Regulation Of PP2A/PHD2/HIF-1α,Signal Axis And Intervention Effects Of DMBT On Vasculogenic Mimicry Under Hypoxia Microenvironment In Breast Cancer

BackgroundBreast cancer,which seriously endangers the lives and health of women,is the main cause of cancer-related death in women.Vasculogenic mimicry(VM),a marker of the aggressive breast tumor cell phenotype,is a highly patterned and functional tubular structure consisted of highly invasive tumor cells.And it may play roles during various phases of tumor progression.At the same time,the tumor cells surrounded by VM were directly exposed to the blood,which accelerated the metastasis of tumor cells.Many factors are necessary in the process of VM formation,including tumor anoxic microenvironment.Hypoxia is a typical feature of many solid tumors.Hypoxia-inducible factors-1α(HIF-1α)is a "starting switch" in VM signaling,which can directly or indirectly regulate the expression of VE-cadherin and activate the phosphatidylinositol 3-kinase/protein kinase B(PI3K/AKT)cascade.Finally,through the expression and activation of matrix metalloproteinase(MMPs),the contractile matrix enters the extracellular microenvironment to form the VM.But how the tumor cells sense the extracellular hypoxia microenvironment and then transmit it to the cells is not clear.Although HIF-1α is an executor of hypoxia,HIF-1α itself does not directly sense changes in oxygen tension(pO2)which is regulated by prolyl 4-hydroxylase 2(PHD2).The hydroxylation of PHD2 can regulate the stability and transcriptional activity of HIF-1α.Under normal oxygen conditions,PHD2 catalyzes the combination of an oxygen atom and a hydrogen atom of proline on HIF-1α to form a hydroxyl group,which is the key to the ubiquitination of HIF-1α.During hypoxia,due to O2 restriction,PHD2 inhibits the hydroxylation of HIF-1α,reduces the degradation of HIF-1α,and then continuously accumulates and enters the nucleus,forming a heterodimer HIF-1 with HIF-1β.This dimer is specific binds to DNA and activates hypoxia signaling pathways.This means that accurate regulation of PHD2 is essential for the development of cancer.Reversible phosphorylation is a common way of post-translation regulation of proteins.Protein phosphatase 2A(PP2A),a serine/threonine phosphatase,can regulate the oxidative metabolism,invasion and metastasis of tumor cells by triggering the dephosphorylation of proteins.At present,the protein structure of PHD2 has been revealed that there are dephosphorylation sites in its nuclear transport sequence,which can be dephosphorylated by phosphatase.PHD2 phosphorylation can promote the death of cancer cells in the hypoxic region of the tumor.Tumor cells tend to dephosphorylate PHD2 by overexpression of phosphatase PP2A/B55,resulting in partial inactivation of PHD2,but the molecular mechanism of PP2A and PHD2 on breast cancer invasion and metastasis is not clear.Especially in the hypoxia microenvironment,how PP2A affects the VM formation of breast cancer through PHD2 has not been reported.In addition,it is not clear whether there is a common target between traditional anti-angiogenic drugs and anti-VM.Therefore,in order to find new targets,we should fully take VM into consideration,and develop therapeutic drugs against VM or both endothelial cells and VM,in order to improve their lethality to tumor cells,which is of great significance for the clinical treatment of breast cancer.Our previous study has found that trehalose derivative DMBT can inhibit VM formation and invasion and migration of human breast cancer cells under hypoxic environment,but the specific mechanisms of DMBT and the roles of PP2A/PHD2/HIF-1α need further to be explored.The main purpose of this study is to explore how PP2A regulates the VM formation of breast cancer by affecting PHD2 and HIF-1α in anoxic microenvironment in vitro and in vivo,and then affects the invasion and metastasis of breast cancer.Clarifying the signal transduction of hypoxia and the regulation mechanism of VM in breast cancer can effectively inhibit the invasion and metastasis of breast cancer cells,which will provide a better theoretical basis for anti-invasion and metastasis therapy of breast cancer.In addition,by investigating the interaction between DMBT and PP2A,the mechanism of DMBT against breast cancer invasion and metastasis and whether it is related to PP2A/PHD2/HIF-1α signal axis were further studied,and the action target of DMBT was identified,which laid a foundation for the development of anti-breast cancer invasion and metastasis drugs with high efficiency and low toxicity.Methods and ResultsIn vitro experimentsHypoxia modeling:CoCl2 treatment and 1%O2 culture were used to create a hypoxic microenvironment for breast cancer cells.MTT was used to detect cells survival rate.And the expression of hypoxia-related proteins HIF-la and PHD2 were detected by Western blotting.Hypoxia models of MDA-MB-231 and MCF-7 cells were established by using 400 μmol/L CoCl2 or 1%O2 for 24 h in vitro.Effect of PP2A on the invasion and migration of breast cancer cells:The effects of PP2A inducer FTY720 and PP2A inhibitor OA on the survival rate of MDA-MB-231 and MCF-7 cells was detected by MTT assay,then low concentrations of FTY720(2 μmol/L)and OA(10 nmol/L)which did not affect cell survival were selected for Wound healing assay.Adhesion assay,Transwell assay and Wound healing assay were used to detect the effects of PP2A on migration,invasion and adhesion of MDA-MB-231 and MCF-7 cells under hypoxia.The results showed that PP2A inducers FTY720 or Ad-PP2A promoted the migration,invasion and adhesion of MDA-MB-231 and MCF-7 cells,and PP2A inhibitors OA or Ad-dn-PP2A inhibited their migration,invasion and adhesion.Effects of PP2A on VM formation of breast cancer cells:VM formation assay was used to detect the effects of Ad-PP2A/Ad-dn-PP2A on the VM formation of MDA-MB-231 cells under hypoxia.And the expression of VM-related proteins PI3K,P-AKT,VE-Cadherin,and MMP-2 in MDA-MB-231 and MCF-7 cells transfected with Ad-PP2A/Ad-dn-PP2A under hypoxia were measured by Western blotting.The results showed that Ad-PP2A promoted VM formation and VM-related protein expression under hypoxia in breast cancer cells,while Ad-dn-PP2A inhibited VM formation and VM-related protein expression.Mechanism of PP2A on VM formation,invasion and metastasis:Western blotting was used to detect the accumulation of HIF-1α after adenovirus transfection under normoxic and hypoxic conditions.The results showed that the accumulation of HIF-1α increased in both normoxic and hypoxic conditions in Ad-PP2A group,while decreased in Ad-dn-PP2A group.The results of PP2A enzyme activity detection showed that the activity of PP2A in anoxic environment was 57.47%,higher than that in normoxic environment,which suggested that the changes of invasion and migration ability of breast cancer cells may be closely related to PP2 A.Western blotting was used to measure the effect of PP2A on PHD2 expression in hypoxic environment.It was found that in hypoxic environment,PHD2 was downregulated.Ad-PP2A further inhibited PHD2,and Ad-dn-PP2A upregulated PHD2 in hypoxic environment.Then,after cells being treated with the PHD2 inhibitor DMOG,Transwell migration assay was used to test the cell migration.It was found that DMOG reversed the inhibitory effect of Ad-dn-PP2A on the migration of MDA-MB-231 cells,and the migration promotion rate reached 18.06%.Western blotting was used to detect the expression of HIF-1α,PHD2,and OH-HIF-1α.The results showed that DMOG reversed the effect of Ad-dn-PP2A,downregulated OH-HIF-1α,and increased the accumulation of HIF-1α.It is proved that the role of PP2A is related to PHD2.Subsequently,immunofluorescence was used to detect the distribution of PHD2 in the nucleus.The results showed that PHD2 in the Hypoxia+LacZ group was significantly more distributed in the nucleus than in the LacZ group.Ad-PP2A further promoted the distribution of PHD2 in the nucleus,while PHD2 was relocated to the cytoplasm in Ad-dn-PP2A group.Next,we further explore the mechanism of DMBT on invasion,metastasis and VM formation of breast cancer cells in hypoxic microenvironment.Computer molecular docking assay showed that DMBT could bind to the active site Mn2+ of the catalytic subunit of PP2A.The phosphatase activity of PP2A assay showed that DMBT significantly reduced the phosphatase activity of PP2A under hypoxia.VM formation test showed that DMBT could effectively inhibit VM formation under hypoxia.DMBT and Ad-dn-PP2A had synergistic effect,while Ad-PP2A could reverse the inhibitory effect of DMBT on VM formation.Western blotting showed that the expression of P-AKT,P-MAPK,mTOR,PI3K,MMP-2 and VE-cadherin was significantly down-regulated in Hypoxia+LacZ+DMBT group,and there was a synergistic effect between DMBT and Ad-dn-PP2A.The expressions of P-AKT,P-MAPK,mTOR,PI3K,MMP-2 and VE-cadherin were further down-regulated in Hypoxia+Ad-dn-PP2A+DMBT group.However,Ad-PP2A and DMBT reversed the effect of DMBT and up-regulated the expression of P-AKT,P-MAPK,mTOR,MMP-2 and VE-cadherin under hypoxia.Immunofluorescence staining showed that there was a synergistic effect between DMBT and Ad-dn-PP2A which made PHD2 relocate in the cytoplasm,while Ad-PP2A reversed the effect of DMBT and made PHD2 more distributed in the nucleus.In vivo experiments18～22 g Female BALB/c-nu nude mice(5～6 weeks of age)were injected with MDA-MB-231-luc-D3H2LN cells transfected with LacZ,Ad-PP2A or Ad-dn-PP2A through tail vein to establish the model of experimental lung metastasis in vivo.DMBT was administered continuously for 20 days,and the body weight and survival time were recorded.In vivo imaging of small animals was used to observe the metastasis of tumor cells in Control group,LacZ group,Hypoxia+LacZ,Hypoxia+Ad-PP2A group,Hypoxia+Ad-dn-PP2A group,Hypoxia+LacZ+DMBT group,Hypoxia+Ad-PP2A+DMBT group and Hypoxia+Ad-dn-PP2A+DMBT group.The VM formation was detected by CD34/PAS double staining.Pulmonary metastatic nodules were observed by dissecting lung tissue.The results showed that compared with Control group,the lung fluorescence intensity of LacZ group decreased slightly,but there was no significant difference.Compared with the LacZ group,the lung fluorescence intensity was significantly enhanced in Hypoxia+LacZ group.Compared with the Hypoxia+LacZ group,the lung fluorescence intensity was significantly enhanced,the pulmonary metastatic nodules were increased,the survival time was shorter,the lung tissue had more VM channels and the body weight was significantly lower in the Hypoxia+Ad-PP2A group,while in the Hypoxia+Ad-dn-PP2A group,the pulmonary fluorescence intensity were weakened,the pulmonary metastatic nodules were less,and the survival time was longer,the formation of VM was difficult to be found in the lung tissue,and the ducts were rare and small.In addition,compared with Hypoxia+LacZ group,Hypoxia+LacZ+DMBT could inhibit the lung fluorescence intensity and reduce lung metastatic nodules.Ad-dn-PP2A and DMBT had a synergistic effect,while Ad-PP2A reversed the effect of DMBT.Lung fluorescence intensity increased and lungmetastatic nodules increased.Conclusions 1.The overexpression of PP2A promoted VM formation,invasion and metastasisof breast cancer cells under hypoxia both in vitro and in vivo.2.MDA-MB-231 and MCF-7 cells experience anoxic microenvironment,which began with the upregulation of PP2A activity,which increased the dephosphorylation of PHD2,promoted the transport of PHD2 into the nucleus,blocked the nuclear space between PHD2 and HIF-1α,reduced the hydroxylation and degradation of HIF-1α,led to the accumulation of HIF-1α,initiated the VE-Cadherin/PI3K/AKT/MMPs signal pathway,promoted the formation of VM in breast cancer cells,and then affected the ability of invasion and metastasis of breast cancer cells.3.DMBT could bind to the active site of PP2A and decrease the activity of PP2A under anoxic environment.Low-expression of PP2A and DMBT synergistically inhibited the formation of VM in MDA-MB-231 and MCF-7 cells,while overexpression of PP2A and DMBT have antagonistic effect.That was to say,PP2A might be the key factor for DMBT to down-regulate HIF-1α,and DMBT might down-regulate HIF-1α through PP2A/PHD2 signal axis,thus reducing the expression of VM-related proteins,inhibiting VM formation,invasion and metastasis of breast cancer cells.SignificanceThis study has investigated the roles and mechanisms of PP2A on VM formation,invasion and metastasis of breast cancer by affecting PHD2 and HIF-1α in anoxic microenvironment and the intervention effect and mechanism of DMBT.The results clarified the regulation mechanism of PP2A and DMBT on VM formation in breast cancer through PP2A/PHD2/HIF-1α signal axis,which will provide better theoretical and practical support for anti-invasion and metastasis treatment of breast cancer.It lays a foundation for the development of anti-breast cancer invasion and metastasis drugs with high efficiency and low toxicity.