KLF11 regulates lung adenocarcinoma ferroptosis and chemosensitivity by suppressing GPX4

Abstract

Ferroptosis, an iron-dependent non-apoptotic cell death, has been shown to play a vital role in tumor proliferation and chemotherapy resistance. Here, we report that KLF11 inhibits lung adenocarcinoma (LUAD) cell proliferation and promotes chemotherapy sensitivity by participating in the GPX4-related ferroptosis pathway. Through an RNA-sequence screen from LUAD cells pretreatment with ferroptosis inducers (FINs), we discovered that KLF11 expression was significantly higher in FINs-treated cells, suggesting that KLF11 may be involved in ferroptosis. Overexpression of KLF11 promoted LUAD cells to undergo ferroptosis alterations. Meanwhile, upregulation of KLF11 expression also inhibited cell proliferation and increased chemosensitivity, whereas knockout of KLF11 did the opposite. With ChIP-Seq and RNA-Seq, we identified GPX4 as a downstream target of KLF11. Through ChIP-qPCR and dual luciferase assay, we clarified that KLF11 binds to the promoter region of GPX4 and represses its transcription. Restored GPX4 expression antagonized the ability of KLF11 to promote ferroptosis, increase chemotherapy sensitivity and inhibit cell proliferation in vitro and in vivo. Clinically, KLF11 declined in LUAD and its low expression was associated with reduced patient survival. Our findings established the function of KLF11 to promote ferroptosis in LUAD, thereby inhibiting cell proliferation and enhancing the efficacy of chemotherapy.

Fig. 1. Identification of KLF11 as involved in ferroptosis in LUAD.

a Schematic diagram of RNA-seq screening workflow in A549 cell line. b, c Volcano plot illustrating KLF11 involved in regulating ferroptosis induced by RSL3 and IKE. d, e KLF11 RNA level was measured after treatment with RSL3 or DFO + RSL3 or Ferr1 + RSL3 in A549 and PC9 cell lines. f, g KLF11 protein level was measured after treatment with RSL3 or DFO + RSL3 or Ferr1 + RSL3 in A549 and PC9 cell lines.

Fig. 2. Dysregulated KLF11 expression affects the cytotoxicity and intracellular ROS levels of LUAD cell lines induced by RSL3 and IKE-induced.

a–d qRT-PCR and western blot confirming the overexpression and knock-out of KLF11 in A549 and PC9 cells. e–h CCK-8 assay to detect the cell activity of A549 and PC9 cells with different levels of KLF11 after 24 h of treatment with different doses of RSL3 and IKE. i–l Lipid peroxidation was measured by flow cytometry after C11-BODIPY staining in KLF11-NC, KLF11-OE, and KLF11-KO treatment by RSL3 or IKE.

Fig. 3. Dysregulation of KLF11 expression causes abnormal mitochondrial morphology in RSL3 or IKE-treated LUAD cells and affects the proliferation ability and sensitivity to chemotherapeutic agents.

a Characteristic alterations of mitochondria in A549 cells treated with RSL3 or IKE analyzed by TEM, Scale bars: 50 nm. b, c Dysregulation of KLF11 expression affects the proliferation ability of A549 and PC9 cells. d–g Dose-toxicity curves showing the viability of A549 and PC9 cells transfected with KLF11-NC, KLF11-OE, and KLF11-KO upon CDDP or Pem treatment at the indicated concentrations for 48 h. h–k The viability of A549 and PC9 cells transfected with KLF11-NC, KLF11-OE, and KLF11-KO were measured after 24 h treatment with RSL3/IKE, RSL3/IKE+Ferr1, or RSL3/IKE+Z-VAD.

Fig. 4. Identification of GPX4 as the target molecule of KLF11 to regulate ferroptosis.

a Schematic diagram of RNA-seq screening workflow in A549 cell line transfected with KLF11-NC and KLF11-OE. b Volcano plot showing differential expression of genes between the A549-KLF11-NC and KLF11-OE. c Venn plot showing the intersection of predicted targets of KLF11. d, e qRT-PCR results showed differences in RNA levels of three key molecules (GPX4, SLC7A11 and ASCL4) of ferroptosis between the KLF11-NC, KLF11-OE and KLF11-KO of A549 and PC9 cell lines. f, g Western blot results showed differences in protein levels of GPX4, SLC7A11, and ASCL4 of ferroptosis between the KLF11-NC, KLF11-OE, and KLF11-KO of A549 and PC9 cell lines.

Fig. 5. KLF11 binds to the GPX4 promoter region and represses its transcription.

a, b Genome-wide data from the ENCODE project and our ChIP-seq results show the solid KLF11-binding peak in the promoter region close to the TSS of GPX4. c Characteristic sequences predicted by JASPAR for transcriptional binding of KLF11. d, e ChIP-qPCR assay depicting the enrichments of KLF11 binding in A549 and PC9. f Schematic diagram showing the possible binding sites of KLF11 to the critical region of the GPX4 promoter are mutated. g Dual luciferase activity assays to analyze the fluorescence intensity of KLF11-NC and KLF11-OE with or without mutations in the GPX4 promoter region.

Fig. 6. Restoring GPX4 expression antagonizes KLF11’s ability to promote ferroptosis and inhibit cell proliferation.

a–f qRT-PCR and western blot confirming the restored GPX4 expression in A549 and PC9 cells transfected with KLF11-NC and KLF11-OE. g–j Dose-toxicity curves showing the viability of A549 and PC9 cells transfected with KLF11-NC, KLF11-OE, KLF11-NC+GPX4, and KLF11-OE+GPX4 upon RSL3 or IKE treatment at the indicated concentrations for 24 h. k–n Lipid peroxidation was measured by flow cytometry after C11-BODIPY staining in KLF11-NC, KLF11-OE, KLF11-NC+GPX4, and KLF11-OE+GPX4 treatment by RSL3 or IKE. o Characteristic alterations of mitochondria in A549 cells treated with RSL3 or IKE analyzed by TEM after restored GPX4 expression, Scale bars: 50 nm. p, q GPX4 expression promotes the proliferation of A549 and PC9 cells inhibited by KLF11.

Fig. 7. GPX4 expression antagonizes KLF11’s ability to increase chemotherapy sensitivity in vitro and in vivo, KLF11 is lowly expressed in LUAD and positively correlates with prognosis.

a–d Dose-toxicity curves showing the viability of A549 and PC9 cells transfected with KLF11-NC, KLF11-OE, KLF11-NC+GPX4, and KLF11-OE+GPX4 upon CDDP or Pem treatment at the indicated concentrations for 48 h. e Schematic diagram showing the process of subcutaneous tumor formation in nude mice and the treatment measures. f Representative tumors formed in nude mice by KLF11-NC(a), KLF11-OE(b), KLF11-NC+GPX4(c), and KLF11-OE+GPX4(d) cells upon IKE or CDDP+Pem treatment. g The weight of the final tumor formed between different groups. h The expression of KLF11 between adjacent normal tissues and tumor tissues from patients with LUAD in our hospital. i The expression of KLF11 was negatively correlated with GPX4. j The low expression of KLF11 in LUAD compared to normal lung tissue was verified in the TCGA database. k, l Both our cohort and the TCGA cohort showed a positive correlation between KLF11 expression and prognosis in LUAD patients. m Mechanism of KLF11 regulation of GPX4 transcription.