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. 2021 Jul 30;13(15):3833.
doi: 10.3390/cancers13153833.

Involvement of HIF-1α in the Detection, Signaling, and Repair of DNA Double-Strand Breaks after Photon and Carbon-Ion Irradiation

Anne-Sophie Wozny  1   2 , Arnaud Gauthier  1   2 , Gersende Alphonse  1   2 , Céline Malésys  1 , Virginie Varoclier  1 , Michael Beuve  3 , Delphine Brichart-Vernos  1 , Nicolas Magné  1   4 , Nicolas Vial  1   4 , Dominique Ardail  1 , Tetsuo Nakajima  5 , Claire Rodriguez-Lafrasse  1   2
Affiliations

Involvement of HIF-1α in the Detection, Signaling, and Repair of DNA Double-Strand Breaks after Photon and Carbon-Ion Irradiation

Anne-Sophie Wozny et al. Cancers (Basel). .

Abstract

Hypoxia-Inducible Factor 1α (HIF-1α), which promotes cancer cell survival, is the main regulator of oxygen homeostasis. Hypoxia combined with photon and carbon ion irradiation (C-ions) stabilizes HIF-1α. Silencing HIF-1α under hypoxia leads to substantial radiosensitization of Head-and-Neck Squamous Cell Carcinoma (HNSCC) cells after both photons and C-ions. Thus, this study aimed to clarify a potential involvement of HIF-1α in the detection, signaling, and repair of DNA Double-Strand-Breaks (DSBs) in response to both irradiations, in two HNSCC cell lines and their subpopulations of Cancer-Stem Cells (CSCs). After confirming the nucleoshuttling of HIF-1α in response to both exposure under hypoxia, we showed that silencing HIF-1α in non-CSCs and CSCs decreased the initiation of the DSB detection (P-ATM), and increased the residual phosphorylated H2AX (γH2AX) foci. While HIF-1α silencing did not modulate 53BP1 expression, P-DNA-PKcs (NHEJ-c) and RAD51 (HR) signals decreased. Altogether, our experiments demonstrate the involvement of HIF-1α in the detection and signaling of DSBs, but also in the main repair pathways (NHEJ-c and HR), without favoring one of them. Combining HIF-1α silencing with both types of radiation could therefore present a potential therapeutic benefit of targeting CSCs mostly present in tumor hypoxic niches.

Keywords: DNA repair; cancer stem cells; carbon ions; double-strand breaks; homologous recombination; hypoxia; hypoxia-inducible factor 1; irradiations; non-homologous end joining pathway; photons.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
HIF-1α nucleoshuttling is induced by photons under normoxia and hypoxia, but only under hypoxia with C-ions (a) Representative images at 4 h of SQ20BCD44Low cells (0 or 2 Gy photons and C-ions, under normoxia or hypoxia) are presented with the indicated biomarkers. (b) Signal intensities of HIF-1α into the nucleus were measured for each condition and normalized to basal signal. The means ± SD were statistically compared to the basal ratio. (Student’s t-Tests using Holm–Sidak method, α = 0.5). (n = 3 photons; n = 2 C-ions). * p < 0.05.
Figure 2
Figure 2
The silencing of HIF-1α combined with photons and C-ions decreases the initiation of the signaling of DSBs under hypoxia. (a) Representative images of silencing of HIF-1α in SQ20BCD44Low cells under hypoxia at 4 h. (b) Kinetics of ATM phosphorylation (P-ATM) after 2 Gy X-rays or C-ions, ± siHIF-1α. The percentages of foci per nucleus were calculated for each condition considering the X-ray normoxic peak as the reference. The symbols and error bars indicate means ± SD values. (c,d) represent respectively the P-ATM peak-rate from basal levels to peak and its decay-rate from peak to 6 h, as determined from the data shown in (b). Each plot represents the mean ± SD values (two-way ANOVA test, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). (e) Effect of HIF-1α silencing on p-ATM expression under hypoxia. The p-ATM percentages between hypoxic conditions were statistically compared (Student’s t-Tests, Holm–Sidak method, α = 0.5) (n = 3 photons; n = 2 C-ions).
Figure 3
Figure 3
Silencing HIF-1α under hypoxia increases the residual detected DSBs in response to both irradiations. (a) Kinetics of γH2AX foci after X-rays and C-ions, ± siHIF-1α. The percentages of foci per nucleus were calculated for each condition considering the X-ray normoxic peak as the reference (means ± SD). (b,c) represent respectively the peak-rate from basal levels and decay-rate from peak to 6 h, as determined from the data shown in (a) (two-way ANOVA test, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns: non-significant). (d) Residual foci were quantified at 24 h and each condition was statically compared to their respective normoxic condition (two-way ANOVA test) (n = 3).
Figure 4
Figure 4
HIF-1α silencing does not impact 53BP1 levels of foci. (a) Kinetics of 53BP1 foci after X-rays and C-ions ± siHIF-1α. The percentages of foci per nucleus were calculated for each condition considering the X-ray normoxic peak as the reference (means ± SD). (b,c) represent respectively the 53BP1 peak-induction from the basal levels and the decay-rate from the peak to 6 h, as determined from the data shown in (a). (d) Residual foci were quantified at 24 h and each condition was compared to their respective normoxic condition (two-way ANOVA test, ns = non-significant). (n = 3 photons; n = 2 C-ions).
Figure 5
Figure 5
HIF-1α silencing under hypoxia decreases the NHEJ-c pathway after both irradiations. (a) Kinetics of P-DNA-PKcs foci after X-rays and C-ions ± siHIF-1α. The percentages of foci per nucleus were calculated for each condition considering the X-ray normoxic peak as the reference (means ± SD). (b) Effect of HIF-1α silencing under hypoxia on P-DNA-PKcs expression. The percentages between hypoxia ± siHIF-1α were statistically compared (Student’s t-Tests, Holm–Sidak method, α = 0.5) (n = 3 photons; n = 2 C-ions). (c,d) represent respectively the induction-rate of the P-DNA-PKcs peak from the basal levels, and its decay-rate from the peak to 6 h, as determined from the data shown in (a) (two-way ANOVA test, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns: non-significant). (e) Residual foci were quantified at 24 h and each condition was compared to their respective normoxic condition (two-way ANOVA test) (n = 3 photons, n = 2 C-ions).
Figure 6
Figure 6
HIF-1α inhibition under hypoxia decreases HR in response to photons and C-ions. (a) Kinetics of RAD51 foci after X-rays and C-ions HIF-1α ± siHIF-1α. The percentages of foci per nucleus were reported to the reference (X-rays-Normoxia) and calculated for each condition (means ± SD). (b) Effect of the silencing of HIF-1α under hypoxia on RAD51 expression. The RAD51 percentages between hypoxia ± siHIF-1α were statistically compared (Student’s t-Tests, Holm–Sidak method, α = 0.5) (n = 3). * p < 0.05.
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