Epigenetic silencing of serine protease HTRA1 drives polyploidy.

BACKGROUND: Increased numbers and improperly positioned centrosomes, aneuploidy or polyploidy, and chromosomal instability are frequently observed characteristics of cancer cells. While some aspects of these events and the checkpoint mechanisms are well studied, not all players have yet been identified. As the role of proteases other than the proteasome in tumorigenesis is an insufficiently addressed question, we investigated the epigenetic control of the widely conserved protease HTRA1 and the phenotypes of deregulation. METHODS: Mouse embryonal fibroblasts and HCT116 and SW480 cells were used to study the mechanism of epigenetic silencing of HTRA1. In addition, using cell biological and genetic methods, the phenotypes of downregulation of HTRA1 expression were investigated. RESULTS: HTRA1 is epigenetically silenced in HCT116 colon carcinoma cells via the epigenetic adaptor protein MBD2. On the cellular level, HTRA1 depletion causes multiple phenotypes including acceleration of cell growth, centrosome amplification and polyploidy in SW480 colon adenocarcinoma cells as well as in primary mouse embryonic fibroblasts (MEFs). CONCLUSIONS: Downregulation of HTRA1 causes a number of phenotypes that are hallmarks of cancer cells suggesting that the methylation state of the HtrA1 promoter may be used as a biomarker for tumour cells or cells at risk of transformation.

Comparing the Prognostic Value of BAP1 Mutation Pattern, Chromosome 3 Status, and BAP1 Immunohistochemistry in Uveal Melanoma.

Uveal melanoma (UM), a tumor of the eye, can be divided into 2 major classes correlating with patients' prognosis. Gene expression profiles and chromosome 3 status are correlated with tumor classification and prognosis. Somatic BAP1 mutations are another feature largely restricted to metastatic UM. Here we performed thorough BAP1 mutation analysis including sequencing and gene dosage analysis of all BAP1 coding exons as well as methylation analysis of the promoter CpG island in a set of 66 UMs. The results were compared with the BAP1 protein expression as determined by immunohistochemistry and the tumor-related survival of the patients. BAP1 sequencing and gene dosage analysis of BAP1 exons by multiplex ligation-dependent probe amplification revealed a mutation in 33 (89%) of 37 tumors with monosomy 3 (M3) or isodisomy 3. BAP1 mutations were not detected in any of the 28 tumors with disomy 3 or partial monosomy 3 (partM3). Most of the sequence mutations (21 of 28) were frame-shift, splice-site, or nonsense mutations leading to a premature termination codon. BAP1 protein as determined by immunohistochemistry was absent in all samples with a BAP1 mutation irrespective of the functional type of mutation. Kaplan-Meier analysis revealed a highly significant association between BAP1 protein staining and patients' survival (P=0.0004). The association between BAP1 mutation status and tumor-related survival was less pronounced but still significant (P=0.0023). We conclude that BAP1 protein staining is favorable over BAP1 mutation screening by Sanger sequencing for prognostic testing of UM patients.

BAP1 germline mutation in two first grade family members with uveal melanoma.

BACKGROUND: Uveal melanoma (UM) is the most common primary cancer of the eye in adults. About half of the patients are at risk of developing metastatic disease resulting in a poor clinical prognosis. Metastatic progression is strongly associated with loss of one chromosome 3 in the tumour (monosomy 3). The tumour suppressor gene BAP1 was found to be recurrently mutated in UM with monosomy 3. Familial UM is rare and amounts to about 0.6-6% of all patients with melanoma. However, BAP1 germline mutations have been identified in rare hereditary tumour syndromes, including cases with UM. One may assume that UM may be part of these hereditary conditions with predisposition to malignant cancers. METHODS: The patients underwent complete ophthalmological workup and enucleation due to UM. Microsatellite analysis was performed to determine the chromosome 3 status of the tumours. Sanger sequencing of all coding exons of the BAP1 gene was performed in blood DNA of the patients. RESULTS: Here we report on two family members (mother and son) diagnosed with UM. In both patients, a cosegregating BAP1 germline mutation (c.299 T>C) was found. The mutant BAP1 allele was retained in the tumour of the son showing monosomy 3. The son further developed urothelial carcinoma and liver metastasis, the mother was affected by the UM and cholangiocellular carcinoma. CONCLUSIONS: [corrected] We detected a cosegregating BAP1 germline mutation in two family members with UM. This suggests that, consistent with a classic tumour suppressor model, carriers of damaging mutations in BAP1 are predisposed to UM. However, as BAP1 germline mutations have been found to cause other cancer syndromes as well, there must be other factors that decide about the type of tumour emerging from BAP1 inactivation.