TESTING THE EFFICACY OF A NOVEL ACIDIC-SENSING PHLIP DELIVERY REAGENT FOR ONCOMIR KNOCKDOWN IN PROSTATE CANCER
Poster #: 128
Session/Time: A
Author:
Alex Tyler James Cain, BS
Mentor:
Aurora Esquela Kerscher, PhD
Research Type: Basic Science
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 nucleotides) noncoding RNAs that act as tumor suppressors and pro-oncogenic factors in the prostate. We noted that miR-888 and miR-891a were enriched in human metastatic PCa cells and prostatic fluids from high-grade compared to low-grade/non-cancer patients. Non-aggressive human PCa cells overexpressing miR-888/miR-891a showed increased proliferation and invasion in vitro and accelerated prostate tumor load in mice. Conversely, prostate cells treated with antisense miRNA oligonucleotides (antimiRs) to block miR-888 or miR-891a reversed these phenotypes. AntimiRs are valuable tools for inhibiting miRNA function; however, current antimiR-based therapeutic regimens require high concentrations and suffer from poor tissue-targeting specificity. To address these shortcomings, we collaborated with chemist Dr. Raman Bahal to test the efficacy of acid-sensing peptides known as pHLIP (pH low insertion peptides) as prostate tumor delivery reagents using chemically optimized peptide nucleic acid (PNA) conjugates designed to bind/inhibit miR-888/miR-891a. pHLIP is a 36-amino acid peptide that adopts an alpha-helical conformation at low pH, facilitating membrane insertion and transport of its cargo into cells. When administered systemically, pHLIP targets solid tumors with inherently high glycolytic rates that create acidic microenvironments. We hypothesized that pHLIP-PNA antimiR conjugates against miR-888/-891a would allow efficient delivery to human PCa cells in a simulated acidic environment and result in suppressed cell growth. More pronounced effects were predicted in castration resistant versus androgen-responsive PCa lines, correlating with miRNA expression. Additionally, this technology enables simultaneous inactivation of multiple miRNAs, so we evaluated whether combined antimiR-888/891a treatment produced additive or synergistic effects. We also aimed to translate this work using a pre-clinical PC3-ML xenograft mouse model.
METHODS:
In vitro experiments: PC3-ML or LNCaP cells were treated with 4 uM pHLIP-PNA-antimiRs (or NC67 controls) in pH 6.0 media for 3 hours, recovered for 24-48 hours in pH 7.4 media, and then harvested for RNA isolation and qRT-PCR analysis. Alternatively, cells were prepared for WST-1 (proliferation) or soft agar assays (anchorage-independent growth). miR-888/891a expression was analyzed in RWPE-1, LNCaP, PC3-ML, NIH-H660, LASCPC-01 lines to correlate expression with castration resistance. Mouse experiments: PC3-ML cells were injected into flanks of male NOD SCID mice. When tumor volume reached ~300 mm³, animals were injected with cy5-pHLIP for imaging, or with pHLIP-PNAs (antimiRs-888/891a/NC67) on days 1, 4, 8, and 11, to measure efficacy.
RESULTS:
miR-888/miR-891a expression correlated with castration resistance, especially neuroendocrine CRPC. In vitro, pHLIP-PNA-antimiR-891a more effectively reduced growth in PC3-ML than LNCaP, while antimiR-888 treatment showed minimal effects. Combined antimiR treatment exhibited more potent growth phenotypes. In vivo, cy5-pHLIP efficiently targeted xenograft tumors in mice, with low signal in kidney and liver. Ongoing mouse efficacy studies suggest antimiR-891a is promising.
CONCLUSION:
Novel acid-sensing pHLIP reagents designed to inactivate miR-888 and miR-891a demonstrate clinical promise for lethal PCa. Further work is needed to address variable in vitro results. Developing luciferase sensor assays to monitor miRNA activity will be useful for batch and formulation testing. This research may improve PCa patient outcomes.
Metastatic prostate cancer (PCa) represents an unmet treatment need, with a 5-year survival rate of only 30%. MicroRNAs (miRNAs) are small (~24 nucleotides) noncoding RNAs that act as tumor suppressors and pro-oncogenic factors in the prostate. We noted that miR-888 and miR-891a were enriched in human metastatic PCa cells and prostatic fluids from high-grade compared to low-grade/non-cancer patients. Non-aggressive human PCa cells overexpressing miR-888/miR-891a showed increased proliferation and invasion in vitro and accelerated prostate tumor load in mice. Conversely, prostate cells treated with antisense miRNA oligonucleotides (antimiRs) to block miR-888 or miR-891a reversed these phenotypes. AntimiRs are valuable tools for inhibiting miRNA function; however, current antimiR-based therapeutic regimens require high concentrations and suffer from poor tissue-targeting specificity. To address these shortcomings, we collaborated with chemist Dr. Raman Bahal to test the efficacy of acid-sensing peptides known as pHLIP (pH low insertion peptides) as prostate tumor delivery reagents using chemically optimized peptide nucleic acid (PNA) conjugates designed to bind/inhibit miR-888/miR-891a. pHLIP is a 36-amino acid peptide that adopts an alpha-helical conformation at low pH, facilitating membrane insertion and transport of its cargo into cells. When administered systemically, pHLIP targets solid tumors with inherently high glycolytic rates that create acidic microenvironments. We hypothesized that pHLIP-PNA antimiR conjugates against miR-888/-891a would allow efficient delivery to human PCa cells in a simulated acidic environment and result in suppressed cell growth. More pronounced effects were predicted in castration resistant versus androgen-responsive PCa lines, correlating with miRNA expression. Additionally, this technology enables simultaneous inactivation of multiple miRNAs, so we evaluated whether combined antimiR-888/891a treatment produced additive or synergistic effects. We also aimed to translate this work using a pre-clinical PC3-ML xenograft mouse model.
METHODS:
In vitro experiments: PC3-ML or LNCaP cells were treated with 4 uM pHLIP-PNA-antimiRs (or NC67 controls) in pH 6.0 media for 3 hours, recovered for 24-48 hours in pH 7.4 media, and then harvested for RNA isolation and qRT-PCR analysis. Alternatively, cells were prepared for WST-1 (proliferation) or soft agar assays (anchorage-independent growth). miR-888/891a expression was analyzed in RWPE-1, LNCaP, PC3-ML, NIH-H660, LASCPC-01 lines to correlate expression with castration resistance. Mouse experiments: PC3-ML cells were injected into flanks of male NOD SCID mice. When tumor volume reached ~300 mm³, animals were injected with cy5-pHLIP for imaging, or with pHLIP-PNAs (antimiRs-888/891a/NC67) on days 1, 4, 8, and 11, to measure efficacy.
RESULTS:
miR-888/miR-891a expression correlated with castration resistance, especially neuroendocrine CRPC. In vitro, pHLIP-PNA-antimiR-891a more effectively reduced growth in PC3-ML than LNCaP, while antimiR-888 treatment showed minimal effects. Combined antimiR treatment exhibited more potent growth phenotypes. In vivo, cy5-pHLIP efficiently targeted xenograft tumors in mice, with low signal in kidney and liver. Ongoing mouse efficacy studies suggest antimiR-891a is promising.
CONCLUSION:
Novel acid-sensing pHLIP reagents designed to inactivate miR-888 and miR-891a demonstrate clinical promise for lethal PCa. Further work is needed to address variable in vitro results. Developing luciferase sensor assays to monitor miRNA activity will be useful for batch and formulation testing. This research may improve PCa patient outcomes.