Abstract
Abstract
The incidence of hepatocellular carcinoma (HCC) is rising in the US with parallel increase in mortality rate. Lack of effective therapy for advanced HCC mandates development of novel targeted therapies to counteract this fatal malady. Astrocyte elevated gene-1 (AEG-1), also known as metadherin (MTDH) and LYRIC, plays a pivotal role in hepatocarcinogenesis and serves as an ideal target for anti-HCC therapy. AEG-1 interacts with retinoid X receptor (RXR) and inhibits retinoic acid-induced gene expression and cell death. Retinoic acid has been evaluated for HCC treatment without promising result. Overexpression of AEG-1 might underlie the poor performance of retinoic acid in HCC clinical trials. We documented that combination of a lentivirus expressing AEG-1 shRNA (lenti.shAEG-1) and all-trans retinoic acid (ATRA) profoundly and synergistically inhibited subcutaneous human HCC xenografts in nude mice. We now have developed liver-targeted nanoplexes by conjugating poly(amidoamine) (PAMAM) dendrimers with polyethylene glycol (PEG) and lactobionic acid (PAMAM-PEG-Gal) which were complexed with AEG-1 siRNA (PAMAM-AEG-1si). PAMAM has a net positive charge that complexes with the net negative charge of the siRNA backbone, PEG increases stability of the nanoplexes and increases circulation time and the galactose binds to asialoglycoprotein receptors that are upregulated on liver cells. The polymer conjugate was characterized by 1H-NMR and optimal nanoplex formulations were characterized for surface charge and size using a zetasizer Nano ZS. We established orthotopic xenografts of human HCC cell QGY-7703 expressing luciferase (QGY-luc) in the livers of athymic nude mice and monitored tumor development by bioluminescence imaging (BLI). One week after tumor establishment mice were treated with PAMAM-siCon, PAMAM-siCon+ATRA, PAMAM-AEG-1si and PAMAM-AEG-1si+ATRA by 8 i.v. injections over 4 weeks, and sacrificed 2 weeks after the last injection. In the control group the tumor developed aggressively. ATRA showed little effect due to high AEG-1 levels in QGY-luc cells. PAMAM-AEG-1si showed significant reduction in tumor growth and the combination of PAMAM-AEG-1si+ATRA showed profound and synergistic inhibition so that the tumors were almost undetectable by BLI. Measurement of liver weight at the end of the experiment corroborated these findings. Analysis of AEG-1 mRNA in tumor samples showed a marked decrease in AEG-1 level by PAMAM-AEG-1si indicating efficacy of in vivo knockdown. The group treated with PAMAM-AEG-1si+ATRA nanoplexes showed increased necrosis, inhibition of proliferation and increased apoptosis when compared to other groups. Liver is an ideal organ for RNAi therapy and ATRA is an approved anti-cancer agent. Our exciting observations suggest that the combinatorial approach might be an effective way to combat HCC and need to be evaluated stringently in endogenous mouse models of HCC for a potential Phase I/II clinical trial.
Citation Format: Jyoti Srivastava, Devaraja Rajasekaran, Ayesha Siddiq, Rachel Gredler, Chadia L. Robertson, Maaged A. Akiel, Xue-Ning Shen, Kareem A.N. Ebeid, Aliasger K. Salem, Paul B. Fisher, Devanand Sarkar. A novel combinatorial therapy for hepatocellular carcinoma (HCC). [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5400. doi:10.1158/1538-7445.AM2015-5400