Endowed Chair for Experimental Pathology, Director- Division of Experimental Pathology (EP), Professor of Pathology (EP) & Medicine (GI, Hepatology and Nutrition), University of Pittsburgh School of Medicine, 200 Lothrop Street S-422 BST, Pittsburgh, PA 15261, USA.
Curr Cancer Drug Targets. 2013 Oct;13(8):867-78. doi: 10.2174/15680096113139990081.
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.
肝细胞癌（HCC）是全球癌症死亡的第三大原因。在目前的研究中，我们针对β-连环蛋白（β-catenin），该基因在 20-30%的 HCC 中发生突变且持续活跃，通过一种新的、细胞可渗透的基于γ-胍基的肽核酸（γGPNA）反义寡核苷酸，针对人类β-连环蛋白基因的转录或翻译起始位点设计。使用 TOPflash，一种荧光素酶报告基因检测，我们表明针对转录起始位点的γGPNA 在携带β-连环蛋白基因突变的肝肿瘤细胞（HepG2 和 Snu-449）中对β-连环蛋白活性显示出更强的活性。我们发现同时抑制β-连环蛋白表达和各种 Wnt 靶标，包括谷氨酰胺合成酶（GS）和细胞周期蛋白 D1。同时，γGPNA 处理降低了 HCC 细胞的增殖、存活和活力。有趣的是，血管生成定量实时 PCR 阵列鉴定出在肝肿瘤细胞中抑制β-连环蛋白后，几种促血管生成分泌因子的表达下调，如 EphrinA1、FGF-2 和 VEGF-A。相反，稳定的β-连环蛋白突变体的转染增强了血管生成因子如 VEGF-A 的表达。用β-连环蛋白而非错配γGPNA 处理的 HepG2 细胞的条件培养基显著减少了 SK-Hep1 细胞（一种与 HCC 相关的内皮细胞系）的球体和小管形成。因此，我们报告了一类新型的细胞可渗透且有效的γGPNAs，可有效靶向β-连环蛋白，这是肝脏中的已知癌基因。我们的研究还确定了β-连环蛋白在肝肿瘤血管生成中的新作用，通过旁分泌机制，除了其在增殖、存活、代谢和癌症干细胞生物学中的作用外，进一步加强了其作为 HCC 治疗靶点的有效性。
