Prostate-targeted radiosensitization via aptamer-shRNA chimeras in human tumor xenografts
Xiaohua Ni, … , Theodore L. DeWeese, Shawn E. Lupold
Xiaohua Ni, … , Theodore L. DeWeese, Shawn E. Lupold
Published May 9, 2011
Citation Information: J Clin Invest. 2011;121(6):2383-2390. https://doi.org/10.1172/JCI45109.
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Research Article Oncology

Prostate-targeted radiosensitization via aptamer-shRNA chimeras in human tumor xenografts

Abstract

Dose-escalated radiation therapy for localized prostate cancer (PCa) has a clear therapeutic benefit; however, escalated doses may also increase injury to noncancerous tissues. Radiosensitizing agents can improve ionizing radiation (IR) potency, but without targeted delivery, these agents will also sensitize surrounding normal tissues. Here we describe the development of prostate-targeted RNAi agents that selectively sensitized prostate-specific membrane antigen–positive (PSMA-positive) cells to IR. siRNA library screens identified DNA-activated protein kinase, catalytic polypeptide (DNAPK) as an ideal radiosensitization target. DNAPK shRNAs, delivered by PSMA-targeting RNA aptamers, selectively reduced DNAPK in PCa cells, xenografts, and human prostate tissues. Aptamer-targeted DNAPK shRNAs, combined with IR, dramatically and specifically enhanced PSMA-positive tumor response to IR. These findings support aptamer-shRNA chimeras as selective sensitizing agents for the improved treatment of high-risk localized PCa.

Authors

Xiaohua Ni, Yonggang Zhang, Judit Ribas, Wasim H. Chowdhury, Mark Castanares, Zhewei Zhang, Marikki Laiho, Theodore L. DeWeese, Shawn E. Lupold

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Figure 4

PSMA selectivity and aptamer-shRNA chimera processing.

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PSMA selectivity and aptamer-shRNA chimera processing. (A and B) PSMA se...
(A and B) PSMA selectivity. (A) PC3-PIP or (B) PC3-Flu cells were treated with 400 nM aptamer-shRNA chimeras for 48 hours, and DNAPK expression was quantified by qRT-PCR. siRNA DNAPK (100 nM transfected) was included as a positive control. Expression is normalized to GAPDH. Mean ± SEM (n = 3). *P < 0.05. (C) Aptamer-shRNA chimera processing by Dicer in vitro. Cleavage products were analyzed by denaturing PAGE and ethidium bromide staining. Image is inverted for clarity. (D) Cell-based RNAi processing assay. LNCaP cells were treated with 400 nM aptamer-shRNA chimeras, and RNA was extracted 48 hours later for Northern blot assay. Probes are specific to corresponding antisense siRNAs. ds, double-stranded; ss, single-stranded. (E and F) Targeted in vivo knockdown. Subcutaneous LNCaP tumors were injected with aptamer-shRNA chimeras (200 pmol/injection) on days –3 and –2 and harvested on day 0, and DNAPK expression was determined. (E) qRT-PCR. Mean ± SEM. *P < 0.05. (F) Immunohistochemistry. Original magnification, ×400. (G and H) 5′-RACE PCR analysis to assess siRNA-mediated cleavage of DNAPK. (G) LNCaP cells transfected with DNAPK siRNA or with A10-3–DNAPK chimeras produced a specific DNAPK cleavage product. (H) In vivo treatment of LNCaP xenografts with A10-3–DNAPK chimera resulted in siRNA-mediated DNAPK cleavage.