Multiomics Reveals Ectopic ATP Synthase Blockade Induces Cancer Cell Death via a lncRNA-mediated Phospho-signaling Network

Abstract

The EGFR tyrosine kinase inhibitor gefitinib is commonly used for lung cancer patients. However, some patients eventually become resistant to gefitinib and develop progressive disease. Here, we indicate that ecto-ATP synthase, which ectopically translocated from mitochondrial inner membrane to plasma membrane, is considered as a potential therapeutic target for drug-resistant cells. Quantitative multi-omics profiling reveals that ecto-ATP synthase inhibitor mediates CK2-dependent phosphorylation of DNA topoisomerase IIα (topo IIα) at serine 1106 and subsequently increases the expression of long noncoding RNA, GAS5. Additionally, we also determine that downstream of GAS5, p53 pathway, is activated by ecto-ATP synthase inhibitor for regulation of programed cell death. Interestingly, GAS5-proteins interactomic profiling elucidates that GAS5 associates with topo IIα and subsequently enhancing the phosphorylation level of topo IIα. Taken together, our findings suggest that ecto-ATP synthase blockade is an effective therapeutic strategy via regulation of CK2/phospho-topo IIα/GAS5 network in gefitinib-resistant lung cancer cells.

Keywords: Multiomics; RNA-binding proteomics; cancer therapeutics; drug resistance; ectopic ATP synthase; gefitinib resistance; lung cancer; micro arrays; phosphoproteome; phosphoproteomics; targeted therapies; transcriptional regulation.

Graphical abstract

Fig. 1.

Expression of ecto-ATP synthase was correlated with gefitinib sensitivity and cell survival. A, Cell viabilities with increasing concentrations of gefitinib throughout the time course (24, 48, and 72 h) were detected in lung cancer cell lines with different EGFR mutations via MTS assay. The relative cell viabilities are presented as the mean ± S.D., n = 3. The average IC₅₀ for each cell line at 48 h is shown. B, The expression levels of ecto-ATP synthase were observed via immunocytochemistry with ATP-synthase–complex antibodies in nonpermeabilized lung cancer cells. DAPI was used to stain nuclei. Scale bars, 10 μm. C, Expression of the ATP synthase complex on the cell surface was analyzed via flow cytometry using antibodies for the ATP synthase complex, or control mouse IgG, in nonpermeabilized lung cancer cells. MFI, represents the mean fluorescent intensity values of ecto-ATP synthase. D, Volcano plot depicting the gene expression differences between cells with high and low levels of ecto-ATP synthase. The orange points represent the significantly differentially expressed genes (q < 10⁻⁶ and fold-change > 4). E, Scatter plot displaying the positive correlation between the abundance of ecto-ATP synthase and cell viability under gefitinib treatment in lung cancer cells. Cell viability is represented as the area under the receiver operating characteristic (ROC) curve (AUC). Larger AUCs indicate greater resistance to gefitinib. F, Kaplan–Meier plot showing overall survival stratified according to ecto-ATP synthase levels in lung cancer patients. P: log-rank test.

Fig. 2.

Targeting ecto-ATP synthase inhibited cell proliferation and colony formation in gefitinib-resistant cells. A, An MTS assay was used to determine the cytotoxic effects of increasing concentrations of eATPi on lung cancer cell lines over the time course (24, 48, and 72 h) (mean± S.D., n = 3). The average IC₅₀ at 48 h for each cell line is shown. B, Numbers of gefitinib-resistant cells treated with the IC₅₀ of eATPi for 48 h were observed using phase-contrast microscopy and calculated via a hemocytometer cell counting assay. Scale bars, 50 μm. C, The effect of eATPi on the colony-forming ability of gefitinib-resistant cells was validated by clonogenic assay. Data in B and C are illustrated using representative images, and quantitative results are presented as the mean ± S.D. in the bar charts, n = 3. * p < 0.05; ** p < 0.01; *** p < 0.001 (two-tailed t test).

Fig. 3.

Identification of an eATPi-induced dynamic molecular network. A, Schematic representation of the multi-omics approach used with gefitinib-resistant lung cancer cells under control or eATPi treatment. Quantitative phosphoproteomic profiling of cells under treatment for 1 min, 10 min, and 60 min and transcriptomic profiling of cells under treatment for 24 h were performed to identify the early and late responses, respectively. For quantitative phosphoproteomic profiling, protein extracts obtained from the cells treated with citreoviridin (eATPi) at IC₅₀ concentration or with the same volume of DMSO were enzymatic digested and differentially stable isotope dimethyl-labeled. L represents light stable isotopes (CH₃), whereas H represents heavy stable isotopes (C¹³D₂H). All omics data were analyzed using bioinformatics platforms to establish a global eATPi-responsive network. B, Dot plot depicting the changes in the phosphorylation level of the proteins between eATPi-treated and control cells. Orange points represent the phosphosites with significant change (p < 0.05, derived from significance B). C, Volcano plot depicting the gene expression differences between eATPi-treated and control cells. The orange points represent the significantly differentially expressed genes (q < 10⁻³ and fold-change > 2). D, Enrichment map of eATPi-responsive genes and proteins. Nodes depict gene sets that were statistically overrepresented in the genes with differential expression or phosphosites with phosphorylation changes. Links connecting nodes indicate the relatedness between nodes.

Fig. 4.

The inhibitor of eATP induced cell death by promoting DNA damage, G1 cell-cycle arrest, and apoptosis. A, Cells were treated with control or the IC₅₀ of eATPi for 24 h and subjected to western blotting to detect the presence of phospho-S139-γH₂AX. B, Relative expression levels of phospho-S139-γH₂AX in control and eATPi-treated cells of each independent biological replicates were quantified and shown as the bar charts. C, Cells treated with control or the IC₅₀ of eATPi for 24 h were immunostained for phospho-S139-γH₂AX antibody (green) and counterstained with DAPI to visualize nuclei. Representative images were taken by confocal laser microscopy. Scale bars, 5 μm. D, Cell-cycle distributions of gefitinib-resistant cells treated with control or the IC₅₀ of eATPi for 24 h, detected via flow cytometry. The percentages of G0–G1 phase, S phase, and G2–M phase in each cell line were analyzed using FlowJo software. E, The percentages of apoptotic cells after eATPi treatment for 24 h were determined via Annexin V-FITC/PI double staining assay and quantified using flow cytometry. Data in D and E are illustrated using representative images, and quantitative results are reported as mean ± S.D. in the histograms, n = 3. * p < 0.05; ** p < 0.01; *** p < 0.001 (two-tailed t test).

Fig. 5.

Ecto-ATP synthase blockade triggered cell death via CK2-mediated phosphorylation of topoisomerase IIα at Ser1106. A, Kinases with significant activity changes in response to eATPi, as inferred using DynaPho software. Kinases are ranked according to p-value, shown on the x-axis. B, Cells were treated with or without the IC₅₀ of eATPi for 48 h in the presence or absence of CK2 inhibitor. Cells were counted via a hemocytometer cell counting assay. Scale bars, 50 μm. Images are representative of triplicate experiments. C, Quantitative results are presented as the bar charts. D, Phosphoproteins significantly regulated by eATPi were extracted to establish the CK2-substrates network. The color of squares indicates the relative levels of phosphoproteins (eATPi treatment/control treatment). E, Phosphorylation of topoisomerase IIα at serine 1106 was validated via western blotting using a topo IIα-pS1106 site-specific antibody. F, Cells were treated with or without the IC₅₀ of eATPi in the presence or absence of CK2 inhibitor. The phosphorylation levels of topoisomerase IIα at serine 1106 were detected via western blotting. The intensities of the bands in d and e are shown relative to β-actin, which was used as a loading control. G, Whole-cell lysates were prepared from cells transfected with HA-tagged plasmids encoding WT or S1106A-mutant topoisomerase IIα. The levels of overexpressed proteins were detected using western blotting. H, Cells were transfected with HA-tagged plasmids encoding WT or S1106A-mutated topoisomerase IIα prior to eATPi treatment. An MTS assay was performed to measure cell viability. Error bars show the mean ± S.D., n = 3. * p < 0.05; ** p < 0.01; *** p < 0.001 (two-tailed t test).

Fig. 6.

Long-term treatment with eATPi-induced programed cell death via the cooperation of GAS5 and the p53 pathway. A, The activity of transcriptional factor TP53 in cells treated with or without eATPi was determined via ISMARA. B, GSEA plot of the gene set of the p53 downstream pathway compared between eATPi-treated and control cells. C–E, The mRNA levels of p53 (in C), of p53-related genes that were responsible for programed cell death (in D) and of GAS5 (in E) were validated via RT-qPCR. Relative expression levels were normalized to the internal control, GAPDH, and relative to the mean expression level of each gene in cells without eATPi treatment (mean ± S.E.M., n = 3). F, A dual luciferase reporter assay was carried out to determine the activity of the GAS5 promoter in control versus eATPi-treated cells. Promoter activity is represented as relative luciferase activity (RLA) compared with the control (mean ± S.D., n = 3). G, Cells were transfected with scrambled (si-control) or GAS5-targeting (si-GAS5) constructs and then treated with or without eATPi. Colony-forming ability was detected using clonogenic assay (upper) and quantified using 595 nm absorbance (lower) (mean ± S.D., n = 9). H, The mRNA expression of GAS5 in cells treated with or without the IC₅₀ of eATPi in the presence or absence of CK2 inhibitor was detected using RT-qPCR. The relative expression levels of genes are expressed as mean ± S.E.M., n = 3. * p < 0.05; ** p < 0.01; *** p < 0.001 (two-tailed t test).

Fig. 7.

The lncRNA GAS5 was associated with topoisomerase IIα and increased the phosphorylation of topoisomerase IIα. A, Venn diagram showing the number of interacting proteins in common to the two lines of gefitinib-resistant cells, A549 and H1975. The proteins were enumerated via RNA pulldown assay with a biotin-labeled GAS5 transcript mixed with cell lysate. Rep1 and Rep2 indicate the first and second biological replicates. B, Enrichment map of GAS5-interacting proteins identified in both A549 and H1975 cells. Nodes represent gene sets that were statistically overrepresented in the GAS5-interacting proteins, and node size represents the number of genes in a set. Color intensity is proportional to the enrichment significance. Links connecting nodes indicate the relatedness between nodes. C, GAS5 pulldown assay followed by western blotting was performed to validate the association between topoisomerase IIα and GAS5. Input: whole-cell lysate prepared from the indicated cell lines without RNA precipitation. D, The binding of topoisomerase IIα with GAS5 was confirmed by RNA immunoprecipitation using topo IIα antibody. The PC is the RT-PCR positive control, for which we used the pBlueScript SK (+)-GAS5 plasmid as a template. E–H The mRNA expression levels of GAS5 and TOP2A were measured via quantitative RT-PCR in GAS5-overexpressing (E and F) or GAS5-knockdown cells (G and H), respectively. Values are normalized to the expression of GAPDH and relative to the expression of each gene in cells transfected with empty-vector or si-control. Data are presented as means ± S.E.M., n = 3. I and J, The protein levels and levels of phosphorylation at Ser1106 of topoisomerase IIα were detected in GAS5-overexpressing cells (I) or GAS5-knockdown cells (J) via western blotting. K and L, Relative expression levels of phospho-S1106 topo IIα and total topo IIα in GAS5-overexpressing cells (K) or GAS5-knockdown cells (L) of each independent biological replicates were quantified and shown as the bar charts. * p < 0.05; ** p < 0.01; *** p < 0.001; N.S., not significant.