PFKFB3 Promotes Vessel Abnormalization Induced HER2 Positive Gastric Cancer Trastuzumab Resistance By Targeting Smad3 S425 Phosphorylation Mediated CXCL8 Extracellular Secretion

Background and purpose:Gastric cancer is the third leading cause of cancer death worldwide.Human epidermal growth factor receptor 2(HER2)is one of the best-characterized targetable alterations in solid tumors.Among the various genomic events,abnormal expression of HER2,a prognostic factor for patients,is involved in as many as 7-34%of gastric cancers.Trastuzumab,the only target agent approved by FDA as a first line treatment of metastatic gastric adenocarcinoma,substantially improves the outcome for patients with HER2 positive gastric cancer.However,acquired resistance to the trastuzumab based treatment within 1-year limit the duration of the response to trastuzumab.Thus,a characterization of the resistance mechanism of trastuzumab is urgently needed to offer an alternative option for patients who would suffer from inevitable resistance.Methods:To explore whether HER2 positive GC glycolysis play a role in response to trastuzumab treatment,we first analyzed the correlation between FDG level of GC patients with trastuzumab treatment response.We divided patient that received trastuzumab treatment into sensitive and resistant group by clinical fellow up data.Then IHC-P staining and TUNEL assay were performed.To investigate the mechanism of trastuzumab resistance by glycolysis activation,we generated HER2 positive gastric cancer cell line(NCI-N87)models with continuously stimulation of trastuzumab over 8 months.To quantify the relative physiological glucose uptake,we performed 2-NBDG uptake assays culturing the cells under normoxia(20%,oxygen)or hypoxia(1%oxygen)for 24hours.Then we applied colorimetry to detect the substrate and the products of glycolysis in cells,including residual glucose and lactate production.RT-qPCR and Western blot assay were used to study mRNA and protein expression of glycolysis enzymes.In comparison with the change of anaerobic metabolism,extracellular acidification rate(ECAR)and oxygen consumption rate(OCR).Besides,we cultured cells under low glucose conditions(0.5mM glucose)for 24hours and apply MTT assays to explore the sensibility to Trastuzumab.Then subcutaneously implanted tumor model in nude mouse were performed 18F-fluorodeoxyglucose positron emission tomography(PET)imaging and IHC-P staining.The relationship between the expression of PFKFB3 with gastric cancer patients'disease-free survival(DFS)and overall survival(OS)were evaluated by KM plotter and our center's data.RT-PCR and Western blot were applied to revealed use of PFKFB3 in regulating glycolysis enzymes in mRNA and protein level.To further evaluated the safety of siPFKFB3,wound healing,CCK8 and colony formation assays.We designed the PFKFB3 adeno-associated virus(PFKFB3-AAV)and treated nude mice model,then 18F-fluorodeoxyglucose positron emission tomography(PET)imaging and IHC-P staining were applied.In vitro,we carried on tube formation assays by using HUVECs cells and found that resistant cells co-cultured group.RT-PCR was used to analyze the angiogenic genes changes of HUVECs cells by co-cultured with cancer cells.Clinical patient's tissue and subcutaneously implanted tumor model in nude mouse were used to evaluate the vessel quality by immunofluorescenceChemokine assay was used to screening CXCL8 expressed higher in resistant cell superannuate compared to sensitive cells but reduced while knocking down PFKFB3 in resistant cells.KEGG pathway enrichment,RT-PCR and Western blot were used to confirmed cytokine and chemokine signal level.In vitro,PFKFB3 deficiency by siRNA interference could lead to the diminish of CXCL8 mRNA.Confocal microscopy and immunoprecipitation analysis were used to confirm location and co-localization.Analysis TCGA database to find the enrichment of SMAD3 signal pathway with the overexpression of PFKBF3.Q-PCR,Western blot and Immunoprecipitation analysis were used to explore interaction between PFKFB3 and SMAD3.JASPAR database,ChIP and COIP assays were used to explore interaction between CXCL8 and SMAD3.Regain assay was used to confirm PFKFB3 promotes trastuzumab resistance by targeting Smad3 S425 phosphorylation mediated CXCL8 extracellular secretionResults:Part1.Increased glucose metabolism fuels trastuzumab resistance in HER2 positive gastric cancer trastuzumab resistant cellsAfter analyzing the correlation between FDG level of GC patients with trastuzumab treatment response,we found the higher FDG results in poorer trastuzumab response.Besides,higher FDG was positively related with poorer DFS and OS of GC patients.IHC-P staining showed that resistant group expressed stronger the glycolysis enzymes,such as PGK1,HK2,ENO1,LDHA and SLC16A1(Fig1C).We then detected the apoptosis difference between two groups and revealed that resistant groups showed lower positive staining rate by TUNEL assay.We observed an increase in glucose consumption and lactate release ability of the NCI-N87TR cells by detecting the supernatant compared with NCI-N87-WT cells.To quantify the relative physiological glucose uptake by resistant cells,we performed 2-NBDG uptake assays culturing the cells under normoxia(20%,oxygen)or hypoxia(1%oxygen)for 24hours.RT-PCR and Western blot confirmed the higher mRNA and protein expression of glycolysis enzymes after resistance.Further analysis revealed an increase in the intermediate metabolites of glycolysis.Specifically,we observed an increase in metabolite upstream of dihydroxyacetone phosphate and glyceradehyde-3-phosphate and increased intracellular pyruvate/lactate levels in resistance cells.In comparison with WT cells,NCI-N87TR demonstrated increased extracellular acidification rate(ECAR)and decreased oxygen consumption rate(OCR).Besides,resistant cells failed to respond to trastuzumab treatment but demonstrated significantly diminished survival under low glucose condition.In vivo,we observed bigger tumor size of resistant groups than sensitive groups.Besides,resistant group tumor showed higher glucose uptake than sensitive groups,by performing 18F-fluorodeoxyglucose positron emission tomography(PET)imaging.IHC-P staining confirmed glycolysis enzymes in resistant groups overexpressed,compared with sensitive groupsPart2.PFKFB3 is the metabolic master regulator of enhanced glucose metabolism and pyrimidine biosynthesis in trastuzumab resistant cellsPFKFB3 is a key enzyme of glycolysis that was overexpressed in NCI-N87TR when compared with WT cells by RNA-Seq.Western blot showed that protein PFKFB3 expressed higher in resistant cells than WT cells.Patient tissues,subcutaneous of nude mice confirmed that PFKFB3 expressed higher in resistant groups than sensitive groups.KM plotter and our center's data indicated that higher expression of PFKFB3 was positively correlated with worse DFS and OS.RT-PCR and Western blot revealed that knocking down of PFKFB3 could significantly decrease the expression of glycolysis enzymes both in mRNA and protein level PFKFB3 knocking down resulted in the decreased ability of glucose consumption,lactate release and 2-NBDG uptake.Further,in comparison with resistant cells,NCI-N87TR-siPFKFB3 demonstrated decreased extracellular acidification rate(ECAR)and increased oxygen consumption rate(OCR).We cultured the siPFKFB3 cells and corresponding resistant cells under low glucose conditions(0.5mM glucose)for 24hours.Resistant cells recover the response to trastuzumab treatment but demonstrated significantly increased survival under low glucose condition.To further evaluated the safety of siPFKFB3,wound healing,CCK8 and colony formation assays confirmed that knock down PFKFB3 could inhibit the metastasis and proliferation of resistant cells simultaneously.Then,we designed the PFKFB3 adeno-associated virus(PFKFB3-AAV)and found that PFKFB3-AAV plus trastuzumab could significantly diminished the tumor size and tumor weight when compared with trastuzumab or PFKFB3-AAV single treatment group.Part3.PFKFB3 deficiency could enhance the sensitivity to trastuzumab treatment by improve the normalization of tumor vesselePFKFB3 is a key enzyme of glycolysis that was overexpressed in NCI-N87TR when compared with WT cells by RNA-Seq.Western blot showed that protein PFKFB3 expressed higher in resistant cells than WT cells.Patient tissues,subcutaneous of nude mice confirmed that PFKFB3 expressed higher in resistant groups than sensitive groups.KM plotter and our center's data indicated that higher expression of PFKFB3 was positively correlated with worse DFS and OS.RT-PCR and Western blot revealed that knocking down of PFKFB3 could significantly decrease the expression of glycolysis enzymes both in mRNA and protein level.PFKFB3 knocking down resulted in the decreased ability of glucose consumption,lactate release and 2-NBDG uptake.Further,in comparison with resistant cells,NCI-N87TR-siPFKFB3 demonstrated decreased ECAR and inc.We cultured the siPFKFB3 cells and corresponding resistant cells under low glucose conditions(0.5mM glucose)for 24hours.Resistant cells recover the response to trastuzumab treatment but demonstrated significantly increased survival under low glucose condition.To further evaluated the safety of siPFKFB3,wound healing,CCK8 and colony formation assays confirmed that knock down PFKFB3 could inhibit the metastasis and proliferation of resistant cells simultaneously.Then,we designed the PFKFB3 adeno-associated virus(PFKFB3-AAV)and found that PFKFB3-AAV plus trastuzumab could significantly diminished the tumor size and tumor weight when compared with trastuzumab or PFKFB3-AAV single treatment groupPart4.PFKFB3 deficiency could enhance the sensitivity to trastuzumab treatment by improve the normalization of tumor vesseleScanning electron microscopy revealed that tumor ECs in resistant groups exhibited signs of a hyperactive,nonquiescent endothelium,with few signs of contact inhibition.Immunofluorescence of clinical patient's tissue revealed that VE-Cadherin positive vessel,Collagen type ? marked BM coverage,a-SMA and PDGFRP marked pericyte coverage reduced significantly in resistant groups,compared to sensitive groups.We therefore analyzed whether an abnormalization might explain tumor trastuzumab resistant in nude mice.Upon injection of Redconjugated dextran and fluorescein isothiocyanate(FITC)-Conjugated lectin,extravasation of dextran was induced in resistant tumors.Those results remind us that glycolysis mediated HER2 positive gastric cancer might promote trastuzumab resistance by reducing tumor vessel normalization.In vitro,we carried on tube formation assays by using HUVECs cells and found that resistant cells co-cultured group.RT-PCR was used to analyze the angiogenic genes changes of HUVECs cells by co-cultured with NCI-N87TR transfected with NC and siPFKFB3.Scanning electron microscopy revealed that tumor ECs in PFKFB3 deficiency groups exhibited signs of a quiescent endothelium,without signs of contact inhibition.ECs were tightly connected from each other,intercellular gaps diminished.In vivo,PFKFB3 deficiency could significantly enrich the VE-Cadherin positive,BM coverage and Pericyte coverage.Besides,PFKFB3 deficiency reduced the leakage of resistant tumors vessels obviously.Part5.PFKFB3 promotes the secretion of CXCL8 by activating SMAD3 signal pathwayChemokine assay found that CXCL8 expressed higher in resistant cell superannuate compared to sensitive cells,but reduced while knocking down PFKFB3 in resistant cells.Western blot evaluated that CXCL8 expressed higher in resistant cells than WT cells.KEGG pathway enrichment indicated that PFKFB3 overexpression was correlated with cytokine receptor and chemokine signal pathway.RT-PCR confirmed that cytokine and chemokine signal mRNA level expressed higher than WT groups.In vitro,PFKFB3 deficiency by siRNA interference could lead to the diminish of CXCL8 mRNA.Confocal microscopy showed that PFKFB3 and CXCL8 expressed higher in resistant cells NCI-N87TR compared to WT cells,besides,two proteins have co-localization in cytosol and cytoplasmic membrane.Additionally,immunoprecipitation analysis of cells showed mutual interaction between PFKFB3 and CXCL8.Immunofluorescent indicated that PFKFB3 and CXCL8 expressed higher in resistant groups than sensitive groups and co-localized in cytosol and cytoplasmic membrane while analyzed clinical patients' tissues.Those data point that CXCL8,interacted with PFKFB3,might play an important role in downstream of PFKFB3 that induced vessel abnormalization.TCGA database analysis found the enrichment of SMAD3 signal pathway with the overexpression of PFKBF3.PFKFB3 mRNA expression was positively correlated with the expression of SMAD3.Western blot confirmed that PFKFB3 knock down could inhibit the activation of SMAD3.Immunoprecipitation analysis showed PFKFB3 and CXCL8 could directly interaction with SMAD3.JASPAR predicted that transcription factor STAT3 could binding at the promotor of CXCL8 and upregulated the expression of CXCL8.ChIP assay confirmed that mutant the binding site could lead to the escape regulation by STAT3.Regain assay found that,while overexpressed PFKFB3 by transfected with plasmid and using SMAD3 signal inhibitors simultaneously,CXCL8 expressed lower than the PFKFB3 overexpressed group.Conclusion:Our collective evidence indicates that PFKFB3-SMAD3 axis induced glycolysis reprogramming and CXCL8 secretion is a promising inducing factor that causes HER2 positive gastric cancer trastuzumab resistance by promoting tumor vessel abnormal.Trastuzumab plus PFKFB3 inhibitor was an adjuvant anti-tumor approach that can maximize the therapeutic efficacy of conventional trastuzumab single method The distinguishing characteristics of CXCL8 inhibitor that favorably alter the whole tumor microenvironment makes it an alternative that can be used to fill in the gaps of current anti-angiogenic therapies.Further work remains to be done to demonstrate its safety and efficacy in clinical use.