Tumor-targeted Nanoparticle Delivery of HuR siRNA Inhibits Lung Tumor Growth In Vitro and In Vivo By Disrupting the Oncogenic Activity of the RNA-binding Protein HuR

Abstract

Selective downregulation of the human antigen R (HuR) protein by siRNA may provide a powerful approach for treating lung cancer. To this end, we investigated the efficacy of transferrin receptor-targeted liposomal nanoparticle-based HuR siRNA (HuR-TfNP) therapy and compared with control siRNA (C)-TfNP therapy both, in vitro and in vivo using lung cancer models. In vitro studies showed HuR-TfNP, but not C-TfNP, efficiently downregulated HuR and HuR-regulated proteins in A549, and HCC827 lung cancer cells, resulting in reduced cell viability, inhibition of cell migration and invasion, and induction of G₁ cell-cycle arrest culminating in apoptosis. However, HuR-TfNP activity in normal MRC-9 lung fibroblasts was negligible. In vivo biodistribution study demonstrated that fluorescently labeled HuR-siRNA or ICG dye-loaded TfNP localized in tumor tissues. Efficacy studies showed intratumoral or intravenous administration of HuR-TfNP significantly inhibited A549 (>55% inhibition) and HCC827 (>45% inhibition) subcutaneous tumor growth compared with C-TfNP. Furthermore, HuR-TfNP treatment reduced HuR, Ki67, and CD31 expression and increased caspase-9 and PARP cleavage and TUNEL-positive staining indicative of apoptotic cell death in tumor tissues compared with C-TfNP treatment. The antitumor activity of HuR-TfNP was also observed in an A549-luc lung metastatic model, as significantly fewer tumor nodules (9.5 ± 3.1; P < 0.001; 88% inhibition) were observed in HuR-TfNP-treated group compared with the C-TfNP-treated group (77.7 ± 20.1). Significant reduction in HuR, Ki67, and CD31 expression was also observed in the tumor tissues of HuR-TfNP-treatment compared with C-TfNP treatment. Our findings highlight HuR-TfNP as a promising nanotherapeutic system for lung cancer treatment. Mol Cancer Ther; 16(8); 1470-86. ©2017 AACR.

Figure 1. Tumor-targeted HuR silencing suppresses lung tumor cell growth

A. Schematic illustration of HuR-siRNA-loaded, Tf-modified liposome (HuR-TfNP) preparation. B. Assessment of transfection efficiency of fluorescently-labeled siRNA (siGLO-) TfNP in lung cancer cell lines (A549, HCC827) and normal lung fibroblasts (MRC-9) showed a time-dependent increase in uptake of the NPs by tumor cells compared to normal cells. Inset shows TfR expression is higher in tumor compared to normal cells. C. Cell growth inhibition induced by HuR-TfNP (targeted delivery) compared with HuR-NP (non- targeted delivery) 24 h after treatment of A549 cells. D. Western blot evaluation of HuR and HuR-regulated proteins 24 h after HuR-TfNP or HuR-NP treatment in A549 cells. The quantification of protein bands (Test protein/Actin ratio) is shown as a bar graph at the bottom. * p<0.05, ** p<0.001.

Figure 2. HuR knockdown reduces HuR-target proteins in tumor cells but not normal cells

A. HuR and HuR-regulated protein expressions in A549, HCC827, and MRC-9 cells treated with C-TfNP or HuR-TFNP at 24 h and 48 h after treatment. Among A549 and HCC 827 groups, HuR-TfNP treatment resulted in better knockdown of HuR protein expression in A549, whereas in MRC-9 when compared to both A549 and HCC827 cancer cells HuR-TfNP induced negligible HuR protein inhibition. HuR knockdown also altered the expression of downstream proteins like BCl2, cyclin D1, P27, and cyclin E in tumor cells but not in normal cells. B. Successful knockdown of HuR resulted in significant growth inhibition in lung cancer cells but not in normal cells, at both 24 h and 48 h after treatment. C. HuR-TfNP induces G1 phase arrest in A549 and HCC827 cells, but not in MRC-9 cells. * p<0.05, “NS” – not significant.

Figure 3. HuR-TfNP inhibits tumor cell migration and invasion

Representative images of A, migration and B, invasion assay for A549 tumor cells are shown for C-TfNP and HuR-TfNP treatment groups. Cells that did not receive any treatment served as controls. Quantification of migration and invasion assays expressed by cell counting of respective assays is also presented. * p<0.05, ** p<0.001, “NS” – not significant.

Figure 4. In vivo biodistribution studies

A. IVIS^® Spectrum live images of (a) A549 and (b) HCC827 tumor bearing mice obtained at different time intervals after i.v. injection of Indocyanin green (ICG)-TfNP showed accumulation in the tumor by 24 h. (c) I.v. administration of fluorescent HuR siRNA loaded nanoparticle (DY-647HuR-TfNP) showed NP accumulation in the tumor by 24 h. White circle indicates the location of the tumor on the lower right flank. B. Graphical representation of siRNA accumulation in tumor tissues of A549 and HCC827 mice models as measured by Digital Droplet PCR at 24 h after treatment. Sample # 1 and # 2 represent tumor tissue from two mice of each treatment group analyzed. C. Individual luminescent (A549 luc), fluorescent (Dy647HuR-TfNP), and overlaid images showing localization of DY647HuR siRNA in A549 lung tumors. * p<0.05, ** p<0.001.

Figure 5. HuR-TfNP treatment suppresses lung tumor growth inhibition in vivo

A, A549 and, B. HCC827 subcutaneous tumor-bearing mice were treated intratumorally with C-TfNP or HuR-TfNP for 6 doses over two weeks of time period and tumor growth measured. Inset shows HuR expression in tumors after two treatments. Arrows denote treatments. In vivo tumor growth inhibition profile, representative tumors harvested from mice sacrificed on final day of experiment, immunohistochemistry, and Hematoxylin & eosin stained tumor tissue sections from sacrificed mice on final day of the experiment are shown. C. Mice bearing subcutaneous A549-luc tumors were treated i.v. with C-TfNP or HuR-TfNP for 6 doses over two weeks of time period and tumor growth measured by calipers and by bioluminescent imaging. A significant inhibition in tumor growth was observed in HuR-TfNP-treated mice compared to C-TfNP treatment. D. Representative gross tumor size harvested from C-TfNP- and HuR-TfNP-treated mice sacrificed on final day of experiment, and western blotting for HuR in two tumor samples (# 1 and # 2) representative of each treatment group analyzed after two treatments is shown. E. Mice bearing subcutaneous HCC287 tumors were treated i.v. with -TfNP or HuR-TfNP for 6 doses over two weeks of time period. A significant inhibition in tumor growth was observed in HuR-TfNP-treated mice compared to C-TfNP treatment. F. Representative tumors harvested from C-TfNP and HuR-TfNP-treated mice sacrificed on final day of experiment, and western blotting for HuR in tumors harvested from treated are shown. Arrows denote treatments. * p<0.05, ** p<0.001.

Figure 6. HuR-TfNP treatment suppresses experimental lung metastasis

Mice bearing A549-luc lung tumors were treated i.v. with C-TfNP or HuR-TfNP for a total of six treatments. Mice were imaged at regular intervals to monitor and measure tumor growth. A. Representative bioluminescence images showing inhibition of experimental lung metastasis in HuR-TfNP-treated mice compared to C-TfNP-treated mice. Bar graph represents the quantitation of bioluminescence intensity as Average Radiance [p/s/cm²/sr]. The gradual decrease in the bioluminescence signals in HuR-TfNP compared to C-TfNP groups suggests significant reduction in tumor growth over the period of treatment. B. Western blot image shows reduced HuR expression in HuR-TfNP-treated A549 lung tumors (upper panel). Sample # 1 and # 2 are representative samples for each treatment group analyzed at end of the study. Lung tumor nodules of a representative animal from each treatment group are also shown (middle panel). Animals were euthanized after six weeks; lungs were inflated with bovine solution and the number of extrapulmonary tumor nodules counted (lower panel). A significant reduction in the number of lung nodules were observed in HuR-TfNP-treated animals compared to control. C. Images represent the IHC staining of lung sections for HuR, CD31, and Ki67. Hematoxylin and eosin (H&E) stain showing the tissue pathology with dashed circle marking tumors in the lung. Bar graph represents the quantitation of IHC stains using. * p<0.05, ** p<0.001.