Developing the miR-888 cluster members as an antimiRNA therapy for aggressive prostate cancer

Metastatic prostate cancer (PCa) represents an unmet treatment need, with a 5-year survival rate of only 30%. MicroRNAs (miRNAs) are small noncoding RNAs that hold therapeutic promise for PCa and act as important tumor suppressors and pro-oncogenic factors in the prostate. The Kerscher lab identified the miR-888 cluster as preferentially enriched in human metastatic PCa cell lines and prostatic fluids from patients with high-grade PCa compared to low-grade or non-cancer patients. We showed that cluster members promoted prostate growth and aggressiveness in vitro, particularly miR-888 and miR-891a, and these miRNAs accelerated tumor load in mice. Reciprocally, inactivation of miR-888 and miR-891a using antisense oligos reversed these effects, highlighting their potential as antimiR targets for PCa. AntimiRs are valuable tools for inhibiting miRNA function, but current antimiR therapies require high dosage and suffer from poor tissue specificity. To address these shortcomings, the Kerscher lab collaborated with Dr. Raman Bahal's group to test the efficacy of their acid-sensing peptide pHLIP (pH low insertion peptide) as a novel prostate tumor delivery reagent conjugated to peptide nucleic acids (PNAs) designed to inhibit miR-888 or miR-891a. We hypothesized that pHLIP-PNA-antimiRs against miR-888/-891a would allow efficient delivery to prostate tumors (with inherently acidic microenvironments) and inhibit progression to lethal disease. We also predicted more pronounced effects in castration-resistant PCa. Indeed, when endogenous miR-888/891a levels were analyzed by qRT-PCR across a panel of human PCa cell lines representing the spectrum of this disease, we noted that miR-888 & miR-891a expression was especially elevated in castration-resistant, neuroendocrine PCa lines. pHLIP-PNA-antimiR-888 and antimiR-891a reagents were further validated in vitro to reduce miRNA levels and suppress cancer phenotypes in PCa cell lines. For these experiments, PCa cells were treated with 4 uM pHLIP-PNA-antimiR or control reagents in pH 6.0 media for 3 hours, recovered for 24-48 hours, and harvested for qRT-PCR, WST-1 (proliferation), and soft agar (anchorage-independent growth) assays. As predicted, pHLIP-PNA-antimiR treatment reduced growth more effectively in metastatic PC3-ML than in indolent LNCaP cells. Ultimately, we sought to determine pHLIP-PNA-antimiR efficacy in a xenograft mouse model. We first validated that cy5-pHLIP alone efficiently targeted PC3-ML xenografts in mice, with low signal in kidney and liver. In a pilot efficacy study, we then injected PC3-ML cells into the flanks of mice. When tumor volume reached ~300 mm³, animals were injected with pHLIP-PNA-antimiR-891a or -NC67 controls by tail vein on days 1, 4, 8, and 11. This preliminary study showed that antimiR-891a reduced tumor load. These novel pHLIP-PNA-antimiR reagents demonstrate clinical promise for late-stage PCa.