Activation of Phosphatidylinositol-3′-kinase/AKT Signaling Is Essential in Hepatoblastoma Survival

Abstract

Purpose: 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. Experimental Design: Immunohistochemical stainings of (Ser473)-phosphorylated (p)-AKT protein, its targets p-(Ser9)-GSK-3β and p-(Ser2448)-mTOR, as well as the cell cycle regulators Cyclin D1, p27KIP1, and p21CIP1 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-3β 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-3β, and p-mTOR; subgroups showed significant Cyclin D1, p27KIP1, and p21CIP1 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-3β 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 p27KIP1 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.

The genetic and epigenetic landscapes of hepatoblastomas

“…In addition, mutations were described in the PIK3CA gene, and in growth factors of this signaling pathway, such as IGF2, showing altered expression in this tumor type [59].…”

Therapeutic Innovations for Targeting Hepatoblastoma

“…This pathway is often deregulated in cancer cells (45) as most of its proteins are tumor suppressor genes or proto-oncogenes whose mutation or anarchic expression can promote the development of a tumor (46,47).…”

“…It induces the activation of many intracellular proteins through cascades of phosphorylations (48). A study conducted by Hartmann et al (45) revealed that point mutation of the effector PI3KCA (p110α subunit of PI3K) was present in 2% of hepatoblastomas resulting in a gain of kinase activity. Moreover, they found that PI3K/AKT/mTOR activation was actually central for hepatoblastoma survival as demonstrated by PI3K inhibition in vitro and subsequent reduction of AKT and GSK3β phosphorylation (45).…”

Frequent epigenetic inactivation of the chaperone SGNE1/7B2 in human gliomas

“…For flow cytometric immunophenotyping, 3 Â 10 6 cells were fixed and stained using an Alexa Fluor 647 labeled phospho-(Ser10)-Histone H3 antibody (Cell Signaling Technology; 1:20) and a PE labeled cleaved-Poly(ADP-ribose)-polymerase (PARP; Asp214) antibody (BD Biosciences; 1:5) as well as DAPI (Sigma) to stain DNA as described. 22 FACS analyses were performed using a three-laser LSRII analytical flow cytometer (BD Biosciences, Heidelberg, Germany). Data were analyzed using Flowjo (Tree Star, OR) analysis software.…”