DCZ0415, a small-molecule inhibitor targeting TRIP13, inhibits EMT and metastasis via inactivation of the FGFR4/STAT3 axis and the Wnt/β-catenin pathway in colorectal cancer

Abstract

Thyroid receptor-interacting protein 13 (TRIP13), a protein of the AAA-ATPase family, is upregulated in various human cancers, including colorectal cancer (CRC). This study focused on the inhibition of TRIP13-induced CRC progression and signalling by DCZ0415, a small molecule targeting TRIP13. It demonstrated potent antitumour activity in TRIP13-deregulated cancer cell lines, regardless of their p53, KRAS, BRAF, epidermal growth factor receptor or microsatellite instability status. The treatment of CRC cells with DCZ0415 resulted in decreased cell proliferation, induced cell cycle arrest in the G2-M phase and increased apoptosis. DCZ0415 diminished xenograft tumour growth and metastasis of CRC in immunocompromised mice. DCZ0415 reduced expression of fibroblast growth factor receptor 4 (FGFR4), signal transducer and activator of transcription 3 (STAT3), and proteins associated with the epithelial-mesenchymal transition and nuclear factor kappa B (NF-κB) pathways in cells and xenografts exhibiting high expression of TRIP13. Additionally, DCZ0415 decreased cyclin D1, β-catenin and T-cell factor 1, leading to the inactivation of the Wnt/β-catenin pathway. In a syngeneic CRC model, DCZ0415 treatment induced an immune response by decreasing PD1 and CTLA4 levels and increasing granzyme B, perforin and interferon gamma. In sum, DCZ04145 inhibits the TRIP13-FGFR4-STAT3 axis, inactivates NF-κB and Wnt/β-catenin signalling, activates antitumour immune response and reduces the progression and metastasis of CRC. This study provides a rationale to evaluate DCZ0415 clinically for the treatment of a subset of CRCs that exhibit dysregulated TRIP13 and FGFR4.

Keywords: DCZ0415; FGFR4; TRIP13; colorectal cancer; granzyme B; metastasis.

Fig. 1

Pharmacologic inhibition of DCZ0415 decreases malignant phenotypes of CRC. (A) MTT assay showing viability of CRC cells exhibiting various genetic alterations after treatment with serially diluted DCZ0415 concentrations. The heat map was plotted to show the viability of CRC cells after treatment with DCZ0415 at 4 days. (B) PCNA protein expression was analysed in CRC cells after treatment with DCZ0415 for 48 h. (C) Colony formation assay with CRC cells treated with 5, 10 or 20 µm DCZ0415. (D) Cell cycle analysis was performed in CRC cells after treatment with DCZ0415 for 24 h. The experiments had three replicates.

Fig. 2

DCZ0415 inhibits the downstream signalling pathway of TRIP13. (A) Western blot analysis of TRIP13, p‐EGFR, FGFR4 and p‐STAT3 protein expressions in CRC cells transfected with TRIP13 shRNA. (B) Western blot analysis showing the levels of FGFR4 in lysates of CRC cells treated with DCZ0415. (C) For various cells, the levels of p‐STAT3, t‐STAT3, p‐EGFR and t‐EGFR, determined by western blot analysis, were assessed after CRC cells were exposed to DCZ0415. (D) For various cells, the levels of p‐IKKα/β and p‐NF‐κBp65, determined by western blot analysis, were assessed after CRC cells were exposed to DCZ0415. (E) PARP protein expression in DCZ0415‐treated CRC cells. (F) Apoptosis was quantified by Annexin V/PI staining after the treatment of CRC cells with DCZ0415 (10 and 20 µm) for 48 h. The experiments had three replicates.

Fig. 3

DCZ0415 reduces CRC tumour growth in immunocompromised mice. (A) Representative photograph showing tumours excised from two different experimental groups: vehicle (n = 5) and DCZ0415 (25 mg·kg⁻¹; n = 5). (B) Graph showing tumour growth measured as tumour volumes for the vehicle and DCZ0415 groups (error bar indicates mean ± SD). DCZ0415 were provided to animals 12 times on alternate days after tumours reached 100–200 mm³ (*P = 0.0002). (C) Dot plot showing tumour growth in vehicle‐ and DCZ0415‐treated mice at day 34 (*P = 0.0002). (D) Weights of the tumours excised from the mice in each treatment group were determined at the end of the experiment on day 34 (error bar indicates mean ± SD). Asterisk (*) showing statistically significant data (*P = 0.015). (E) Representative microphotographs of the liver and kidney sections after administration of vehicle or DCZ0415 to show signs of toxicity (Scale bar = 100 µm). (F) Protein levels of FGFR4, p‐STAT3 and t‐STAT3 in lysates of xenografts treated with DCZ0415.

Fig. 4

DCZ0415 reduces metastasis of CRC cells. (A) Luciferase‐transduced RKO cells were injected into the tail veins of NSG mice, which were treated with DCZ0415 (alternate days injection of 10 mg·kg⁻¹ i.p.) or vehicle. (B) Representative photograph showing the liver and kidney from mice. (C) H&E images of the lung, liver, kidney and bone in vehicle‐ or DCZ0415‐treated tumours. Arrows indicate metastatic lesions; Lu—lung; Li—liver; Ki—kidney; B—bone; BM—bone marrow. 2×; scale bar, 500 µm.

Fig. 5

DCZ0415 modulates the EMT and Wnt signalling of CRC cells. (A) Western blot analysis showing the expression of EMT proteins in lysates of DCZ0415‐treated CRC cells. (B) Vimentin, N‐cadherin and Snai1 protein expression in DCZ0415‐treated xenografts. (C) Immunoblot analysis for TRIP13, FGFR4 and N‐cadherin levels in corresponding normal colon (N) and CRC (T) tissue samples of patients. Samples from four CRC patients were utilized for this experiment. Expression of Wnt/β‐catenin pathway target molecules in DCZ0415‐treated (D) cells and (E) xenograft lysates as determined by western blotting. (F) Immunoblot analysis for molecules of the Wnt/β‐catenin signalling pathway in corresponding normal colon (N) and CRC (T) samples.

Fig. 6

DCZ0415 increases immune infiltration in an immunocompetent model. (A) Western blot analysis to show the expression of GZMB and TRIP13 in protein lysates of CRC cells exhibiting TRIP13 knockdown (B) Photographs of syngeneic mouse tumours generated by implanting MC38 cells into C57/BL6 mice after treatment with vehicle and DCZ0415 (n = 5). (C) Tumour weights are shown for vehicle‐ and DCZ0415‐treated tumours (error bar indicates mean ± SD, *P = 0.008). (D) Kinetics of tumour growth treated with vehicle and DCZ0415 (error bar indicates mean ± SD, P = 0.01). (E) Dot plot showing tumour growth in vehicle‐ and DCZ0415‐treated mice at day 26 (error bar indicates mean ± SD, *P = 0.01). (F) H&E and IHC analysis to show the infiltration of immune cells and expression of T‐cell markers in DCZ0415‐treated immunocompetent tumours, Scale bar‐ 20 µm. Arrows indicate positive staining. (G) qPCR analysis to show the levels of PD1 and CTLA4 in DCZ0415‐treated tumours, relative to vehicle control (error bar indicates mean ± SD; P = 0.031 and P = 0.26 respectively). (H) Protein levels of GZMB, perforin, PD1, IFN‐γ and TRIP13 are shown in vehicle‐ and DCZ0415‐treated tumours.