Abstract
Background and objectiveKRAS mutation is an early event in colorectal cancer carcinogenesis. We previously reported that a recombinant adenovirus, carrying a pro-apoptotic gene (PUMA) under the regulation of Ets/AP1 (RAS-responsive elements) suppressed the growth of cancer cells harboring hyperactive KRAS. We propose to exploit the hyperactive RAS pathway, rather than to inhibit it as was previously tried and failed repeatedly. We aim to improve efficacy by substituting PUMA with a more potent toxin, the bacterial MazF-MazE toxin-antitoxin system, under a very tight regulation.ResultsA massive cell death, in a dose-dependent manner, reaching 73% at MOI 10 was seen in KRAS cells as compared to 22% in WT cells. Increase expression of MazE (the anti-toxin) protected normal cells from any possible internal or external leakage of the system and confirmed the selectivity, specificity and safety of the targeting system. Considerable tumor shrinkage (61%) was demonstrated in vivo following MazEF-encoding adenovirus treatment without any side effects.DesignEfficient vectors for cancer-directed gene delivery were constructed; “pAdEasy-Py4-SV40mP-mCherry-MazF”“pAdEasy-Py4-SV40mP-mCherry-MazF-IRES-TetR-CMVmp-MazE-IRES-EGFP“,“pAdEasy-ΔPy4-SV40mP-mCherry-MazF-IRES-TetR-CMVmp-MazE-IRES-EGFP “and “pAdEasy-mCherry”. Virus particles were produced and their potency was tested. Cell death was measured qualitatively by using the fluorescent microscopy and colony formation assay, and was quantified by MTT. FACS analysis using annexin V and RedDot2 dyes was performed for measuring apoptotic and dead cells, respectively. In vivo tumor formation was measured in a xenograft model.ConclusionsA proof of concept for a novel cancer safe and effective gene therapy exploiting an aberrant hyperactive pathway is achievable.
Highlights
Colorectal cancer (CRC) is a major health concern in the Western world [1]
We previously reported that a recombinant adenovirus, carrying a proapoptotic gene (PUMA) under the regulation of Ets/AP1 (RAS-responsive elements) suppressed the growth of cancer cells harboring hyperactive KRAS
A massive cell death, in a dose-dependent manner, reaching 73% at multiples of infection (MOI) 10 was seen in KRAS cells as compared to 22% in wild type (WT) cells
Summary
Colorectal cancer (CRC) is a major health concern in the Western world [1]. CRC patients carrying KRAS mutations are of particular therapeutic challenge, due to their resistance to anti- EGFR therapies. Aberrant activation of the RAS pathway plays an important role in the multistep process of CRC carcinogenesis. Oncogenic RAS stimulates a number of downstream effectors that activate several transcription factors that bind to the RAS-responsive DNA element and induce early response gene expression. The polyoma (Py) virus enhancer consists flanking overlapping binding sites of the Ets and AP1 transcription factors that are essential for oncogene transcriptional activation [2]. KRAS mutation is an early event in colorectal cancer carcinogenesis. We previously reported that a recombinant adenovirus, carrying a proapoptotic gene (PUMA) under the regulation of Ets/AP1 (RAS-responsive elements) suppressed the growth of cancer cells harboring hyperactive KRAS. We aim to improve efficacy by substituting PUMA with a more potent toxin, the bacterial MazF-MazE toxin-antitoxin system, under a very tight regulation
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