Methylation-Mediated Silencing of ATF3 Promotes Thyroid Cancer Progression by Regulating Prognostic Genes in the MAPK and PI3K/AKT Pathways

Background: Aberrant expression of oncogenes and/or tumor suppressor genes (TSGs) drives the tumorigenesis and development of thyroid cancer. We investigated the expression and function of a member of the activating transcription factor (ATF)/cAMP-responsive element-binding protein (CREB) transcription factor (TF) family, ATF3, in thyroid cancer.Methods: Data from 80 patients with papillary thyroid cancer (PTC) in the First Affiliated Hospital of Sun Yat-sen University and 510 PTC samples in The Cancer Genome Atlas thyroid cancer database were utilized for gene expression and prognosis analyses. The survival data were analyzed by Kaplan-Meier curves and Cox regression with adjustment for age, sex, multilocality, extrathyroidal extension, lymph metastases, and history of neoadjuvant treatment. DNA methylation was analyzed by methylation-specific polymerase chain reaction (PCR) and bisulfite sequencing PCR. TFs binding to ATF3 promoter were identified by DNA pull-down combined with mass spectrum assay, and confirmed by quantitative PCR (qPCR), luciferase reporter assay, and chromatin immunoprecipitation (ChIP)-qPCR. We conducted functional assays in vitro and in a xenograft mouse model to evaluate the function of ATF3 in thyroid cancer. Integrated analyses based on RNA sequencing, ChIP-seq, and CUT&Tag assays were performed to explore the mechanisms underlying the function of ATF3.Results: ATF3 was significantly downregulated in PTC and patients with low ATF3 expression had reduced progression-free survival (adjusted hazard ratio = 0.50 [CI 0.26-0.98], p = 0.043). DNA hypermethylation in ATF3 promoter disrupted the binding of SP1 and MYC-MAX, leading to inactivation of the gene. ATF3 functioned as a TSG by inhibiting the proliferation and mobility of thyroid cancer cells. And ATF3 regulated the expression of a number of genes by binding to the regulatory elements of them, particularly for genes in MAPK and PI3K/AKT pathways. Among these target genes, filamin C was positively regulated by ATF3 and associated with a more favorable thyroid cancer prognosis, while dual specificity phosphatase 10, fibronectin-1, tenascin C, and CREB5 were negatively regulated by ATF3 and associated with a poorer prognosis.Conclusions: We observed that the promoter DNA hypermethylation decreased the expression of ATF3, which in turn promoted the progression of thyroid cancer, at least partially, by directly regulating prognosis-related genes in the MAPK and PI3K/AKT pathways.