β-Catenin Knockdown in Liver Tumor Cells by a Cell Permeable Gamma Guanidine-based Peptide Nucleic Acid
Jung M. Lee,
Danith H Ly,
Satdarshan P. S. Monga.
Hepatocellular cancer (HCC) is the third cause of death by cancer worldwide. In the current study we target β-
catenin, an oncogene mutated and constitutively active in 20-30% of HCCs, via a novel, cell permeable gamma
guanidine-based peptide nucleic acid (γGPNA) antisense oligonucleotide designed against either the transcription or the
translation start site of the human β-catenin gene. Using TOPflash, a luciferase reporter assay, we show that γGPNA
targeting the transcription start site showed more robust activity against β-catenin activity in liver tumor cells that harbor
β-catenin gene mutations (HepG2 & Snu-449). We identified concomitant suppression of β-catenin expression and of
various Wnt targets including glutamine synthetase (GS) and cyclin-D1. Concurrently, γGPNA treatment reduced
proliferation, survival and viability of HCC cells. Intriguingly, an angiogenesis quantitative Real-Time-PCR array
identified decreased expression of several pro-angiogenic secreted factors such as EphrinA1, FGF-2, and VEGF-A upon
β-catenin inhibition in liver tumor cells. Conversely, transfection of stabilized-β-catenin mutants enhanced the expression
of angiogenic factors like VEGF-A. Conditioned media from HepG2 cells treated with β-catenin but not the mismatch
γGPNA significantly diminished spheroid and tubule formation by SK-Hep1 cells, an HCC-associated endothelial cell
line. Thus, we report a novel class of cell permeable and efficacious γGPNAs that effectively targets β-catenin, a known
oncogene in the liver. Our study also identifies a novel role of β-catenin in liver tumor angiogenesis through paracrine
mechanisms in addition to its roles in proliferation, survival, metabolism and cancer stem cell biology, thus further
strengthening its effectiveness as a therapeutic target in HCC.
Keywords: Angiogenesis, β-Catenin, liver cancer, proliferation and antisense, Wnt signaling.
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