Presentations

Abstract

Introduction: Prostate cancer (PCa) is the most prevalent male-related cancer and the second leading cause of male cancer-related deaths in the United States. Approximately 20% of men diagnosed with PCa progress to metastatic disease, of which the 5-year survival rate is only 30%, indicating a need for better therapeutic options to increase patient survivorship. MiRNAs are small noncoding RNAs (~22-24 nucleotides) that have emerged as important tumor suppressors and pro-oncogenic factors of PCa and metastasis. MiRNAs negatively regulate gene expression by binding to their messenger RNA targets in a sequence-specific manner and recruiting the RNA-induced silencing complex (RISC) to the site, resulting in mRNA degradation and/or blocking protein translation. The Kerscher lab identified the miR-888 cluster of seven miRNA genes (including miR-888 and miR-891a) as increased in human metastatic PCa cells and prostatic fluids from high-grade PCa patients. We tested if miR-888 and miR-891a played functional roles as pro-oncogenic factors in the prostate. Indeed, treatment of non-aggressive human PCa cell lines with miRNA mimics to overexpress miR-888 or miR-891a resulted in increased cell proliferation and invasion. Conversely, prostate cells treated with antisense miRNA oligonucleotides (antimirs) to block miR-888 or miR-891a activity showed slowed growth and invasion. This indicated the clinical potential of antimiR drugs for advanced PCa. We aim to test a novel acidic-sensing tumor delivery reagent called pH Low Insertion Peptide (pHLIP), a 36 amino acid peptide that adopts an α-helical conformation at low pH (< pH 6.5) facilitating the insertion of its C-terminus across the cell membrane. Systemic administration of pHLIP specifically targets tumors (possessing acidic microenvironments due to high glycolytic rates) and has entered clinical trials as a breast tumor imaging biomarker. AntimiR delivery reagents synthesized by the Bahal lab for this study are composed of pHLIP conjugated to Peptide Nucleic Acid (PNA) antimir-888 or antimiR-891a. We sought to determine if pHLIP-PNA-antimiR delivery to human PCa cells in a simulated acidic tumor environment blocked cell growth and invasion. We predicted that co-treatment of antimir-888 and antimir-891a reagents would have additive or synergistic effects in vitro.

Methods: 2X105 PC3-ML (metastatic, castration-resistant) or LNCaP (low malignancy, hormone-sensitive) cells were plated per well of a 6-well plate. The next day, cells were treated with 4 uM pHLIP-PNA antimir-888, antimir-891a, or in combination in pH 6.0 serum-free DMEM media for 3 hours. As a negative control, cells were also treated with 4 uM pHLIP-PNA antimir-67 (NC67), which targets C. elegans miR-67 that shares no homology in humans. Cells were washed in PBS and recovered for 24 hours in pH 7.4 serum-free DMEM media. For Proliferation Assays; 3000 PC3-ML and LNCaP cells were plated per well of a 96-well plate. Four hours prior to the 24-, 48-, 72-, and 96-hour time points, the WST-1 reagent (Roche) was added to the cells. Cleavage of colorless tetrazolium salt WST-1 to purple formazan by cellular enzymes is proportional to the number of viable cells in the well. Absorbance was measured with a BioTek Synergy HT plate reader (450 nm, 620 nm). Results were quantified in triplicate. For Invasion Assays, 3X105 PC3-ML cells in 500 µL of media with 0.5% FBS media were added to the upper portion of a Boyden Matrigel chamber and traversed across Matrigel and an 0.8 mm polycarbonate insert towards the chemoattractant (15% FBS) for 48 hrs. Cells that traversed the insert were fixed in 4% paraformaldehyde, permeabilized in methanol, stained with 1% crystal violet, and photographed. Wells were destined with 10% acetic acid, 30% methanol. Results were quantified in triplicate (BioTek, 595 nm).

Results: Individual treatment of antimiR-888 and anti-miR-891a to PC3-ML cells showed significantly decreased proliferation and invasion compared to controls. Drug treatment of LNCaP cells showed similar inhibitory growth effects implying that these reagents are effective on hormone-sensitive and castration-resistant PCa cells. Cotreatment of 4 uM antimiR-888 and 4 uM antimiR-891a reagents did not show additive or synergistic effects.

Conclusion: Our results indicated that these novel acidic-sensing pHLIP reagents to inactivate miR-888 and miR-891a show clinical promise in reducing tumor load and metastasis. More work needs to be done to verify the efficacy of combination treatment, and we will test if reducing treatment concentrations to 2 uM antimiR-888 and 2 uM antimiR-891a results in better anti-proliferative effects. This work will lead to new strategies to improve PCa patient survival.