Ferroptosis Boosts Anti-Tumor Immunity by Degrading the Immune Suppressor SHP2
RSL3-induced ferroptosis degrades SHP2 via chaperone-mediated autophagy, amplifying IFN-γ/STAT1 signaling and enhancing anti-PD-1 immunotherapy efficacy.
Summary
Researchers discovered that inducing ferroptosis (iron-dependent cell death) in colorectal cancer cells using the compound RSL3 triggers a previously unknown immune-boosting mechanism. RSL3 activates chaperone-mediated autophagy to degrade SHP2, a protein that normally suppresses immune signaling. Without SHP2, cancer cells become far more sensitive to IFN-γ, ramping up STAT1 activity and increasing surface expression of MHC I molecules that flag cancer cells for destruction by T cells. In mouse models, combining RSL3 with anti-PD-1 antibodies produced superior tumor control compared to either treatment alone, suggesting ferroptosis inducers could overcome resistance to checkpoint immunotherapy, particularly in tumors with high SHP2 expression.
Detailed Summary
Colorectal cancer (CRC) remains the fifth leading cause of cancer mortality in China, with over 592,000 new cases in 2022 alone. Despite advances in immunotherapy using anti-PD-1 checkpoint inhibitors, many tumors resist treatment. This study explores a novel mechanism by which ferroptosis — a regulated, iron-dependent form of cell death driven by lipid peroxidation — can potentiate anti-tumor immune responses and sensitize tumors to immunotherapy.
To establish the biological connection, researchers analyzed a single-cell transcriptome dataset of 48,640 cells from 10 untreated human CRC tumor samples (GSE205506). Using AUCell scoring of 484 ferroptosis-related genes from the FerrDb V2 database, they defined FerrOptic high- and low-activity cancer cell populations. Gene set enrichment analysis (GSEA) revealed that ferroptosis-high cells showed markedly elevated Interferon Gamma Response and JAK-STAT signaling pathway activity compared to ferroptosis-low cells. This correlation was independently confirmed in a ferroptosis-induced myocardial infarction model (GSE114695), where STAT1 downstream targets were substantially upregulated.
In vitro, RSL3-treated SW480 colon cancer cells underwent proteomic mass spectrometry, revealing immune pathway enrichment and upregulation of antigen-presentation proteins HLA-A, HLA-C, and B2M. Critically, SHP2 — a non-receptor tyrosine phosphatase that negatively regulates STAT1 signaling — was significantly reduced after RSL3 treatment in SW480 and SW620 cells in a dose- and time-dependent manner. This reduction was ferroptosis-dependent: the specific ferroptosis inhibitor Ferrostatin-1 reversed SHP2 downregulation caused by both RSL3 and erastin across multiple CRC cell lines and in HeLa cervical cancer cells. Importantly, RSL3 did not reduce SHP2 mRNA; instead, SHP2 mRNA showed a slight increase, ruling out transcriptional suppression.
The mechanism of SHP2 degradation was traced to chaperone-mediated autophagy (CMA). RSL3 treatment caused SHP2 to co-localize with LAMP-2A on lysosomal membranes, and the interaction between SHP2 and the CMA chaperone HSC70 increased significantly. A KFERQ-like motif at SHP2 residues 530–534 was identified as essential: mutation of this motif abolished RSL3-induced SHP2 degradation. Blocking CMA genetically (LAMP-2A knockdown) or pharmacologically preserved SHP2 levels and negated the immunostimulatory effects of ferroptosis induction.
Functionally, RSL3 combined with IFN-γ significantly amplified p-STAT1 phosphorylation, nuclear translocation, and downstream expression of MHC I (HLA-ABC), PD-L1, and CXCL10 at both mRNA and protein levels compared to IFN-γ alone. This heightened sensitivity translated to increased susceptibility of RSL3-treated cancer cells to T cell-mediated killing in co-culture experiments. In vivo, mouse tumor models confirmed that RSL3 reduced intratumoral SHP2, increased CD8+ IFN-γ+ T cell infiltration, and enhanced tumor cell apoptosis. The RSL3 plus anti-PD-1 antibody combination produced superior tumor growth control versus either monotherapy, offering a compelling preclinical rationale for this combination approach.
The study carries important caveats. All in vivo data are from mouse syngeneic tumor models, and the clinical translatability of RSL3 itself remains uncertain given its pharmacological profile. The relationship between SHP2 overexpression, ferroptosis resistance, and immunotherapy non-response deserves prospective clinical validation. Nonetheless, this work identifies a clear mechanistic axis — ferroptosis → CMA-mediated SHP2 degradation → enhanced IFN-γ/STAT1 signaling → improved T cell cytotoxicity — that could guide the rational design of combination cancer immunotherapy strategies.
Key Findings
- FerrOptic high-activity CRC cells showed significantly upregulated Interferon Gamma Response and JAK-STAT pathway activity versus low-activity cells across 48,640 single cells (GSEA, adjusted p < 0.05)
- RSL3 reduced SHP2 protein levels in SW480 and SW620 cells in a dose- and time-dependent manner; Ferrostatin-1 fully reversed this reduction, confirming ferroptosis dependence
- SHP2 mRNA levels were not decreased — they showed a slight increase — after RSL3 treatment, ruling out transcriptional suppression as the mechanism of protein loss
- A KFERQ-like motif at SHP2 residues 530–534 was identified as essential for CMA-mediated degradation; mutation of this motif abolished RSL3-induced SHP2 loss
- RSL3 pretreatment followed by IFN-γ significantly amplified p-STAT1 levels, nuclear translocation, and surface MHC I (HLA-ABC) expression compared to IFN-γ alone (p < 0.01)
- RSL3 combined with IFN-γ increased mRNA levels of HLA-ABC, PD-L1, and CXCL10 in tumor cells, with both surface and total protein levels of HLA-ABC and PD-L1 elevated (p < 0.01)
- In vivo, RSL3 plus anti-PD-1 antibody combination produced superior tumor growth control compared to monotherapy, with increased intratumoral CD8+ IFN-γ+ T cells and enhanced tumor cell apoptosis
Methodology
The study combined bioinformatic analysis of a human CRC single-cell RNA sequencing dataset (GSE205506, n=48,640 cells from 10 patients) with in vitro proteomic mass spectrometry, western blotting, flow cytometry, confocal immunofluorescence, and co-culture T cell cytotoxicity assays across SW480, SW620, HT29, and HeLa cell lines. Ferroptosis dependence was confirmed using Ferrostatin-1 rescue experiments. In vivo efficacy was assessed in syngeneic mouse tumor models using RSL3 alone and in combination with anti-PD-1 antibody. Statistical analysis used unpaired Student's t-tests with results presented as mean ± SEM from three independent experiments; significance thresholds were p < 0.05 and p < 0.01.
Study Limitations
The study relies primarily on murine syngeneic tumor models for in vivo validation, which may not fully recapitulate the complexity of human colorectal cancer immunobiology and tumor microenvironments. RSL3 is a research-grade tool compound with limited pharmacological suitability for direct clinical translation, and no patient-derived data or clinical cohort validation was performed. The authors did not explicitly disclose conflicts of interest in the available text, and whether ferroptosis-CMA-SHP2 crosstalk generalizes to non-CRC cancer types requires further study.
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