Mechanism Of EGFR/PPARδ Pathway-mediated Colon Cancer Cell Metabolism

Background: PPARδ belongs to PPARs(peroxisome-proliferator-activated receptors)family members that consist of PPARα,PPARδ,and PPARγ.PPARδ plays an important role in regulating inflammation,metabolism,and tumorigenesis,while it is still unclear that PPARδ transcriptional modification on cancer progression.As a membrane tyrosine kinase,EGFR(epidermal growth factor receptor)can be activated by ligands such as EGF resulting in activation of downstream signaling cascades such as RAS/RAF/MEK/ERK,PLCγ/PKC,PI3k/Akt,and STATs,which are involved in tumorigenesis.Our previous investigation shows that EGFR induces PPARγ phosphorylation leading to inhibition of PPARγ antitumor function,while it is unclear the interaction of EGFR with PPARδ.Methods: Immunoprecipitation analysis was used to detect the interaction of PPARδ with EGFR.LC-MS/MS analysis was used to identify the phosphorylation site of PPARδ.In vitro kinase was used to analyze EGFR-induced PPARδ phosphorylation.Dual-luciferase analysis was used to detect PPARδ,Glut1 and SLC1A5 transcription activity.qPCR and Western blot were used to assay gene and protein expressions.Soft agar and xenograft model were used to detect cancer cell proliferation and tumor growth.MTT assay was used to detect cell survival.Results: PPARδ protein contains a motif of EGFR phosphorylation by alignment analysis.LC-MS/MS analysis showed that activated EGFR induced PPARδ-Y108 phosphorylation,which was further determined by in vitro kinase analysis.Immunoprecipitation and Western blot analysis showed that EGFR induced PPARδ but not the PPARδ-Y108 A mutant tyrosine phosphorylation.Dual-luciferase analysis showed that overexpression of PPARδ increased its activity,while PPARδ-Y108 A mutant reduced this event.Further analysis showed that PPARδ but not the PPARδ-Y108 A mutant significantly increased Glut1 and SLC1A5 gene transcription activity and protein expressions,leading to promotion of glucose consumption,lactate release,and glutamine uptake in cancer cells.Soft agar analysis showed that stable expression of PPARδ but not the PPARδ-Y108 A mutant significantly enhanced cancer cell proliferation.In addition,PPARδ but not the PPARδ/Y108 A mutant promoted tumor growth in xenograft model,which was involved in PPARδ but not the Y108 A mutant-mediated Glut1 and SLC1A5 protein expression in tumors.In response to chemotherapy drugs,PPARδ but not the Y108 A mutant significantly increased the resistance of the chemotherapy drugs(camptothecin,cisplatin,taxol,etoposide)by MTT analysis.Conclusions: EGFR-induced PPARδ-Y108 phosphorylation enhanced PPARδ activity leading to increased PPARδ-mediated Glut1 and SLC1A5 gene and protein expressions,subsequently,promoted glucose consumption and glutamine uptake resulting in tumor growth and chemo-resistance.