Melanoma cells replicate through chemotherapy by reducing levels of key homologous recombination protein RAD51 and increasing expression of translesion synthesis DNA polymerase ζ.

BACKGROUND: The global incidence of melanoma has been increasing faster than any other form of cancer. New therapies offer exciting prospects for improved survival, but the development of resistance is a major problem and there remains a need for additional effective melanoma therapy. Platinum compounds, such as cisplatin, are the most effective chemotherapeutics for a number of major cancers, but are ineffective on metastatic melanoma. They cause monofunctional adducts and intrastrand crosslinks that are repaired by nucleotide excision repair, as well as the more toxic interstrand crosslinks that are repaired by a combination of nuclease activity and homologous recombination. METHODS: We investigated the mechanism of melanoma resistance to cisplatin using a panel of melanoma and control cell lines. Cisplatin-induced changes in levels of the key homologous recombination protein RAD51 and compensatory changes in translesion synthesis DNA polymerases were identified by western blotting and qRT-PCR. Flow cytometry, immunofluorescence and western blotting were used to compare the cell cycle and DNA damage response and the induction of apoptosis in cisplatin-treated melanoma and control cells. Ectopic expression of a tagged form of RAD51 and siRNA knockdown of translesion synthesis DNA polymerase zeta were used to investigate the mechanism that allowed cisplatin-treated melanoma cells to continue to replicate. RESULTS: We have identified and characterised a novel DNA damage response mechanism in melanoma. Instead of increasing levels of RAD51 on encountering cisplatin-induced interstrand crosslinks during replication, melanoma cells shut down RAD51 synthesis and instead boost levels of translesion synthesis DNA polymerase zeta to allow replication to proceed. This response also resulted in synthetic lethality to the PARP inhibitor olaparib. CONCLUSIONS: This unusual DNA damage response may be a more appropriate strategy for an aggressive and rapidly growing tumour like melanoma that enables it to better survive chemotherapy, but also results in increased sensitivity of cultured melanoma cells to the PARP inhibitor olaparib.

Inhibition of the ERCC1-XPF structure-specific endonuclease to overcome cancer chemoresistance.

ERCC1-XPF is a structure-specific endonuclease that is required for the repair of DNA lesions, generated by the widely used platinum-containing cancer chemotherapeutics such as cisplatin, through the Nucleotide Excision Repair and Interstrand Crosslink Repair pathways. Based on mouse xenograft experiments, where ERCC1-deficient melanomas were cured by cisplatin therapy, we proposed that inhibition of ERCC1-XPF could enhance the effectiveness of platinum-based chemotherapy. Here we report the identification and properties of inhibitors against two key targets on ERCC1-XPF. By targeting the ERCC1-XPF interaction domain we proposed that inhibition would disrupt the ERCC1-XPF heterodimer resulting in destabilisation of both proteins. Using in silico screening, we identified an inhibitor that bound to ERCC1-XPF in a biophysical assay, reduced the level of ERCC1-XPF complexes in ovarian cancer cells, inhibited Nucleotide Excision Repair and sensitised melanoma cells to cisplatin. We also utilised high throughput and in silico screening to identify the first reported inhibitors of the other key target, the XPF endonuclease domain. We demonstrate that two of these compounds display specificity in vitro for ERCC1-XPF over two other endonucleases, bind to ERCC1-XPF, inhibit Nucleotide Excision Repair in two independent assays and specifically sensitise Nucleotide Excision Repair-proficient, but not Nucleotide Excision Repair-deficient human and mouse cells to cisplatin.

The toxicity of nitrofuran compounds on melanoma and neuroblastoma cells is enhanced by Olaparib and ameliorated by melanin pigment.

Nitrofurans are commonly used for the treatment of trypanosomal diseases including Chagas disease. More recently, following the fortuitous discovery that nifurtimox was clinically active against neuroblastoma, nitrofuran compounds are being investigated for activity against cancer. Herein, we show that nitrofuran compounds are similarly potent to human malignant melanoma and neuroblastoma cells. Furthermore, a recently discovered nitrofuran compound, NFN1, was 50- to 175-fold more potent than nifurtimox against human melanoma and neuroblastoma cell lines. As nitrofuran compounds are known to act as pro-drugs, producing DNA-damaging reactive intermediates upon activation, we investigated the DNA repair pathways involved. We show that, contrary to research in Escherichia coli, the Nucleotide Excision Repair pathway is not required to repair nitrofuran-induced DNA damage in mammalian cells. Instead, we show that inhibiting repair of single-strand DNA breaks with the poly(ADP-ribose) polymerase (PARP) inhibitor, Olaparib, enhances nitrofuran toxicity in melanoma and neuroblastoma cells. We propose that this is due to mammalian cells utilising Type 2 nitroreductases for nitrofuran activation producing Reactive Oxygen Species which cause DNA damage that is repaired by the Single Strand Break Repair and/or Base Excision Repair pathways, whereas in bacteria and trypanosomes, Type 1 nitroreductases are also utilised resulting in different DNA lesions. In addition we show that, consistent with Reactive Oxygen Species being formed upon nitrofuran activation and the ability of melanin to absorb Reactive Oxygen Species, production of melanin in melanoma cells offers some protection from NFN1- and hydrogen peroxide-induced toxicity. Our data suggest that combinations of Olaparib and nitrofuran compounds may be advantageous for the treatment of melanoma and neuroblastoma, but that the protection offered to melanoma cells by their melanin pigment must be taken into account.

DNA repair endonuclease ERCC1-XPF as a novel therapeutic target to overcome chemoresistance in cancer therapy.

The ERCC1-XPF complex is a structure-specific endonuclease essential for the repair of DNA damage by the nucleotide excision repair pathway. It is also involved in other key cellular processes, including DNA interstrand crosslink (ICL) repair and DNA double-strand break (DSB) repair. New evidence has recently emerged, increasing our understanding of its requirement in these additional roles. In this review, we focus on the protein-protein and protein-DNA interactions made by the ERCC1 and XPF proteins and discuss how these coordinate ERCC1-XPF in its various roles. In a number of different cancers, high expression of ERCC1 has been linked to a poor response to platinum-based chemotherapy. We discuss prospects for the development of DNA repair inhibitors that target the activity, stability or protein interactions of the ERCC1-XPF complex as a novel therapeutic strategy to overcome chemoresistance.

ALDH2 mediates 5-nitrofuran activity in multiple species.

Understanding how drugs work in vivo is critical for drug design and for maximizing the potential of currently available drugs. 5-nitrofurans are a class of prodrugs widely used to treat bacterial and trypanosome infections, but despite relative specificity, 5-nitrofurans often cause serious toxic side effects in people. Here, we use yeast and zebrafish, as well as human in vitro systems, to assess the biological activity of 5-nitrofurans, and we identify a conserved interaction between aldehyde dehydrogenase (ALDH) 2 and 5-nitrofurans across these species. In addition, we show that the activity of nifurtimox, a 5-nitrofuran anti-trypanosome prodrug, is dependent on zebrafish Aldh2 and is a substrate for human ALDH2. This study reveals a conserved and biologically relevant ALDH2-5-nitrofuran interaction that may have important implications for managing the toxicity of 5-nitrofuran treatment.

Identification of DNA repair gene Ercc1 as a novel target in melanoma.

Increased expression of DNA repair genes contributes to the extreme resistance shown by melanoma to conventional DNA-damaging chemotherapeutics. One such chemotherapeutic effective against a range of other cancers, but not melanoma, is cisplatin. The DNA repair protein, ERCC1, is needed to remove cisplatin-induced DNA damage. We have shown that ERCC1 is essential for melanoma growth and resistance to cisplatin in a mouse xenograft model. Untreated xenografts of our transformed Ercc1-proficient melanocyte cell line grew very rapidly as malignant melanoma. Cisplatin treatment caused initial shrinkage of xenografts, but cisplatin-resistant regrowth soon followed. Cells reisolated into culture had twofold elevated levels of ERCC1 compared to both input cells and cells reisolated from untreated xenografts. An isogenic Ercc1-deficient derivative grew equally well in vitro as the Ercc1-proficient melanocyte cell line. However, in xenografts, the Ercc1-deficient melanomas were much slower to establish and were completely cured by just two cisplatin treatments.