Vitamin C enhances cisplatin sensitivity in bladder cancer via 5hmC-mediated epigenetic modulation of ATF4
Chunru Xu1,2,3, Wenwei Ying1,2,3, Yuhui He1,2,3
1Department of Urology, Peking University First Hospital, No 8 Xishiku Street, Xicheng District, Beijing, 100034, China.
Abstract:
Cisplatin resistance remains a major challenge in the clinical treatment of bladder cancer (BC), and the epigenetic regulation of this resistance, particularly involving 5-hydroxymethylcytosine (5hmC), has not been fully elucidated. Here, we investigated the role of 5hmC and vitamin C (VC) in modulating cisplatin sensitivity in BC. Clinical analyses of 36 BC patients receiving cisplatin-based neoadjuvant chemotherapy showed that reduced 5hmC levels in pre-chemotherapy tumor tissues were significantly associated with cisplatin resistance (CR-BC) and poor prognosis, with low 5hmC correlating with shorter progression-free survival (PFS). In vitro, we established two cisplatin-resistant cell lines (T24-CR, UMUC-3-CR) that exhibited reduced 5hmC compared to parental cells. Treatment with 100 μM VC significantly restored 5hmC levels in CR-BC cells by activating TET enzymes, inhibited cell proliferation, and enhanced cisplatin sensitivity; these effects were abrogated by the TET inhibitor Bobcat339, confirming VC acts in a TET-dependent manner. Mechanistically, genome-wide 850 K methylation array and RNA-seq analyses revealed that VC upregulated methylation specifically at the promoter of ATF4, a downstream effector of the MAPK pathway, thereby downregulating ATF4 expression. ATF4 knockdown in CR-BC cells increasing cisplatin sensitivity, while Bobcat339 reversed VC-induced ATF4 downregulation. In vivo, VC combined with cisplatin significantly inhibited tumor growth in T24-CR xenografts, and co-treatment with ATF4 knockdown further enhanced this effect, accompanied by elevated 5hmC and reduced Ki67 in tumors. Collectively, our findings identify reduced 5hmC as a hallmark of cisplatin-resistant BC and reveal a novel mechanism by which VC enhances cisplatin sensitivity. VC activates TET enzymes to increase 5mC at the ATF4 promoter, downregulating ATF4 and modulating the MAPK pathway. This highlights VC as a potential epigenetic adjuvant to overcome cisplatin resistance in BC.
Insights
Reduced 5-hydroxymethylcytosine (5hmC) in bladder cancer (BC) predicts cisplatin resistance. Vitamin C (VC) boosts cisplatin sensitivity by increasing 5hmC via TET enzymes, downregulating ATF4, and improving patient outcomes.
Area of Science:
- Epigenetics and Cancer Biology
- Oncology
- Biochemistry
Background:
- Cisplatin resistance is a significant clinical challenge in bladder cancer (BC).
- The role of 5-hydroxymethylcytosine (5hmC) in BC cisplatin resistance is not fully understood.
- Epigenetic modifications are crucial in cancer treatment resistance.
Purpose of the Study:
- To investigate the role of 5hmC and vitamin C (VC) in modulating cisplatin sensitivity in BC.
- To elucidate the epigenetic mechanisms underlying VC's effect on cisplatin resistance.
- To assess the clinical relevance of 5hmC levels in BC patients undergoing chemotherapy.
Main Methods:
- Clinical analysis of 36 BC patients' tumor tissues.
- Establishment and treatment of cisplatin-resistant BC cell lines (T24-CR, UMUC-3-CR) with VC and TET inhibitors.
- Genome-wide methylation array and RNA-seq analyses.
- In vivo xenograft studies with VC, cisplatin, and ATF4 knockdown.
Main Results:
- Reduced pre-chemotherapy 5hmC levels in tumor tissues correlated with cisplatin resistance (CR-BC) and poor prognosis.
- VC treatment restored 5hmC levels in CR-BC cells, inhibited proliferation, and enhanced cisplatin sensitivity via TET enzyme activation.
- VC upregulated methylation at the ATF4 promoter, downregulating ATF4 expression, which was linked to increased cisplatin sensitivity.
- Combined VC and cisplatin treatment significantly inhibited tumor growth in vivo, with further enhancement by ATF4 knockdown.
Conclusions:
- Reduced 5hmC is a hallmark of cisplatin-resistant BC.
- VC enhances cisplatin sensitivity by activating TET enzymes, increasing 5hmC, downregulating ATF4, and modulating the MAPK pathway.
- VC represents a potential epigenetic adjuvant therapy to overcome cisplatin resistance in bladder cancer.
