Dados do Trabalho

Título

Exploring Dysregulated Translation of Primary Cilia-Associated mRNAs as a Potential Strategy in Glioblastoma Development

Introdução

Glioblastoma (GBM) is one of the most aggressive and least responsive tumor types to therapies. Currently, gene expression data for these tumors relies on total mRNA expression, but this approach offers limited insight into the cellular proteome. To address this, we focused on actively translated mRNAs (polysome-associated mRNA), which more accurately reflects protein expression and provides a truer measure of gene expression.

Objetivo

We aimed to analyze gene expression profiles by categorizing three distinct molecular groups, identified through polysome-associated mRNAs from GBM patient samples. Specifically, we assessed how translation control of these mRNAs in the group with poor survival (enriched for primary cilia mRNAs) could influence primary cilia (PC) biology in GBM cells.

Métodos

We initiated this study by conducting poly-mRNA enrichment for the group with the poorest survival, utilizing GSEA analysis. Subsequently, polysome-associated mRNAs (poly-mRNA) were isolated from both GBM cell lines (U-87 and GBM33) through polysome profiling. In order to elucidate the translation regulation of these poly-mRNAs, a range of conditions were systematically tested. To inhibit protein synthesis, we administered rapamycin, with the effectiveness of this inhibition being validated via western blot analyses. To gain insights into the translational profiles of enriched genes linked to primary cilia, we employed RT-qPCR. Furthermore, we explored the potential impact of protein synthesis regulation on PC biology within the respective cell lines and patient-derived xenografts cells, utilizing an immunofluorescence assay to identify and quantify these organelles.

Resultados

Differentially expressed genes, as identified through the GSEA analysis of the cohort characterized by poorer survival, encompass DHX9, SAMARCA5, SRPK2, KIAA1429, METTL14, and PCM1. Polysome profiling analyses have unveiled a notable influence of protein synthesis inhibition on translation levels, particularly when contrasted with treatment conducted under standard or stationary conditions. The translational profile of the PCM1 gene, assessed utilizing RT-qPCR, lends further support to the observation of mRNA shifting towards diminished translation rates under conditions of protein synthesis inhibition, thus accentuating its potential for translational dysregulation. In the context of comprehensively evaluating the orchestration of protein synthesis regulation within the realm of PC biology, the application of protein synthesis inhibitors as a modality has substantiated a discernible reduction in the proportion of ciliated cells. This outcome has been consistently noted across both cell lines derived from patients and those originating from patient-derived xenografts (PDXs).

Conclusões

Translational dysregulation was observed in patient-derived cell lines. Dysregulation of genes linked to primary cilia, like PCM1, may crucially contribute to GBM's malignancy in the poor survival group. Next, we investigate if ciliogenesis modulation (using inhibitors and CRISPR-Cas9) reveals treatment resistance mechanisms, unveiling new therapies.

Palavras-chave

Primary cilia, Glioblastoma, translation control

Financiador do resumo

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and, Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Área

Estudo Clínico - Tumores do Sistema Nervoso Central