The Function And Mechanism Of PP2AC In The Positive Regulation Of MTORC1 In Colorectal Cancer

Objective To investigate the expression and function of PP2 AC in human colorectal cancer.To investigate the effect and regulatory mechanism of PP2 AC on mTORC1 signaling under amino acid deprivation condition.Methods1.Tissue microarray(Hcol-Ade150CS-01)was used to detect the expression level of PP2 AC in colorectal cancer tissue and its paired adjacent normal mucosal tissue.We assessed the H-Score of PP2 AC protein and then analyzed the statistical differences of PP2 AC expression between colorectal cancer tissue and adjacent normal mucosal tissue.The clinical pathological data of these patients were collected and sorted.The relationships between expression level of PP2 AC and common clinical pathological parameters were analyzed.We also downloaded the PP2 AC mRNA data of colorectal cancer from TCGA and GEO databases,and performed meta analysis.2.Colorectal cancer cell lines SW480 and RKO with low PP2 AC expression and colorectal cancer cell lines HCT116 and DLD1 with high PP2 AC expression were used to reconstruct stable PP2 AC overexpression cell lines SW480-PP2 AC,RKO-PP2 AC and stable PP2 AC knockdown cell lines HCT116-PP2AC-KD,DLD1-PP2AC-KD.The effect of PP2 AC on the growth of colorectal cancer cells was studied by cell line-derived xenograft tumors in vivo.3.mTORC1 signaling pathway is dependent on amino acids,which is important for colorectal cancer cells survival.To further explore the mechanism by which PP2 AC regulates the mTORC1 signaling pathway,we further studied the effect of overexpression and knockdown PP2 AC on mTORC1 signaling pathway under amino acid deprivation,by using immuno-blotting assay.Sulfonyl rhodamine B colorimetric,colony formation and anchorage-independent growth experiments were used to further validate the function of PP2 AC in colorectal cancer under amino acids deprivation conditions.4.By using co-immunoprecipitation and western blotting assay,we investigated the binding of PP2 AC and c-Myc under the condition of amino acid deprivation.To further elucidate the specific mechanism of PP2 AC regulating c-Myc,cycloheximide chase assay was used to examine c-Myc protein turn-over under amino acid deprivation,in cells with stable overexpression of PP2 AC..The effect of PP2 AC on c-Myc phosphorylation was analyzed by western blotting assay.Cells were transfected with wildtype or mutant(T58A,which could not be phosphorylated)c-Myc,and then the effect of c-Myc phosphorylation on PP2 AC induced mTORC1 activation was detectedunder amino acid deprivation condition.Results1.Compared with adjacent noncancerous tissues,PP2 AC was significantly overexpressed in colorectal cancer.Results from meta analysis of GEO and TCGA database demonstrated that,PP2 AC mRNA level was significant higher in cancerous tissues than in normal mucosal tissues.The PP2 AC level was significantly correlated to poorpathological parameters,including high T stage(P=0.008)and advanced AJCC pathological stage(P=0.015).2.PP2 AC knockdown(KD)significantly inhibited tumor growth in xenograft mouse model.Immunohistochemical analysis showed that KD PP2 AC has a marked reduction in ki67 staining.3.Under the condition of amino acid deprivation,overexpression of PP2 AC significantly enhanced the activity of mTORC1 pathway and promoted the ability of proliferation,colony formation and anchorage-independent growth ability,indicating PP2 AC overexpression elicits cells resistance to amino acid deprivation.Knockdown of PP2 AC further inhibited the activity of mTORC1 pathway,and inhibited the proliferation,colony formation and anchorage-independent growth ability of colorectal cancer cells,indicating PP2 AC depletion leads cells to be more sensitive to unfavorable nutrient condition.Meanwhile,in the amino acid recovery experiment,in PP2 AC overexpression cells,amino acid refeeding after a period of amino acid starvation reinstated mTORC1 signaling to a level much greater than that seen in control cells,indicating PP2 AC overexpression drives cells to be more sensitive to amino acid re-supplement.4.Under the condition of amino acid deprivation,PP2 AC overexpression dramatically increased c-Myc protein level in CRC cells.Suppression of c-Myc abolished PP2 AC overexpression-induced mTORC1 activation,indicating that c-Myc is involved in PP2AC-mediated mTORC1 activation under nutrient-deprived condition.In the co-IP experiment,PP2 AC directly binds with c-Myc,and the presence of PP2 AC dramatically increased the half-life of c-Myc when compared to the absence of PP2 AC.PP2AC overexpression led to dephosphorylation of p T58 on c-MYC,while loss of PP2 AC increased the level of the p T58 c-MYC.In contrast,p S62 c-MYC levels were increased in the presence of PP2 AC,but decreased in PP2 AC knockdown cells.Transfection with c-Myc(T58A)completely rescued the mTORC1 inhibiton resulted from PP2 AC KD.These data suggest that PP2 AC upregulates c-Myc by increasing its stability in CRC cells,and this correlates with increased phosphorylation at S62 and decreased phosphorylation at T58.ConclusionsPP2AC is significantly overexpressed in colorectal cancers,with the upreguated expression of PP2 AC being related to poor pathological parameters.Under amino acid deprivation,PP2 AC is tumor promotional by positively regulating the mTORC1 signaling via c-Myc,and then conferring cells resistant to amino acid deprivation,thus promoting the progression of colorectal cancer.