Initial Exploration Of The Anti-proliferative Effect Of Thiostrepton-mediated Targeting Of The FOXM1 Transcription Factor On Human Liver Cancer Cells

Liver cancer is one of the most common malignancies in China. Being highly malignant with rapid progression, the treatment for human liver cancer is difficult and the therapeutic effect is usually poor. Chemotherapy is the traditional choice for unresectable or metastatic disease; however, HCC is highly resistant to almost all kinds of chemotherapeutic drugs. Thus, there is an urgent need to develop novel therapeutic agents for the treatment of HCC.The development of liver cancer is a multi-factor synergized process. A variety of elements inclucing virus infection, carcinogens, oncogene activation, inactivation of tumor suppressor genes, dysregulation of apoptosis and proliferation acted together, among which dysregulation of proliferation plays a key role during hepatocarcinogenesis. Novel therapeutic strategies derived from increased knowledge of molecular oncology underlying hepatocarcinogenesis are constantly being developed to cure this disease.FOX (forkhead/winged helix) transcription factor, known as the forkhead box gene family with more than 50 transcription factors, is defined by a homologous conserved winged helix DNA binding domain with more than 100 amino acids, which demonstrated its functions in DNA binding, transcriptional activation and transcriptional repression. Fox protein family members play important roles in cell growth, proliferation, differentiation, longevity determination and cellular transformation. FOXM1 transcription factor, an important member of the FOX gene family and a proliferation-specific gene, is involved in a variety of patho-physiological processes such as embryonic development, aging, tissue regeneration and cancinogenesis. Numerous studies have shown that the FOXM1 oncogenic transcription factor can promote cell proliferation through its effect on different phases of cell cycle progression. For instance, an increase in FOXM1 activity may promote tumorigenesis through driving mitosis and cell cycle progression at both G1-S and G2-M transitions by regulating downstream targets such as SKP2, CyclinD1, CDK2 and CDK4. These results indicated that: the FOXM1 transcription factor may play an important role in hepatocarcinogenesis through its regulation on cell proliferation. Meanwhile, over-expressions of FOXM1 oncogenic transcription factor have been identified in many human malignancies including HCC, breast cancer, lung cancer, prostate cancer, gastric cancer, pancreatic cancer and so on. Moreover, FOXM1 was also demonstrated to confer an acquired cisplatin resistance in breast cancer cells. Because of their important roles in mitotic control, tumorigenesis and drug resistance, FOXM1 has attracted much attention as potential cancer therapeutic targets.Recently, FOXM1 up-regulation has been demonstrated to be associated with the acquisition of a susceptible HCC phenotype in rats; on the other hand, both siRNA- and p19ARF- mediated FOXM1 depletions led to proliferation inhibition and resistance to hepatocarcinogenesis in rodents. However, FOXM1 expression in clinical HCC tissues and its clinical significance have not been well investigated. The present study was therefore initiated to (1) demonstrate the frequent over-expression of FOXM1 in clinical HCC samples, and (2) to investigate the potential effect of targeted inhibition of FOXM1 in the treatment of liver cancer.Thiostrepton is a natural cyclic oligopeptide antibiotic, which can be derived from several strains of strepromycetes. Among which Thiostrepton has been used in veterinary medicine in mastitis caused by gram-negative organisms and in dermatologic disorders. It is also active against gram-positive bacteria. Recently, Thiostrepton has been reported to selectively target breast cancer cells through inhibition of FOXM1 expression. Other research also shows that thiostrepton specifically inhibited FOXM1 transcription factor with no effect on other transcription factors. In addition, Thiostrepton was shown to induce apoptosis after its inhibition on FOXM1 expression in a variety of human tumor cell lines with different origines.38 HCC clinical samples and 2 human liver cancers cells, HepG2 and HepG2.2.15 were included into the present study. After demonstrating that FOXM1 was immunochemically over-expressed in both clinical HCC tissues and HCC cell lines, MTT assay was employed for the detection of cellular proliferation before and after Thiostrepton treatment; cell cycle profiles were assessed by flow cytometry; and the cell culture supernatants from both HepG2 and HepG2.2.15 cells were collected and AFP was quantified before and after Thiostrepton treatment; and the possible molecular targets were explored through semi-quantitative RT-PCR, real time quantitative RT-PCR and Western blot, focusing on Thiostrepton-mediated FOXM1 inhibition and its downstream proliferation-associated targets such as CyclinD1, CDK2, CDK4 and SKP2, and its induction of the FOXM1 up-stream FOXO3a transcription factor.The results showed that at 24h and 48h after treatment,TST induced inhibition of cellular proliferation and cell cycle block at G1/S transition in both HepG2 and HepG2.2.15 HCC cells,concomitant with down-regulated AFP secretions. Exploration of the molecular mechanisms revealed that Thiostrepton effectively inhibited FOXM1 expression at both mRNA and protein level(p<0.05), furthermore, its expression dynamics were similar to those of its downstream proliferation-associated targets including CyclinD1, CDK2, CDK4 and SKP2; while on the other hand, the expression of its up-stream FOXO3a was significantly up-regulated(P<0.05). All these results suggested that FOXM1 is an attractive therapeutic target in the fight against HCC. Thiostrepton may specifically target FOXM1 and could be potentially developed as a novel anticancer drug against HCC.