Abstract
The inactivation of p53, a tumor suppressor, and the activation of the RAS oncogene are the most frequent genetic alterations in cancer. We have shown that a unique E. coli MazF-MazE toxin–antitoxin (TA) system can be used for selective and effective eradication of RAS-mutated cancer cells. This out of the box strategy holds great promise for effective cancer treatment and management. We provide proof of concept for a novel platform to selectively eradicate cancer cells using an adenoviral delivery system based on the adjusted natural bacterial system. We generated adenoviral vectors carrying the mazF toxin (pAdEasy-Py4-SV40mP-mCherry-MazF) and the antitoxin mazE (pAdEasy-RGC-SV40mP-MazE-IRES-GFP) under the regulation of RAS and p53, resp. The control vector carries the toxin without the RAS-responsive element (pAdEasy-ΔPy4-SV40mP-mCherry-MazF). In vitro, the mazF-mazE TA system (Py4-SV40mP-mCherry-MazF+RGC-SV40mP-MazE-IRES-GFP) induced massive, dose-dependent cell death, at 69% compared to 19% for the control vector, in a co-infected HCT116 cell line. In vivo, the system caused significant tumor growth inhibition of HCT116 (KRASmut/p53mut) tumors at 73 and 65% compared to PBS and ΔPY4 control groups, resp. In addition, we demonstrate 65% tumor growth inhibition in HCT116 (KRASmut/p53wt) cells, compared to the other two control groups, indicating a contribution of the antitoxin in blocking system leakage in WT RAS cells. These data provide evidence of the feasibility of using mutations in the p53 and RAS pathway to efficiently kill cancer cells. The platform, through its combination of the antitoxin (mazE) with the toxin (mazF), provides effective protection of normal cells from basal low activity or leakage of mazF.
Highlights
In normal tissue, cell proliferation is a strictly regulated process
Once RAS is activated, it proceeds to stimulate downstream effectors that eventually bind to the RAS-responsive DNA element (RRE) and induces the transcription of early response genes involved in cell survival, proliferation, and differentiation [8]
We have successfully shown that the RRE, denoted as PY2 and consisting of Ets and AP-1-binding sites, is capable of selectively activating the expression of a destructive element called the p53 upregulated modulator of apoptosis (PUMA, kindly provided by Bert Vogelstein) that is able to kill cancer cells [16,17,18,19]
Summary
Normal tissue architecture and function rely on accurate cell number homeostasis. This homeostasis is a result of a dynamic balance between growth-promoting and growth-inhibiting signals [1]. The promoting signals are transduced mainly by growth factors that bind cell-surface receptors, commonly containing intracellular tyrosine kinase. The initiating step of the RAS pathway is the binding of extracellular ligands to cognate tyrosine kinase receptors that recruit guanine nucleotide exchange factors (GEFs), which mediate RAS activation in the cytosol. Once RAS is activated, it proceeds to stimulate downstream effectors that eventually bind to the RAS-responsive DNA element (RRE) and induces the transcription of early response genes involved in cell survival, proliferation, and differentiation [8]. The proto-oncogene RAS leads to the constitutive activation of downstream effectors due to a lack of GTPase activity [1, 8,9,10]
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