Tyrosyl DNA phosphodiesterase 1 as a putative target for camptothecin-based antitumor treatment

Camptothecins are cytotoxic agents with a wide spectrum of antitumor activity and are endowed with a unique mechanism of action because they inhibit DNA topoisomerase IB (Topo I), an enzyme implicated in crucial cellular processes such as DNA replication, transcription, recombination and repair. Topo I catalyzes changes in the DNA topology by transiently breaking and resealing the nucleic acid backbone. The enzyme forms a covalent bond at the 3’ terminus of the nicked DNA. Such a reaction leads to the formation of a Topo I covalent complex (i.e cleavable complex) through a tyrosine hydroxyl group of the enzyme. Camptothecins poison Topo I by stabilizing the DNA-Topo I complex, thereby leading - in proliferating tumor cells – to replication fork arrest and generation of irreversible DNA strand breaks. Because of the features of the Topo I-DNA lesions, Tyrosyl DNA phosphodiesterase 1 (TDP1), which catalyses the hydrolysis of 3’ phosphotyrosyl bonds is implicated in repair of Topo I-DNA covalent complexes and represents a putative target for modulating the activity of camptothecins. Thus, the aim of the present study was to define the potential interest of TDP1 as a target in antitumor therapy with particular reference to its role in cellular resistance to gimatecan, a camptothecin currently undergoing clinical evaluation. Since in ovarian carcinoma cell lines selected for resistance to gimatecan (IGROV-1CPT/H and IGROV-1CPT/L), resistance was associated with increased levels of TDP1, the significance of this factor has been initially explored in these ovarian carcinoma cell lines. The selected sublines exhibited around 6-fold resistance to gimatecan, cross-resistance to other camptothecins (topotecan, SN38) and to DNA topoisomerase II inhibitors. The resistant cells displayed an increased capability to repair gimatecan-induced single-strand breaks and a reduced amount of double-strand breaks. TDP1 silencing resulted in an increased amount of gimatecan-induced double-strand breaks, as assessed by staining of phosphorylated H2AX histone in resistant cells. The U2-OS cell line that is very suitable for loss and gain of function studies was then employed to investigate whether TDP1 may be a target for modulating the efficacy of camptothecins. Knocking down of TDP1 either by transfection of synthetic small interfering RNAs or by stable expression of exogenous TDP1 tailored miRNAs did not produce a change in cell sensitivity to gimatecan. However, co-targeting of other pathways cooperating with TDP1 in cell response to Topo I poisons indicated that XRCC1 – a scaffold protein implicated in DNA strand break repair - may cooperate with TDP1 at least in the presence of low levels of DNA damage. Mild resistance to gimatecan was observed in cells transfected with TDP1 cDNA using clonogenic assays, and this feature was associated with a decreased level of single-strand breaks after drug exposure. Overall, our findings support that TDP1 participates in cellular resistance to camptothecins as suggested by increased expression in resistant cells, and as supported by reduced levels of damage in cells transfected with TDP1 full-lenght cDNA. However, TDP1 alone cannot account for relevant levels of resistance as documented by loss and gain of function approaches. Its role in repair of Topo I-mediated DNA lesions appears dispensable. The cooperation between TDP1 and other pathways in regulating sensitivity to camptothecins is the most likely event, but critical inter-players of TDP1 in cell response to gimatecan still need to be identified. In conclusion, TDP1 per se does not appear to be an attractive target for camptothecin-based antitumor treatment. However, it is conceivable that inhibition of TDP1 in tumor cells exhibiting defects in other pathways playing a role in repair of Topo I-mediated damage could favour cell death after exposure to camptothecins. Since tumor cells may exhibit checkpoint defects, the interest of co-targeting of TDP1 and other putative co-targets should be pursued and is expected to further dissect out the most critical aspects of repair of Topo I-mediated damage.