The Role of PTEN Protein Phosphatase in Nasopharyngeal Carcinoma ： The Ability of TGF-β1 Inducing Migration and Invasion was Inhibited by PTEN Protein Phosphatase through Down-regulating p-P38

Background ： The phosphatase and tensin homolog gene(PTEN) is a crucial ca ncersuppressor gene in nasopharyngeal carcinoma(NPC),which encodes the prote in i-ncluding lipid phosphatase and protein phosphatase.p-P38 plays a vital role in the development process of cancers.However,whether and how PTEN protein phosphatase inhibit p-P38 expression and regulate migration and invasion in nasopharyngeal carcinoma is still fully elucidated. Methods: The abilities of migration and invasion were analyzed using Scratch, Transwell,Boyden experiments in NPC cells.Westere Blot was utilized to explore the expression of E-caherin,N-caherin and VIMENTIN in Epithelial-mesench-ymal transition(EMT),P38,p-P38(Thr180/Tyr182),AKT,p-AKT(Ser473),PTEN and its mutants.qPCR was done to detect the mRNA expression of PTEN and PTEN mutants.Immunofluorescence localization assay and Co-immunoprecipitatio-n assay was used to explore the mutual combination of PTEN and P38. Results: We verified that transforming growth factor-β1(TGF-β1) could induce migration,invasion,and EMT in NPC again,and appropriate concentration was 5ng/ml.Interesting,we demonstrated that 5ng/ml p-AKT （ Ser473 ） and p-P38(Thr180/Tyr182).Further more,after overexpression of PTEN WT and various mutants including PTEN-C124S(lacking of both lipid phosphatase and protein phosphatase),PTEN-G129E(only lacking of lipid phosphatase but remaining protein phosphatase),PTEN-Y138L(lacking of protein phosphatase but remaining lipid phosphatase),and PTEN 1-353(Lack of c-tail structur-e),we observed that PTEN protein phosphatase reduced the ability of migration, invasion by inhibiting TGF-β1-induced p-P38,but the PTEN c-tail did not invo-lvein this process.At last,we confirmed PTEN could bind to P38 each other. Conclusions: The asssys in the study indicated that PTEN protein phosphatase might be anticancer,where it was able to inhibit the ability of TGF-β1 inducing migration and invasion by reducing p-P38 in NPC cells.

TGF-β is a secreted cytokine and exists in three isomeric forms,from β1 to β3,in hunman [19].TGF-β1 plays a pleiotropic regulator in tumorigenesis and development such as metastasis,proliferation,survival,etc [20].
Thus,P38 can also regulate the progress of nasopharyngeal carcinoma [32], which is serine and tyrosine kinase,and can be phosphorylated [33].In fact,p-P38 for malignant tumor growth,metastasis and apoptosis also plays a very important role [34][35].
Therefore,we speculate that PTEN might directly regulate activation of P38 and affect migration and invation in NPC cells.

Materiais and methods
Cell culture.NPC cell lines including HONE1,SUNE-1,CNE-1,CNE-2,5-8F and nasopharyngeal epithelial immortalized cell line NP69 were obtained from Central Laboratory of TCM-Integrated Cancer of Southern Medical University.
Plasmids transfection.NC,PTEN WT,PTEN C124S,PTEN G129E and PTEN Y138L plasmids(Addgene) were transfected into 5-8F and HONE1 cells with Lipofectamine 2000(Invitrogen) in serum-free conditions.6~8h later,the medium was changed to the 10% serum medium with TGF-β1.Transfected cells were cultured 24~48h before qPCR,and 48h before Western Blot. Migration and invasion assay.Scratch asasay was performed using a 200μl pipette tip and ruler to obtain the straight lane in cell-culture dish.Next,the cells were washed twice with D-hanks(8.0g NaCl,0.4g KCl,0.08g Na2HPO4·12H2O,0.06g KH2PO4,0.35g NaHCO3,ddH2O 1000ml,PH 7.2),and the cells was cultured with serum-free medium.12h,24h,36h and 48h later,the change of the straight lane was observed and photographed under a inverted microscope(Nikon,To-kyo,Japan) at a specific location of cell-culture dish.Migration and invasion assay was carried out using Standard 24-well chemotaxis chamber (8µm pore size;Corning).For cell invasion assay,Matrigel(BD Biosciences) was added to the transwell chamber,which was as upper well.Briefly,2×10 5 cells in 100μl serum-free media were moved to the upper chamber and 500μl media containing 10% FBS was put into the lower well.The cells were cultured at 37°C for 16-20h for migration assay and 36-40h for invasion assay.Those cells passed through the polycarbonate membrane were signed by Giemsa stain,and photographed by the inverted fluorescence microscope(Nikon).In five random highpower felds,thenumber of cells was calculated.All assays were repeated independently at least for three times. Co-Immunoprecipitation(co-IP) Assay.In brief,PTEN WT plasmids were transfected into 5-8F cells.48h later,D-hanks was used to wash the cells,twice.Then, 6 those cells were lysed in 800 µL of cell Lysis/Washing buffer(ybiotech) at 4℃ for 30 minutes.After cell lysates was centrifugated at 12,000×g at 4℃ for 10 minutes,the supernatant was taken as IP,and the rest was used as Input.The PTEN interacting proteins were enriched by co-immunoprecipitation using 10µl PTEN antibody(Cell Signaling Technology).The protein complex was analyzed by Western Blot using corresponding antibodies.
Statistical analysis.All statistical data were gained from at least three independent experiments and analyzed by SPSS(version 20.0,IBM,USA).Student T test, One-way ANOVA test and Dunnett's multiple comparison test was performed for two groups and multiple groups,respectively.Two-way ANOVA test was performed for two factors data.Data were presented as the mean±sem.P<0.05 was considered statistically significant.

TGF-β1 induces metastasis of NPC cells .
TGF-β1 plays a key role in the control of metastatic capacity of many malignancies [30,36].To demostrate the function of TGF-β1 on NPC cells,5-8F and HONE1 cells were cultured in 10% FBS medium with 0ng/ml,1ng/ml,5ng/ml,10 ng/ml of TGF-β1,respectively.After 48h,Scratch,Treanswell and Boyden asasay was used to detect the change in ability of migration and invasion( Figure.2A,B, C,D,E,F).As for 5-8F and HONE1 cells,metastatic capacity was enhanced in a concentration-dependent behavior.But,the diference between the experimental groups and the control group(0ng/ml TGF-β1 group) was statistical significance when the concentration of TGF-β1 was≥5ng/μl.And Western Blot was used to prove the change of EMT transformation,we found that the expression of E-cadherin was reduced,whereas,N-cadherin and VIMENTIN was increased( Figure.3 A,B).The results showed,compared to 0ng/ml TGF-β1,5ng/ml or 10ng/ml TGF-β1 promoted migration,invasion and EMT in NPC cells,and we gave that appropriate stimulus concentration of TGF-β1 was 5ng/μl. 7 The metastasis ability of malignant tumor cells is correlated with the phosphorylation levels of AKT(Ser473)and P38(Thr180/Tyr182)kinase [37].To confirm whether TGF-β1 could induce the increasing level of p-AKT(Ser473) and p-p38 (Thr180/Tyr182),we need the 5ng/μl TGF-β1 to stimulate 5-8F and HONE1 for 48h,Western Bolt was used to detect p-AKT(Ser473) and p-p38(Thr180/Tyr182) ( Figure.4).And the result demonstrated that TGF-β1 enhanced the expression of p-AKT(Ser473) and p-P38(Thr180/Tyr182).
We overexpressed PTEN WT,mutant PTEN C124S without lipid phosphatase and protein phosphatase activity,PTEN G129E with only protein phosphatase activity,PTEN Y138L with only lipid phosphatase activity,and PTEN 1-353 lacking c-tail structure in NPC cells.As shown in Figure.

PTEN protein phosphatase inhibits TGF-β1-induced migration,invasion and EMT in NPC.
In order to verify the functions of different enzyme activities and structures of PTEN,on the basis of various PTEN overexpression,5-8F and HONE1 cells were teated in 10% FBS medium with 5ng/μl TGF-β1 for 48h.Transwell and Boyden assyas were used to test the migration and invasion ability. Those results proved that both the protein phosphatase and lipid phosphatase of PTEN could inhibit migration,invasion and EMT in NPC.And c-tail of PTEN did not participate in those processes.

PTEN protein phosphatase reduced TGF-β1-induced p-p38.
To further clarify the mechanism of PTEN both lipid phosphatase and protein phosphatase inhibiting metastasis of NPC cells,Western Blot was used to detect the expressions of AKT,p-AKT(Ser473),P38 and p-P38(Thr180/Tyr182).The increase of TGF-β1-induced p-P38 was prevented by PTEN WT,PTEN G129E and PTEN 1-353 compared with the NC,but PTEN C124S not.The expression of TGF-β1-induced p-AKT was declined by PTEN WT,PTEN Y138L and PTEN 1-353.Therefore,those results explained that PTEN protein Phosphatase could lower the expression of TGF-β1-induced p-p38,but its lipid phosphatase did not.On the contrary,PTEN lipid phosphatase only could inhibit the expression of p-AKT,but its protein phosphatase didn't( Figure.7 A,B).So we argued that TGF-β1 inducing migration and invasion was inhibited by PTEN protein phosphatase through reducing p-P38 in NPC.

PTEN could bind to P38 each other
Yet,How do PTEN and P38 directly interact?We conducted immunofluorescence localization experiments in 5-8F,which confirmed the localization of PTEN was in cytoplasm and nucleus.And P38 was in the nucleus,the two proteins co-located with the nucleus (Fig.8 A).Further more,PTEN and P38 were detected simultaneously by co-immunoprecipitation (Fig.8 B).Those results shown that PTEN could bind to P38 each other.

Discussion
Witte D.etl found that TGF-β1-induced cell migration requires RAC1 and NOX4-dependent activation of P38 in pancreatic carcinoma cells [30].Interesting, Liliental J.etl think that genetic deletion of the PTEN promotes cell motility by activation of Rac1 and Cdc42 GTPases [31].Perhaps,PTEN could down-regulate TGF-β1-induced p-P38 by inhibiting the activity of RAC1,thereby inhibit the metastasis ability of tumor cells.We illuminate PTEN protein can directly act on P38 and regulate the migration and invasion ability of cancer cells under the stimulation of TGF-β1.
We confirmed under the stimulus of TGF-β1,PTEN protein phosphatase can inhibit NPC cell metastasis,and PTEN c-tail does not seem to be involved in the metastasis process of nasopharyngeal carcinoma cells,but this paper was lacking some experiments,for instance,we are unable to determine the definited working location of PTEN protein and P38,and our animal experiments also failed to undertake.

Conclusion
In this study,we determined PTEN protein phosphatase could inhibit the migration and invasion of NPC cells in TGF-β1 microenvironment,which correlated with PTEN combined with P38 and down-regulated p-P38(Thr180/Tyr182).To our knowledge,this is the first report to describe p-P38 is the downstream substr ate of PTEN.
To better understand the influence of different enzyme activities of PTEN on the migration,invasion and EMT of NPC and possible mechanisms,we did those assays.There is a growing realization about the metastasis process of NPC.May-be we can use this as a breakthrough point to guide clinicians in the diagnosis and treatment of tumors.

Ethics approval and consent to participate
Not applicable.

Consent for publication
Written informed consent for publication was obtained from all participants.

Availability of data and materials
The datasets used or analysed during the current study are available from the corresponding author on reasonable request.

Competing interests
The authors declare that they have no competing interests.

Funding
The study was supported by the National Natural Science Foundation of    When TGF-β1 was at 5ng/ml or 10ng/ml,the expression of E-cadherin was significantly enhanced than 0ng/ml.However,the levels of N-cadherin and VIMENTIN were lower.The data were shown as the mean±sem,One-way ANOVA,*P<0.05,**P<0.01.