title: Functional Role of mTORC2 versus Integrin-Linked Kinase in Mediating Ser473-Akt Phosphorylation in PTEN-Negative Prostate and Breast Cancer Cell Lines.
abstract: Although the rictor-mTOR complex (mTORC2) has been shown to act as phosphoinositide-dependent kinase (PDK)2 in many cell types, other kinases have also been implicated in mediating Ser473-Akt phosphorylation. Here, we demonstrated the cell line specificity of integrin-linked kinase (ILK) versus mTORC2 as PDK2 in LNCaP and PC-3 prostate and MDA-MB-468 breast cancer cells, of which the PTEN-negative status allowed the study of Ser473-Akt phosphorylation independent of external stimulation. PC-3 and MDA-MB-468 cells showed upregulated ILK expression relative to LNCaP cells, which expressed a high abundance of mTOR. Exposure to Ku-0063794, a second-generation mTOR inhibitor, decreased Ser473-Akt phosphorylation in LNCaP cells, but not in PC-3 or MDA-MB-468 cells. In contrast, treatment with T315, a novel ILK inhibitor, reduced the phosphorylation of Ser473-Akt in PC-3 and MDA-MB-468 cells without affecting that in LNCaP cells. This cell line specificity was verified by comparing Ser473-Akt phosphorylation status after genetic knockdown of rictor, ILK, and other putative Ser-473-Akt kinases. Genetic knockdown of rictor, but not ILK or the other kinases examined, inhibited Ser473-Akt phosphorylation in LNCaP cells. Conversely, PC-3 and MDA-MB-468 cells were susceptible to the effect of ILK silencing on Ser473-Akt phosphorylation, while knockdown of rictor or any of the other target kinases had no appreciable effect. Co-immunoprecipitation analysis demonstrated the physical interaction between ILK and Akt in PC-3 cells, and T315 blocked ILK-mediated Ser473 phosphorylation of bacterially expressed Akt. ILK also formed complexes with rictor in PC-3 and MDA-MB-468 cells that were disrupted by T315, but such complexes were not observed in LNCaP cells. In the PTEN-functional MDA-MB-231 cell line, both T315 and Ku-0063794 suppressed EGF-induced Ser473-Akt phosphorylation. Inhibition of ILK by T315 or siRNA-mediated knockdown suppressed epithelial-mesenchymal transition in MDA-MB-468 and PC-3 cells. Thus, we hypothesize that ILK might bestow growth advantage and metastatic potential in the course of tumor progression.
authors.affiliation: Division of Medicinal Chemistry, College of Pharmacy, The Ohio State University, Columbus, Ohio, United States of America.
authors.forename: Su-Lin
authors.initials: SL
authors.lastname: Lee
authors.forename: Chih-Chien
authors.initials: CC
authors.lastname: Chou
authors.forename: Hsiao-Ching
authors.initials: HC
authors.lastname: Chuang
authors.forename: En-Chi
authors.initials: EC
authors.lastname: Hsu
authors.forename: Po-Chen
authors.initials: PC
authors.lastname: Chiu
authors.forename: Samuel K
authors.initials: SK
authors.lastname: Kulp
authors.forename: John C
authors.initials: JC
authors.lastname: Byrd
authors.forename: Ching-Shih
authors.initials: CS
authors.lastname: Chen
mesh_terms: D000813:Anilides / Q000494:pharmacology; D001565:Benzoates / Q000494:pharmacology; D001943:Breast Neoplasms / Q000378:metabolism*; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D058750:Epithelial-Mesenchymal Transition / Q000187:drug effects; D005260:Female; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006801:Humans; D008297:Male; D000076225:Mechanistic Target of Rapamycin Complex 2; D009025:Morpholines / Q000494:pharmacology; D046912:Multiprotein Complexes / Q000378:metabolism*; D051059:PTEN Phosphohydrolase / Q000172:deficiency; D010766:Phosphorylation / Q000187:drug effects; D011471:Prostatic Neoplasms / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism*; D011743:Pyrimidines / Q000494:pharmacology; D000076226:Rapamycin-Insensitive Companion of mTOR Protein; D012694:Serine / Q000378:metabolism*; D058570:TOR Serine-Threonine Kinases / Q000378:metabolism*; D015854:Up-Regulation; D000099200:Scaffold Protein ILK
chemical_list: D000813:Anilides; D001565:Benzoates; D002352:Carrier Proteins; D000076225:Mechanistic Target of Rapamycin Complex 2; D009025:Morpholines; D046912:Multiprotein Complexes; D051059:PTEN Phosphohydrolase; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; D011743:Pyrimidines; D000076226:Rapamycin-Insensitive Companion of mTOR Protein; D012694:Serine; D058570:TOR Serine-Threonine Kinases; D000099200:Scaffold Protein ILK; C492571:RICTOR protein, human; C533995:T 3157; C541932:Ku 0063794; C494929:PTEN protein, human