ZSWIM4 inhibition improves chemosensitivity in epithelial ovarian cancer cells by suppressing intracellular glycine biosynthesis

Abstract

Background: Zinc finger SWIM-type containing 4 (ZSWIM4) induces drug resistance in breast cancer cells. However, its role in epithelial ovarian cancer (EOC) remains unknown. In this study, we aimed to investigate the clinical significance of ZSWIM4 expression in EOC and develop new clinical therapeutic strategies for EOC.

Methods: ZSWIM4 expression in control and EOC tumor tissues was examined using immunohistochemistry. Lentiviral transduction, Cell Counting Kit-8 assay, tumorsphere formation assay, flow cytometry, western blotting, and animal xenograft model were used to assess the role of ZSWIM4 in chemotherapy. Cleavage Under Targets and Tagmentation (CUT&Tag) assays, chromatin immunoprecipitation assays, and luciferase reporter assays were used to confirm FOXK1-mediated upregulation of ZSWIM4 expression. The mechanism by which ZSWIM4 inhibition improves chemosensitivity was evaluated using RNA-sequencing. A ZSWIM4-targeting inhibitor was explored by virtual screening and surface plasmon resonance analysis. Patient-derived organoid (PDO) models were constructed from EOC tumor tissues with ZSWIM4 expression.

Results: ZSWIM4 was overexpressed in EOC tumor tissues and impaired patient prognoses. Its expression correlated positively with EOC recurrence. ZSWIM4 expression was upregulated following carboplatin treatment, which, in turn, contributed to chemoresistance. Silencing ZSWIM4 expression sensitized EOC cells to carboplatin treatment in vitro and in vivo. FOXK1 could bind to the GTAAACA sequence of the ZSWIM4 promoter region to upregulate ZSWIM4 transcriptional activity and FOXK1 expression increased following carboplatin treatment, leading to an increase in ZSWIM4 expression. Mechanistically, ZSWIM4 knockdown downregulated the expression of several rate-limiting enzymes involved in glycine synthesis, causing a decrease in intracellular glycine levels, thus enhancing intracellular reactive oxygen species production induced by carboplatin treatment. Compound IPN60090 directly bound to ZSWIM4 protein and exerted a significant chemosensitizing effect in both EOC cells and PDO models.

Conclusions: ZSWIM4 inhibition enhanced EOC cell chemosensitivity by ameliorating intracellular glycine metabolism reprogramming, thus providing a new potential therapeutic strategy for EOC.

Keywords: Carboplatin; Chemosensitivity; Epithelial ovarian cancer; FOXK1; Glycine biosynthesis; ZSWIM4.

Fig. 1

ZSWIM4 is highly expressed in epithelial ovarian cancer (EOC) and indicates poor prognosis. A IHC staining for ZSWIM4 in EOC tumor tissues and adjacent tissues; representative graphs are presented. B Statistical analysis of ZSWIM4 IHC score in EOC tumor tissues (n = 15) and adjacent tissues (n = 15). C IHC staining of ZSWIM4 in primary tumor tissues from patients with recurrent and non-recurrent EOC; representative graphs are presented. D IHC scores of ZSWIM4 in the recurrent and non-recurrent groups. Re, recurrence. E Statistical analysis of ZSWIM4 protein expression in primary tumor tissues from patients with recurrent (n = 91) and non-recurrent (n = 23) EOC. Re, recurrence. F, G Survival analysis of 114 EOC patients based on ZSWIM4 expression in TMA. Overall survival F. Relapse-free survival G. H Survival analysis of ZSWIM4 expression in OC patients treated with platinum-based drugs using the Kaplan–Meier plotter website

Fig. 2

Elevated ZSWIM4 resists chemotherapy in epithelial ovarian cancer (EOC) cells. A ZSWIM4 protein expression levels in the vector control and ZSWIM4-overexpressing IOSE-80 and ES-2 cells. B IC₅₀ curves for vector control and ZSWIM4-overexpressing IOSE-80 and ES-2 cells following incubation with a gradient concentration CBP for 72 h. C Immunofluorescence detection of ZSWIM4 protein expression in OVCAR8 and SKOV3 cells after CBP (75 μM) treatment for 48 or 72 h. D ZSWIM4 protein expression in the vector control and ZSWIM4-overexpressing OVCAR8 and SKOV3 cells. E IC₅₀ curves for vector control and ZSWIM4-overexpressing OVCAR8 and SKOV3 cells following incubation with CBP for 72 h. F Statistical data (mean ± SD) of the apoptosis rate in ZSWIM4-overexpressing and vector control OVCAR8 and SKOV3 cells treated with the vehicle control or CBP (50 μM) treatment for 72 h. G Cleaved caspase-3 protein levels in ZSWIM4-overexpressing OVCAR8 and SKOV3 cells treated with CBP (50 μM) for 72 h detected via western blotting

Fig. 3

Silencing ZSWIM4 enhances the sensitivity of epithelial ovarian cancer (EOC) cells to CBP. A ZSWIM4 protein expression levels in the vector control and shRNA-mediated ZSWIM4-knockdown EOC clones. Representative flow cytometry histograms (left) and statistical data (mean ± SD) are shown (right). B Killing effects in ZSWIM4-knockdown and shRNA control OVCAR8 cells treated with CBP (100 μM) for 72 h and ZSWIM4-knockdown and shRNA control SKOV3 cells treated with CBP (50 μM) for 72 h; CCK-8 assays. C CBP sensitivity of ZSWIM4-knockdown OVCAR8 and SKOV3 cells. D The statistical data (mean ± SD) of tumorspheres of control and ZSWIM4-knockdown EOC cells incubated in 50 μM CBP for 5 days. E The statistical data (mean ± SD) of apoptosis rate of ZSWIM4-knockdown and control EOC cells with or without CBP (50 μM) treatment for 72 h. F The protein expression levels of cleaved caspase-3 in CBP-treated ZSWIM4-knockdown EOC cells. shC, shCTR. G–I The shRNA control and ZSWIM4-knockdown SKOV3-derived xenograft mice were treated with vehicle control or CBP (20 mg/kg thrice weekly) for two weeks (n = 8/group). Photographs of tumors G. Growth curves of xenograft tumors H. Tumor weight I. Veh, vehicle

Fig. 4

FOXK1-dependent ZSWIM4 transcription contributes to epithelial ovarian cancer (EOC) cell chemotherapy resistance. A FOXK1 protein levels in both EOC cell lines treated with vehicle or CBP (75 μM) for 0, 48, and 72 h. B CUT&Tag-seq analyses of FOXK1 binding peaks on the promoter regions of ZSWIM4 in OVCAR8 and SKOV3 cells. Rep-1, replicated sample-1, Rep-2, replicated sample-2. C Enrichment of ZSWIM4 promoter fragments using an antibody against FOXK1 was assessed by ChIP-qPCR analysis. D Agarose electrophoresis analysis of PCR products of the ZSWIM4 promoter after ChIP-PCR assays. E ZSWIM4 promoter activity in EOC cells transfected with wild-type or mutant plasmids in the presence or absence of 100 μM CBP for 24 h. F FOXK1 knocked down in OVCAR8 and SKOV3 cells. G ZSWIM4 promoter activity in FOXK1 silencing EOC cells treated with vehicle or CBP (75 μM) for 48 h. H Immunofluorescence analysis of ZSWIM4 expression in control and FOXK1-knockdown OVCAR8 cells treated with CBP (75 μM) for 48 h. I ZSWIM4 promoter activity in OVCAR8 cells transfected with wild-type ZSWIM4 promoter-luciferase plasmid with or without FOXK1 co-transfection. The cells were treated with vehicle or CBP (75 μM) for 48 h

Fig. 5

Glycine metabolism reprogramming is triggered after knocking down ZSWIM4 in EOC cells. A Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis in OVCAR8 cells after ZSWIM4 knockdown. B The mRNA expression levels of genes encoding key enzymes for glycine synthesis in vector control and ZSWIM4-knockdown OVCAR8 cells. C, D Protein expression levels of PHGDH and SHMT2 in ZSWIM4-knockdown EOC clones C and ZSWIM4-overexpressing EOC clones D. E Intracellular glycine content of ZSWIM4-knockdown OVCAR8 and SKOV3 cells. F Viability of cells following SHMT2 knockdown in vector control and ZSWIM4-overexpressing EOC cells treated with vehicle or CBP (100 μM) for 72 h; CCK-8 assay. G Rescue experiments using exogenous supplementation of hypoxanthine and GSH in ZSWIM4-knockdown OVCAR8 cells. H ROS levels in vector control and ZSWIM4-knockdown OVCAR8 cells after CBP (50 μM) treatment for 72 h. Representative histograms (upper) and statistical data (mean ± SD) are shown (lower). I Flow cytometry detection of ROS levels in vector control and ZSWIM4-overexpressing OVCAR8 cells treated with vehicle or CBP (50 μM) for 72 h. Representative histograms (upper) and statistical data (mean ± SD) (lower) are shown

Fig. 6

IPN60090, a ZSWIM4-targeting inhibitor, enhances the chemotherapeutic sensitivity of EOC cells. A 3D structure of human ZSWIM4. B Structural simulation of ZSWIM4 and IPN60090. IPN60090 is depicted as a gray-white stick, with the red dashed line indicating the length of the hydrogen bond. C–D The affinity of anti-ZSWIM4 antibody C and IPN60090 D for ZSWIM4 protein determined by SPR analyses. KD, dissociation constant. E Detection of intracellular glycine content in OVCAR8 and SKOV3 cells treated with 10 μM IPN60090 for 48 h. F ROS levels in EOC cells following IPN60090 (10 μM) treatment for 48 h. G IC₅₀ curves for OVCAR8 and SKOV3 cells following treatment with CBP and/or IPN60090 (10 μM) for 72 h. H Tumorsphere formation in OVCAR8 cells treated with vehicle control, CBP (50 μM), IPN60090 (10 μM), and CBP (50 μM) combined with IPN60090 (10 μM) for 5 day. Representative images (left) and statistical data (mean ± SD) (right) are shown. I Viability of ZSWIM4-knockdown and vector control EOC cells following treatment with CBP (25 μM) with or without IPN60090 (10 μM) for 72 h; CCK-8 assay. J–L SKOV3-derived xenograft mice were divided into four groups (n = 4/group) and administered the vehicle control, CBP (15 mg/kg, i.p.) thrice weekly, IPN60090 (30 mg/kg/day, p.o.), and CBP combined with IPN60090. Photographs of tumors J. Tumor weight K. Growth curves of xenograft tumors L

Fig. 7

ZSWIM4 inhibitor enhances chemotherapy sensitivity in CBP-resistant SKOV3 cells. A Drug sensitivity of CBP in parental SKOV3 and SKOV3/CBP cells. B The protein expression of FOXK1 in parental SKOV3 and SKOV3/CBP cells. C Immunofluorescence detection of ZSWIM4 protein expression in parental SKOV3 and SKOV3/CBP cells. D ZSWIM4 gene promoter-luciferase reporter activity in parental SKOV3 and SKOV3/CBP cells. E IC₅₀ curves of SKOV3/CBP cells with a gradient concentration of CBP with or without IPN60090 (50 μM) treatment for 72 h. (F–I) SKOV3/CBP cells treated with vehicle, CBP (200 μM), IPN60090 (50 μM), and CBP (200 μM) combined with IPN60090 (50 μM) for 72 h. ROS levels F, and statistical data (mean ± SD) G. Flow cytometry analysis of cell apoptosis rate (mean ± SD) H. Cleaved caspase-3 protein abundance I

Fig. 8

Patient-derived organoid (PDO) models with ZSWIM4 expression are sensitive to combined therapy. A Immunohistochemical analysis of ZSWIM4 protein expression in EOC tissues from two patients. B, C PDOs were treated with vehicle, CBP (100 μM) combined with PTX (10 nM), IPN60090 (50 μM), or CBP combined with PTX plus IPN60090, and the cell viability was measured on days four and seven using an ATP detection kit. PDO #1 B; PDO#2 C. Typical images (left) and statistical data (mean ± SD) (right). Veh, vehicle. IPN, IPN60090

Fig. 9

Graphic model of ZSWIM4 inhibition enhancing the chemosensitivity of EOC cells Epithelial ovarian cancer (EOC) cells survive in the presence of carboplatin by inducing ZSWIM4 expression via FOXK1-mediated transcription. Elevated ZSWIM4 levels upregulate the glycine biosynthesis-associated rate-limiting enzymes represented by SHMT2 and PHGDH, leading to increased intracellular glycine content and decreased levels of reactive oxygen species, further reducing the sensitivity of EOC cells to chemotherapy. IPN60090, a ZSWIM4-targeting inhibitor, enhances the chemosensitivity of EOC cells.