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. 2019 Apr;1(4):460-474.
doi: 10.1038/s42255-019-0052-9. Epub 2019 Apr 8.

miR-147b-mediated TCA Cycle Dysfunction and Pseudohypoxia Initiate Drug Tolerance to EGFR Inhibitors in Lung Adenocarcinoma

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Free PMC article

miR-147b-mediated TCA Cycle Dysfunction and Pseudohypoxia Initiate Drug Tolerance to EGFR Inhibitors in Lung Adenocarcinoma

Wen Cai Zhang et al. Nat Metab. .
Free PMC article

Abstract

Drug-tolerance is an acute defense response prior to a fully drug-resistant state and tumor relapse, however there are few therapeutic agents targeting drug-tolerance in the clinic. Here we show that miR-147b initiates a reversible tolerant-state to the EGFR inhibitor osimertinib in non-small cell lung cancer. With miRNA-seq analysis we find that miR-147b is the most upregulated microRNA in osimertinib-tolerant and EGFR mutated lung cancer cells. Whole transcriptome analysis of single-cell derived clones reveals a link between osimertinib-tolerance and pseudohypoxia responses irrespective of oxygen levels. Further metabolomics and genetic studies demonstrate that osimertinib-tolerance is driven by miR-147b repression of VHL and succinate dehydrogenase linked to the tricarboxylic acid cycle and pseudohypoxia pathways. Finally, pretreatment with a miR-147b inhibitor delays osimertinib-associated drug tolerance in patient-derived three-dimensional (3D) structures. This link between miR-147b and tricarboxylic acid cycle may provide promising targets for preventing tumor relapse.

Conflict of interest statement

Competing interests The authors declare no competing financial interests.

Figures

Figure 1
Figure 1. NSCLC cells adopt a tolerance strategy against EGFR-TKIs.
a, Representative phase contrast images of 3D structures from AALE cells cultured according to the protocol at top of the panel. Scale bar, 50 μm. Repeated six times with similar results. b, Top, the scenario of anti-EGFR tolerance and resistance in lung cancer. The tumor cells treated with the EGFR-TKI gefitinib or osimertinib enter a reversible drug-tolerant cycle (in green arrows, 1° Tolerant) with a brief therapy withdrawal (up to 21 days) followed by reinstatement of the 160 nM dose (2° Tolerant). Alternatively, the tumor cells treated continuously with gefitinib or osimertinib without therapy interruption undergo drug-tolerance briefly and go into a drug-resistance state in which cells do not respond to gefitinib (1° Resistant)/osimertinib (2° Resistant). Bottom, osimertinib treatment response on HCC827 3D structures. Representative images of Parental cells, 1° Tolerant cells (derived from the Parental cells treated with 160 nM osimertinib for 11 days), Recovered cells (derived from the 1° Tolerant cells with a therapy withdrawal up to 21 days) and 2° Tolerant cells (derived from the Recovered cells by reinstatement of the 160 nM dose for 11 days). Scale bar, 200 μm. Repeated six times with similar results. c, Representative phase contrast microscopy (left panel) and H&E staining of HCC827 3D structures derived from parental (top) and osimertinib-tolerant (bottom) cells. Images in blue dotted squares (middle panel) were amplified (right panel) and shown. Scale bar, 50 μm. Repeated six times with similar results. d, qRT-PCR analysis of SFTPC, HOPX, ID2 and CEACAM5 expression in single cell clone HCC827-derived 3D structures in the presence of osimertinib. Single cell clone derived cells were plated with geltrex and treated with 100 nM osimertinib (tolerant) or vehicle (parental) for 24 days. Gene expression for surviving 3D structures were analyzed. n=3 independent biological replicates. e, Single-cell clonogenicity of PC9 cells treated with gefitinib. A single cell was sorted by FACS into a 96-well plate and treated with 0.1, 0.4, and 2 μM gefitinib or the vehicle for 14 days. The frequency of colony formation was calculated as a ratio of the total number of colonies to the total number of wells plated with a single cell in a 96-well plate. n=3 independent biological replicates. f, qRT-PCR analysis of top upregulated and downregulated genes in gefitinib-tolerant clones (n=2) compared with vehicle-treated parental single cell clone (n=1) in PC9. The gene expression in parental sensitive clone was calibrated as 1. ACTB was used as endogenous control. n=4 independent biological replicates. g, Whole transcriptome and gene ontology analysis of gefitinib-tolerant clones compared with the parental single cell clone in PC9. n=4 independent biological replicates. h, qRT-PCR analysis of genes in top regulated signaling pathways including Wnt planar cell polarity signaling, glutamine metabolic process, cellular response to hypoxia, and tricarboxylic acid cycle in gefitinib-tolerant clones compared with parental the single cell clone in PC9. The gene expression in parental sensitive clone was calibrated as 1. ACTB was used as endogenous control. n=4 independent biological replicates. Data are mean ± s.e.m. and were analysed with unpaired two-tailed t-test with Welch’s correction (d); modified one-tailed Fisher’s Exact test (g).
Figure 2
Figure 2. miR-147b initiates drug-tolerance.
a, A heat map showing top upregulated and downregulated miRNAs in two paired osimertinib-tolerant (OTR) and parental cells in PC9 and HCC827 by miRNA-seq analysis. b, qRT-PCR analysis of miR-147b expressions in parental, recovered, primary and secondary osimertinib-tolerant cells in PC9. The parental tumor cells treated with 160 nM EGFR-TKI osimertinib for 6 days enter a drug-tolerant state (primary tolerant cells) with a brief therapy withdrawal up to 18 days (recovered cells) followed by reinstatement of the 160 nM dose for 11 days (secondary tolerant cells). The relative miR-147b expression level in the parental cells were calibrated as 1. miR-423 was used as endogenous control. n=3 independent biological replicates. c-d, Osimertinib (c) and gefitinib (d) treatment response on scrambled control (Scr) and miR-147b-overexpressing cells (147b) in HCC827 for 3 days. n=3 independent biological replicates. e, Osimertinib (40 nM) and gefitinib (40 nM) treatment response on scrambled control and miR-147b-overexpressing cells in HCC827 by colony formation assay. 20, 40 and 80 cells were plated in 10-cm dish and the colonies were stained with Giemsa on day 10 and the total number of colonies were quantified. n=3 independent biological replicates. f, Osimertinib treatment response on H1975 cells with miR-147b knockdown (anti147b) and scrambled control (antictrl). The cell viability was measured on day 4. n=3 independent biological replicates. g, Osimertinib (160 nM) treatment response on H1975 cells with miR-147b knockdown. Left, the monolayer colonies were treated for 10 days and stained with Giemsa. Right, the 3D structures were treated for 14 days. -, vehicle; +, osimertinib. Scale bar, 1000 μm. n=3 independent biological replicates. Data are mean ± s.e.m. and were analysed with one-way ANOVA (b); unpaired two-tailed t-test with Holm-Sidak’s correction (e,g).
Figure 3
Figure 3. miR-147b-VHL axis mediates drug-tolerance through impaired VHL activity.
a, Left, gene candidates predicted for miR-147b by the TargetScan tool were shown in signaling pathways enriched for gefitinib-tolerance in PC9 single-cell clones in fig. 1f. Right, qRT-PCR analysis for the predicted gene candidates for miR-147b in H1975 cells with miR-147b knockdown compared with scrambled control. n=3 independent biological replicates. b, Left, computational prediction of RNA duplex formation between miR-147b and the 3’UTR (untranslated region) of VHL mRNA. Mutations generated within the 3’UTR for the luciferase assay are shown in red. Right, dual-luciferase reporter assay in miR-147b-overexpressing AALE cells. The Firefly luciferase and Renilla luciferase activities were measured 48 hours post co-transfection with miR-147b or control vector and wild-type (WT) or mutant (Mut) VHL 3’UTR. n=3 independent biological replicates. c, Western blot analysis and quantification of VHL in miR-147b-overexpressing AALE cells. β-Actin was used as loading control. n=3 independent biological replicates. d, qRT-PCR analysis for fold change of hypoxia gene expression in AALE cells with miR-147b overexpression relative to scrambled control (147b/Scr) and cells with co-overexpression of miR-147b and VHL relative to scrambled control (147b+VHL/Scr). ACTB was used as endogenous control. n=3 independent biological replicates. e, Fractional viability of HCC827 cells treated with vehicle, osimertinib (20 nM), miR-147b vector, VHL vector or combinations. The cell viability was measured on day 3. The relative cell viability treated with vehicle on day 3 was calibrated as 1. n=7 independent biological replicates. Data are mean ± s.e.m. and were analysed with unpaired two-tailed t-test (a,b,c,d); Kruskal-Wallis test (e).
Figure 4
Figure 4. miR-147b-SDH axis mediates drug tolerance through SDH enzyme activity in the TCA cycle.
a, Left, computational prediction of RNA duplex formation between miR-147b and the 3’UTR of SDHD mRNA. Mutations generated within the 3’UTR for the luciferase assay are shown in red. Right, dual-luciferase reporter assay in miR-147b-overexpressing AALE cells. The Firefly luciferase and Renilla luciferase activities were measured 48 hours post co-transfection with miR-147b or control vector and wild-type (WT) or mutant (Mut) SDHD 3’UTR. n=3 independent biological replicates. b, Principal component analysis (PCA) of parental cells, osimertinib-tolerant cells (H1975OTR) and tolerant cells with miR-147b knockdown (H1975OTR-anti147b) in H1975 cell monolayers. The tolerant cells were derived from the parental cells treated with 100 nM osimertinib continuously for 21 days. n=5 independent biological replicates. c, A heat map showing top metabolites levels across cells of H1975, H1975OTR and H1975OTR-anti147b. n=5 independent biological replicates. d, Levels of succinate, 2-oxoglutarate, fumarate, and malate in cells of H1975, H1975OTR and H1975OTR-anti147b. The relative levels in the parental H1975 cells were calibrated as 1. n=5 independent biological replicates. e, Schematic of the interaction among miR-147b and SDH enzyme leading to dysregulated TCA cycle metabolites for drug-tolerance to EGFR tyrosine kinase inhibitors. Upregulated levels of oxoglutarate and succinate (in red) as well as downregulated levels of fumarate and malate (in green) in drug-tolerant cells are highlighted. f, SDH inhibitor promotes drug-tolerance to osimertinib in H1975 cells. Vehicle or 100 nM osimertinib (osim)-treated cells were co-incubated with 0, 0.03 and 0.1 mM membrane-permeable dimethyl malonate (DMM) for 3 days. The cell viability was measured on day 4. n=3 independent biological replicates. Data are mean ± s.e.m. and were analysed with unpaired two-tailed t-test (a,f); one-way ANOVA (d).
Figure 5
Figure 5. Blocking miR-147b overcomes drug-tolerance.
a, Fractional viability of H1975 3D structures treated with osimertinib (25 nM), LNA miR-147b inhibitor (LNA-anti147b, 90 nM), DMOG (10 μM) or combinations for 14 days. n=3 independent biological replicates. b, qRT-PCR analysis for hypoxia gene expression in H1975 cells treated with 90 nM LNA miR-147b inhibitor (LNA-anti147b) and 10 µM DMOG or vehicle for three days. The relative gene expression in scrambled control cells treated with vehicle was calibrated as 1. n=3 independent biological replicates. c, Fractional viability of H1975 3D structures treated with 25 nM osimertinib, 90 nM LNA-anti147b, 30 µM R59949 or combinations for 14 days. n=7 independent biological replicates. d, qRT-PCR analysis of HIF1A in H1975 cells with shRNAs against HIF1A. H1975 cells were transfected with shRNAs against HIF1A (shHIF1A-1 and -2) or scrambled control (shCtrl) and selected with 0.5 μg/ml puromycin. GAPDH was used as endogenous control. n=3 independent biological replicates. e, Cell viability of H1975 cells with HIF1A knockdown treated with osimertinib. The cells with shRNAs against HIF1A (shHIF1A-1 and shHIF1A-2) and scrambled control cells (shCtrl) were treated with 100 nM osimertinib or vehicle for 3 days. The cell viability was analyzed on day 4. n=4 independent biological replicates. f, Cell viability of H1975 cells with constitutive active HIF1A mutant treated with osimertinib. The cells were transfected with HIF1A A588T and scrambled control cells (Scr) followed by 600 μg/ml neomycin selection. Then the cells were treated with 100 nM osimertinib or vehicle for 3 days. The cell viability was analyzed on day 4. n=4 independent biological replicates. g, Derivation and growth of 3D structures from lung PDX tumors. (top) Representative phase contrast microscopy for parental EGFR mutant lung PDX-derived 3D structures in PDX_LU_10 3D structures. Repeated six times with similar results. (Bottom) growth curve of PDX 3D structures. The 3D structures size was measured every two days. The media were replenished every three days till day 14. n=3 independent biological replicates. Scale bar, 50 µm. h, Pretreatment response on lung PDX_LU_10 3D structures with LNA miR-147b inhibitor (anti147b) and osimertinib. The 3D structures were established at medium size seven days after seeding 2000 single-cells into 3D cultures in 96-well plate. This timepoint was recorded as day 0. Then the 3D structures were administrated with LNA anti147b or antictrl (90 nM) on day 0 and day 2 or osimertinib (25 nM) on day 1 and day 4. The vehicle treated group did not receive treatments with LNA or osimertinib. The 3D structures’ size was measured every two days. The media were replenished every three days till day 14. n=3 independent biological replicates. i, Schematic for miR-147b-driven drug-tolerance model. miR-147b is enriched in a subpopulation of parental lung cancer cells entering drug-tolerant status when they are treated with EGFR-TKIs. miR-147b mediates drug-tolerance through repressing activities of VHL and SDH leading to activated pseudohypoxia response. TKI, tyrosine kinase inhibitor; SDH, succinate dehydrogenase; TCA, tricarboxylic acid; PHD, prolyl-hydroxylase. Data are mean ± s.e.m and were analysed with Kruskal-Wallis test (a,c); unpaired two-tailed t-test (b,d,e,f,h).

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