Fig 1: SIPA1 alters ATP production source from respiration to aerobic glycolysis. (A–B) ATP levels in MDA-MB-231 (A) and MCF7 (B) with various SIPA1 expression levels after treatment with oligomycin or not. A mitochondria ATP level was calculated by the following formula (ATP(m) = ATP(T) - ATP(g)), in which total ATP level (ATP(T)) was measured after incubating cells with 15 mM glucose and the glycolytic ATP (ATP(g)) were measured after incubating cells with 15 mM glucose and 100 nM oligomycin for 5 h. Yellow bars: control; green bars: oligomycin (left panel). Based on the left panel, the contribution of each ATP source on cellular ATP production was estimated. Red: aerobic glycolysis; white: mitochondrial respiration (right panel). (C–D) Effect of sodium pyruvate as a sole carbon source on ATP production in MDA-MB-231 (C) and MCF7 cells (D) with various SIPA1 expression levels. Cells were grown in the medium containing glucose or sodium pyruvate as a sole carbon source, and the cellular ATP level was measured. Yellow bars, glucose; green bars, sodium pyruvate (left panel). Based on the left panel, the contribution of each ATP source on cellular ATP production was estimated. Red: aerobic glycolysis; white: mitochondrial respiration (right panel). (E–F) Effect of oxamate and oligomycin on the proliferation of MDA-MB-231 (E) and MCF7 cells (F). (G) mRNA levels of PDK1 were determined by qRT-PCR. (H) Protein levels of PDK1 were determined by Western blotting. Data were shown as mean ± s.d of triplicate measurements (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001, ns, no significance. (Student’s t-test).
Fig 2: SIPA1/EPAS1 axis is a poor prognosis predictor of breast cancer. (A–B) mRNA level of SIPA1 in breast cancer subtypes in TCGA (A) and GSE41313 dataset (B). (C–D) The relationship between SIPA1 and EPAS1 was analyzed based on TCGA (C) and GSE41313 datasets (D). Pearson correlation coefficients (Pr) for SIPA1 and EPAS1 gene expression levels were shown. (E) Kaplan-Meier survival analysis for survival probability of breast cancer patient in TCGA according to EPAS1 expression status. The p value was presented using the log-rank test. (F) A proposed diagram for SIPA1-mediated aerobic glycolysis and breast cancer cell metastasis.
Fig 3: SIPA1 enhances aerobic glycolysis in breast cancer cells under a normoxic condition. (A) SIPA1 protein expression levels in breast cancer cells were determined by Western blotting analysis. 231si (SIPA1-knocked down MDA-MB-231); 231si + SIPA1 (SIPA1-resumed 231si); MCF7/SIPA1 (SIPA1 overexpressed MCF7). β-tubulin was included as a loading control. (B–C) Effect of SIPA1 on the lactate production (B) and the glucose consumption (C) in breast cancer cell lines. (D) Heatmap of mRNA levels of major glycolysis-related genes in three independent parental and SIPA1-knockdown MDA-MB-231 cells, and two independent MCF7 and MCF/SIPA1 cells were presented with Log2 FPKM by transcriptome sequencing and analysis. (E) mRNA levels of glycolysis-related genes were determined by qRT-PCR. Data were shown as mean ± s.d. The experiments were conducted in triplicate. Mitochondrial succinate dehydrogenase cytochrome b560 subunit (SDHC) was included as an endogenous control. (F) Expression of HK2, LDHA, HIF-1α and c-myc were determined by Western blotting. β-tubulin was included as a control. (G–H) the extracellular lactate dehydrogenase (LDH) activity (G) and intracellular LDH activity (H) were determined. Data are shown as mean ± s.d triplicate measuements (n = 3). **p < 0.01, ***p < 0.001. (Student’s t-test).
Fig 4: SIPA1 upregulates EPAS1 transcription by binding to its promoter region. (A) Transcriptome sequencing datasets of two pairs of cells (MDA-MB-231 vs 231si, MCF7 vs MCF7/SIPA1) were analyzed and 432 overlapping candidate genes which were positively regulated by SIPA1 were selected in the Venn diagram. (B) GO biological process enrichment analyses of the 432 genes positively correlated with SIPA1. Top nine GO terms were listed with p values, counts and enrichment scores. (C) Genes allocated to “responses to hypoxia” in (B) were plotted as a protein-protein interaction network. (D) qRT-PCR analyses of EPAS1 and its downstream gene candidates, TGFB1, VEGFA and CA9. Data were shown as mean ± s.d. The experiments were conducted in triplicate. SDHC was included as an endogenous control. (E) Western blotting analyses of HIF-2α expression in breast cancer cells with various SIPA1 levels. β-tubulin was included as an internal control. (F) The effect of SIPA1 on EPAS1 promoter activity were accessed by a luciferase reporter assay. (G) Interaction of SIPA1 with EPAS1 gene promoter revealed by ChIP-PCR. Seg 1, Seg 2 and Seg 3 represented the indicated promoter regions. TSS, transcription start site. (H) Interactions of truncated SIPA1 protein (540-1042aa) with EPAS1 gene promoter segment 2 were revealed by EMSA. Data shown are mean ± s.d. of triplicate measurements (n = 3).
Fig 5: SIPA1/HIF-2α axis blockade suppresses aerobic glycolysis and breast cancer growth and metastasis in a xenograft mouse model. (A) The schematic overview of the xenograft experimental workflow. Oxamate treatment on 231si cell xenografted tumor formation experiment was done twice (1# and 2#). (B) Tumor volumes in the four groups of mice were measured from day 5 to day 19. (C) The tumors in each group were photographed and weighed. (D) Lactate levels in tumor tissues were determined in each group, n = 3. (E) mRNA levels of glycolytic rate-limiting genes in tumor tissues of mice (n = 3 in each group) were determined by qRT-PCR. Data are shown as mean ± s.d. The experiments were done in triplicate. (F) Immunohistochemical analyses of HIF-2α, SLC2A1 and PDK1 in tumor tissues derived from two groups of mice were performed. (G) Lungs from four groups of mice were photographed (left panel) and the number of nodules observed on the lung surface were counted (right panel). (H) Representative histopathological images of HE staining in the lungs were presented. Scale bar: 1 mm. Data are mean ± s.d. of triplicate measurements. *p < 0.05, **p < 0.01, ***p < 0.001. (Student’s t-test).
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