Curative-intent radiation therapy relies mainly on the generation of DNA damages lethal for cancer cells. Paradoxically, because DNA damages are mutagenic, ionizing radiations also have tumorigenic effects. A severe complication of radiation therapy is therefore the induction of second primary cancers (SPCs), also called radiotherapy-induced second cancers, second cancers after radiation therapy, or cancers in irradiated fields (Berrington de Gonzalez et al., 2013; Doyen et al., 2010; Travis, 2006; Trott, 2017; Trott, 2017; Tubiana, 2009; Vautravers et al., 2010). SPCs are not a recurrence of the initial cancer but are neo-formed from normal cells affected by the radiations. Data on the increased risk of developing an SPC after radiation therapy is quite divergent in the available studies: it ranges from 0 in a cohort of 13,457 patients with rectal cancer having received radiation therapy after surgery compared to surgery alone (Martling et al., 2017) to 14 in a cohort of 1136 patients diagnosed with Hodgkin lymphoma at a median age of 11 years (Holmqvist et al., 2019).

Although the causal relationship between the initial radiation therapy and the induction of the SPC is impossible to definitely prove, SPCs are highly suspected to be radiation-induced. However, lifestyle, exogenous stressors, and genetic factors could also be contributing factors (Doyen et al., 2010). Amazingly, in radiation therapy using external X-rays, SPCs develop not preferentially inside the planning target volume (PTV), that is, the volume having received 100% of the therapeutic dose, but preferentially close to the edge of the beams, or farther around, on several centimeters (Diallo et al., 2009; Dörr and Herrmann, 2002). In a clinical linear accelerator, the region surrounding a beam (which will be called the margin in this article) receives photons leaking and scattering from the primary and secondary collimators, scattering from the flattening filter and scattering inside the patient from the PTV toward normal peritumoral tissues (Chofor et al., 2012). Although the marginal dose (also called peripheral dose or out-of-field dose) emanating from the collimator and the flattening filter could be reduced by optimizing the accelerator’s technology, the scattering inside the patient is unavoidable. This internal scatter component was shown to be the major determinant of the dose deposited in the most proximal margin (Chofor et al., 2012). The marginal radiation has three characteristics: (i) the deposited dose exponentially decreases with the distance, (ii) the deposited dose is approximately proportional to the PTV size, and (iii) the photon’s spectral energy fluence distribution is shifted to lower energies compared with those inside the PTV (Chofor et al., 2012; Chofor et al., 2011; Kirkby et al., 2007; Kry et al., 2007; Wiezorek et al., 2009). It was suggested that this photon quality change has the potential to increase the relative biological effectiveness (RBE) (Chofor et al., 2011; Kirkby et al., 2007), but the underlying specific cellular damages, if any, are unknown.

X-rays induce, among others, DNA single-strand breaks (SSBs) and double-strand breaks (DSBs) (Azzam et al., 2012; Jonathan et al., 1999; Panganiban et al., 2013). DSBs are defined as two breaks in the sugar-phosphate backbone on each DNA strand, at less than 10 bp. Their signalization and repair involve the activation of the DNA damage response (DDR) pathway with, downstream, the activation of the tumor suppressor TP53 that induces either a transitory cell cycle arrest favoring DNA repair or apoptosis. An SSB is a break in the sugar-phosphate backbone on only one DNA strand, often accompanied by a nucleotide loss and often displaying abnormal 5′ and 3′ ends (Caldecott, 2008). The SSB repair (SSBR) pathway involves a first step of break detection initiated by the poly(ADP)ribose polymerase (PARP) 1, which, once activated by its linkage to the broken DNA, synthetizes long chains of poly(ADP)ribose (PAR). The accumulated PAR chains favor the recruitment of the X Ray Repair Cross-Complementing Group (XRCC) 1 scaffold protein. XRCC1 is then phosphorylated and recruits the repair enzymes themselves, including the polynucleotide kinase phosphatase (PNKP) and the polymerase β. These last steps are common with the base exchange pathway (BER) (Caldecott, 2014). It is known for a long time that the curative effect of ionizing radiations results from the formation of DSBs too many to be repaired, leading to cancer cell death by apoptosis or mitotic catastrophe (Jonathan et al., 1999; Norbury and Hickson, 2001; Panganiban et al., 2013). SSBs are less detrimental than DSBs, but 1 Gy deposited in the PTV generates 10–25 more SSBs than DSBs (Dahm-Daphi et al., 2000). Presently, nothing is known about the respective quantities of SSBs and DSBs generated in the margin of the PTV and the consequences on the outcome of the affected normal cells.

Besides their spatial distribution related to the PTV, another amazing property of SPCs is their long latency period before emergence, ranging from 1 to 40 years after the treatment of the first cancer (Berrington de Gonzalez et al., 2013; Dörr and Herrmann, 2002; Doyen et al., 2010; Sheppard and Libshitz, 2001; Tubiana, 2009; Vautravers et al., 2010). Moreover, the cumulative incidence of SPCs increases with time (Doyen et al., 2010; Friedman et al., 2010; Olsen et al., 2009; Storm, 1988): for instance, the cumulative incidence of second sarcoma in a cohort of 16,705 patients treated by radiation therapy for nonmetastatic breast carcinoma was calculated at 0.7% at 5 years, at 0.27% at 10 years, and at 0.48% at 15 years after the initial treatment time (Kirova et al., 2005). This suggests that the normal cells that were affected by the scattered photons remain dormant in the organism while maintaining a constant neoplastic transformation potential. However, the precise biological form of this dormant cell state is completely unknown. We assayed in this study whether it was senescence.

Indeed, although initially described as the state reached by normal human fibroblasts after a finite doubling number, senescence is now recognized as a more general reprogrammed state established to adapt to several stresses, such as telomere shortening, DNA damages, oxidative damages, or hyperactivation of some oncogenes (Abbadie et al., 2017; Ben-Porath and Weinberg, 2005). By producing DNA damages and oxidative stress, ionizing radiations are senescence inducers (Cmielová et al., 2011; Panganiban et al., 2013; Seol et al., 2012; Suzuki et al., 2003). The senescence program includes an increase in cell size, complex epigenetic changes, an increase in reticulum endoplasmic stress, an increase in autophagy, changes in the composition of the secretome, and above all a long-term cell cycle arrest in G1 associated with a resistance to apoptosis, making senescent cells nonproliferating long-lived cells in vivo (Abbadie et al., 2017; Martien and Abbadie, 2007; Pluquet et al., 2015). The senescent cell cycle arrest results from the persistence of unrepaired DNA damages. When these damages are of a type signalized through the DDR pathway, such as DSBs or shortened telomeres, the cell cycle arrest is very stable, almost irreversible, considered as tumor suppressor (Campisi and d’Adda di Fagagna, 2007). In contrast, when the unrepaired damages are SSBs, as we have shown in a seminal work on endogenous oxidative stress-induced senescence, about one senescent cell on 10,000 systematically and spontaneously re-enters in cell cycle to give rise to a progeny of cells we named post-senescent neoplastic emergent (PSNE) cells. These cells display mutations, markers of transformation, markers of epithelial-to-mesenchyme transition, and are able to form small hyperplasias or carcinomas in in vivo assays (Deruy et al., 2010; Gosselin et al., 2009a; Malaquin et al., 2013; Martin et al., 2014; Nassour et al., 2016).

We investigated in this study whether SSB-induced senescence could be a step in the generation of SPCs after conformational 3D radiotherapy using X-rays. As SPC type, we focused on sarcomas that represent a large part of SPCs (Diallo et al., 2009), and whose outcome is very poor with a survival rate at 5 years of 10–40% (Depla et al., 2014; Vautravers et al., 2010). We report that normal fibroblasts located in the margin of a PTV receiving 2 Gy/day as patients enter after 2 weeks of treatment in premature senescence. This senescence induction is associated with an accumulation of SSBs in the almost absence of DSBs. We also report that about 10 days after the end of radiation fractions a few senescent cells re-enter cell cycle and generate a progeny of cells resuming proliferation and displaying mutations and increased invasive capacities. These findings lead us to propose for the first time a mechanism explaining how photons scattering inside the patient from the PTV toward the normal peritumoral tissues could induce a delayed development of sarcomas. They also pave the way for using senolytics to prevent second sarcoma development.

Second sarcomas are a rare but very severe late side effect of radiation therapy. Several studies conclude that they could be due to the out-of-field dose affecting normal cells surrounding the treated volume (Chofor et al., 2012; Chofor et al., 2011). In this study, we investigated the effects of this out-of-field dose on normal fibroblasts in terms of DNA damage and cell outcome. We show that the out-of-field dose induces the formation of SSBs, but nearly no DSBs. Importantly, the SSBs accumulate during a mimicked standard therapeutic protocol because of a lack of repair due to a decrease in the PARylation capacity that establishes along the successive daily radiation fractions. The fibroblasts affected by this specific accumulation of unrepaired SSBs do not undergo cell death but enter in premature senescence. They remain stably arrested in this senescent state for a while, and then a few of them re-enter cell cycle to generate a progeny of daughter cells displaying hallmarks of cancerous transformation, but which have not become fully tumorigenic. This scenario was established by robust in vitro experiments using proliferating or quiescent primary human fibroblasts derived from different donors, as well as by in vivo experiments in mice. It is, to our knowledge, the first scenario that could explain at the same time the latency period of second sarcoma emergence, underpinned by the long life of senescent cells, and the preferential location of second sarcomas around the treated volume, underpinned by the specific accumulation in this area of nonlethal but mutagenic unrepaired DNA damages.

It is known that the marginal dose exponentially decreases with the distance from the limit of the beam (Chofor et al., 2012; Kry et al., 2007; Wiezorek et al., 2009). In accordance, we found that the quantity of DSBs dramatically decreases from a few millimeters from the limit of the beam. In contrast and surprisingly, SSBs were almost as numerous at several centimeters from the limit of the beam as inside the PTV. Moreover, we demonstrated that their formation necessitates continuity in the material from the PTV. This indicates that SSBs are induced by photons propagating inside the material, confirming several studies showing that the internal scattering is the major source of the dose deposited in the most proximal margin (Chofor et al., 2012; Lee et al., 2016). Moreover, it can be hypothesized that the preferential generation of SSBs on DSBs in the margin could be the consequence of the tightening of the scattered photon spectral energy fluence distribution around lower energies (Chofor et al., 2012; Chofor et al., 2011; Kirkby et al., 2007). The experimental setup of this study was designed to only address the question of the potential role of photons scattering from the PTV, without mixing with the bystander effect relying on molecules secreted by the irradiated cancer and normal cells present inside the PTV. Of course, in the patient context, these two mechanisms could add, even synergize, or perhaps oppose the induction of DNA damages and the full transformation of the cells.

The results of this study also suggest that the ratio of DSBs/SSBs is very important in the cell outcome determination. Indeed, our data show that cells, whether they are cancerous or normal, developed both DSBs and SSBs when positioned inside the PTV, as already known (Reisz et al., 2014), and consequently either underwent apoptosis in a few days, or more slowly entered a senescent-like state and then died. In contrast, normal cells positioned at the margin, which developed almost exclusively SSBs, or normal cells in which we induced almost exclusively SSBs by a co-treatment with a mild concentration of H₂O₂ associated with a PARP inhibitor did not die at a significant level but entered senescence. Similarly, in normal human epithelial cells (keratinocytes and mammary epithelial cells), senescence occurs following an increase in endogenous oxidative stress, which also results in an accumulation of unrepaired SSBs, in the absence of DSBs or telomere shortening (Nassour et al., 2016). Very importantly, in all these situations where senescence is induced by SSBs in the almost absence of DSBs, telomere shortening or any other DNA damage able to induce the DDR pathway, a few cells systematically re-enter in S-phase to generate a progeny of daughter cells displaying neoplastic or pre-neoplastic properties (Deruy et al., 2010; Gosselin et al., 2009b; Martin et al., 2014; Nassour et al., 2016 and results herein). In contrast, NHDFs (the same as those used in the irradiation experiments of this study), having entered in replicative senescence because of shortened telomeres, never undergo apoptosis and remain very stably arrested in G1 without ever escaping (d’Adda di Fagagna et al., 2003; Nassour et al., 2016). This state of replicative senescence fits very well with the dogma equating senescence with tumor suppression (Campisi, 2005; Campisi, 2001; Kirkland and Tchkonia, 2017; Shay and Roninson, 2004). In contrast, SSB-only-induced senescence, which harbors a cell cycle arrest that is not so stable for a few cells and source of cells harboring cancerous hallmarks, has to be considered as a tumor-promoting state.

Senescence establishment implies the accumulation of DNA breaks, some of which have to remain unrepaired to sustain a prolonged activation of the cell cycle arrest pathways. This is true for replicative senescence in which shortened telomeres, which are assimilated to unrepaired DSBs, constantly activate the DDR pathway and downstream the tumor suppressor TP53 and its target p21, a CKI (Rossiello et al., 2014). This is also true for SSB-only-induced senescence of human keratinocytes and mammary epithelial cells where unrepaired SSBs activate the upregulation of p16 (Nassour et al., 2016), a major in vivo and in vitro senescence-associated CKI (He and Sharpless, 2017; Rayess et al., 2012). We now demonstrate this mechanism in this study also for fibroblasts in which SSBs were induced by a mild H₂O₂ treatment associated with a PARP inhibitor. And most importantly, we now demonstrate this mechanism for cells submitted to out-of-field radiations in a radiotherapy-mimicking context. But why SSBs are not repaired in this radiotherapy context is not yet completely established. We established in this study that the SSB repair capacity declines with the daily irradiations of a fractioned protocol, in correlation with a decline in the PARylation capacity. However, the precise mechanism of the PARylation capacity decline is presently unknown and is under investigation.

One major characteristic of cancer cells is their multiple mutations affecting oncogenes and tumor suppressor genes. The (pre-)neoplastic cells that are formed by SSB-only-induced senescence evasion display mutations and transformed characteristics including morphological change and increased invasion capacity (Nassour et al., 2016 and results herein). The accumulation of unrepaired SSBs is the inducer of senescence, but paradoxically it is also the fuel for post-senescence evasion (Nassour et al., 2016), probably because of the mutagenic potential of unrepaired DNA breaks. However, the precise mechanisms by which SSBs induce senescence and (pre)neoplastic evasion remain to be investigated.

The results of this study open at least two therapeutic avenues to decrease the risk of developing a second cancer after radiotherapy. One could be to decrease the accumulation of unrepaired SSBs in peri-PTV cells by supplementing the patient with NAD+ precursors. However, this also should decrease the accumulation of SSBs in cancer cells, consequently decreasing their rate of death and thereby compromising the cure. The other avenue could be to try to eliminate the senescent cells after the end of the radiotherapy. It is now widely established that senescence contributes to multiple age-related dysfunctions and diseases, and eliminating senescent cells in mice delays or decreases the incidence of these disorders, including cancer (Baker et al., 2011; Wissler Gerdes et al., 2020). This prompted several teams to develop drugs, called senolytics, specifically targeting senescent cells, to alleviate age-related disorders. There are presently two major senolytics whose efficacy was demonstrated in vitro and in mouse models, and which are subject to clinical trials (Chang et al., 2016; Pan et al., 2017): (i) the BH3 mimetics ABT-737 and ABT-263 (navitoclax) and (ii) the association of dasatinib + quercetin. Interestingly, navitoclax, dasatinib, and quercetin are or were used in anticancer treatments, presupposing that their use after radiation therapy to prevent from a second cancer emergence should not hamper the efficacy of the radiotherapy, even they could act additionally or synergistically with the treatment.

To conclude, in addition to have a clinical interest, studying post-radiotherapy second sarcoma is a model to study the very initial steps of tumorigenesis. Indeed, most often, these steps are inaccessible because of affecting too few cells, of unknown type, altered by an unknown stressing or damaging agent. In the context of post-radiotherapy second sarcomas, the affected cells and the inducer are known. The very first tumorigenic event we highlight in this study involves the formation of discreet DNA damages – SSBs – often assumed to be of no consequence because these are easily repaired and do not affect the genome stability. We show here that cells adapt to these damages and survive for a long time by adopting the senescent phenotype. They thereby become a reservoir of potentially tumorigenic mutations.

All data generated or analyzed during this study are included in the manuscript and supporting files. Source data files have been provided for all the figures where it was needed.

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Author details

1. Erwan Goy

   Univ. Lille, CNRS, Inserm, CHU Lille, Institut Pasteur de Lille, UMR9020-U1277 - CANTHER - Cancer Heterogeneity, Plasticity and Resistance to Therapies, F-59000 Lille, France

   Contribution

   Conceptualization, Data curation, Formal analysis, Funding acquisition, Methodology, Supervision, Writing – original draft

   Competing interests

   No competing interests declared

2. Maxime Tomezak

   1. Univ. Lille, CNRS, Inserm, CHU Lille, Institut Pasteur de Lille, UMR9020-U1277 - CANTHER - Cancer Heterogeneity, Plasticity and Resistance to Therapies, F-59000 Lille, France
   2. Univ. Lille, CNRS, UMR8520, Institut d'Electronique, Microélectronique et Nanotechnologie, F-59652 Villeneuve d'Ascq, France

   Contribution

   Conceptualization, Data curation, Formal analysis, Methodology, Writing – original draft

   Competing interests

   No competing interests declared

3. Caterina Facchin

   Univ. Lille, CNRS, Inserm, CHU Lille, Institut Pasteur de Lille, UMR9020-U1277 - CANTHER - Cancer Heterogeneity, Plasticity and Resistance to Therapies, F-59000 Lille, France

   Contribution

   Conceptualization, Data curation, Formal analysis, Funding acquisition, Methodology, Supervision, Writing – original draft

   Competing interests

   No competing interests declared

4. Nathalie Martin

   Univ. Lille, CNRS, Inserm, CHU Lille, Institut Pasteur de Lille, UMR9020-U1277 - CANTHER - Cancer Heterogeneity, Plasticity and Resistance to Therapies, F-59000 Lille, France

   Contribution

   Conceptualization, Data curation, Formal analysis, Funding acquisition, Methodology, Supervision, Writing – original draft

   Competing interests

   No competing interests declared

5. Emmanuel Bouchaert

   1. Oncovet Clinical Research, Plateforme PRECI, F-59120 Loos, France
   2. Oncovet, Plateforme PRECI, F-59650 Villeneuve d’Ascq, France

   Contribution

   Conceptualization, Data curation, Formal analysis, Funding acquisition, Methodology, Supervision, Writing – original draft

   Competing interests

   No competing interests declared

6. Jerome Benoit

   1. Oncovet Clinical Research, Plateforme PRECI, F-59120 Loos, France
   2. Oncovet, Plateforme PRECI, F-59650 Villeneuve d’Ascq, France

   Contribution

   Conceptualization, Data curation, Formal analysis, Funding acquisition, Methodology, Supervision, Writing – original draft

   Competing interests

   No competing interests declared

7. Clementine de Schutter

   Univ. Lille, CNRS, Inserm, CHU Lille, Institut Pasteur de Lille, UMR9020-U1277 - CANTHER - Cancer Heterogeneity, Plasticity and Resistance to Therapies, F-59000 Lille, France

   Contribution

   Investigation, Methodology

   Competing interests

   No competing interests declared

8. Joe Nassour

   Univ. Lille, CNRS, Inserm, CHU Lille, Institut Pasteur de Lille, UMR9020-U1277 - CANTHER - Cancer Heterogeneity, Plasticity and Resistance to Therapies, F-59000 Lille, France

   Contribution

   Conceptualization, Data curation, Formal analysis, Funding acquisition, Methodology, Supervision, Writing – original draft

   Competing interests

   No competing interests declared

9. Laure Saas

   Univ. Lille, CNRS, Inserm, CHU Lille, Institut Pasteur de Lille, UMR9020-U1277 - CANTHER - Cancer Heterogeneity, Plasticity and Resistance to Therapies, F-59000 Lille, France

   Contribution

   Conceptualization, Data curation, Formal analysis, Funding acquisition, Methodology, Supervision, Writing – original draft

   Competing interests

   No competing interests declared

10. Claire Drullion

    Univ. Lille, CNRS, Inserm, CHU Lille, Institut Pasteur de Lille, UMR9020-U1277 - CANTHER - Cancer Heterogeneity, Plasticity and Resistance to Therapies, F-59000 Lille, France

    Contribution

    Methodology

    Competing interests

    No competing interests declared

11. Priscille M Brodin

    Univ. Lille, CNRS, Inserm, CHU Lille, Institut Pasteur de Lille, U1019 - CIIL - Centre d'Infection et d'Immunité de Lille, F-59000 Lille, France

    Contribution

    Conceptualization, Data curation, Formal analysis, Funding acquisition, Methodology, Supervision, Writing – original draft

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0003-0991-7344

12. Alexandre Vandeputte

    Univ. Lille, CNRS, Inserm, CHU Lille, Institut Pasteur de Lille, U1019 - CIIL - Centre d'Infection et d'Immunité de Lille, F-59000 Lille, France

    Contribution

    Conceptualization, Data curation, Formal analysis, Funding acquisition, Methodology, Supervision, Writing – original draft

    Competing interests

    No competing interests declared

13. Olivier Molendi-Coste

    Univ. Lille, Inserm, CHU Lille, Institut Pasteur de Lille, U1011 - EGID, F-59000 Lille, France

    Contribution

    Conceptualization, Data curation, Formal analysis, Funding acquisition, Methodology, Supervision, Writing – original draft

    Competing interests

    No competing interests declared

14. Laurent Pineau

    Univ. Lille, Inserm, CHU Lille, Institut Pasteur de Lille, U1011 - EGID, F-59000 Lille, France

    Contribution

    Conceptualization, Data curation, Formal analysis, Funding acquisition, Methodology, Supervision, Writing – original draft

    Competing interests

    No competing interests declared

15. Gautier Goormachtigh

    Univ. Lille, CNRS, Inserm, CHU Lille, Institut Pasteur de Lille, UMR9020-U1277 - CANTHER - Cancer Heterogeneity, Plasticity and Resistance to Therapies, F-59000 Lille, France

    Contribution

    Conceptualization, Data curation, Formal analysis, Funding acquisition, Methodology, Supervision, Writing – original draft

    Competing interests

    No competing interests declared

16. Olivier Pluquet

    Univ. Lille, CNRS, Inserm, CHU Lille, Institut Pasteur de Lille, UMR9020-U1277 - CANTHER - Cancer Heterogeneity, Plasticity and Resistance to Therapies, F-59000 Lille, France

    Contribution

    Conceptualization, Data curation, Formal analysis, Funding acquisition, Methodology, Supervision, Writing – original draft

    Competing interests

    No competing interests declared

17. Albin Pourtier

    Univ. Lille, CNRS, Inserm, CHU Lille, Institut Pasteur de Lille, UMR9020-U1277 - CANTHER - Cancer Heterogeneity, Plasticity and Resistance to Therapies, F-59000 Lille, France

    Contribution

    Conceptualization, Data curation, Formal analysis, Funding acquisition, Methodology, Supervision, Writing – original draft

    Competing interests

    No competing interests declared

18. Fabrizio Cleri

    Univ. Lille, CNRS, UMR8520, Institut d'Electronique, Microélectronique et Nanotechnologie, F-59652 Villeneuve d'Ascq, France

    Contribution

    Conceptualization, Data curation, Formal analysis, Funding acquisition, Methodology, Supervision, Writing – original draft

    Competing interests

    No competing interests declared

19. Eric Lartigau

    Lille University, Medical School and Centre Oscar Lambret, Lille, France

    Contribution

    Conceptualization, Data curation, Formal analysis, Funding acquisition, Methodology, Resources, Supervision, Writing – original draft

    Competing interests

    No competing interests declared

20. Nicolas Penel

    Lille University, Medical School and Centre Oscar Lambret, Lille, France

    Contribution

    Conceptualization

    Competing interests

    No competing interests declared

21. Corinne Abbadie

    Univ. Lille, CNRS, Inserm, CHU Lille, Institut Pasteur de Lille, UMR9020-U1277 - CANTHER - Cancer Heterogeneity, Plasticity and Resistance to Therapies, F-59000 Lille, France

    Contribution

    Conceptualization, Data curation, Formal analysis, Funding acquisition, Methodology, Resources, Supervision, Writing – original draft

    For correspondence

    corinne.abbadie@ibl.cnrs.fr

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0002-8174-2393

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