Abstract 5418: Tumor-targeted nanoparticle delivery of HuR-RNAi suppresses lung cancer cell proliferation and cell migration

Abstract

Abstract Objective. HuR is an mRNA-binding protein that specifically binds to AU rich (ARE) sites at the 3′end of the mRNA of several growth factors, cell-cycle regulators, and transcription-regulating proteins and transports the mRNA to the cytoplasm for protein translation. HuR overexpression has been demonstrated to be a poor prognostic marker in a spectrum of human cancers and correlate with chemotherapy resistance. On the basis of these reports we hypothesized HuR is a druggable target for cancer therapy and its inhibition will down-regulate multiple oncoproteins resulting in efficient tumor cell killing. To test our hypothesis, we developed and tested the efficacy of a nanoparticle (NP) containing HuR-specific siRNA and targeted towards folate receptor (Fr) overexpressing lung cancer cells in vitro. Methods. Human lung cancer (H1299, A549) and normal fibroblast (MRC-9) cell lines were used in the present study. Folate receptor (Fr) overexpressing KB cell line was used as positive control. Tumor targeted nanoparticle was synthesized by mixing cationic lipid (DOTAP) with a neutral lipid (cholesterol) in equimolar ratio. The NPs thus formed were used to encapsulate fluorescently labeled siRNA for determining transfection efficiency; scrambled (control) siRNA or HuR-specific siRNA (100 nm) for efficacy studies. The control- and HuR-siRNA-containing NPs were subsequently decorated with DSPE-PEG5000-folate and labeled as C-FNP and HuR-FNP respectively. Transfection efficiency, NP specificity, cell viability, cell migration and HuR knock down studies were performed using standard laboratory assay protocols. Results. HuR protein expression in H1299 and A549 was higher than in MRC-9 cell line. Folate receptor (Fr) expression from highest to lowest was in the following order KB> H1299>MRC9. Fr expression was not detected in A549 cells. Transfection efficiency study showed FNP uptake was highest in KB cells followed by H1299 cells with lowest uptake by A549 cells. Addition of exogenous folic acid resulted in reduced FNP uptake by H1299 cells demonstrating NP specificity towards Fr. Treatment of H1299 cells with HuR-FNP significantly reduced cell viability at 48h compared to C-FNP-treatment and correlated with a marked reduction in HuR mRNA and protein expression levels. Analysis for proteins whose mRNAs are targets for and regulated by HuR (Bcl2, Cyclin D1, HIF-1α) showed diminished expression in HuR-FNP-treated cells. Finally, cell migration was significantly inhibited in HuR-FNP-treated H1299 cells compared to C-FNP treatment. Conclusions. Tumor-targeted nanoparticle delivery of HuR-RNAi in lung cancer cells selectively inhibited HuR and HuR-regulated oncoproteins resulting in diminished cell proliferation and cell migration in vitro. Acknowledgement. This study was funded by a grant (R01CA167516-01) from the National Cancer Institute. Citation Format: Ranganayaki Muralidharan, Anish Babu, Kanthesh Basalingappa, Anupama Munshi, Rajagopal Ramesh. Tumor-targeted nanoparticle delivery of HuR-RNAi suppresses lung cancer cell proliferation and cell migration. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5418. doi:10.1158/1538-7445.AM2014-5418