Characterization of AKT independent effects of the synthetic AKT inhibitors SH-5 and SH-6 using an integrated approach combining transcriptomic profiling and signaling pathway perturbations.
BACKGROUND
Signal transduction processes mediated by phosphatidyl inositol phosphates affect a broad range of cellular processes such as cell cycle progression, migration and cell survival. The protein kinase AKT is one of the major effectors in this signaling network. Chronic AKT activation contributes to oncogenic transformation and tumor development. Therefore, analogs of phosphatidyl inositol phosphates (PIAs) were designed as new small drugs to block AKT activity for cancer treatment. Here we characterize the biological effects of the PIAs SH-5 and SH-6 in colorectal cancer cell lines.


METHODS
Serum-starved or serum-supplemented human colorectal cancer cell lines SW480, HT29 and HCT116 were exposed to SH-5 and SH-6. AKT activation was determined by western blotting. Cell viability was assessed using a colorimetric XTT-based assay, apoptosis and cell cycle changes were monitored by FACS analysis. The dynamics of cell morphology alterations was evaluated by confocal and time-lapse microscopy. Transcriptional changes due to inhibitor treatment were analyzed using Affymetrix HG-U133A microarrays and RT-PCR.


RESULTS
While the PIAs clearly reduce AKT phosphorylation in serum starved cells, we did not observe a significant reduction under serum supplemented conditions, giving us the opportunity to analyze AKT independent effects of these compounds. Both inhibitors induce broadly the same morphological alterations, in particular changes in cell shape and formation of intracellular vesicles. Moreover, we observed the induction of binucleated cells specifically in the SW480 cell line. Gene expression analysis revealed transcriptional alterations, which are mostly cell line specific. In accordance to the phenotype we found a gene group associated with mitosis and spindle organization down regulated in SW480 cells, but not in the other cell lines. A bioinformatics analysis using the Connectivity Map linked the gene expression pattern of the inhibitor treated SW480 cells to PKC signaling. Using confocal laser scanning microscopy and time lapse recording we identified a specific defect in the last step of the cytokinesis as responsible for the binucleation.


CONCLUSIONS
The PIAs SH-5 and SH-6 impinge on additional cellular targets apart from AKT in colorectal cancer cells. The effects are mostly cell line specific and have an influence at the outcome of the treatment. In view of potential clinical trials it will be necessary to take these diverse effects into consideration to optimize patient treatment.
Krech
Till
T
Laboratory of Molecular Tumor Pathology, Institute of Pathology, Charité, Universitätsmedizin Berlin, Charitéplatz 1, D-10117 Berlin, Germany.
Thiede
Margarethe
M
Hilgenberg
Ellen
E
Schäfer
Reinhold
R
Jürchott
Karsten
K
D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D015153:Blotting, Western; D002469:Cell Separation; D048430:Cell Shape / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D015179:Colorectal Neoplasms / Q000201:enzymology* / Q000235:genetics / Q000473:pathology; D019295:Computational Biology; D048749:Cytokinesis / Q000187:drug effects; D004789:Enzyme Activation; D005434:Flow Cytometry; D020869:Gene Expression Profiling / Q000379:methods; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D045325:HCT116 Cells; D019073:HT29 Cells; D006801:Humans; D007295:Inositol Phosphates / Q000494:pharmacology*; D018613:Microscopy, Confocal; D018715:Microscopy, Video; D020411:Oligonucleotide Array Sequence Analysis; D010716:Phosphatidylinositols / Q000494:pharmacology*; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors / Q000494:pharmacology*; D051057:Proto-Oncogene Proteins c-akt / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000187:drug effects*; D013997:Time Factors
C435044:1-((1-O-octadecyl-2-O-methylglycero)phospho)-3-deoxy-myo-inositol; D000970:Antineoplastic Agents; D007295:Inositol Phosphates; D010716:Phosphatidylinositols; D047428:Protein Kinase Inhibitors; C516024:SH-6 compound; D051057:Proto-Oncogene Proteins c-akt