FoxM1 Is a General Target for Proteasome Inhibitors

Proteasome inhibitors are currently in the clinic or in clinical trials, but the mechanism of their anticancer activity is not completely understood. The oncogenic transcription factor FoxM1 is one of the most overexpressed genes in human tumors, while its expression is usually halted in normal non-proliferating cells. Previously, we established that thiazole antibiotics Siomycin A and thiostrepton inhibit FoxM1 and induce apoptosis in human cancer cells. Here, we report that Siomycin A and thiostrepton stabilize the expression of a variety of proteins, such as p21, Mcl-1, p53 and hdm-2 and also act as proteasome inhibitors in vitro. More importantly, we also found that well-known proteasome inhibitors such as MG115, MG132 and bortezomib inhibit FoxM1 transcriptional activity and FoxM1 expression. In addition, overexpression of FoxM1 specifically protects against bortezomib-, but not doxorubicin-induced apoptosis. These data suggest that negative regulation of FoxM1 by proteasome inhibitors is a general feature of these drugs and it may contribute to their anticancer properties

Overexpression of FoxM1 partially protects cancer cell lines from thiazole antibiotic-induced apoptosis.

<p>(A) Immunoblot analysis after treatment with Siomycin A revealed close correlation between downregulation of FoxM1 and induction of apoptosis. (B) Following treatment with thiostrepton, thiopeptide-induced apoptosis and inhibition of FoxM1 protein expression are more prominent in the presence of cyclohexamide (Chx) as depicted by immunoblotting for FoxM1 and cleaved caspase-3. (C) The expression of endogenous FoxM1 decreased in a time-dependent fashion in the presence of thiostrepton and Chx, while the levels of exogenous FoxM1 were not affected. Overexpression of FoxM1 protected against cell death induced by thiostrepton as detected by immunoblotting for cleaved caspase-3. (D) FoxM1 overexpressing cells were resistant to the treatment with increasing amount of Siomycin A as analyzed by immunoblotting for cleaved caspase-3. (E) Immunoblot analysis revealed that overexpression of FoxM1 also protected against Siomycin A-induced apoptosis in the presence of Chx.</p

Proteasome inhibitors target FoxM1 protein and overexpression of FoxM1 partially protects cancer cell lines from proteasome inhibitor-induced apoptosis.

<p>(A) Proteasome inhibitors induce apoptosis and inhibit FoxM1 protein expression in multiple myeloma, leukemia and osteosarcoma cell lines. U266 and RPMI8226 multiple myeloma, HL-60 leukemia and U2OS-C3 osteosarcoma cell lines were treated with proteasome inhibitors MG115, MG132 and bortezomib (BOR). Apoptosis was assessed by immunoblotting for cleaved caspase-3 and FoxM1 protein levels were detected by immunoblotting. β-actin was used as the loading control. (B) Proteasome inhibitor-induced apoptosis was further quantified by using Annexin V -PE/7AAD staining. U266 and RPMI8226 multiple myeloma, HL-60 leukemia and U2OS-C3 osteosarcoma cell lines were grown in the absence or presence of indicated concentrations of proteasome inhibitors MG115, MG132 and bortezomib (BOR) for 24 hours. Following drug treatment cells were stained with Annexin V- PE/7AAD and then analyzed by flow cytometry. Percentage of apoptotic cells is shown in brackets. Concentrations of drugs are the same as in the Fig 2A. (C) Overexpression of FoxM1 specifically protects against cell death induced by Bortezomib. Overexpression of FoxM1 protected against cell death induced by increasing amount of bortezomib (BOR), but not that of doxorubicin. Immunoblotting was carried out with antibodies specific for cleaved caspase-3 and β-actin.</p

Thiazole antibiotics inhibit FoxM1-dependent transcription and FoxM1 expression.

<p>(A) The chemical structure of the thiazole antibiotic, thiostrepton that differs from Siomycin A by only two residues (Thiostrepton-R1-R2: Isoleucine-alanine; Siomycin- R1-R2: valine-dehydroalanine). (B) Luciferase assays after treatment of the C3-Luc2.3-FoxO1 cell line with the combination of either 1 µg/mL doxycycline (Doxy) or 300 nM tamoxifen (Tam) and 3 µM of Siomycin A (Sio) or thiostrepton (Tio), respectively revealed that thiostrepton is also a negative regulator of FoxM1 transcriptional activity and thiazole antibiotics inhibit FoxM1 transcriptional activity among the Forkhead family members. (C) Thiazole antibiotics downregulated FoxM1 protein levels, but not FoxA1, FoxO1 and FoxO3a levels as detected by immunoblotting. (D) The HCT116-p53RE-Luc cell line, which stably expressing firefly luciferase under the control of multiple p53 response elements treated with the indicated concentration of Siomycin A or thiostrepton. After overnight treatment the luciferase activity was measured. (E) SW480 colon cancer cell line was transiently transfected with the Tcf/Lef-dependent TOPFlash and the control FOPFlash constructs. Twenty-four hrs following transfection the cells were treated with 3 µM of Siomycin A or thiostrepton. The next day the luciferase activity was measured. (F) A549 lung cancer cells were transiently transfected with the GLI-dependent GLIBS-Luc, the control miniTK reporter constructs and the GLI expression plasmid. Cells were treated with the indicated concentration of the thiopeptides 24 hrs after transfection and the luciferase activity was measured the following day. Bars in B, D–F are representative mean values of triplicate experiments+/−SD.</p

Evaluation of the effects of the thiopeptides on a panel of human cancer cell lines.

<p>(A) Treatment of caspase-8 deficient and reconstituted NB7 neuroblastoma cell lines with the thiazole antibiotics revealed that thiopeptide-induced apoptosis mainly involves the intrinsic apoptotic pathway. (B) Leukemia cancer cell lines CEM [IC₅₀/µM: Sio-0.73 (0.08); Thio-1.47 (0.1)], HL60 [IC₅₀/µM: Sio-0.68 (0.06); Thio-1.78 (0.4)], and U937 [IC₅₀/µM: Sio-0.53 (0.1); Thio-0.73 (0.3)], and liver cancer cell lines Hep-3B [IC₅₀/µM: Sio-3.6 (1.3); Thio-2.3 (0.8)], Huh7 [IC₅₀/µM: Sio-2.3 (0.5); Thio-1.8 (0.2)], and SK-Hep [IC₅₀/µM: Sio-3.7 (0.4); Thio-6.0 (1.4)], showed sensitivity in low micromolar range to the thiazole antibiotics as determined by growth inhibition assays. (C–D) Siomycin A and thiostrepton inhibit FoxM1 expression and induce potent apoptosis in leukemia and liver cancer cells.</p