PRMT5 reduces immunotherapy efficacy in triple-negative breast cancer by methylating KEAP1 and inhibiting ferroptosis

Abstract

Background: As an emerging treatment strategy for triple-negative breast cancer (TNBC), immunotherapy acts in part by inducing ferroptosis. Recent studies have shown that protein arginine methyltransferase 5 (PRMT5) has distinct roles in immunotherapy among multiple cancers by modulating the tumor microenvironment. However, the role of PRMT5 during ferroptosis, especially for TNBC immunotherapy, is unclear.

Methods: PRMT5 expression in TNBC was measured by IHC (immunohistochemistry) staining. To explore the function of PRMT5 in ferroptosis inducers and immunotherapy, functional experiments were conducted. A panel of biochemical assays was used to discover potential mechanisms.

Results: PRMT5 promoted ferroptosis resistance in TNBC but impaired ferroptosis resistance in non-TNBC. Mechanistically, PRMT5 selectively methylated KEAP1 and thereby downregulated NRF2 and its downstream targets which can be divided into two groups: pro-ferroptosis and anti-ferroptosis. We found that the cellular ferrous level might be a critical factor in determining cell fate as NRF2 changes. In the context of higher ferrous concentrations in TNBC cells, PRMT5 inhibited the NRF2/HMOX1 pathway and slowed the import of ferrous. In addition, a high PRMT5 protein level indicated strong resistance of TNBC to immunotherapy, and PRMT5 inhibitors potentiated the therapeutic efficacy of immunotherapy.

Conclusions: Our results reveal that the activation of PRMT5 can modulate iron metabolism and drive resistance to ferroptosis inducers and immunotherapy. Accordingly, PRMT5 can be used as a target to change the immune resistance of TNBC.

Keywords: Breast Neoplasms; Immunotherapy.

Figure 1

PRMT5 overexpression prevents the ferroptosis of TNBC cells. (A) After knockdown or overexpression of PRMT5, HCC1937 and MDA-MB-231 cells were treated with 0, 1, 2, 4, 8, and 16 μM erastin or 0, 0.5, 1, 2, 4, and 8 μM RSL3 for 24 hours. The killing efficiency was examined using the MTT assay. (B,C) PRMT5 overexpressing or PRMT5-deficient HCC1937 and MDA-MB-231 cells were treated with 3 μM RSL3 (3 μM) for 12 hours, and then BODIPY 581/591 C11 staining (B) and MDA assays (C) were conducted to detect the content of cellular lipid ROS (B) and MDA (C), respectively. (D) After 24 hours of treatment with 1 μM RSL3 in combination with 60 μM 3-MA, 10 μM Z-VAD-FMK, 10 μM necrostatin-1, or 1 μM Fer-1, the death of PRMT5-deficient HCC1937 and MDA-MB-231 cells was examined by flow cytometry. Average of three experiments. *p<0.05; **p<0.01; NS, no significance. PRMT5, protein arginine methyltransferase 5; TNBC, triple-negative breast cancer; DMSO, dimethyl sulfoxide; ROS, reactive oxygen species.

Figure 2

PRMT5 suppresses the ferroptosis of TNBC cells by downregulating HMOX1. (A,B) After 6 hours of treatment with 3 μM RSL3, the expression of HMOX1 in differentially treated MDA-MB-231 and HCC1937 cells at the mRNA (B) and protein (C) levels was examined by RT-qPCR (B) and western blotting (C), respectively. (D–F) HCC1937 and MDA-MB-231 cells overexpressing or not overexpressing HMOX1 were transfected with PRMT5 and then treated with 1 μM RSL3. After 12-hour and 24-hour treatments, MTT and MDA assays were performed to determine cell viability and intracellular MDA, respectively. Intracellular ferrous content was assessed by an Iron Assay Kit after 6 hours. Average of three experiments. **p<0.01. PRMT5, protein arginine methyltransferase 5; TNBC, triple-negative breast cancer; DMSO, dimethyl sulfoxide; JNJ, PRMT5 inhibitor, Onametostat (JNJ-64619178).

Figure 3

PRMT5 inhibits NRF2/HMOX1 by methylating and stabilizing KEAP1. (A,B) PRMT5 overexpression promotes the translation of KEAP1 and suppresses the expression of NRF2 and its targets (HMOX1, SLC7A11, and GPX4) in the presence of RSL3 and vice versa. (C,D) Representative blots showing the interaction of PRMT5 and KEAP1, as well as the methylation of KEAP1. (E) Deficiency of PRMT5 decreases the methylation of KEAP1 in MDA-MB-231 and HCC1937 cells. (F) Deficiency of PRMT5 significantly accelerated KEAP1 degradation in both cell lines. (G) Overexpression of PRMT5 increased the half-life of KEAP1. Representative graph showing the summarized results of the left panel. (H) The decreased expression of KEAP1 in HCC1937 and MDA-MB-231 cells caused by PRMT5 deficiency was blocked by MG132. (I) The positive correlation of PRMT5 expression with KEAP1 expression in human breast cancer specimens was determined by immunohistochemistry. Representative staining results are shown. Average of three experiments. *p<0.05. PRMT5, protein arginine methyltransferase 5.

Figure 4

PRMT5 inhibits TRIM25-mediated KEAP1 ubiquitination by inducing KEAP1R596me2. (A) Overexpression of PRMT5 suppresses the ubiquitination of KEAP1 in an arginine methyltransferase activity-dependent manner. (B) PRMT5 overexpression interfered with TRIM25 binding to KEAP1 in an arginine methyltransferase activity-dependent manner. The cells transfected with the indicated plasmids were extracted following IP and IB analysis. (C) The level of KEAP1 protein was restored by the double silencing of PRMT5 and TRIM25. (D) Representative graph showing the regions in KEAP1 that interact with PRMT5. KEAP1 179-321 aa and 322-624 aa could bind to PRMT5 while KEAP1 322-624 aa can be methylated. (E) Identification of the domain in PRMT5 involved in the interaction with KEAP1. The 1-324 aa region participates in the interaction of PRMT5 with KEAP1. (F) Representative graph showing the putative methylated residues of KEAP1. (G) R596 of KEAP1 can be methylated by PRMT5. After transfection with different plasmids, HEK293T cells were collected, and co-IP was conducted to determine methylation at the indicated site. (H) The level of ubiquitination of KEAP1-R596K was significantly lower than that of the other mutants. Average of three experiments. IB, immunoblotting; IP, immunoprecipitation; PRMT5, protein arginine methyltransferase 5; HA, anti-HA tag.

Figure 5

Cellular ferrous levels might be the key to influencing cell fate in the case of alterations in PRMT5 or NRF2 protein levels. (A) Different breast cancer cell lines were transfected with PRMT5 or NRF2 plasmids and then treated with RSL3 for 24 hours. Then, an MTT assay was conducted to determine cell viability. (B) NRF2 expression-related survival curves of all patients with breast cancer or patients with triple negative breast cancer. (C) Cellular ferrous levels of a panel of breast cancer cell lines were determined using FerroOrange. Scale bar, 100 μm. Average of three experiments. *p<0.05. PRMT5, protein arginine methyltransferase 5; TNBC, triple-negative breast cancer.

Figure 6

PRMT5 inhibitors potentiated the therapeutic efficacy of immunotherapy. (A) PRMT5 overexpression prevents the cytotoxic effect of IFN-γ, RSL3 and erastin on murine 4T1 mammary carcinoma cells. (B) Representative blots showing the expression of NRF2, HMOX1, and KEAP1 in 4T1 cells treated with RSL3 (3 µM), erastin (8 µM) or IFN-γ (20 ng/mL) in combination with or without PRMT5 inhibitors. (C) Schematic showing the animal anti-PD1 administration scheme. Tumor-bearing mice were given anti-PD1 antibody or IgG as a control on the indicated days for a total of five treatments. (D) Excised tumors collected on day 19. (E) Representative graph showing the calculated tumor volume at the indicated time points. (F) The tumor weights of excised tumors were measured at the end of the experiment. (G) Representative graph showing the content of MDA in the tumors collected at day 19. (H) PRMT5 inhibitors in combination with anti-PD1 significantly decreased the translation of KEAP1 and increased the production of NRF2 and HMOX1 at the protein level in the indicated tumor tissues. (I) The HMOX1 expression-related survival curves of patients with cancer who received immunotherapy. (J) Expression of PRMT5, NRF2 and HMOX1 protein in human specimens derived from eight patients with TNBC who received immunotherapy. *p<0.05. CR, complete response; IFN, interferon; PD, progressive disease; PR, partial response; PRMT5, protein arginine methyltransferase 5; SD, stable disease.