New Discovery in the Mechanism of Radioresistance in Esophageal Squamous Cell Carcinoma: The Role of the RFC4 Gene

Academic Background

Esophageal squamous cell carcinoma (ESCC) is one of the most common gastrointestinal malignancies in China, and radiotherapy is a crucial treatment modality. However, radioresistance in tumor cells is a major cause of treatment failure and tumor recurrence. The molecular mechanisms underlying radioresistance remain unclear, especially regarding the role of genes related to DNA damage repair. DNA double-strand breaks (DSBs), the most severe form of DNA damage induced by radiotherapy, are repaired through the activation of the DNA damage response (DDR), leading to radioresistance in tumor cells. Therefore, studying the role of DNA damage repair-related genes in ESCC radioresistance is of great significance for developing new radiosensitization strategies.

Replication Factor C4 (RFC4), a key gene in the DNA damage repair process, has been found to be abnormally expressed in various cancers in recent years, closely associated with tumor cell proliferation, apoptosis, and chemoresistance. However, the specific role of RFC4 in ESCC radioresistance remains unclear. This study aims to explore the role and molecular mechanisms of RFC4 in ESCC radioresistance, providing new therapeutic targets for overcoming radioresistance in ESCC.

Source of the Paper

This paper was jointly completed by Tao Yang, Yue Fan, Guang Bai, and Yinpeng Huang from the Department of General Surgery at the First Affiliated Hospital of Jinzhou Medical University, supported by the Joint Funds of the Natural Science Foundation of Liaoning Province. The paper was first published on December 13, 2024, in the journal American Journal of Physiology - Cell Physiology, with the DOI: 10.1152/ajpcell.00533.2024.

Research Workflow and Results

1. Analysis of RFC4 Expression in ESCC

The study initially analyzed the expression of RFC4 in various cancers using bioinformatics tools, finding it significantly upregulated in esophageal cancer (ESCA) tissues. Further analysis using data from TCGA and GEO databases confirmed high RFC4 expression in ESCC tissues and cells, particularly in radioresistant cases. Through qRT-PCR, immunohistochemistry, and Western blotting experiments, the research team validated the high expression of RFC4 in ESCC tissues and found that high RFC4 expression correlates with poor patient prognosis.

2. Functional Studies of RFC4

To investigate the function of RFC4 in ESCC, the research team constructed cell lines with varying levels of RFC4 expression and performed cell proliferation, apoptosis, and cell cycle analyses. MTT and EdU assays showed that high RFC4 expression promoted ESCC cell proliferation, while its knockdown significantly inhibited cell proliferation. Flow cytometry analysis revealed that high RFC4 expression induced S-phase cell cycle arrest, whereas its knockdown promoted apoptosis. These results indicate that RFC4 plays an oncogenic role in ESCC.

3. Relationship Between RFC4 and Radioresistance

To study the role of RFC4 in ESCC radioresistance, the research team exposed ESCC cells to different doses of radiation and found that high RFC4 expression enhanced cell survival and clonogenic ability, while its knockdown significantly reduced radioresistance. Further studies revealed that RFC4 enhances the DNA damage repair capacity of ESCC cells by regulating the expression of DNA damage repair-related proteins (such as γ-H2AX and DNA-PKcs), thereby leading to radioresistance.

4. RFC4 Promotes Radioresistance by Inhibiting the p53 Signaling Pathway

To further elucidate the molecular mechanisms of RFC4-mediated radioresistance, the research team examined the expression levels of p53 signaling pathway-related proteins. Western blotting analysis showed that high RFC4 expression inhibited the expression of p53 and p21 while increasing the expression of Cyclin D1 and DNA repair protein RAD51. By co-expressing RFC4 and p53, the research team found that p53 activation partially reversed RFC4-mediated radioresistance. These results suggest that RFC4 promotes radioresistance in ESCC cells by inhibiting the p53 signaling pathway and enhancing DNA damage repair.

5. Animal Experiment Validation

To verify the role of RFC4 in vivo, the research team constructed an ESCC xenograft mouse model. The results showed that RFC4 knockdown or combination with radiotherapy significantly inhibited tumor growth. Immunohistochemical analysis further confirmed that RFC4 knockdown reduced Ki-67 expression in tumor cells while increasing the expression of p53 and γ-H2AX. These results indicate that RFC4 also promotes radioresistance in vivo.

Conclusions and Implications

This study is the first to reveal the critical role of RFC4 in ESCC radioresistance and elucidates its molecular mechanism of enhancing DNA damage repair by inhibiting the p53 signaling pathway. This discovery provides a new therapeutic target for overcoming radioresistance in ESCC, with significant scientific and clinical application value. In the future, targeted therapy against RFC4 may become an effective strategy to improve the efficacy of ESCC radiotherapy.

Highlights of the Study

1. Key Findings: RFC4 is highly expressed in ESCC and correlates with poor patient prognosis; RFC4 enhances DNA damage repair by inhibiting the p53 signaling pathway, leading to radioresistance.
2. Novelty: This is the first study to reveal the specific role and molecular mechanisms of RFC4 in ESCC radioresistance.
3. Application Value: RFC4 may serve as a new target for overcoming radioresistance in ESCC, providing a theoretical basis for the development of radiosensitizing drugs.

Other Valuable Information

The research team also conducted functional enrichment analysis, discovering that RFC4 and its co-expressed genes are mainly involved in cell cycle and DNA damage repair pathways, further supporting the role of RFC4 in ESCC radioresistance. Additionally, the bioinformatics analysis methods and experimental models developed by the research team provide important references for similar studies.