Abstract
Introduction
Metastatic prostate cancer (PCa) represents an unmet treatment need, with a 5-year survival rate of only 30%. MicroRNAs (miRNAs) are small ~24-nucleotide noncoding RNAs with potential as clinical tools for PCa. The Kerscher lab identified miR-888 and miR-891a as preferentially enriched in metastatic PCa cell lines and prostatic fluids from patients with high-grade PCa. These miRNAs promote PCa growth and aggressiveness in vitro and tumor growth in mice, while inhibition reverses these activities, highlighting their potential as anti-miRNA targets for PCa. Synthetic antisense oligonucleotides designed to bind and sequester miRNAs (antimiRs) are valuable tools for inhibiting miRNA function, but current antimiR therapies require high doses and suffer from poor tissue specificity. To improve tumor-specific antimiR delivery and efficacy, we collaborated with Dr. Raman Bahal (UConn) to test peptide nucleic acid (PNA) antimiRs targeting miR-888 and miR-891a conjugated to the acid-sensing pH low insertion peptide (pHLIP). This work tested the hypothesis that pHLIP-PNA antimiRs would facilitate delivery to PCa cells under acidic conditions and to prostate tumors in vivo, resulting in inhibition of aggressive phenotypes and tumor growth. This work also evaluated the potential of miR-888/891a as targets for treatment in the neuroendocrine (NE) PCa pathology, which represents a major unmet need.
Methods
qRT-PCR was carried out to establish miR-888/891a expression levels across a panel of PCa cell lines including RWPE-1 (nonmalignant), LNCaP (low aggression), PC3-N (low aggression), PC3-ML (high aggression), NCI-H660 (low aggression, NE), and LASCPC-01(high aggression, NE). Cell culture assays were performed to evaluate the effects of pHLIP-PNA-antimiR-888/891a treatment on PC3-ML proliferation (WST-1) and anchorage-independent growth (soft agar). NOD/SCID mice were inoculated with PC3-ML cells; following tumor formation, mice were injected via tail vein with either CY5-labeled pHLIP delivery peptide (1 mg/kg) or pHLIP-PNA-antimiR-888/891a (1 mg/kg). pHLIP-CY5-injected mice were sacrificed 24h post-injection and organs and tumors were harvested and imaged to determine drug localization. Mice receiving pHLIP-PNA-antimiR treatment were monitored for tumor growth rate until day 25 of the study, after which they were sacrificed. Organs and tumors were harvested for downstream analysis. qRT-PCR was carried out to determine tumor miR-888/891a expression and western blotting was carried out to determine expression levels of miR-888/891a target TIMP2.
Results
qRT-PCR analysis showed that miR-888/891a expression increases in step with aggressiveness, with NE lines showing highly elevated expression. AntimiR-888 alone had minimal effects (contrary to previous experiments with other batches), indicating potential variability in reagent potency, whereas combined antimiR-888/891a treatment produced additive growth inhibition. Colony formation assays showed similar trends for individual antimiRs, while combined treatment demonstrated synergy (Bliss analysis, p0.001). In vivo, Cy5-pHLIP efficiently localized to PC3-ML xenograft tumors, with additional retention in kidney and liver.
AntimiR-891a showed a nonsignificant trend toward reduced overall tumor burden (Mann-Whitney U, p=0.190), while linear mixed-effects modeling demonstrated significantly reduced tumor growth rate over time (p=0.00847).
Conclusion
Together, these studies demonstrate the feasibility of pHLIP-mediated PNA-antimiR delivery to prostate tumors and provide evidence that targeting miR-891a can inhibit aggressive PCa phenotypes and tumor growth, supporting further reagent development for advanced and castration-resistant PCa.