In vivo model shows knocking down DNA repair gene boosts cancer-killing effects of ionizing radiation.
Scientists have developed a chimeric molecule comprising a gene-inhibiting short hairpin RNA (shRNA) bound to a tumor-targeting RNA aptamer that they claim significantly improves the sensitivity of prostate cancer to radiation therapy. The Johns Hopkins University School of Medicine team has tested the new approach in cell models, mouse xenograft models, and in normal human prostatic tissue models. They report their research in the Journal of Clinical Investigation, in a paper titled “Prostate-targeted radiosensitization via aptamer-shRNA chimeras in human tumor xenografts.”
Dose-escalated radiation therapy for localized prostate cancer (PCa) has well-established therapeutic benefits, but high doses can also harm noncancer tissues, report Shawn E. Leopold, Ph.D., and colleagues. Chemical or siRNA-based radiosensitizing agents that block cancer cells’ DNA repair mechanisms can boost the effects of radiotherapy, but it isn’t possible to selectively target cancer cells or specific tissues, and so sensitization also results in increased injury to noncancerous tissues.
Dr. Leopold’s team has previously developed prostate-specific membrane antigen-targeted (PSMA-targeted) RNA aptamers that can target drugs, nanoparticles, and toxins to PSMA-expressing prostate cancer (PSa) cells and tumors. The researchers claim that conjugating these aptamers to siRNAs and shRNAs provides the ability to knock down genes in the targeted cells specifically. Because PSMA is highly expressed in nearly all localized prostate tumors, they hypothesized that PSMA-targeted aptamer-shRNA chimeras could be used to inhibit DNA repair pathways in prostatic cells subjected to radiation therapy, and effectively boost the effects of treatment on locally advanced PCa, without simultaneously sensitizing normal tissues.
The researchers screened a custom siRNA library against mRNAs that encoded critical DNA repair proteins in order to identify potential radiosensitizing target genes and corresponding siRNAs. They then linked shRNAs constructed against the most promising genes to the PSMA-targeting aptamer. When tested in different PSMA-expressing cancer cell lines, they found that a construct targeting DNAPK was most effective at knocking down its target gene.
The effect of the DNAPK-targeting chimera on the effectiveness of radiotherapy was compared to that of a chimera comprising a control shRNA, in cultured cancer cells and in mice bearing human prostate cancer tumors. The results showed that treating cultured cancer cells with the DNAPK chimera significantly boosted the cell-killing effects of subsequent radiotherapy. Importantly, in the xenograft mice, tumors treated using the DNAPK chimera plus radiotherapy took 10 weeks to quadruple in volume, compared with just one week for tumor-bearing mice treated with radiotherapy and the aptamer-shRNA chimera. “Thus, in cell and tumor models, aptamer-targeted knockdown of DNAPK selectively enhanced radiosensitivity and increased therapeutic effect,” the authors claim.
The team then tested the DNAPK-knockdown aptamer in an ex vivo-cultured human tissue model comprising fresh sections of histologically normal human prostate obtained from radical prostatectomy sections. Using immunostaining techniques they showed that even in the apparently normal prostate tissue DNAPK production was decreased by 25% just two days after treatment. “We anticipate that knockdown in cancer specimens would be much more substantial because of known elevated PSMA expression in primary prostate tumors,” the researchers suggest.
Double-stranded breaks are generally regarded as the most lethal of all DNA lesions, and so mechanisms involved in the repair of such DNA damage represent promising targets for RNAi-induced radiosensitization therapy, the authors suggest. Interestingly, of the 249 mRNAs screened by Dr. Leopold’s team, only six potential candidates were identified and validated. “These target genes are likely good candidates for radiosensitization in other tissue and cancer types,” the researchers note, although they point out that aptamer-shRNA chimeras could theoretically be developed for virtually any target gene. “Some advantages of aptamer-shRNA chimeras are their simplicity, potential for chemical synthesis, safety, and low toxicity,” the team concludes. “Inhibition of DNA repair pathways can also sensitize cells to chemotherapeutics, such as alkylating agents and topoisomerase inhibitors, therefore providing a potential mechanism for systemic chemosensitization.”