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. 2022 Oct 25;23(21):12876.
doi: 10.3390/ijms232112876.

PTEN Loss Enhances Error-Prone DSB Processing and Tumor Cell Radiosensitivity by Suppressing RAD51 Expression and Homologous Recombination

Xile Pei  1   2 , Emil Mladenov  1   2 , Aashish Soni  1   2 , Fanghua Li  2 , Martin Stuschke  1   3 , George Iliakis  1   2
Affiliations

PTEN Loss Enhances Error-Prone DSB Processing and Tumor Cell Radiosensitivity by Suppressing RAD51 Expression and Homologous Recombination

Xile Pei et al. Int J Mol Sci. .

Abstract

PTEN has been implicated in the repair of DNA double-strand breaks (DSBs), particularly through homologous recombination (HR). However, other data fail to demonstrate a direct role of PTEN in DSB repair. Therefore, here, we report experiments designed to further investigate the role of PTEN in DSB repair. We emphasize the consequences of PTEN loss in the engagement of the four DSB repair pathways-classical non-homologous end-joining (c-NHEJ), HR, alternative end-joining (alt-EJ) and single strand annealing (SSA)-and analyze the resulting dynamic changes in their utilization. We quantitate the effect of PTEN knockdown on cell radiosensitivity to killing, as well as checkpoint responses in normal and tumor cell lines. We find that disruption of PTEN sensitizes cells to ionizing radiation (IR). This radiosensitization is associated with a reduction in RAD51 expression that compromises HR and causes a marked increase in SSA engagement, an error-prone DSB repair pathway, while alt-EJ and c-NHEJ remain unchanged after PTEN knockdown. The G2-checkpoint is partially suppressed after PTEN knockdown, corroborating the associated HR suppression. Notably, PTEN deficiency radiosensitizes cells to PARP inhibitors, Olaparib and BMN673. The results show the crucial role of PTEN in DSB repair and show a molecular link between PTEN and HR through the regulation of RAD51 expression. The expected benefit from combination treatment with Olaparib or BMN673 and IR shows that PTEN status may also be useful for patient stratification in clinical treatment protocols combining IR with PARP inhibitors.

Keywords: DDR; DSBs; HR; PARP inhibitors; PTEN; SSA; alt-EJ; c-NHEJ; ionizing radiation.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
PTEN knockdown radiosensitizes RPE-1 hTert and M059K cells. (A) Western blot analysis of PTEN in RPE-1 hTert and M059K cells transfected with specific siRNA targeting PTEN protein; β-actin serves as loading control. (B) Flow cytometry histograms of RPE-1 hTert and M059K cells after transfection with PTEN siRNA. (C) Distribution of PTEN knock-down RPE-1 hTert and M059K cells in the different cell cycle phases. The analysis shows no significant differences between negative control (siNC) and PTEN knock-down (siPTEN) cells. (D) Clonogenic survival experiments of RPE-1 hTert and M059K cells transfected or not with PTEN siRNA. Data represent the mean ± SD from three independent experiments. The significance level, or p-value, is calculated using the two-tailed, Student’s t-test: ns (not significant), * p < 0.05, ** p < 0.01, *** p < 0.001.
Figure 2
Figure 2
Effect of PTEN deficiency on HR, SSA, NHEJ and alt-EJ. The established GFP-reporter assays in U-2 OS cells, specifically designed to report the repair of I-SceI-induced DSBs by HR (DR-GFP), SSA (SA-GFP), NHEJ (EJ5-GFP) and alt-EJ (EJ2-GFP) were utilized. (A) Percentage of GFP positive cells (GFP+), in the negative control (siNC) and PTEN knock-down (siPTEN) of DR-GFP cells. Bar plots (right panel) reflect the siNC-normalized GFP+ cells. (B) Same as panel (A), but for SA-GFP cells. (C) Same as panel (A), but for EJ5-GFP cells. (D) Same as panel (A), but for EJ2-GFP cells. Data represent the mean ± SD from three independent experiments. The significance level, or p-value, is calculated using the two-tailed, Student´s t-test: ns (not significant), * p < 0.05, ** p < 0.01, *** p < 0.001.
Figure 3
Figure 3
PTEN deficiency results in decreased number of IR-induced RAD51 repair foci. (A) Representative images of RAD51 foci in RPE-1 hTert cells, irradiated with 2 Gy of X-rays and collected at the indicated times after irradiation. Forty-eight hours before irradiation, cells were transfected with siNC or siPTEN, siRNA. (B) Quantification of RAD51 foci in siNC and siPTEN, RPE-1 hTert, EdU−, G2-cells. (C) Same as panel (A), but for M059K cells. (D) Same as panel (B), but for M059K cells. Cell cycle-specific RAD51 foci analysis was performed in EdU negative, G2-phase cells (EdU-, G2-cells), as described in Materials and Methods. The scale bar is 30 µm for all images. Data represent the mean and ± SD from three independent determinations. The significance level, or p-value, is calculated using the two-tailed Student´s t-test: ns (not significant), * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.
Figure 4
Figure 4
PTEN knock-down fails to change DNA-end-resection in RPE-1 hTert or M059K cells. (A) Representative flow cytometry histograms of RPA70 intensity signal in EdU-negative, G2-phase RPE-1 hTert cells (EdU−, G2-cells). (B) Quantification of the data shown in panel (A). The radiation effect on DNA end resection is shown after normalization of RPA70 signal, by dividing the mean signal intensity of irradiated cells by that of non-irradiated cells. (C) Same as panel (A), but for M059K cells. (D) Same as panel (B), but for M059K cells. (E) Representative IF images of RPA70 foci in exponentially growing RPE-1 hTert cells exposed to 2 Gy of X-rays. RPA70 foci are scored in EdU-negative, G2-phase cells (EdU-, G2-cells), as described in Materials and Methods. (F) Same as panel (E), but for M059K cells. The scale bar is 20 µm for all images. Data are means ± SD from three independent determinations. The significance level, or p-value, is calculated using the two-tailed Student´s t-test: ns (not significant), * p < 0.05.
Figure 5
Figure 5
PTEN knock-down suppresses RAD51 expression. (A) Effect of PTEN deficiency on RAD51 expression in RPE-1 hTert cells. β-Actin serves as loading control. (B) Same as panel (A), but for M059K cells. (C) Western blot analysis of AKT-pS473, PTEN and RAD51 protein levels in RPE-1 hTert cells treated with the indicated concentrations of bpV(HOpic) or SF1670 (PTEN inhibitors) for the indicated times. (D) Same as panel (C), but for M059K cells. (E) Same as panel (C), but for U-2 OS cells. Densitometry analysis of the gels are shown in Figure S4. The β−actin normalized values obtained from densitometry analysis are indicated in magenta below each lane.
Figure 6
Figure 6
PTEN inhibition by bpV(HOpic) or SF1670 leaves unaffected the levels of HR or SSA determined by GFP-reporter assays. (A) DR-GFP and SA-GFP reporter U-2 OS cells treated with the indicated concentrations of PTEN inhibitors. GFP-positive cells (GFP+) are measured 48 h after transfection with I-SceI expression plasmid. (B) Clonogenic survival assays with RPE-1 hTert cells treated with the indicated concentrations of PTENi. (C) Same as panel (B), but for M059K cells. Data represent the means ± SD from three independent experiments. ANOVA analysis with Tukey HSD post hoc test is used to calculate the statistical significance for the data plotted in Figure 6A, while the significance level in Figure 6B,C is calculated using the two-tailed Student´s t-test: ns (not significant), * p < 0.05.
Figure 7
Figure 7
PTEN knock-down impairs G2-checkpoint activation in RPE-1 hTert and M059K cells. The checkpoint activated in cells that are in G2-phase at the time of irradiation is measured by analyzing the mitotic index (MI), using two-parameter flow cytometry detecting DNA through PI staining and phosphorylated H3 at Serine 10 (H3pS10), a specific marker of mitotic cells, by antibody staining. (A) Normalized MI in siNC and siPTEN transfected RPE-1 hTert cells. Normalized MI is calculated by dividing the MI measured in irradiated cells by that of non-irradiated cells. (B) Same as in panel (A), but for M059K cells. (C) Single parametric flow cytometry analysis of siNC and siPTEN transfected RPE-1 hTert cells showing the activation of the G2-checkpoint of S-phase irradiated cells. (D) Same as in panel (C), but for M059K cells. Data from three independent experiments are presented as mean ± SD. The significance level, or p-value, is calculated using the two-tailed Student´s t-test: ns (not significant), * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.
Figure 8
Figure 8
PTEN knock-down suppresses HR and renders non-irradiated and irradiated cells sensitive to PARPi, Olaparib and BMN673. (A) Effect of Olaparib and BMN673 on the survival of RPE-1 hTert and M059K cells. Cells are treated with the indicated PARPi concentrations for 24 h. (B) Clonogenic survival assays of RPE-1 hTert and M059K cells exposed to increasing doses of X-rays and treated with PARPi (Olaparib—3 μM or BMN673—50 nM). Data show the mean ± SD from three independent experiments. The significance level, or p-value, is calculated using the two-tailed Student´s t-test: ns (not significant), * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Note that the significance of the observed effect is cell line dependent.
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