Dados do Trabalho

Título

PIWIL4 acts as an oncogene and regulates the expression of transposable elements and interferon-mediated viral mimicry in gastric cancer

Introdução

PIWIL4 is a member of the PIWIL argonaut family of proteins that acts along with small piwi-RNAs to repress transposable elements (TEs) at transcriptional and translational levels. TEs are spread across the genome, and their expression is repressed by epigenetic mechanisms to avoid genomic instability in normal cells, but their aberrant expression in tumors is associated with increased immunogenicity. PIWIL4 is upregulated in gastric cancer (GC), but its involvement in tumor biology, regulation of TEs and immunogenicity is not characterized.

Objetivo

This study aimed to investigate the influence of PIWIL4 in GC, specially its potential role in the regulation of TEs and activation of type I and III interferon (IFN) pathways in human GC molecular datasets and in cellular models of GC.

Métodos

We evaluated PIWIL4 expression and methylation in GC data from The Cancer Genome Atlas (TCGA). Using CRISPR and lentiviral overexpression systems, we generated GC cellular models (from the AGS cell line) permanently lacking (PIWIL4-KO) or overexpressing PIWIL4, which were characterized by growth kinetics analysis, proliferation assay (cell trace dilution analysis) and cell cycle analysis by flow cytometry, clonogenic assays and spheroid formation in low-attachment plates. Also, the response to the demethylating agent decitabine (DAC) was evaluated by MTT assay, quantification of IFNα and IFNλ1 in culture supernatant by ELISA and gene expression analysis of TEs (HERV-K and LINE-1), type I (IFNB1) and type III (IFNL1) IFNs and interferon stimulated genes (ISGs: DDX58 and ISG15) by qPCR. Finally, the immune stimulatory capacity of KO cells was assessed by stimulating peripheral blood mononuclear cells (PBMCs) with anti-CD28 and anti-CD3 in the presence of conditioned culture medium and evaluating their profile by flow cytometry.

Resultados

Analysis of TCGA data reveal that PIWIL4 was upregulated at the mRNA and protein level in GC compared to tumor-adjacent tissue, and patients showing high PIWIL4 expression or low gene methylation had worse prognosis. PIWIL4 positively correlated with the expression of TEs, and these were highly correlated with the expression of ISGs. PIWIL4-overexpressing GC cells only had slower growth of spheroids, while PIWIL4-KO cells showed impaired growth kinetics, explained by their reduced proliferative capacity, cell cycle arrest and higher basal apoptosis rate. Also, they had limited tumor formation capacity in clonogenic assays and could not form spheroids. These cells also expressed more TEs, IFNB1 and IFNL1 and secreted more IFNα. Under DAC treatment, PIWIL4-KO cells showed resistance to death/cytostasis. Finally, PBMCs stimulated in the presence of conditioned medium from PIWIL4-KO cells, upregulated markers for activation, tissue residency, exhaustion and cytotoxicity within lymphoid compartment.

Conclusões

Therefore, PIWIL4 may act as an oncogene in GC, once it is overexpressed and associated with worse prognosis in patients and that its deletion in GC cells impairs their growth. Also, it may regulates the expression of TEs and the viral mimicry response. In the next steps, we will characterize transcriptomic alterations promoted by PIWIL4 KO to gain deeper insights on its role in GC and further dissect the impact of PIWIL4 on tumor evolution in xenograft models.

Palavras-chave

PIWIL4
Transposable Elements
Gastric Cancer

Financiador do resumo

FAPESP, grants 18/14034-8 (to TSM) and 20/10299-7 (to GAFV)

Área

Estudo Clínico - Tumores do Aparelho Digestivo Alto