PLK1 inhibition by volasertib suppresses key transcriptional regulators underlying fibroblast activation and pulmonary fibrosis

Human Samples

IPF and healthy donor lung samples were obtained with assistance from the Translational Pulmonary Science Center at the University of Cincinnati Medical Center. This center maintains a repository of tissue and primary cells from patients with chronic lung diseases. All human IPF (n = 6 male) and normal control lung explants (n = 5 male, n = 1 female) used in this study were predominantly from male donors aged 50–75 yr. Lung explants used as controls were obtained from donor lungs that exhibited no evidence of lung disease and were considered unsuitable for transplantation, including size mismatch, logistical constraints, or accidental death. IPF lung explants were collected from individuals undergoing therapeutic lung transplantation. Procedures for collecting and maintaining samples are approved by the University of Cincinnati Institutional Review Board (IRB; Protocol No. 2013-8157), ensuring the protection of human subjects’ privacy and confidentiality in compliance with HIPAA regulations.

Mouse Model of TGFα-Induced Pulmonary Fibrosis

We used a transgenic mouse model of pulmonary fibrosis that overexpresses TGFα on the FVB/NJ inbred strain background, as previously described (30). Homozygous CCSP^rtTA mice were crossed with heterozygous TetO-cmv-TGFα mice to generate bitransgenic TGFα^OE (CCSP/TGFα) mice and littermate controls (CCSP^/−). Both male and female mice aged 12–16 wk were used for the experiments. TGFα expression was induced by administering doxycycline (DOX; 62.5 mg/kg) through food and water, resulting in severe fibrotic lung disease after 4 wk of treatment (31). All mice were housed under specific pathogen-free conditions at the University of Cincinnati. For in vivo experiments, volasertib (Selleckchem, Cat. No. S2235) was freshly formulated in 0.5% carboxy methyl cellulose (Sigma, Cat. No. C5678) in PBS. Mice were anesthetized with isoflurane before drug administration. For oral administration, 10 mg/kg dosage of volasertib was used for each mouse on every other day for 4 wk.

Histology and Immunohistochemistry

Histological procedures were performed, as mentioned previously (31). In brief, paraffin-embedded tissues were cut into 5-μm sections and deparaffinized for Masson’s trichrome staining. For immunohistochemistry, lung sections were deparaffinized, followed by citric acid antigen retrieval (pH 6.0) and methanol treatment, blocked in 5% donkey serum, incubated with the primary antibody overnight, incubated with species-specific secondary peroxidase antibodies, and then stained with diaminobenzidine. Isotype control antibodies matching the host species and the IgG subclass of the primary antibodies were used to confirm the specificity of immunohistochemical staining. Antibody details are provided in Table 3. Bright-field images were captured using a Keyence BZ series microscope and analyzed using BZ-X Analyzer software (Keyence Corporation, Osaka, Japan). Immunohistochemistry was performed simultaneously using slides from the control and treatment groups. All images were acquired using identical imaging settings to ensure consistency across samples. Image acquisition was performed randomly across comparable anatomical regions, and subsequent analyses were conducted in a blinded manner to minimize experimental bias.

Hydroxyproline Content Estimation

Total lung hydroxyproline content was measured using a colorimetric assay (Abcam, Cat. No. ab222941), following the manufacturer’s protocol and as previously described (32). In brief, lung tissue was hydrolyzed in 10 N sodium hydroxide (NaOH), followed by neutralization with 10 N hydrochloric acid (HCl). The released hydroxyproline was then oxidized to form a pyrrole intermediate, which subsequently reacted with Ehrlich’s reagent in perchloric acid to produce a chromogenic complex. Absorbance was measured at 560 nm, and hydroxyproline concentrations were determined based on a standard curve.

RNA Preparation and Real-Time PCR

RNA was isolated from lung tissue homogenates using an RNAeasy mini kit (Qiagen, Cat. No. 74106) following tissue homogenization with beads and a high-speed homogenizer (Qiagen, TissueLyzer II), as previously described (33). cDNA synthesis was performed using Superscript III (Invitrogen, Cat. No. 56575), and real-time PCR was conducted using SYBR Green Select Master Mix (Applied Biosystems, Cat. No. 4309155) in CFX Opus 384 Touch Real-Time PCR System (Bio-Rad Laboratories, Hercules, CA). Target gene expression was normalized to β-actin for human samples and hypoxanthine-guanine phosphoribosyltransferase (HPRT) for mouse samples (Tables 1 and 2). For each gene and experimental run, a single control replicate was designated as the calibrator (assigned a value of 1); consequently, the remaining control replicates were not constrained to 1, allowing for the assessment of biological variance within the control group.

Primary Fibroblast Cultures and Treatments

Primary fibroblasts were isolated from the distal subpleural fibrotic parenchyma of the right lower lobe from both patients with IPF and control donor lungs. Tissue was processed via collagenase digestion, followed by the depletion of hematopoietic cells through negative selection for CD45 using MACS (human CD45 microbeads, Cat. No. 130-045-801, Miltenyi Biotec), as previously described (31). Similarly, mouse fibroblasts were negatively selected for CD45 using MACS (mouse CD45 microbeads, Cat. No. 130-052-301, Miltenyi Biotec). Fibroblasts were cultured in DMEM supplemented with 10% FBS and antibiotics for human or IMDM supplemented with 5% FBS for mouse. The human fibroblasts of passages 1 to 4 were used for all in vitro experiments. Cells were treated with TGFα (100 ng/mL) (PeproTech, Cat. No. 100-16A) overnight at low serum conditions. Fibroblasts were treated with vehicle (DMSO) or volasertib for 48 h. For siRNA-mediated knockdown studies, IPF fibroblasts were transfected with control or MYCN-specific siRNA from Invitrogen for 72 h using a Lipofectamine 3000 transfection kit (Life Technologies, Cat. No. L3000015), as described previously (34). Each sample (n) represents a biological replicate, defined as an independently derived fibroblast culture from a unique donor lung.

Western Blot

Western blotting was performed, as previously described (35). The membranes were blocked with 5% BSA in 0.1% Tween phosphate-buffered saline and incubated overnight with primary antibodies at 4°C. Protein bands were visualized using ECL Prime Western Blotting Detection Reagent (Thermo Fisher, Cat. No. RPN2232) in ChemiDoc MP Imaging System (Bio-Rad Laboratories, Hercules, CA) according to the manufacturer’s instructions. All immunoblot images were processed and analyzed using Image Lab software (version 6.1, Bio-Rad). Protein expression levels were quantified and normalized to GAPDH, which served as a loading control. Normalized values were expressed as the ratio of the target protein to GAPDH. Details of all antibodies used in this study, including target, source, and dilution, are provided in Table 3. Full blot images are shown in Supplemental Figs. S2–S5.

Quantification of Apoptosis

Quantification of apoptotic cell death was measured either by using the Caspase 3/7 Green apoptosis assay or the Tunnel assay. Apoptotic cell death measurement using Caspase 3/7 activity was described previously using the IncuCyte ZOOM real-time imaging system (31). In brief, primary cells were incubated in Caspase 3/7 Green apoptosis assay reagent (Caspase 3/7 substrate conjugated to green fluorophore; Essen BioScience). Time-lapse fluorescence imaging was performed using the IncuCyte ZOOM system (Essen BioScience, Ann Arbor, MI); nine images per well were collected at ×20 magnification every 2 h for 14 h. The average number of green objects produced by the apoptotic cells was measured using IncuCyte ZOOM software 2015 A (Essen BioScience, Ann Arbor, MI). For the tunnel assay, human IPF fibroblasts were seeded at 3 × 10⁴ cells/well in an eight-well chambered slide and incubated overnight. Cells were then treated with either vehicle or volasertib for 24 h. Human-specific anti-Fas antibody (250 ng/mL, Millipore Sigma) was added along with vehicle or volasertib and further incubated for 24 h. Nuclei were stained using DAPI, and staining of apoptotic cells was performed using the terminal deoxynucleotidyl TUNEL method using an In Situ Cell Death Detection Kit, TMR Red (Roche Diagnostics, Cat. No. 12156792910). All images were captured at ×20 magnification using a Keyence BZ series microscope (Keyence Corporation, Osaka, Japan) and quantified using a BZ-X Analyzer software (Keyence). Image acquisition and analysis were performed randomly and in a blinded manner to minimize bias.

BrdU Incorporation Assay

Cell proliferation assay was performed using a BrdU incorporation assay kit (Cat. No. 6813S, Cell Signaling Technology, Denver, CO), following the manufacturer’s protocol, and as previously described (19). Lung-resident fibroblasts were isolated and cultured for 48 h under low serum conditions (0.5% FBS) in 96-well plates. After the initial 24 h, cells were pulsed with a BrdU labeling solution and incubated for an additional 24 h. Following incubation, the cells were fixed, and the DNA was denatured to expose incorporated BrdU. Proliferation was quantified by incubating with a mouse anti-BrdU monoclonal antibody, followed by a horseradish peroxidase-conjugated secondary antibody and TMB substrate development. The absorbance at 450 nm was measured, which is directly proportional to the magnitude of BrdU incorporation and DNA synthesis.

Statistics

All data were analyzed using GraphPad Prism (version 10.5; GraphPad Software, San Diego, CA). Statistical tests were selected based on the distribution characteristics of the data and the experimental design. Depending on the comparison, data were analyzed using the Student’s two-tailed t test, one-way ANOVA or two-way ANOVA. The specific statistical test used is indicated in each figure legend. Exact P values are reported to denote statistical significance. All data are presented as means ± standard deviation (SD). All measurements were performed in a blinded manner to minimize experimental bias.

Ethics Statement

Human lung tissue samples were collected and used in accordance with the University of Cincinnati Institutional Review Board (IRB) guidelines (Protocol No. 2013-8157), ensuring participant protection and confidentiality. All animal experiments were performed in an AAALAC-accredited, specific pathogen-free facility at the University of Cincinnati, under protocols approved by the Institutional Animal Care and Use Committee (IACUC).

MYCN Is Upregulated in Fibroblasts during Pulmonary Fibrosis

To determine whether MYCN is upregulated in the lungs of IPF, we immunostained lung sections with an antibody against MYCN and observed a marked increase in MYCN staining in spindle-shaped fibroblasts located in fibroblastic foci and subpleural regions of IPF lung tissue compared with normal donor lungs (Fig. 1A and Supplemental Fig. S1). Also, we quantified MYCN transcript levels in lung-resident fibroblasts isolated from IPF and control donor lungs and observed a significant increase in MYCN transcripts in IPF fibroblasts compared with fibroblasts from normal lungs (Fig. 1B).

MYCN is highly expressed in lung development and plays a critical role during embryonic lung development (6, 7). We assessed the kinetics of MYCN expression during lung development and postnatal stages of lung development in mice. As expected, we observed high levels of Mycn transcripts in the early stages of lung development, and subsequently declined, with the lowest expression observed in the adult lungs (Fig. 1C). To determine whether MYCN is upregulated during pulmonary fibrosis in adult lungs, we quantified Mycn transcripts in total lung RNA of mice during TGFα-induced pulmonary fibrosis. We observed a significant increase in Mycn transcript levels in the lungs during TGFα-induced pulmonary fibrosis (Fig. 1D). To determine the direct effects of TGFα on MYCN expression, we have quantified Mycn transcripts in lung-resident fibroblasts treated with media or TGFα for 16 h and observed a significant increase in Mycn transcripts with TGFα treatment compared with media in fibroblasts from mouse lungs (Fig. 1E). Consistently, we observed a significant increase in MYCN transcript levels with TGFα treatment in fibroblasts isolated from normal human donor lungs (Fig. 1F).

TGFα promotes pulmonary fibrosis by activating epidermal growth factor receptor signaling, which induces the profibrotic transcription factor WT1, leading to sustained fibroblast activation and ECM deposition (21, 30). To assess whether WT1 promotes MYCN expression, fibroblasts isolated from fibrotic lung lesions of TGFα overexpressing mice were treated with control or WT1-specific siRNA for 72 h, and the knockdown of WT1 was sufficient to reduce Mycn transcripts level (Fig. 1G). Conversely, overexpression of WT1 using adenovirus resulted in a significant increase in Mycn transcripts in lung-resident fibroblasts isolated from normal mice (Fig. 1H). Together, these new findings suggest that MYCN is upregulated in fibroblasts via the TGFα-WT1 axis in the pathogenesis of pulmonary fibrosis.

MYCN Functions as a Positive Regulator of ECM Gene Expression in IPF Fibroblasts

The activated fibroblast, particularly the myofibroblast, is the major cell type responsible for collagen deposition, including collagen types I and III, contributing to the scarring and stiffening of lung tissue in severe fibrotic lung disease (8, 9). Given that WT1 regulates the expression of both MYCN and ECM genes, we assessed whether MYCN alters ECM gene expression in IPF fibroblasts (2). Notably, the knockdown of MYCN in fibroblasts isolated from the lungs of patients with IPF was sufficient to attenuate expression of ECM genes, including COL1A1, COL3A1, FN1, LOXL2, and ACTA2 (Fig. 2, A–F). We next confirmed the knockdown effects of MYCN on ECM protein levels through Western blot analysis of IPF fibroblasts treated with MYCN-specific siRNA compared with those treated with control siRNA for 72 h. Consistent with changes in gene transcripts, we observed a significant decrease in ECM-associated protein levels (COL1A1, COL3A1, FN1, and ELN) with the loss of MYCN in IPF fibroblasts (Fig. 2, G–L). In summary, these results indicate positive regulation of ECM production and remodeling by MYCN in IPF fibroblasts.

MYCN Promotes the Proliferation and Survival of IPF Fibroblasts

To determine whether MYCN contributes to fibroblast activation, we treated IPF fibroblasts with control or MYCN-specific siRNA for 72 h and quantified transcripts using real-time PCR for proliferation genes (PLK1, CDK1, CCNE1, and AURKB) and survival genes (BCL2 and BAD) (Fig. 3, A–F). The loss of MYCN resulted in a significant decrease in gene transcripts associated with fibroblast proliferation and survival (PLK1, CDK1, CCNE1, AURKB, and BCL2), as well as an increase in proapoptotic gene BAD transcripts (Fig. 3, A–F). We next confirmed the knockdown effects of MYCN on PLK1 and BCL2 protein levels through Western blot analysis of IPF fibroblasts treated with MYCN-specific siRNA compared with those treated with control siRNA for 72 h. Consistent with changes in gene transcripts, we observed a decrease in PLK1 and BCL2 protein levels with the loss of MYCN in IPF fibroblasts (Fig. 3, G–I).

To test whether MYCN induces fibroblast proliferation, we assessed the impact of MYCN knockdown on BrdU incorporation in fibroblasts isolated from the lungs of IPF. We observed a significant decrease in BrdU incorporation in fibroblasts treated with MYCN-specific siRNA compared with control siRNA (Fig. 3J). These findings establish that MYCN functions as a positive regulator of fibroblast proliferation in IPF.

In progressive fibrosis, fibroblasts and myofibroblasts develop resistance to apoptosis, thereby promoting excessive ECM deposition and the expansion of fibrotic lung lesions (11, 41). To test whether MYCN induces fibroblast survival, the mature fibrotic lesions of IPF were cultured to isolate fibroblasts, and apoptosis was quantified in fibroblasts treated with either control or MYCN-specific siRNA. Treatment of Fas ligand (FasL) agonist significantly induced apoptosis in IPF fibroblasts. Notably, the loss of MYCN transcripts in the presence of FasL agonist further enhanced the apoptosis induction in fibroblasts from IPF (Fig. 3, K–M). Together, the above findings establish that increased MYCN expression contributes to proliferation and apoptosis resistance in IPF fibroblasts.

Volasertib Inhibits the Proliferation and Survival of IPF Fibroblasts

To test whether the inhibition of PLK1 activity alters MYCN expression, we assessed the effects of volasertib treatment on the expression of PLK1 and MYCN in fibroblasts isolated from the lungs of IPF. Upon treatment with volasertib, a known PLK1 inhibitor, we observed a significant reduction in the expression of PLK1 and MYCN (Fig. 4, A–C). Volasertib treatment also reduced WT1 protein levels in IPF fibroblasts (Fig. 4, A and D). Similar to the loss of MYCN expression, inhibition with volasertib reduced the proliferation of IPF fibroblasts (Fig. 4E). To assess the effect of volasertib on fibroblast survival, we treated fibroblasts isolated from IPF lungs with volasertib in the presence and absence of FAS ligand and evaluated the percentage of TUNEL-positive apoptotic cells. Treatment with volasertib resulted in a significant increase in TUNEL-positive cells compared with vehicle control (Fig. 4, F and G). Thus, either the loss of MYCN expression or inhibition of PLK1 with volasertib is sufficient to mitigate fibroblast activation, including proliferation and survival in IPF fibroblasts.

Volasertib Inhibits Fibroblast Activation and Pulmonary Fibrosis

To assess the effect of volasertib on ECM gene expression, IPF fibroblasts were treated with vehicle or increasing doses of volasertib, and ECM gene transcripts were quantified by real-time PCR. Volasertib treatment resulted in dose-dependent reduction in the expression of COL1A1, COL3A1, FN1, and ACTA2 (Fig. 5, A–D). Consistent with RT-PCR results, volasertib treatment reduced the protein levels of key ECM components, including COL1A1, COL3A1, ELN, and FN1, confirming its inhibitory effect on their expression at both the transcript and protein levels (Fig. 5, E–H).

To test if treatment with volasertib prevents the development of lung fibrosis, TGFα mice were concomitantly treated with DOX to induce TGFα expression and either volasertib (10 mg/kg body wt) or vehicle on alternate days for 4 wk (Fig. 6A). Induction of TGFα caused extensive subpleural, perivascular, and peribronchial fibrosis (Fig. 6B). TGFα-over-expressing mice that were concomitantly treated with volasertib showed reduced collagen staining, with infrequent, scattered, and small fibrotic areas. Furthermore, we observed a significant decrease in the fibrotic lesions and hydroxyproline levels in TGFα mice treated with volasertib compared with vehicle-treated mice (Fig. 6, C and D). In agreement with histological and biochemical changes, we observed a significant decrease in lung collagen (Col1A1, Col3a1, and Col5A1), and Eln levels in TGFα mice treated with volasertib compared with vehicle-treated TGFα mice on DOX for 4 wk (Fig. 6, E–H). Also, we observed that volasertib treatment attenuated the expression of Plk1, Mycn, and Wt1 in TGFα overexpression mice (Fig. 6, I–K). These findings collectively suggest that pharmacological inhibition of PLK1 by volasertib attenuates fibroblast activation and pulmonary fibrosis.

Perspectives and Significance

Pulmonary fibrosis remains a devastating disease with limited therapeutic options and poor prognosis. Understanding the molecular mechanisms driving fibroblast activation is essential for developing effective therapeutic interventions. In this study, we uncovered a previously unrecognized role for MYCN and PLK1 as key positive regulators of fibroblast proliferation, survival, and extracellular matrix production. Importantly, we demonstrate that WT1 drives MYCN expression, thereby establishing a WT1-MYCN-PLK1 signaling axis that promotes fibroblast activation. These findings not only provide new mechanistic insights into fibroblast biology in pulmonary fibrosis but also highlight this signaling pathway as a promising therapeutic target to mitigate disease progression.