Low Expression of SLC7A11 Confers Drug Resistance and Worse Survival in Ovarian Cancer via Inhibition of Cell Autophagy as a Competing Endogenous RNA

Abstract

Drug resistance is the main cause of chemotherapy failure in ovarian cancer (OC), and identifying potential druggable targets of autophagy is a novel and promising approach to overcoming drug resistance. In this study, 131 genes associated with autophagy were identified from three autophagy-related databases, and of these, 14 were differentially expressed in 90 drug-resistant OC tissues versus 197 sensitive tissues according to the Cancer Genome Atlas ovarian cancer cohort. Among these 14 genes, SLC7A11 was significantly decreased in two paclitaxel-resistant OC cells (HeyA8-R and SKOV3-R) and in 90 drug-resistant tissues compared with their controls. In vitro overexpression of SLC7A11 significantly increased the sensitivity of HeyA8-R cells to paclitaxel, inhibited colony formation, induced apoptosis, and arrested cell cycle. Further, low SLC7A11 expression was correlated with poor overall survival (OS), progression-free survival (PFS), and post-progression survival (PPS) in 1815 OC patients. Mechanistically, SLC7A11 strongly regulated cell autophagy as a competing endogenous RNA (ceRNA) based on pan-cancer analyses of 32 tumor types. Specifically, as a ceRNA for autophagy genes STX17, RAB33B, and UVRAG, SLC7A11 was strongly and positively co-expressed with these three genes in 20, 12, and 12 different tumors, respectively, in 379 OC tissues and in 90 drug-resistant OC tissues, and the former two were significantly upregulated in SLC7A11-overexpressed HeyA8-R cells. Further, SLC7A11 induced the protein expression of other autophagy genes, such as LC3, Atg16L1, and Atg7, and the expression of the respective proteins was further increased when the cells were treated with paclitaxel. The results strongly suggest that SLC7A11 regulates autophagy via ceRNA interactions with the three abovementioned genes in pan-cancer and in drug-resistant OC. Moreover, low expression of STX17 and UVRAG also significantly predicted low OS, PFS, and PPS. The combination of SLC7A11 with STX17 was more predictive of OS and PFS than either individually, and the combination of SLC7A11 with UVRAG was highly predictive of OS and PPS. The above results indicated that decreased SLC7A11 resulted in drug resistance and effected low rates of survival in OC patients, probably via ceRNA interactions with autophagy genes, and thus the gene could serve as a therapeutic target and potential biomarker in OC.

Keywords: SLC7A11; autophagy; ceRNA; drug resistance; ovarian cancer; prognosis.

Figure 1

Identification of genes potentially related to autophagy and drug resistance in ovarian cancer (OC). (A) The 131 overlapping genes involved in autophagy and drug resistance were retrieved from three independent databases. A total of 786 and 1086 autophagy-related genes were retrieved from database Autophagy DB and Thanatos, respectively. 868 genes related to OC drug resistance and autophagy were selected through text mining conducted by Coremine (P < 0.05); (B) Fourteen of the above 131 genes were significantly dysregulated in chemoresistant OC tissues (Resistant, n=90) compared with the sensitive tissues (Sensitive, n=197) according to the TCGA ovarian cohort (*P < 0.05; **P < 0.01).

Figure 2

Kaplan–Meier analysis of gene expression with OC survival in a large sample of 1815 OC patients using the Kaplan–Meier Plotter tool. The auto-selected best cutoff value was used to dichotomize gene expression into high (H) and low (L). (A) Low expression of OPA1, VCP and SLC7A11 predicted poor OS, PFS and PPS; (B) As ceRNA target of SLC7A11, STX17 and UVRAG suppression predicted poor OS, PFS and/or PPS; (C) The combination of SLC7A11 expression with STX17 or UVRAG was associated with poor OS, PFS and/or PPS. OS, overall survival; PFS, progression-free survival; PPS, post-progression survival.

Figure 3

Expression of SLC7A11 in ovarian cancer cell lines. (A) RT-qPCR analysis of SLC7A11 mRNA levels in paclitaxel resistant cells HeyA8-R (H-R), SKOV3-R (S-R) and their parental cells HeyA8 (H), SKOV3 (S); (B) Western blotting analysis of SLC7A11 expression levels in H-R, H, S-R and S cells; (C) Analysis of SLC7A11 mRNA expression in SLC7A11-overexpressed HeyA8-R-SLC7A11 (H-R-SLC7A11) cells and the control HeyA8-R-eGFP (H-R-eGFP) cells by RT-qPCR; (D) Western blotting analysis of SLC7A11 expression levels in HeyA8-R-SLC7A11 (H-R-SLC7A11) cells and the control HeyA8-R-eGFP (H-R-eGFP) cells. Values represent the mean ± SD (**P < 0.01; ***P < 0.001).

Figure 4

SLC7A11-overexpressed HeyA8-R-SLC7A11 (H-R-SLC7A11) cells were more sensitive to paclitaxel than H-R-eGFP cells. (A, B) H-R-SLC7A11 and H-R-eGFP cells were treated with various doses of paclitaxel for 72 hours. The CCK-8 assay was used to reveal the cell viability and the IC₅₀ of paclitaxel. Values represented the mean ± SD of four independent experiments (**P < 0.01; ***P < 0.001); (C) Representative scan images of the colony formation assay. H-R-SLC7A11 and H-R-eGFP cells were treated with gradient of paclitaxel for 8 days. Scale bar: 4 mm; (D) The relative colony formation rates of H-R-SLC7A11 and H-R-eGFP were analyzed by Image J. Values represented the mean ± SD of three independent experiments (*P < 0.05; **P < 0.01).

Figure 5

Effects of paclitaxel on the apoptosis of H-R-eGFP and H-R-SLC7A11 cells by PE Annexin V/7-AAD-double staining flow cytometry analysis. (A) Representative cell-culture images and flow cytometric findings of H-R-eGFP and H-R-SLC7A11 cells treated with gradient concentrations of paclitaxel for 72 hours. The lower left quadrant shows living cells, the lower right quadrant early apoptotic cells, the upper left quadrant non-apoptotic dead cells, and the upper right quadrant shows late apoptotic cells and dead cells; (B) The histogram of apoptosis experiments. Statistical analysis was performed to compare the living cell rate of H-R-eGFP to H-R-SLC7A11 (lower left quadrant), early apoptotic cell rate (lower right quadrant), late apoptotic cell rate, and dead cell rate (upper right quadrant), and total apoptosis and death cell rates (upper left + upper right + lower right quadrant). The statistical analysis in the figure was a pair-wise comparison between H-R-eGFP and H-R-SLC7A11 cells treated with the same paclitaxel concentration and the same state of cells (*P < 0.05, **P < 0.01, ***P < 0.001).

Figure 6

Effects of paclitaxel on the cell cycle distribution of H-R-eGFP and H-R-SLC7A11 by PI staining and the flow cytometry analysis. (A) Representative cell-culture photos and cell-cycle images for H-R-eGFP and H-R-SLC7A11 cells treated with gradient paclitaxel for 72 h; (B) The percentage of cells in G0/G1 phase, S phase and G2/M phase of H-R-eGFP and H-R-SLC7A11 cells treated with gradient concentrations of paclitaxel for 72 hours. The statistical analysis in the figure was a pair-wise comparison between H-R-eGFP and H-R-SLC7A11 cells data with the same paclitaxel concentration and the same cell-cycle stage (**P < 0.01, ***P < 0.001).

Figure 7

Effects of SLC7A11 and paclitaxel on the expression of related proteins analyzed by western blotting. The H-R-eGFP and H-R-SLC7A11 cells were treated with gradient concentration of paclitaxel for 72 hours. (A) Proteins were extracted and detected by western blotting to analyze the expression of p21 Waf1/Cip1, p27 Kip1, CDK2, CDK7, Cyclin A2, Cyclin B1 and Cyclin D3; (B) Expression of LC3 -II, LC3 -I, STX17, RAB33B, UVRAG, Atg7, Atg16L1 and Akt proteins were analyzed by western blotting. Representative blots were shown with GAPDH and β-Tubulin as loading control.

Figure 8

Protein interaction network of SLC7A11 created by the GeneMANIA online tool. The 31 drug resistance-related proteins were text-mined by Coremine Medical (P < 0.001). The blue circles indicate the 16 proteins which interacted with SLC7A11 directly; the grey circles indicate the proteins which interacted with SLC7A11 indirectly. The interaction types between proteins indicated as networks legend.

Figure 9

The relationship between SLC7A11 and eight autophagy related genes. SLC7A11 act as a ceRNA for eight autophagy genes and positively co-expressed with these genes in 379 ovarian cancer tissues of TCGA cohort according to StarBase (P<0.05). The gene expression values from RNA-seq data were presented as log2 (FPKM + 0.01).

Figure 10

The distribution of the eight genes in autophagy pathway (map04140). The eight genes (indicated as orange oval) were the potential targets of SLC7A11 as a ceRNA in 379 ovarian cancers which significantly and positive co-expressed with the SLC7A11, and the three genes (indicated in red) were those co-expressed in 90 drug resistant ovarian cancer tissues.

Figure 11

SLC7A11 is positively correlated with the expression of STX17, UVRAG, and RAB33B in 90 drug resistant ovarian cancer tissues based on TCGA cohort. The correlation of genes was analyzed by bivariate correlation (P < 0.05).

Figure 12

As ceRNA targets of SLC7A11, (A) STX17, (B) UVRAG, and (C) RAB33B were positively co-expressed in 12, 20 and 12 kinds of cancers according to pan-cancer analyses. The other 2 cancers that SLC7A11 negatively co-expressed with RAB33B was excluded. The gene expression values from RNA-seq data were presented as log2 (FPKM + 0.01) (P<0.05). BRCA, Breast Invasive Carcinoma; THCA, Thyroid Carcinoma; COAD, Colon Adenocarcinoma; PRAD, Prostate Adenocarcinoma; UCEC, Uterine Corpus Endometrial Carcinoma; READ, Rectum Adenocarcinoma; HNSC, Head and Neck Squamous Cell Carcinoma; LGG, Brain Lower Grade Glioma; ACC, Adrenocortical Carcinoma; UVM, Uveal Melanoma; KICH, Kidney Chromophobe; KIRC, Kidney Renal Clear Cell Carcinoma; SKCM, Skin Cutaneous Melanoma; LUSC, Lung Squamous Cell Carcinoma; TGCT, Testicular Germ Cell Tumors; BLCA, Bladder Urothelial Carcinoma; LUAD, Lung Adenocarcinoma; LAML, Acute Myeloid Leukemia; CESC, Cervical Squamous Cell Carcinoma and Endocervical Adenocarcinoma; KIRP, Kidney Renal Papillary Cell Carcinoma; UCS, Uterine Carcinosarcoma; THYM, Thymoma; LIHC, Liver Hepatocellular Carcinoma.

Figure 13

ceRNA network of SLC7A11 with STX17, UVRAG and RAB33B. The ceRNA pairs were determined by StarBase and the network was constructed by Cytoscape. Acting as a ceRNA, SLC7A11 shared miRNAs with the target genes as indicated in the network. The light green oval indicate the miRNAs for SLC7A11 with RAB33B, the purple oval indicates the miRNAs for SLC7A11 with STX17, the rose-colored oval indicates the miRNAs for SLC7A11 with UVRAG, the green oval indicate the common miRNAs for SLC7A11 with RAB33B and STX17, the mauve oval indicates the common miRNAs for SLC7A11 with STX17 and UVRAG, the deep yellow oval indicates the common miRNAs for SLC7A11 with UVRAG and RAB33B, the sky-blue oval indicates the common miRNAs for SLC7A11 with STX17, UVRAG and RAB33B.