Breast cancer cells have an increased ferroptosis risk induced by system xc- blockade after deliberately downregulating CYTL1 to mediate malignancy

Abstract

Cytokine-like protein 1 (CYTL1) expression is deliberately downregulated during the progression of multiple types of cancers, especially breast cancer. However, the metabolic characteristics of cancer progression remain unclear. Here, we uncovered a risk of breast cancer cells harboring low CYTL1 expression, which is metabolically controlled during malignant progression. We performed metabolism comparison and revealed that breast cancer cells with low CYTL1 expression have highly suppressed transsulfuration activity that is driven by cystathionine β-synthase (CBS) and contributes to de novo cysteine synthesis. Mechanistically, CYTL1 activated Nrf2 by promoting autophagic Keap1 degradation, and Nrf2 subsequently transactivated CBS expression. Due to the lack of cellular cysteine synthesis, breast cancer cells with low CYTL1 expression showed hypersensitivity to system x_c^- blockade-induced ferroptosis in vitro and in vivo. Silencing CBS counteracted CYTL1-mediated ferroptosis resistance. Our results show the importance of exogeneous cysteine in breast cancer cells with low CYTL1 expression and highlight a potential metabolic vulnerability to target.

Keywords: Breast cancer; CBS; CYTL1; Ferroptosis; Transsulfuration pathway.

Graphical abstract

Fig. 1

The transsulfuration activity is suppressed in breast cancer with low level of CYTL1. (A) Metabolomic comparison of MDA-MB-231 cells with intact or stably overexpressed CYTL1. Six biological replicates are shown as separate columns for each cell type. (B) Schematic of the serine-glycine and one-carbon pathways. Metabolic enzymes are in red. Folate cycle is in green. Methionine cycle is in orange. Transsulfuration pathway is in blue. (C) Abundance of intracellular primary one-carbon metabolites as determined by LC-MS, normalized to the abundance in MDA-MB-231 cells with intact CYTL1. (D) GSH levels in the indicated breast cancer cell lines with different levels of intact CYTL1 expression were determined using kit assays. (E) GSH levels in MDA-MB-231 cells transfected with HA-tagged CYTL1 expressing plasmids. (F) GSH levels in ZR-75-1 cells transfected with CYTL1 shRNAs. (G) GSH levels in breast cancer samples in different clinical stages. Each stage shows three samples. CYTL1 protein expression was determined by Western blot. The statistical significance of the differences between groups was determined by (C–G) two-tailed Student's t-test (*P < 0.05, ***P < 0.001 versus NC, ZR-75-1 or shNC). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 2

CBS expression is positively associated with CYTL1 abundance in breast cancer cells. (A) CBS protein expression as determined by Western blot in the indicated breast cancer cell lines. GAPDH was used as a loading control. The densitometry of the immunoblots was performed with image J software and is presented in the histograms (right panel). (B, C) The mRNA (B) and protein (C) levels of CBS were detected in MDA-MB-231 cells transfected with HA-tagged CYTL1 expressing plasmids by real-time PCR and Western blot, respectively. (D, E) The mRNA (D) and protein (E) levels of CBS were detected in ZR-75-1 cells transfected with CYTL1 shRNAs. (F) CBS protein expression as determined by Western blot in breast cancer samples in different clinical stages. Each stage shows three samples. The statistical significance of the differences between groups was determined by two-tailed Student's t-test (*P < 0.05, **P < 0.01, ***P < 0.001 versus ZR-75-1, NC or shNC).

Fig. 3

CYTL1 stabilizes Nrf2 by preventing Keap1-mediated proteasomal degradation to upregulate CBS expression. (A–C) CYTL1 stably expressing MDA-MB-231 cells were transfected with Nrf2 siRNAs. The mRNA (A) and protein (B) levels of CBS were detected by real-time PCR and Western blot, respectively. (C) Cellular cysteine levels were detected using kit assays. (D) Nrf2 protein expression in MDA-MB-231 cells transfected with HA-tagged CYTL1 expressing plasmids. (E) Nrf2 protein expression in ZR-75-1 cells transfected with CYTL1 shRNAs. (F) The degradation of Nrf2 over time in the presence of cycloheximide (CHX) was monitored by Western blot in CYTL1 stably expressing MDA-MB-231 cells. (G) Co-IP analysis of the interaction between Nrf2 and HA-Ub in HEK293T cells cotransfected with Myc-NC or Myc-CYTL1 plasmids. (H) The protein expression of Keap1, p62, LC3B was determined by Western blot in CYTL1 stably expressing MDA-MB-231 cells. (I) Immunocytochemical detection of auotophagosome in CYTL1 stably expressing MDA-MB-231 cells fused with lysosomes. Left panel: representative images. Scale bars, 5 μm. Right panel: quantification of co-localization of LC3 with Lyso Tracker. Ten random fields were photographed per coverslip. The statistical significance of the differences between groups was determined by (A-E, G-H) two-tailed Student's t-test (*P < 0.05, ***P < 0.001 versus NC or shNC).

Fig. 4

Inhibition of extracellular cystine import facilitates ferroptosis in breast cancer cells with low level of CYTL1. (A) Cell viability of MDA-MB-231 and ZR-75-1 cells was detected by MTT assay after treatment with 20 μM erastin for the indicated periods. (B, C) Cell viability of MDA-MB-231 cells transfected with HA-tagged CYTL1 expressing plasmids after treatment (B) with various concentrations of erastin for 48 h or (C) with 20 μM erastin for the indicated periods. (D) Cell viability of ZR-75-1 cells transfected with CYTL1 shRNAs after treatment with 20 μM erastin for the indicated periods. (E) Cell viability of ZR-75-1 cells transfected with CYTL1 shRNAs after treatment with 20 μM erastin for 24 h in the absence or presence of 2 μM ferroststin-1. (F–J) CYTL1-overexpressing MDA-MB-231 cells were treated with 20 μM erastin for 48 h. (F) ROS levels were measured by flow cytometry using MitoSOX Red. Quantification of ROS production was analyzed using mean fluorescence intensity (MFI). (G) Intracellular Fe²⁺, (H) GSH and (I) MDA levels were determined using kit assays. (J) The protein expression of GPX4 and p53 as determined by Western blot. The statistical significance of the differences between groups was determined by two-tailed Student's t-test (*P < 0.05, **P < 0.01, ***P < 0.001 versus ZR-75-1, NC or shNC). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 5

Knockdown of CBS counteracts system x_c⁻ blockade-mediated ferroptosis resistance by CYTL1. (A) Western blot demonstrating CBC silence in CYTL1 stably expressing MDA-MB-231 cells transfected with siRNAs specific for CBS. (B–E) The resulting cells were treated with 20 μM erastin for 48 h. (B) Cell viability. (C) ROS levels. (D) GSH levels. (E) MDA levels. The statistical significance of the differences between groups was determined by two-tailed Student's t-test (*P < 0.05, **P < 0.01, ***P < 0.001 versus siNC).

Fig. 6

Low expression of CYTL1 renders breast cancer cells preferentially sensitive to system x_c⁻ blockade-mediated ferroptosis. (A–F) Female C57BL/6 mice were subjected to orthotopic injection with E0771 cells stably expressing vector control (NC) or CYTL1, and administered with olive oil or erastin (20 mg/kg, twice each other day) for 20 days. Some mice were sacrificed on day 21 after administration. Some mice were monitored until they died. (A) Tumor growth curves (n = 6). Left panel: representative images of the tumors at the end of the experiments. (B) Tumor weight. (C) GSH levels in tumor tissues. (D) MDA levels in tumor tissues. (E) The protein expression of GPX4, p53, Nrf2 and CBS in tumor tissues. (F) Survival curve of mice (n = 6). (G–K) Female nude mice were subjected to orthotopic injection with MDA-MB-231 cells stably expressing NC or CYTL1, and administered for 16 days as described above. The mice were sacrificed on day 17 after administration. (G) Tumor growth curves (n = 5). (H) Tumor weight. (I) GSH levels in tumor tissues. (J) MDA levels in tumor tissues. (K) The protein expression of GPX4 and p53 in tumor tissues. Animal experiments were performed twice independently. The statistical significance of the differences between groups was determined by one-way ANOVA with post hoc Tukey HSD test (*P < 0.05, **P < 0.01, ***P < 0.001 versus NC or CYTL1 group).