Mild hyperthermia inhibits homologous recombination, induces BRCA2 degradation, and sensitizes cancer cells to poly (ADP-ribose) polymerase-1 inhibition

Abstract

Defective homologous recombination (HR) DNA repair imposed by BRCA1 or BRCA2 deficiency sensitizes cells to poly (ADP-ribose) polymerase (PARP)-1 inhibition and is currently exploited in clinical treatment of HR-deficient tumors. Here we show that mild hyperthermia (41-42.5°C) induces degradation of BRCA2 and inhibits HR. We demonstrate that hyperthermia can be used to sensitize innately HR-proficient tumor cells to PARP-1 inhibitors and that this effect can be enhanced by heat shock protein inhibition. Our results, obtained from cell lines and in vivo tumor models, enable the design of unique therapeutic strategies involving localized ondemand induction of HR deficiency, an approach that we term induced synthetic lethality.anti-cancer treatment | RAD51 | double-strand break M any anti-cancer therapies are based on cytotoxicity of DNA double strand breaks (DSBs) induced by ionizing radiation or, indirectly, by chemical agents. However, efficient DSB repair mechanisms protect cells from the genotoxic effects of DSBs, thereby reducing the effectiveness of the therapies. Two major pathways are involved in DSB repair in mammalian cells: homologous recombination (HR) and nonhomologous end joining (NHEJ). HR uses intact homologous DNA sequences, usually the sister chromatid in postreplicative chromatin, to faithfully restore DNA breaks (1), whereas NHEJ operates throughout the entire cell cycle and does not require a DNA template (2). Agents inhibiting DNA repair processes potentiate the cytotoxicity of DSBs in cancer therapy (3). Elevated temperature is one such agent that, via unclear mechanisms, interferes with multiple pathways of DNA repair (4-6) and is clinically applied (7). ResultsTo investigate if HR, among other processes and DSB repair pathways, is influenced by elevated temperature, we used an isogenic set of mouse embryonic stem (ES) cells that are either HR proficient (wild-type) or HR deficient (Rad54 −/− ) due to the disruption of the Rad54 gene, which is important for HR activity (1). We compared radiosensitization of these cells by incubating them at 37°C or 41°C before irradiation. For this and subsequent experiments we chose temperatures below 43°C, because they are relevant in clinical practice (8). Interestingly, we observed that wild-type but not Rad54 −/− cells were radiosensitized by preincubation at 41°C compared with cells incubated at 37°C (Fig. 1A). Similarly, HeLa cells, in which the important HR factors XRCC3 or BRCA2 were down-regulated using siRNA, were refractory to further temperature-mediated radiosensitization (Fig. 1B and Fig. S1). These results suggest that elevated temperature inactivates HR. To directly measure the effect of temperature on HR, we quantitated HR-mediated gene targeting in ES cells (9) and found that the efficiency of gene targeting was significantly reduced by preincubation at 41°C compared with 37°C (Fig. 1C). Similarly, preincubation at 41°C reduced the frequency of spontaneous and mitomycin C-induced sister chromatid exchanges in SW-1573 cells (Fig. S2A), w...

Regulation of Recombination and Genomic Maintenance

“…Such positivistic projections of data and interpretations from diverse models and organisms into a single model have accelerated progress in the field but also bear the danger of ignoring informative species-specific differences. The recent literature highlights the tremendous progress in our understanding of recombination and illuminates a vibrant field that is in the process of making growing contributions in the diagnosis and treatment of human disease, specifically cancer (Barker and Powell 2010;Moynahan and Jasin 2010;Krawczyk et al 2011;Chernikova et al 2012;Carvalho and Kanaar 2014).…”

Cell survival and radiosensitisation: Modulation of the linear and quadratic parameters of the LQ model

“…Mild temperatures may have more subtle effects than high temperatures, such as tumour reoxygenation (23)(24)(25)(26). We recently discovered that mild HT (42˚C for 1 h) transiently breaks down the BRCA2 protein (27). In the following sections, the effects of HT for 1 h at 41 or 43˚C on the LQ parameters are summarized.…”

Impact of silicone and metal port-a-cath implants on superficial hyperthermia treatment quality

“…Although only a small number of these patient have a port-a-cath in the re-irradiation field, a good knowledge on how and to what extent a metal or silicone port-a-cath disturbs the SAR distribution of an applicator is important for a safe and effective superficial hyperthermia treatment. Further, we notice there is a growing discussion [18] towards the combination of hyperthermia with chemotherapy [19][20][21][22][23]. In that patient group, inclusion of a porta-cath in the hyperthermia treatment field might be a more regular occurrence, and hence stresses the need to know how close to the port-a-cath we can heat.…”