Activation of phosphatidylinositol-3'-kinase/AKT signaling is essential in hepatoblastoma survival.
OBJECTIVE
Hepatoblastoma represents the most frequent malignant liver tumor in childhood. The phosphatidylinositol-3'-kinase (PI3K)/AKT pathway is crucial in downstream signaling of multiple receptor tyrosine kinases of pathogenic importance in hepatoblastoma. Increased PI3K/AKT signaling pathway activity and activating mutations of PIK3CA, encoding a PI3K catalytic subunit, have been reported in different childhood tumors. The current study was done to analyze the role of PI3K/AKT signaling in hepatoblastoma.


METHODS
Immunohistochemical stainings of (Ser473)-phosphorylated (p)-AKT protein, its targets p-(Ser9)-GSK-3beta and p-(Ser2448)-mTOR, as well as the cell cycle regulators Cyclin D1, p27(KIP1), and p21(CIP1) were done and the PIK3CA gene was screened for mutations. In vitro, two hepatoblastoma cell lines treated with the PI3K inhibitor LY294002 were analyzed for AKT and GSK-3beta phosphorylation, cell proliferation, and apoptosis. Additionally, simultaneous treatments of hepatoblastoma with LY294002 and cytotoxic drugs were carried out.


RESULTS
Most tumors strongly expressed p-AKT, p-GSK-3beta, and p-mTOR; subgroups showed significant Cyclin D1, p27(KIP1), and p21(CIP1) expression. One hepatoblastoma carried an E545A mutation in the PIK3CA gene. In vitro, PI3K inhibition diminished hepatoblastoma cell growth being accompanied by reduced AKT and GSK-3beta phosphorylation. Flow cytometry and 4', 6-diamidino-2-phenylindole stainings showed that PI3K pathway inhibition leads to a substantial increase in apoptosis and a decrease in cellular proliferation linked to reduced Cyclin D1 and increased p27(KIP1) levels. Simultaneous treatment of hepatoblastoma cell lines with LY294002 and cytotoxic drugs resulted in positive interactions.


CONCLUSIONS
Our findings imply that PI3K signaling plays an essential role in growth control of hepatoblastoma and might be successfully targeted in multimodal therapeutic strategies.
Hartmann
Wolfgang
W
Department of Pathology and Neuropathology, University of Bonn Medical Center, Sigmund-Freud-St. 25, Bonn D-53105, Germany. wolfgang.hartmann@uni-bonn.de
Küchler
Jan
J
Koch
Arend
A
Friedrichs
Nicolaus
N
Waha
Anke
A
Endl
Elmar
E
Czerwitzki
Jacqueline
J
Metzger
Dagmar
D
Steiner
Susanne
S
Wurst
Peter
P
Leuschner
Ivo
I
von Schweinitz
Dietrich
D
Buettner
Reinhard
R
Pietsch
Torsten
T
D000293:Adolescent; D000328:Adult; D017209:Apoptosis / Q000187:drug effects; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D002648:Child; D002675:Child, Preschool; D002867:Chromones / Q000494:pharmacology; D002945:Cisplatin / Q000494:pharmacology; D004357:Drug Synergism; D005434:Flow Cytometry; D038362:Glycogen Synthase Kinase 3 / Q000378:metabolism; D000071679:Glycogen Synthase Kinase 3 beta; D018197:Hepatoblastoma / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D006801:Humans; D007150:Immunohistochemistry; D008875:Middle Aged; D009025:Morpholines / Q000494:pharmacology; D009154:Mutation; D009687:Nuclear Proteins / Q000037:antagonists & inhibitors / Q000235:genetics*; D010766:Phosphorylation / Q000187:drug effects; D018807:Polymorphism, Single-Stranded Conformational; D011494:Protein Kinases / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism*; D034622:RNA Interference; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D058570:TOR Serine-Threonine Kinases; D014157:Transcription Factors / Q000037:antagonists & inhibitors / Q000235:genetics*; D055815:Young Adult
D002867:Chromones; D009025:Morpholines; D009687:Nuclear Proteins; C503859:PI3KCA protein, human; D014157:Transcription Factors; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; D011494:Protein Kinases; C546842:MTOR protein, human; C000605506:GSK3B protein, human; D000071679:Glycogen Synthase Kinase 3 beta; D051057:Proto-Oncogene Proteins c-akt; D058570:TOR Serine-Threonine Kinases; D038362:Glycogen Synthase Kinase 3; D002945:Cisplatin