Login to Access Video or Poster Abstract: MP83-15
Sources of Funding: This work was supported by National Institutes of Health Grants U01CA166905 and U01CA152758 (to C.C. and D.P.) and the Pelotonia Intramural Research Program (D.P.).

Introduction

Recent studies have shed light on the role of microRNAs (miRs) in prostate cancer (PCa). miR-21 is among the most frequently deregulated miRs in cancer, and has been implicated in a castration-resistant prostate cancer (CRPC) phenotype. In addition to miR-21's many oncogenic effects, a positive feedback loop between miR-21 and the androgen receptor (AR) has been reported in PCa. Separately, miRs -221 and -222 have been shown to target tumor suppressor p27Kip1, leading to inhibition of apoptosis and aberrant proliferation. Nucleolin (NCL) is a multifunctional protein found in the nucleocytoplasm of most human cells, and is abnormally translocated to the cell membrane in cancer. One of the mechanisms by which NCL exerts its oncogenic activity is through the biogenesis of miRs -21, -221, and -222. 4LB5 is a novel single-chain fragment variable (scFv) antibody that binds preferentially to cancer cell membranes via the RNA-binding domain of NCL and inhibits production of these oncogenic miRs. The therapeutic and diagnostic potential of 4LB5 has been described in breast and hepatocellular carcinoma cells in previous studies. Given the supporting evidence, we aim to characterize the effects of 4LB5 on CRPC and compare its efficacy among androgen-dependent and androgen-independent cells.

Methods

Levels of NCL expression in PCa cell lines DU145, PC3, and LNCaP were investigated with western blot (WB). Cell surface ELISA was performed to compare 4LB5 binding across PCa cell lines. Cell survival assay was performed using 50 nM 4LB5 or control buffer. Levels of mature and precursor forms of miRs in 4LB5-treated cells were assessed using quantitative real-time polymerase chain reaction. MDA-MB-231 cells were used a positive control in all experiments.

Results

All cell lines expressed NCL as evidenced by WB, and downregulation of NCL was observed with siNCL transfection. ELISA results showed a strong exponential association between 4LB5 concentration and binding activity in DU145 (R² = 0.9972), PC3 (R² = 0.9887), and LNCaP (R² = 0.9897) cells, with more binding units observed in PC3 and DU145 compared to LNCaP. 4LB5 significantly inhibited proliferation of DU145 (p = 0.000016) and PC3 (p = 0.0074) cells, but not LNCaP (p = 0.45). Treatment with 4LB5 caused downregulation of mature forms of miRs 21, 221, and 222 with expected upregulation of precursor forms in PC3 cells, but LNCaP cells exhibited the opposite effect.

Conclusions

4LB5 binds CRPC cells and inhibits proliferation through abrogation of oncogenic miR production. 4LB5 could represent a novel therapeutic option in CRPC patients.

Funding

This work was supported by National Institutes of Health Grants U01CA166905 and U01CA152758 (to C.C. and D.P.) and the Pelotonia Intramural Research Program (D.P.).