Germline Mutation in MUS81 Resulting in Impaired Protein Stability is Associated with Familial Breast and Thyroid Cancer.

Multiple primary thyroid cancer (TC) and breast cancer (BC) are commonly diagnosed, and the lifetime risk for these cancers is increased in patients with a positive family history of both TC and BC. Although this phenotype is partially explained by TP53 or PTEN mutations, a significant number of patients are negative for these alterations. We judiciously recruited patients diagnosed with BC and/or TC having a family history of these tumors and assessed their whole-exome sequencing. After variant prioritization, we selected MUS81 c.1292G>A (p.R431H) for further investigation. This variant was genotyped in a healthy population and sporadic BC/TC tissues and investigated at the protein level and cellular models. MUS81 c.1292G>A was the most frequent variant (25%) and the strongest candidate due to its function of double-strand break repair. This variant was confirmed in four relatives from two families. MUS81 p.R431H protein exhibited lower expression levels in tumors from patients positive for the germline variant, compared with wild-type BC, and normal breast and thyroid tissues. Using cell line models, we showed that c.1292G>A induced protein instability and affected DNA damage response. We suggest that MUS81 is a novel candidate involved in familial BC/TC based on its low frequency in healthy individuals and proven effect in protein stability.

Polysome Profiling of a Human Glioblastoma Reveals Intratumoral Heterogeneity.

Glioblastoma (GBM) is one of the most aggressive cancers, with median survival of less than 2 years. Despite of considerable advance in molecular classification of GBMs, no improvements in therapy have been described. The scenario is further complicated by tumor heterogeneity and the relationship among genetic, transcriptional and functional findings. Classically, gene expression has been evaluated by steady-state mRNA, however, this does not take translational control into consideration, which contributes considerably to the composition of the proteome. In this study, we evaluated the transcriptomic and translatomic signature of a GBM obtained from a single patient focusing in tumor heterogeneity. In a sampling of eight fragments, we investigated the translation rates, mTORC1 and ERK1/2 pathways and identified both total and polysome associated mRNAs. An increased translation rate was observed in fragments with high-grade histological features. High-grade histology was also associated with the expression of genes related to extracellular matrix (ECM) and angiogenesis, in both transcriptomes and translatomes. However, genes associated with epithelial to mesenchymal transition and stress response, were observed only in translatomes from high-grade fragments. Overall, our results demonstrate that isolation of translated mRNA can be used to identify biomarkers and reveal previously unrecognized determinants of heterogeneity in GBMs.

Translatômica aplicada ao descobrimento de alterações moleculares em gliomas / Translatomic aplied to the discovery of molecular alterations in gliomas

O glioblastoma (GBM) está entre os tipos tumorais mais agressivos e de menor resposta a terapias, o que requer, portanto, uma melhor compreensão sobre o comportamento deste tipo tumoral, auxiliando no desenvolvimento de novos tipos de tratamento para a doença. Atualmente, dados de expressão gênica em tumores baseiam-se na população de mRNA total. No entanto, essa abordagem fornece pouca informação sobre os mediadores moleculares das alterações tumorais, pois o nível de expressão de mRNAs não necessariamente reflete os níveis de proteínas expressos. Por outro lado, a população de mRNAs ativamente traduzidos reflete, de maneira mais fidedigna, a expressão proteica, sendo, dessa forma, mais próxima à real medida da expressão gênica. Dito isso, tem-se como objetivo estabelecer a técnica de identificação de RNAs diferencialmente traduzidos (translatômica) e utilizá-la em glioblastomas humanos, contribuindo, assim, para um avanço no conhecimento sobre essa doença. Indica-se com sucesso, como resultado desse estudo, a técnica de translatômica, com o desenvolvimento de um novo gradiente de sacarose que permitiu maior rendimento às preparações e à padronização da identificação de RNAs diferencialmente traduzidos, utilizando tanto microarrays quanto sequenciamento de nova geração. A translatômica foi primeiramente utilizada para comparar a heterogeneidade intratumoral. Comparando áreas histologicamente de maior e menor graus provenientes de um mesmo tumor, observa-se a tradução diferencial de vias de TGF-b e de ciclo celular. Em um segundo momento, a translatômica foi realizada em um número maior de amostras, sendo capaz de classificar três subgrupos moleculares, com um impacto notável na sobrevida: o grupo 1 foi caracterizado pelo aumento das vias associadas à mTORC2, às mitocôndrias à ciliogênese, com sobrevida média de 5 meses; o grupo 2 foi caracterizado por tradução dependente de mTORC1, baixa presença de mitocôndrias e aumento da angiogênese, com uma sobrevida média de 6,3 meses; e o grupo 3 teve uma sobrevida mediana de 21,1 meses e é caracterizado pela alta presença de mitocôndrias, baixa cíliogênese e baixas vias mTORC1 e 2 associadas. Esses grupos não podem ser definidos pela expressão gênica baseada no RNA total, pois não correspondem a classificações moleculares anteriores. Logo, este estudo foi capaz de desenvolver, com sucesso, a técnica de translatômica e aplicá-la ao descobrimento de vias moleculares biologicamente relevantes no processo de tumorigênese em glioblastomas

Glioblastoma (GBM) is among the most aggressive tumor types and the least response to therapy, so better understanding the behavior of this tumor type could help to develop new types of treatment for this disease. Currently, gene expression data on tumors are based on the total mRNA population. However, this approach provides little information on the molecular mediators of tumor changes, since the level of mRNA expression does not necessarily reflect expressed protein levels. Therefore, the population of actively translated mRNAs reflects more accurately the protein expression, being closer to the real measurement of the gene expression. Establish the technique of the identification of differentially translated RNAs (translatomics) and apply it in human glioblastomas, contributing to an advance in the knowledge about this disease. We successfully established the translatomic technique, with the development of a new sucrose gradient that allowed higher yields to the preparations and standardization of the identification of differentially translated RNAs using both microarrays and next generation sequencing. Translatomics was first used to compare intratumoral heterogeneity. Comparing histologically areas of higher and lower degrees from the same tumor, we observed the differential translation of the TGF-b and cell cycle pathways. Afterwards, translatomics were performed in a larger number of samples, being able to classify GBMs into three molecular subgroups, with a remarkable impact on survival. Group 1 was characterized by increased pathways associated with mTORC2, mitochondria and cilia, with an average survival of 5 months. Group 2 was characterized by mTORC1-dependent translation, low mitochondria and increased angiogenesis, with a mean survival of 6.3 months. Group 3 had a median survival of 21.1 months and is characterized by the high presence of mitochondria, low cilia and low associated mTORC1 and 2 pathways. These groups can not be defined using gene expression based on total RNA and thus do not correspond to prior molecular classifications. This study was able to successfully develop the translatomic technique and to apply it to the discovery of biologically relevant molecular pathways in the process of tumorigenesis in glioblastomas

Evaluation of Akt and RICTOR Expression Levels in Astrocytomas of All Grades.

The mammalian target of rapamycin (mTOR) binds to several protein partners and forms two complexes, termed mTOR complexes 1 and 2 (mTORC1/2), that differ in components, substrates, and regulation. mTORC2 contains the protein Rapamycin-insensitive companion of mTOR (RICTOR); phosphorylates kinases of the AGC family, such as Akt; and controls the cytoskeleton. Even though the regulation of mTORC2 activity remains poorly understood, the hyperactivation of this signaling pathway has been shown to contribute to the oncogenic properties of gliomas in experimental models. In this work, we evaluated expression and phosphorylation of Akt, and expression of RICTOR and Ki-67 in 195 human astrocytomas of different grades (38 cases of grade I, 49 grade II, 15 grade III, and 93 grade IV) and 30 normal brains. Expression and phosphorylation of Akt increased with histological grade and correlated with a worse overall survival in glioblastomas (GBMs). RICTOR was overexpressed in grade I and II astrocytomas and demonstrated a shift to nuclear localization in GBMs. Nuclear RICTOR was associated to increased proliferation in GBMs. Our results point to an increase in total and phosphorylated Akt in high-grade gliomas and to a possible role of RICTOR in proliferations of high-grade GBM cells.