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. 2022 Feb;10(4):224.
doi: 10.21037/atm-21-6909.

SENP1 inhibition suppresses the growth of lung cancer cells through activation of A20-mediated ferroptosis

Chuancheng Gao #  1   2 , Fengjun Xiao #  2 , Lin Zhang  1   2 , Yang Sun  1 , Lei Wang  3 , Xiang Liu  4 , Huiyan Sun  5 , Zhidan Xie  6 , Yaqi Liang  6 , Qinqin Xu  3 , Lisheng Wang  1   2
Affiliations

SENP1 inhibition suppresses the growth of lung cancer cells through activation of A20-mediated ferroptosis

Chuancheng Gao et al. Ann Transl Med. 2022 Feb.

Abstract

Background: Ferroptosis is a type of cell death driven by iron accumulation and lipid peroxidation, which is involved in the pathogenesis of various tumors. Small ubiquitin-like modifier (SUMO)-specific protease 1 (SENP1) is a critical SUMO-specific protease, which controls multiple cellular signaling processes. However, the roles and mechanisms of SENP1-mediated protein SUMOylation in the regulation of cell death and ferroptosis remain unexplored.

Methods: The gene expression of SENP1 and ferroptosis-related genes in samples of lung cancer patient and cells were determined by immunohistochemical staining, real-time polymerase chain reaction (RT-qPCR) and Western blot. The association of gene expression with the survival rate of lung cancer patients was analyzed from public database. The erastin and cisplatin was used to induce ferroptosis, and cell ferroptosis were determined by evaluated lipid-reactive oxygen species (ROS), cell viability and electron microscopy. The protein interaction was determined by immunoprecipitation (IP) and shotgun proteomics analysis. An in vivo tumor transplantation model of immunodeficient mice was used to evaluate the effect of SENP1 on tumor growth in vivo.

Results: SENP1 is aberrantly overexpressed in lung cancer cells and is associated with the low survival rate of patients. SENP1 inhibition by short hairpin RNA transduction or a specific inhibitor suppressed the proliferation and growth of lung cancer cells both in vitro and in vivo. SENP1 overexpression protected lung cancer cells from ferroptosis induced by erastin or cisplatin. Transcriptome and proteomics profiles revealed the involvement of SUMOylation regulation of the inflammation signal A20 in SENP1 inhibition-induced ferroptosis. Functional studies proved that A20 functions as a positive inducer and enhances the ferroptosis of A549 cells. A20 was shown to interact with ACSL4 and SLC7A11 to regulate the ferroptosis of lung cancer cells.

Conclusions: SENP1 was identified as a suppressor of ferroptosis through a novel network of A20 SUMOylation links ACSL4 and SLC7A11 in lung cancer cells. SENP1 inhibition promotes ferroptosis and apoptosis and represents a novel therapeutic target for lung cancer therapy.

Keywords: A20; Lung cancer; SENP1; ferroptosis; target therapy.

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Conflict of interest statement

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://atm.amegroups.com/article/view/10.21037/atm-21-6909/coif). The authors have no conflicts of interest to declare.

Figures

Figure 1
Figure 1
SENP1 expression in lung cancer and its correlation with the survival of lung cancer patients. (A) The Gene Expression profiling interactive analysis (GEPIA) database analyzed the overall gene expression in lung cancer (with partial results). (B) The GEPIA database analysis showed that SENP1 was highly expressed in lung cancer (the red boxplot is tumor, and the black boxplot is normal). (C) Immunohistochemical (IHC) staining for SENP1 in 35 pairs of lung cancer samples (left), and the H-score statistical analysis was conducted for SENP1 (right). (D) Oncomine analyzed the association of SENP1 expression with overall survival (OS) and free progress (FP) in patients with lung cancer (n=1,145). (E) Oncomine analyzed the association of SENP1 expression with OS and FP in patients with lung cancer after cisplatin chemotherapy (n=478).
Figure 2
Figure 2
Effects of silencing SENP1 on the biological characteristics of lung cancer cells in vitro. (A,B) The mRNA and protein levels of SENP1 of A549 cells transduced with SENP1-shRNA or a control vector were detected by reverse transcription polymerase chain reaction (RT-PCR) (A) and western blotting (B). (C) The apoptotic cells were stained with Annexin V and detected by flow cytometry. (D) The proliferation of A549 cells transduced with SENP1-shRNA or a control vector were detected by CCK8 assay. (E) The cell viability of A549 cells treated with Momordin at concentrations indicated was detected by CCK8 assay; 0.5 µm Fer-1 was unable to resist the action of Momordin. (F) The clonal formation (×20) of A549 cells transduced with SENP1-shRNA or treated with 5 µm Momordin. *, P<0.05 vs. control; **, P<0.01 vs. control; ***, P<0.001 vs. control.
Figure 3
Figure 3
SENP1 affects the growth and cisplatin resistance of lung cancer cells in vivo. (A) SENP1-overexpression in A549 cells was detected by reverse transcription polymerase chain reaction (RT-PCR). (B) The survival rate of nude mice grafted with SENP1-overexpressed A549 cells. (C) The tumor volume of nude mice grafted with SENP1-overexpressed A549 cells. (D) The tumor volume of nude mice grafted with SENP1-overexpressed A549 cells with or without cisplatin treatment. (E) The body weight of nude mice grafted with SENP1-overexpressed A549 cells with or without cisplatin treatment. (F,G) Comparison of tumor weight (F) and representative volume (G) in each group. *, P<0.05 vs. control; **, P<0.01 vs. control; ***, P<0.001 vs. control.
Figure 4
Figure 4
SENP1 silencing enhances the erastin-induced ferroptosis of A549 cells. (A) The cell viability of A549 cells treated with erastin at different concentrations for 24 hours. The concentration of Fer-1 was 0.5 µm. (B) The cell viability of SENP1-silenced A549 cells treated with erastin at different concentrations for 24 hours. (C) The ROS production in A549 cells treated with erastin in the presence or absence of 0.5 µm Fer-1. (D) The ROS production in SENP1-silenced A549 cells treated with erastin. (E) The cell viability of SENP1-silenced A549 cells treated with ACSL4 and GPX4 inhibitors (SENP1-silenced A549 cells treated with 5 µm erastin). (F) Morphological observation of SENP1-silenced A549 cells treated with erastin (2.5 µm) or H2O2 (1 µm) in the presence or absence of 0.5 µm Fer-1, magnification ×40. *, P<0.05 vs. control; **, P<0.01 vs. control; ***, P<0.001 vs. control.
Figure 5
Figure 5
SENP1 suppresses cisplatin-induced ferroptosis of lung cancer cells. (A) The cell viability of A549 cells treated with cisplatin in the presence or absence of 0.5 µm Fer-1. (B) A549 cells were treated with 5 µm erastin and 5 µm cisplatin for 24 h. Observation under transmission electron microscopy (TEM) at 500 nm and ×40,000. (C,D) The cell viability of SENP1-silenced A549 cells treated with cisplatin at different concentrations in the absence (C) or presence (D) of 0.5 µm Fer-1. (E,F) The cell viability of SENP1-overexpressed A549 cells treated with cisplatin at different concentrations in the absence (E) or presence (F) of 0.5 µm Fer-1. *, P<0.05; **, P<0.01; ***, P<0.001.
Figure 6
Figure 6
Silencing SENP1 upregulates the expression of A20 and ferroptosis-related genes in A549 cells. Transcriptomic profile of SENP1-silenced A549 cells compared to control cells (A). (B) Western blot was used to detect the ferroptosis-related proteins ACSL4, GPX4, A20 and P53 in SENP1-silenced A549 cells or control cells treated with 5 µM erastin for 24 h. (C) Western blot was used to detect the ferroptosis-related proteins ACSL4, GPX4, A20, and P53 in SENP1-silenced A549 cells treated with cisplatin for 24 h. (D) Immunohistochemical staining assay (×100): the tissues were blocked and incubated with the primary antibody, second antibody and several staining steps. the expression of ACSL4, GPX4, A20, and SLC7A11 in tumor tissues of nude mice grafted with SENP1 overexpressed A549 cells treated with cisplatin.
Figure 7
Figure 7
Expression of A20 and ferroptosis-related genes in lung cancer specimens. (A) The expression data of TNFAIP3 (A20), ACSL4, GPX4, and SLC7A11 expression in the GEPIA database. (B-E) Expressions of A20, ACLSL4, GPX4, and SLC7A11 were detected by immunohistochemical staining in 20 pairs of lung cancer samples. The H-score statistical analysis was conducted for A20, ACSL4, GPX4, and SLC7A11 after immunohistochemical (IHC) detection.
Figure 8
Figure 8
A20 SUMOylation and its effect on growth of lung cancer cells. (A) A20 and SUMO-1 in SENP1-silenced A549 cells were detected by western blotting. (B) A20 and SUMO-1 in A20-overexpressed A549 cells were detected by western blotting. (C) A20 immunoprecipitation of A549 cells was detected for SUMO1 by western blotting. (D) The proliferation of A549 cells transduced with A20 or a control vector was detected by CCK8 assay. (E) The ROS generation of A549 cells transduced with A20 or a control vector treated with erastin and 0.5 µm Fer-1. (F) The cell viability of A549 cells transduced with A20 or a control vector treated with erastin was detected by CCK8 assay. *, P<0.05 vs. control vector; **, P<0.01 vs. control vector.
Figure 9
Figure 9
A20 interacts with ACSL4 and SLC7A11. (A) Immune co-precipitation A20 and shotgun proteomics was used to detect ferroptosis-related proteins. (B) A gene regulatory network (A20, ACSL4 and SLC7A11 marked as Red) associated with ferroptosis. (C) A20 immunoprecipitation was used to detect ACSL4 and SLC7A11 by western blotting. (D) The ACSL4 and SLC7A11 levels in A20-overexpressed A549 cells were detected by western blot. *, P<0.05; **, P<0.01.
Figure 10
Figure 10
Schematic model showing that SENP1 regulates A20 by deSUMOylation to inhibit ferroptosis by affecting the interaction of A20 with ACSL4 and SLC7A11.
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