Can RNAi-mediated hsp90α knockdown in combination with 17-AAG be a therapy for glioma?

Adi Mehta,Amal Shervington,John Howl,Sarah Jones,Leroy Shervington

doi:10.1016/j.fob.2013.06.002

Adi Mehta, Amal Shervington + Show 3 more

Open Access

https://doi.org/10.1016/j.fob.2013.06.002

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Abstract

Heat shock protein 90 promotes tumor progression and survival and has emerged as a vital therapeutic target. Previously we reported that the combinatorial treatment of 17AAG/sihsp90α significantly downregulated Hsp90α mRNA and protein levels in Glioblastoma Multiforme (GBM). Here we investigated the ability of cell penetrating peptide (Tat48–60 CPP)-mediated siRNA-induced hsp90α knockdown as a single agent and in combination with 17-allylamino-17-demethoxygeldanamycin (17-AAG) to induce tumor growth inhibition in GBM and whether it possessed therapeutic implications. GBM and non-tumorigenic cells exposed to siRNA and/or 17-AAG were subsequently assessed by qRT-PCR, immunofluorescence, FACS analysis, quantitative Akt, LDH leakage and cell viability assays. PAGE was performed for serum stability assessment. A combination of siRNA/17-AAG treatment significantly induced Hsp90α gene and protein knockdown by 95% and 98%, respectively, concomitant to 84% Akt kinase activity attenuation, induced cell cycle arrest and tumor-specific cytotoxicity by 88%. Efficient complex formation between CPP and siRNA exhibited improved serum stability of the siRNA with minimal intrinsic toxicity in vitro. The preliminary in vivo results showed that combination therapy induced hsp90α knockdown and attenuated Akt kinase activity in intracranial glioblastoma mouse models. The results imply that RNAi-mediated hsp90α knockdown increases 17-AAG treatment efficacy in GBM. In addition, the cytotoxic response observed was the consequence of downregulation of hsp90α gene expression, reduced Akt kinase activity and S-G2/M cell cycle arrest. These results are novel and highlight the ability of Tat to efficiently deliver siRNA in GBM and suggest that the dual inhibition of Hsp90 has therapeutic potentials.

Highlights

Heat shock protein 90 is a vital molecular chaperone involved in regulating cellular processes by chaperoning its oncogenic clientele including EGFR, PDGFR, Cdks, Raf, Akt and p53 [1,2]
U87-MG cells treated with siRNA/CPP complexes (SCC) showed significant hsp90α downregulation after 48 h, exhibiting a Cell penetrating peptides (CPP) concentration-dependent knockdown of up to 30% (Fig. 1A)
After 72 h, significant gene knockdown was achieved at a lower CPP concentration, increasing the CPP concentration weakened the RNAi response

Summary

Introduction

Heat shock protein 90 is a vital molecular chaperone involved in regulating cellular processes by chaperoning its oncogenic clientele including EGFR, PDGFR, Cdks, Raf, Akt and p53 [1,2]. Many of these signalling proteins are overexpressed or mutated in GBM, indicating Hsp90-dependent tolerance to genetic alterations that would otherwise be fatal. A combination of RNAi and 17-AAG manifested significant gene and protein knockdown, concomitant to suppressed Akt levels and reduced GBM growth [6]. Successful RNAi response is relatively straight forward in vitro, achieving high transfection is challenging in vivo [7]

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