LACTB suppresses liver cancer progression through regulation of ferroptosis

Abstract

Ferroptosis, driven by iron-dependent phospholipid peroxidation, is emerging as an intrinsic cancer defense mechanism. However, the regulatory networks involved in ferroptosis remain largely unknown. Here, we found that serine beta-lactamase-like protein (LACTB) inhibits liver cancer progression by regulating ferroptosis. LACTB is downregulated in liver cancer, and the ectopic expression of LACTB markedly inhibits cell viability, colony formation, and tumour growth. LACTB knockout exerts the opposite effects. Further investigation revealed that LACTB blocks HSPA8 transcription in a p53-dependent manner, resulting in the elevation of NCOA4-mediated ferritinophagy and inhibition of SLC7A11/GSH/GPX4 signalling, thereby triggering ferroptosis and suppressing liver cancer progression. Liver cancer cells with an endogenous mutation of p53 binding site in the HSPA8 promoter exhibited increased resistance to ferroptosis inducers, and the ferroptosis-promoting effect of LACTB was significantly weakened in these mutant cells. Importantly, LACTB is identified as a downstream target of lenvatinib, and adeno-associated virus-mediated overexpression and knockdown of LACTB notably enhance and attenuate the anti-tumour efficacy of lenvatinib in vivo, respectively. Taken together, our study reveals a novel action of LACTB and provides potential therapeutic strategies for enhancing the efficacy of lenvatinib in liver cancer.

Keywords: Ferroptosis; LACTB; Lenvatinib; Liver cancer.

Fig. 1

LACTB inhibits liver cancer progression. A. Western blot testing the protein levels of LACTB in ten paired liver cancer and normal tissues. B. IHC staining of LACTB in 12 normal and 31 liver cancer tissues. Scale bar = 50 μm. C. CPTAC database showing LACTB protein expression in normal and liver cancer tissues. D. Western blot verifying LACTB overexpression in SK-HEP-1 cells. E. CCK-8 assay testing cell viability in LACTB-expressing SK-HEP-1 cells. F. Colony formation assay testing the cloning ability of LACTB-expressing HepG2 and SK-HEP-1 cells. G. Western blot verifying LACTB knockout in SK-HEP-1 cells. H, I. CCK-8 and colony formation assays testing cell viability and cloning ability in LACTB^−/− cells, respectively. J. Xenograft tumour model testing the in vivo effects of LACTB overexpression on SK-HEP-1 cell growth. Scale bar = 1 cm. Paired Student's t-test was used for A, Student's t-test was used for B, F, J (right panel), two-way ANOVA with Sidak post-hoc test was used for E, H, J (middle panel), and one-way ANOVA with Dunnett post-hoc test was used for I.

Fig. 2

LACTB as a driver of ferroptosis. A. The heat map of differentially expressed genes in control and LACTB-expressing SK-HEP-1 cells. B. MA plot of the differentially expressed genes, red denotes upregulation, blue-green denotes downregulation, and gray denotes no difference. C. KEGG enrichment analysis showing signalling pathways for differential gene enrichment. D. GSEA showing the link between LACTB and ferroptosis. E. FerroOrange staining testing Fe²⁺ levels in LACTB-expressing HepG2 and SK-HEP-1 cells. Scale bar = 25 μm. F. Liperfluo staining testing lipid peroxidation in LACTB-expressing cells. G, H. Evaluation of the effects of LACTB overexpression on MDA levels and GSH/GSSG ratio. I. qRT-PCR analysis of PTGS2 mRNA expression in LACTB-expressing HepG2 and SK-HEP-1 cells. J. Western blot testing 4HNE protein expression in LACTB-expressing SK-HEP-1 cells. K-M. Detection of effects of LACTB knockout on Fe²⁺, lipid peroxidation, MDA, GSH/GSSG ratio, PTGS2 mRNA and 4HNE protein levels in cells. Scale bar = 25 μm. N, O. CCK-8 assay testing the viability of SK-HEP-1 cells expressing LACTB after treatment with erastin or RSL3. P, Q. CCK-8 assay testing the viability of LACTB^−/− cells treated with erastin or RSL3. R. CCK-8 assay testing the viability of cells expressing LACTB after treatment with the indicated chemicals. S. Tumour weight in vector and LACTB groups in the established PDX models. T-V. Detection of Fe2+, GSH/GSSG ratio and PTGS2 mRNA levels in vector and LACTB groups. W. IHC staining of PDX tumour tissues using anti-4HNE antibody. Scale bar = 50 μm. Student's t-test was used for E-I, S–W, and one-way ANOVA with Dunnett post-hoc test was used for K-M. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 3

HSPA8 is a target of LACTB. A. qRT-PCR testing the effects of LACTB overexpression and knockout on HSPA8 mRNA levels. B. Western blot testing the indicated protein levels after LACTB overexpression in SK-HEP-1 cells. C. Western blot testing the indicated protein levels in LACTB^−/− cells reexpressing LACTB. D. Western blot testing the indicated protein levels in LACTB-expressing cells transfected with HSPA8-expressing plasmid. E. Western blot testing the indicated protein levels in LACTB^−/− cells transfected with si-HSPA8. F–I. Detection of effects of LACTB overexpression or in combination with HSPA8 overexpression on Fe²⁺, lipid peroxidation, MDA levels and GSH/GSSG ratio in cells. Scale bar = 25 μm. J,K. Detection of effects of LACTB knockout or in combination with HSPA8 silencing on Fe²⁺ and lipid peroxidation levels in HepG2 and SK-HEP-1 cells. Scale bar = 25 μm. L. Kaplan-Meier plotter database showing the overall survival rate of liver cancer patients with high or low HSPA8 expression. M. Xenograft tumour model testing the in vivo effects of LACTB or LACTB + HSPA8 on SK-HEP-1 cell growth. Scale bar = 1 cm. Student's t-test was used for A (left panel), one-way ANOVA with Tukey post-hoc test was used for A (right panel), F–K, M (right panel), and two-way ANOVA with Tukey post-hoc test was used for M (middle panel).

Fig. 4

LACTB drives ferroptosis via regulating the p53/HSPA8 axis. A. qRT-PCR testing HSPA8 mRNA levels in LACTB-expressing Huh7 and Hep3B cells. B, C. qRT-PCR and Western blot testing HSPA8 mRNA and protein levels in LACTB-expressing cells transfected with si-p53. D. Western blot testing p53 protein expression in LACTB-expressing SK-HEP-1 cells treated with 100 μg/mL cycloheximide for the indicated time. E. Western blot testing p53 ubiquitination levels in LACTB-expressing SK-HEP-1 cells. F. Western blot testing HSPA8 protein expression in LACTB-expressing p53^+/+ Hep3B cells. G. Luciferase reporter assay testing HSPA8 promoter activity in LACTB-expressing cells transfected with si-p53. H. p53 binding sites on HSPA8 promoter among different species. I. The schema showing the wild-type or mutant p53 binding motifs on HSPA8 or p21 promoter, and primer design positions for ChIP assay. J. ChIP assay testing the binding of p53 on the indicated regions of HSPA8 promoter. K. DNA pull-down assay using wild-type or mutant HSPA8 promoter probe, followed by Western blot analysis of p53 protein expression. L. Generation of SK-HEP-1 cells with endogenous mutation of p53 binding site in HSPA8 promoter using CRISPR/Cas9 gene editing technology with the indicated ssODN and gRNA. M. CCK-8 testing the viability of wild-type or mutant SK-HEP-1 cells treated with erastin and RSL3. N, O. Liperfluo and FerroOrange staining testing lipid peroxidation and Fe²⁺ levels in wild-type or mutant SK-HEP-1 cells. Scale bar = 25 μm. P. Western blot testing HSPA8 protein levels in wild-type or mutant SK-HEP-1 cells with LACTB or p53 overexpression. Q. IHC staining of LACTB, wild-type (wt) p53 and HSPA8 in liver cancer tissue microarray, followed by analysis of their expression correlations. Scale bar = 50 μm. One-way ANOVA with Tukey post-hoc test was used for B and G. Student's t-test was used for J, N, O.

Fig. 5

LACTB potentiates the response of liver cancer to lenvatinib. A, B. FerroOrange and liperfluo staining testing Fe²⁺ and lipid peroxidation levels in HepG2 and SK-HEP-1 cells treated with lenvatinib, respectively. Scale bar = 25 μm. C. Western blot testing LACTB protein expression in HepG2 and SK-HEP-1 cells treated with lenvatinib. D-G. FerroOrange, liperfluo staining, CCK-8 and colony formation assays respectively testing Fe²⁺, lipid peroxidation levels, viability and cloning ability in lenvatinib-treated cells with LACTB overexpression or knockout. H, I. Xenograft tumour model testing the in vivo effects of LACTB overexpression or knockout on the anti-tumour effect of lenvatinib. Scale bar = 1 cm. J. Western blot testing the indicated protein expression in the indicated groups. K. The proposed model showing that LACTB induced by lenvatinib promotes ferroptosis by increasing p53 protein stability and inhibiting HSPA8-mediated anti-ferroptosis pathway. One-way ANOVA with Tukey post-hoc test was used for A, B, D-G, I.