A marine-derived small molecule induces immunogenic cell death against triple-negative breast cancer through ER stress-CHOP pathway

Abstract

Although triple-negative breast cancer (TNBC) is the most refractory subtype among all breast cancers, it has been shown to have higher immune infiltration than other subtypes. We identified the marine-derived small molecule MHO7, which acts as a potent immunogenic cell death (ICD) inducer through the endoplasmic reticulum (ER) stress-C/EBP-homologous protein (CHOP) pathway, to treat TNBC. MHO7 exerted cytostatic and cytotoxic effects on TNBC cells at an IC₅₀ of 0.96-1.75 µM and suppressed tumor growth with an approximately 80% inhibition rate at a dose of 60 mg/kg. In 4T1 cell tumor-bearing mice, 30 mg/kg MHO7 inhibited pulmonary metastasis with an efficacy of 70.26%. Transcriptome analyses revealed that MHO7 changed the transcription of genes related to ribosome and protein processes in the ER. MHO7 also triggered reactive oxygen species (ROS) generation and attenuated glutathione (GSH) levels, which caused excessive oxidative stress and ER stress via the PERK/eIF2α/AFT4/CHOP pathway and led to cell apoptosis. ER stress and ROS production facilitated the release of ICD-related danger-associated molecular patterns (DAMPs) from TNBC cells, which activated the immune response in vivo, as indicated by the release of antitumor cytokines such as IL-6, IL-1β, IFN-γ, and TNF-α, increases in CD86⁺ and MHC-II dendritic cells and CD4⁺ and CD8⁺ T cells and a decrease in regulatory T cells (Tregs). These results reveal that MHO7 triggers an aggressive stress response to amplify tumor immunogenicity and induce a robust immune response. This synergistic effect inhibits primary breast cancer growth and spontaneous metastasis in TNBC, providing a new strategy for TNBC treatment.

Keywords: C/EBP-homologous protein; endoplasmic reticulum stress; immunogenic cell death; oxidative stress; sesterterpene; triple-negative breast cancer.

Figure 1

Antitumor effect of MHO7 on TNBC in vitro and in vivo. (A) The chemical structure of MHO7. (B) The IC₅₀ value of MHO7 on TNBC cell lines at 12, 24, and 48 h. (C) The percentages of apoptosis cells were measured by flow cytometry under MHO7 treatment at 24 h. (D) Tumor growth was measured in mice. Data was presented as mean ± SEM. (n=6). (E) Final tumor weight was measured in each group. Data was presented as mean ± SEM. (n=6). (F) Tumor tissue was analyzed by H&E staining and immunohistochemistry analysis of Ki67. Scale bars 50 µm. (n=3). *P <0.05; **P < 0.01; ***P <0.001; and**** P < 0.0001.

Figure 2

MHO7 induced ER stress and cell cycle arrest in TNBC cells. (A) The top KEGG pathway enrichment was analyzed by RNA-seq. The red highlighted upregulated pathways, and the blue highlighted downregulated pathways. (B) The heat map of DEGs related to ER stress. (C) ER stress-regulated genes in MDA-MB-231 cells were detected by qRT-PCR under MHO7 treatment. (D) The expression of BiP/p-PERK/p-eIF2α/ATF4/CHOP were measured by western blot under MHO7 treatment in MDA-MB-231 cells. (E) Bcl-2 was detected by qRT-PCR when si-CHOP or MHO7 (3 µM) treatment. (F) Bax was detected by qRT-PCR when si-CHOP or MHO7 (3 µM) treatment. (G) Representative apoptosis rate was measured by flow cytometry under si-CHOP or MHO7 (3 µM) treatment. *P <0.05; **P < 0.01; ***P <0.001; and ****P < 0.0001; ns=no significant.

Figure 3

MHO7-induced ROS generation contributed to ER stress-mediated apoptosis. (A) The ROS level was detected by flow cytometry when treated with MHO7 for 24 h or pretreated with NAC (4 mM) for 1 h in MDA-MB-231 cells. (B) GSH /GSSG ratio of MDA-MB-231 cells was detected under the treatment of MHO7 (2, 3 µM) and pretreatment of NAC (4 mM). (C) The percentages of apoptosis cells were measured by flow cytometry when treated with MHO7 for 24 h or pretreated with NAC (4 mM) for 1 h in MDA-MB-231 cells. (D) ER stress-related proteins were measured by western blot under the treatment of MHO7 (+: 2 µM; ++:3 µM) or pretreated with NAC (4 mM) in MDA-MB-231 cells. (E) Intracytoplasmic calcium ionic was detected by flow cytometry after the treatment of MHO7 in MDA-MB-231 and 4T1 cells. *P <0.05; **P < 0.01; ***P <0.001; and ****P < 0.0001.

Figure 4

MHO7 induced the release of DAMPs from TNBC cells. (A) CRT translocation was analyzed by immunofluorescence staining after exposure to MHO7 or DOX (2 µM). Scale bars 25 µm. (B) Expression of CRT and HSP70 proteins in the cell cytosol and the cell membrane were measured by western blot under treatment with MHO7. (C) HMGB1 exposure was detected by immunofluorescence staining after MHO7 or DOX (2 µM) treatment. Scale bars 50 µm. (D) ATP was detected in the cell supernatant of MDA -MB-231 and 4T1 cells after MHO7 or DOX (2 µM) treatment for 24 h. *P <0.05; **P < 0.01; ns: no significance.

Figure 5

MHO7 suppressed tumor growth and pulmonary metastases in tumor-bearing mice. (A) Diagram of procedure for mouse tumor model. (B) The Observation of the primary tumors. (C) Tumor growth and the final tumor weights were measured in mice. (n=6). Data was presented as mean ± SEM. (D) Ki67 and cleaved caspase 3 expression in tumor tissues were analyzed by H&E staining and IHC staining. Scale bar, 50 µm. (E) Lung tissues in mice was analyzed by H&E staining, and the tumor areas were indicated by the black circle. *P <0.05; **P < 0.01; ***P <0.001; and ****P < 0.0001.

Figure 6

MHO7 activated the antitumor immune response in mice. (A) KEGG pathway enrichment of DEGs was analyzed by RNA-seq in 4T1 tumor tissue. (B) GO enrichment of DEGs was analyzed by RNA-seq in 4T1 tumor tissue. (C) Percentages of CD11C⁺/CD86⁺ DCs were measured by flow cytometry in spleens. (D) Percentages of CD11C⁺/ MHC II⁺ DCs were measured by flow cytometry in spleens. (E) Percentages of CD3⁺/CD8⁺ T cells were measured by flow cytometry in spleens. (F) Percentages of CD4⁺/FOXP3⁺ T cells were measured by flow cytometry in spleens. (n=6) The level of (G) IFN-γ, (H) TNF-α, (I) IL-1β, and (J) IL-6 were measured by ELISA in the serum on day 7 after treatment. Data was presented as mean ± SEM. *P <0.05; **P < 0.01; ***P <0.001; and ****P < 0.0001; ns=no significance.

Figure 7

MHO7 induced ICD in vivo. (A) Tumor sections were stained with CRT (green) and DAPI (blue). (B) Tumor sections were stained with HMGB1 (red) and DAPI (blue). (C) Tumor sections were stained with CD8⁺ (green) and DAPI (blue). (D) Tumor sections were stained with FOXP3⁺ (red) and DAPI (blue). Scale bar, 20 µm. (E) Diagram of procedure for vaccination assay in mice. (F) Tumor growth was measured in mice. Data was presented as mean ± SEM. (G) Tumor-free progression was measured in mice. Log rank test. (H) Overall survival was measured of mice. Log rank test. *P <0.05; **P < 0.01; ***P <0.001; ns=no significant.