The role of p53 expression in the glioblastoma microenvironment

Download
Favorite this paper
How to cite this paper?
Details
  • Presentation type: MS - Master's student
  • Track: 2. Cellular Biology
  • Keywords: Glioblastoma; p53; Tumor microenvironment;

Authors:

  • 1 Universidade Federal do Rio de Janeiro

Please log in to watch the video

Log in
Abstract

INTRODUCTION AND OBJECTIVES: Glioblastoma (GBM) is the most common and aggressive primary brain tumor, with patients displaying median overall survival of only 19 months. The high infiltration rate of mesenchymal-like GBM cells and interactions with the brain stroma are thought to contribute towards therapy resistance and disease recurrence. TP53, the most frequently altered gene in human cancers, is known to regulate the crosstalk between tumor and stromal cells in the tissue microenvironment (TME). However, how p53 regulates malignant cell phenotypes in GBM is not clearly understood. Our group has previously shown that murine astrocytes heterozygous for Trp53 (p53het) deposit an extracellular matrix that is enriched for fibronectin and laminin compared to their wild-type counterpart (p53wt). Importantly, the p53het matrix protected GBM cells from cell death and increased their motility. This suggests that, in the TME, decreased p53 expression in astrocytes promotes the GBM phenotype towards higher malignancy. The current work aims to further dissect the role of stromal p53 expression in regulating GBM cell behavior by assaying murine knockout astrocytes (p53ko), which represent loss of stromal p53 expression in our model. MATERIAL AND METHODS: Primary astrocyte cultures were established from the brain cortex of p53wt, p53het and p53ko mice. Cells were cultured in DMEM/F12 cell medium supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin and 0.1% fungizone. Western blotting was performed for detection of fibronectin and laminin protein levels. RESULTS AND CONCLUSION: Protein level analysis showed that p53ko and p53het murine astrocytes have increased deposition of fibronectin and laminin in their respective extracellular matrices compared to p53wt astrocytes. Both these proteins have been implicated in chemoresistance and highly mesenchymal phenotypes in GBM cells. We will perform transwell and dose-response assays to further analyze the p53-dependent role of astrocytic extracellular matrix in GBM cell invasion and chemoresistance using the T98G and U87 GBM cell lines.

Questions (2 topics)

Share your ideas or questions with the authors!

Did you know that the greatest stimulus in scientific and cultural development is curiosity? Leave your questions or suggestions to the author!

Sign in to interact

Have a question or suggestion? Share your feedback with the authors!

Author

Leonel Cardozo de Menezes e Souza

Bom dia, Sara! Obrigado pelas perguntas. Nosso foco é no papel da matriz extracelular dos astrócitos sobre o comportamento das linhagens de GBM. Para isso, isolamos a matriz depositada pelos astrócitos de diferentes genótipos (p53+/+, p53+/- ou p53-/-) através de um protocolo de lise, e então cultivamos as células de GBM por cima dessas matrizes. Para detalhes do protocolo, por favor ver o trabalho publicado pelo nosso grupo nessa linha de pesquisa: https://doi.org/10.1038/oncsis.2014.36. Atualmente, estamos avaliando o efeito da cultura sobre as matrizes na resposta das linhagens U87MG e T98G ao tratamento com temozolomida. O próximo passo imediato é a avaliação da migração/invasão por transwell. Nesses experimentos, as matrizes de astrócitos serão novamente usadas como substratos, mas especificamente no ensaio de invasão elas serão depositadas em gel (semelhante ao Matrigel) para avaliar a capacidade invasiva das linhagens de GBM. O efeito do sobrenadante de astrócitos sobre GBM é interessantíssimo e já foi analisado antes (por favor ver este trabalho: https://doi.org/10.1371/journal.pone.0054752), mas acreditamos que a interação entre a matriz extracelular de astrócitos e células de GBM ainda é pouco explorada! Estou à disposição para demais dúvidas, sugestões e comentários.           

Sara Santos Bernardes

Irei checar os artigos sugeridos, para entender melhor os protocolos. Vocês pensam em avaliar alguma droga que iniba a produção de laminina e/ou fibronectina, até mesmo para futuramente pensarem em ensaios in vivo?

Mais uma vez, parabéns pelo trabalho!

Author

Leonel Cardozo de Menezes e Souza

Oi novamente! Não conhecemos drogas que inibam especificamente a produção de laminina e/ou fibronectina, mas já existem compostos e anticorpos específicos para bloquear integrinas, que são a classe de receptores que se ligam a proteínas de matriz e comunicam informações da matriz extracelular para o interior da célula. Acredito que a inibição farmacológica desses receptores possa ser uma via mais interessante. É importante identificar a combinação de integrinas que são especificamente expressas no tumor de GBM e que podem ser usadas como alvos terapêuticos com segurança. Essa é com certeza uma sugestão muito interessante que vamos considerar explorar para avançar o trabalho, muito obrigado!  

Author

Leonel Cardozo de Menezes e Souza

Hello Pedro, thank you for your questions and comments! I will answer them in order: 1) It's important to point out that we are not evaluating oncogenic gain-of-function of mutant p53 in this model. Our primary astrocytes carry deletion of either one (Trp53+/-) or both (Trp53-/-) alleles. So, we are really focusing on the effect of loss of normal tumor suppression function exerted by p53, due to reduction or loss of its expression. In order to draw any conclusions for mutational gain-of-function, we would need to analyze mutant p53-expressing astrocytes, which is definitely very relevant and is a potential future approach. 2) Recent work in ovarian cancer cells has characterized FN1, the fibronectin gene, as a direct target of wild-type p53, which represses its promoter (please check: https://doi.org/10.1186/s12964-020-00580-3). In line with our study, knockdown of p53 expression greatly enhanced FN1 mRNA and protein levels in these cells! I am not aware of similar evidence for the laminin gene family, which encodes many laminin subunits and has a more complex regulation. However, p53 has been seen to indirectly modulate laminin expression in a number of contexts (please check: https://doi.org/10.1073/pnas.1808314115). Many microRNAs which are p53 targets regulate fibronectin/laminin expression, so it's totally plausible that p53 indirectly regulates expression of these proteins through miRNA-mediated mechanisms. 3) Finally, we will validate fibronectin and laminin expression in the astrocytes of different genotypes also by immunofluorescence. I believe reintroducing normal levels of p53 expression in either Trp53+/- or Trp53-/- astrocytes with transfection of p53wt, and then checking fibronectin and laminin expression, could further confirm our hypothesis.  Additionally, we aim in the future to analyze the global profile of proteins in astrocytes by mass spectrometry-based proteomics, which is expected to give us a wider view on the level of changes in the astrocytic extracellular matrix due to loss of p53 expression and GBM cell contact.