Abstract
Cisplatin is widely used against various tumors, but resistance is commonly encountered. By inducing DNA crosslinks, cisplatin triggers DNA damage response (DDR) and cell death. However, the molecular determinants of how cells respond to cisplatin are incompletely understood. Since ubiquitination plays a major role in DDR, we performed a high-content siRNA screen targeting 327 human ubiquitin ligases and 92 deubiquitinating enzymes in U2OS cells, interrogating the response to cisplatin. We quantified γH2AX by immunofluorescence and image analysis as a read-out for DNA damage. Among known mediators of DDR, the screen identified the ubiquitin ligase G2E3 as a new player in the response to cisplatin. G2E3 depletion led to decreased γH2AX levels and decreased phosphorylation of the checkpoint kinase 1 (Chk1) upon cisplatin. Moreover, loss of G2E3 triggered apoptosis and decreased proliferation of cancer cells. Treating cells with the nucleoside analogue gemcitabine led to increased accumulation of single-stranded DNA upon G2E3 depletion, pointing to a defect in replication. Furthermore, we show that endogenous G2E3 levels in cancer cells were down-regulated upon chemotherapeutic treatment. Taken together, our results suggest that G2E3 is a molecular determinant of the DDR and cell survival, and that its loss sensitizes tumor cells towards DNA-damaging treatment.
Highlights
Cancer cells are sensitive to genotoxic stress, at least in part due to a loss of checkpoints that would otherwise prevent the unscheduled proliferation of cells that suffered DNA damage [reviewed in 1, 2]
We found that phosphoChk1 (Ser317) levels were decreased upon G2E3 knockdown in U2OS cells treated with gemcitabine (Fig. 6A)
The amount of overexpressed, HA-tagged G2E3 was strongly decreased upon cisplatin treatment (Fig. 7D). These results suggest that G2E3 does regulate parameters within the DNA damage response (DDR), but that it is itself regulated by DNA damage, apparently at transcriptional as well as posttranscriptional levels
Summary
Cancer cells are sensitive to genotoxic stress, at least in part due to a loss of checkpoints that would otherwise prevent the unscheduled proliferation of cells that suffered DNA damage [reviewed in 1, 2]. Along with oxaliplatin and carboplatin, is one of the most frequently prescribed chemotherapeutics for treating solid tumors. It induces intra- and interstrand crosslinks of the DNA, resulting in the accumulation of single-stranded DNA and DNA DSBs [3, 5]. We still lack detailed understanding of how DNA damage by cisplatin treatment is transduced to a signaling cascade and which components determine whether this response leads to cell cycle arrest, repair, or apoptosis. Our aim was to find new regulators of the DNA damage response (DDR) to cisplatin and potential targets whose inhibition could sensitize cancer cells to chemotherapeutic treatment
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