Functional Role of mTORC2 versus Integrin-Linked Kinase in Mediating Ser473-Akt Phosphorylation in PTEN-Negative Prostate and Breast Cancer Cell Lines
Authors: En-Chi Hsu
Po-Chen Chiu
Su-Lin Lee
Chih-Chien Chou
John C Byrd
Samuel K Kulp
Hsiao-Ching Chuang
Ching-Shih Chen
Journal: PLoS ONE (2013)
Discipline: Multidisciplinary
Keywords: rictor-mTOR complex; PDK2; Ser473-Akt phosphorylation; integrin-linked kinase (ILK); LNCaP
Affiliations: Division of Medicinal Chemistry, College of Pharmacy, The Ohio State University, Columbus, Ohio, United States of America.

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. 

Retracted: Yes

PubPeer comments:
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 August 2018 retraction notice. Paper reported in 2015.  The Ohio State University Office of Research Compliance notified PLOS ONE that the institution investigated the work reported in this article [1] and found evidence of data falsification in Figures 1B, 3A, and 3B. In Figure 1B, the p70S6K and u03b2-actin Western blot data were replaced with unidentified blots, and the Akt blot for LNCaP cells was incorrectly labeled when compared to the original research record. No supporting data for the published blots for p70S6K or u03b2-actin panels were identified in the investigation, while the published blot for the Akt panel was shown to be generated under different experimental conditions than as reported. These changes resulted in a figure that shows equal expression of p70S6K where the original data for the described experiment showed unequal expression. In Figure 3A, Western blot data in the MK2 blot (lanes 1, 3) and the PAK1 blot (lanes 1, 3, 8) were replaced and relabeled when compared to the original research record. This changed the reported results by showing all the untreated samples in the MK2 and PAK1 blots had the same expression level, while the original research record showed unequal expression. In Figure 3B, Western blot data for the p308-T-Akt, ILK, Akt and u03b2-actin blots were incorrectly labeled as being treated with or without Ku-0063794 and or ILK siRNA when compared to the original research record. This changed the reported results by showing that ILK knockdown had no effect on the expression of pSer473AKT on untreated cells, but that phosphorylation decreased with KU-0063794 treatment along with IL siRNA treatment. In light of these concerns, and in line with the institutional recommendation, the PLOS ONE Editors retract this article. SLL, ECH and SKK agree with the retraction. CCC, HCC, PCC, JCB and CSC did not respond. ,