The Mechanism Underlying PKC-ε And Related Signaling Molecules Contribute To Warburg Effect And Invasion In Prostate Cancer Cells

Part1.The mechanism of PKC epsilon-Smad3 axis regulates theWarburg effect and tumor cell growth in prostate cancer cells1.IntroudctionMetabolic alteration and energy requirement is one of hallmark of the tumor cells,which are closely associated with tumor development and progession.Tumor cells,whether in both aerobic and anaerobic conditions,are required to take up glucose through Glycolysis,to meet the energy production and tumor growth,which was well known as Warburg effect[1,2],Current research showed that the metabolic change of tumor cells are not only limited to switch of glycolysis and three carboxylic acid(TCA)cycle,but also many metabolism pathways in tumor cell are occurred metabolic reprogramming,and what’s more,related metabolic disease such as obesity,and diabetes are also closely associated with the tumor development and progression[3].Therefore,further studies of the Warburg effect mechanism and its association with tumor development not only contribute to reveal the intrinsic relationship of tumor metabolism and tumor progression,but also provide new insight into specific tumor metabolic markers for clinical diagnosis and strategy for tumor-targeted therapy based on tumor metabolism.PKC belongs to a family of serine/threonine kinase activated by tyrosine kinase receptor(RTK)and G protein coupled receptor(GPCR),including three subfamily:typical PKC of Ca2+and DAG-dependent(PKC-α,-β,-γ),DAG-dependent and Ca2+non-dependent PKC(PKC-PKC(PKC-KC(PKCn-θ),DAG and Ca2+ non-dependent atypical PKC(PKC-ξon-de).Current studies showed that overexpression of PKC-ur the most carcinogenic activity of oncogenes,was found in numerous tumors including prostate cancer,breast cancer and lung cancer among various isoforms of PKC family,which was an important poorly prognostic marker for tumor recurrence and short survival in patients[20,21].In recent years,functional role of PKC-ε in prostate cancer development and progression have received considerable attentions:(a)overexpression of PKC-c contributed to androgen-independent growth of androgen-dependent LNCaP prostate cancer cells and tumor growth in nude mice with castration treatment;(b)overexpression of PKC-ε had resistance to TNF-alpha induced apoptosis of LNCaP cells;(c)knockout of PKC-εhin transgenic TRAMP mouse mode of prostate cancer inhibited prostate cancer tumoregenesis and metastasis,on the other hand,mice of prostate specific PKC-ε expression developed prostate hyperplasia as well as prostate intraepithelial neoplasia(PIN)that displayed enhanced phospho-Akt and phospho-Stat3 levels,as well as enhanced resistance to apoptotic stimuli.Although these studies suggested that PKC-ε are closely related to the development and progression of prostate cancer,the key signaling pathways and mechanisms contribution to tumor development and progression are still unclear.Phosphorylation activation of linker region of Smad3 is the key to promote tumor growth and progression.Smad3,as a critical molecule mediated by TGF-βsignaling pathway,consists of a conserved N-terminal MH1 domain(binding to DNA),C-terminal MH2 domain(interaction with TGF-β1 receptor)and central linker region.Multiline of evidence showed that Smad2/3 mediated by TGF-βsignaling pathway inhibit early tumor growth,while,it promotes the tumor growth and metastasis through interaction with Ras pathway-related kinases in late stage of human cancers.Current data found that the C-terminal phosphorylation of S423/425 sites(pSmad2/3C(S423/425)is a inhibitory signal of tumor growth mediated by TGF-GF-β signaling pathway,consists of a conservedsites(Thr179,Ser204,Ser208,Ser213)of linker region can be activated by multiple kinase such as MAPK,JNK,CDK4,CamKII),especially,phosphorylation of serine 213 site(pSmad3L(Ser213))is critical for tumor growth and progression,clinical data also showed that phosphorylation of serine 213 site is an important poorly prognostic marker for the recurrence and short survival of liver and gastric cancers.Interestingly,androgen-independent cells expressed much higher levels of Smad3 than did androgen-dependent prostate cancer cells.Moreover,Smad3 was overexpressed in human prostate cancer,which correlated with Gleason score and expression of proliferating cell nuclear antigen.Overexpression of dominant-negative mutant of Smad3(D)in prostate cancer cells significantly reduced tumor growth in nude mice.These results suggest the Smad3 is correlated with prostate cancer growth and progression,however,which site of linker region was activated in prostate cancer?What did clinical relevance with prostate cancer progression exsit?Whether is PKC-ε involved in linker region phosphorylation of Smad3 and its functional implications?All of the questions are still not clear.2.Objectives(1)Determine whether PKC-ε promotes Warburg effect of prostate cancer cells,lactate secretion and tumor growth through activation of pSmad3L in the linker region of Smad3;(2)Explore the mechanism underlying PKC-ε-Smad3 signaling axis contributions to Warburg effect,lactate secretion and tumor growth in prostate cancer cells.These data will provide not only new insights into the intrinsic correlation of tumor glucose metaoblic switch with the tumor growth and progression in prostate cancers,but also clinical diagnosis and strategy for tumor-targeted therapy based on tumor metabolism.3.MethodsThis research mainly explore the mechanism underlying PKC-ich further contribute tumor Warburg effect as well as tumor progression.h and progressioexpression and cell growth in prostate cancer cells through different strategies such as inhibitor,the overexpression of plasmids,small interference(siRNA),Immunofluorescence staining and immunoprecipitation,ChIP assay.4.Results1.Knockdown of Protein Kinase C epsilon(PKC-εn reduced aerobic glycolysis and inhibited prostate cancer cells growthThe biochemical analysis of susperantant from prostate cancer showed that activity inhibition of PKC treated by PKC inhibitor Go6983 or knockdown of PKC-s hcould significantly reduce the glucose uptake and lactic acid secretion(5.69±0.37 vs 3.91 ±0.45,P<0.050),meanwhile,depletion of PKC-ε reduced glucose uptake(0.96±0.04 vs 0.65±0.03,t=9.11,P<0.001)and lactate production(0.88±0.10 vs 0.48±0.05,t=6.17,P<0.001);Western blotting also indicated that knockdown of PKC-ernlactic acid secretionat activity inhibition of PKC treated by PKC inhibitor Go6983 ond progresrostate cancer cells,and inhibited proliferation of prostate cancer cells.2.Aerobic glycolysis in prostate cancer cells upregulated by PKC-ε was independent of PKDsWhether PKD inhibitor Go 6976 treatment or Silencing of PKC-ytixpression in prostate cancer cells could not decrease the glucose uptake and lactic acid secretion(5.69±0.37 vs 5.18±0.38,P>0.050)as well as related protein expression of glycolysis.These data suggested that PKC-ε may contribute to the glucose uptake and lactic acid secretion in PKDs-independent manner.3.PKC-ε activated phosphorylation of linker region in Smad3 and colocalized in nucleaus,endogenous PKC-ε interacted with Smad2/3 in prostate cancer cells.Silencing of PKC-n by siRNA significantly decreased pSmad3L(Ser213)phosphorylation of linker region in Smad3,moreover,immunofluoresence staining showed that endogenous PKC-ε was colocalized with Smad2/3 in nucleus of prostate cancer cells.In addition,endogenous endogenous PKC-ε interacted with Smad2/3 both in DU 145 and PC-3M cells.4.Smad2/3 promoted to the related genes expression and prostate cancer cells growth in TGF-β1-independent way.Overexpressions of Smad3 in prostate cancer cells DU145 and PC-3M increased the glucose uptake and lactate secrection,whereas knockdown of Smad3 by siRNA transfection decreased the glucose uptake(3.23±0.25 vs 2.60±0.20,t=3.41,P=0.030)and lactate secrection(7.59±0.37 vs 5.51 ±0.52,t=-5.66,P=0.005).Meanwhile,silencing of Smad2 or Smad3 could reduce glycolytic related genes expression(HKII,PFKP,MCT4 and HIF-1α)as well as prostate cancer cells growth.In addition,whether TGF-or TGF-p receptor inhibitor SB431542 treatment could not reduce glucose uptake and lactate secrection in prostate cancer cells(p>0.050).5.Overexpression of Smad3 antagonized the inhibition of aerobic glycolysis and tumor growth triggered by silencing of PKC-ε in prostate cancer cells.Knockdown of PKC-ε remarably inhibited glycolysis and cell growth in DU145,on the contrary,overexpression of Smad3 promoted aerobic glycolysis and tumor growth.Cotransfection with si-PKC-ε and Smad3 plasmid further showed that overexpression of Smad3 antagonized the inhibition of aerobic glycolysis and tumor growth induced by silencing of PKC-ε in prostate cancer cells6.PKC-ε contributed to the binding of Smad2/3 to glycolytic related genes promoter.The Chromatin immunoprecipitation showed that endogenous PKC-s silencing by siRNA obviously decreased binding of Smad2/3 to the promoter of glycolytic related genes PFKP(0.13±0.00 vs 0.07±0.02,t=6.42,P=0.003),MCT4(0.62±0.09 vs 0.23±0.03,t=7.02,P=0.002),HKII(0.46±0.08 vs 0.11 ±0.00,t=7.74,P=0.018).5.Conclusion1.Knockdown of Protein Kinase C epsilon(PKC-ε)reduced aerobic glycolysis and inhibited prostate cancer cells growth2.Expression and activation of PKC-s promoted Aerobic glycolysis and glycolytic related proteins expression in prostate cancer cells in PKDs-independent way.3.PKC-ε regulated phosphorylation of linker region in Smad3 and endogenous PKC-ε interacted with Smad2/3 in prostate cancer cells.4.Overexpression of Smad3 antagonized the inhibition of aerobic glycolysis and tumor growth triggered by silencing of PKC-s in prostate cancer cells.5.PKC-ε contributed to the binding of Smad2/3 to glycolytic related genes promoter.Part2.PKD2 and PKD3 promote prostate cancer cell invasion via uPA modulated by NF-κB1.IntroductionProstate cancer is the most common malignancy and the second mortality of aged men in the United States.In China,with the expectation increasing of lifespan and improvement of the living standards,the morbidity of prostate cancer increased significantly.Although surgical operation or castration was still the preferred treatment of advanced prostate cancer,patients who were almost sensitive to the initial endocrine treatment eventually gained hormone resistance and distal tissue metastasis,as well as clinical irreversible deterioration,and caused the death of the patient.Therefore,it was great of importance to investigate the mechanism of prostate cancer metastasis and the identify the key signaling pathways for the development and prognosis of the prostate cancer treatment.Multiline of evidences showed that the prostate cancer progressed from prostate intraepithelial neoplasia and localized adenocarcinoma to hormone resistant metastatic prostate cancer.Prostate cancer,similar to other tumor metastasis,was a complex process including " dissemination-movement-adhesion " three steps.Although some proteins such as matrix metalloproteinase 2,9(MMP2,9),IKKa,Maspin associated with tumor invasion and metastasis have been identified in prostate cancers,the target molecules of clinical diagnosis and treatment are still poorly understood.Protein kinase D(PKD)belongs to a family of serine/threonine protein kinases that is now classified as a subfamily of the Ca2+/calmodulin-dependent kinase(CaMK)superfamily.Three members of the PKD family have been identified to date,namely,PKD1(PKCμ),PKD2,and PKD3(PKCvD,PKD has been implicated in various biological processes,such as cell proliferation,survival,cell migration,differentiation,inflammation,and vesicle transportation.The role of PKDs in cancer invasion/metastasis has been increasingly recognized,and,of the three PKD isoforms,PKD1 has been the focus of most studies in this context.In breast cancer cells,the expression of PKD 1 has been shown to be inversely correlated to invasive potential,impeding this process through downregulating the expression of matrix metalloproteinase(MMP-2,MMP-7,MMP-9,MMP-10,MMP-11,MMP-13,MMP-14).Furthermore,overexpression of PKD 1 increased aggregation,but reduced motility and invasiveness of PCa cell lines through interaction with and phosphorylation of E-cadherin.In our previous study,PKD3 levels were substantially increased in human prostate tumors compared with normal tissues,as revealed by immunohistochemistry.Moreover,PKD3 exhibited a marked increase in nuclear localization in tumor tissues,correlating with tumor grade.Increased protein expression and nuclear accumulation of PKD3 were observed in the more invasive and metastatic PC-3 and DU 145 cell lines compared to the less aggressive LNCaP cell line,and this was shown to contribute to the growth and survival of prostate cancer cells.However,the exact role of PKD2 and PKD3 in tumor cell invasion has not been characterized.2.ObjectsThe primary goal of this study was to investigate the function and regulatory mechanisms of PKD2-and PKD3-mediated pathways in prostate cancer cell invasion.The study will provide the new insights into the mechanism of prostate cancer invasion and metastasis.3.MethodsThis research mainly explore the mechanism underlying PKD2 and PKD3 controbute to uPA activity and expression in prostate cancer cells through different strategies such as transfection with siRNA targeted PKD2 and PDK3,the overexpression of plasmids,Real-time RT-QPCR,gelatin zymography,Luciferase assay,Immunofluorescence staining and immunoprecipitation,ChIP assay.4.Results1.Knockdown of PKD2 and PKD3 inhibited migration/invasion of prostate cancer PC-3M and DU 145 cellsCell migration and Boden Chamber invasion assay showed that depletion of PKD2 or PKD3 significantly decreased cell migration,although depletion of PKD3 resulted in a greater effect than the depletion of PKD2.Moreover,depletion of both PKD2 and PKD3 resulted in greater inhibitory effects.2.PKD2 and PKD3 regulated the expression of invasion/metastasis-related genes of uPA/uPAR and MMPs,contributed to uPA and MMP-9 gelatinase activityReal time RT-PCR showed that depletion of PKD2,PKD3 or combined silencing of PKD2 and PKD3 led to significant down-regulation of Activin-A,MT-MMP1,uPA,uPAR,and up-regulation of PAI-2 at mRNA level compared to siRNA control in PC-3M cells,yet.no significant difference of osteopontin and VEGF was observed.Similarly,protein levels of uPA,uPAR and MT1-MMP in PC-3M and DU 145 cells were significantly downregulated by silencing of PKD2 or PKD3.In addition,knockdown of either PKD2 or PKD3 significantly reduced uPA activity as well as MMP-9 gelatinase activity induced by PMA treatment in in DU145 and PC-3M cells.3.PKD2 regulated p65 nuclear translocation,PKD2 and PKD3 contributed to the binding of p65 NF-κB to uPA promoter and subsequent uPA transactivationSubcellular fractionation and immunofluorescent staining indicated that PMA-induced p65 NF-xB nuclear translocation was substantially reduced by PKD2 depletion.Futhermore,PMA or TNF-a induced DNA binding activity of NF-κB to uPA specific promoter sequence(uPA-luc)declined dramatically in PC-3M cells with PKD2,PKD3 or combined depletion.Chromatin IP assay demonstrated that both baseline and PMA-triggered binding of p65 NF-κB to uPA promoter region was drastically decreased in response to PKD2 or PKD3 depletion(si-CTL vs si-PKD2:4.88±0.06 vs 1.68±0.02,P<0.001;si-CTL vs si-PKD3:4.88±0.06vs 1.80±0.06,P<0.001)与 uPA(si-CTL vs si-PKD2:8.04±0.09 vs 3.56±0.08,P<0.001;si-CTL vs si-PKD3:8.04±0.09 vs 2.14±0.11,P<0.001).4.PKD2 and PKD3 interacted with IKK β,contributing to different site phosporylation of p65Endogenous PKD2 and PKD3 co-precipitated with IKKβ in PC-3M cells,and was significantly enhanced by PMA stimulation.Moreover,IKK,I就B and p65 phosphorylation at Ser276 was reduced by PKD2 knockdown.Whereas PMA and TNF-a phosphorylate p65 phosphorylation at Ser536 was reduced mainly by PKD3 knockdown in PC-3M cells.5.Enforced expression of wild-type and constitutive active mutant(S536D)of p65 rescued uPA transactivity inhibiton caused by PKD3 depletion as well as tumor invasion downregulated by PKD2 or PKD3 depletion in DU145 cells.Overexpression of wild-type p65(p65WT)or constitutive active p65(S536D)recovered uPA transactivity downregulated by PKD3 depletion.In contrast,constitutive inactive p65(S536A)showed far less rescue effect on uPA transactivity.over-expression of wild-type p65(p65WT)or constitutive active p65(S536D)recovered uPA transactivity after PKD3 depletion(81.54±1.35 vs 38.43±0.45,P<0.001).In contrast,constitutive inactive p65(S536A)showed far less rescue effect on uPA transactivity.Meanwhile,overexpression of wild-type p65 rescued DU 145 cell invasive potential reduced by knockdown of PKD2 or PKD3.5.Conclusion1.PKD2 and PKD3 promote nuclear factor-kappaB(NF-showed far less rescue effect on uPA transactivity.Meanwhile,overexpression of wild-type p65 rescued DU 145 cell invasive ell invasion.2.PKD2 mainly regulated the phosphorylation of Ser276 on p65,while PKD3 was responsible for the phosphorylation of Ser536 on p65.3.Depletion of PKD2 and/or PKD3 decreased the binding of p65 NF-De to the uPA promoter,suppressing transcriptional activation of uPA.