In silico investigation of FOXM1 binding and novel inhibitors in epithelial ovarian cancer.

OBJECTIVE: Using TCGA database, we had demonstrated that aberrantly activated Forkhead box M1 (FOXM1) correlates to worse overall survival in a subgroup of platinum resistant patients. Application of thiostrepton, a natural thiazole antibiotics that inhibits FOXM1 transcription activity in the clinic is hampered by difficulties in synthesis, degradation potential, and solubility. In this study, we aim to identify potential FOXM1 small molecule inhibitors to develop a new class of therapeutic agents to address the challenges in treating chemotherapy resistant EOC. METHODS: We used in silico screening of compounds against a solved structure of FOXM1 and subsequently to derive a list of possible compounds that could inhibit FOXM1. Three compounds were tested for in vitro cytotoxicity and FOXM1 expression level was confirmed by RT-PCR and Western blot in EOC cell lines. RESULTS: The FOXM1 structure obtained from 3G73 represented the DNA binding region of FOXM1 and possessed the winged helix fold representative of the Forkhead family of enzymes with two wings in direct contact with DNA. For ease of representation, we described both wings as a dimer and a single wing as a monomer. From this structure, we hypothesized two main models of how thiostrepton binding to FOXM1 could possibly curtail its transcriptional activity. In the first model thiostrepton could bind either of the wings or both wings and prevent association to DNA. In the second model thiostrepton bind the FOXM1/DNA complex and weaken association of FOXM1 to DNA. Subsequently, small molecular inhibitors could also use either of the models to inhibit transcription. To account for both models, the NCI diversity set was screened against the FOXM1 dimer:DNA complex (39 hits), dimer (11 hits) and monomer (14 hits). Those hits were further classified by chemical structure, biological function and chemical similarities to known molecules that target FOXM1. In cellular cytotoxicity assays, N-phenylphenanthren-9-amine (related to hit #225) successfully showed cytotoxicity to all three cell lines with IC50 around 1µM, and downregulate FOXM1 and transcription of its downstream molecules such as CCNB1. CONCLUSION: By a combination of in silico screening coupled to cellular cytotoxicity studies, we have taken the first step towards identifying potential inhibitors of FOXM1 that can replace thiostrepton.

Effects of FOXM1 inhibition and ionizing radiation on melanoma cells.

Metastatic melanoma can be highly refractory to conventional radiotherapy and chemotherapy but combinatorial-targeted therapeutics are showing greater promise on improving treatment efficacy. Previous studies have shown that knockdown of Forkhead box M1 (FOXM1) can sensitize various tumor types to radiation-induced cell death. The effect of combining radiation with a small molecule FOXM1 inhibitor, Siomycin A, on growth, death and migration of a metastatic melanoma cell line (SK-MEL-28) that overexpresses this pleiotropic cell cycle regulator was investigated. Siomycin A (SIOA) was found to be a strong inducer of apoptosis, and inhibitor of proliferation and migration in a scratch wound assay in this cell line. Induction of apoptosis occurred at concentrations >1 µM in association with reductions in the constitutive FOXM1 and anti-apoptotic B-cell lymphoma 2 protein levels found in these cells. Single doses of ionizing radiation (0-40 Gy) delivered by linear accelerator caused inhibition of growth and migration without significant induction of cell death. Pretreatment with SIOA did not increase the sensitivity of this melanoma cell line to radiation as observed in other tumor types. These data confirm that as a single agent, SIOA is an effective inducer of cell death and inhibitor of migration in metastatic melanoma cells expressing constitutive FOXM1. In combination with radiation, SIOA pre-treatment, however, may not be of added benefit.

FOXM1 in sarcoma: role in cell cycle, pluripotency genes and stem cell pathways.

FOXM1 is a pro-proliferative transcription factor that promotes cell cycle progression at the G1-S, and G2-M transitions. It is activated by phosphorylation usually mediated by successive cyclin - cyclin dependent kinase complexes, and is highly expressed in sarcoma. p53 down regulates FOXM1 and FOXM1 inhibition is also partly dependent on Rb and p21. Abnormalities of p53 or Rb are frequent in sporadic sarcomas with bone or soft tissue sarcoma, accounting for 36% of index cancers in the high penetrance TP53 germline disorder, Li-Fraumeni syndrome.FOXM1 stimulates transcription of pluripotency related genes including SOX2, KLF4, OCT4, and NANOG many of which are important in sarcoma, a disorder of mesenchymal stem cell/ partially committed progenitor cells. In a selected specific, SOX2 is uniformly expressed in synovial sarcoma. Embryonic pathways preferentially used in stem cell such as Hippo, Hedgehog, and Wnt dominate in FOXM1 stoichiometry to alter rates of FOXM1 production or degradation. In undifferentiated pleomorphic sarcoma, liposarcoma, and fibrosarcoma, dysregulation of the Hippo pathway increases expression of the effector co-transcriptional activator Yes-Associated Protein (YAP). A complex involving YAP and the transcription factor TEAD elevates FOXM1 in these sarcoma subtypes. In another scenario 80% of desmoid tumors have nuclear localization of ß-catenin, the Wnt pathway effector molecule. Thiazole antibiotics inhibit FOXM1 and because they have an auto-regulator loop FOXM1 expression is also inhibited. Current systemic treatment of sarcoma is of limited efficacy and inhibiting FOXM1 represents a potential new strategy.