title: Folate receptor-targeted nanoparticle delivery of HuR-RNAi suppresses lung cancer cell proliferation and migration.
abstract: BACKGROUND
  Human antigen R (HuR) is an RNA binding protein that is overexpressed in many human cancers, including lung cancer, and has been shown to regulate the expression of several oncoproteins. Further, HuR overexpression in cancer cells has been associated with poor-prognosis and therapy resistance. Therefore, we hypothesized that targeted inhibition of HuR in cancer cells should suppress several HuR-regulated oncoproteins resulting in an effective anticancer efficacy. To test our hypothesis, in the present study we investigated the efficacy of folate receptor-α (FRA)-targeted DOTAP:Cholesterol lipid nanoparticles carrying HuR siRNA (HuR-FNP) against human lung cancer cells.
  
  
  RESULTS
  The therapeutic efficacy of HuR-FNP was tested in FRA overexpressing human H1299 lung cancer cell line and compared to normal lung fibroblast (CCD16) cells that had low to no FRA expression. Physico-chemical characterization studies showed HuR-FNP particle size was 303.3 nm in diameter and had a positive surface charge (+4.3 mV). Gel retardation and serum stability assays showed that the FNPs were efficiently protected siRNA from rapid degradation. FNP uptake was significantly higher in H1299 cells compared to CCD16 cells indicating a receptor-dose effect. The results of competitive inhibition studies in H1299 cells demonstrated that HuR-FNPs were efficiently internalized via FRA-mediated endocytosis. Biologic studies demonstrated HuR-FNP but not C-FNP (control siRNA) induced G1 phase cell-cycle arrest and apoptosis in H1299 cells resulting in significant growth inhibition. Further, HuR-FNP exhibited significantly higher cytotoxicity against H1299 cells than it did against CCD16 cells. The reduction in H1299 cell viability was correlated with a marked decrease in HuR mRNA and protein expression. Further, reduced expression of HuR-regulated oncoproteins (cyclin D1, cyclin E, and Bcl-2) and increased p27 tumor suppressor protein were observed in HuR-FNP-treated H1299 cells but not in C-FNP-treated cells. Finally, cell migration was significantly inhibited in HuR-FNP-treated H1299 cells compared to C-FNP.
  
  
  CONCLUSIONS
  Our results demonstrate that HuR is a molecular target for lung cancer therapy and its suppression using HuR-FNP produced significant therapeutic efficacy in vitro.
authors.affiliation: Departments of Pathology, University of Oklahoma Health Sciences Center, Oklahoma City, OK, 73104, USA.|Stephenson Cancer Center, University of Oklahoma Health Sciences Center, Oklahoma City, OK, 73104, USA.
authors.forename: Ranganayaki
authors.initials: R
authors.lastname: Muralidharan
authors.affiliation: Departments of Pathology, University of Oklahoma Health Sciences Center, Oklahoma City, OK, 73104, USA.|Stephenson Cancer Center, University of Oklahoma Health Sciences Center, Oklahoma City, OK, 73104, USA.
authors.forename: Anish
authors.initials: A
authors.lastname: Babu
authors.affiliation: Departments of Pathology, University of Oklahoma Health Sciences Center, Oklahoma City, OK, 73104, USA.|Stephenson Cancer Center, University of Oklahoma Health Sciences Center, Oklahoma City, OK, 73104, USA.
authors.forename: Narsireddy
authors.initials: N
authors.lastname: Amreddy
authors.affiliation: Departments of Radiation Oncology, University of Oklahoma Health Sciences Center, Oklahoma City, OK, 73104, USA.|Stephenson Cancer Center, University of Oklahoma Health Sciences Center, Oklahoma City, OK, 73104, USA.
authors.forename: Kanthesh
authors.initials: K
authors.lastname: Basalingappa
authors.affiliation: Departments of Radiation Oncology, University of Oklahoma Health Sciences Center, Oklahoma City, OK, 73104, USA.|Stephenson Cancer Center, University of Oklahoma Health Sciences Center, Oklahoma City, OK, 73104, USA.
authors.forename: Meghna
authors.initials: M
authors.lastname: Mehta
authors.affiliation: Departments of Epidemiology and Statistics, University of Oklahoma Health Sciences Center, Oklahoma City, OK, 73104, USA.
authors.forename: Allshine
authors.initials: A
authors.lastname: Chen
authors.affiliation: Departments of Epidemiology and Statistics, University of Oklahoma Health Sciences Center, Oklahoma City, OK, 73104, USA.|Stephenson Cancer Center, University of Oklahoma Health Sciences Center, Oklahoma City, OK, 73104, USA.
authors.forename: Yan Daniel
authors.initials: YD
authors.lastname: Zhao
authors.affiliation: Department of Pharmaceutical Sciences and Opthalmology, University of Colorado, Denver, CO, 80045, USA.
authors.forename: Uday B
authors.initials: UB
authors.lastname: Kompella
authors.affiliation: Departments of Radiation Oncology, University of Oklahoma Health Sciences Center, Oklahoma City, OK, 73104, USA.|Stephenson Cancer Center, University of Oklahoma Health Sciences Center, Oklahoma City, OK, 73104, USA.
authors.forename: Anupama
authors.initials: A
authors.lastname: Munshi
authors.affiliation: Departments of Pathology, University of Oklahoma Health Sciences Center, Oklahoma City, OK, 73104, USA. rajagopal-ramesh@ouhsc.edu.|Stephenson Cancer Center, University of Oklahoma Health Sciences Center, Oklahoma City, OK, 73104, USA. rajagopal-ramesh@ouhsc.edu.|Graduate Program in Biomedical Sciences, University of Oklahoma Health Sciences Center, Oklahoma City, OK, 73104, USA. rajagopal-ramesh@ouhsc.edu.|Department of Pathology, Stanton L. Young Biomedical Research Center, Suite 1403, 975 N.E., 10th Street, Oklahoma City, OK, 73104, USA. rajagopal-ramesh@ouhsc.edu.
authors.forename: Rajagopal
authors.identifier: http://orcid.org/0000-0002-7658-5338
authors.initials: R
authors.lastname: Ramesh
mesh_terms: D045744:Cell Line, Tumor; D002465:Cell Movement; D049109:Cell Proliferation; D002784:Cholesterol / Q000737:chemistry / Q000378:metabolism; D016503:Drug Delivery Systems; D000067780:ELAV-Like Protein 1 / Q000235:genetics*; D058975:Folate Receptor 1 / Q000235:genetics / Q000378:metabolism*; D005492:Folic Acid / Q000737:chemistry / Q000378:metabolism; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D008168:Lung / Q000378:metabolism / Q000473:pathology; D008175:Lung Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology / Q000628:therapy*; D053758:Nanoparticles / Q000737:chemistry / Q000378:metabolism; D034741:RNA, Small Interfering / Q000008:administration & dosage / Q000235:genetics / Q000627:therapeutic use*; D000067492:RNAi Therapeutics
chemical_list: D000067780:ELAV-Like Protein 1; C098770:ELAVL1 protein, human; D058975:Folate Receptor 1; D034741:RNA, Small Interfering; D005492:Folic Acid; D002784:Cholesterol
keywords: Folate; HuR; Lung cancer; Nanoparticle; Oncoprotein; Targeting; siRNA