A new perspective: Acyl-CoA synthetase long-chain family member 4 inhibits ubiquitin-specific protease 7-induced epithelial ovarian cancer progression by inducing ferroptosis and M1 macrophage polarization

Abstract

Objective: Epithelial ovarian cancer (EOC) is the most common and lethal type of ovarian cancer, and the cross-talk between tumor cell ferroptosis and macrophages is essential to cancer progression. This study aims to investigate the roles of ubiquitin-specific protease 7 (USP7) and acyl-CoA synthetase long-chain family member 4 (ACSL4) in the pathogenesis of EOC.

Material and methods: The expression patterns of USP7 and ACSL4 in EOC cell lines were first determined by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and Western blot. ACSL4 recombinant protein was applied alone or in conjunction with a USP7 overexpression plasmid in EOC cells, and the effects of USP7 and ACSL4 on EOC cell proliferation and apoptosis were assessed using colony formation assays and terminal deoxynucleotidyl transferase deoxyuridine triphosphate (dUTP) nick end labeling staining. The effects of USP7 and ACSL4 on ferroptosis in EOC cells were evaluated by measuring reactive oxygen species (ROS) fluorescence intensity, malondialdehyde (MDA), glutathione (GSH) levels, and glutathione peroxidase 4 (GPX4) messenger RNA (mRNA) levels. Co-culture of EOC cell-conditioned medium treated with ACSL4 recombinant protein or USP7 overexpression plasmid was performed with Human Acute Monocytic Leukemia Cell Line (THP-1) macrophages, and the expression levels of cluster of differentiation 86 and cluster of differentiation 206 were analyzed by flow cytometry. The expression levels of M1 polarization markers and M2 markers in macrophages were measured by qRT-PCR.

Results: ACSL4 was expressed at low levels in the EOC cell lines, whereas USP7 was expressed at high levels. Treatment with ACSL4 recombinant protein reduced colony formation and increased apoptotic cell levels in the EOC cells (P < 0.001). In addition, ACSL4 treatment increased ROS fluorescence intensity and MDA levels while decreasing GSH levels and GPX4 expression (P < 0.001). Furthermore, ACSL4 treatment promoted the polarization of THP-1 macrophages toward M1, increasing the expression of M1 markers (P < 0.001). USP7 overexpression exerted the opposite effect (P < 0.001).

Conclusion: This study reveals the critical role of USP7 in the progression of EOC. ACSL4 inhibits EOC growth and anti-apoptosis by inhibiting USP7-induced antiferroptosis and anti-M1 macrophage polarization, highlighting this mechanism as a potential therapeutic target in EOC.

Keywords: Acyl-CoA synthetase long-chain family member 4; Epithelial ovarian cancer; Ferroptosis; Macrophage; Ubiquitin-specific protease 7.

Figure 1:

ACSL4 inhibits EOC cell proliferation. (a-c) mRNA and protein levels of ACSL4 in ovarian epithelial cells (IOSE-80) and EOC cells (SKOV-3) were detected by qRT-PCR and Western blot. (d and e) The assessed effect of ACSL4 recombinant protein on colony formation in SKOV-3 cells. (f and g) The effect of ACSL4 recombinant protein on apoptosis in SKOV-3 cells was measured by TUNEL staining. Magnification 200×. n = 6. ^✶✶✶P < 0.001. ACSL4: Acyl-CoA synthetase long-chain family member 4, TUNEL: Terminal deoxynucleotidyl transferase dUTP nick end labeling, DAPI: 4',6-diamidino-2-phenylindole, qRT-PCR: quantitative reverse transcription polymerase chain reaction, EOC: Epithelial ovarian cancer.

Figure 2:

ACSL4 induces ferroptosis in EOC Cells. (a and b) Assessment of the effect of ACSL4 on ROS levels in SKOV-3 cells by ROS staining. Magnification 200×. (c and d) Measurement of MDA and GSH levels in SKOV-3 cells following ACSL4 treatment by spectrophotometric method. (e) Detection of the effect of ACSL4 on the mRNA expression level of GPX4 in SKOV-3 cells according to qRT-PCR. n = 6. ^✶✶✶P < 0.001. ROS: Reactive oxygen species, MDA: Malondialdehyde, GSH: Glutathione, GPX4: Glutathione peroxidase 4, qRT-PCR: quantitative reverse transcription polymerase chain reaction, EOC: Epithelial ovarian cancer.

Figure 3:

ACSL4 promotes the M1 polarization of macrophages. (a) THP-1 macrophages were treated with conditioned media from differently treated SKOV-3 cells, and the expression levels of CD86 and CD206 were measured by flow cytometry. (b) CD86/CD206 ratio. (c-f) THP-1 macrophages were treated with conditioned media from differently treated SKOV-3 cells, and the mRNA levels of iNOS, TNF-α, Arg-1, and IL-10 were measured by qRT-PCR. n = 6. ^✶✶✶P < 0.001. EOC: Epithelial ovarian cancer, THP-1/EOC-CM: THP-1 cells treated with EOC cell culture medium, THP-1/EOC (ACSL4)-CM: THP-1 cells treated with EOC cell culture medium pretreated with ACSL4, CD86: Cluster of differentiation 86, CD206: Cluster of differentiation 206, iNOS: Inducible nitric oxide synthase, TNF-α: Tumor necrosis factor-α, Arg-1: Arginase-1, IL-10: Interleukin-10, qRT-PCR: quantitative reverse transcription polymerase chain reaction, EOC: Epithelial ovarian cancer.

Figure 4:

ACSL4 Reverses USP7-Induced Proliferation in EOC Cells. (a) The mRNA levels of USP7 in ovarian epithelial cells (IOSE-80) and EOC cells (SKOV-3) were measured by qRT-PCR. (b) The transfection of SKOV-3 cells with USP7 overexpression vector and treatment with ACSL4 recombinant protein, showing the mRNA levels of USP7 in the SKOV-3 cells. (c) The mRNA levels of ACSL4 in the SKOV-3 cells was detected by qRT-PCR. (d and e) The assessed effects of ACSL4 and USP7 on colony formation in the SKOV-3 cells. (f and g) The effects of ACSL4 and USP7 on apoptosis in the SKOV-3 cells were measured by TUNEL staining. Magnification 200×. n = 6. ^✶✶✶P < 0.001. USP7: Ubiquitin-specific protease 7, Ov-NC: Overexpression-negative control, Ov-USP-7: Overexpression-USP7, qRT-PCR: quantitative reverse transcription polymerase chain reaction, EOC: Epithelial ovarian cancer.

Figure 5:

ACSL4 Reverses USP7-Induced Ferroptosis Resistance in EOC Cells. (a and b) Assessment of the effects of ACSL4 and USP7 on ROS levels in the SKOV-3 cells by ROS staining, magnification 200×. (c and d) The measurement of MDA and GSH levels in the SKOV-3 cells treated with ACSL4 and USP7 through ELISA. (e) The evaluation of the effects of ACSL4 and USP7 on the mRNA expression levels of GPX4 in SKOV-3 cells through qRT-PCR. Magnification 200×. n = 6. ^✶✶✶P < 0.001. qRT-PCR: Quantitative reverse transcription polymerase chain reaction, EOC: Epithelial ovarian cancer.

Figure 6:

ACSL4 reverses USP7-induced resistance to M1 macrophage polarization. (a and b) USP7 was overexpressed in the SKOV-3 cells with or without the addition of ACSL4 recombinant protein for treatment. The THP-1 macrophages were treated with conditioned media from differently treated SKOV-3 cells, and flow cytometry was used in measuring the expression levels of CD86 and CD206. (c-f) THP-1 macrophages were treated with conditioned media from differently treated SKOV-3 cells, and the mRNA levels of iNOS, TNF-α, Arg-1, and IL-10 were assessed through qRT-PCR. n = 6. ^✶✶✶P < 0.001. THP-1/EOC(Ov-NC)-CM group: THP-1 cells treated with EOC cell culture medium pretreated with Ov-NC, THP-1/EOC(Ov-USP7)-CM group: THP-1 cells treated with EOC cell culture medium pretreated with Ov-USP7, THP-1/EOC(Ov-USP7+ACSL4)-CM group: THP-1 cells treated with EOC cell culture medium pretreated with Ov-USP7 and ACSL4, qRT-PCR: Quantitative reverse transcription polymerase chain reaction, EOC: Epithelial ovarian cancer.