Elsevier

DNA Repair

Volume 9, Issue 7, 1 July 2010, Pages 745-753
DNA Repair

Downregulation of XPF–ERCC1 enhances cisplatin efficacy in cancer cells☆

https://doi.org/10.1016/j.dnarep.2010.03.010Get rights and content

Abstract

Bulky cisplatin lesions are repaired primarily by nucleotide excision repair (NER), in which the structure specific endonuclease XPF–ERCC1 is a critical component. It is now known that the XPF–ERCC1 complex has repair functions beyond NER and plays a role in homologous recombination (HR). It has been suggested that expression of ERCC1 correlates with cisplatin drug resistance in non-small cell lung cancer (NSCLC). In our study, using NSCLC, ovarian, and breast cancer cells, we show that the XPF–ERCC1 complex is a valid target to increase cisplatin cytotoxicity and efficacy. We targeted XPF–ERCC1 complex by RNA interference and assessed the repair capacity of cisplatin intrastrand and interstrand crosslinks by ELISA and alkaline comet assay, respectively. We also assessed the repair of cisplatin-ICL-induced double-strand breaks (DSBs) by monitoring γ-H2AX focus formation. Interestingly, XPF protein levels were significantly reduced following ERCC1 downregulation, but the converse was not observed. The transcript levels were unaffected suggesting that XPF protein stability is likely affected. The repair of both types of cisplatin-DNA lesions was decreased with downregulation of XPF, ERCC1 or both XPF–ERCC1. The ICL-induced DSBs persist in the absence of XPF–ERCC1. The suppression of the XPF–ERCC1 complex significantly decreases the cellular viability which correlates well with the decrease in DNA repair capacity. A double knockdown of XPF–ERCC1 displays the greatest level of cellular cytotoxicity when compared with XPF or ERCC1 alone. The difference in cytotoxicity observed is likely due to the level of total protein complex remaining. These data demonstrate that XPF–ERCC1 is a valid target to enhance cisplatin efficacy in cancer cells by affecting cisplatin-DNA repair pathways.

Introduction

Cisplatin [cis-diammine-dichloroplatinum (II)] is one of the most widely used platinum containing chemotherapeutic agents. It is an alkylating agent that acts by forming cisplatin-DNA adducts which include monoadducts, intrastrand DNA adducts and DNA interstrand crosslinks (ICLs). These adducts inhibit and block DNA replication, which leads to cell death [1], [2]. Cisplatin is used clinically to treat a wide variety of tumors such as ovarian, testicular, head and neck, and NSCLC. Randomized clinical trials have confirmed that adjuvant chemotherapy with platinum-based (carboplatin or cisplatin) drug combinations for NSCLC significantly increases survival [3].

Despite its success with testicular cancer, its effectiveness in the treatment of other cancers is limited. This is due to the development of resistance over time. There are multiple mechanisms of cisplatin resistance but increased DNA repair is proposed to be one of the major reasons of resistance development [4]. Studies with a series of cisplatin-resistant cells in ovarian cancer cell lines show a clear relation between adduct removal and cisplatin cytotoxicity in a cisplatin-resistant model [5]. A major pathway involved in the repair of cisplatin-DNA adducts is the nucleotide excision repair (NER) pathway [6]. While the intrastrand DNA lesions and monoadducts are repaired by NER, the exact mechanism and events occurring during ICL repair are poorly understood [7].

The NER pathway has several steps: DNA damage recognition, dual incision/excision, repair synthesis and ligation. An important member of NER, the excision repair cross-complementing group 1 protein (ERCC1), forms a heterodimer with XPF and together they perform a critical incision step in the NER reaction. The XPF–ERCC1 complex is responsible for the incision 5′ to the lesion to cleave the damaged strand during NER [8].

It has been suggested that ERCC1 levels influence and correlate with DNA repair capacity [9], [10], [11]. ERCC1 expression has been associated with cellular and clinical resistance to platinum-based chemotherapy. There is much speculation about the importance of ERCC1 as prognostic indicator of cisplatin response in different cancer types [12]. Testicular cancer is generally responsive to cisplatin and has low levels of XPF–ERCC1, raising the possibility that XPF–ERCC1 levels influence the response to platinum therapy [13]. XPF–ERCC1 also has specific roles in ICL repair, recombination and regulates telomere integrity [14], [15], [16], [17], [18], [19]. It has been shown that XPF–ERCC1 facilitates the repair of DSBs induced by cisplatin-ICL processing, and it has been proposed that the key role of XPF–ERCC1 is unhooking the crosslink in ICL repair [14]. It has also been shown that the complex plays a role in completion of homologous recombination during ICL repair [20].

Thus, we rationalize that XPF–ERCC1 complex is an important molecular target in cancer chemotherapy to potentiate cisplatin cytotoxicity as decreased levels of XPF–ERCC1 can increase responses to cisplatin treatment. In our study, we used RNA interference to downregulate the XPF–ERCC1 complex in addition to individual suppression of XPF and ERCC1 proteins in cancer cell lines to compare their DNA repair capacity and test the effect on cisplatin sensitivity.

Section snippets

Chemicals

Cisplatin [cis-diammine-dichloroplatinum (II)] was purchased from Sigma–Aldrich. For StaRT-PCR, primers that amplify ERCC1, XPF and β-actin (control) were obtained from Gene Express (Toledo, OH). ERCC1 forward primer 5′-CTGGAGCCCCGAGGAAGC-3′; reverse primer 5′-CACTGGGGGTTTCCTTGG-3′. XPF forward primer 5′-AGTGCATCTCCATGTCCCGCTACTA-3′; reverse primer 5′-CGATGTTCTTAACGTGGTGCATCAA-3′. β-Actin forward primer 5′-CCCAGATCATGTTTGAGACC-3′; reverse primer 5′-CCATCTCTTGC TCGAAGTCC-3′. TRIzol reagent was

siRNA mediated knockdown of ERCC1, XPF and XPF–ERCC1 in cancer cells

We have chosen different NSCLC, ovarian and breast cancer cells to downregulate XPF–ERCC1. NSCLC cells, H1299 and H1355 cells were transiently transfected with smartpool siRNA's either directed individually against XPF and ERCC1 or at XPF–ERCC1 simultaneously (Fig. 1, Supplemental Fig. 1 and Table 1). Protein extracts from 72 to 120 h post-initial transfection were analyzed for ERCC1 and XPF expression with α-tubulin as a loading control in each cell line (Fig. 1, Supplemental Fig. 1 and Table 1

Discussion

Cisplatin cytotoxicity is believed to be attributed to the formation of platinum-DNA adducts which are primarily repaired by NER [5], [7]. As NER is a major player in the mechanism of cisplatin-induced DNA repair, targeting key NER components represents a molecular approach to enhance cisplatin efficacy for cancer treatment. Clinical investigations suggest that ERCC1 expression is a useful marker or predictor of response to cisplatin chemotherapy [34]. This has led to our evaluation of the

Conflict of interest

None declared.

Acknowledgements

We gratefully acknowledge Erin L. Crawford and Dr. James C. Willey (University of Toledo) for helping with technical procedures for StaRT-PCR/the transcript abundance data. We also thank Akshada Sawant for helping with the γ-H2AX foci images.

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    ☆

    Financial support: American Cancer Society - (ACS, RSG-06-163-01-GMC) to S.M.P.

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