Allele-specific silencing of mutant p53 attenuates dominant-negative and gain-of-function activities

// Swathi V. Iyer 1 , Alejandro Parrales 1 , Priya Begani 1 , Akshay Narkar 1 , Amit S. Adhikari 2 , Luis A. Martinez 3 and Tomoo Iwakuma 1 1 Department of Cancer Biology, The University of Kansas Cancer Center, University of Kansas Medical Center, Kansas City, KS, USA 2 Center for Advanced Preclinical Research, Center for Cancer Research, National Cancer Institute, Frederick, MD, USA 3 Department of Pathology, Stony Brook School of Medicine, Stony Brook, NY, USA Correspondence to: Tomoo Iwakuma, email: // Keywords : mutant p53, allele-specific siRNA silencing, gain of function, oncogene addiction, dominant negative Received : June 19, 2015 Accepted : December 12, 2015 Published : December 16, 2015 Abstract Many p53 hotspot mutants not only lose the transcriptional activity, but also show dominant-negative (DN) and oncogenic gain-of-function (GOF) activities. Increasing evidence indicates that knockdown of mutant p53 (mutp53) in cancer cells reduces their aggressive properties, suggesting that survival and proliferation of cancer cells are, at least partially, dependent on the presence of mutp53. However, these p53 siRNAs can downregulate both wild-type p53 (wtp53) and mutp53, which limits their therapeutic applications. In order to specifically deplete mutp53, we have developed allele-specific siRNAs against p53 hotspot mutants and validated their biological effects in the absence or presence of wtp53. First, the mutp53 -specific siRNAs selectively reduced protein levels of matched p53 mutants with minimal reduction in wtp53 levels. Second, downregulation of mutp53 in cancer cells expressing a mutp53 alone (p53 mut ) resulted in significantly decreased cell proliferation and migration. Third, transfection of mutp53 -specific siRNAs in cancer cells expressing both wtp53 and mutp53 also reduced cell proliferation and migration with increased transcripts of p53 downstream target genes, which became further profound when cells were treated with an MDM2 inhibitor Nutlin-3a or a chemotherapeutic agent doxorubicin. These results indicate that depletion of mutp53 by its specific siRNA restored endogenous wtp53 activity in cells expressing both wtp53 and mutp53. This is the first study demonstrating biological effects and therapeutic potential of allele-specific silencing of mutp53 by mutp53 -specific siRNAs in cancer cells expressing both wtp53 and mutp53, thus providing a novel strategy towards targeted cancer therapies.