HSP90 inhibition induces cytotoxicity via down-regulation of Rad51 expression and DNA repair capacity in non-small cell lung cancer cells

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Abstract

Heat shock protein 90 (HSP90) is an exciting new target in cancer therapy. Repair protein Rad51 is involved in protecting non-small cell lung cancer (NSCLC) cell lines against chemotherapeutic agent-induced cytotoxicity. This study investigated the role of Rad51 expression in HSP90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG)-induced cytotoxicity in two NSCLC cell lines, A549 and H1975. The 17-AAG treatment decreased cellular Rad51 protein and mRNA levels and phosphorylated MKK1/2-ERK1/2 protein levels, and disrupted the HSP90 and Rad51 interaction. This triggered Rad51 protein degradation through the 26S proteasome pathway. The 17-AAG treatment also decreased the NSCLC cells’ DNA repair capacity, which was restored by the forced expression of the Flag-Rad51 vector. Specific inhibition of Rad51 expression by siRNA further enhanced 17-AAG-induced cytotoxicity. In contrast, enhanced ERK1/2 activation by the constitutively active MKK1/2 (MKK1/2-CA) vector significantly restored the 17-AAG-reduced Rad51 protein levels and cell viability. Arachidin-1, an antioxidant stilbenoid, further decreased Rad51 expression and augmented the cytotoxic effect and growth inhibition of 17-AAG. The 17-AAG and arachidin-1-induced synergistic cytotoxic effects and decreased DNA repair capacity were abrogated in lung cancer cells with MKK1/2-CA or Flag-Rad51 expression vector transfection. In conclusion, HSP90 inhibition induces cytotoxicity by down-regulating Rad51 expression and DNA repair capacity in NSCLC cells.

Highlights

HSP90 inhibition by 17-AAG decreases cellular Rad51 expression. ► 17-AAG downregulates the DNA repair capacity of human lung cancer cells. ► Rad51 plays a protective role in 17-AAG-induced cytotoxicity.

Introduction

Homologous recombination repair pathway represents a novel target for cancer therapy. Rad51 is the major strand-transferase required for recombination (Helleday et al., 2008). Many different cancer-derived cell lines and primary tumors have been shown to overexpress Rad51 (Klein, 2008), which lead to worse clinical outcomes in lung cancer (Qiao et al., 2005). At the same time, the Rad51 down-regulated human lung cancer cells show increased sensitivity to DNA damaging drugs like mitomycin C, cisplatin, and gemcitabine (Ashworth, 2008, Bristow and Hill, 2008, Helleday et al., 2007, Ko et al., 2008). Very high levels of mutagenesis have been observed in Rad51d-deleted Chinese hamster ovarian cells (CHO) cells (Hinz et al., 2006). Thus, defects in Rad51 play an important role in carcinogenesis.

Heat shock protein 90 (HSP90) is a major molecular chaperone that promotes the maturation and conformational stabilization of many cellular proteins, collectively referred to as “client proteins”, which are involved in tumor progression and proliferative and survival signals (Pearl and Prodromou, 2006, Whitesell and Lindquist, 2005). The HSP90 inhibitor, 17-allylamino-17-demethoxygeldanamycin (17-AAG), a geldanamycin derivative (Sharp and Workman, 2006), binds a conserved ATP interaction pocket in the HSP90 NH2-terminal domain (Isaacs et al., 2003). In pre-clinical models, 17-AAG has activity against various human cancers, where it is highly selective in degrading HSP90-client oncoproteins (Waza et al., 2005).

The present study aimed to determine whether or not 17-AAG affected Rad51 expression in NSCLC, and the role of Rad51 in 17-AAG-induced cytotoxicity. The 17-AAG-mediated Rad51 down-regulation caused decreased DNA repair ability in two NSCLC cell lines, whereas the over-expression of Rad51 restored cell viability and DNA repair capacity in 17-AAG-treated human lung cancer cells. These suggest that Rad51 plays a protective role in 17-AAG-induced cytotoxicity. As such, this study also posited the hypothesis that silencing Rad51 expression might be a new target for sensitization of NSCLC cells to HSP90 inhibition.

Section snippets

Drugs and reagents

The 17-AAG was purchased from Sigma Chemical (St. Louis, MO), while U0126 was purchased from Calbiochem-Novabiochem (San Diego, CA). The specific phospho-ERK1/2(Thr202/Tyr204) and phospho-MKK1/2(Ser217/Ser221) antibodies were purchased from Cell Signaling (Beverly, MA). Rabbit polyclonal antibodies against HSP90(H-114) (sc-7947), ERK2(C-14) (sc-154), Rad51(H-92) (sc-8349), and Actin(I-19) (sc-1616) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Cell lines and culture

Human lung cell carcinoma A549 and

17-AAG decreased Rad51 protein and mRNA in a time- and dose-dependent manner

HSP90 inhibition caused the depletion of HSP90 clients by disrupting client chaperoning and targeting the clients for proteasomal degradation. Treatment of cells with an HSP90 inhibitor, 17-AAG, provided a rapid and simple assay to assess whether a given protein depended either directly or indirectly on HSP90 chaperoning activity. To evaluate whether components of the MKK1/2-ERK1/2-Rad51 signaling pathway required HSP90, sub-confluent A549 or H1975 human lung cancer cells were treated with

Discussion

HSP90 expression is increased in a number of malignancies (Tsutsumi and Neckers, 2007) and it is an important molecular chaperone for protein folding and proteolytic turnover of key regulators of cell growth and survival (Whitesell and Lindquist, 2005). In the present study, the results illustrate for the first time that 17-AAG-decreased cellular Rad51 expression plays a role in decreasing the DNA repair capacity and cell survival in NSCLC cells. Enforced Rad51 expression can restore cell

Conflicts of interest statement

None declared.

Acknowledgements

The authors wish to thank Dr. Jia-Ling Yang for the expression plasmids for transfection and Dr. Chiou for with the arachidin-1. This work was supported by a grant from the National Science Council of Taiwan (Grant NSC 99-2320-B-415-001-MY3) and the National Taiwan University Hospital, Hsin-Chu Branch, Taiwan (Grant HCH101-05).

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