Mechanistic investigations of the liver toxicity of the free fatty acid receptor 1 agonist fasiglifam (TAK875) and its primary metabolites.

For fasiglifam (TAK875) and its metabolites the substance-specific mechanisms of liver toxicity were studied. Metabolism studies were run to identify a putatively reactive acyl glucuronide metabolite. In vitro cytotoxicity and caspase 3/7 activation were assessed in primary human and dog hepatocytes in 2D and 3D cell culture. Involvement of glutathione (GSH) detoxication system in mediating cytotoxicity was determined by assessing potentiation of cytotoxicity in a GSH depleted in vitro system. In addition, potential mitochondrial liabilities of the compounds were assessed in a whole-cell mitochondrial functional assay. Fasiglifam showed moderate cytotoxicity in human primary hepatocytes in the classical 2D cytotoxicity assays and also in the complex 3D human liver microtissue (hLiMT) after short-term treatment (24 hours or 48 hours) with TC50 values of 56 to 68 µM (adenosine triphosphate endpoint). The long-term treatment for 14 days in the hLiMT resulted in a slight TC50 shift over time of 2.7/3.6 fold lower vs 24-hour treatment indicating possibly a higher risk for cytotoxicity during long-term treatment. Cellular GSH depletion and impairment of mitochondrial function by TAK875 and its metabolites evaluated by Seahorse assay could not be found being involved in DILI reported for TAK875. The acyl glucuronide metabolites of TAK875 have been finally identified to be the dominant reason for liver toxicity.

A cell-penetrating ARF peptide inhibitor of FoxM1 in mouse hepatocellular carcinoma treatment.

The forkhead box m1 (Foxm1) transcription factor is essential for initiation of carcinogen-induced liver tumors; however, whether FoxM1 constitutes a therapeutic target for liver cancer treatment remains unknown. In this study, we used diethylnitrosamine/phenobarbital treatment to induce hepatocellular carcinomas (HCCs) in either WT mice or Arf(-/-)Rosa26-FoxM1b Tg mice, in which forkhead box M1b (FoxM1b) is overexpressed and alternative reading frame (ARF) inhibition of FoxM1 transcriptional activity is eliminated. To pharmacologically reduce FoxM1 activity in HCCs, we subjected these HCC-bearing mice to daily injections of a cell-penetrating ARF(26-44) peptide inhibitor of FoxM1 function. After 4 weeks of this treatment, HCC regions displayed reduced tumor cell proliferation and angiogenesis and a significant increase in apoptosis within the HCC region but not in the adjacent normal liver tissue. ARF peptide treatment also induced apoptosis of several distinct human hepatoma cell lines, which correlated with reduced protein levels of the mitotic regulatory genes encoding polo-like kinase 1, aurora B kinase, and survivin, all of which are transcriptional targets of FoxM1 that are highly expressed in cancer cells and function to prevent apoptosis. These studies indicate that ARF peptide treatment is an effective therapeutic approach to limit proliferation and induce apoptosis of liver cancer cells in vivo.

Forkhead box M1 regulates the transcriptional network of genes essential for mitotic progression and genes encoding the SCF (Skp2-Cks1) ubiquitin ligase.

The Forkhead box m1 (Foxm1) gene is critical for G(1)/S transition and essential for mitotic progression. However, the transcriptional mechanisms downstream of FoxM1 that control these cell cycle events remain to be determined. Here, we show that both early-passage Foxm1(-)(/)(-) mouse embryonic fibroblasts (MEFs) and human osteosarcoma U2OS cells depleted of FoxM1 protein by small interfering RNA fail to grow in culture due to a mitotic block and accumulate nuclear levels of cyclin-dependent kinase inhibitor (CDKI) proteins p21(Cip1) and p27(Kip1). Using quantitative chromatin immunoprecipitation and expression assays, we show that FoxM1 is essential for transcription of the mitotic regulatory genes Cdc25B, Aurora B kinase, survivin, centromere protein A (CENPA), and CENPB. We also identify the mechanism by which FoxM1 deficiency causes elevated nuclear levels of the CDKI proteins p21(Cip1) and p27(Kip1). We provide evidence that FoxM1 is essential for transcription of Skp2 and Cks1, which are specificity subunits of the Skp1-Cullin 1-F-box (SCF) ubiquitin ligase complex that targets these CDKI proteins for degradation during the G(1)/S transition. Moreover, early-passage Foxm1(-)(/)(-) MEFs display premature senescence as evidenced by high expression of the senescence-associated beta-galactosidase, p19(ARF), and p16(INK4A) proteins. Taken together, these results demonstrate that FoxM1 regulates transcription of cell cycle genes critical for progression into S-phase and mitosis.

ARF directly binds DP1: interaction with DP1 coincides with the G1 arrest function of ARF.

The tumor suppressor ARF inhibits cell growth in response to oncogenic stress in a p53-dependent manner. Also, there is an increasing appreciation of ARF's ability to inhibit cell growth via multiple p53-independent mechanisms, including its ability to regulate the E2F pathway. We have investigated the interaction between the tumor suppressor ARF and DP1, the DNA binding partner of the E2F family of factors (E2Fs). We show that ARF directly binds to DP1. Interestingly, binding of ARF to DP1 results in an inhibition of the interaction between DP1 and E2F1. Moreover, ARF regulates the association of DP1 with its target gene, as evidenced by a chromatin immunoprecipitation assay with the dhfr promoter. By analyzing a series of ARF mutants, we demonstrate a strong correlation between ARF's ability to regulate DP1 and its ability to cause cell cycle arrest. S-phase inhibition by ARF is preceded by an inhibition of the E2F-activated genes. Moreover, we provide evidence that ARF inhibits the E2F-activated genes independently of p53 and Mdm2. Also, the interaction between ARF and DP1 is enhanced during oncogenic stress and "culture shock." Taken together, our results show that DP1 is a critical direct target of ARF.

The Forkhead Box m1 transcription factor stimulates the proliferation of tumor cells during development of lung cancer.

The proliferation-specific Forkhead Box m1 (Foxm1 or Foxm1b) transcription factor (previously called HFH-11B, Trident, Win, or MPP2) regulates expression of cell cycle genes essential for progression into DNA replication and mitosis. Expression of Foxm1 is found in a variety of distinct human cancers including hepatocellular carcinomas, intrahepatic cholangiocarcinomas, basal cell carcinomas, ductal breast carcinomas, and anaplastic astrocytomas and glioblastomas. In this study, we show that human Foxm1 protein is abundantly expressed in highly proliferative human non-small cell lung cancers (NSCLC) as well as in mouse lung tumors induced by urethane. To determine the role of Foxm1 during the development of mouse lung tumors, we used IFN-inducible Mx-Cre recombinase transgene to delete mouse Foxm1 fl/fl-targeted allele before inducing lung tumors with urethane. We show that Mx-Cre Foxm1-/- mice exhibit diminished proliferation of lung tumor cells causing a significant reduction in number and size of lung adenomas. Transient transfection experiments with A549 lung adenocarcinoma cells show that depletion of Foxm1 levels by short interfering RNA caused diminished DNA replication and mitosis and reduced anchorage-independent growth of cell colonies on soft agar. Foxm1-depleted A549 cells exhibit reduced expression of cell cycle-promoting cyclin A2 and cyclin B1 genes. These data show that Foxm1 stimulates the proliferation of tumor cells during progression of NSCLC.

Increased levels of the FoxM1 transcription factor accelerate development and progression of prostate carcinomas in both TRAMP and LADY transgenic mice.

The proliferation-specific Forkhead Box M1 (FoxM1 or FoxM1b) transcription factor is overexpressed in a number of aggressive human carcinomas. Mouse hepatocytes deficient in FoxM1 fail to proliferate and are highly resistant to developing carcinogen-induced liver tumors. We previously developed a transgenic (TG) mouse line in which the ubiquitous Rosa26 promoter was used to drive expression of the human FoxM1b cDNA transgene in all mouse cell types. To investigate the role of FoxM1b in prostate cancer progression, we bred Rosa26-FoxM1b mice with both TRAMP and LADY TG mouse models of prostate cancer. We show that increased expression of FoxM1b accelerated development, proliferation, and growth of prostatic tumors in both TRAMP and LADY double TG mice. Furthermore, development of prostate carcinomas in TRAMP/Rosa26-FoxM1b double TG mice required high levels of FoxM1 protein to overcome sustained expression of the alternative reading frame tumor suppressor, a potent inhibitor of FoxM1 transcriptional activity. Depletion of FoxM1 levels in prostate cancer cell lines PC-3, LNCaP, or DU-145 by small interfering RNA transfection caused significant reduction in proliferation and anchorage-independent growth on soft agar. This phenotype was associated with increased nuclear levels of the cyclin-dependent kinase inhibitor protein p27(Kip1) and diminished expression of S-phase promoting cyclin A2 and M-phase promoting cyclin B1 proteins. Finally, we show that elevated levels of FoxM1 protein correlate with high proliferation rates in human prostate adenocarcinomas. Our results suggest that the FoxM1 transcription factor regulates development and proliferation of prostate tumors, and that FoxM1 is a novel target for prostate cancer treatment.

FoxM1 regulates transcription of JNK1 to promote the G1/S transition and tumor cell invasiveness.

The Forkhead box M1 (FoxM1) protein is a proliferation-specific transcription factor that plays a key role in controlling both the G(1)/S and G(2)/M transitions through the cell cycle and is essential for the development of various cancers. We show here that FoxM1 directly activates the transcription of the c-Jun N-terminal kinase (JNK1) gene in U2OS osteosarcoma cells. Expression of JNK1, which regulates the expression of genes important for the G(1)/S transition, rescues the G(1)/S but not the G(2)/M cell cycle block in FoxM1-deficient cells. Knockdown of either FoxM1 or JNK1 inhibits tumor cell migration, invasion, and anchorage-independent growth. However, expression of JNK1 in FoxM1-depleted cells does not rescue these defects, indicating that JNK1 is a necessary but insufficient downstream mediator of FoxM1 in these processes. Consistent with this interpretation, FoxM1 regulates the expression of the matrix metalloproteinases MMP-2 and MMP-9, which play a role in tumor cell invasion, through JNK1-independent and -dependent mechanisms in U2OS cells, respectively. Taken together, these findings identify JNK1 as a critical transcriptional target of FoxM1 that contributes to FoxM1-regulated cell cycle progression, tumor cell migration, invasiveness, and anchorage-independent growth.

Myocardium defects and ventricular hypoplasia in mice homozygous null for the Forkhead Box M1 transcription factor.

The Forkhead Box m1 (Foxm1) transcription factor is expressed in cardiomyocytes and cardiac endothelial cells during heart development. In this study, we used a novel Foxm1 -/- mouse line to demonstrate that Foxm1-deletion causes ventricular hypoplasia and diminished DNA replication and mitosis in developing cardiomyocytes. Proliferation defects in Foxm1 -/- hearts were associated with a reduced expression of Cdk1-activator Cdc25B phosphatase and NFATc3 transcription factor, and with abnormal nuclear accumulation of the Cdk-inhibitor p21(Cip1) protein. Depletion of Foxm1 levels by siRNA caused altered expression of these genes in cultured HL-1 cardiomyocytes. Endothelial-specific deletion of the Foxm1 fl/fl allele in Tie2-Cre Foxm1 fl/fl embryos did not influence heart development and cardiomyocyte proliferation. Foxm1 protein binds to the -9,259/-9,288-bp region of the endogenous mouse NFATc3 promoter, indicating that Foxm1 is a transcriptional activator of the NFATc3 gene. Foxm1 regulates expression of genes essential for the proliferation of cardiomyocytes during heart development.