Alternative splicing-regulated protein of hepatitis B virus hacks the TNF-α-stimulated signaling pathways and limits the extent of liver inflammation.

Hepatitis B splicing-regulated protein (HBSP) of the hepatitis B virus (HBV) was uncovered a few years ago but its function remains unknown. HBSP expression occurs from a spliced viral transcript that increases during the course of liver disease. This study aimed at characterizing the impact of HBSP on cellular signaling pathways in vitro and on liver pathogenesis in transgenic (Tg) mice. By RT-qPCR array, NF-κB-inducible genes appeared modulated in HepG2 cells transduced with a HBSP-encoding lentivirus. Using luciferase and Western blot assays, we observed a decreased activation of the NF-κB pathway in HBSP-expressing cells following TNF-α treatment, as illustrated by lower levels of phosphorylated IκB-α. Meanwhile, the level of phosphorylated JNK increased together with the sensitivity to apoptosis. The contrasting effects on JNK and IκB-α activation upon TNF-α stimulation matched with a modulated maturation of TGF-ß-activated kinase 1 (TAK1) kinase, assessed by 2-dimensional SDS-PAGE. Inhibition of the NF-κB pathway by HBSP was confirmed in the liver of HBSP Tg mice and associated with a significant decrease of chemically induced chronic liver inflammation, as assessed by immunohistochemistry. In conclusion, HBSP contributes to limit hepatic inflammation during chronic liver disease and may favor HBV persistence by evading immune response.

Epithelial to mesenchymal transition is activated in metastatic pheochromocytomas and paragangliomas caused by SDHB gene mutations.

CONTEXT: Pheochromocytoma and paraganglioma are rare neural-crest-derived tumors. They are metastatic in 15% of cases, and the identification of a germline mutation in the SDHB gene is a predictive risk factor for malignancy and poor prognosis. To date, the link between SDHB mutations and malignancy is still missing. OBJECTIVE: Epithelial to mesenchymal transition (EMT) is a developmental event, reactivated in cancer cells to promote cell mobility and invasiveness. The aim of this study was to address the participation of EMT in the metastatic evolution of pheochromocytoma/paraganglioma. DESIGN AND PATIENTS: Transcriptomic profiling of EMT was performed on 188 tumor samples, using a set of 94 genes implicated in this pathway. Activation of EMT was further confirmed at protein level by immunohistochemistry in a second set of 93 tumors. RESULTS: Hierarchical unsupervised classification showed that most SDHB-metastatic samples clustered together, indicating that EMT is differently regulated in these tumors. Major actors of EMT, metalloproteases and components of cellular junctions, were either up-regulated (LOXL2, TWIST, TCF3, MMP2, and MMP1) or down-regulated (KRT19 and CDH2) in SDHB-metastatic tumors compared with nonmetastatic ones. Interestingly, within metastatic tumors, most of these genes (LOXL2, TWIST, TCF3, MMP2, and KRT19) also allowed us to discriminate SDHB-mutated from non-SDHB-related tumors. In the second set of tumors, we studied Snail1/2 expression by immunohistochemistry and observed its specific nuclear translocation in all SDHB-metastatic tumors. CONCLUSION: We have identified the first pathway that distinguishes SDHB-metastatic from all other types of pheochromocytomas/paragangliomas and suggest that activation of the EMT process might play a critical role in the particularly invasive phenotype of this group of tumors.

A novel TMEM127 mutation in a patient with familial bilateral pheochromocytoma.

OBJECTIVE: In this report, we describe a new patient with unexplained familial bilateral pheochromocytoma. Following the recent description of TMEM127 as a new pheochromocytoma susceptibility gene, the aim of this study was to test the hypothesis of a causative TMEM127 gene mutation in this patient. DESIGN: Pheochromocytoma susceptibility genes were analyzed in germline DNA and losses of heterozygosity (LOH) assessed by BAC array comparative genomic hybridization in tumor DNA. SDHB expression and S6 kinase (S6K) phosphorylation were analyzed by immunohistochemistry. Genome-wide expression microarray studies were performed, and vascular density was quantified after CD34 immunohistochemistry. RESULTS: A first germline variant was identified in the SDHB gene (c.158G>A; p.Gly53Glu). However, a positive SDHB immunostaining in the tumor indicated that this SDHB variant was a non-functional polymorphism. A novel TMEM127 germline mutation (c.140C>A, p.Ala47Asp) associated with a 2q11 LOH was found. Transcriptome and immunohistochemical analyses showed that TMEM127-related pheochromocytoma clusterized with NF1-related and RET-related tumors in a large series of pheochromocytomas and paragangliomas, exhibited a reduced TMEM127 mRNA expression and displayed a low vascularization. The phosphorylation of S6K observed in this tumor was suggestive of an activation of the MTOR pathway. CONCLUSIONS: Pathological and genomic data demonstrated that a TMEM127 gene mutation not previously described was causative of a new case of familial bilateral pheochromocytoma. This report highlights the importance of supplementary analyses on tumor tissue to provide an accurate pheochromocytoma/paraganglioma genetic testing result to affected patients.

SDHA immunohistochemistry detects germline SDHA gene mutations in apparently sporadic paragangliomas and pheochromocytomas.

CONTEXT: Pheochromocytoma-paraganglioma syndrome is caused by mutations in SDHB, SDHC, and SDHD, encoding subunits of succinate dehydrogenase (SDH), and in SDHAF2, required for flavination of SDHA. A recent report described a patient with an abdominal paraganglioma, immunohistochemically negative for SDHA, and identified a causal germline mutation in SDHA. OBJECTIVE: In this study, we evaluated the significance of SDHA immunohistochemistry in the identification of new patients with SDHA mutations. SETTING: This study was performed in the Erasmus Medical Center in Rotterdam (The Netherlands) and the Université Paris Descartes in Paris (France). METHODS: We investigated 316 pheochromocytomas and paragangliomas for SDHA expression. Sequence analysis of SDHA was performed on all tumors that were immunohistochemically negative for SDHA and on a subset of tumors immunohistochemically positive for SDHA. RESULTS: Six tumors were immunohistochemically negative for SDHA. Four tumors from Dutch patients showed a germline c.91C → T SDHA gene mutation (p.Arg31X). Another tumor (from France) carried a germline SDHA missense mutation c.1753C → T (p.Arg585Trp). Loss of the wild-type SDHA allele was confirmed by loss of heterozygosity analysis. Sequence analysis of 35 SDHA immunohistochemically positive tumors did not reveal additional SDHA mutations. CONCLUSIONS: Our results demonstrate that SDHA immunohistochemistry on paraffin-embedded tumors can reveal the presence of SDHA germline mutations and allowed the identification of SDHA-related tumors in at least 3% of patients affected by apparently sporadic (para)sympathetic paragangliomas and pheochromocytomas.