Article Figures & Data
Figures
- Fig. 1.
Eribulin but not paclitaxel induces expression of interferon-β and interferon-stimulated genes in THP-1 cells. (A) Soluble (S) or polymerized (P) β-tubulin in THP-1 cells treated with 100 nM eribulin (ERB) or paclitaxel (PTX) for 2, 6, or 24 hours as compared with DMSO (Veh). (B) Cell cycle analysis of THP-1 cells treated with 100 nM eribulin or paclitaxel for 2, 6, or 24 hours compared with DMSO (Veh) as percentage of cells in G1 (black), S (gray), or G2/M (white). (C) Caspase 3/7 cleavage in THP-1 cells treated with 100 nM eribulin or paclitaxel for 2, 6, or 24 hours compared with DMSO as percentage of live (white) or apoptotic (gray) cells from two independent experiments with errors denoting range. (D) IFNβ mRNA in THP-1 cells treated with 100 nM eribulin or paclitaxel for 2, 6, or 24 hours as compared with DMSO. Significance determined by vehicle compared one-way ANOVA with Dunnett’s post hoc test compared with vehicle. (E) IFNβ intracellular protein in live cells treated with DMSO or 100 nM eribulin for 2 or 6 hours. Individual data points represent three independent experiments, and significance was determined by two-way ANOVA (time * drug) with Tukeys’s post hoc test. (F and G) IFIT1 mRNA in THP-1 cells (F) pretreated with 1 µM of ruxolitinib (JAKi) or vehicle for 4 hours and then treated with DMSO or 100 nM eribulin for 24 hours with the inhibitor still present or (G) with siRNA to IFNAR1/2 (siIFNAR1/2) or a scrambled sequence (siCtrl) for 48 hours followed by treatment with DMSO or 100 nM eribulin for 24 hours. Significance was determined by two-way ANOVA (drug * inhibitor/siRNA) with Tukeys’s post hoc test. qRT-PCR data are shown as individual points from two independent biologic replicates with error bars denoting range. *P < 0.05, **P < 0.01, ****P < 0.0001. Veh, vehicle.
- Fig. 2.
Eribulin-mediated interferon-β expression occurs independently of mitotic arrest and caspase cleavage. (A) Immunofluorescence images of microtubules (green) and DNA (blue) in BMDM cells treated with 100 nM eribulin (ERB) for 2, 6, or 24 hours as compared with DMSO (Veh). Scale bar, 10 µm. (B) Cell cycle analysis of BMDM cells treated with 100 nM eribulin for 2, 6, or 24 hours as compared with DMSO as percentage of cells in G1 (black), S (gray), or G2/M (white) from two independent experiments, with error bars denoting range. (C) Caspase 3/7 cleavage in wild-type BMDM cells treated with 100 nM eribulin for 2, 6, or 24 hours compared with DMSO as percentage of live (white) or apoptotic (gray) cells from two independent experiments with errors denoting range. IFNβ (D) and IFIT1 (E) mRNA in BMDM cells treated with 100 nM ERB for 2, 6, or 24 hours compared with DMSO. qRT-PCR data are shown as individual points from two independent biologic replicates with error bars denoting range. Significance was determined by vehicle-compared one-way ANOVA with Dunnett’s post hoc test. **P < 0.01, ****P < 0.0001. Veh, vehicle.
- Fig. 3.
Eribulin-dependent upregulation of interferon-β and interferon-stimulated genes is dependent on the STING pathway. IFNβ (A) and IFIT1 (B) mRNA in BMDM cells pretreated with 1 µM of the indicated inhibitor for 4 hours and then treated with 100 nM eribulin (ERB) for 2, 6, or 24 hours as compared with DMSO with the inhibitor still present from two independent experiments with error bars denoting range. IFNβ (C and E) and IFIT1 (D and F) mRNA from BMDM and THP-1 cells pretreated with 1 µM H-151 (STINGi) for 4 hours and then treated with 100 nM eribulin for 2, 6, or 24 hours as compared with DMSO with the inhibitor still present. IFNβ (G) and IFIT1 (H) mRNA in wild-type and Sting gt/gt BMDM cells treated with 100 nM eribulin for 2, 6, or 24 hours. Data are shown as individual points from two independent biologic replicates with error bars denoting range. Significance was determined by two-way ANOVA (time * inhibitor) using a Tukey’s post hoc test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Veh, vehicle.
- Fig. 4.
Expression of interferon-β by eribulin in TNBC cell lines requires the DNA sensor cGAS. (A) Immunoblot analysis of cGAS, STING, and β-tubulin expression in THP-1 and TNBC cells. (B) IFNβ mRNA in TNBC cells transfected with 1 µg of HT-DNA for 24 hours or mock-transfected. (C and D) IFNβ mRNA in TNBC cells treated with 100 nM eribulin (ERB) for 2 hours (C) or 6 hours (D) as compared with DMSO controls. Significance was determined by two-way ANOVA (cell line * drug) with Tukey’s post hoc test. (E) IFIT1 mRNA in CAL-51 cells transfected with RFP-cGAS or mock-transfected and treated with DMSO (Veh) or 100 nM ERB for 24 hours. Significance was determined by two-way ANOVA (cGAS * drug) with Tukey’s post hoc test. (F) HCC1937 IFNβ intracellular protein in live cells treated with DMSO or 100 nM eribulin for 6 hours. Significance determined by an unpaired two-tailed t test. (G) Human IFIT1 mRNA in HCC1937 cells treated with 100 nM eribulin for 2, 6, or 24 hours compared with DMSO. Significance determined by vehicle-compared one-way ANOVA with Dunnett’s post hoc test. Data are shown as individual points from two independent biologic replicates with error bars denoting range. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Veh, vehicle.
- Fig. 5.
Eribulin promotes cytoplasmic accumulation of mtDNA. (A) qRT-PCR analysis of relative abundance of genomic DNA (gDNA) (ACTb, GAPDH, HPRT, PGK, RPS18, and TBP) and mitochondrial DNA (mtDNA) (ATP6, ATP8, COX-1, ND1, ND4, and ND6) sequences present in the cytoplasm of HCC1937 cells treated with 100 nM eribulin (ERB) for 6 hours. Data are shown as the ratio of gDNA or mtDNA in the cytoplasmic fraction as compared with the organelle-enriched fraction and normalized to the vehicle control as fold change. Significance was determined by one-way ANOVA with Dunnett’s post hoc test. (B and C) COX-1 mtDNA present in the cytoplasm of HCC1937 (B) or BMDM (C) cells treated with 100 nM eribulin or paclitaxel (PTX) for 6 hours as compared with vehicle. Significance determined by vehicle-compared one-way ANOVA with Dunnett’s post hoc test compared with vehicle. (D) IFNβ mRNA in THP-1 cells treated with 10, 100, or 1000 nM ERB, vinorelbine (VNR), ixabepilone (IXA), PTX, or docetaxel (DTX) for 24 hours. Significance determined by vehicle-compared two-way ANOVA (drug * concentration) with Tukey’s post hoc test compared with vehicle. (E) IFIT1 mRNA in THP-1 cells treated with 10, 100, or 1000 nM ERB, VNR, or PTX for 24 hours. Significance determined by vehicle-compared two-way ANOVA (drug * concentration) with Tukey’s post hoc test compared with vehicle. (F) COX-1 mRNA in control and ethidium bromide cultured (Rho0) HCC1937 cells treated with 100 nM eribulin for 2 or 6 hours as compared with DMSO. Significance was determined by two-way ANOVA (Rho status * drug) with Tukey’s post hoc test. (G) IFNβ mRNA in control and Rho0 HCC1937 cells treated with 100 nM eribulin for 2 or 6 hours as compared with DMSO. Significance was determined by two-way ANOVA (Rho status * drug) with Tukey’s post hoc test. (H) Cytoplasmic COX-1 DNA present in control and Rho0 HCC1937 cells treated with 100 nM eribulin or paclitaxel for 6 hours as compared with DMSO. Significance was determined by two-way ANOVA (Rho status * drug) with Tukey’s post hoc test. Data are shown as individual points from two independent biologic replicates with error bars denoting range. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Veh, vehicle; ctrl, control.
Additional Files
Data Supplement
- Supplemental Data -
Figure S1. The effects of eribulin and paclitaxel on cytokine expression.
Figure S2. Eribulin-mediated expression of interferon stimulated genes is dependent on canonical IFN-mediated JAK signaling.
Figure S3. Pharmacological inhibitors do not increase apoptosis in BMDMs.
Figure S4. Inhibition of STING suppresses eribulin-mediated interferon β expression.
Figure S5. Eribulin-mediated expression of interferon stimulated genes in CAL-51 cells is dependent on the DNA sensor cGAS.
Figure S6. DNA sensing by the cGAS-STING pathway is retained in HCC1937 Rho0 cells.
Table S1. DNA oligonucleotides used in this study.
- Supplemental Data -
In this issue
Eribulin Activates cGAS-STING via mtDNA Accumulation
