en utilisant des siRNA ciblant les ARNm des différents gènes candidats. J’ai tout d’abord validé l’efficacité des différents siRNA ciblant les gènes candidats par RT-qPCR. La diminution d’expression observée pour chaque candidat par rapport au contrôle est supérieure à 50% pour les 3 candidats (Figure 19).
J’ai transfecté les cellules de neuroblastome IGR-N91::GFP avec un siRNA contrôle ou un siRNA ciblant le gène candidat (siRNA candidat). J’ai par la suite greffé ces cellules chez l’embryon de poulet en suivant la technique expérimentale mise en place lors de l’établissement du modèle. 48h après greffe, j’ai récolté les embryons, et ai analysé la localisation des cellules de neuroblastome dans l’embryon de poulet ainsi que la taille des tumeurs formées. J’ai réalisé cette analyse sur coupes, en utilisant le marqueur HNK1 pour localiser les dérivés sympathiques des cellules de la crête neurale.
Pour les différents candidats étudiés, j’ai pu constater que l’interférence avec leur expression n’altère pas la capacité des cellules de neuroblastome à former des tumeurs au sein des ganglions sympathiques. En effet, 100% des embryons présentent des tumeurs au sein des dérivés sympathiques, dans les conditions siRNA Scr et siRNA candidats.
** * ns
Stades 1 à 3 Stade 4 Stade 4S 8 10 12 14 **** *** ns
Stades 1 à 3 Stade 4 Stade 4S 0 5 10 20 15 **** **** ns
Stades 1 à 3 Stade 4 Stade 4S 14 16 18 20 1.0 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0 0 24 48 72 96 120 144 168 192 216 Durée en mois p 0.286 bonf p 1.000 Forte expression de PATZ1 (n=35) Faible expression de PATZ1 (n=441)
Pr obabilit é de survie sans r echut e Pr obabilit é de survie sans r echut e 24 48 72 96 120 144 168 192 216 p 2.5e-15 bonf p 1.1e-12 Forte expression de TMEFF2 (n=272) Faible expression de TMEFF2 (n=204) 1.0 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0 0 Durée en mois Pr obabilit é de survie sans r echut e 24 48 72 96 120 144 168 192 216 raw p 5.1e-12 bonf p 2.4e-9 Forte expression de TMSB4X (n=394) Faible expression de TMSB4X (n=82) 1.0 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0 0 Durée en mois A. B. C..
Figure 18. Expression des différents candidats dans les cohortes de neuroblastomes humains. Les panels de gauche représentent, pour chaque candidat, le taux d’expression du gène dans les neuroblastome de stades 1 à 3, de stade 4 et de stade 4S dans la cohorte publiée de Koçak et al. (GEO ID : GSE45547). Les panels de droite présentent les courbes Kaplan-Meier de survie sans rechute en fonction des taux d’expression de chaque gène candidat. A. Analyse de l’expression du gène PATZ1. B. Analyse de l’expression du gène TMEFF2. C. Analyse de l’expression du gène TMSB4X.
n=357 n=214 n=78
n=357 n=214 n=78
J’ai mesuré par la suite l’aire maximale des tumeurs présentes dans les ganglions sympathiques pour chacun des candidats. J’ai ainsi pu constater que l’interférence avec l’expression des gènes PATZ1 et TMEFF2 n’impacte pas le développement des masses tumorales car il n’y a pas de différence significative de taille des masses au sein des ganglions sympathiques, dans la condition où ces candidats sont invalidés par le siRNA. A l’inverse, l’interférence avec l’expression de TMSB4X conduit à une augmentation significative de la taille des tumeurs sympathiques. TMSB4X semble donc être impliqué dans la croissance tumorale, et représente de ce fait un candidat intéressant à étudier dans la tumorigénèse neuroblastique. Cependant, TMSB4X étant une petite protéine liée au cytosquelette, la mise en place de traitements ciblés représente un défi. De plus, l’interférence avec l’expression de cette protéine conduit à une augmentation du diamètre des tumeurs.
Les résultats les plus probants ont été obtenus avec le candidat HDGF (résultats présentés dans l’article ci-après). Les patients présentant un fort taux d’expression d’HDGF présentent également un taux de survie sans rechute significativement plus faible. De plus, l’interférence avec l’expression de HDGF conduit à la diminution de l’aire moyenne des tumeurs dans les embryons de poulet. Enfin, HDGF représente actuellement un espoir thérapeutique dans les cas de cancer du poumon. J’ai donc poursuivi ma thèse par l’étude fonctionnelle de HDGF. Cette étude a conduit à mettre en évidence HDGF comme un gène fortement exprimé dans le neuroblastome agressif de stade 4. L’effet anti-tumoral d’une diminution de HDGF observé in vivo serait lié à l’impact de cette manipulation sur la dynamique du cycle cellulaire des cellules neuroblastiques confrontées au microenvironnement embryonnaire.
Un article concernant ces résultats est en cours d’écriture. Des expériences complémentaires sont en cours et sont planifiées afin de préciser les mécanismes cellulaires et moléculaires impliqués dans le rôle de la protéine HDGF sur le cycle des cellules de neuroblastome. De plus, l’étude de HDGF comme potentielle cible thérapeutique dans le neuroblastome est en cours.
siRNA Scr siRNA PATZ1 0 50 100 150 siRNA TMEFF2 siRNA Scr 0 50 100 150 200 250 siRNA TMSB4X siRNA Scr 0 200 100 400 300 500 A. B. C. 150 100 50 0 siRNA Scr siRNA PATZ1 150 100 50 0 150 100 50 0 siRNA Scr siRNA TMEFF2 siRNA Scr siRNA TMSB4X % d’e xpr ession de TMEFF2 % d’e xpr ession de P ATZ1 % d’e xpr ession de TMSB4X Air
es normalisées des masses sy
m pa th iq ue s (% ) Air
es normalisées des masses sy
mpathiques (%)
Air
es normalisées des masses sy
mpathiques (%) TN GS No AD Gms Me IGR-N91 - GFP::siRNA Scr Ƚ-HNK1 Hoechst TN GS No AD Gms Me
IGR-N91 - GFP::siRNA PATZ1
Ƚ-HNK1 Hoechst TN GS No AD Gms Me IGR-N91 - GFP::siRNA Scr Ƚ-HNK1 Hoechst TN GS No AD Gms Me
IGR-N91 - GFP::siRNA TMEFF2
Ƚ-HNK1 Hoechst TN GS No AD Gms Me IGR-N91 - GFP::siRNA Scr Ƚ-HNK1 Hoechst TN GS No AD Gms Me IGR-N91 - GFP::siRNA TMSB4X Ƚ-HNK1 Hoechst
A’. A’’. A’’’.
B’. B’’. B’’’. C’. C’’. C’’’. * *** *** *** ns ns
Figure 19. Impact d’une diminution d’expression des différents candidats sur la tumorigénèse neu-roblastique in vivo. Les panels de gauche présentent le taux d’expression du gène candidat 48h après trans-fection avec un siRNA scr ou un siRNA ciblant le candidat. Le panel central présente des coupes transversales ǯͷͲ°Ǥ±ϐ ǯ tumeurs sympathiques, exprimée en pourcentage.
n=10 n=10
n=6 n=5
Microenvironment-dependent induction of Hepatoma-derived growth factor is a pro-mitogenic trigger in neuroblastoma tumors
Lorette Bertin1, Karine Thoinet1, Thomas Buffet1, Stéphane Ansieau2, Edmund Derrington1, Céline Delloye-Bourgeois1& Valérie Castellani1
1, University of Lyon, University of Lyon 1 Claude Bernard Lyon1, NeuroMyoGene Institute, CNRS UMR5310, INSERM U1217, 16 rue Raphael Dubois, F-69000 Lyon, France
2, Cancer research Center of Lyon; INSERM UMR-S1052, CNRS UMR5286, UNIV UMR1052; Centre Léon Bérard, 28 rue Laennec, F-69008 Lyon, France.
ABSTRACT
Neuroblastoma (NB) is a pediatric cancer arising from a transitory embryonal cell population: sympatho-adrenal neural crest cells. This particularity has limited the feasibility of studies that aim at deciphering the impact of microenvironment-driven events on NB tumorigenesis. We exploited a recently developed embryonic model, driving human NB tumorigenesis in tissues homolog to patients, to extract novel tumor players related to NB emergence in an immature context. We found that, upon embryonic microenvironmental influences, Hepatoma-Derived Growth Factor (HDGF) is induced in NB sympathetic tumors, triggering a pro-mitogenic program within NB cells. Targeting HDGF in the NB avian model counteracted this effect and impaired cell proliferation in tumor masses. Together with patient cohort analysis, these data point at HDGF as a new potential therapeutic target for aggressive NB.
INTRODUCTION
Neuroblastoma (NB) is a highly heterogeneous pediatric cancer that, in its aggressive form, accounts for 15% of all cancer-related deaths in childhood (Brodeur & Bagatell, 2014; Maris, 2010). Despite drastic treatment protocols administered to young patients, the overall survival rate for aggressive cases is still no more than 60%, with only few relevant therapeutic innovations in the last ten years (Barone, Anderson, Pearson, Petrie, & Chesler, 2013; Brodeur et al., 2014; Matthay, George, & Yu, 2012). Indeed, as more than half of childhood cancers, neuroblastoma is suspected to have an embryonal origin. Making case of this particularity is crucial to decipher the mechanisms of NB tumors etiology and biology (Marshall et al., 2014) but also complicates access to early NB tumorigenic events and thus limits therapeutic breakthrough.
To address these key questions, we recently developed a novel NB avian model that consists in placing NB cells back to their original environment: the embryonic sympatho-adrenal neural crest (NC). Confronting NB cells to this particular immature mcicroenvironment lead them to undergo extensive NC cells-like migration towards sympatho-adrenal derivatives where they coalesce and switch on a pro-tumorigenic program implicating sustained proliferation and dissemination towards secondary metastatic sites (Delloye-Bourgeois et al., in revision).
We exploited this new paradigm to extract a list of genes differentially expressed in tumors formed in the avian model as compared to NB cells that had not been confronted to the embryonic environment (Delloye-Bourgeois et al., in revision). Here we concentrated on those genes impacted by the embryonic environment that could participate to a pro-tumorigenic switch, leading NB cells that coalesce into sympathetic derivatives to engage a pro-tumoral program. We focused on Hepatocyte-Derived Growth Factor (HDGF), a factor identified in the supernatant of human hepatoma cells HuH-7 (Klagsbrun, Sasset, Sullivant, & Smitht, 1986), recently involved in several cancer, with partially described functions in mitogenesis, apoptosis, migration and angiogenesis (Bao, Wang, Ma, Wang, & Cheng, 2014a; Enomoto, Nakamura, Liu, & Nishiguchi, 2015a; Okuda et al., 2003). We demonstrate that HDGF expression is induced in NB cells confronted to and settled in the embryonic sympathoadrenal microenvironment, and triggers a pro-mitogenic program in NB tumors. Together with NB patient cohorts analysis, these results identify HDGF as a promising and previously inaccessible therapeutic target in aggressive neuroblastoma.
RESULTS
We recently set up a NB model that consists in grafting human fluorescent NB cells within HH14 chick embryo sympatho-adrenal neural crest (Delloye-Bourgeois et al., in revision). To identify new players involved in NB tumorigenesis, we explored whether changes of gene expression in NB primary tumors could reflect microenvironment-related pro-tumorigenic mechanisms. We grafted IGR-N91 cells stably expressing GFP in the avian model and micro-dissected tumoral masses in sympathetic ganglia of HH25 chick embryos. The transcriptome of the latter samples was compared with that of naive IGR-N91 cells having not experienced tumorigenesis in the embryonic environment, using RNAseq approach (Delloye-Bourgeois et al., in revision). Among the 3920 genes expressed above background, 384 of them were related to embryonic development, and 604 to cell growth and proliferation functions (Figure 1A). Interestingly, 297 of them were connected to both items and were differentially expressed in NB tumors as compared to NB naive cells. We focused on those genes to identify NB players that could be implicated in microenvironment-driven pro-tumorigenic programs. Among promising candidates, we further concentrated on HDGF (Hepatoma-Derived Growth Factor), a growth factor known to be implicated in the proliferation of several embryonal cell populations –i.e: fetal hepatocytes, smooth muscle cells, aortic cells (Enomoto et al., 2002; Everett, Lobe, Matsumura, Nakamura, & McNamara, 2000; Everett, Stoops, & McNamara, 2001)- and found overexpressed in different cancer types (Bao et al., 2014a). In RNASeq experiments, HDGF expression was increased by 3.83 folds ±0.76 (p=1.89E-2). We confirmed in two independent qRT-PCR experiments performed on IGR-N91 tumor masses that HDGF expression was upregulated in NB tumors (Figure 1B). Moreover, we detected HDGF in most stage 4 neuroblastoma cell lines (Figure 1C) Remarkably, we found that HDGF expression was significantly higher in stage 4 NBs as compared to localized stages in two independent patients cohorts (Figure1D, p<0.0001 in Wolf and Koçak’s cohort and Figure1E, p<0.0001 in Shi and Fischer’s cohort). Moreover, HDGF high expression was
associated with a lower event-free survival probability in both cohorts (Kaplan-Meier analysis, p=6.6E-11, LogRank test (Figure 1F) and p=4.2E-9 LogRank test (Figure 1G) respectively for Wolf and Koçak’s and Shi and Fischer’s cohorts). Thus, regulating HDGF expression may impact on NB tumorigenic program.
HDGF expression is targeted in vitroandin vivo by specific siRNAs
To functionally study HDGF role in NB tumorigenesis, we used a siRNA approach to experimentally achieve robust HDGF downregulation. We first observed by quantitative PCR that HDGF mRNA expression was decreased by 72.3% (±4.1%, p<0.0001) / 69.0% (±6.4%, p=0.001) and by 51.9% (±8.2% p=0.006) / 60.9% (±5.6%, p=0.003), respectively in IGR-N91 and SH-SY5Y cell lines with two distinct HDGF siRNAs (siRNA HDGF #1/siRNA HDGF #2) (Figure 2A). We further confirmed that both HDGF siRNAs also impacted on HDGF protein expression by western blot analysis (decreased by 63.1% (±6.4 %, p=0.0022) / 78.1% (±7.4%, p=0.022), and by 52.5% (±9.6%, p=0.0079) / 49.5% (±11.7, p=0.0179) in IGR-N91 cells and SH-SY5Y cells respectively,Figures 2B and C) and by immunofluorescence on cultured cells (decreased by 32.4% (p=0.0023) / 16.2% (p=0.3052) in IGR-N91 cells and SH-SY5Y cells respectively, Figures 2D and 2E). Finally, transitory transfection of NB cells with HDGF siRNAs prior to their graft in the avian model led to a significant decrease in HDGF expression in NB sympathetic tumors formed 48 hours after the graft (Figures 2F and 2G, p=0.0006). Thus, HDGF expression is significantly reduced by a siRNA strategy, both in vitroandin vivo.
Targeting HDGF inhibits neuroblastoma tumorigenesis
Next we explored the impact of HDGF expression on NB tumor formation in the avian model. To do so, we transfected either IGR-N91::GFP cells or SH-SY5Y::GFP cells with control or HDGF siRNA prior to their graft in the avian model and monitored the formation
of NB sympathetic tumors 48 hours later. While the formation of dense tumor masses was not impacted by HDGF downregulation (100% of NB tumors in sympathetic derivatives both in siRNA scr- and in siRNA HDGF-transfected conditions), we observed that the tumor size was significantly reduced upon HDGF downregulation with both cell lines tested in the model (Figure 3, tumor area decreased by 41.5%, p<0.0001 and 59.5%, p=0.0053 with IGR-N91 cells and SH-SY5Y cells respectively). Thus, counteracting microenvironment-driven induction of HDGF expression in NB cells decreases their tumorigenic potential.
Targeting HDGF does not directly affect cell survival
To further decipher HDGF mode of action in NB tumorigenesis, we first looked whether HDGF downregulation could impact on cell apoptosis, as it has been described for several cancer types (Bao et al., 2014a; Marubuchi et al., 2006; Tsang et al., 2009; Yee, Wan, Tang, & Pui, 2008; Zhou et al., 2004). We performed in vitro TUNEL assay on IGR-N91 cultured cells transfected either with control or HDGF siRNA. In these settings, we did not observe any effect of HDGF downregulation on cell apoptosis (Figures 4A and 4B, p=0.3874). We confirmed these results in vivo, since apoptosis rate in NB sympathetic tumors was similar upon control or HDGF siRNA transfection (Figures 4C and 4D, p=0;0848). As HDGF is also described to have a direct mitogenic activity in several cancers (Bao, Wang, Ma, Wang, & Cheng, 2014b; Enomoto, Nakamura, Liu, & Nishiguchi, 2015b; Okuda et al., 2003; Zhang et al., 2006), we next assess whether HDGF expression could impact on NB cell proliferation. Upon HDGF siRNA transfection, IGR-N91 cells proliferation index, measured by detection of Ki67 antigen, was slightly but significantly decreased in vitro (Figures 5A and 5B, p=0;0490). The latter effect was also observed in vivo by measuring the percentage of proliferating IGR-N91 cells in sympathetic tumors formed in the avian model (Figures 5C and 5D, p=0.0496). Thus, induction of HDGF
expression in NB sympathetic tumors could increase the mitogenic potential of NB cells within primary tumors.
HDGF expression boosts cell cycle dynamics in neuroblastoma tumors
To more formerly study HDGF effect on NB mitogenic potential, we analyzed phase-specific markers in NB cells upon HDGF siRNA transfection. We observed that the fraction of phosphor-histone3 (PH3) positive cells in NB sympathetic tumors was significantly reduced for HDGF siRNA-transfected cells (Figures 6A and 6B, p=0.0001), indicating a lower mitotic rate. In the same line, we assessed the effect of HDGF downregulation on NB cell propensity to undergo S phase. We first performed BrdU staining on cultured IGR-N91 cells transfected with control or HDGF siRNA and observed a 7% decrease of S-phase-labeled cells upon HDGF downregulation both by immunofluorescence on cultured cells and by cell cycle analysis by FACS (Figures 6C and 6D, p=0.0191 and Figure 6E). This effect was further confirmed in vivo: a single BrdU injection in the veins of chick embryos grafted with IGR-N91::siRNA HDGF induced a 7% decrease of S-phase-labeled NB cells within sympathetic tumors as compared to IGR-N91::siRNA scr. cells (Figure6F and 6G, p=0.0286). This slight decrease is to considerer at a single time point and could thus reflect a more robust effect on cell cycle dynamics that will be studied by successive BrdU/EdU injections. Therefore, microenvironment-dependent increase of HDGF expression could contribute to boost NB cell cycle dynamics within the primary tumor site.
DISCUSSION
In this study, we exploited the data extracted from our novel NB model to identify new NB tumorigenic players related to the embryonal origin of this pediatric disease. We show that HDGF, a growth factor involved in different physiologic and tumor contexts, is
induced upon NB tumor formation within embryonic sympatho-adrenal structures, and mediates an early pro-mitogenic program in NB cells.
The strategy used here allows identifying key NB genes having a dynamic regulated expression that crucially depends on NB cell interaction with an immature environment. Such hits are not accessible via classical screening methods that do not integrate dynamic gene expression changes of tumor cells sequentially confronted to specific microenvironmental components. Microenvironmental partners implicated in HDGF upregulation in NB tumors will have to be further investigated. However, RNASeq data point at several known HDGF molecular interactors whose regulated expression in NB tumors suggests a global impact on HDGF-related signaling pathways. Notably, HDGF direct interactors SMYD2, SMYD3 and PSIP1 are co-regulated together with HDGF and are known to be implicated in the regulation of stem cell differentiation (Du, Tan, & Zhang, 2014; Yang & Everett, 2009). Future studies will allow determining whether specific HDGF contributions -nuclear, secreted or both- mediate, directly or not, the pro-mitogenic program induced in NB tumors.
Together with experimental data obtained here, patient cohorts analyses point at HDGF as a marker specifically overexpressed in stage 4 aggressive NB, with high expression levels associated with a poor outcome. Interestingly, Maschietto and collaborators reported a gene signature, comprising HDGF, potentially involved in the interruption of blastemal cell differentiation during Wilms’Tumor (WT) onset (Maschietto et al., 2011). While the cell of origin is distinct between NB and WT, both are pediatric cancers related to kidney structures and originating from an embryonal cell population. An interesting question would be to evaluate whether HDGF expression observed in sympatho-adrenal embryonal structures (Allen Spinal Cord Atlas, 2008) reflects a similar role in neural crest cells and as such, a similar deregulation during NB onset.
Further functional studies will allow assessing whether HDGF is a suitable therapeutic target in NB. Indeed, several cancers do show an aberrant expression of HDGF that is associated with a bad prognosis and that, for some of them, has been related to crucial tumorigenic processes –proliferation, angiogenesis, anti-apoptosis- (Bao et al., 2014b; Enomoto et al., 2015b). In this line, therapeutic strategies targeting HDGF have been patented in the last years (WO2008054431 A2). Interestingly, antibodies blocking HDGF have recently been shown to be promising therapeutic approaches in non-small cell lung cancer, with a particular impact on cancer stem cells that are involved in relapse (Ren, Chu, & Mao, 2009; Zhao et al., 2013). Thus, HDGF could be an interesting therapeutic entry point to target NB resting cells involved in NB relapse for which no therapeutic option is available to date.
FUNDINGS
This work was supported by a grant from the INCa (PLBIO14-306). This work was conducted within the framework of the LABEX CORTEX and Labex DevWeCAN of Université de Lyon, within the programme ‘Investissements d'Avenir’ (ANR-11-IDEX-0007) operated by the French National Research Agency (ANR).
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