| Background and objectiveNasopharyngeal carcinoma(NPC)is a malignant carcinoma that occurs in the epitheliallining of the nasopharynx and bilateral pharyngeal recess.Histopathologically,NPC is frequently a squamous cell carcinoma.In most parts of the world NPC is a rare disease with an incidence well under 1 per 100 000 population per year.But there is a high rate of incidence in Southern China,northern Africa and Alaska,especially in Guangdong Province of Southern China.Since the complication of anatomy and no typical early symptoms,it is difficult to make an early diagnosis of NPC.Most of the people are advanced nasopharyngeal carcinoma patients when diagnosed.At present,radiotherapy with concomitant chemotherapy has increased survival,and improved techniques such as intensity-modulated radiotherapy,early detection of recurrence,and application of appropriate surgical salvage procedures have contributed to improved prognosis.However,recurrence and distant metastasis remain the leading causes of treatment failure of nasopharyngeal carcinoma.A good understanding of pathogenesisis is necessary to find clinical biomarkers of early diagnosis and therapeutic targets of nasopharyngeal carcinoma.PTEN is an important tumor suppressor gene,the protein which encoded is a dual specificity phosphatase,including protein phosphatase activity and lipid phosphatase activity.Ddeletion or dysfunction of PTEN is closely related to the development of human malignancies.Previous studies have show the tumor suppressor function of PTEN mainly depends on its lipid phosphatase activity.PTEN can transfer PIP3 to PIP2 by dephosphorylating PIP3 at the D3 phosphate.Thereby reduced the level of PIP3 and inhibited the activation of PI3K/Akt signaling pathway.PTEN inhibits cell survival,induces programmed cell apoptosis and arrests cell cycle progression by regulating negatively PI3K/Akt signaling pathway.It also regulates cell adhesion migration and differentiation.As reported,the loss of PTEN plays an important role in maintenance and viability of stem-like tumor cell in prostatic cancer and glioma by mediating PI3K/Akt signaling pathway.Besides,the sensitivities of prostatic cancer cell and breast cancer cell to chemotherapeuticswere decreased after loss of PTEN.In recent years,numerous studies have showed that protein interaction is another important mechanism to regulatethe functions and activities of PTEN except its lipid phosphatase activity.Such as,protein phosphatase activity of PTEN also has an important tumor suppressor function.PTEN also has many functions that are independent of its phosphatase activity.Besides,although mutations and deletions of PTEN are frequently involved in the development of cancer,post-translational modificationaffects the stablility and activity of PTEN.These maybe involved in the protein interactions of PTEN.Therefore,overall study about PTEN function and regulation mechanisms contributes to find new therapies for cancinoma.The structure of PTEN lays the foundation of PTEN protein interaction.Catalytic phosphatase domain(PD)is necessary for PTEN tumor suppressor functions.C2 domains contributes to the membrane-binding of PTEN and C-tail are involved in the regulation of combination of PTEN with other proteins.As reported,PTEN crystal structure revealed that the PD and membrane-binding C2 domains are ordered,the C-tail of PTEN is an intrinsically disordered region(IDR)which enhances PTEN's protein-protein interactions.Structural plasticity leads to PTEN versatile functions through interaction with various proteins.Targeting PTEN IDR and its interactors emerges as a new strategy for treatment of PTEN related pathologies.Researchers have predicted PTEN can interact with thounsands of signaling proteins by bioinformatics analysis,most of which are related to cancer.Researchers also have revealed the different interaction proteins between wild PTEN and mutant PTENG20E,which has the lipid phosphatase activity,but lost protein phosphatase activity.For example,researchers have found PTEN can combine with Receptor for activated protein kinase C by reliable method which was based on stable isotope labeling by amino acids in cell culture,SILAC,then affinity purification and mass spectrometry were conducted.But there is little study to prove the interaction of the both and the specific mechanism.Receptor for activated protein kinase C(RACK1)is an adaptor protein with seven WD40 motifs,36 kDa,mainly allocated in cytosol and cytoplasma membrane,providing a scaffold for multiple protein-protein interactions,thus involved in various signaling pathways.As reported,RACK1 influences the binding parters through shuttling partners from one site to another,modifying the activity of the partners,changing intermolecular interaction(enhancing association/dissociation),and modulating the stability of binding proteins.RACK1 can regulate the phosphorylation modification of interaction proteins or the activity of Phosphatases and kinases.In addition,phosphorylation is the only definite post-translational modification of RACKlwhich is emerging as an important factor that modulates the binding of proteins to RACK1 and RACK1 location.RACKlcan regulate many cellular actions including cell growth,differentiation,adhesion,migration and immunity.Recently,researchers have found the high expression level of RACK1 in a multitude of tumors such as melanomas,breast cancer,colorectal cancer,pulmonary adenocarcinoma,hepatoma,esophageal squamous cell carcinoma,and oral squamous cell carcinomas.With regard to its roles in tumorigenesis,RACK1 mainly contributes to tumor growth,invasion and metastasis.However,some contradictory results exist in its oncogenic property.It can exert a negative control on Src activity and an inhibitory effect on Ki-Ras-mediated morphological cell transformation.It is down-regulated and acts as a tumor suppressor negatively regulating the Wnt signaling in gastric cancer.Interestingly,more and more studies have been recently indicated that RACK1 is interacted with some proteins(such as,ZEBRA,LMP1 and A73)encoded by Epstein-Barr virus(EBV)which has been implicated in the molecular abnormalities leading to NPC,implying that RACK1 probably plays roles in NPC development and progression.However,there is little report about the expression and function of RACK1 in NPC.Numerous studies have demonstrated PTEN plays an important tumor suppressor function in NPC.But the molecular mechanism of PTEN in nasopharyngeal carcinoma is not comprehensive.Therefore,this paper explores the combination of PTEN with RACK1 and the molecular mechanism of interaction in nasopharyngeal carcinoma.This lays the foundation for the research of PTEN'sprotein phosphatase and protein-protein interactions in nasopharyngeal carcinomaand is beneficial to find new diagnosis and therapy targets.for NPC.Methods1.Identification RACK1 asthe potential interaction protein of PTEN.1)Based on the results reported and bioinformational analysis,we select the potential proteins that interact with PTEN as the following conditions.First,the protein binds toPTEN.Second,the protein is related to the progression of cancers.Third,phosphorylation modification is one of the most important methods to regulat the function and activation of the protein.2)Detect the expression level of RACK1 in nasopharyngeal carcinoma tissues and cells byImmunohistochemistry,RT-PCR and Western Blot.3)Topredict the function of RACK1 in NPC through clinical data analysis.2.To explore the functions of potential protein interacting with PTEN(RACK1)in NPC through experiments in vitro.1)To construct plasmids and siRNA2)To test successfully overexpression and downregulation of the protein in NPC cells.3)To detect the regulation of protein on NPC cells proliferation by MTT assay,EdU assay and plate clone formation assay.4)To detect the regulation of protein on NPC cells migration and invasion by transwell assay and boyden assay respectively.3.To prove the binding of PTEN with RACK1 and the combination domains.1)Construct the HA-PTEN,c-Myc RACK1,c-Myc RACK1WD3-4,c-Myc RACK1WD5-7 plasmids.2)To prove PTEN associated with RACK1 with immunoprecipitation experiments.3)To detect the interaction of PTEN with RACK1 by proximity ligation in situ assay(PLA)in NPC cells.4)To discover the combination domains by immunoprecipitation experiments.4.Molecular mechanism involved in the interaction of PTEN with RACK1 in NPC.1)Examine the changes of PI3K/Akt and FAK activities after overexpression or downregulation of RACK1 in NPC cells.2)Examine the changes of PI3K/Akt and FAK activities after overexpression PTEN to verify the important role of PTEN in NPC cells.Stimulate the PTEN overexpression NPC cells with Phorbol-]2-myristate 13-acetate(PMA),as reported,which can activateprotein kinase C(PKC)and RACK1,then examination the changes of Akt and FAK activities.3)Examination the influence on the expression of PTENor RACK1 total protein by each other.ResultsPart 1 Identification the expression of potential PTEN interaction protein in NPC.1)Select the RACK1 protein as an potential interactor with PTEN in NPC based on the literature and reports2)Expression of RACK1 in NPC tissues and NP tissuesWeinitially detected the protein expression level of RACK1 in 58 paraffin-embedded NPC samples and 37 non-cancerous nasopharyngeal(NP)samples using immunohistochemical staining.RACK1 protein was detectable in 98%(57/58)of NPC samples and in 86%(32/37)of NP samples.Notably,RACK1 protein expression was considerably higher in NPC samples than NP samples(P<0.001).76%(44/58)of NPC samples showed high expression level of RACK1,while only 30%(11/37)of NP samples showed a relatively high expression level of RACK.3)Identification the expression of RACK1 in NPC cellsQuantitative real-time PCR were used to identify the level of RACK1 mRNA in NPC cells and immortalized nasopharyngeal epithelial cell NP69.The results indicated that compared to NP69,RACK1mRNA was not obviously increased in NPC cells,even there appeared a reduced tendency in some NPC cells.Western Blotwere used to identify the level of RACK1 protein in NPC cells and NP69.The results showed that the highly invasive NPC cells(5-8F,CNE2,CNE1)showed higher expression levels of RACK1 than relatively low malignant NPC cells(HONE1,6-10B)and NP69.4)Identification of the RACK1 location in NPC by confocal microscope and immunofluorescence experiment.Confocal microscope was performed to detect the subcellular compartmentalization of RACKlin NPC.Claudin-1 was used as a cell membrane marker.The nuclei were stained with DAPI.The merged images showed RACK1 was mainy located in cell cytoplasm of NPC tissues.Immunofluorescence images showed the similar localization of RACK1 in NPC cells to tissue samples.5)Relationship between RACK1 expression and clinicopathological variables in NPC patientsTo further support our above findings,58 NPC patients were recruited for in vivo evaluatingthe correlation between RACK1 protein expression level and clinicopathologic features.We did not find a significant association of RACK1 expression with patients' age,sex,and tumor size,but we observed significant correlations of RACK 1 expression with lymph node invasion and clinical stage.These data provide further evidence that RACK1,correlating with advanced NPC,plays an important role in NPC progression.Part 2Exploreeffects of RACK1 on proliferation,migration and invasion of NPC cells through in vitro experiments1)RACK1 promotes proliferation of NPC cells.After plasmid transfection,MTT assays,colony formation assays and EdU assays were carried out to determine the effect of RACK1 on cell viability and proliferation ability.Notably,overexpressed RACK1 appeared to significantly increase cell growth,EdU-positive cell number,and colony formation of NPC cells compared with control cells.Subsequently,NPC cells(5-8F and CNE1)expressing a relatively high level of RACK1 were also chosen to be transfected with si-RACK1 or scrambled siRNA.Relative to scrambled control,RACK1 protein expression was obviously inhibited in 5-8F and CNE1 cells treated with RACK1-siRNA.Notably,after transfection with si-RACK1,5-8F and CNE1 cells displayed cell growth inhibition,less EdU positive cells as well as fewer and smaller colonies compared with scrambled control.Taken together,these data indicate the promotion of cell proliferation by RACK1 in NPC2)RACK1 prompts NPC cells migration and invasionFurthermore,the effect of RACK1 on NPC cell migration and invasion was determined using Transwell assays and Boyden assays.As shown in,overexpressed RACK1 stimulated cell migration in NPC cells(HONE1 and 6-10B)that were relatively low malignant.The cell invasion abilities were also significantly increased as assessed using modified Boyden assays.Next,the migration and invasion phenotypes were compared between high-invasive NPC cells treated with si-RACK1 and control cells.Similarly,down-regulation of RACK1 inhibited the migration and invasion of NPC cells(5-8F and CNE1).Thus,these data demonstrate that RACK1 also enhances NPC cell migration and invasion.Part 3 PTEN interacts with RACK1 in nasopharyngeal carcinoma1)PTEN can combine with RACK1 in nasopharyngeal carcinoma cells.First,we detected the interaction of PTEN with RACK1 by co-immunoprecipitation experiment.CO-IP was performed with anti-HA antibody and anti-RACK1 antibodyrespectively,anti-HA antibody was used to detect PTEN protein as the PTEN plasmid with a HA tag.IgG antibody was used as a control.We found both the PTEN and RACK1 proteins in the products of immunoprecipitation by Western Blot.This indicated PTEN interacts with RACK1 in NPC cell.What's more,we further confirmed the interaction of PTEN with RACK1 by proximity ligation in situ assay(PLA)in NPC cells.NPC cell CNE1 was transfected with HA-PTEN plasmid,anti-HA antibody and anti-RACK1 antibody together were used to do the PLA,just incubated with anti-HA antibody or anti-RACK1 respectively as the controls.We observed red fluorescence dots in the experimental group,while the control goups didn't appear.The result also demonstrated that PTEN interacts with RACK1 in NPC cell.2)The combination domains of RACK1 with PTEN.We constructed the wild HA-PTEN plasmid and the wild RACK1 and various mutants,such as Myc-RACK1,Myc-RACK1WD3-4 and Myc-RACK1WD5-7,to research the combination domains of RACK1 with PTEN by co-immunoprecipitation experiments.Results showed that group transfected with Myc-RACK1 or Myc-RACK1WD5-7 detected the bands of PTEN and Myc together,while the Myc-RACK1 WD3-4 group just appeared Myc,this demonstrated that RACK1 interacts with PTEN through the WD5-7 domain.3)The molecular mechanism of PTEN interacting with RACK1 in NPCour research showed PTEN can inhibit the activation of Akt and FAK in nasopharyngeal carcinoma cells.Overexpression of RACK1 promoted the activation of Akt and FAK,while downregulation of RACK1 reduced the level of p-Akt and p-FAK.Stimulated the PTEN overexpression NPC cells with Phorbol-12-myristate 13-acetate(PMA),as reported,which can activate protein kinase C(PKC)and RACK1,the level of p-Akt and p-FAK were improved.All results demonstrated that RACK1can promot the activiation of PI3K/Akt and FAK pathway in NPC.The activation of PKC/RACK1 can counteract the negative regulation of PTEN to PI3K/Akt/FAK pathway.The interaction of PTEN and RACK1 was not involved in the regulation of protein expression.We detected the protein expression of PTEN and RACK1 in NPC cells,there was not obvious relation between two proteins.Overexpression of PTENdid not affect the expression of RACK1,and there was no influence on the expression of PTEN protein after knockdown of RACK1 in NPC cells.All those indicated that the interaction of PTEN and RACK1 was not involved in the regulations of protein expression.The mechanisms of RACK 1 interacting with PTEN to regulate the progression of nasopharyngeal carcinoma maybe involved in protein modification,change of the protein location or influence of interaction with other proteins and so on.Conclusions1.RACK1 is relative high expression in NPC tissueses and correlates with the advance NPC.2.RACK1 promptes the proliferation,migration and invasion of NPC cells.3.PTEN interacts with RACK1 to regulate the progression of nasopharyngeal carcinoma through PI3K/Akt/FAK pathway. |
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