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

. 2011 Jun 14;108(24):9851-6.

doi: 10.1073/pnas.1101053108. Epub 2011 May 9.

Berina Eppink, Jeroen Essers, Jan Stap, Hans Rodermond, Hanny Odijk, Alex Zelensky, Chris van Bree, Lukas J Stalpers, Marrije R Buist, Thomas Soullié, Joost Rens, Hence J M Verhagen, Mark J O'Connor, Nicolaas A P Franken, Timo L M Ten Hagen, Roland Kanaar, Jacob A Aten

Affiliations

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

Przemek M Krawczyk et al. Proc Natl Acad Sci U S A. 2011.

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 on-demand induction of HR deficiency, an approach that we term induced synthetic lethality.

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Conflict of interest statement

Conflict of interest statement: Mark J. O'Connor is an employee of AstraZeneca.

Figures

Fig. 1.

Fig. 1.

Mild increases in temperature radiosensitizes HR-proficient but not HR-deficient cells and inhibits HR repair. (A) Cloning efficiency of wild-type (Left) and Rad54−/− (Right) mouse ES cells incubated for 75 min at 37 °C (□) or 41 °C (formula image) and subsequently exposed to the indicated dose of γ-rays. (B) Cloning efficiency of HeLa cells transfected with siRNA directed against luciferase (squares) or XRCC3 (circles), incubated for 75 min at 37 °C (open symbols) or 42.5 °C (filled symbols), and exposed to the indicated dose of γ-rays. Inset shows reduction of XRCC3 protein levels in HeLa cells transfected with siRNA directed against XRCC3. Cell lysates were analyzed by immunoblotting with antibodies against XRCC3. Equal sample loading was verified by probing for ORC2. (C) Efficiency of HR-mediated gene targeting in mouse ES cells. Cells were incubated for 7 h at 37 °C or 41 °C. At 2 h into this incubation period, cells were transfected with the hRad54GFP-puro knock-in targeting construct. Cells containing integrated construct were then selected in medium containing puromycin and analyzed by FACS. In this assay, GFP-positive cells arise when the targeting construct integrates in the mRad54 locus via HR. FACS profiles obtained in a single representative experiment show the percentage of GFP-positive cells upon incubation at the indicated conditions. The bar graph in Fig. 4_A_ shows the relative average percentage of GFP-positive cells obtained from three independent experiments with the error bars representing SEM.

Fig. 2.

Fig. 2.

Mild temperature increase interferes with the function and stability of HR proteins. (A) Visualization of accumulation of repair proteins at DSB sites in cells preincubated at 37 °C or 41 °C. U2OS cells were incubated at 37 °C or 41 °C for 60 min, irradiated with α-particles from a source positioned alongside the cells, resulting in linear arrays of DSBs, and then incubated for 15 min at 37 °C and fixed. Cells were stained for DNA (blue), γH2AX, or MDC1 (red), which were used as markers of the DSBs induced by α-particles, and either of the following proteins: MRE11, RPA34, BRCA2, or RAD51 (green). (Scale bar: 5 μm.) (B) Quantification of accumulation of repair proteins at DSB sites in cells preincubated at 37 °C or 41 °C. Cells treated and prepared as in A were scored as positive if they contained at least three IRIF of the indicated repair protein colocalizing with either γH2AX or MDC1 IRIF. Graphs represent average percentages of positive cells. Error bars represent the range of percentages obtained from two independent experiments. At least 50 cells containing damage induced by α-particles were scored per experiment. (C) Immunoblotting of cells subjected to mild heat and/or proteasome inhibitor. HeLa and BRO cells were incubated at 37 °C or 42.5 °C for 75 min in the presence or absence of 10 μM MG132. Next, cells were lysed and the lysates were analyzed by immunoblotting with antibodies against RAD51 (Upper, HeLa cells) or BRCA2 (Lower, BRO cells). (D) Immunoblotting of cells subjected to mild heat. BRO cells were incubated at 37 °C or 42.5 °C for 75 min. Next, cells were incubated for the indicated period at 37 °C and lysed. Lysates were analyzed by immunoblotting with antibodies against BRCA2 (Upper). Equal sample loading was verified by probing for ORC2 (Lower). (E) Visualization of accumulation of RAD51 at DSB sites in cells isolated from cervix carcinoma biopsies preincubated at 37 °C or 41 °C for 60 min, then irradiated with α-particles from a source positioned above the cells, incubated for 30 min at 37 °C or 41 °C, and fixed. Cells were stained for DNA (blue), γH2AX (red), and RAD51 (green). (Scale bar: 5 μm.)

Fig. 3.

Fig. 3.

A mild increase in temperature is toxic to cells lacking PARP-1 functionality. Cloning efficiency of U2OS (A) and R-1 (B) cells incubated at 41 °C for the indicated period in the absence (open symbols) or presence (filled symbols) of 100 μM NU1025. Graphs represent average cloning efficiencies corrected for the toxicity of NU1025. Error bars represent SEM from three independent experiments. (C) Cloning efficiency of BRO cells exposed to heat as described above, except for the use of 5 μM PJ-34 instead of 100 μM NU1025. Cloning efficiency of HeLa (D) and BRO (E) cells with reduced levels of PARP-1 protein subjected to mild heat. Cells were transfected with siRNA directed against luciferase (○) or PARP-1 (siRNA #1 △, siRNA #2 □) and incubated at 42.5 °C for the indicated period. Error bars represent SEM in a single experiment. The figure is representative for three independent experiments. Inset shows the efficiency of siRNA-induced reduction of PARP-1 protein levels. Cell lysates were analyzed by immunoblotting with antibodies against PARP-1. Equal sample loading was verified by probing for ORC2.

Fig. 4.

Fig. 4.

An HSP90 inhibitor enhances temperature-induced degradation of BRCA2, reduction of HR efficiency, sensitivity of heat-treated cells to PARP-1 inhibitors and to ionizing radiation, and reduction in in vivo tumor outgrowth. (A) Immunoblotting of cells subjected to a mild temperature increase and/or HSP90 inhibitor. BRO cells were incubated at 37 °C or 42.5 °C for 75 min in the presence or absence of 100 nM 17-DMAG. Next, cells were lysed and lysates were analyzed by immunoblotting with antibodies against BRCA2. Equal sample loading was verified by probing for ORC2. (B) Effect of 17-DMAG on the efficiency of HR-mediated gene targeting. Mouse ES cells were incubated for 7 h at 37 °C or 41 °C in the presence of 100 nM 17-DMAG. Transfection with the targeting construct and analysis by FACS was done as described in the legend to Figure 1_C_. FACS profiles obtained in a single representative experiment (Left) show the percentage of GFP-positive cells upon incubation at the indicated conditions. The bar graph (Right) shows the relative average percentage of GFP-positive cells obtained from three independent experiments. Error bars represent SEM. (C) Cloning efficiency of R-1 cells incubated for 4 h at 41 °C in the presence or absence of 100 μM NU1025 and/or 100 nM 17-DMAG. Error bars represent the range of cloning efficiencies obtained in two independent experiments. (D) Cloning efficiency of HeLa cells incubated for 75 min at 42.5 or 37 °C and subsequently exposed to the indicated dose of γ-rays in the presence or absence of 100 nM 17-DMAG. Error bars represent SEM from three independent experiments. (E) Temperature-controlled sensitization of tumors to PARP-1 inhibition. BRO derived-tumor bearing animals were treated with either PJ-34 or vehicle and subsequently exposed to elevated temperature (1 h at 42 °C) on the days marked with arrows. The number of animals per group is indicated in brackets. Error bars represent SEM (P < 0.002). (F) R-1 rhabdomyosarcoma-bearing rats were subjected to the indicated treatment on the days marked with arrows. Heat treatment of tumor-bearing hind limb was applied for 1.5 h at 42 °C. Olaparib and 17-DMAG were used at 50 and 10 mg/kg, respectively. The number of tumors analyzed/number of animals per group are indicated in brackets. Error bars represent SEM (P < 0.0001). (G) Survival of rats during the experiment shown in E. Animals were killed when the tumors exceeded a volume of 3,500 mm3. The number of animals per group is indicated in brackets.

Comment in

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