Inhibition of Aurora Kinase B attenuates fibroblast activation and pulmonary fibrosis

Abstract

Fibroblast activation including proliferation, survival, and ECM production is central to initiation and maintenance of fibrotic lesions in idiopathic pulmonary fibrosis (IPF). However, druggable molecules that target fibroblast activation remain limited. In this study, we show that multiple pro-fibrotic growth factors, including TGFα, CTGF, and IGF1, increase aurora kinase B (AURKB) expression and activity in fibroblasts. Mechanistically, we demonstrate that Wilms tumor 1 (WT1) is a key transcription factor that mediates TGFα-driven AURKB upregulation in fibroblasts. Importantly, we found that inhibition of AURKB expression or activity is sufficient to attenuate fibroblast activation. We show that fibrosis induced by TGFα is highly dependent on AURKB expression and treating TGFα mice with barasertib, an AURKB inhibitor, reverses fibroblast activation, and pulmonary fibrosis. Barasertib similarly attenuated fibrosis in the bleomycin model of pulmonary fibrosis. Together, our preclinical studies provide important proof-of-concept that demonstrate barasertib as a possible intervention therapy for IPF.

Keywords: Aurora Kinase B; Barasertib; Wilms’ tumor 1; fibroproliferation; pulmonary fibrosis.

Figure 1. Multiple growth factors upregulate AURKB in severe fibrotic lung disease

1. A

   Quantification of AURKB transcripts in non‐IPF lung‐derived resident fibroblasts treated with indicated mitogens for 16 h. *P < 0.05, ***P < 0.0005, and ****P < 0.00005, 1‐way ANOVA (n = 4).

2. B

   Quantification of AURKB gene transcripts in isolated fibroblasts from non‐IPF and IPF lung stromal cell cultures. **P < 0.005, unpaired t‐test (n = 6).

3. C

   IPF and non‐IPF lung sections were immunostained using AURKB antibody (n = 4). Representative images were obtained at 40× magnification. Scale bar: 40 μm. Dotted area: black; myofibroblastic core, blue; active fibrotic front. Arrows indicate AURKB positive cells.

4. D

   Immunoblots of Aurkb and Aurka in total lung lysates of normal (CCSP/−) and fibrotic (CCSP/TGFα) mice fed with Dox for 6 weeks. Quantification was performed using phosphor imager software and normalization was done using loading control Gapdh. *P < 0.05, unpaired t‐test (n = 4).

5. E

   Immunostaining was performed using AURKB antibody in lung sections of control (CCSP/−) and TGFα (CCSP/TGFα) mice on Dox for 6 weeks (n = 6). Representative images were obtained at 20× (low; Scale bar: 100 μm) and 63× (high; Scale bar: 30 μm) magnification. Dashed box indicates area of the section showing in high magnification.

6. F

   Western blot analysis of Aurkb in total lung lysates from saline and bleomycin‐treated mice. Quantification was performed using phosphor imager software, and normalization was done using loading control Gapdh. **P < 0.005, unpaired t‐test (n = 4–5).

Data information: All data were presented as mean ± SEM. Exact P values are shown in Appendix Table S6. Source data are available online for this figure.

Figure EV1. Lung‐resident fibroblasts show AURKB localized in the nucleus

Primary lung‐resident fibroblasts were isolated from lung fibroblast cultures of TGFα mice on Dox for 4 weeks. Co‐immunostaining was performed using antibodies against AURKB (Green) and Vimentin (Red). All images were obtained at 40× magnification. Scale bar, 50 μm.

Figure 2. WT1 regulates AURKB expression

1. A

   Human IPF lung fibroblasts were transiently transfected with control or WT1 siRNA for 72 h and AURKB transcripts were quantified. ****P < 0.00005, unpaired t‐test (n = 4).

2. B

   Lung‐resident fibroblasts from TGFα mice on Dox for 4 weeks were transiently transfected with control or WT1 siRNA for 72 h, and AURKB transcripts were quantified. ****P < 0.00005, unpaired t‐test (n = 4).

3. C

   Immunoblot analysis of AURKB and WT1 in the lysates of non‐IPF fibroblasts transduced with control or WT1‐adenoviral particles for 72 h. **P < 0.005, unpaired t‐test (n = 3).

4. D

   Quantification of AURKB transcripts in primary fibroblasts from IPF lung treated with either control or WT1 siRNA and stimulated with media or TGFα (100 ng/ml) for 16 h. ****P < 0.00005, unpaired t‐test (n = 4).

5. E

   Primary lung‐resident fibroblasts were isolated from stromal cultures of TGFα mice placed on Dox for 8 weeks. Cell lysates were prepared, and the ChIP assay was performed with anti‐WT1 antibody or normal rabbit IgG as a negative control using AURKB gene promoter‐specific PCR primers. Non‐immunoprecipitated DNA is represented as input DNA (product size, 104 bp). ***P < 0.0005, unpaired t‐test (n = 2).

6. F

   AURKB promoter luciferase activity was measured in HEK293 cells transfected with control or WT1 overexpressing (OE) vector. ****P < 0.00005, one‐way-ANOVA (n = 6).

Data information: All data were presented as mean ± SEM. The above data were a representative of two independent experiments with similar results. P values were shown in Appendix Table S6. Source data are available online for this figure.

Figure EV2. WT1 increases AURKB expression

1. A

   Quantification of AURKB transcripts in non‐IPF fibroblasts transduced with control or WT1 adenoviral particles for 72 h (n = 4). Data were presented as mean ± SEM. **P < 0.005, unpaired t‐test.

2. B

   Schematic illustration of AURKB gene with location of the putative WT1 binding sites that were conserved among mammals including humans and mice.

Data information: All data are presented as mean ± SEM.

Figure EV3. The knockdown of AURKB alters IPF‐specific gene network

1. A

   Primary lung‐resident fibroblasts isolated from TGFα mice on Dox for 10 days. Cells were transiently transfected with control or Aurkb siRNA, and transcripts were quantified by RT–PCR after 72 h. Data were presented as mean ± SEM. ****P < 0.00005, unpaired t‐test (n = 3).

2. B

   Heat map shows two clusters of differentially expressed genes up‐ or downregulated (indicated with color key) by twofold or more upon genetic knockdown of Aurkb compared to control siRNA. (n = 3).

Data information: All data were presented as mean ± SEM.

Figure 3. AURKB functions as a positive regulator of fibroproliferation and survival

1. A

   Venn diagram depicting the comparison and overlap of differentially expressed genes in IPF lungs and AURKB siRNA‐treated fibrotic fibroblasts. The dashed box indicates genes that were up‐ (141 genes) or downregulated (DN, 50 genes) in IPF lungs compared with AURKB siRNA knockdown gene expression signatures.

2. B

   AURKB‐driven genes activated in IPF were analyzed using ToppFun and visualized using Cytoscape. Red‐ and blue‐colored circles represent genes that are up or downregulated, respectively, in IPF lungs. The turquoise‐colored circle represents enriched biological processes for the inversely correlated genes between AURKB siRNA knockdown and IPF.

3. C

   Lung sections from control and TGFα mice fed with Dox food for 4 weeks were stained for AURKB (green) and Ki‐67(red). Merged image shows cells that co‐express AURKB and Ki‐67 (yellow).

4. D

   Quantification of Ki‐67⁺ and AURKB⁺ double‐positive cells was performed using five confocal images per mice in control and TGFα mice. Images were obtained at 40× magnification. Scale bar: 50 μm. ****P < 0.00005, unpaired t‐test (n = 4).

5. E

   Proliferation was measured in primary lung‐resident fibroblast isolated from either IPF lung or TGFα mice and treated with either control or AURKB siRNA. ****P < 0.00005, unpaired t‐test (n = 9–11).

6. F

   Proliferation was measured in primary lung‐resident fibroblasts isolated from stromal cultures of TGFα mice on Dox for 2 weeks and transiently transfected with control or AURKB siRNA and stimulated with TGFα (20 ng/ml) for 24 h. ****P < 0.00005, 1‐way ANOVA (n = 9–11).

7. G

   Quantification of CCNA2 and PLK1 transcripts in human IPF fibroblasts transiently transfected with control or AURKB siRNA for 72 h. **P < 0.005, ***P < 0.0005, unpaired t‐test (n = 3).

8. H

   Lung sections from control and TGFα mice fed with Dox food for 6 weeks were stained for AURKB (green) and αSMA (red). Images were obtained at 40× magnification. Scale bar: 50 μm. (n = 4).

9. I

   Quantification of apoptotic cells using Incucyte ZOOM (caspase 3/7-positive cells) in lung‐resident fibroblasts isolated from IPF and TGFα mice on Dox for 6 weeks and treated with control or AURKB siRNA for 72 h. **P < 0.005, ****P < 0.00005, 2‐way ANOVA (n = 4).

10. J

    Quantification of Bak, Bax, and Fas gene transcripts in human IPF lung‐resident fibroblasts treated with either control or AURKB siRNA for 72 h. **P < 0.005, ***P < 0.0005, unpaired t‐test (n = 3).

11. K

    Quantification of Bak, Bax, and Fas gene transcripts in lung‐resident fibroblasts isolated from TGFα mice and treated with either control or AURKB siRNA for 72 h. *P < 0.005, **P < 0.005, unpaired t‐test (n = 3).

Data information: All data were presented as mean ± SEM. The above data were a representative of two independent experiments with similar results. Exact P values are shown in Appendix Table S6.

Figure 4. Blockade of AURKB activity impacts fibroblast proliferation and survival

1. A

   Proliferation was assessed using BrdU incorporation assay in human IPF fibroblasts treated with indicated doses of barasertib for total of 48 h. ****P < 0.00005, 1‐way ANOVA (n = 9–11).

2. B

   Proliferation was assessed using BrdU incorporation assay in fibroblasts isolated from TGFα mice lung and treated with indicated doses of barasertib for total of 48 h. ***P < 0.0005, 1‐way ANOVA (n = 9–11).

3. C

   Primary lung‐resident fibroblasts isolated from TGFα mice on Dox for 4 weeks were treated with vehicle or 5 μM barasertib for 48 h and immunostained using PCNA antibody. Images were obtained at 40× magnification. Scale bar: 50 μm. The number of PCNA‐positive cells and total DAPI‐positive cells was quantified using MetaMorph image analysis software. Proliferation is indicated as the percentage of proliferating cells in total DAPI‐positive cells. ****P < 0.00005, unpaired t‐test (n = 3–4).

4. D

   Proliferation was measured in primary fibroblasts treated with TGFα (20 ng/ml) and indicated doses of barasertib for 48 h. **P < 0.005, 1‐way ANOVA (n = 9–11).

5. E

   Quantification of apoptotic cells using Incucyte ZOOM (caspase 3/7‐positive cells) in resident fibroblasts isolated from IPF lung stromal cultures and treated with vehicle or 5 μM Barasertib. ****P < 0.00005, 2‐way ANOVA (n = 4).

Data information: All data were presented as mean ± SEM. The above data were a representative of two independent experiments with similar results. P values were shown in Appendix Table S6. Source data are available online for this figure.

Figure EV4. Barasertib treatment attenuates the expression of fibroproliferative genes

Quantification of CCNA2 and PLK1 transcripts in IPF fibroblasts treated with vehicle or barasertib (1 μM) for 16 h. Data were presented as mean ± SEM. (n = 4). *P < 0.05, unpaired t‐test. Data information: All data were presented as mean ± SEM.

Figure 5. In vivo Barasertib treatment prevents from TGFα induced lung fibrosis

1. A

   Schematic illustration of barasertib preventive treatment protocol. Control and TGFα mice were treated with either vehicle or barasertib (40 mg/kg; twice a day) for 4 weeks, while they were fed with Dox‐containing food.

2. B

   Representative images of Masson's trichrome‐stained lung sections from the vehicle‐ and barasertib‐treated mice. Images were obtained at 10× magnification. Scale bar: 200 μm.

3. C

   Quantification of right lung weight of mice treated with vehicle or barasertib. ***P < 0.0005, ****P < 0.00005, 1‐way ANOVA (n = 8–10 mice/group).

4. D

   Quantification of total lung hydroxyproline levels in mice treated with vehicle or barasertib. *P < 0.05, ***P < 0.0005, 1‐way ANOVA (n = 8–10 mice/group).

5. E

   Quantification of Col1α, Col5α, and Fn1 gene transcripts in total lung of mice treated with vehicle or barasertib. ***P < 0.0005, ****P < 0.00005, 1‐way ANOVA (n = 8 mice/group).

Data information: All data were presented as mean ± SEM. P values were shown in Appendix Table S6.

Figure 6. In vivo barasertib treatment attenuates mesenchymal proliferation

1. A

   Lung sections from vehicle‐ and barasertib‐treated mice were immunostained using Ki‐67 antibody. Representative images were obtained at 20× magnification. Scale bar: 50 μm.

2. B

   Immunoblot analysis in lung lysates from vehicle‐ and barasertib‐treated mice using PCNA antibody. Gapdh is used as loading control. **P < 0.005, 1‐way ANOVA (n = 4–5 mice/group).

3. C

   Quantification of Aurkb, Plk1, and CcnA2 gene transcripts in total lungs of mice treated with vehicle or barasertib. **P < 0.005, ***P < 0.0005, ****P < 0.00005, 1‐way ANOVA (n = 8 mice/group).

Data information: All data were presented as mean ± SEM. P values were shown in Appendix Table S6. Source data are available online for this figure.

Figure EV5. Barasertib inhibits Wt1 expression in the lungs of TGFα mice

Quantification of Wt1 gene transcripts in total lung transcripts of mice treated with vehicle or barasertib for 4 weeks. Data were presented as mean ± SEM. *P < 0.05, ****P < 0.00005, 1‐way ANOVA, (n = 8 mice/group).

Figure 7. Therapeutic barasertib treatment reduces established and ongoing lung fibrosis

1. A

   Schematic illustration of barasertib treatment protocol. Control and TGFα mice were treated with either vehicle or barasertib (40 mg/kg; twice a day) for last 3 weeks, while they were fed with Dox‐containing food for total of 6 weeks.

2. B

   Quantification of right lung weight of mice treated with vehicle and barasertib. **P < 0.005, ****P < 0.00005, 1‐way ANOVA (n = 9–10 mice/group).

3. C

   Western blot analysis in lung lysates from vehicle‐ and barasertib‐treated mice using Col1α and Fn1 antibodies. Gapdh is used as loading control. *P < 0.05, **P < 0.005, ***P < 0.0005, 1‐way ANOVA (n = 4–5 mice/group).

4. D

   Representative images of Masson's trichrome‐stained lung section from mice treated with vehicle and barasertib. Images were obtained at 10× magnification. Scale bar: 200 μm.

5. E

   Quantification of lung mechanics in mice treated with vehicle and barasertib. *P < 0.05, **P < 0.005, ***P < 0.0005, ****P < 0.00005, 1‐way ANOVA (n = 9–10 mice/group).

Data information: All data were presented as mean ± SEM. P values were shown in Appendix Table S6.

Figure 8. Therapeutic barasertib treatment attenuates bleomycin‐induced lung fibrosis

1. A

   Schematic illustration of barasertib treatment protocol. Mice were treated with either vehicle or barasertib (40 mg/kg; twice a day) for last 2 weeks, while they were injected intradermally with either saline or bleomycin 5 days per week for total of 4 weeks.

2. B

   Representative images of Masson's trichrome‐stained lung section from mice treated with vehicle and barasertib. Images were obtained at 10× magnification. Scale bar: 200 μm.

3. C

   Quantification of total lung hydroxyproline in mice treated with vehicle and barasertib. *P < 0.05, ***P < 0.0005, 1‐way ANOVA (n = 7–8 mice/group).

4. D

   Quantification of Col1α1, Col3α, Col5α, Col14, Col15, αSma, Ccna2, and Fas gene transcripts in total lungs of mice treated with vehicle or barasertib. *P < 0.05, **P < 0.005 ***P < 0.0005, ****P < 0.00005, 1‐way ANOVA (n = 6 mice/group).

5. E

   Quantification of αSma protein levels in lung lysates from vehicle‐ and barasertib‐treated mice. Gapdh is used as loading control. Data are presented as mean ± SEM. **P < 0.005, unpaired t‐test (n = 5 mice/group).

Data information: All data were presented as mean ± SEM. P values were shown in Appendix Table S6.