Novel FOXM1 inhibitor STL001 sensitizes human cancers to a broad-spectrum of cancer therapies

Abstract

Forkhead box protein M1 (FOXM1) is often overexpressed in human cancers and strongly associated with therapy resistance and less good patient survival. The chemotherapy options for patients with the most aggressive types of solid cancers remain very limited because of the acquired drug resistance, making the therapy less effective. NPM1 mutation through the inactivation of FOXM1 via FOXM1 relocalization to the cytoplasm confers more favorable treatment outcomes for AML patients, confirming FOXM1 as a crucial target to overcome drug resistance. Pharmacological inhibition of FOXM1 could be a promising approach to sensitize therapy-resistant cancers. Here, we explore a novel FOXM1 inhibitor STL001, a first-generation modification drug of our previously reported FOXM1 inhibitor STL427944. STL001 preserves the mode of action of the STL427944; however, STL001 is up to 50 times more efficient in reducing FOXM1 activity in a variety of solid cancers. The most conventional cancer therapies studied here induce FOXM1 overexpression in solid cancers. The therapy-induced FOXM1 overexpression may explain the failure or reduced efficacy of these drugs in cancer patients. Interestingly, STL001 increased the sensitivity of cancer cells to conventional cancer therapies by suppressing both the high-endogenous and drug-induced FOXM1. Notably, STL001 does not provide further sensitization to FOXM1-KD cancer cells, suggesting that the sensitization effect is conveyed specifically through FOXM1 suppression. RNA-seq and gene set enrichment studies revealed prominent suppression of FOXM1-dependent pathways and gene ontologies. Also, gene regulation by STL001 showed extensive overlap with FOXM1-KD, suggesting a high selectivity of STL001 toward the FOXM1 regulatory network. A completely new activity of FOXM1, mediated through steroid/cholesterol biosynthetic process and protein secretion in cancer cells was also detected. Collectively, STL001 offers intriguing translational opportunities as combination therapies targeting FOXM1 activity in a variety of human cancers driven by FOXM1.

Fig. 1. STL001 causes dose-dependent suppression of FOXM1 protein levels in a variety of solid tumor-derived cancer cell lines.

A The structural formula of novel FOXM1 inhibitor STL001 and its precursor molecule STL427944, modified from source [5, 37]. B–G Various cancer cell lines were treated with increasing concentrations of STL427944 (B) and STL001 (C–G) for 24 h. Total protein samples obtained from treated cells were analyzed for FOXM1 protein levels via immunoblotting, and β-actin was used as internal loading control (n = 3 for each group). The STL001 was 25–50 times more efficient in reducing cellular FOXM1 protein levels in a variety of solid cancers as compared to its parental compound STL427944.

Fig. 2. FOXM1 suppression by STL001 is the autophagy-mediated process and is prevented by nuclear-export arrest.

A U2OS-C3-luc cells expressing doxycycline (Doxy)-inducible EGFP-FOXM1 fusion protein and FLO-1 cells were treated with STL001 for 24 h. Total protein samples were analyzed via immunoblotting for FOXM1 and LC3 expression, and β-actin was used as an internal loading control. B The bar graph represents the differential expression of FOXM1 and the ratio of LC3 II/I, analyzed by blot densitometry and presented as means ± S.D from three independent experiments (n = 3; **p < 0.001, ***p < 0.0001 using two-tailed Student’s t-tests). C U2OS-C3-luc cells were treated with indicated concentrations of doxycycline, STL001, chloroquine, and leptomycin B for 24 h. Total protein samples obtained from treated cells were analyzed for FOXM1 and LC3 expression levels via immunoblotting, and β-actin was used as an internal loading control (n = 3 for each group).

Fig. 3. RNA-Seq analysis of the gene expression by STL001 treatment in esophageal cancer cells.

A Heatmap of mean gene expression levels in control and STL001 (5 µM) for 947 differentially expressed genes (DEG). B Gene enrichment analysis of the 947-DEG in Gene Ontology biological processes. The size of points is proportional to the number of overlapped differential genes in the biological processes. C The network output of the most enriched biological processes and associated proteins was generated by the GOnet tool. Arrows indicate a direct link between most enriched biological processes according to the GOnet annotation. Round nodes represent proteins; square nodes represent enriched biological processes. D Gene set enrichment analysis of differential gene expression using Pathway Interaction Database (PID) collection of gene signature [PMID: 18832364].

Fig. 4. Differential gene expression shared between STL001 treatment and FOXM1-KD in ovarian cancer.

A Heatmap of mean gene expression changes relative to Control in STL001 treatment and FOXM1-KD for 14,980 analyzed genes. B Venn diagrams of up-and down-regulated genes in STL001 treatment and FOXM1-KD. C Scatter plot of log2 gene expression changes in STL001 treatment and FOXM1-KD, for 3018 genes differentially expressed in either STL001 treatment or FOXM1-KD. D Gene set enrichment analysis of differential gene expression using Pathway Interaction Database (PID) collection of gene signature [PMID: 18832364]. Presented are pathways significant at FDR < 0.1 in one condition and with nominal P-value < 0.01 in the other condition.

Fig. 5. STL001-mediated FOXM1 suppression sensitizes esophageal cancer (FLO-1) cells to a broad spectrum of chemotherapeutic agents with different mechanisms of action.

A, C, E, G FLO-1 cells were treated with indicated concentrations of cisplatin, irinotecan, 5’FU, paclitaxel, and STL001 alone or in combination with STL001 for 24 h. In all cases, total protein samples were obtained from cells immediately after treatment and analyzed for FOXM1, cleaved caspase-3 levels via immunoblotting, and β-actin was used as internal loading control (n = 3 for each group). B, D, F, H Percent (%) dead cells in FLO-1 cells treated with indicated concentrations of cisplatin, irinotecan, 5’FU, paclitaxel, and STL001 alone or in combination with STL001 for 24 h. The results shown are the mean ± SEM of three independent experiments performed in triplicate (**p < 0.001 vs control, using two-tailed Student’s t-tests; n = 3). I FLO-1 cells with stable shRNA-mediated FOXM1-KD were treated with paclitaxel alone or in combination with STL001 for 24 h and compared to parental cells under the same treatment conditions. In all cases, total protein samples were obtained from cells immediately after treatment and analyzed for FOXM1 and cleaved caspase-3 levels via immunoblotting, and β-actin was used as internal loading control (n = 3 for each group).

Fig. 6. STL001 enhances the cytotoxic effect of conventional chemotherapies through suppression of FOXM1 in a variety of solid cancers.

A Colon cancer (HCT-116 and FET) cells were treated with indicated concentrations of 5-FU and STL001 alone or in combination for 24 h. B HCT-116 cells with stable shRNA-mediated FOXM1-KD were treated with 5-FU alone or in combination with STL001 for 24 h and compared to parental cells under the same treatment conditions. C Prostate cancer (22RV1 and LNCaP) cells were treated with indicated concentrations of paclitaxel and STL001 alone or in combination for 24 h. D Tamoxifen resistance MCF-7 breast cancer cells (TAM-R) were treated with indicated concentrations of tamoxifen and STL001 alone or in combination for 24 h. In all cases, total protein samples were obtained from cells immediately after treatment and analyzed for FOXM1 and cleaved caspase-3 levels via immunoblotting, β-actin was used as internal loading control (n = 3 for each group). E Percent (%) dead cells in TAM-R cells treated with indicated concentrations of tamoxifen alone or in combination with STL001 for 24 h. The results shown are the mean ± SEM of three independent experiments performed in triplicate (**p < 0.001 vs control, using two-tailed Student’s t-tests; n = 3). F Ovarian cancer (OVCAR-8 and ES-2) cells were treated with indicated concentrations of doxorubicin (Doxo) and STL001 alone or in combination for 24 h. G OVCAR-8 cells with stable shRNA-mediated FOXM1-KD were treated with doxorubicin alone or in combination with STL001 for 24 h and compared to parental cells under the same treatment conditions. H, I Triple-negative breast cancer cells (HCC1143) were treated with indicated concentrations of doxorubicin (Doxo) (H) and cisplatin (I) alone or in combination with STL001 for 24 h. J HCC1143 cells with stable shRNA-mediated FOXM1-KD were treated with doxorubicin alone or in combination with STL001 for 24 h and compared to parental cells under the same treatment conditions. In all cases, total protein samples were obtained from cells immediately after treatment and analyzed for FOXM1, cleaved caspase-3 levels via immunoblotting, and β-actin was used as internal loading control (n = 3).