Rôle d’ATG9A dans la lignée de cancer du sein triple négatif MDA-MB-436 La protéine ATG9A étant impliquée dans l’induction du processus autophagique, nous

avons souhaité caractériser son rôle dans la lignée cellulaire de cancer du sein triple négatif MDA-MB-436. Pour cela, nous avons mis au point deux modèles d’extinction de l’expression d’ATG9A dans cette lignée: 1) à l’aide de l’expression stable de sh-ARN ciblant la partie 5’

de l’ARNm d’ATG9A (Figure 18A) ou, 2) de la technique CRISPR-Cas9 en utilisant un ARN guide ciblant 19 nucléotides au niveau de l’exon 5 du gène ATG9A (Figure 18B).

Figure 18 : Séquences des sh-ARN et ARN guide utilisées pour nos modèles d’extinction de l’expression d’ATG9A par sh-ARN et la technique CRISPR-Cas9, respectivement. (A) Le plasmide MissionTM _{sh-RNA (Sigma)}

utilisé pour l’extinction de l’expression d’ATG9A est clivé par Dicer pour obtenir un siRNA. Le siRNA est pris en charge par le complexe RISC (RNAi-induced silencing complex) pour permettre sa liaison au niveau de l’ARNm d’ATG9A (entre les nucléotides 3419 et 3439) et permettre ensuite son clivage et sa dégradation pour inhiber son expression. (B) La séquence d’ARN guide utilisée cible le gène d’ATG9A entre les nucléotides 2782 et 2800. L’endonucléase Cas9 induit alors une coupure d’ADN double brin au niveau de cette séquence cible. L’insertion ou la délétion de nucléotides au niveau du site de cassure est réalisé par les mécanismes de réparation de l’ADN double brin, pouvant résulter à une modification du cadre de lecture et la survenue précoce d’un codon stop.

Nous avons ainsi montré que l’extinction d’ATG9A par sh-ARN dans les cellules MDA- MB-436 diminue significativement leurs capacités de prolifération et d’invasion in vitro et de croissance tumorale in vivo. Cependant, l’extinction d’ATG9A par la technique CRISPR-Cas9 ne nous a pas permis d’observer de différence significative pour ces mêmes phénotypes cancéreux, comparé aux clones contrôles. Le séquençage de nos clones CRISPR a mis en évidence qu’un ou deux nucléotides adénines avaient été insérés dans la séquence du gène

ATG9A, au niveau de la séquence ciblée par l’ARN guide utilisé. Des études bioinformatiques

ont mis en évidence que ces mutations pourraient conduire à la synthèse d’une protéine ATG9A tronquée de 68 ou 98 acides aminés, respectivement. Ces protéines possèderaient la même séquence qu’ATG9A jusqu’à l’acide aminé 63, puis présenteraient un décalage du cadre de lecture jusqu’à un codon stop en position 68 ou 98. La partie N-terminale d’ATG9A pourrait donc être impliquée dans la régulation des caractéristiques cancéreuses des cellules MDA-MB-436.

Afin de déterminer si l’extinction d’ATG9A a un effet sur le processus autophagique, nous avons analysé les niveaux de LC3B-II, la forme lipidée de LC3B qui est associée aux autophagosomes, ainsi que la présence de vésicules acides par marquage Lysotracker®_{. Nous}

avons ainsi montré que l’extinction d’ATG9A par la méthode sh-ARN ou CRISPR-Cas9 conduit à l’accumulation de LC3B-II et de vésicules acides dans les cellules, comparé aux cellules contrôle. Ces données suggèrent que la perte d’ATG9A dans les cellules MDA-MB- 436 conduit à l’accumulation d’autophagosomes ne pouvant pas être éliminés par fusion avec des lysosomes, au vu de l’accumulation massive de vésicules acides dans nos clones n’exprimant pas ATG9A.

Publication II:

ATG9A is overexpressed in triple negative breast cancer and its in vitro extinction leads

to the inhibition of pro-cancer phenotypes.

Aurore Claude-Taupin,Leïla Fonderflick, Thierry Gauthier, Laura Mansi, Jean-René Pallandre, Christophe Borg, Valérie Perez, Franck Monnien, Marie-Paule Algros, Marc

“ATG9A is overexpressed in triple negative breast cancer

and its in vitro extinction leads to the inhibition of pro-cancer phenotypes.”

Aurore Claude-Taupin1_{, Leïla Fonderflick}1_{, Thierry Gauthier}2_{, Laura Mansi}3_{, Jean-René}

Pallandre2, Christophe Borg3, Valérie Perez1, Franck Monnien4, Marie-Paule Algros4, Marc Vigneron5, Régis Delage-Mourroux1, Michaël Boyer-Guittaut1 and Eric Hervouet1*

1 _{Univ. Bourgogne Franche-Comté, Laboratoire de Biochimie, EA3922 « Estrogènes,}

Expression Génique et Pathologies du Système Nerveux Central », SFR IBCT FED4234, UFR Sciences et Techniques, 16 route de Gray, 25030 Besançon Cedex, France.

2 _{INSERM UMR1098, EFS Bourgogne Franche Comté, Univ. Bourgogne Franche-Comté, 8}

rue du Dr Girod, 25020 Besançon, France.

3 _{INSERM UMR1098, EFS Bourgogne Franche Comté, Univ. Bourgogne Franche-Comté,}

Department of Medical Oncology, University Hospital of Besançon, Besançon, France.

4 _{Service de pathologie, CHRU de Besançon, 2, boulevard A.-Fleming, 25000 Besançon,}

France.

5 _{Ecole Supérieure de Biotechnologie de Strasbourg, UMR 7242, CNRS/Université de}

Strasbourg, boulevard Sébastien Brant, 67412 Illkirch, France. *Corresponding author: eric.hervouet@univ-fcomte.fr

Univ. Bourgogne Franche-Comté, Laboratoire de Biochimie, EA3922 « Estrogènes, Expression Génique et Pathologies du Système Nerveux Central », SFR IBCT FED4234, UFR Sciences et Techniques, 16 route de Gray, 25030 Besançon Cedex, France.

Key words: autophagy, triple negative breast cancer, ATG9, LC3, lysosome, MDA-MB-436 Running Title: ATG9A role in autophagy and cancer development

ABBREVIATIONS

ATG, autophagy-related protein; BAFA1, Bafilomycin A1; BC, breast cancer; DMEM, dulbecco’s minimum essential medium; EBSS, earle’s balanced salt solution; ECM, extra cellular matrix; ER, estrogen receptor; FBS, fetal bovine serum; HER2, Human Epidermal Growth Factor Receptor 2; HRP, horseradish peroxidase; IHC, Immunohistochemistry; LC3, microtubule-associated protein 1 light chain 3; mTORC1, mammaliam target of rapamycin complexe 1; MTT, 3-(4,5-dimethylthiazol- 2-yl)-2,5-diphenyltetrazolium bromide; P62/SQSTM1, sequestosome 1; PBS, phosphate buffer saline; PBS-T, PBS triton-X-100; pN, lymph node status; pT, tumor size; PVDF, polyvinylidene difluoride; RT-qPCR, reverse transcription – quantitative polymerase chain reaction; SDS, sodium dodecylsulfate; SISH, silver in situ hybridization; TN, triple negative; TNBC, triple negative breast cancer

ABSTRACT

Even if earlier detection and targeted treatments has led to a significant decrease in mortality linked to breast cancer (BC), important issues will still need to be addressed in the future. One of them will be to find new triple negative breast cancer (TNBC) therapeutic strategies since none are currently efficiently targeting this subtype of BC. Amongst the potential pathways, numerous studies have reported the possibility to target the autophagy pathway. In our study, we identified ATG9A, a transmembrane protein involved in autophagosome formation, as a potential marker of TNBC. Indeed, we analyzed the expression of 6 autophagy genes (ATG9A, ATG9B, BECLIN1, LC3B, NIX and P62/SQSTM1) by qRT-PCR in breast cancer tissues and compared their expression with healthy adjacent tissues and we showed an increase of ATG9A mRNA expression in TNBC samples from our breast cancer cohort. To corroborate these in vivo data, we have designed shRNA-driven inhibition of ATG9A in the MDA-MB-436 TNBC cell line in order to determine its role in the regulation of cancer phenotypes in vitro but also in xenografts models in vivo. We found that

ATG9A inhibition led to an inhibition of in vitro and in vivo cancer features together with an

accumulation of autophagosomes and lysosomes. On the opposite, when we designed CRISPR-driven inhibition of ATG9A in the MDA-MB-436 cell line, we also observed an accumulation of autophagosome or lysosome but did not inhibit in vitro cell proliferation or in

vivo tumorigenesis in nude mice. This difference of cancer phenotypes could be linked to the

expression of a short cytosolic N-terminus fragment of ATG9A still present in our CRISPR clones. Our results revealed that the localization of ATG9A in membranes is essential for its function in cancer but that the cytosolic N-terminus fragment is sufficient to regulate the autophagy process. This study showed, for the first time, that ATG9A could be considered as a new marker of TNBC and might be targeted in the future to develop new specific TNBC therapies.

INTRODUCTION

Breast cancer (BC) is one of the three most commonly diagnosed cancers in women and 246,660 new cases of female invasive BC are expected to be diagnosed in 2016 in the United States (1). Thanks to earlier detection and targeted treatments, mortality has declined about 30 % over the past two decades (2). Nevertheless, the American Cancer Society estimates 40,450 deaths linked to breast cancers in the United States in 2016 (1), rending it a major health problem. The triple-negative breast cancers (TNBC) represents approximately 10 % of BC and are defined as tumors lacking estrogen receptor (ER), progesterone receptor (PR) and HER2 (Human Epidermal Growth Factor Receptor 2) expression (ER-, PR-, HER2-). TNBC present a poor prognosis mainly because of the limited treatment options. In fact, no specific therapy targeting TNBC is currently available and cytotoxic chemotherapy still remains the current treatment strategy (3).

During the course of development of new therapeutic options for the treatment of BC, numerous studies have reported the possibility to target the autophagy pathway. For example, autophagy inhibition has been shown to improve chemotherapy efficiency in TNBC presenting high levels of LC3B, the main protein marker of autophagy (4), and to enhance the therapeutic response in both anthracycline-sensitive and resistant TNBC cells (5). Autophagy has also been proposed to protect TNBC cells from damaged mitochondria accumulation induced by chemotherapies (6). Furthermore, a phase II clinical trial is underway to determine the efficiency and safety of chloroquine, a lysosomal inhibitor, in combination with taxane chemotherapies, in advanced and metastatic BC patients, who have previously failed anthracycline therapy (NCT01446016, clinicaltrials.gov).

Autophagy is a cellular process involved in the maintenance of cell homeostasis and cell survival by inducing the degradation and/or the recycling of intracellular components such as protein aggregates or organelles (e.g. damaged mitochondria). This process is mediated by more than 30 core ATG (AuTophaGy-related) proteins and characterized by the formation of a double-membrane structure, called phagophore, which elongates and closes to generate an autophagosome. The autophagosome, which engulfs cytosolic material as well as organelles, later fuses with a lysosome to form an autophagolysosome, leading to the degradation of its content by lysosomal proteases (for a review, see (7)). The initiation and elongation of this structure requires several ATG proteins, one of them being the ATG9 protein. Two isoforms of ATG9, A and B, are expressed in mammals and these proteins are the only transmembrane ATG proteins identified so far. ATG9A is ubiquitously expressed while ATG9B is enriched in placenta and the pituitary gland, suggesting that ATG9B may

play a specific role during embryogenesis (8). While the specific function of these two isoforms still remains unclear, it has been proposed that ATG9B may complement ATG9A role during autophagy. ATG9A presents six conserved transmembrane domains with both N and C-terminus localized on the cytosolic side of the vesicle. ATG9A intracellular trafficking between the endosome or Golgi and the elongating phagophore is essential for autophagosome formation and ATG9A has therefore been proposed as the main candidate for delivering lipids to growing phagophores (9). Indeed, under nutrient-rich conditions, ATG9A is found in the trans-Golgi network, recycling and late endosomal compartments (9–12) but upon amino-acid starvation, it is relocalized towards the autophagosome initiation site, where it dynamically interacts with phagophores and autophagosomes without being incorporated into them (10).

ATG9A deregulation has already been linked to cancer since ATG9A protein overexpression has been associated with poor survival in patients with oral squamous cell carcinoma (13) whereas ATG9A inhibition was associated to the apparition of trastuzumab resistance in HER2+ BC cells (14). Furthermore, a recent study reported a decrease of ATG9A mRNA expression in breast invasive ductal carcinomas, compared to adjacent normal tissues, and this decrease of expression was linked with increased promoter methylation (15). More interestingly, the authors also demonstrated a significant decrease of ATG9A mRNA expression in HER2+ BC but, to our knowledge, no published study has already characterized the expression and the role of ATG9A in TNBC.

In our study, we analyzed the expression of six ATG genes (ATG9A, ATG9B, BECLIN1,

LC3B, NIX and SQSTM1/P62) using quantitative real-time PCR (qRT-PCR) in BC tissues and

compared their expression with healthy adjacent tissues. Our data clearly demonstrated that only ATG9A mRNA levels were significantly higher in TNBC tissues compared to healthy adjacent tissues. We then inhibited ATG9A expression in the TNBC cell line MDA-MB-436 using specific sh-RNA and found that ATG9A inhibition led to an inhibition of in vitro and in

vivo cancer features, proliferation, invasion and tumor growth, together with an accumulation

of autophagosomes and lysosomes. On the opposite, the inhibition of ATG9A expression using a CRISPR-Cas9 strategy in MDA-MB-436 cells also led to an accumulation of autophagosomes or lysosomes but did not inhibit in vitro cell proliferation or in vivo tumorigenesis in nude mice. The analysis of the mutations created in the ATG9A gene in our CRISPR clones showed that these cells could still produce a truncated form of ATG9A, including the 63 first amino-acids of the wild-type protein, but which will no longer be a

essential for its function in cancer but that the short cytosolic N-terminus fragment produced in the CRISPR clones is sufficient to regulate the autophagy process.

MATERIALS AND METHODS