Dual Ferroptosis Induction in N2-TANs and TNBC Cells via FTH1 Targeting: A Therapeutic Strategy for Triple-Negative Breast Cancer

Dual Ferroptosis Induction via FTH1 Targeting: A New Therapeutic Strategy for Triple-Negative Breast Cancer (TNBC)

Background: Why Conduct This Study?

Breast cancer is one of the most common cancer types worldwide, accounting for 11.7% of all new cancer cases globally. Among its several subtypes, triple-negative breast cancer (TNBC) stands out for its aggressive nature, poor prognosis, and high metastatic potential. The five-year survival rate for TNBC patients is only 11%. Traditional chemotherapy and targeted drugs show limited efficacy in treating TNBC due to the absence of specific molecular targets found in other breast cancer subtypes. Therefore, developing more effective therapeutic strategies and novel drugs to improve TNBC patient survival is a pressing priority in cancer research.

Furthermore, tumor-associated neutrophils (TANs) play a critical role in the progression and prognosis of TNBC. Among TANs, N2-type TANs possess pro-tumorigenic characteristics, promoting cancer cell proliferation, angiogenesis, and metastasis. While suppressing neutrophil infiltration into tumors can partially control TNBC progression, neutrophils are the most abundant immune cells in the body, and their full suppression may severely compromise overall immune defense. Consequently, selectively targeting N2-type neutrophils has become a crucial solution.

At the same time, ferroptosis—a non-apoptotic form of cell death—has emerged as a research hotspot due to its potential role in cancer therapy. Ferroptosis is characterized by excessive polyunsaturated lipid peroxidation and is closely related to iron, amino acid, and lipid metabolism. TNBC cells and N2-type TANs, due to their high iron content and lipid enrichment characteristics, exhibit heightened sensitivity to ferroptosis inducers. Researchers hypothesize that targeting a molecule capable of simultaneously inducing ferroptosis in TNBC cells and N2-type TANs could create a novel therapeutic strategy for TNBC.


Study Source and Researchers

This study was conducted by Yichen Liu et al., involving researchers from several distinguished Chinese institutions, including the Shanghai Frontiers Science Center of TCM Chemical Biology at Shanghai University of Traditional Chinese Medicine and the National Key Laboratory of Lead Druggability Research (Shanghai Institute of Pharmaceutical Industry). The research was published under the title “Dual Ferroptosis Induction in N2-TANs and TNBC Cells via FTH1 Targeting: A Therapeutic Strategy for Triple-Negative Breast Cancer” in Cell Reports Medicine on January 21, 2025.


Research Process and Methods

Overall Workflow and Technical Details

The core of this study is the discovery of CT-1, a small-molecule cryptotanshinone derivative, and exploration of its mechanism in inducing dual ferroptosis of N2-type TANs and TNBC cells via ferritin heavy chain 1 (FTH1) targeting. The detailed workflow is as follows:

  1. Establishment and Characterization of N2 and N1 TANs
    Using the HL-60 model cell line, TANs were polarized into N1-type TANs (anti-tumor) and N2-type TANs (pro-tumor) under different stimulation conditions. Immunofluorescence and RNA sequencing verified the presence of abundant N2-type TANs in TNBC tissues and explored their characteristics, such as high lipid content and sensitivity to ferroptosis.

  2. Discovery and Structural Optimization of CT-1
    Based on the natural compound cryptotanshinone (CTS), the researchers developed CT-1. Advanced imaging techniques revealed that CT-1 significantly inhibited TNBC cell proliferation while selectively deactivating N2-type TANs.

  3. Verification of Ferroptosis-Inducing Mechanism via FTH1

    • Utilizing Western blot, drug affinity responsive target stability (DARTS) assays, and molecular dynamics modeling, FTH1 was identified as the direct target of CT-1. Mutagenesis experiments confirmed that Pro128 and Thr123 are the critical binding sites for CT-1 interaction with FTH1.
    • Electron microscopy (TEM) displayed ferroptotic features in cells treated with CT-1, such as loss of mitochondrial cristae and increased mitochondrial membrane density.
    • Measurements of ferroptosis markers (e.g., elevated Fe²⁺ levels, increased reactive oxygen species [ROS], and lipid peroxidation) further demonstrated CT-1’s effectiveness.
  4. Validation in vitro
    Co-culture experiments revealed that the synergistic interaction between N2-type TANs and TNBC cells was disrupted by CT-1. CT-1 effectively induced N2-type neutrophil ferroptosis while enhancing TNBC cell inhibition.

  5. Evaluation of Anti-Tumor Effects in vivo
    Mouse TNBC models confirmed the significant anti-tumor efficacy of CT-1. Intravenous administration of CT-1 inhibited tumor growth and improved mice survival rates. Immunohistochemical analysis demonstrated CT-1’s role in reducing FTH1 and antioxidant enzyme GPX4 expression, leading to increased ROS levels.

  6. Testing in Patient-Derived Organoids (PDOs)
    CT-1 suppressed the survival and growth of organoids derived from TNBC patients in a concentration-dependent manner. Live-dead staining confirmed CT-1’s superior effects compared to the parent compound, cryptotanshinone.

Data Analysis and Algorithmic Tools

The study incorporated RNA-Seq transcriptome analysis, immunofluorescence, mass spectrometry flow cytometry (CyTOF), and statistical analyses to ensure the precision and reliability of the results.


Results and Analysis

  1. High Expression of FTH1 in N2 TANs Makes Them Susceptible to Ferroptosis
    Single-cell sequencing and lipid staining revealed that N2 TANs exhibited high lipid content and elevated FTH1 expression, making them more sensitive to CT-1-induced ferroptosis.

  2. CT-1 Selectively Targets FTH1 and Promotes NCOA4-Mediated Ferritinophagy
    In N2 TANs and TNBC cells, CT-1 enhanced the interaction between FTH1 and NCOA4, promoting lysosomal degradation of FTH1 and inducing ferroptosis.

  3. CT-1 Demonstrates Significant Anti-Tumor Effects in vivo
    In mouse TNBC models, CT-1 reduced tumor growth and the proportion of N2 TANs in tumor tissue, sparing N1 TANs and other immune cells.

  4. CT-1 is Safe and Exhibits Favorable Pharmacokinetics
    Toxicity tests showed that high doses of CT-1 had no significant impact on mouse body weight, organ indices, or blood parameters, indicating a good safety profile.


Significance and Applications

This study establishes the critical role of FTH1 in both N2 TANs and TNBC cells, introducing CT-1 as a novel dual-targeting ferroptosis therapy. The discovery not only provides a potential clinical solution for TNBC but also opens new directions in cancer immunotherapy research.


Key Highlights

  1. Novel Mechanism
    The study unveils CT-1’s mechanism of triggering NCOA4-mediated ferritinophagy via FTH1, selectively inducing ferroptosis in both N2 TANs and TNBC cells.

  2. Selective Therapy
    CT-1 preserves the anti-tumor effects of N1 TANs while avoiding the immune suppression associated with traditional ferroptosis inducers.

  3. Translational Potential
    Structural optimization enhanced drug properties, laying a foundation for clinical application in TNBC treatment.


Future Directions

While successful in mouse models, further validation is needed using patient-derived xenograft models to confirm CT-1’s clinical viability. Additionally, the impact of CT-1 on other immune cell types, such as dendritic cells and myeloid-derived suppressor cells, warrants further investigation.