Iron (Ferric Ion, Fe3+)-Dependent Telomerase Reactivation Drives Colorectal Cancer Development

I. Research Background

Colorectal Cancer (CRC) is the third most common cancer globally, involving both genetic and non-genetic factors in its pathogenesis. Existing research shows a significant association between high iron content, hereditary iron overload, and the occurrence of CRC. However, how metal-mediated signaling pathways contribute to carcinogenesis remains a mystery. Heterocyclic amines and polycyclic aromatic hydrocarbons found in red meat are recognized as carcinogenic, but few studies explain how elevated iron levels drive CRC. While iron plays a crucial role in essential cellular functions, abnormal iron levels can lead to tissue damage and DNA damage, promoting cancer cell growth. Therefore, understanding the specific molecular mechanisms of iron in CRC pathogenesis is vital.

II. Research Source

This study was a collaborative effort by researchers from various institutions, including the Institute of Molecular and Cell Biology (IMCB) under the Agency for Science, Technology and Research (A*STAR) of Singapore. The main authors include Raghuvaran Shanmugam, Prativa Majee, Wei Shi, and others. The study was published in the journal “Cancer Discovery,” highlighting the critical role of iron in the progression of colorectal cancer.

III. Research Workflow

1. Detailed Description of the Research Procedure

The study initially identified that the reactivation of telomerase reverse transcriptase (hTERT) under iron (ferric ion, Fe3+) dependency is a critical event in CRC pathogenesis from fresh CRC patient samples, patient-derived xenografts (PDX), primary CRC organoids, and cell lines. Subsequently, through chemical genetic screening combined with isothermal dose-response fingerprinting and mass spectrometry analysis, researchers discovered a small molecule SP2509 that specifically inhibits Pirin-mediated hTERT reactivation by competitively binding with ferric ions (Fe3+).

The detailed workflow included several major steps:

a. Screening of Telomerase Regulatory Factors

Researchers constructed a nanoluciferase reporter system controlled by the hTERT promoter. Using this system for high-throughput screening of multiple small molecule libraries, SP2509 was identified as one of the strongest hTERT transcription inhibitors.

b. Experimental Validation and Molecular Target Confirmation

Through thermal melting assay and mass spectrometry analysis, SP2509’s molecular target was confirmed to be Pirin, an iron-binding protein. Supplementary experiments revealed that SP2509 inhibits Pirin function by competitively binding with iron, leading to decreased hTERT expression and telomerase activity.

c. Study on the Correlation Between Ferric Ion (Fe3+) and hTERT Activity

Using 40 fresh CRC patient samples, telomerase repeated amplification protocol (TRAP) and Prussian blue staining methods demonstrated a significant correlation between ferric ion (Fe3+) levels and telomerase activity in tumor samples, indicating iron’s key role in hTERT transcription.

d. Positive Regulatory Role of Pirin in hTERT Transcription

Through transgenic and small molecule inhibitor experiments, Pirin’s regulatory role on hTERT transcription under Fe3+ dependence was validated. Overexpression of Pirin restored telomerase activity inhibited by SP2509, while knockdown or mutated Pirin significantly reduced hTERT expression and cell proliferation.

e. Further In Vivo Studies

In patient-derived xenograft (PDX) models with high and low Pirin expression, iron supplementation significantly promoted tumor growth, whereas iron chelators (e.g., DFO) and SP2509 inhibited the rapid growth of Pirin high-expressing PDX, supporting Pirin’s key role in iron-dependent hTERT transcription activation.

2. Research Results

The study uncovered several key points:

• Pirin is a linear iron-dependent transcription factor that promotes hTERT transcription and drives CRC occurrence in the presence of ferric ions (Fe3+).
• SP2509 acts as a small molecule inhibitor that significantly suppresses telomerase activity by competitively binding to Pirin binding sites, thereby inhibiting CRC cell proliferation.
• Reactivation of hTERT transcription is particularly significant in CRC samples with high Pirin expression and high Fe3+ levels, indicating the interaction of these factors in CRC pathogenesis and progression.

IV. Research Significance and Value

1. Scientific Value

This study is the first to reveal the molecular mechanism of iron in the development of colorectal cancer, providing a new perspective on the relationship between iron and cancer. It also identified Pirin as a key regulatory factor in hTERT transcription and demonstrated that inhibiting Pirin function with SP2509 effectively reduces telomerase activity and cancer cell proliferation, offering a new therapeutic target for cancer treatment.

2. Application Value

The study’s results are significant for developing new cancer therapeutics. Small molecules like SP2509 hold promise as new treatment options, especially for CRC patients with high telomerase activity and high ferric ion (Fe3+) levels. Additionally, iron chelators can potentially manage iron-dependent cancer progression to some extent.

3. Highlights and Innovations

The major highlights of this study include:

• First demonstration of the relationship between iron-mediated Pirin-driven hTERT reactivation and colorectal cancer.
• Discovery of SP2509, which effectively inhibits Pirin’s function by competing for iron binding sites, thereby suppressing telomerase activity.
• Proposal of iron chelators as a potential therapeutic strategy for CRC patients with high iron indices to inhibit tumor growth.

Conclusion

By thoroughly analyzing the mechanism of iron in colorectal cancer, this study demonstrates the importance of iron-dependent Pirin in telomerase reactivation and cancer development. It showcases the efficacy and safety of SP2509 as a potential therapeutic agent. Future studies should explore SP2509’s application in other cancer types and investigate how to best combine it with other treatments to enhance its clinical efficacy.