Abstract
c-Met, a cognate receptor tyrosine kinase of hepatocyte growth factor, is overexpressed and/or mutated in number of tumors. Therefore, abrogation of c-Met signaling may serve as potential therapeutic targets. In this study, we generated Ads expressing single shRNA specific to c-Met (shMet) (dl/shMet4 and dl/shMet5) or dual shRNAs specific to c-Met (dl/shMet4+5); and examined the therapeutic potential of these newly engineered Ads in targeting c-Met, and delineated their mechanism of action in vitro and in vivo. Ads expressing shMet induced knock-down in c-Met, and phenotypically resulted in autophagy-like features including appearance of membranousvacuoles, formation of acidic vesicular organelles, and cleavage and recruitment of microtubule-associated protein1 light chain 3 to autophagosomes. Ads expressing shMet also suppressed Akt phosphorylation and increased number of senescence-related gene products including SM22, TGase II, and PAI-1. These changes resulted in inhibition of cell proliferation and G2/M arrest of U343 cells. In vivo, intratumoral injection with dl/shMet4+5 resulted in a significant reduction of tumor growth with corresponding increasing overall survival. Histopathological analysis of these treated tumors revealed that Atg5 was highly up-regulated, indicating the therapeutic induction of autophagy. In sum, these results reveal that autophagic cell death induced by shMet-expressing Ads provide a novel strategy for targeting c-Met-expressing tumors through non-apoptotic mechanism of cell death.
Highlights
Receptor tyrosine kinases (RTKs) regulate many key processes in mammalian cell growth and survival, organ morphogenesis, neovascularization, and tissue repair and regeneration
We show for the first time that inhibition of c-Met by Ad-mediated short hairpin RNA (shRNA) expression results in robust anti-tumor efficacy via autophagic cell death in various cancer cells
The expression level of total c-Met was not reduced by shMet-expressing Ads in human dermal fibroblast (HDF) normal cells (Supplementary Figure S2A). These results demonstrate that c-Met-specific shRNA-expressing Ads can significantly suppress the level of c-Met expression as well as downstream signaling of c-Met in cancer cells, and further suggest that dual shRNA expression system is more effective in suppressing the expression of c-Met than single shRNA expression system
Summary
Receptor tyrosine kinases (RTKs) regulate many key processes in mammalian cell growth and survival, organ morphogenesis, neovascularization, and tissue repair and regeneration. Another common mutation occurs at the juxtamembrane domain, which plays a critical role in regulation of its catalytic function These activating mutations result in driving the activity and progression of c-Met driven tumors. HGF neutralizing monoclonal antibodies [8], c-met antisense oligonucleotides [9], dominant-negative forms of the c-Met protein [10], ribozyme-mediated reduction of c-Met expression [11, 12], and dual-function decoy c-Met receptor [13] are being investigated as possible strategies to block c-Met activation and suppress tumor growth, invasion, and metastasis. The growth of established U343 human glioma xenograft was significantly suppressed by dl/shMet4+5 These observations strongly suggest that inhibition of c-Met via dual c-Met specific shRNAexpressing Ad is a viable approach to the treatment of c-Met driven tumor types and warrants further testing in the clinic
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