Fosciclopirox suppresses growth of high-grade urothelial cancer by targeting the γ-secretase complex

Abstract

Ciclopirox (CPX) is an FDA-approved topical antifungal agent that has demonstrated preclinical anticancer activity in a number of solid and hematologic malignancies. Its clinical utility as an oral anticancer agent, however, is limited by poor oral bioavailability and gastrointestinal toxicity. Fosciclopirox, the phosphoryloxymethyl ester of CPX (Ciclopirox Prodrug, CPX-POM), selectively delivers the active metabolite, CPX, to the entire urinary tract following parenteral administration. We characterized the activity of CPX-POM and its major metabolites in in vitro and in vivo preclinical models of high-grade urothelial cancer. CPX inhibited cell proliferation, clonogenicity and spheroid formation, and increased cell cycle arrest at S and G0/G1 phases. Mechanistically, CPX suppressed activation of Notch signaling. Molecular modeling and cellular thermal shift assays demonstrated CPX binding to γ-secretase complex proteins Presenilin 1 and Nicastrin, which are essential for Notch activation. To establish in vivo preclinical proof of principle, we tested fosciclopirox in the validated N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN) mouse bladder cancer model. Once-daily intraperitoneal administration of CPX-POM for four weeks at doses of 235 mg/kg and 470 mg/kg significantly decreased bladder weight, a surrogate for tumor volume, and resulted in a migration to lower stage tumors in CPX-POM treated animals. This was coupled with a reduction in the proliferation index. Additionally, there was a reduction in Presenilin 1 and Hes-1 expression in the bladder tissues of CPX-POM treated animals. Following the completion of the first-in-human Phase 1 trial (NCT03348514), the pharmacologic activity of fosciclopirox is currently being characterized in a Phase 1 expansion cohort study of muscle-invasive bladder cancer patients scheduled for cystectomy (NCT04608045) as well as a Phase 2 trial of newly diagnosed and recurrent urothelial cancer patients scheduled for transurethral resection of bladder tumors (NCT04525131).

Conflict of interest statement

M.T. and S.J.W. are co-inventors of fosciclopirox composition of matter patents issued in the US, Europe, and Japan as well as a methods of use patents issued in the US. S.A. and S.J.W. are inventors on an issued patent in Europe, and a pending application in China. The exclusive, non-terminable rights to intellectual property generated by the inventors, as employees of the University, were licensed by the University of Kansas to CicloMed LLC in 2015. CicloMed is developing fosciclopirox for the treatment of bladder cancer. Kansas Life Sciences Development Company, a subsidiary of the University of Kansas Medical Center Research Institute, Inc. (which is a private, not-for-profit 501(c)(3) corporation established to promote and support medical research and faculty invention disclosures), possesses a financial interest in CicloMed.

Figures

Fig. 1. CPX inhibits proliferation and colony formation in bladder cancer cells.

A Chemical structures of CPX and CPX-POM. B CPX inhibits proliferation of bladder cancer cell lines (T24, UM-UC-3, HTB-9, HTB-5, HT-1376, and RT-4). Cells were incubated with increasing concentration (0–40 μM) of CPX for up to 72 h. The treatment showed a significant dose and time-dependent decrease in cell proliferation when compared with untreated controls in the bladder cancer cell lines. C CPX inhibits colony formation. Cells were incubated with 0–20 μM CPX for 48 h and allowed to grow into colonies for 10 d. Incubation with CPX inhibits clonogenicity and number of colonies in bladder cancer cell lines (D).

Fig. 2. CPX induces cell cycle arrest in bladder cancer cell lines.

A Cell cycle analysis of CPX-treated cells. T24 and UM-UC-3 cells were treated with 4 µM concentrations of CPX, and examined by flow cytometry. CPX treatment-induced G0/G1 arrest in T24 cells and S-phase arrest in UM-UC-3 cells. The percent cells in each phase are presented graphically (B). C CPX downregulates cyclin D1 and B1 expression in T24 and UM-UC-3 cells as assessed by western blot. D Immunofluroscence analysis shows downregulation of cyclin D1 expression after CPX treatment in T24 cells.

Fig. 3. CPX induces apoptosis in bladder cancer cell lines.

A T24 and UM-UC-3 cells were treated with CPX stained with Annexin V (FITC) and PI, and analyzed by flow cytometry. CPX treatment induced significant early and late apoptosis in T24 and UM-UC-3 cells. B The flow cytometric quantification of early and late apoptotic cells after CPX treatment over a period of 72 h in T24 and UM-UC-3 cells in Annexin-PI assay. C CPX induces apoptosis in T24 cells. T24 cells treated with CPX (4 µM) for 48 h were stained with Annexin V-FITC/PI solution and studied using immunofluorescence. CPX induced apoptosis in T24 cells. D Lysates from T24 and UM-UC-3 cells treated with CPX were studied by western blot for evaluating the effect on proteins involved in apoptosis. CPX treatment reduced the levels of antiapoptotic marker proteins Bcl2 and Bcl-XL. CPX treatment also increases the PARP cleavage compared to untreated controls. E CPX induces autophagy early which then followed by apoptosis. Lysates from T24 and UM-UC-3 cell treated with CPX (4 µM) for 24–48 h were analyzed by western blot. CPX treatment increased the expression of LC3B at 24 h and cleaved caspase-3 expression at 48 h. The data suggest that at early time points, CPX induces autophagy, which switches to apoptosis in T24 and UM-UC-3 cells.

Fig. 4. CPX inhibits bladder cancer spheroid growth and cell migration.

A, B CPX inhibits spheroid growth. CPX suppressed the size (A) and number (B) of spheroids of bladder cancer cell lines. Cells were grown in spheroid growth media in ultralow adherent plates and treated with CPX 2 µM and 4 µM concentrations for 5 days. CPX significantly decreased bladdosphere formation in the T24, UM-UC-3, HT-1376, HTB-9, and RT-4 cell lines. C CPX inhibits the expression of bladder cancer stem cell marker proteins. Lysates from T24 and UM-UC-3 cell lines after CPX treatment were subjected to western blot analysis. CPX treatment inhibited the expression of SOX9 and CD44 in both cell lines. D CPX inhibits migration of T24 and UM-UC-3 cells. Both cell lines were grown to 95–100% confluency and a scratch was made to study the cell movement over a period of 12 h. CPX treatment reduced cell migration. E CPX inhibited the percent migration in both cell lines. F CPX inhibits migration of T24 and UM-UC-3 cells. CPX treatment significantly reduced the migration of both cell lines through transwell cell culture inserts (Boyden’s chamber). G The percent migraition is represented in graphical format.

Fig. 5. CPX targets the Notch signaling pathway, through inhibition of the γ-secretase complex.

A CPX inhibits mRNA expression of genes involved in Wnt, Hedgehog, and Notch cell signaling pathways. RNA extracted from T24 cells treated with and without CPX for 48 h was used to generate cDNA. The samples were analyzed using a Human stem cell RT2 profiler PCR array to identify signaling pathways that important for stem cell maintenance like Notch signaling and Wnt signaling. Expression of Hes5, Lfng, Myc, and Notch 1 mRNAs were significantly downregulated after CPX treatment. B CPX affects the expression of Notch receptors. Western blot analysis demonstrates that CPX treatment increased the expression of Notch 1 and suppressed the activation of Notch 2 and Notch 3, while CPX has little to no effect on Notch 4. C CPX suppresses γ-secretase pathway proteins. Western blot analysis of CPX-treated T24 and UM-UC-3 cell lysates showed CPX treatment reduced levels of Nicastrin, Presenilin 1, APH-1, and PEN-2. D CPX suppresses notch signaling pathway ligand and downstream proteins. CPX increased the expression of ligand Jagged 1 and inhibited the expression of downstream targets Hes1, cMYC, and Cyclin D1 in T24 and UM-UC-3 cells.

Fig. 6. CPX-POM treatment suppresses bladder tumorigenesis in vivo in the validated BBN mouse model of bladder cancer.

A Mean (±SD) plasma ciclopirox (CPX) concentration-time profiles following IV administration of 13.4 mg/kg CPX-POM and 18.1 mg/kg CPX-O to three C57BL/6 mice per serial blood collection time point per treatment group demonstrating rapid and complete metabolism of CPX-POM to its active metabolite, CPX. B Mean (±SD) plasma CPX concentration-time profiles following administration of 23.4 mg/kg IV and 117.5 mg/kg IP CPX-POM to three C57BL/6 mice per serial blood collection time point per treatment group demonstrating acceptable bioavailability of CPX following IP administration of CPX-POM. C Male C57BL/6 mice received 0.05% of N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN) in the drinking water for 16 weeks. Mice were treated with vehicle, 235 mg/kg CPX-POM (½MTD), or 470 mg/kg (MTD) CPX-POM IP once daily for 4 weeks (weeks 17–20). After 20 weeks, mice were euthanized, bladder was collected weighed, and subjected to further analysis. D CPX-POM treatment significantly reduced the bladder weight as compared to the vehicle-treated cohort at well-tolerated doses (p < 0.05). There were no statistically significant differences in bladder weights between the 235 mg/kg (½ MTD) and 470 mg/kg (MTD) CPX-POM treatment cohorts. E Immunohistochemistry analysis of bladder tumors obtained from CPX-POM-treated mice show a lower number of PCNA-positive nuclei compared to bladder tumors obtained from vehicle-treated mice. Bladder tumors obtained from CPX-POM treated mice also showed reduced expression of Notch 1, Presenilin 1, Hes1, and Nicastrin and increased levels of cleaved caspase-3 compared bladder tumors obtained from vehicle-treated mice.

Fig. 7. CPX targets γ-secretase complex proteins Presenilin 1 and Nicastrin.

A Schematic representation showing the Notch signaling pathway. CPX targets γ-secretase target proteins Presenilin 1 and Nicastrin in bladder cancer cells. B mRNA expression of Presenilin 1 and Nicastrin in different cancer patients in the Cancer Genome Atlas database, extracted from Timer 2.0. (Bladder Urothelial Carcinoma [BLCA], Breast invasive carcinoma [BRCA], Cervical squamous cell carcinoma and endocervical adenocarcinoma [CESC], Colon Adenocarcinoma [COAD], Esophageal Squamous Cell Carcinoma [ESCA], Glioblastoma multiforme [GBM], Head and Neck squamous cell carcinoma [HNSC], Kidney renal clear cell carcinoma [KIRC], Liver hepatocellular carcinoma [LIHC], Lung adenocarcinoma [LUAD], Lung squamous cell carcinoma [LUSC], Pancreatic adenocarcinoma [PAAD], Prostate adenocarcinoma [PRAD], Rectum adenocarcinoma [READ], Stomach adenocarcinoma [STAD], Thyroid carcinoma [THEA], Uterine Corpus Endometrial Carcinoma [UCEC]). C Kaplan–Meier survival analysis showed that higher levels of Presenilin 1 or Nicastrin expression (p < 0.05) were significantly associated with poor overall survival in patients with bladder cancer. D The molecular docking analysis predicted the binding of CPX in the protein cavity of Presenilin 1 and Nicastrin. AutoDock Vina software program was used for molecular docking. E Cellular thermal shift assay (CETSA). T24 cell lysates were treated with CPX and subjected to differential temperature treatment for 3 mins and evaluated using western blot analyses. CPX protected Presenilin 1 and Nicastrin against thermal degradation, suggesting the active metabolite of CPX-POM interacts with these proteins. F Cumulative results from the densitometric evaluation of CETSA assay.