Yan Sun

Ambroise Manceau

Lisa Frydman

Lucie Cappuccio

David Neves

Valeria Basso

Hong Wang

Joanna Fombonne

Carine Maisse

Patrick Mehlen
email: patrick.mehlen@lyon.unicancer.fr.

Andrea Paradisi
email: andrea.paradisi@lyon.unicancer.fr

pro-oncogenic activity

Keywords: BIOLOGICAL SCIENCES, Cell Biology apoptosis, netrin-1, p53 isoform

Introduction

Several studies have highlighted the crucial role of the secreted protein netrin-1 as an original cancer biomarker and therapeutic target (1)(2)(3). Initially discovered as an axon guidance cue during neuronal development, netrin-1 is also involved in cell survival and tumorigenesis (4). Indeed, netrin-1 receptors Deleted in Colorectal Carcinoma (DCC) and UNC5 homologue (UNC5H, i.e. UNC5A-D), belonging to the growing family of dependence receptors [START_REF] Goldschneider | Dependence receptors: a new paradigm in cell signaling and cancer therapy[END_REF], induce "positive" signals upon ligand binding leading to differentiation and cell survival, and trigger apoptosis when unbound [START_REF] Paradisi | Netrin-1, a missing link between chronic inflammation and tumor progression[END_REF]. Hence, cell survival depends on the presence of ligands of dependence receptors, the latter representing ideal tumor suppressors candidates. Indeed, the current hypothesis is that the expression of such a receptor represents a protective mechanism that limits tumor development. This could be achieved via the induction of apoptosis of tumor cells that would grow in or migrate to regions of limited ligand availability [START_REF] Mehlen | Metastasis: a question of life or death[END_REF]. Consequently, cell transformation towards a malignant or metastatic phenotype likely relies either on the loss of dependence receptors expression or on the constitutive inhibition of apoptosis induced by these receptors via ectopic ligand expression. Consistently, netrin-1 up-regulation has been described in several aggressive cancers [START_REF] Fitamant | Netrin-1 expression confers a selective advantage for tumor cell survival in metastatic breast cancer[END_REF][START_REF] Paradisi | NF-kappaB regulates netrin-1 expression and affects the conditional tumor suppressive activity of the netrin-1 receptors[END_REF][START_REF] Delloye-Bourgeois | Netrin-1 acts as a survival factor for aggressive neuroblastoma[END_REF][START_REF] Delloye-Bourgeois | Interference with netrin-1 and tumor cell death in nonsmall cell lung cancer[END_REF], and is associated with tumor progression [START_REF] Fitamant | Netrin-1 expression confers a selective advantage for tumor cell survival in metastatic breast cancer[END_REF][START_REF] Delloye-Bourgeois | Interference with netrin-1 and tumor cell death in nonsmall cell lung cancer[END_REF][START_REF] Paradisi | Netrin-1 up-regulation in inflammatory bowel diseases is required for colorectal cancer progression[END_REF]. The identification of netrin-1 as a tumor biomarker led to the development of an innovative personalized anti-cancer strategy. Indeed, interference of netrin-1 and its receptor binding induces apoptosis and favors tumor regression in several aggressive cancer models, expressing high netrin-1 levels (1,[START_REF] Fitamant | Netrin-1 expression confers a selective advantage for tumor cell survival in metastatic breast cancer[END_REF][START_REF] Delloye-Bourgeois | Netrin-1 acts as a survival factor for aggressive neuroblastoma[END_REF][START_REF] Delloye-Bourgeois | Interference with netrin-1 and tumor cell death in nonsmall cell lung cancer[END_REF][START_REF] Paradisi | Netrin-1 up-regulation in inflammatory bowel diseases is required for colorectal cancer progression[END_REF]. According to these findings, a humanized anti-netrin-1 antibody, called NP137, was developed (2) and was assessed in a phase I clinical trial (https://clinicaltrials.gov/ct2/show/NCT02977195) and is now moving to a phase II clinical trial.

Despite the importance of netrin-1 in cell survival and cancer progression, little is known about the regulation of its expression. We formerly demonstrated that the transcription factor p53 was involved in the regulation of netrin-1 gene expression, following chemotherapy (1). p53 is a well-known tumor suppressor gene, through its ability to regulate the expression of different genes involved in a broad range of cellular responses, including apoptosis, cell cycle arrest, senescence and metabolism [START_REF] Vousden | Blinded by the Light: The Growing Complexity of p53[END_REF]. p53 functions mainly depend on its ability to induce or repress transcription of target genes.

However, transcription-independent activities of p53 have been described [START_REF] Moll | Transcription-independent pro-apoptotic functions of p53[END_REF][START_REF] Green | Cytoplasmic functions of the tumour suppressor p53[END_REF]. The transactivation activity of p53 requires two distinct transcriptional activation domains (TAD), encompassing residues 1-40 (TAD1) and 41-83 (TAD2) [START_REF] Candau | Two tandem and independent sub-activation domains in the amino terminus of p53 require the adaptor complex for activity[END_REF]. Experiments in mice bearing inactivating point mutations in one or both TADs demonstrated a functional specialization of each TAD, that could regulate different target genes and effector pathways [START_REF] Brady | Distinct p53 transcriptional programs dictate acute DNA-damage responses and tumor suppression[END_REF][START_REF] Sullivan | Mechanisms of transcriptional regulation by p53[END_REF]. Interestingly, in the last few years, several alternative p53 transcripts were identified [START_REF] Bourdon | p53 isoforms can regulate p53 transcriptional activity[END_REF][START_REF] Marcel | Biological functions of p53 isoforms through evolution: lessons from animal and cellular models[END_REF]. These protein isoforms are generated by different mechanisms, such as alternative splicing sites, promoters and translational initiation sites [START_REF] Khoury | The isoforms of the p53 protein[END_REF][START_REF] Marcel | p53 isoforms -a conspiracy to kidnap p53 tumor suppressor activity? Cellular and molecular life sciences[END_REF]. Some of these p53 isoforms lack TAD1 (∆40p53) or both transactivation domains (∆133p53) [START_REF] Marcel | Biological functions of p53 isoforms through evolution: lessons from animal and cellular models[END_REF]. The ∆40p53 isoform can be produced by alternative splicing, leading to the retention of intron 2 [START_REF] Ghosh | Regulation of human p53 activity and cell localization by alternative splicing[END_REF][START_REF] Courtois | DeltaN-p53, a natural isoform of p53 lacking the first transactivation domain, counteracts growth suppression by wild-type p53[END_REF][START_REF] Matlashewski | Alternative splicing of human p53 transcripts[END_REF], or through the use of an alternative internal ribosomal entry sequence (IRES) at codon 40 [START_REF] Courtois | DeltaN-p53, a natural isoform of p53 lacking the first transactivation domain, counteracts growth suppression by wild-type p53[END_REF]. Conversely, the ∆133p53 isoform is generated from an internal promoter, located between intron 1 and exon 5, controlling the expression of a p53 mRNA sequence starting from intron 4 [START_REF] Bourdon | p53 isoforms can regulate p53 transcriptional activity[END_REF][START_REF] Marcel | p53 regulates the transcription of its Delta133p53 isoform through specific response elements contained within the TP53 P2 internal promoter[END_REF].

Even though we previously showed that the full-length p53 (FLp53) directly binds to and activates the netrin-1 promoter (1), the molecular mechanisms underlying this regulation remain unclear. We reveal here that the regulation of netrin-1 and its main dependence receptor UNC5B requires the transactivation activity of p53 in tumor cells.

Notably, netrin-1 transcriptional activation requires only p53 TAD2, unlike the majority of p53 target genes [START_REF] Brady | Distinct p53 transcriptional programs dictate acute DNA-damage responses and tumor suppression[END_REF]. Consequently, we show that ∆40p53, and not ∆133p53, can positively regulate netrin-1 and UNC5B gene expression, in a FLp53-independent manner. Moreover, we demonstrate that the expression of ∆40p53 and netrin-1 are correlated in human tumors. Finally, we speculate that treatment of tumor cells expressing ∆40p53 with neutralizing anti-netrin-1 antibody could be an innovative cancer therapeutic strategy.

Results

Netrin-1 regulation by p53 requires the transcriptional activity of the TAD2 domain

To unravel p53 regulation of netrin-1 expression, in particular the involvement or not of its transcriptional activity [START_REF] Moll | Transcription-independent pro-apoptotic functions of p53[END_REF][START_REF] Green | Cytoplasmic functions of the tumour suppressor p53[END_REF], and possibly of its N-terminal transactivation domains (TADs) [START_REF] Brady | Distinct p53 transcriptional programs dictate acute DNA-damage responses and tumor suppression[END_REF], we generated stable lung cancer H1299 cells bearing point mutations using sleeping beauty-based vectors. Indeed, site-specific L22Q and W23S mutations in TAD1 significantly inhibit the transcriptional activation of the p53 protein [START_REF] Lin | Several hydrophobic amino acids in the p53 amino-terminal domain are required for transcriptional activation, binding to mdm-2 and the adenovirus 5 E1B 55-kD protein[END_REF], whereas W53Q and F54S mutations inactivate TAD2 (Fig. 1A) [START_REF] Candau | Two tandem and independent sub-activation domains in the amino terminus of p53 require the adaptor complex for activity[END_REF]. This methodology enabled us to activate wild-type or transcriptionally inactivated p53 mutants upon treatment with doxycycline of this p53-null cell line. Interestingly, basal protein levels of p53 mutants, in particular TAD1 mutants, were elevated compared to wild-type p53, likely due to the inhibition of Mdm2 ubiquitin ligase binding to p53 following the mutation of residues 22/23 [START_REF] Brady | Distinct p53 transcriptional programs dictate acute DNA-damage responses and tumor suppression[END_REF][START_REF] Lin | Several hydrophobic amino acids in the p53 amino-terminal domain are required for transcriptional activation, binding to mdm-2 and the adenovirus 5 E1B 55-kD protein[END_REF][START_REF] Chi | Structural details on mdm2-p53 interaction[END_REF]. As previously shown (1), over-expression of wild-type p53 strongly induced netrin-1, as well as p21, a p53 target gene (Fig. 1B). However, while inactivation of p53 TAD1 (p53 22,23 ) did not affect netrin-1 protein expression, overexpression of the TAD2 mutant, alone (p53 53,54 ) or in combination with the TAD1 mutant (p53 22,23,53,54 ), blocked netrin-1 expression and that of its target gene p21 (Fig. 1B). This finding was further confirmed by quantitative RT-PCR (Fig. 1C), suggesting that netrin-1 up-regulation requires the transcriptional activity of p53, in particular the TAD2 domain.

∆40p53, but not ∆133p53, regulates netrin-1 and UNC5B gene expression Some p53 alternative transcripts, generated by 5' splicing, IRES or alternative promoter, lack TAD1 (∆40p53) or both TADs (∆133p53) (Fig. 2A), similarly to the transcriptional mutants generated above. We wondered whether different domains of the p53 N-terminal could regulate netrin-1 gene expression using known p53 isoforms. To this purpose, we generated stable lung cancer A549 lines, harboring wild-type p53, inducible for ∆40p53 or ∆133p53 isoforms and we analyzed gene expression by quantitative RT-PCR. Upon treatment with doxycycline, ∆40p53 over-expression strongly activated netrin-1 gene expression, whereas ∆133p53 induction had no effect (Fig. 2B, left panel), confirming results obtained with p53 transcriptional mutants. Interestingly, ∆40p53, but not ∆133p53, also up-regulated the main netrin-1 receptor, UNC5B (Fig. 2B, middle panel), and, despite the fact that ∆40p53 was reported to negatively modulate p21 [START_REF] Takahashi | Dominant effects of Delta40p53 on p53 function and melanoma cell fate[END_REF][START_REF] Hafsi | Effects of Delta40p53, an isoform of p53 lacking the N-terminus, on transactivation capacity of the tumor suppressor protein p53[END_REF], we observed a positive regulation of p21 by ∆40p53 in our inducible cell lines (Fig. 2B, right panel). We confirmed the positive regulation of netrin-1 and p21 by ∆40p53 induction at the protein level by Western blot (Fig. 2C). It is of interest to note that in this model, ∆40p53 over-expression seemed to rather promote the p53 cell cycle arrest activity, as evidenced by p21 expression, compared to its pro-apoptotic function, since the BAX protein (Fig. 2C), another p53 target gene, remained unaffected.

To confirm that netrin-1 induction by ∆40p53 depends on its transcriptional activity, we conducted point mutations of residues W14 and F15 of ∆40p53 (∆40p53 14,15 ), equivalent to positions 53 and 54 in FLp53 (see Fig. 2A). We then generated stable A549 cells inducible for wild-type ∆40p53 and ∆40p53 14,15 upon treatment with doxycycline, and we assessed protein and transcript levels by Western blot and quantitative RT-PCR analyses. Dysregulation of ∆40p53 transcriptional activity strongly inhibited netrin-1 and p21 protein expression upon doxycycline induction (Fig. 2D). Moreover, netrin-1, UNC5B and p21 gene expression was very weak following ∆40p53 14,15 over-expression compared to ∆40p53 (SI appendix, Fig. S1).

Unlike H1299 cells, A549 cells harbor wild-type p53. We thus wondered whether the expression of ∆40p53 and ∆133p53 could affect endogenous FLp53 accumulation and activation upon Doxorubicin treatment and in turn affect netrin-1, UNC5B and p21 gene expression. We treated sleeping-beauty-modified A549 cells with doxycycline (to produce ∆40p53 or ∆133p53) and doxorubicin, and analyzed gene expression by quantitative RT-PCR. In ∆40p53-A549 cells, netrin-1, UNC5B and p21 gene expression was strongly upregulated upon treatment with doxycycline and doxorubicin separately (Fig. 2E), confirming that ∆40p53 positively regulates these genes, and that endogenous FLp53, induced by doxorubicin, triggers netrin-1 and UNC5B gene expression (1). Interestingly, netrin-1 and UNC5B up-regulation increased further upon induction of ∆40p53, while in ∆133p53-A549 cells doxycycline treatment strongly inhibited netrin-1 and UNC5B induction by doxorubicin (Fig. 2E), confirming a dominant-negative role for ∆133p53 against FLp53 [START_REF] Bourdon | p53 isoforms can regulate p53 transcriptional activity[END_REF][START_REF] Horikawa | Delta133p53 represses p53-inducible senescence genes and enhances the generation of human induced pluripotent stem cells[END_REF]. Intriguingly, p21 gene expression following doxorubicin treatment was suppressed by doxycycline in both ∆40p53-and ∆133p53-A549 cells (Fig. 2E), suggesting that, at least for p21, ∆40p53 may also act as a dominant-negative effector of FLp53.

FLp53 is not necessary for netrin-1 regulation by ∆40p53

Even though increasing evidence suggests that ∆40p53 may regulate gene expression independently of FLp53 [START_REF] Yin | p53 Stability and activity is regulated by Mdm2-mediated induction of alternative p53 translation products[END_REF][START_REF] Candeias | Expression of p53 and p53/47 are controlled by alternative mechanisms of messenger RNA translation initiation[END_REF][START_REF] Powell | Stress-dependent changes in the properties of p53 complexes by the alternative translation product p53/47[END_REF], this isoform is also known to function as a FLp53 regulator, since ∆40p53 can oligomerize with FLp53, regulating its activities and functions [START_REF] Courtois | DeltaN-p53, a natural isoform of p53 lacking the first transactivation domain, counteracts growth suppression by wild-type p53[END_REF]. To assess the involvement of FLp53 in ∆40p53-dependent regulation of netrin-1, we generated stable p53-null H1299 cells, inducible for ∆40p53, FLp53 or both proteins. Gene expression analysis upon doxycycline treatment revealed a robust induction of netrin-1 gene expression following over-expression of both p53 proteins (Fig. 3A). Moreover, co-expression of FLp53 and ∆40p53 did not significantly change netrin-1 expression, compared to separate inductions. We confirmed these results at the protein level by Western blot analysis (Fig. 3B).

To ascertain that ∆40p53 regulates netrin-1 per se, regardless of FLp53 expression, we silenced endogenous FLp53 expression in A549 cells inducible for ∆40p53, using a specific siRNA targeting exon 2 of the p53 gene -i.e., thus avoiding degradation of ∆40p53 mRNA (Fig. 3C)-. Cells were treated with doxorubicin, in addition to doxycycline to induce ∆40p53 expression, and FLp53 silencing was then verified by western blot and quantitative RT-PCR (Fig. 3C and SI appendix, Fig. S2A). Western blot and quantitative RT-PCR analysis showed a strong accumulation of netrin-1, UNC5B and p21 upon induction of ∆40p53 or doxorubicin treatment in cells transfected with control siRNA (Fig. 3 C and D, and SI appendix, Fig. S2A). Moreover, although FLp53 silencing completely abolished the increase in target gene expression observed in doxorubicintreated cells, ∆40p53 was able to trigger netrin-1, UNC5B and p21 gene expression in these cells (Fig. 3 C and D, and SI appendix, Fig. S2A).

Finally, to validate the independence of ∆40p53 activity on netrin-1 with respect to FLp53 without relying on siRNA transfections, we stably repressed endogenous FLp53 in A549 cells inducible for ∆40p53, using CRISPR-dCas9-KRAB, a modified CRISPR/Cas9based system [START_REF] Thakore | Highly specific epigenome editing by CRISPR-Cas9 repressors for silencing of distal regulatory elements[END_REF]. We designed small guide RNAs (sgRNAs), targeting the first canonical p53 promoter, able to direct the binding of a nuclease defective Cas9 (dCas9) enzyme fused to KRAB (Krüppel-associated box) domain of Kox1 [START_REF] Thakore | Highly specific epigenome editing by CRISPR-Cas9 repressors for silencing of distal regulatory elements[END_REF][START_REF] Gilbert | CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes[END_REF]. The KRAB domain leads to transcription repression of p53 isoforms whose expression is regulated by this promoter -i.e., full-length and ∆40p53 isoforms (α, β and γ)-by mediating local epigenetic reprogramming of histone modifications [START_REF] Adli | The CRISPR tool kit for genome editing and beyond[END_REF][START_REF] Urrutia | KRAB-containing zinc-finger repressor proteins[END_REF]. FLp53 down-regulation was verified by Western blot analysis (Fig. 3E) in stably transfected A549 cells treated with doxorubicin. sgp53A completely inhibited basal and drug-induced FLp53 expression, while sgp53B showed a robust repression of FLp53 expression. Accordingly, netrin-1 up-regulation following doxorubicin treatment was completely inhibited in FLp53-repressed cells at protein and transcriptional levels (Fig. 3E and SI appendix, Fig. S2B), as well as its receptor UNC5B (SI appendix, Fig. S2C). However, ∆40p53 induction by doxycycline, not affected by dCas9-KRAB expression since it was regulated by a tetracycline-dependent CMV promoter, was still sufficient to trigger netrin-1 and UNC5B gene expression, even in the absence of FLp53 (Fig. 3E). Collectively, these results demonstrate that netrin-1 induction by ∆40p53 is independent of FLp53.

∆40p53 directly binds and activates the netrin-1 promoter

We then assessed whether ∆40p53 regulated netrin-1 via its promoter. Indeed, we previously cloned two different netrin-1 promoters (9, 39), and confirmed the presence of a p53 binding site in the internal netrin-1 promoter (1). Induction of FLp53 or ∆40p53 using doxycycline triggered transcription of firefly luciferase reporter gene placed under the control of the internal netrin-1 promoter or p21 promoter (Fig. 4A). However, site-direct mutagenesis of p53 binding site in the netrin-1 promoter completely abolished promoter transactivation upon induction of both FLp53 and ∆40p53 (Fig. 4A).

As ∆40p53, similarly to FLp53, can activate the netrin-1 promoter using the same p53 binding site, we wondered whether ∆40p53 interacted with the netrin-1 promoter in the same region. To ascertain this, we performed a chromatin-immunoprecipitation assay, using stable H1299 cells inducible for ∆40p53. PCR results show that, upon immunoprecipitation of over-expressed ∆40p53, using anti-HA-tagged antibody, we amplified a DNA fragment surrounding the p53 binding site in the netrin-1 promoter (Fig. 4B). Moreover, we confirmed that ∆40p53 was also able to interact with the p21 promoter (Fig. 4B). Altogether, these results demonstrate that ∆40p53 can directly bind the netrin-1 promoter at the same binding site as FLp53, favoring its transcriptional activation.

Endogenous ∆40p53 regulates netrin-1 expression

Having shown that over-expression of ∆40p53 induces the expression of netrin-1 and its receptor UNC5B, we next studied the role of endogenously-produced ∆40p53 on netrin-1 regulation. To achieve this, we used immortalized human skeletal myoblasts LHCN-M2, harboring wild-type p53. We designed two different sgRNAs, targeting "canonical" ATG in p53 exon 2 (sgFLp53) or the ATG codon 40 in p53 exon 4 (sgTOTp53), in order to inactivate FLp53 (i.e., p53α, β and γ) or FL/∆40p53 (including ∆40p53α, β and γ) isoforms, respectively (Fig. 5A). Moreover, both sgRNAs should not affect the expression of ∆133p53 and ∆160p53 isoforms. Then, using the CRISPR-Cas9 technique, we generated stable LHCN-M2 cell lines. Western blot analysis revealed a complete knock-out of p53 isoforms in stable cells generated with sgTOTp53, whereas in cells set-up with sgFLp53 only FLp53 was deleted (Fig. 5B). Moreover, in these cells a band corresponding to ∆40p53α appeared, demonstrating that this CRISPR-Cas9 strategy allowed us to force LHCN-M2 cells to produce endogenous ∆40p53 instead of FLp53.

Interestingly, netrin-1 and UNC5B proteins were strongly expressed in LHCN-M2-sgFLp53 cells, harboring ∆40p53 instead of FLp53, compared to control or sgTOTp53 cells (Fig. 5B). Moreover, quantitative RT-PCR analysis confirmed an increase in netrin-1 expression in LHCN-M2 cells expressing endogenous ∆40p53 (Fig. 5C). Intriguingly, gene expression of p21 and GADD45 (p53 target genes involved in cell cycle arrest) strongly decreased in both FLp53 knock-out cells, compared to parental or control LHCN-M2 cells, while only complete p53 knock-out drove a decrease in BAX (involved in apoptotic function of p53) gene expression (Fig. 5C and SI appendix, Fig. S3).

∆40p53 expression intensifies netrin-1 dependence and sensitizes cells to netrin-1 silencing-induced apoptosis

Since the over-expression or forced expression of ∆40p53 triggered an increase in the expression of netrin-1 and its receptor UNC5B, we hypothesized that in these cells survival dependency on netrin-1 expression may be amplified. To confirm this, we transfected stable LHCN-M2 cells with siRNA specific for netrin-1 (1). Netrin-1 silencing induced an increase in DNA fragmentation exclusively in sgFLp53 cells expressing endogenous ∆40p53 and consequently netrin-1 and UNC5B (Fig. 6A). Moreover, in these cells netrin-1 silencing induced a robust caspase-3 activation, compared to parental or control LHCN-M2 cells (Fig. 6B). Furthermore, complete p53 knock-out desensitized sgTOTp53 cells to netrin-1 silencing (Fig. 6 A andB). DNA fragmentation and caspase-3 activation observed in LHCN-M2 cells suggest that cell death triggered by netrin-1 silencing is apoptosis. Along the same line, transfection of siRNA targeting netrin-1 induced a consistent increase of the percentage of Annexin V positive cells only in sgFLp53 cells expressing endogenous ∆40p53α (Fig. 6C and SI appendix, Fig. S4). In line with these results, silencing netrin-1 increased DNA fragmentation upon induction of ∆40p53 in stable A549 cells (Fig. 6D), confirming the amplification of netrin-1 dependency in cells expressing high levels of ∆40p53. Interestingly, netrin-1 silencing had no effect on the induction of apoptosis in cells not treated with doxycycline (Fig. 6D).

Netrin-1 interference inhibits tumor growth in tumors cells expressing ∆40p53 in vivo

Because we observed that over-expression of ∆40p53, by inducing both netrin-1 and UNC5B, renders cancer cells more prone to die upon netrin-1 silencing, we next look whether this can be associated with tumor growth inhibition in vivo, using a more therapeutically-relevant approach. For this purpose, we evaluated the effect of NP137, a neutralizing anti-netrin-1 antibody able to induce cancer cell death and tumor regression in several preclinical models (2), upon ∆40p53 induction using the chicken chorioallantoic membrane (CAM) xenograft model. This technique is well accepted to study primary tumor growth on the highly vascularized CAM of chicken embryo [START_REF] Ménard | Hey1-and p53-dependent TrkC proapoptotic activity controls neuroblastoma growth[END_REF][START_REF] Genevois | Dependence receptor TrkC is a putative colon cancer tumor suppressor[END_REF]. Stable A549 cells inducible for ∆40p53 were engrafted into the CAM of E10 chicken embryos and primary tumors at the inoculation site were resected seven days after (Fig. 7A). In presence of doxycycline, ∆40p53α was induced (SI appendix, Fig. S5A) and anti-netrin-1 antibody (NP137) strongly reduced both the size and the weight of the tumors, compared to isotypic antibody (Iso-mAb)-treated engrafted cells (Fig. 7 B andC). Moreover, treatment with NP137 antibody did not affect CAM tumor growth in PBS-treated cells, indicating that the antitumoral effect of netrin-1 interfering is dependent on ∆40p53 expression. Interestingly, production of ∆40p53 upon doxycycline treatment slightly sustained tumor growth, even though this increase was not significant, suggesting a pro-tumoral effect of ∆40p53/netrin-1 axis, that could be reverted by netrin-1 interference.

The antitumoral effect of anti-netrin-1 antibody was also confirmed in a more conventional preclinical model of nude mice, engrafted with stable A549 cells inducible for ∆40p53α. As observed for the CAM model, while we noticed a substantial tumor growth in mice treated with PBS or isotypic control, anti-netrin-1 antibody considerably inhibited tumor progression in mice watered with doxycycline (Fig. 7D and SI appendix, Fig. S5B).

This effect was associated with apoptosis induction, as indicated by the increase of cleaved caspase-3 positive cells in engrafted tumors treated with both doxycycline and NP137 antibody (Fig. 7E and SI appendix, Fig. S5C). Finally, we tested how endogenous ∆40p53 protein could affect sensitivity to netrin-1 inhibition. For this purpose, we used colorectal cancer HCT116 cells wild-type (HCT116 p53+/+ ) or deleted for FLp53 (HCT116 p53-/-) [START_REF] Bunz | Requirement for p53 and p21 to sustain G2 arrest after DNA damage[END_REF]. Similar to the sgFLp53 LHCN-M2 cells we described above (see Fig. 5A), HCT116 p53-/-cells were generated by deletion of exon 2 on p53 gene, resulting in FLp53 knock-out. So, in this setting the HCT116 p53+/+ parental cell line is expressing low level of wild-type FLp53 but no ∆40p53 protein, whereas HCT116 p53-/-cells express no FLp53 but ∆40p53 protein (SI appendix, Fig. S6A).

Of interest, HCT116 p53-/-cells showed a significant increase of netrin-1 and UNC5B transcripts (SI appendix, Fig. S6B). Accordingly, transfection of siRNA targeting netrin-1 had no effect on cell viability in wild-type cells -i.e. supporting the view that low endogenous FLp53 does not regulate netrin-1/UNC5B activity-while it triggers DNA fragmentation exclusively in HCT116 p53-/-cells (SI appendix, Fig. S6C). Moreover, NP137 antibody nicely decreased the size and the weight of tumors obtained from HCT116 p53-/- cells on chicken CAMs (Fig. 7F and SI appendix, Fig. S6D).

Netrin-1 and ∆40p53 gene expression is correlated in human tumor biopsies.

We thus wondered whether ∆40p53 could positively regulate netrin-1 in a panel of human tumors. Indeed, conversely to FLp53, the activity of which is mainly regulated at a post-translational level in human cancers, ∆40p53 could be transcriptionally regulated and contribute to the neoplastic phenotype [START_REF] Takahashi | Dominant effects of Delta40p53 on p53 function and melanoma cell fate[END_REF][START_REF] Hofstetter | The N-terminally truncated p53 isoform Delta40p53 influences prognosis in mucinous ovarian cancer[END_REF][START_REF] Avery-Kiejda | Small molecular weight variants of p53 are expressed in human melanoma cells and are induced by the DNA-damaging agent cisplatin[END_REF][START_REF] Avery-Kiejda | The relative mRNA expression of p53 isoforms in breast cancer is associated with clinical features and outcome[END_REF]. Moreover, netrin-1 is often dysregulated in cancers. For this purpose, we analyzed mRNA levels of netrin-1 and ∆40p53 gene expression in melanoma and colorectal cancer biopsies by quantitative RT-PCR. As shown in Fig. 8A, in both cohorts analyzed, we found a positive and significant correlation between netrin-1 and ∆40p53. Moreover, netrin-1 appeared more expressed in human tumor materials presenting high levels of ∆40p53 (Fig. 8B). These results support the view that in human tumors, netrin-1 could be positively regulated by ∆40p53.

Discussion

Netrin-1 is a protein frequently up-regulated in aggressive human cancers [START_REF] Fitamant | Netrin-1 expression confers a selective advantage for tumor cell survival in metastatic breast cancer[END_REF][START_REF] Paradisi | NF-kappaB regulates netrin-1 expression and affects the conditional tumor suppressive activity of the netrin-1 receptors[END_REF][START_REF] Delloye-Bourgeois | Netrin-1 acts as a survival factor for aggressive neuroblastoma[END_REF][START_REF] Delloye-Bourgeois | Interference with netrin-1 and tumor cell death in nonsmall cell lung cancer[END_REF].

The pro-tumoral function of netrin-1 mainly results from its ability to bind dependence receptors DCC and UNC5H, inhibiting their pro-apoptotic activity [START_REF] Goldschneider | Dependence receptors: a new paradigm in cell signaling and cancer therapy[END_REF][START_REF] Mazelin | Netrin-1 controls colorectal tumorigenesis by regulating apoptosis[END_REF][START_REF] Llambi | Netrin-1 acts as a survival factor via its receptors UNC5H and DCC[END_REF][START_REF] Mehlen | The DCC gene product induces apoptosis by a mechanism requiring receptor proteolysis[END_REF][START_REF] Castets | DCC constrains tumour progression via its dependence receptor activity[END_REF]. Within this context, several studies have focused on inhibiting the binding of netrin-1 to its receptors to induce cancer cell death and tumor regression (2,[START_REF] Fitamant | Netrin-1 expression confers a selective advantage for tumor cell survival in metastatic breast cancer[END_REF][START_REF] Delloye-Bourgeois | Netrin-1 acts as a survival factor for aggressive neuroblastoma[END_REF][START_REF] Delloye-Bourgeois | Interference with netrin-1 and tumor cell death in nonsmall cell lung cancer[END_REF][START_REF] Paradisi | Netrin-1 up-regulation in inflammatory bowel diseases is required for colorectal cancer progression[END_REF][START_REF] Mille | Interfering with multimerization of netrin-1 receptors triggers tumor cell death[END_REF]. These attempts led to the development of a netrin-1 neutralizing antibody, currently in Phase Ib/II clinical trial (2). However, netrin-1 inhibition may only be applied to the fraction of tumors expressing high levels of netrin-1. Therefore, the knowledge of which cancer cells rely on this strategy to survive and what mechanisms underlie netrin-1 up-regulation in tumor cells could be important in developing personalized therapeutic treatments. Having shown that the transcription factor p53 positively regulates netrin-1 (1), we investigated the underlying mechanisms, as such knowledge could increase the fraction of cancer patients who may be eligible for a netrin-1 interference-based treatment during early clinical evaluation.

Here we demonstrate that ∆40p53, a N-terminal truncated isoform of p53, transcriptionally regulates the gene expression of netrin-1 and its receptor UNC5B, and unexpectedly, it does so independently of the presence of FLp53. Indeed, early studies reported ∆40p53 as a dominant-negative regulator of FLp53 [START_REF] Marcel | Biological functions of p53 isoforms through evolution: lessons from animal and cellular models[END_REF], since its overexpression down-regulates FLp53-induced target genes, counteracting FLp53-dependent growth suppression [START_REF] Ghosh | Regulation of human p53 activity and cell localization by alternative splicing[END_REF][START_REF] Courtois | DeltaN-p53, a natural isoform of p53 lacking the first transactivation domain, counteracts growth suppression by wild-type p53[END_REF]. However, ∆40p53 was more recently described as a positive regulator of FLp53 functions [START_REF] Hafsi | Effects of Delta40p53, an isoform of p53 lacking the N-terminus, on transactivation capacity of the tumor suppressor protein p53[END_REF], since Δ40p53 over-expression induces apoptosis in melanoma cells in cells expressing wild-type FLp53 exclusively [START_REF] Takahashi | Dominant effects of Delta40p53 on p53 function and melanoma cell fate[END_REF]. These paradoxical results have been attributed to dose-dependent effects of hetero-oligomerization of p53 isoforms, dependent on the relative expression levels [START_REF] Anbarasan | The Emerging Landscape of p53 Isoforms in Physiology, Cancer and Degenerative Diseases[END_REF]. For instance, it has been demonstrated that low ∆40p53 expression enhances FLp53 transactivation activity, whereas increasing levels suppress anti-proliferative effects of FLp53 [START_REF] Hafsi | Effects of Delta40p53, an isoform of p53 lacking the N-terminus, on transactivation capacity of the tumor suppressor protein p53[END_REF]. As a consequence, because netrin-1 was previously described as a FLp53 target gene (1), its regulation by ∆40p53 expression could have been due to p53 stabilization and activation by hetero-oligomers ∆40p53/FLp53. However, our results, showing netrin-1 up-regulation by ∆40p53 in H1299 p53-null cells or upon FLp53 knock-out in A549 p53-wild-type cells, suggest the formation of a ∆40p53-only homo-tetramer, binding to and activating netrin-1 promoter. On the other hand, binding of ∆40p53 to p53 responsive elements located in MDM2, GADD45 and BAX genes, leading to FLp53-independent transactivation, has been already reported [START_REF] Yin | p53 Stability and activity is regulated by Mdm2-mediated induction of alternative p53 translation products[END_REF]. Interestingly, our data also show that the CRISPR-Cas9 technique, using specific sgRNAs, enabled us to generate stable primary cells expressing ∆40p53 instead of FLp53. In this way, we were also able to confirm the up-regulation of netrin-1 and UNC5B by endogenously-expressed ∆40p53. This information, together with gene expression analysis on inducible stable cell lines, suggests that ∆40p53 can establish homo-tetramers, transactivating the netrin-1 promoter, in the context of both high and low ∆40p53 expression levels, at least in the absence of FLp53. p53 isoforms are differentially expressed in several human cancers [START_REF] Khoury | The isoforms of the p53 protein[END_REF][START_REF] Vieler | p53 Isoforms and Their Implications in Cancer[END_REF], such as triple-negative breast and mucinous ovarian cancers and melanoma [START_REF] Hofstetter | The N-terminally truncated p53 isoform Delta40p53 influences prognosis in mucinous ovarian cancer[END_REF][START_REF] Avery-Kiejda | Small molecular weight variants of p53 are expressed in human melanoma cells and are induced by the DNA-damaging agent cisplatin[END_REF][START_REF] Avery-Kiejda | The relative mRNA expression of p53 isoforms in breast cancer is associated with clinical features and outcome[END_REF]. However, the high expression of ∆40p53 and correlated cancer aggressiveness has been associated with the dominant-negative function of this isoform on FLp53 activity. Interestingly, we show here a positive correlation between ∆40p53 and netrin-1 gene expression in melanoma and colorectal cancer cohorts. Moreover, netrin-1 and its dependence receptors are involved in melanoma and colorectal cancer progression [START_REF] Paradisi | NF-kappaB regulates netrin-1 expression and affects the conditional tumor suppressive activity of the netrin-1 receptors[END_REF][START_REF] Castets | DCC constrains tumour progression via its dependence receptor activity[END_REF][START_REF] Bernet | Inactivation of the UNC5C Netrin-1 receptor is associated with tumor progression in colorectal malignancies[END_REF][START_REF] Boussouar | Netrin-1 and Its Receptor DCC Are Causally Implicated in Melanoma Progression[END_REF]. Our data thus suggest for the first time a FLp53-independent pro-tumoral role for ∆40p53, owing to its ability to transactivate an anti-apoptotic protein such as netrin-1.

Regulation of netrin-1 and UNC5B receptor by the ∆40p53 isoform could have significant therapeutic consequences. Indeed, the fact that both the ligand and its dependence receptor are up-regulated in human cancers co-expressing ∆40p53 could enhance dependency of tumor cell survival on netrin-1 expression. Specifically, inhibition of interference of netrin-1 in these cancer cells should lead to dependence receptorinduced apoptosis and tumor regression. Our results demonstrate that silencing of netrin-1 expression by RNA interference induces cell death only in cells that are forced to produce ∆40p53 and consequently express high levels of netrin-1 and UNC5B. From a therapeutic point of view, we show here that, upon induction of ∆40p53, interference of the binding between netrin-1 and UNC5B with an anti-netrin-1 neutralizing antibody is sufficient to inhibit tumor growth in two different preclinical models of chicken CAM and nude mice.

In conclusion, we show here for the first time that the ∆40p53 isoform could positively regulate an anti-apoptotic protein, such as netrin-1, in a FLp53-independent way. In addition, this regulation of ∆40p53 could explain the increased transcript level of this survival protein, observed in several human cancers. Indeed, each mutation changing the ratio between FLp53 and ∆40p53 generation (i.e., non-sense mutations in exon 2/3) could confer a selective advantage to cancer cells. ∆40p53 could inhibit the tumor suppressor activity of FLp53, acting as a dominant-negative protein, but could also actively transactivate pro-tumoral factors, such as shown here for netrin-1. In this context, a promising therapeutic strategy could be to inhibit the pro-tumoral activity, as we propose for netrin-1, instead of targeting the dominant-negative function of ∆40p53.

Materials and Methods

Generation of stable cell lines, reagents and transfection procedures

A549 and H1299 cell lines were obtained from the ATCC (Manassas, VA, USA).

hTERT and cdk4-immortalized LHCN-M2 cells were a kind gift from Dr Vincent Mouly, (Center for research In Myology, Paris, France) [START_REF] Zhu | Cellular senescence in human myoblasts is overcome by human telomerase reverse transcriptase and cyclin-dependent kinase 4: consequences in aging muscle and therapeutic strategies for muscular dystrophies[END_REF]. HCT116 p53-/-cells were generated previously [START_REF] Bunz | Requirement for p53 and p21 to sustain G2 arrest after DNA damage[END_REF] and were kindly provided by B. Vogelstein (Ludwig Center at Johns Hopkins, Baltimore, MD). N-terminal HA-tagged human FLp53, ∆40p53 and ∆133p53 coding sequences were cloned at SfiI sites in the pITR plasmid [START_REF] Wang | The Proto-oncogene c-Kit Inhibits Tumor Growth by Behaving as a Dependence Receptor[END_REF]. Detailed cell culture conditions, reagents, transfection procedures, and protocols for generation of stable cell lines are provided in SI Appendix, Supplementary Materials and Methods. siRNA and small guide cloning sequences are listed in SI Appendix, Fig. S7.

Quantitative RT-PCR and western blot analysis

Total RNAs were extracted using the NucleoSpin® RNA Plus Kit (Macherey Nagel, Düren, Germany) according to the manufacturer's protocol. RT-PCR reactions were performed with the PrimeScript RT Reagent Kit (Takara Bio Europe, Saint-Germain-en-Laye, France). For immunoblotting analysis, cells were lysed by sonication in SDS buffer (10 mM Tris-HCl pH 7.4, 10% glycerol, 5% SDS, 1% TX-100, 100 mM DTT) in the presence of protease inhibitor cocktail (Roche Applied Science). Antibodies and detailed protocols are supplied in SI Appendix, Supplementary Materials and Methods. Primer sequences are available in SI Appendix, Fig. S7. Expression of endogenous FLp53 and over-expressed ∆40p53α was also evaluated by Western blot, using Pab 1801 p53 antibody (C). (E) ∆40p53-induced netrin-1 gene expression is not altered by knocking-down endogenous p53. A549 cells were infected using CRISPR/Cas9-modified pLV-hU6-sgRNA-hUbC-dCas9-KRAB-T2a-Puro plasmid, and stable cell lines were generated using firefly luciferase-control (sgLUC) or p53specific (sgp53A and sgp53B) small guide RNAs. Endogenous p53-repressed cell lines were then transfected with empty (pITR) or ∆40p53 sleeping-beauty plasmids. Stable A549 cell lines knockdown for p53 and inducible for ∆40p53 were treated with doxorubicin and doxycycline for 24 h. Efficacy of endogenous p53 repression was evaluated by Western blot, using p53-DO1 antibody, while anti-HA-tagged antibody provided data on ∆40p53 expression. In addition, netrin-1 and p21 protein levels were assessed by Western blot. Immunoblots in B, C and E are representative of three independent experiments. Quantitative PCR results in A and D are presented as mean ± SEM. ns, non-significant; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001. NT, not treated; Dox, doxycycline; DoxoR, doxorubicin; NTN1, netrin-1; HA, influenza hemagglutinin; sg, small guide; FLp53, fulllength p53; sgLUC, small guide targeting luciferase. Fig. 4. ∆40p53 directly binds to and activates the netrin-1 promoter. (A) ∆40p53 activates the netrin-1 promoter, binding to canonical p53 responsive elements. Stable H1299 cells inducible for ∆40p53 or full-length p53 (FLp53) were transfected with wild-type (NetP) or mutated for p53 binding site (NetP mut) promoters, as well as for the p21 promoter (p21P). 24 h after transfection, cells were treated with 2 µg/mL of doxycycline for a further 24 h and promoter activities were assessed by luciferase assay. Values represent mean ± SEM (n = 3). Induction of ∆40p53 and FLp53 was verified by Western blot, using α-HA antibody.

Reporter assay and chromatin immunoprecipitation assay

(B) ∆40p53 directly binds to the netrin-1 promoter. Stable H1299 cells inducible for ∆40p53-HA were treated with 2 µg/mL of doxycycline for 24 h. Chromatin immunoprecipitation (ChIP) was performed using α-HA antibody, as described in the Material and Methods section. ∆40p53-co-precipitated DNA was analyzed by quantitative PCR, using two different primer couples surrounding the p53 binding site in the netrin-1 promoter (p53RE-1 and -2) (1). A couple of primers, amplifying a not-p53-related fragment in the netrin-1 promoter, was used as negative control (Neg CTRL). p21 primers, surrounding the p53 binding site -1354 contained in the p21 promoter [START_REF] Macleod | p53-dependent and independent expression of p21 during cell growth, differentiation, and DNA damage[END_REF], were used to confirm activation and binding of p21 promoter upon ∆40p53 induction. Values represent mean ± SEM (n = 4). Western blot in the inset, performed with α-HA antibody, confirmed ∆40p53 expression in immunoprecipitated samples. **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; ns, non-significant. CTRL, control; Dox, doxycycline; FLp53, full-length p53; NetP, netrin-1 promoter; p21P, p21 promoter. Weight and size of CAM-engrafted tumors. Primary tumors were treated with 10 µg/mL of neutralizing anti-netrin-1 antibody (NP137) or isotypic control (Iso-mAb), in presence of 2 µg/mL of doxycycline. After seven days, primary tumors were imaged using Zeiss expression does not affect ∆40p53-dependent UNC5B up-regulation. Stable A549 cell lines, inducible for ∆40p53, were transfected with a scramble siRNA (siCTRL) or with a specific siRNA targeting exon 2 of the p53 gene (sip53). 24 h after transfection, cells were treated with doxorubicin or doxycycline for a further 24 h. UNC5B and p21 transcript levels were quantified by quantitative RT-PCR. p53 silencing efficacy was evaluated by quantitative RT-PCR, using full-length p53-specific primers (FLp53), designed in exon 2 of p53 gene. Gene expression was normalized using TATA-binding protein (TBP) and beta-glucuronidase (GUSB) genes, used as housekeeping genes. (B, C) ∆40p53-induced netrin-1 and UNC5B gene expression is not altered by knocking-down endogenous p53. A549 cells were infected using CRISPR/Cas9-modified pLV-hU6-sgRNA-hUbC-dCas9-KRAB-T2a-Puro plasmid, and stable cell lines were generated using firefly luciferase-control (sgLUC) or p53-specific (sgp53A and sgp53B) small guide RNAs. Endogenous p53 knocked out cell lines were then transfected with empty (pITR) or ∆40p53 sleeping-beauty plasmids. Stable A549 cell lines knocked out for p53 and inducible for ∆40p53 were treated with doxorubicin and doxycycline for 24 h. Netrin-1 (B) and UNC5B (C) transcript levels were assessed by quantitative RT-PCR. Data are presented as mean ± SEM (n = 3). ns, non-significant; *, p < 0.05; ***, p < 0.001; ****, p < 0.0001. CTRL, not treated, control cells; Dox, doxycycline; DoxoR, doxorubicin; NTN1, netrin-1; FLp53, full-length p53; sg, small guide.

Fig. 1 .

 1 Fig. 1. Netrin-1 regulation by p53 requires the transcriptional activity of the TAD2 domain. (A) Schematic representation of the structure of p53 domains. The structure can be

Fig. 2 .

 2 Fig. 2. ∆40p53, but not ∆133p53, regulates netrin-1 and UNC5B expression. (A)

Fig. 3 .

 3 Fig. 3. Full-length p53 is not necessary for netrin-1 regulation by ∆40p53. (A, B) Over-

Fig. 5 .

 5 Fig. 5. Endogenous ∆40p53 regulates netrin-1 expression. (A) Schematic representation

Fig. 6 .

 6 Fig. 6. ∆40p53 expression sensitizes cells to netrin-1 silencing-induced apoptosis. (A, B,

Fig. 7 .

 7 Fig. 7. ∆40p53 expression sensitizes cells to netrin-1 interference-induced tumor growth

  Microscope and tumor areas were measured with AxioVision Rel Software. Moreover, tumors were resected and weighted. n indicated the number of samples for each condition. (C) Representative images of A549 primary tumors, treated with PBS or doxycycline (dox), in combination with anti-netrin-1 (NP137) or isotypic antibodies (Iso-mAb). Red lines represent tumor volume automatically measured by AxioVision Software (scale bar = 1 mm). (D) Neutralizing netrin-1 antibody (NP137) inhibits tumor growth in nude mice engrafted with stable A549 inducible for ∆40p53 and watered with doxycycline, compared to animals treated with isotypic antibody (Iso-mAb) (n = 10 for each group).Tumor volume were measured at the indicated days after the first treatment with antibodies. (E) Anti-tumoral effect of NP137 antibody is associated with apoptosis induction. Engrafted tumors were resected 36 days after beginning treatments and apoptosis was evaluated by immunohistochemical staining of active caspase-3. (F) ∆40p53 expression in colorectal cancer cells sensitizes cells to netrin-1 inhibition in the CAM model. Wild-type or FLp53-knockout HCT116 cells were seeded in the CAM of E10 chick embryo, together with 10 µg/mL of anti-netrin-1 (NP137) or control (Iso-mAb) antibodies. Tumors were imaged after seven days and tumor areas were measured, as well as their weight. n indicated the number of samples for each condition. Data are represented as mean ± SEM. ns, non-significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; Dox, doxycycline; CAM, chorioallantoic membrane; mAb, monoclonal antibody; Iso, isotypic; cCaspase-3, cleaved caspase-3.

Fig. 8 .

 8 Fig. 8. Netrin-1 and ∆40p53 gene expression is correlated in human melanoma and

  Figure 5

Fig. S2 .

 S2 Figure 6

Fig. S3 .

 S3 Fig. S3. Endogenous ∆40p53 affects GADD45 and BAX gene expression. Stable LHCN-M2 cells knocked out for p53 gene were generated using firefly luciferase-control (sgLUC), ATG1-(sgFLp53) or ATG40-(sgTOTp53) targeting sgRNAs. Expression levels of Growth Arrest and DNA Damage 45 (GADD45) and BAX transcripts were analyzed by quantitative RT-PCR. Results are presented as mean ± SEM (n = 3). ns, non-significant; *, p < 0.05.

Acknowledgements

We wish to thank Brigitte Manship for critical reading of the manuscript, Dr Jean-Christophe Bourdon for p53-related materials and Nicolas Gadot (Plateforme Anatomopathologie Recherche, Université de Lyon, Université Claude Bernard Lyon 1, INSERM 1052, CNRS 5286, Centre Léon Bérard, Centre de recherche en Cancérologie de Lyon (CRCL), Lyon, 69373, France) for histological examination. This work was supported by institutional grants from CNRS, INSERM, University of Lyon, Centre Léon Bérard and from the Ligue Contre le Cancer, INCA, ANR, ERC and Fondation Bettencourt. Yan Sun and Hong Wang have been supported by a fellowship from China Scholarship Council, Ambroise Manceau and Lisa Frydman from Ligue Contre le Cancer, Valeria Basso from Nuovo-Soldati Cancer Research Foundation.

Stable A549 cells inducible for FLp53 or ∆40p53 were plated in 12-well plates and transfected with the different firefly luciferase reporters containing wild-type or p53mutated netrin-1 promoter constructs, described before (1,[START_REF] Paradisi | NF-kappaB regulates netrin-1 expression and affects the conditional tumor suppressive activity of the netrin-1 receptors[END_REF][START_REF] Delloye-Bourgeois | Nucleolar localization of a netrin-1 isoform enhances tumor cell proliferation[END_REF] (SI Appendix, Supplementary Materials and Methods). Chromatin immunoprecipitation assay was performed as previously described (1), with some minor changes, described in SI Appendix, Supplementary Materials and Methods. Sequences of primers used for ChIP analysis are illustrated in SI Appendix, Fig. S7.

Cell Death Assays

For DNA fragmentation analysis (SubG1), DNA content was evaluated using the NucleoCounter NC-3000 system (ChemoMetec A/S, Allerød, Denmark). For AnnexinV and propidium iodide (PI) double staining, Annexin V positive cells were then evaluated by fluorescence activated cell sorting (FACS) on FACSCanto II (BD Biosciences, San Jose, CA, USA). Caspase-3 activity assay was performed as described previously [START_REF] Wang | The Proto-oncogene c-Kit Inhibits Tumor Growth by Behaving as a Dependence Receptor[END_REF].

Detailed protocols are available on SI Appendix, Supplementary Materials and Methods.

Xenografts in ovo and in nude mice

For chick chorioallantoic membrane (CAM) assay, cells were seeded on 10 daysold CAMs, together with netrin-1 neutralizing antibody NP137 and relative control NP001 (10 µg/mL) (Netris Pharma, Lyon, France) and doxycycline (2 µg/mL). For xenografts in nude mice, A549 cells were implanted in 5-week-old female athymic nude Foxn1 nu/nu mice.

Procedures and treatment administration are detailed in SI Appendix, Supplementary Materials and Methods.

All experiments were performed in accordance with relevant guidelines and regulations of animal ethics committee (Authorization APAFIS n°: 28723; accreditation of laboratory animal care by CECCAP, ENS Lyon-PBES, France).

Patients and tumor samples

24 melanoma samples were collected as previously described [START_REF] Boussouar | Netrin-1 and Its Receptor DCC Are Causally Implicated in Melanoma Progression[END_REF], whereas 19 primary colorectal cancer tissue samples were obtained from the Biological Resource Centre (CRB, Centre de Resource Biologique) of Léon Bérard Centre (protocol number: BB-0033-00050) (Lyon, France), as previously described [START_REF] Ruocco | Alu RNA accumulation induces epithelial-to-mesenchymal transition by modulating miR-566 and is associated with cancer progression[END_REF] (SI Appendix, Supplementary Materials and Methods).

Authors Contribution

Investigation, Y.S., A.M., L.F., L.C., D.N., V.B., H.W. and A.P.; Conceptualization, Y.S., P.M. and A.P.; Resources, L.C. and C.M.; Supervision, J.F., C.M., P.M. and A.P.; Writing -Original Draft, P.M. and A.P.; Writing -Review & Editing, P.M. and A.P.; Funding Acquisition, P.M.

Conflict of Interest

A B

Supplementary Information for

Δ40p53 isoform up-regulates netrin-1/UNC5B expression and potentiates netrin-1 pro-oncogenic activity c Netris Pharma, 69008 Lyon.

1 A.M. and L.F. contributed equally to this work. 2 To whom correspondence may be addressed. Email: andrea.paradisi@lyon.unicancer.fr or patrick.mehlen@lyon.unicancer.fr.

Generation of stable cell lines

For the generation of inducible stable cell lines, N-terminal HA-tagged human

Quantitative RT-PCR

Total RNAs were extracted using the NucleoSpin® RNA Plus Kit (Macherey Nagel, Düren, Germany) according to the manufacturer's protocol. RT-PCR reactions were performed with the PrimeScript RT Reagent Kit (Takara Bio Europe, Saint-Germain-en-Laye, France). 500 ng total RNA was reverse-transcribed using the following program: 37°C for 15 min and 85°C for 5 sec. For expression studies, the target transcripts were amplified on the LightCycler®2.0 apparatus (Roche Applied Science, Indianapolis, IN, USA), using the Premix Ex Taq probe qPCR Kit (Takara Bio Europe), according to the manufacturer's instructions. Expression of target genes was normalized against TATA binding protein (TBP) and beta-glucuronidase (GUSB) genes, used as housekeeping genes. The amount of target transcripts, normalized against the housekeeping gene, was calculated using the comparative CT method. A validation experiment was performed, in order to demonstrate that the efficacy of target and housekeeping genes was almost equivalent. Primer sequences are available in SI Appendix, Fig. S7.

Western blot analysis

For immunoblotting analysis, cells were lysed by sonication in SDS buffer

Reporter assay

Stable A549 cells inducible for FLp53 or ∆40p53 were plated in 12-well plates and transfected with the different firefly luciferase reporters containing wild-type or p53mutated netrin-1 promoter constructs. 24 h after transfection, cells were treated with 2 µg/mL doxycycline and further incubated for 24 h. All transfections were performed in triplicate and the Dual-Luciferase Reporter Assay system (Promega, Charbonnieres, France) was carried out 48 h after transfection according to the manufacturer's protocol, using the Infinite F500 apparatus (Tecan, Männedorf, Switzerland). As an internal control of transfection efficacy, the Renilla luciferase encoding plasmid (pRL-CMV, Promega) was co-transfected and for each sample firefly luciferase activity was normalized against the Renilla luciferase activity.

Chromatin immunoprecipitation assay

Stable H1299 cells inducible for ∆40p53 were treated with doxycycline for 24 h and collected by enzymatic detachment. Cells were then cross-linked with 1% formaldehyde/methanol solution (Sigma-Aldrich) in PBS. After 30 min incubation at 4°C, the cross-link reaction was blocked by centrifugation and three washes with PBS.

After lysis with buffer L1 (50 mM Tris-HCl pH 8.0, 2 mM EDTA, 0.1% NP40, Glycerol

Cell Death Assays

For DNA fragmentation analysis (SubG1), LHCN-M2 cell lines were plated onto 6-well plates at a density of 1.5*10 5 cells per well. The following day, cells were transfected with siRNA, as described above, and incubated in serum-free medium for 48 h. For SubG1 analysis of A549 cell lines, cells were plated onto 6-well plates at a density of 5.0*10 5 cells per well and transfected with siRNA the day after. 24 h later, transfected cells were plated again in complete medium onto 6-well plates at a density of 1.5*10 5 cells per well. The following day, cells were treated with doxycycline in serum-free medium and further incubated for 48 h. Cells were then collected, fixed in 70% ethanol and incubated at -20°C for at least one night. Subsequently, cells were washed twice with PBS and incubated for 5 min at 37°C in PBS containing 0.1% TX-100 and 2 µg/mL 4′,6-diamidino-2-phenylindole (DAPI). DNA content was evaluated using the NucleoCounter NC-3000 system (ChemoMetec A/S, Allerød, Denmark).

For AnnexinV and propidium iodide (PI) double staining, LHCN-M2 cell lines were seeded onto 6-well-plates at a density of 2.5*10 5 cells per well. The following day, cells were transfected with siRNA and incubated in serum-free media for 72 h. Cells were then harvested, washed with PBS and incubated for 15 minutes in Annexin V buffer containing allophycocyanin (APC) conjugated to Annexin V and PI (BioLegend, San Diego, CA, USA). Annexin V positive cells were then evaluated by fluorescence activated cell sorting (FACS) on FACSCanto II (BD Biosciences, San Jose, CA, USA).

Caspase-3 activity assay was performed as described previously (1) and was calculated from a 1 h kinetic cycle reading on a spectrofluorometer (405 nm / 510 nm, Infinite F500, Tecan).

Xenografts in ovo

Fertilized white Leghorn chicken eggs were incubated at 38°C with 60% humidity. A small window was made in the shell on day 3 of chick embryo development under aseptic conditions, in order to aspire liquid inside. Windows were then sealed and eggs incubated for further seven days. For stable A549 cells, inducible for ∆40p53, Tumor size was measured three times per week with a caliper. Tumor volume was calculated according to the formula v= 0.5 x (l x w2), where v is volume, l is length, and w is width. All experiments were performed in accordance with relevant guidelines and regulations of animal ethics committee (Authorization APAFIS n°: 28723; accreditation of laboratory animal care by CECCAP, ENS Lyon-PBES, France).

Immunohistochemical staining

For histological examination of tumors engrafted in nude mice, endpoint tumors were fixed in 10% buffered formalin and embedded in paraffin. 4-µm-thick tissue sections of formalin-fixed, paraffin-embedded tissue were prepared according to conventional procedures. Sections were then stained with hematoxylin and eosin and examined with a light microscope. Immunohistochemistry was performed on an automated immunostainer (Ventana Discovery XT, Roche, Meylan, France) using

Omnimap DAB Kit according to the manufacturer's instructions. Sections were incubated with a rabbit anti-cleaved caspase-3 antibody (diluted at 1:500, #9661S, Cell Signaling). HRP-conjugated anti-rabbit antibody was applied on sections. Staining was visualized with DAB solution with 3,3′-diaminobenzidine as a chromogenic substrate.

Then, the sections were counterstained with Gill's hematoxylin. Finally, sections were scanned with panoramic scan II (3D Histech, Budapest, Hungary) at 20X.

Patients and tumor samples

For the melanoma cohort, 24 tumor samples were collected as previously described ( 4), from patients who had undergone surgery for skin melanocytic lesions between 2007 and 2011. The research protocol was approved by the Ethics committee of Saint Louis Hospital (Paris, France). 19 primary colorectal cancer tissue samples were obtained from the Biological Resource Centre (CRB, Centre de Resource Biologique) of Léon Bérard Centre (protocol number: BB-0033-00050) (Lyon, France).

The research protocol was approved by CRB medical and scientific committee. All of the patients signed informed consent letters, as required by the French Bioethics law, approving the use of their samples for research.

Statistical analysis

The data reported are expressed as the mean ± S.D. of at least three independent determinations, each performed in triplicate. Statistical analysis was performed using the Two-way ANOVA or linear regression tests, using GraphPad Prism version 6.01 for Windows (GraphPad Software, La Jolla, CA, USA), except where indicated differently.