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. 2020 May 25;10(15):6928-6945.
doi: 10.7150/thno.43811. eCollection 2020.

Aurora-A/SOX8/FOXK1 signaling axis promotes chemoresistance via suppression of cell senescence and induction of glucose metabolism in ovarian cancer organoids and cells

Huizhen Sun  1 , Husheng Wang  1 , Xue Wang  1 , Yoichi Aoki  2 , Xinjing Wang  1 , Yufei Yang  3 , Xi Cheng  3 , Ziliang Wang  1 , Xipeng Wang  1
Affiliations

VSports最新版本 - Aurora-A/SOX8/FOXK1 signaling axis promotes chemoresistance via suppression of cell senescence and induction of glucose metabolism in ovarian cancer organoids and cells

Huizhen Sun et al. Theranostics. .

Abstract

Rationale: Cisplatin derivatives are first-line chemotherapeutic agents for epithelial ovarian cancer. However, chemoresistance remains a major hurdle for successful therapy and the underlying molecular mechanisms are poorly understood at present VSports手机版. Methods: RNA sequencing of organoids (PDO) established from cisplatin-sensitive and -resistant ovarian cancer tissue samples was performed. Glucose metabolism, cell senescence, and chemosensitivity properties were subsequently examined. Immunoprecipitation, mass spectrometry, Fӧrster resonance energy transfer-fluorescence lifetime imaging (FRET-FLIM), luciferase reporter assay, ChIP and animal experiments were conducted to gain insights into the specific functions and mechanisms of action of the serine/threonine kinase, Aurora-A, in ovarian cancer. Results: Aurora-A levels were significantly enhanced in cisplatin-resistant PDO. Furthermore, Aurora-A promoted chemoresistance through suppression of cell senescence and induction of glucose metabolism in ovarian cancer organoids and cells. Mechanistically, Aurora-A bound directly to the transcription factor sex determining region Y-box 8 (SOX8) and phosphorylated the Ser327 site, in turn, regulating genes related to cell senescence and glycolysis, including hTERT, P16, LDHA and HK2, through enhancement of forkhead-box k1 (FOXK1) expression. Conclusions: Aurora-A regulates cell senescence and glucose metabolism to induce cisplatin resistance by participating in the SOX8/FOXK1 signaling axis in ovarian cancer. Our collective findings highlight a novel mechanism of cisplatin resistance and present potential therapeutic targets to overcome chemoresistance in ovarian cancer. .

Keywords: Aurora-A; Chemoresistance; Ovarian cancer; PDOs; SOX8 V体育安卓版. .

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interest exists.

Figures (V体育官网)

Figure 1
Figure 1
Cisplatin-resistance is closely associated with cell senescence and glucose metabolism in organoids of ovarian cancer. (A) Images of cisplatin-sensitive and -resistant PDOs of ovarian cancer with 3ug/L cisplatin treatment on days 0 and 21. (B) Cell viability assay of PDOs treated with 3 µg/L cisplatin in different time intervals. (C) GSEA analysis was performed using cisplatin-sensitive and cisplatin-resistant PDOs of ovarian cancer. The signature was defined by genes with significant expression changes. (D) Representative images of β-galactosidase staining in cisplatin-sensitive and cisplatin-resistant ovarian cancer tissues. (E and F) The relationship between SUVmax value of PET/CT image and chemosensitivity in ovarian cancer patients. SUVmax value of PET/CT scan in 100 cisplatin-sensitive and 100 cisplatin-resistant ovarian cancer patients were analyzed. (G) The association between SUVmax value of PET/CT image and overall survival in ovarian cancer patients. SUVmax value of PET/CT scan in 200 ovarian cancer patients were analyzed. (H) Metabolites in glycolysis pathway. (I) and (J) Heat map (I) and histogram (J) of qRT-PCR analysis of indicated genes in cisplatin-sensitive and cisplatin-resistant ovarian cancer organoids. Data were shown as mean ± SD. Significance was calculated using the Student t test. *P < 0.05, **P < 0.01.
Figure 2
Figure 2
Aurora-A regulates chemoresistance and cell senescence in ovarian cancer. (A) GSEA analysis was performed using cisplatin-sensitive and cisplatin-resistant PDOs of ovarian cancer. The signature was defined by genes with significant expression changes. (B) Immunofluorescence assay detected the relation of Aurora-A and chemosensitivity in PDOs of ovarian cancer. (C) Aurora-A knockdown cell lines were established by immunoblotting. (D and E) Values of IC50 of cisplatin. Aurora-A silencing cells, 0.5 nM MLN8237-treated cells and their controls were treated with cisplatin in different concentrations for 48h. **P < 0.01. (F) and (G) Percentage of apoptotic cells. Except the blank group, cells were treated with 5 µg/mL cisplatin, 0.5nM MLN8237, or 5 µg/mL cisplatin plus 0.5 nM MLN8237 for 48 h, respectively. Apoptosis was measured by flow cytometry in cells stained with annexin V and propidium iodide. **P < 0.01. (H and I) β-galactosidase staining. **P < 0.01. Cells were treated with the same method as Figure 2F-G. (J) Immunoblotting analysis of proteins associated with the cell senescence and apoptosis.
Figure 3
Figure 3
Aurora-A modulated the glucose metabolism in ovarian cancer cells. (A-D) Glucose uptake (A), ATP (B), lactate (C) and NADPH (D) production were determined as described in Methods. “CON” was the cells transfected with scrambled shRNA expression vector. Data shown were mean ± SD of triplicate measurements repeated 3 times with similar results. Statistical significance was assessed by two-tailed Student's t test. (E and F) ECAR (E) and OCR (F) were determined as described in Methods. (G) Immunoblotting analysis of glycolytic gene expression in Aurora-A knockdown ovarian cancers and the controls. (H) Immunofluorescence assay detected the relationship between Aurora-A and glycolysis associated proteins.
Figure 4
Figure 4
Aurora-A combined with SOX8 directly and activated it by phosphorylation. (A) Cellular extracts from SKOV3-CisR cells were immunopurified with anti-Aurora-A affinity columns and eluted with Aurora-A peptide. The eluates were resolved by SDS-PAGE and silver stained. (B) Mass spectrometry analysis of the SDS-PAGE above. The detected peptides spectra of SOX8 were listed. (C) Co-IP analysis demonstrated an interaction between Aurora-A and SOX8 protein in OVCA429-CisR and SKOV3-CisR cell lines. (D and E) Interaction between Aurora-A and SOX8 confirmed by FRET-FLIM upon transient co-expression. FE, FRET efficiency. **P < 0.01. (F) Aurora-A silencing significantly changed the distribution and expression of SOX8 and p-SOX8 (Ser327) in cytoplasm and nucleus of ovarian cancer cells. (G and H) Immunofluorescence assay detected the relation of Aurora-A and p-SOX8 (Ser327) in ovarian cancer cell lines (G) and PDOs (H). (I) In vitro kinase assay to detect the phosphorylation of recombinant GST-SOX8 protein by wild-type or mutant Aurora-A coprecipitates.
Figure 5
Figure 5
FOXK1 is the downstream targeting molecule of SOX8. (A) GSEA analysis was performed using SOX8 knockdown cell line and the control (SKOV3-CisR/SOX8-i1 and SKOV3-CisR/Con). The signature was defined by genes with significant expression changes. (B and C) Immunoblotting and immunofluorescence assay detected the relation of SOX8 and FOXK1 in ovarian cancer cells. (D) FOXK1 mRNA expression level in Aurora-A knockdown ovarian cancer cell lines and SOX8 transfected cell lines in rescue experiment. (E) FOXK1 promoter activity affected by Aurora-A knockdown and SOX8 overexpression. (F) FOXK1 promoter activity affected by SOX8 silencing. (G) The region of SOX8 binding sites within the FOXK1 promoter. (H) ChIP results of the binding of SOX8 to the promoter of FOXK1. (I) The map of SOX8 binding sits in the promoter region of FOXK1. (J) Luciferase reporter assay was used for the detection of mutant sites in the promoter region of FOXK1. **P < 0.01.
Figure 6
Figure 6
Aurora-A knockdown inhibited the progression of ovarian cancer and sensitized cancer cells response to cisplatin in vivo. (A) Flow chart of the in vivo experiment. (B) Representative image of nude mice bearing tumors formed by SKOV3-CisR/Aur-i1 and their control cells with cisplatin treated or untreated. (C) Growth curve of the xenograft tumors burdened in mice. (D) The average tumor weight of nude mice. (E) Average SUVmax values of nude mice bearing tumors. (F) Representative image of PET-CT, which was used for the detection of glucose uptake. (G) Representative image of β-galactosidase staining of the nude mice tumor tissues with or without cisplatin treatment. (H) Immunofluorescence assay detected the relation among Aurora-A, SOX8, and FOXK1 in the nude mice tumor tissues with cisplatin treatment. (I) The mRNA expression levels of Aurora-A, SOX8, and FOXK1 were tested by qRT-PCR assay in tumors in vivo. Error bars: 95% Cis; **P < 0.01. (J) Heat map showed the gene expression associated with cell senescence and glycolysis in the nude mice tumor tissues with cisplatin treatment.
Figure 7
Figure 7
Immunohistochemistry staining and immunofluorescence of Aurora-A, SOX8, FOXK1 and proteins related to glycolysis and cell senescence. (A) Representative images of immunohistochemistry staining of Aurora-A, SOX8, and FOXK1 in cisplatin-sensitive and cisplatin-resistant ovarian cancer tissues. (B) Relatively high or low expression ratio of Aurora-A, SOX8 and FOXK1 in cisplatin-sensitive and cisplatin-resistant ovarian cancer samples. (C) Immunofluorescence staining of Aurora-A, SOX8 and FOXK1. (D) Immunohistochemistry of Aurora-A and the proteins associated with cell senescence and glycolysis. (E) The mRNA expression levels of genes associated with cell senescence and glycolysis were tested by qRT-PCR assay. (F) Schematic model showing the role of the Aurora-A/SOX8/FOXK1 signaling axis in the regulation of cell senescence, glycolysis and chemosensitivity.
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