The Interaction Between CDK4 And PFKFB3 Is Crucial For The Palbociclib Sensitivity

Background and ObjectiveProtein interaction has a very important role in the biological behavior of tumor cells.PFKFB3,a key enzyme in the regulation of cell glycolysis,has become a target of tumor energy metabolism.CDK4 is a key protein in regulating cell cycle and proliferation.Palbociclib as selective CDK4 / 6 inhibitor can block the cell cycle from the G1 phase to S phase transition and inhibit the proliferation of a variety of breast cancer cell lines,in particular ER + breast cancer cells.In addition,Palbociclib in combination with letrozole or Fulvestrant(estrogen antagonist)treating breast cancer is significantly better than letrozole or Fulvestrant monotherapy and significantly improve the prognosis of patients with breast cancer.Therefore,Palbociclib in 2015 was approved by the FDA.Many researches report the possible mechanisms of influencing Palbociclib sensitivity,such as the lose of p16,the function of pRb and the expression level of CDK4.The study through a series of vitro experiments finds that CDK4 interacts with PFKFB3 and further explore the underlying molecular mechanism of the Palbociclib sensitivity.MethodAfter constructing the HA-PFKFB3 plasmid and using the HA-beads to gather PFKFB3 protein,we use the mass spectrometry method to screen possible proteins interacting with PFKFB3;By co-immunoprecipitation and GST-Pulldown assays,we further verify that whether the CDK4 protein and PFKFB3 protein exits the direct interaction;According to the domain of PFKFB3,PFKFB3 was segmented to researchthe PFKFB3 binding fragment with CDK4.In addition,according to the PFKFB3 bioinformatics analysis,we comparise the man,mouse,zebrafish,xenopus and leaves kiss chimaeras cognate amino acids and build 11 mutants of PFKFB3 to determine CDK4 and PFKFB3 binding site.After co-transfecting with HA-PFKFB3 and FLAG-CDK4 plasmid to Hela cells,immunofluorescence and confocal microscopy observe localization between PFKFB3 and CDK4.The specific small interfering RNA targeting CDK4 gene and negative control were transfected into the A549 cell.The efficiency of CDK4 gene silencing in mRNA and protein levels was detected by real-time fluorescent quantitative PCR and Western blot,respectively.The expression level of PFKFB3 was detected by Western blot.The changes in glucose metabolism and oxygen consumption were detected by 18 F-FDG uptake assay,lactate detection assay and Seahorse XF24 analyzer assay.The changes in proliferation,colony formation and cell cycle were observed by cell count,colony formation assay and flow cytometry,respectively.By the immunohistochemical assay,we detect PFKFB3 protein and CDK4 protein expression levels in breast cancer,and analyze the correlation of PFKFB3 and CDK4 expression levels in breast cancer.We explicit the chang of PFKFB3 protein impact on the CDK4 protein stability by ubiquitin proteasome and cycloheximide assays.We build a MCF-7 cell line with stable knockdown of PFKFB3 gene and detect the PFKFB3 gene affect the Palbociclib sensitivity by CD-DST and CCK-8assays.ResultPFKFB3 mass spectrometry results analyze that CDK4 and PFKFB3 may exist interactions.CO-IP and GST-Pulldown technology further verify that PFKFB3 directly interacts with CDK4 and K147 site is a key binding site between PFKFB3 and CDK4.Immunofluorescence confocal test detects that PFKFB3 and CDK4co-localized in Hela cell nucleus.After the siRNA-CDK4 was successfully transfected into A549 cells,the expression levels of CDK4 mRNA and protein were significantly lower than that ofthe negative control(P < 0.01).Compared with the negative control(siRNA-NC)group,the group transfected with siRNA-CDK4 showed that the expression level of PFKFB3 was declined.The 18F-FDG uptake was decreased(42.21±1.90)%(P <0.05),and lactate production was reduced(29.39±5.35)%(P < 0.05),but basic oxygen consumption was increased(67.17±3.58)%(P < 0.01).The cell proliferation in 48,72 and 96 h was decreased(P < 0.05),the cell cycle was arrested in G1 stage and the percentage of S stage was decreased(both P < 0.05).Immunohistochemistry detects that PFKFB3 protein and CDK4 protein showed high expression in breast cancer tissue(P < 0.001),and showed a positive correlation.After overexpression HA-PFKFB3 in MCF-7 cell,we found that the expression level of CDK4 protein increase;After small interfering RNA technology knockdown PFKFB3,we found that CDK4 protein expression levels decreased,but CDK4 mRNA levels did not change.In MCF-7 cells or 293 T cells,We found that after knockdown PFKFB3,ubiquitination level of CDK4 increased significantly and CDK4 protein expression levels decreased,while added the proteasome inhibitor(MG132),CDK4 protein levels did not decline.MCF-7 cells were transfected with HA-PFKFB3 and HA-Vector,added cycloheximide(CHX),after 0 h,1 h and 3 h,we found overexpression PFKFB3 group,the degradation rate of CDK4 protein was significantly lower than the control group.CD-DST assay results show that1 uM 5uM 10 uM 15 uM 20 uM Palbociclib(CDK4 inhibitor)were added to MCF-7 cells,with the increase of drug concentration,MCF-7 cell survival rates were 81.56%,51%,27.83%,2.38% and 0.69%,respectively.The same concentration gradient Palbociclib were treated stable knockdown PFKFB3 of MCF-7 cells and control group,stable knockdown PFKFB3 of MCF-7 cells decreased sensitivity for the Palbociclib.ConclusionWe show that PFKFB3 can interact directly with CDK4,promote the stability of CDK4 in protein level,suppress CDK4 degradation in an ubiquitination-proteasome dependent manner and increase the Palbociclib sensitivity of MCF-7 cells.