Quantitative Proteomic Profiling Identifies SOX8 as Novel Regulator of Drug Resistance in Gestational Trophoblastic Neoplasia

Abstract

The development of drug resistance remains one of the major challenges to current chemotherapeutic regimens in gestational trophoblastic neoplasia (GTN). Further understanding on the mechanisms of drug resistance would help to develop more effective therapy to treat GTN. Herein, tandem mass tag-based (TMT) quantitative proteomic technique was used to establish drug resistance-related proteomic profiles in chemoresistant GTN cell models (JEG3/MTX, JEG3/VP16, JEG3/5-Fu). In total, we identified 5,704 protein groups, among which 4,997 proteins were quantified in JEG3 and its chemoresistant sublines. Bioinformatics analysis revealed that multiple biological processes/molecular pathways/signaling networks were involved in the regulation of drug resistance in chemoresistant JEG3 sublines. SOX8 was upregulated in all the three chemoresistant sublines, and its function was further investigated. Knockdown of SOX8 significantly reduced cell viability, impaired soft agar clonogenesis, and increased caspase-3 activities after drug treatment in JEG3 chemoresistant sublines. In addition, over-expression of SOX8 promoted cell survival, enhanced soft agar clonogenesis, and attenuated caspase-3 activities after drug treatment in GTN cells. Importantly, SOX8 might be a potential regulator of reactive oxygen species (ROS) homeostasis, as SOX8 regulated the expression of antioxidant enzymes (GPX1, HMOX1) and reduced drug-induced ROS accumulation in GTN cell models. Collectively, SOX8 might promote drug resistance through attenuating the accumulation of ROS induced by chemotherapeutic drugs in GTN cells. Targeting SOX8 might be useful to sensitize GTN cells to chemotherapy.

Keywords: SOX8; drug resistance; gestational trophoblastic neoplasia; quantitative proteomics; reactive oxygen species.

Figure 1

(A) IC₅₀ concentrations of JEG3 and its chemoresistant sublines treated with MTX, 5-Fu, or VP16, respectively. n = 3, *P < 0.05, JEG3 sublines vs. JEG3. (B) Experimental scheme for the quantitative proteomic analysis on JEG3 and its chemoresistant sublines. (C) The number of differentially expressed proteins identified by TMT labeling and LC-MS/MS in JEG3 and its chemoresistant sublines. Criteria were set for up-regulation (JEG3 sublines vs. JEG3, fold change ≥ 1.5) and down-regulation (JEG3 sublines vs. JEG3, fold change ≤ 0.67). (D) Top enriched molecular pathways/biological processes of up-regulated or down-regulated proteins in JEG3/MTX. (E) Top enriched molecular pathways/biological processes of up-regulated or down-regulated proteins in JEG3/5-Fu. (F) Top enriched molecular pathways/biological processes of up-regulated or down-regulated proteins in JEG3/VP16.

Figure 2

(A) Relative expression levels of known drug resistance-related proteins in JEG3 and its chemoresistant sublines (JEG3 sublines vs. JEG3). (B) Venn diagram of up- or down-regulated drug resistance-related proteins across JEG3 chemoresistant sublines. (C) Top five commonly enriched molecular pathways/biological processes of up-regulated (n = 52) or down-regulated (n = 33) drug resistance-related proteins in JEG3 chemoresistant sublines.

Figure 3

Signaling networks based on Protein-Protein interaction enrichment analysis on drug resistance-related proteins. (A) Signaling networks identified in up-regulated drug resistance-related proteins in JEG3 chemoresistant sublines. (B) Signaling networks identified in down-regulated drug resistance-related proteins in JEG3 chemoresistant sublines.

Figure 4

(A) Relative protein levels of top 10 up-regulated drug resistance-related proteins in JEG3 and its chemoresistant sublines (JEG3 sublines vs. JEG3). (B) Western blotting validation on protein levels of SLAMF1, TTN, GRIA2, UBIAD1, and SOX8 in JEG3 and its chemoresistant sublines. β-actin was used as loading control. (C) The mass spectrum of SOX8 unique peptide (TELQQAGAK) identified by TMT labeling and LC-MS/MS.

Figure 5

Knockdown of SOX8 attenuated drug resistance in JEG3 chemoresistant sublines. (A) Western blotting analysis on protein expression of SOX8 following lentivirus-mediated shRNA knockdown in JEG3 sublines. β-actin was used as loading control. (B) IC₅₀-values of chemotherapeutic drugs in JEG3 chemoresistant sublines expressing Scr or shSOX8 shRNAs. n = 3, *P < 0.05. (C) Knockdown of SOX8 impaired soft agar clonogenesis after drug treatment (MTX: 10 μg/mL; 5-Fu: 50 μg/mL; VP16: 10 μg/mL) in JEG3 sublines. The colony formation in Scr group without drug treatment was regarded as 100%, respectively. n = 4, *P < 0.05. (D) Knockdown of SOX8 increased caspase-3 activity following drug treatment (MTX: 10 μg/mL; 5-Fu: 50 μg/mL; VP16: 10 μg/mL) for 48 h in JEG3 sublines. The caspase-3 activity in Scr group without drug treatment was regarded as 100%, respectively. n = 4, *P < 0.05.

Figure 6

Over-expression of SOX8 promoted drug resistance in GTN cell lines. (A) Lentiviral transduction of SOX8 considerably increased their protein expression in JEG3 and JAR cells. β-actin was used as loading control. (B) IC₅₀-values of chemotherapeutic drugs in JEG3 and JAR cells expressing SOX8 or EV. n = 3, *P < 0.05. (C) Over-expression of SOX8 rescued soft agar clonogenesis after drug treatment (MTX: 1 μg/mL; 5-Fu: 5 μg/mL; VP16: 2 μg/mL) in JEG3 and JAR cells. The colony formation in EV of each cell line without drug treatment was regarded as 100%, respectively. n = 4, *P < 0.05. (D) Over-expression of SOX8 attenuated caspase-3 activity following drug treatment (MTX: 1 μg/mL; 5-Fu: 5 μg/mL; VP16: 2 μg/mL) for 48 h in JEG3 and JAR cells. The caspase-3 activity in EV of each cell line without drug treatment was regarded as 100%, respectively. n = 4, *P < 0.05.

Figure 7

Attenuation of ROS induced by drugs might be associated with drug resistance in GTN cells. (A) ROS levels of JEG3 and JEG3 chemoresistant sublines after drug treatment. JEG3 and JEG3 sublines were treated with MTX (1 μg/mL), 5-Fu (5 μg/mL), or VP16 (2 μg/mL) for 48 h, respectively. ROS levels in JEG3 without drug treatment were regarded as 100%. n = 4, *P < 0.05. (B) Cytotoxicity induced by drugs was associated with ROS accumulation in JEG3 cells. JEG3 cells were treated with MTX (1 μg/mL), 5-Fu (5 μg/mL), or VP16 (2 μg/mL) alone or with NAC (5 mM) for 48 h, followed by CCK-8 assay to assess the cell viability. Cell viability in JEG3 without drug treatment was regarded as 100%. n = 4, *P < 0.05. (C) Cytotoxicity induced by drugs was associated with ROS accumulation in JAR cells. JAR cells were treated with MTX (1 μg/mL), 5-Fu (5 μg/mL), or VP16 (2 μg/mL) alone or with NAC (5 mM) for 48 h, followed by CCK-8 assay to assess the cell viability. Cell viability in JAR without drug treatment was regarded as 100%. n = 4, *P < 0.05.

Figure 8

SOX8 regulated the expression of antioxidant enzymes and reduced the drug-induced ROS accumulation in GTN cells (A) Knockdown of SOX8 increased drug-induced ROS in JEG3 sublines. JEG3 sublines expressing Scr or shSOX8 shRNAs were treated with MTX (10 μg/mL), 5-Fu (50 μg/mL), or VP16 (10 μg/mL) for 48 h. The DCFDA fluorescence in Scr group of three JEG3 sublines without drug treatment was regarded as 100%, respectively. n = 4, *P < 0.05. (B) Over-expression of SOX8 attenuated drug-induced ROS in GTN cell lines. GTN cells (JEG3, JAR) expressing EV or SOX8 were treated with MTX (1 μg/mL), 5-Fu (5 μg/mL), or VP16 (2 μg/mL) for 48 h. The DCFDA fluorescence in EV group of JEG3 or JAR cells without drug treatment was regarded as 100%, respectively. n = 4, *P < 0.05. (C) Knockdown of SOX8 reduced the expression of antioxidant enzymes GPX1 and HMOX1 in JEG3 chemoresistant sublines. The gene expression of GPX1 or HMOX1 in Scr group of three JEG3 sublines was regarded as 100%, respectively. n = 3, *P < 0.05. (D) over-expression of SOX8 increased GPX1 and HMOX1 expression in JEG3 and JAR cells. The gene expression of GPX1 or HMOX1 in EV group of JEG3 or JAR cells was regarded as 100%, respectively. n = 3, *P < 0.05.