PROTAC‑Mediated HMGCR Depletion Reprograms Lipid Metabolism in Breast Cancer to Potentiate Photoimmunotherapy via Ferroptosis

Abstract

Aberrant lipid metabolism characterizes the progression of breast cancer. Statins, the canonical agents for modulating this pathway, have been associated with improved overall survival in patients with triple-negative breast cancer (TNBC). However, their clinical benefit remains limited because the reversible inhibition of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) elicits a rebound in the mevalonate pathway and enables evasion of ferroptosis. Therefore, we developed a 170 nm self-assembled nanomedicine (PRO-P) that integrates an HMGCR-targeting PROTAC (PRO) with a disulfide-linked Pyropheophorbide-a (Ppa) photosensitizer, enabling laser-gated protein HMGCR degradation and photodynamic stress within one formulation. Under laser irradiation, PRO-P catalytically depletes HMGCR while generating reactive oxygen species (ROS), collapsing the mevalonate/CoQ10-GPX4 axis and redirecting lipids into ferroptosis. In 4T1 cells, PRO-P enhanced cellular uptake by 1.34-fold and elevated ROS by 9.5-fold. Following intravenous administration in TNBC xenografts, PRO-P achieved 92.5% tumor regression, eradicated pulmonary metastases, and elicited no systemic toxicity after single laser exposure. Immune profiling revealed remodeling of the microenvironment, with 2.6-fold more CD8⁺ Granzyme-B⁺ T cells, 4.3-fold more mature dendritic cells, and fewer Tregs, thereby establishing durable memory. PRO-P exploits multi-omics-guided HMGCR targeting to convert lipid addiction into a redox-immunologic vulnerability, yielding a low-toxicity therapy for TNBC and other lipid-driven cancers.

Keywords: PROTAC (proteolysis‐targeting chimera); breast cancer; ferroptosis; lipid metabolism; mevalonate pathway; photoimmunotherapy.

FIGURE 1

Schematic overview of the study workflow and therapeutic cascade for PROTAC‐mediated HMGCR depletion, which reprograms TNBC lipid metabolism and to potentiate photoimmunotherapy via ferroptosis.

FIGURE 2

Bulk RNA‐seq and scRNA‐seq analysis revealed that HMGCR promotes tumor growth by establishing an immunosuppressive microenvironment. (a) GeneMANIA analysis related to HMGCR. (b) GSEA Ridge Map related to HMGCR. (c) Volcano plot of differentially expressed genes between TNBC and normal sample groups. (d,e) Expression of HMGCR in TNBC cancer tissue and surrounding tissues. (f) The precision of HMGCR expression in patient diagnosis and prognostic outcome prediction was assessed using the TimeROC curve. (g,h) IPS score of high and low HMGCR expression groups. (i) UMAP diagram for cell annotation. (j) UMAP clustering diagram of cells across various TNBC groups. (k) Various categories consist of subsets of TNBC cells. (l) The expression differences of the HMGCR gene in TNBC among various cells. (m,n) Diagram of dimensionality reduction and grouping of T cell clusters. (o) Cytotoxicity functional score.

FIGURE 3

Synthesis and Physicochemical Characterization of PRO‐P. (a) Molecular structural formulas of PROTAC and AA‐SS‐Ppa. (b–d) Diameter dimensions and transmission electron microscopy images of PRO‐P, PRO, and Ppa, scale bar: 200 µm. (e) Energy‐dispersive X‐ray spectroscopy elemental mapping images of PRO‐P, scale bar: 100 µm. (f) Atomic Force Microscopy of PRO‐P. (g) Polymer dispersibility index of PRO‐P in water. (h) Zeta potential of PRO‐P. (i) UV–vis absorption of PRO‐P, PRO, and Ppa in water. (j,k) Hemolysis experiments of different drugs (10 µg/mL) and various concentrations of PRO‐P.

FIGURE 4

PRO‐P and statins related antitumor effects in vitro studies. (a) Confocal uptake images of 4T1 cells by PROTAC, Ppa, or PRO‐P, scale bar: 100 µm. (b) Flow cytometry uptake analysis of 4T1 cells by PROTAC, Ppa, or PRO‐P. (c) IC₅₀ of PRO‐P. (d) Flow cytometry analysis of the effects of different drug treatments on cell apoptosis. (e,f) Evaluation of drug cytotoxicity using Live‐Dead cell staining and (g) CCK‐8 assay in 4T1 cells, scale bar: 200 µm. (h,i) Scratch experiment analysis of the effect of different drug treatments in 4T1 cells, scale bar: 100 µm. Data are presented as mean ± SD (n = 3).

FIGURE 5

PRO‐P and statins targeting HMGCR to induce ferroptosis and antitumor effects in vitro studies. (a) Schematic illustration related to MVA and ferroptosis. (b–d) Expression of HMGCR with lovastatin and PROTAC, and expression of GPX4 with different drugs, as well as their (e–g) semi‐quantitative western blot analysis, respectively. (h,i) Confocal microscopy analysis of GPX4 protein expression after 4T1 treatment with different drugs. (j) GSH detection after treatment of 4T1 cells with different drugs. (k) CoQ10 synthesis in 4T1 cells treated with different drugs. (l,m) Confocal microscopy analysis of LPO accumulation after 4T1 treatment with different drugs. (n,o) Confocal microscopy analysis of mitochondrial membrane potential after 4T1 treatment with different drugs. (p,q) Confocal microscopy analysis of TFR‐1 after 4T1 treatment with different drugs. Data are presented as mean ± SD (n = 3), scale bar: 100 µm. Data are presented as mean ± SD (n = 3), scale bar: 100 µm.

FIGURE 6

Laser‐activated PRO‐P‐associated antitumor immunotherapy in vitro studies. (a) Schematic representation of the cellular immunological response triggered by ICD. (b) Cell survival rate after treatment with PROTAC, Ppa, or PRO‐P with or without laser irradiation. (c) Flow cytometry analysis of ROS generated. (d,f) Confocal microscopy analysis of ROS generated. (e) Assessment of intracellular ATP levels. (g–j) Confocal microscopy analysis of CRT and HMGB1 was generated. (k,l) GO and KEGG enrichment analysis charts of the TCGA database related to TNBC. (m,n) Co‐culture of 4T1 cells with mouse spleen lymphocytes after treatment with different drugs, and population expression of mature DCs (CD11c⁺CD40⁺CD86⁺), (o,p) cytotoxic T cells (CD45⁺CD3⁺CD8⁺), and (q,r) Treg cells (CD3⁺CD4⁺CD25⁺). Data are presented as mean ± SD (n = 3), scale bar: 100 µm.

FIGURE 7

Laser‐activated PRO‐P exerts antitumor therapeutic effects in vivo studies. (a) Schematic diagram of treatment in 4T1 tumor‐bearing mice. (b) in vivo NIR fluorescence imaging of the breast cancer tumor model after intravenous administration of Ppa or PRO‐P. Data are presented as mean ± SD (n = 3). (c) Semi‐quantitative analysis of in vitro organs. (d) Tumor size curves of mice within 18 days of treatment. (e) Body weight curves of mice within 18 days of treatment. (f) Tumor image after 18 days of treatment. (g) Tumor weight of mice. (h) Tumor burden of mice. Data are presented as mean ± SD (n = 6). (i) H&E, Tunel, Ki67, and GPX4 staining of tumor tissues, scale bar: 100 µm.

FIGURE 8

Laser‐activated PRO‐P demonstrates antitumor immune efficacy in vivo studies. (a) Variations in immune cell infiltration between groups with high and low expression of HMGCR. (b) Spearman analysis. (c) ESTIMATE analysis. (d) Variations in TIP scores across populations with high and low expression of HMGCR. (e) Flow cytometry detection of the effects of different drug treatments on cytotoxic T cells (CD8⁺ and CD4⁺) and (f) cytotoxic infiltrating lymphoid T cells (CD8⁺and Granzyme B⁺) generated in tumors. (g) Flow cytometry detection of the effects of different drug treatments on the production of Treg (CD25⁺) in tumors. (h) Flow cytometry detection of the effects of different drug treatments on NK cells (CD49b⁺ and CD3⁻) produced in tumors. (i) Flow cytometry detection of the effects of different drug treatments on cytotoxic T cells (CD8⁺) in the spleen. (j) Flow cytometry detection of the effects of different drug treatments on mature DCs (CD80⁺and CD86⁺) and (k) memory T cells (CD44⁺ and CD69L⁺) in the spleen. Data are presented as mean ± SD (n = 3).

FIGURE 9

Laser‐activated PRO‐P demonstrates anti‐metastatic ability in vivo studies. (a) Schematic diagram of distant tumor inoculation. (b) Distant tumor image after 17 days of treatment. (c) Distant tumor size curves of mice. (d) Weight of mice. Data are presented as mean ± SD (n = 4). (e) Schematic diagram of lung metastasis inoculation. (f) Baseline image of PET/MRI. (g‐h) PET/MRI images of whole body, in situ tumors, and lung metastases in PBS and PRO‐P‐L groups of mice. (i) SUV_bw calculation formula. (j) Quantification of TLG and MTV in situ tumors and lungs of mice in PBS and PRO‐P‐L groups. (k) Lung images and H&E after different drug treatments. Data are presented as mean ± SD (n = 3), scale bar: 100 µm.

FIGURE 10

Comparative proteomics between PRO‐P‐L and PBS groups. (a) Principal component analysis (PCA) of the quantitative proteomics (n = 4). (b) The volcano plot of differentially expressed proteins (DEPs) between PRO‐P‐L and PBS groups. (c) The heatmap of significant differential proteins (354 species). (d) GO enrichment of DEPs in the term of biological process (BP). (e) Chord diagram of the enriched BP terms with the associated DEPs. (f) The box plots of the differentially expressed proteins between PRO‐P‐L and PBS groups.