Upregulated Foxm1Expression Induced By TNF-α/ROS/HIF-1α Pathway Promotes HCC Proliferation And Resistance To Apoptosis

Liver cancer is one of the most common cancers in the world. The increasingepidemiological evidence has shown that the inflammatory microenvironmentplays a critical role in promoting tumor progression certainly including livercancer. Among factors secreted by tumor-associated-inflammatory cells, TNF-α isidentified as a master one. However, the detailed mechanism TNF-α facilitatingtumor progression is far from having been clarified.It is suggested that HIF-1α might be involved in the regulation of geneexpression during inflammation. In our previous finding, hypoxia could induceFoxM1overexpression which accelerating growth and survival of hepaticcarcinoma cells.FoxM1(Forkhead box M1) belongs to the Fox transcription factor familysharing winged helix DNA-binding domain. FoxM1is ubiquitously expressed inproliferating mammalian cells. It is a key regulator of cell cycle influencing boththe transition G1to S and G2to mitosis phases. Knockout of FoxM1results incell cycle arrest, mitotic spindle defects and chromosomal mis-aggregation.It is reported recently that FoxM1is involved in cancer progression besides cell proliferation. Take an example, FoxM1overexpression is shown to beassociated with poor prognosis in HCC patients. Deletion of FoxM1results infailure of mouse hepatocyte proliferation and HCC tumorigenesis in response todiethylnitrosamine (DEN). Furthermore, the p19ARF peptide, significantlyinhibiting FoxM1transcriptional activity, could efficiently reduce HCCproliferation as well as induce apoptosis in mice. These results suggest thatFoxM1is an important regulator of hepatoma cell growth and survival. However,the molecular mechanism regulating FoxM1expression under inflammatorytumor microenvironment is still unclarified.【Aims】1. To investigate the expression of FoxM1after TNF-α induction in hepatomacells and the possible underlying pathway.2. To study the influence of FoxM1onproliferation and apoptosis of hepatoma cells induced by TNF-α.3. To explorethe clinical significance of FoxM1in hepatocellular carcinoma.【Methods】1. Using quantitative PCR, western blot and luciferase reporter assay todetermine the expression and promoter activity of FoxM1after treatment ofTNF-α.2. Using luciferase reporter assay to investigate the role of promotercis-elements in the regulation of TNF-α-induced FoxM1transcription.3. Usingbinding/competition ELISA and ChIP assay to determine whether HIF-1α bindsto the FoxM1promoter after TNF-α treatment.4. Using siRNA technique andwestern blot assay to confirm the essential role of HIF-1α in TNF-α-inducedFoxM1expression.5. Using MTT assay, colony formation assay and FACSanalysis to investigate the functional role of FoxM1in hepatoma cells induced byTNF-α.6. Using immunohistochemistry assay to determine the clinicalsignificance of FoxM1in hepatocellular carcinoma. 【Results】1. TNF-α induces FoxM1expression and transactivates its promoter activity inHCC cells.It was found that TNF-α significantly increased FoxM1mRNA level in HepG2and Huh7cells with concentration dependence. We next investigated the proteinlevel of FoxM1after treatment of TNF-α. Western blot assay demonstrated thatTNF-α treatment also enhanced FoxM1protein level in hepatoma cells.Furthermore, we found that the protein level of two FoxM1target genes, cyclinB1and cyclin D1, was also upregulated. Then luciferase reporter assay found thatFoxM1promoter activity was significantly enhanced after TNF-α treatment, alsowith concentration dependence.2. HIF-1α binding element is involved in TNF-α-induced FoxM1expression.In our previous study, it was found that the HIF-1α binding sites on the FoxM1promoter are critical for the transcription of FoxM1induced by hypoxia. In thepresent study we found that the sequence between nt–330and-58was criticalfor the activation of FoxM1promoter by TNF-α. Four HIF-1α binding sites inthis area, predicted by informatics software, were then mutated respectively.Luciferase reporter assay showed that the mutation of HIF1α-1or HIF1α-3/4significantly decreased the transactivation of FoxM1promoter by TNF-α.Furthermore, mutation with both sites HIF1α-1and HIF1α-3/4showed lowerluciferase activity than respectively. These results suggest that HIF-1α bindingsites (HIF1α-1and HIF1α-3/4) are required for the activation of FoxM1promoterby TNF-α. 3. HIF-1α directly binds to FoxM1promoter.Binding/competition ELISA assay was performed to determine whetherHIF-1α bound to the FoxM1promoter after TNF-α treatment. Increased HIF-1αDNA binding activity was found in HepG2cells treated with TNF-α comparedwith untreated ones. Moreover, increasing binding activity was observed with theaddition of increasing amounts of nuclear extracts. These data suggest that TNF-αincreases the HIF-1α DNA binding activity to FoxM1promoter in HepG2cells.Further, binding/competition assay indicated that HIF-1α bound to the HIF1α-1and HIF1α-3/4sites in FoxM1promoter. In addition, these results were furtherconfirmed by ChIP assay. Taken together, it is suggested that HIF-1α directlybinds to FoxM1promoter after TNF-α treatment in HepG2cells.4. HIF-1α activation is required for TNF-α-induced FoxM1expression.HepG2cells were co-transfected with pGL-FoxM1and HIF-1α siRNA.24hpost transfection, luciferase reporter assay indicated that the activation of FoxM1promoter induced by TNF-α was significantly inhibited by depletion of HIF-1α,which was further confirmed by western blot. Similar results were obtained bytreatment with HIF-1α inhibitor (YC-1). Quantitative PCR assay showed thatTNF-α treatment did not alter the mRNA level of HIF-1α. However, the proteinlevel of HIF-1α was significantly increased after treatment with TNF-α. Thesedata suggests that TNF-α treatment stabilizes HIF-1α which is required forTNF-α-induced FoxM1expression.5. TNF-α-induced HIF-1α activation and FoxM1expression is ROS-dependent.Several studies have demonstrated that HIF-1α stabilization is regulated by ROS, which has been shown to be induced by TNF-α. In our study, the luciferasereporter assay showed that inhibition of ROS product with BHA significantlydecreased the FoxM1promoter activity. Furthermore, BHA significantlydecreased HIF-1α and FoxM1expression induced by TNF-α. Taken together,these data suggests that TNF-α-induced HIF-1α activation and FoxM1expressionis ROS-dependent.6. TNF-α-induced FoxM1increases the proliferation and dereases the apoptosisof HepG2cellsTo investigate the functional role of FoxM1induced by TNF-α, we establishedfour stable cell lines denoted LV-shcontrol, LV-shFoxM1, LV-control andLV-FoxM1by lentivirus infection with HepG2cells. After TNF-α treatment, thecell viability of LV-shcontrol and LV-control cells by MTT assay was similarlylow. LV-FoxM1cells showed highest surviving rate, while LV-shFoxM1cellslowest. Furthermore, treatment with YC-1or BHA besides TNF-α in the stablecell lines results in further inhibited proliferation of hepatoma cells whose FoxM1was depleted. In addition, FoxM1re-overexpression by LV-FoxM1could reversethe inhibition of hepatoma cells proliferation by TNF-α and YC-1/BHA. Analysisrevealed that FoxM1expression is positively correlated with proliferation ofhepatoma cells by MTT assay. Next, it was found that LV-shFoxM1cells showedfewer colony formations compared with LV-shcontrol cells. However, LV-FoxM1cells could increase the ability of colony formation which can be inhibited byTNF-α and YC-1/BHA. Besides, Annexin-V/PI staining assay showed thatinhibition of FoxM1expression could significantly increase the apoptosis ofhepatoma cells, while facilitation of FoxM1expression could resist to apoptosisinduced by TNF-α and YC-1/BHA. Hochest33258staining assay showed a similar result. These data suggests that FoxM1expression is critical for HCCproliferation.7. HIF-1α and FoxM1coexpression indicates poor prognosis of human HCC aftersurgical resection.Immunohistochemistry revealed that there was a significant positivecorrelation between HIF-1α and FoxM in406human HCC tissues (P=0.044).Overexpression of HIF-1α was associated with poor prognosis. Combinedexpression of HIF-1α and FoxM1, patients were subsequently divided into4groups. Kaplan-Meier analysis showed statistically distinct recurrence andsurvival patterns within4subgroups, among which patients with coexpression ofHIF-1α and FoxM1endured the highest recurrence and shortest lifetime. Inaddition, in both positive and negative HIF-1α tumors, patients with positiveFoxM1expression had higher recurrence and shorter lifetime. Taken together,these results suggest that HIF-1α and FoxM1coexpression indicates poorprognosis in HCC patients.【Conclusions】1. We for the first time found that TNF-α induces FoxM1expression andtransactivates its promoter activity in HCC cells. Furthermore, two FoxM1target genes, cyclin B1and cyclin D1, was also upregulated in response toTNF-α.2. Using luciferase reporter, ChIP assay and siRNA technique, we found thatHIF-1α directly binds to FoxM1promoter after TNF-α treatment in HepG2cells. HIF1α-1and HIF1α-3/4sites are critical to this binding.3. It is suggested that TNF-α-induced HIF-1α activation and FoxM1expression is ROS-dependent.4. Using lentivirus interference with FoxM1and HIF-1α expression, chemicalinhibitors to HIF-1α and ROS product, we found that overexpression ofFoxM1induced by TNF-α could promote HCC growth in dependence withROS/HIF-1α pathway.5.406tissue immunohistochemistry analysis showed that coexpression ofHIF-1α and FoxM1indicates poor prognosis in HCC patients.