The Research Of Impact And Mechanism Via FOXL1-PP2A Co-expression On Proliferation And Metastasis For Pancreatic Cancer PANC-1 Cells

Objective: Pancreatic cancer is a common malignant tumor.The incidence is rising in recent years.Most of the pathological type is the pancreatic ductal adenocarcinoma cells cancer.Since the deep anatomical location of the pancreas and prone to early metastasis into the surrounding tumor,its prognosis is extremely poor after diagnosed.Although pancreaticoduodenectomy is feasible for early pancreatic cancer,most patients are not eligible for surgery at the time of diagnosis,with a 5-year survival rate of less than 7%.Even combined with chemotherapy,the therapeutic effect is still not satisfactory.Therefore,there is great practical significance to explore new and effective targets to study the mechanism of carcinogenesis and cancer-promoting for the treatment of pancreatic cancer.Forkhead box L1(FOXL1)belongs to a forkhead/winged helix-box(FOX)family of transcription factors.All Fox members,being classified as FOXA to FOXR,Fox molecules play critical roles in a variety of physiologic or pathologic processes such as cancer,as tumor suppressors.Particularly,FOXM1 was identified to be oncogenic in pancreatic cancer,and is associated with poor prognosis and pathologic stage of PADC,whereas,FOXL1 has recently been recognized as a tumor suppressor in PADC.Therefore,FOXL1 might be another target for the treatment of pancreatic cancers.Protein phosphatase 2A(PP2A)is a large collection of oligomeric protein serine/threonine phosphatases and accounts for a large fraction of phosphatase activity in eukaryotic cells.PP2 A is a critical tumor suppressor,via controlling a number of cellular processes,including cell cycle progression.Key signaling pathways that are negatively regulated by PP2 A include members of the MAPK/ERK pathways,NF-?B,and C-MYC signaling.PP2 A inhibitor CIP2 A,have an nagative correlation with survival time after surgery in pancreatic cancer patients.In particular,PP2 A suppresses the oncogenic activity of C-MYC via specifically dephosphorylating the key serine 62(S62)in C-MYC,stimulating its ubiquitination and thus accelerating the degradation of C-MYC.Methods : FOXL1 and PP2 A coexpression adenovirus vectors were constructed the expressions of FOXL1 and PP2 A in three groups of FOXL1,PP2 A and FOXL1+PP2A were detected by immunoblotting and real-time fluorescent quantitative PCR,and the expression of TRAIL,MYC,CASPASE3 and PARP in downstream proteins was detected.Cell counting assay was used to detect the proliferation of three groups of cells at 0,3,5 and 7 days.The cell transfer ability of the three groups was detected by scratch test,and the drug sensitivity of the three groups was verified after induction of apoptosis by 5-fu.T-test and variance analysis were used to detect the difference of the three groups of cells treated by different methods.Results: After the infection of Ad(FOXL1)or Ad(FOXL1+PP2A)(both P< 0.001,1or 3 MOI),the m RNA level of FOXL1 was significantly increased,there was no significant difference between Ad(FOXL1)and Ad(FOXL1+PP2A);the PP2 A of m RNA was significantly up-regulated in Ad(PP2A)or Ad(FOXL1+PP2A)group(P<0.001,P< 0.0001).The FOXL1 protein level in Ad(FOXL1+PP2A)is higher than Ad(CON)(P< 0.001),The PP2 A protein level in Ad(FOXL1+PP2A)is higher than Ad(CON)(P< 0.0001).Cell counting experiments showed that the growth efficiency of PANC-1 cells in Ad(FOXL1+PP2A)group decreased(P< 0.05 for 5 D.P.I.,or P< 0.01 for 7 D.P.I.)after infection with adenovirus 5 or 7 days.Cell cloning experiments showed that Ad(FOXL1),Ad(PP2A)or Ad(FOXL1+PP2A)formed less cell communities(P<0.05,P< 0.01 or P< 0.001).There was significant difference in clone size between the four groups(P<0.001).The clones in Ad(FOXL1),Ad(PP2A)or Ad(FOXL1+PP2A)groups were significantly smaller than those in Ad(CON)groups.The migration assay of PANC-1 cells indicated that in contrast to the Ad(CON)virus infection,the infection with either Ad(FOXL1),Ad(PP2A)or Ad(FOXL1 + PP2A)at 3 MOI significantly reduced the migration of PANC-1 cells,there were less migratory cells in the three groups(P<0.01)for Ad(FOXL1)or Ad(PP2A;P<0.001 for Ad(FOXL1 + PP2A).In PANC-1 cells,the protein levels of TRAIL increased significantly after infection with 3MOI Ad(FOXL1)and Ad(FOXL1+PP2A)(P < 0.01),but there was no significant difference in the protein levels of MYC in each group.After infection with Ad(PP2A)and Ad(FOXL1+PP2A),the level of MYC phosphorylation(S62)was significantly inhibited(P < 0.05).Conclusion: The adenovirus-mediated co-expression of FOXL1 and PP2 A with the 2A peptide linker exterts synergistic suppression of pancreatic cancer cells via inhibiting the growth and promoting apoptosis of cancer cells.Coexpression of FOXL1 and PP2 A sensitized PANC-1 cells to 5'-FU via enhancing apoptosis induction.The coexpressed FOXL1 and PP2 A functions independently via upregulating TRAIL(by FOXL1)and reducing the phosphorylation of MYC(by PP2A).Our findings reconfirmed the tumor suppressive role of PP2 A and FOXL1 in pancreatic combined expression of FOXL1 and PP2 A inhibits proliferation and metastasis of pancreatic ductal carcinoma cell line PANC-1 and inhibits apoptosis induced by MYC phosphorylation by increasing the expression of TRAIL protein,cancer cells,with an enhanced antitumor effect via co-expression of both molecules.