenance and epithelial differentiation. In addition, the TAp63 isoform with an N-terminal transactivation domain functions as an inducer of apoptosis during the development of sympathetic neurons. Previous work has indicated that the co-activator and histone acetyltransferase (HAT), p300, can bind to TAp63 and stimulate TAp63-dependent transcription of the p21Cip1 gene. Novel INHAT Repressor (NIR) is an inhibitor of HAT. Here, we report that the central portion of NIR binds to the transactivation domain and the C-terminal oligomerization domain of TAp63. NIR is highly expressed in G2/M phase of the cell cycle and only weakly expressed in G1/S. Furthermore, except during mitosis, NIR is predominantly localized in the nucleolus; only a small portion co-localizes with TAp63 in the nucleoplasm and at the p21 gene promoter. Consistent with NIR acting as a repressor, the induced translocation of NIR from the nucleolus into the nucleoplasm resulted in the inhibition of TAp63-dependent transactivation of p21. Conversely, knockdown of NIR by RNAi stimulated p21 transcription in the presence of TAp63. Thus, NIR is a cell-cycle-controlled, novel negative regulator of TAp63. The low levels of nucleoplasmic NIR might act as a buffer toward potentially toxic TAp63.