Cbx4 Increased Activity Of HIF-1 And Establishing The System Of Screening Compounds Targeting Transcription Activity Of HIF-1

Cbx4(Chromobox 4),also known as Pc2(Polycomb 2)is not only a component of PRC1(polycomb repressive complex 1),but also an E3 ligase for several SUMO substrates.Members of the polycomb repressive complex 1,such as BMI1,MEL18 and RING1 B,has an important role in the process of cancer.On the other hand,many proteins associated with cancer can be sumoylated,for instance,P53,RB,PTEN and MYC,so SUMOylation has aroused increasingly attention about its role in tumorigenesis.However,it was little-known about the function of Cbx4 in cancer.Hypoxia-inducible factor-1(HIF-1)is a key transcription factor for the cellular adaptive response to hypoxia.HIF-1 heterodimer comprises a oxygen-sensitive HIF-1α subunits and a constitutively expressed HIF-1β subunit.HIF-1,as a transcription factor,promotes the expression of VEGF(vascular endothelial growth factor),which is involved in the regulation of angiogenesis.We found that Cbx4 contributes to enhanced transcriptional activity of HIF-1 through sumoylation of HIF-1α,thus elevating hypoxia-induced VEGF production and angiogenesis,The result follows:1)Cbx4 significantly increased VEGF expression under hypoxia.Overexpression of Cbx4 significantly increased VEGF expression under hypoxia,while knocked down Cbx4 inhibited the hypoxia-induced VEGF expression.2)HIF-1 is possibly required for Cbx4 action on hypoxia-induced VEGF expression.The HIF-1α or HIF-1β silencing almost completely abrogated VEGF expression under hypoxia.Under these conditions,Cbx4 overexpression also failed to increase VEGF expression.3)Cbx4 interaction and sumoylation of HIF-1α.By IP,GST pull-down etc.we found that Cbx4 interacts with HIF-1α directly and identified the Cbx4/HIF-1α interaction domains.Cbx4 dose dependently enhance HIF-1α sumoylation.The in vitro sumoylation assay confirm that Cbx4 promotes HIF-1α sumoylation at K391 and K477.4)Cbx4 enhances transcriptional activity of HIF-1 and VEGF expression through sumoylation of HIF-1α.Through the Luciferase,q RT-PCR and ELISA tests we found Cbx4 cannot increase the transcription activity of mutations sumoylation sites of HIF-1α.5)Cbx4 increases VEGF expression,promotes the growth of orthotopically transplanted HCC cells depends on its SUMO E3 ligase activity.In conclusion,Cbx4 contributes to enhanced transcriptional activity of HIF-1 through sumoylation of HIF-1α at K391 and K477,thus elevating hypoxia-induced VEGF production and angiogenesis.Targeting SUMO E3 ligase activity of Cbx4 and/or the interaction between Cbx4 and HIF-1α is a potential therapeutic strategy for HCC patients.