m6A-modified circCD2AP suppressed ferroptosis in bladder cancer by upregulating FOXC1 to promote PARK7 transcriptional activity.

Abstract

Background: Bladder cancer (BCa) is the most common genitourinary malignancy and causes substantial economic losses worldwide. Recent studies suggest overexpression of circCD2AP in BCa, but its mechanistic contributions to carcinogenesis and ferroptosis regulation remain unclear.

Methods: From August 2021 to January 2024, 98 pairs of BCa and adjacent normal tissue samples were collected from the Third Xiangya Hospital, Central South University. Gene/protein expression, lipid ROS levels, and cell viability were analyzed. Interactions among circCD2AP/YTH domain-containing protein 1 (YTHDC1), circCD2AP, insulin-like growth factor 2 mRNA-binding protein 3 (IGF2BP3), and IGF2BP3/forkhead box C1 (FOXC1) were validated, along with circCD2AP-IGF2BP3 colocalization. FOXC1 binding sites in the Parkinson's disease protein 7 (PARK7) promoter were mapped. M6A modifications on circCD2AP were quantified via methylated RNA immunoprecipitation polymerase chain reaction.

Results: circCD2AP was significantly upregulated in BCa and positively correlated with PARK7 expression (R2 = 0.1357, P = 0.0002). Patients with high circCD2AP expression showed poorer survival rates. CircCD2AP knockdown sensitized BCa cells to ferroptosis inducers (erastin and Ras-selective lethal small molecule 3). Methyltransferase-like 3 (METTL3) knockdown inhibited circCD2AP m6A modification and relative expression. YTHDC1 mediated m6A-dependent cytoplasmic export of circCD2AP (2.8-fold increase). Besides, circCD2AP stabilized FOXC1 mRNA, and FOXC1 bound the PARK7 promoter to enhance its expression. Knockdown of FOXC1 or PARK7 blocked the antiferroptotic effect of circCD2AP overexpression in BCa cells. Knockdown of METTL3 or circCD2AP reduced tumor burden in mice, decreasing both volume (approximately 48.3% or 56.4%) and weight (approximately 65.3% or 58.7%) through FOXC1/PARK7-mediated ferroptosis.

Conclusions: METTL3-mediated m6A modification drives circCD2AP overexpression, which is subsequently exported to the cytoplasm with enhanced efficiency mediated by YTHDC1. Cytoplasmic circCD2AP stabilizes FOXC1 mRNA, enabling FOXC1-dependent transcriptional activation of PARK7. This axis suppresses ferroptosis and promotes BCa progression, revealing a novel therapeutic target.