Study Of The Role And Mechanism Of PFKFB4 In The Adaptation To Hypoxic Microenvironment In Multiple Myeloma

BackgroundMultiple myeloma(MM)is a malignant monoclonal plasma cell disease,characterized by terminal organ damage,including renal damage,hypercalcemia,osteolytic lesions and anemia.It ranks second among common hematological malignancy with high morbidity and mortality.Recent advances have significantly reduced mortality and improved life quality of MM patients but resistance/relapse often still occur after individual treatment with immunomodulatory agents,proteasome inhibitors,histone deacetylation inhibitors,monoclonal antibodies,or bone marrow transplants.Hypoxic bone marrow microenvironment plays a crucial role in the development,treatment resistance and poor prognosis of hematological tumors.Therefore,searching for genes related to the promotion of adaptation of hypoxic microenvironment can provide new potential targets for clinical MM treatment,improving the therapeutic effect of patients and the prognosis of the MM.Glycolysis is a crucial enzymatic process in human cellular metabolism,and it is involved in the production of substrates required for many biochemical pathways.In order to obtain sufficient energy and nutrients for growth and reproduction,tumor cells often show abnormal plasticity to the microenvironment and then reprogram their metabolic process,as characterized by abnormal hypermetabolism.Studies have proved that the activity and protein expression levels of key enzymes involved in glycolysis are upregulated in various cancer cells.Among them,phosphofructokinase-1(PFK-1)is one of the major regulatory sites in glycolysis.Fructose 2,6-bisphosphate(F-2,6-BP)is the strongest allosteric activator of PFK-1,which is regulated by 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase(PFKFB) family isoenzymes.As a subtype of PFKFB protein family,PFKFB4 regulates the synthesis and hydrolysis of F-2,6-BP.Therefore,PFKFB4 is an important enzyme that regulates glycolysis,and further participates in the growth,reproduction,invasion and metastasis of cancer cells,leading to tumor progression and recurrence.So far,PFKFB4 has been proved to be aberrantly highly expressed in a variety of diseases including solid and non-solid tumors,and it is closely associated with poor prognosis of tumor diseases.The purpose of this study is to explore whether PFKFB4,a glycolysis-related gene,is involved in the adaptation of hypoxia microenvironment of MM bone marrow and the underlying mechanisms.We believe that this work is helpful to seek new targets for MM treatment and provide new theoretical basis for clinical treatment.Methods1.Suitable datasets GSE80140 and GSE80545 were selected after searching the Biotechnology Information-Gene Expression Omnibus Database(NCBI-GEO)according to keywords,"hypoxia" and " multiple myeloma ".Glycolysis-related gene PFKFB4 was selected as the potential key gene related to hypoxic adaptation of MM.The relationship between PFKFB4 and MM prognosis was further analyzed by online survival analysis.2.Considering the results of bioinformatics analysis,two MM cell lines(RPMI-8226 and U266)were separately cultured under normoxic and hypoxic conditions for different time,and the state of MM cells was observed.The transcription level of PFKFB4 mRNA was detected by quantitative reverse transcription-PCR(qRT-PCR)to determine the optimal time for hypoxic culture.Then,transcriptome sequencing was performed,and the mRNA transcription and protein expression levels of PFKFB4 were detected by qRT-PCR and Western blot,respectively.3.CCK8,flow cytometry(FCM),and Western blot were used to detect the proliferation,apoptosis,and cycle arrest of MM cells after inhibiting PFKFB4 under hypoxia.In addition,human F-2,6-BP assay kit was used to detect the effects of PFKFB4 on the glycolysis level of MM cells.4.CHIP kit and qRT-PCR were used to detect the ability of the transcription factor hypoxia inducible factor-1 alpha(HIF-1α)to bind to the promoter region of PFKFB4 to promote its transcription under normoxic and hypoxic conditions.5.MM cells were separately treated with specific PFKFB4 inhibitor 5MPN,PI3 K inhibitor LY294002,and HIF-1α inhibitor YC-1.Thereafter,Western blot was used to detect the phosphorylation level of PI3K/Akt signaling pathway molecules and the change of PFKFB4 protein expression level.6.NOD-SCID mice were subcutaneously injected with U266 cells to establish a subcutaneous tumor model.After tumor formation in NOD-SCID mice,they were divided into four groups randomly,including saline control group,dexamethasone(Dex)group,5-(n-(8-methoxy-4-quinolyl)amino)pentyl nitrate(5MPN)group,and Dex + 5MPN group.After administration of experimental Dex or/and 5MPN for 10 days,the mice were sacrificed and the tumor was removed from each mouse to measure the tumor volume and weight.Simultaneously,immunofluorescence was used to detect the changes of tumor cell proliferation,cycle,apoptosis and signaling pathway-related proteins to observe the inhibitory effect of PFKFB4 in vivo.7.Bone marrow specimens of primary MM patients were collected and grouped to analyze the correlation between baseline data and PFKFB4 expression levels in MM patients according to the ΔCt value of PFKFB4 derived from CD138+ primary cells by qRT-PCR.Meanwhile,survival analysis was also performed to observe the effect of PFKFB4 expression levels on the survival of MM patients.Results1.Bioinformatics analysis showed that the PFKFB4 expression level increased significantly under hypoxia in both MM cell lines and primary cells.In addition,online survival analysis suggested that high expression level of PFKFB4 was associated with poor prognosis of MM patients.2.With the prolongation of hypoxia time,the expression level of PFKFB4 increased in MM cells.Of note,the expression level of PFKFB4 in U266 cells was slightly lower in the 48-hour group than that in the 36-hour group.Interestingly,the expression levels of PFKFB4 in all hypoxic groups were significantly higher than those in normoxic control groups(p < 0.05).3.MM cells transcriptome sequencing results indicated that the transcription level of glycolysis-related gene PFKFB4 mRNA in the hypoxic group was remarkably higher than that in the normoxic group.Meanwhile,qRT-PCR and Western blot assay further proved that hypoxia could induce the upregulation of PFKFB4 mRNA transcription and protein expression levels.4.CCK8 assay showed that inhibition of PFKFB4 could suppress the viability of MM cells and reduce the proliferation rate.FCM analysis results indicated that compared with control group,inhibition of PFKFB4 could induce MM cell cycle arrest at G0/G1 phase(p < 0.05),but could not induce significantly apoptosis in MM cells(p > 0.05).Western blot assay confirmed a decreased expression of cell cycle-related protein Cyclin D1 and an increased expression of P21,but there were no significant changes in the expression of Bax,Cleaved Caspase-3 and Bcl-2 proteins(p > 0.05).And the experimental results of human 2,6-diphosphate fructose detection suggested that inhibition of PFKFB4 could significantly reduce the level of cellular glycolysis.5.CHIP-qPCR results showed that HIF-1α could promote the transcription of PFKFB4 mRNA via binding to the promoter region of PFKFB4 under both normoxia and hypoxia.The ability of HIF-1α to bind to the CR3 fragment in the promoter region of PFKFB4 increased significantly under hypoxia(p < 0.05).6.Under hypoxic condition,inhibition of PFKFB4 reduced the phosphorylation level of PI3K/Akt signaling pathway and the PI3 K inhibitor LY294002 had no significant effect on the expression level of PFKFB4.All these results suggested that PFKFB4 affected the activation of PI3K/Akt signaling pathway.After incubation of HIF-1α inhibitor YC-1 with MM cells under hypoxia,the expression levels of HIF-1α and PFKFB4 protein decreased compared with control group(all p < 0.05),indicating that HIF-1α could promote the transcription level of PFKFB4 to a certain extent under hypoxia.But the expression level of PFKFB4 in MM cells did not completely depend on the transcription of HIF-1α under hypoxia.7.The in vivo experimental study showed that subcutaneous tumor weight and volume were reduced to some extent,but the differences were not statistically significant between the 5MPN group and the control group(p > 0.05).Notably,the additional Dex significantly improved the anti-tumor effect of 5MPN(p < 0.05).In addition,the immunofluorescence results indicated that compared with the control group,the expressions of Ki67,PCNA and Cyclin D1 decreased significantly,whereas the expression of P21 increased in the 5MPN group.At the same time,the phosphorylation levels of PI3 K and Akt remarkably decreased.8.A higher expression level of PFKFB4 was associated with higher R-ISS staging,β2-MG levels and cytogenetic abnormalities in primary cells derived from clinical MM patients(p < 0.05).Clinically,survival analysis results showed a higher expression level of PFKFB4 with shorter OS in MM patients.Conclusions1.Expression level of PFKFB4 increases in MM cell lines under hypoxia.2.Under hypoxia,inhibition of PFKFB4 results in a decreased cell viability,cell cycle arrest at G0/G1 phase,and a decreased glycolysis level in MM.3.Under both normoxic and hypoxic conditions,the transcription factor HIF-1α promotes its transcription via binding to the promoter region of PFKFB4 and the transcription level is further enhanced by the hypoxia.4.Under hypoxia,PFKFB4 promotes the cell proliferation and cell cycle arrest by activating the PI3K/Akt signaling pathway in MM cells.5.Inhibition of PFKFB4 reduces tumor tissue cell cycle progression in mice bearing MM,and the combination of 5MPN with Dex exhibits a more inhibitory effect on tumor-bearing mice.6.High expression of PFKFB4 is associated with poor prognosis of MM patients.