Les fibroblastes associés aux cancers (CAFs) jouent un rôle primordial dans la progression tumorale, et notamment dans la résistance aux thérapies. Dans ce contexte, nous avons étudié l’effet des CAFs sur la réponse de cellules cancéreuses mammaires non seulement à une chimiothérapie conventionnelle mais aussi à des inducteurs directs de la voie mitochondriale de l’apoptose : les inhibiteurs BH3-mimétiques de BCL-2 et BCL-xL. Nous avons montré que des cultures primaires de CAFs, provenant de résections tumorales de cancer du sein de patientes naïves de traitement, diminuent la sensibilité à l’apoptose des cellules cancéreuses. Les mécanismes de résistance mis en jeu sont davantage influencés par le sous type moléculaire des cellules tumorales mammaires que par l’origine des CAFs. Ainsi, dans des lignées de type luminal, la résistance induite par le secrétome des CAFs implique un ou des facteurs thermosensibles, dépendant de l’autocrinie de la cytokine IL-6 dans les CAFs, et permettant l’activation de la voie de signalisation ERK dans les cellules cancéreuses où elles conduisent à la stabilisation de la protéine anti-apoptotique MCL-1.A l’inverse, dans des lignées de type triple négatif, la résistance est induite par un facteur thermorésistant, dépendant de l’autocrinie de la prostanglandine E2 dans les CAFs, et modifiant le métabolisme de ces cellules cancéreuses. Dans tous les cas, notre travail montre que le ciblage de la protéine MCL-1, à la fois dans les cellules cancéreuses mais aussi dans leurs cellules de soutien, a un potentiel thérapeutique. En effet, le stroma amplifie le phénomène de dépendance à MCL-1 dans les cancers du sein. La combinaison d’un traitement ciblant MCL-1 à une thérapie conventionnelle apparaît donc comme une option valide pour contrer l’influence du stroma sur les phénomènes de résistance. / Cancer-associated fibroblasts (CAFs) play a key role in tumor progression and resistance to therapy. Here, we studied in breast cancer cell the effects of CAFs on response to conventional chemotherapy and to BH3-mimetics, which directly induce mitochondrial apoptosis by targeting the antiapoptotic proteins BCL-2 and BCL-xL. We have shown that primary culture of CAFs protects breast cancer cell against drug-induced apoptosis. We demonstrated that CAF-linked protection mechanisms is influenced by the molecular subtypes of breast cancer cell lines, but not by the molecular subtypes of CAFs. In luminal breast cancer cells, apoptosis protection is linked to CAF secreted factors, involves one or more thermo-labile factors. It relies on CAFs IL-6 autocrine loop, which activates ERK signaling pathway in breast cancer cells, leading then to stabilization of the antiapoptotic protein MCL-1. In contrast, in Triple Negative breast cancer cells, CAFs-induced apoptosis resistance is mediated by thermoresistant factors, and relies on CAFs prostaglandin E2 autocrine loop that leads to a metabolic shift in cancer cells. In any case, our work shows that the targeting of MCL-1 both in cancer cells and their supporting cells could lead to new therapeutic options. Indeed, knowing that stroma promotes the MCL-1 dependency in breast cancers, a combination of MCL-1 inhibitor with conventional therapies appears as a promising option to counteract the stromal influence on cancer cells apoptosis resistance.

Regulation of apoptosis is critical for cell survival during mild stress and for proper removal of damaged cells during severe stress including hyperthermia. Previous studies have shown that knockdown of the BH3-only protein Noxa prevents hyperthermia-induced Mcl-1 degradation and activation of apoptosis. Noxa is a pro-apoptotic BH3-only protein that is able to selectively bind to and disable anti-apoptotic Mcl-1. Phosphorylation of Noxa on serine-13 by the cyclin-dependent kinase CDK5 inhibits the apoptotic function of Noxa. In this study I investigated whether hyperthermia is able to induce apoptosis by preventing Noxa phosphorylation, due to reduced CDK5 activity, leading to activation of Noxa. I was able to demonstrate that both the phosphorylation status and solubility of CDK5 is reduced during hyperthermia. Furthermore, overexpression of a non-phosphorylatable Noxa (S13A) resulted in a significant decrease in cell viability and increase in caspase-3 activity compared to overexpression of wild-type Noxa at 37°C. However, I was unable to detect in vivo phosphorylation of Noxa serine-13 in lymphoid cells and therefore was unable to conclude whether or not hyperthermia affects the phosphorylation status of Noxa.

京都大学 / 新制・課程博士 / 博士(生命科学) / 甲第24755号 / 生博第496号 / 新制||生||66(附属図書館) / 京都大学大学院生命科学研究科高次生命科学専攻 / (主査)教授 北島 智也, 教授 井垣 達吏, 教授 鈴木 淳 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

La macro-autophagie est une voie catabolique conservée dans l’évolution permettant la dégradation des organites endommagés ou vieux, des protéines à longue durée de vie ou agrégées et des portions du cytosol pour le recyclage métabolique afin de maintenir l'homéostasie cellulaire. L'absence d'autophagie est fréquemment observée dans de nombreuses pathologies incluant les cancers et les maladies neurodégénératives. Beclin 1, un suppresseur de tumeur,est une protéine clé dans la régulation de l’autophagie et participe à la nucléation de l’autophagosome. Beclin 1 est une protéine “BH3-only” pouvant interagir avec le site de fixation au domaine BH3 présent dans la protéine Bcl-2 et ses homologues. Cette interaction inhibe l’autophagie. Certains agents pharmacologiques tels qu’ABT737, appelés«BH3 mimetics», occupent le site de fixation du domaine BH3 de façon compétitive pour perturber l'interaction inhibitrice entre Beclin 1 et Bcl-2/Bcl-XL. Ceci permet à Beclin 1 de maintenir l’activité classe IIIphosphatidylinositol-3-kinase de Vps34 pour la formation du phagophore. L'autophagie est un processus finement régulé par de nombreux complexes protéiques. Les senseurs de la charge énergétique comme l’AMP-dependant kinase(AMPK), la cible mammalienne de la rapamycine (mTOR), la Sirtuin1 (SIRT1) ou les voies d’intégration du stress telles que celles impliquant l'inhibiteur des kinases NF-κB (IκBα) (IKK) et le suppresseur de tumeur p53, ont tous un impact majeur dans la régulation de l'autophagie. Dans de nombreux paradigmes de stimulation autophagique, ils semblent tous agir en amont de la dissociation Beclin 1-Bcl-2. Nos résultats révèlent qu’ABT737 stimule plusieurs voies pro-autophagiques pour obtenir une efficacité optimale. Ces résultats placent la SIRT1, AMPK / mTOR, HDM2et IKK en aval de la dissociation du complexe Beclin 1-Bcl-2. Cette étude démontre que les BH3-mimetics activent des voies multiples de stimulation de l’autophagie, peut-être en raison du degré élevé de connectivité qui existe entre les complexes protéiques de régulation de l’autophagie. Cela signifie qu’un effet spécifique sur l’interactome de Beclin 1 peut affecter d'autres voies dans le réseau du contrôle autophagique. Ces voies ne semblent pas suivre une hiérarchie linéaire, mais doivent être plutôt interconnectées dans un circuit complexe dans lequel la stimulation de l'autophagie par des déclencheurs physiologiques (tels que la carence en nutriments ou le stress des organites) induit un ensemble de changements intimement liés et impliqués dans une boucle de régulation positive qui constituerait un ensemble indissociable composant l’«autophagy switch». / Macro-autophagy is a conserved catabolic pathway that culminates in the degradation of old/damaged organelles,long-lived/aggregated proteins and portions of the cytosol for metabolic recycling to maintain cellular homeostasis.The absence of autophagy is frequently observed in many pathologies including cancers and neurodegenerative diseases. Beclin 1, a bona fide tumour suppressor, is the key autophagy regulatory protein that participates in autophagosome nucleation. Infect, Beclin 1 is a BH3-only protein that can interacts with the BH3 receptor domain contained within Bcl-2 and its homologues. This interaction functions as a inhibitory check on autophagy. Some pharmacological agents such as ABT737, referred to as ‘BH3 mimetics’, occupy the BH3-binding grooves to competitively disrupt the inhibitory interaction between Beclin 1 and Bcl-2/Bcl-XL allowing Beclin 1 to maintain the class III phosphatidylinositol-3-kinase activity of Vps34 for the phagophore formation. Autophagy is a complex process that is regulated by multiple protein complexes beyond that organized around Beclin 1. The energy sensors including AMP-dependent kinase (AMPK), mammalian target of rapamycin (mTOR), Sirtuin1 (SIRT1) as well as stress-integrating pathways such as those involving the inhibitor of NF-κB (IκB) kinases (IKK) and the tumour suppressor protein p53, all have a major impact on the regulation of autophagy. In many paradigms of autophagic stimulation, they all seem to act upstream of the dissociation of Beclin 1-Bcl-2. Our results reveal that ABT737stimulate multiple pro-autophagic pathways to be optimally efficient. These results place SIRT1, AMPK/mTOR,HDM2 and IKK downstream of the dissociation of the Beclin 1-Bcl-2 complex. This study advocates that BH3mimetics trigger multiple autophagy-stimulatory pathways maybe due to the high degree of connectivity that exists among autophagy-regulatory protein complexes meaning that a specific effect on the Beclin 1-interactome might affect other nodes in the autophagy-controlling network. These pathways cannot follow a linear hierarchy and rather must be interconnected in a complex circuitry, in which stimulation of autophagy by physiological triggers (such as starvation or organelle stress) induce an ensemble of intimately linked changes that are coupled to each other in positive feed forward loops constituting an indissociable ensemble that composes the “autophagic switch”.

The resistance of cancer cells to traditional chemotherapeutic agents is a major obstacle in the successful treatment of cancer. Cancer cells manipulate a variety of signaling pathways to enhance resistance to anticancer agents; such mechanisms include disrupting the DNA damage response and hyperactivating survival signaling pathways. In an attempt to better understand the molecular mechanisms that underlie resistance to chemotherapeutic agents, we investigated multiple processes regulated by the Rad9-Rad1-Hus1 (9-1-1) complex and Bif-1. The 9-1-1 complex plays an integral role in the response to DNA damage and regulates many downstream signaling pathways. Overexpression of members of this complex has been described in several types of cancer and was shown to correlate with tumorigenicity. In this study, we demonstrate that disruption of the 9-1-1 complex, through loss of Hus1, sensitizes cells to DNA damaging agents by upregulating BH3-only protein expression. Moreover, loss of Hus1 results in release of Rad9 into the cytosol, which enhances the interaction of Rad9 with Bcl-2 to potentiate the apoptotic response. We also provide evidence that disruption of the 9-1-1 complex sensitizes cells to caspase-independent cell death in response to DNA damage. Furthermore, we found that loss of Hus1 enhances DNA damage-induced autophagy. As autophagy has been implicated in caspase-independent cell death, these data suggest that the enhanced autophagy observed in Hus1-knockout cells may act as an alternate cell death mechanism. However, inhibition of autophagy, through knockdown of Atg7 or Bif-1, did not suppress, but rather promoted DNA damage-induced cell death in Hus1-deficient cells, suggesting that in apoptosis-competent cells autophagy may be induced as a cytoprotective mechanism. The aberrant activation of survival signals, such as enhanced EGFR signaling, is another mechanism that provides cancer cells with resistance to DNA damage. We found that knockdown of Bif-1 accelerated the co-localization of EGF with late endosomes/lysosomes thereby promoting EGFR degradation. Our results suggest that Bif-1 may enhance survival not only by inducing autophagy, but also by regulating EGFR degradation. Taken together, the results from our studies indicate that the 9-1-1 complex and Bif-1 may be potential targets for cancer therapy as they both regulate sensitivity to DNA damage.

Gain- and loss-of-function studies of the BCL-2 family of proteins have shown that they can impact chemotherapeutic sensitivity. However, cells contain myriad anti-apoptotic and pro-apoptotic BCL-2 family members making it difficult to predict cell fate decisions based on the initial conditions of these proteins. BH3 profiling is a tool that measures mitochondrial priming, the readiness of a cell to die through the intrinsic (or mitochondrial) apoptotic pathway. Priming is due to the cumulative effect of the BCL-2 family of proteins that act as the gate keepers of the mitochondrial apoptotic pathway. Priming is measured by determining the sensitivity of mitochondria to perturbation by peptides derived from the BH3 domains of pro-apoptotic proteins. Using BH3 profiling, we now have a functional readout that can quantify priming and assess its contribution to drug sensitivity. Here we show that priming affects the sensitivity of acute myeloid leukemia (AML) cell lines to various standard chemotherapeutics, especially topoisomerase II inhibitors. Priming predicts clinical response to conventional induction chemotherapy as well as the long term maintenance of remission in AML patients. Interestingly, the priming of normal hematopoietic stem cells (HSCs) sits at the boundary line between the priming of cured and refractory patient AML. This HSC priming likely defines the therapeutic index since AML that are lower primed than HSCs are often refractory and cannot be cured without transplantation. Additionally, our BH3 profiles revealed that AML cells are more sensitive to BCL-2 antagonism than normal HSCs, which are primarily dependent on MCL-1. Indeed, we were able to kill primary refractory AML cells in vitro with the BCL-2 antagonist ABT-737 at doses that left HSCs unharmed. Cumulatively, these findings show that priming is a major mechanistic determinant of AML response in vitro and in the clinic to standard induction chemotherapy. With the ability to predict outcome, BH3 profiling may offer physicians and patients a promising tool for treatment decision-making.

The cJUN NH2-terminal kinase (JNK) pathway responds to environmental stresses and participates in many cellular processes, including cell death, survival, proliferation, migration, and genome maintenance. Importantly, genes that encode components of the JNK signaling pathway are frequently mutated in human breast cancer, but the functional consequence of these mutations in mammary carcinogenesis is unclear. Anoikis – suspension-induced apoptosis – has been implicated in oncogenic transformation and tumor cell metastasis. Anoikis also contributes to lumen formation during mammary gland development and epithelial cell clearance during post-lactational involution. JNK is known to contribute to certain forms of cell death, but the role of JNK during anoikis was unclear. I examined the requirement of JNK in anoikis and discovered that JNK promotes cell death by transcriptional and post-translational regulation of pro-apoptotic BH3-only proteins. This conclusion suggested that JNK signaling may contribute to mammary gland remodeling during involution. Indeed, JNK deficiency in mammary epithelial cells disrupted the remodeling program of gene expression and delayed involution. Finally, I sought to understand the importance of JNK in mammary carcinogenesis. I found that JNK loss in the mammary epithelium was sufficient for genomic instability and tumor formation. Moreover, JNK loss in a model of breast cancer resulted in significantly accelerated tumor development. Collectively, these studies advance our understanding of the JNK pathway and breast biology, and provide insight that informs the design of therapeutic approaches that target the JNK signal transduction pathway.

The regulated cell death apoptosis enables redundant or compromised cells in ontogeny and homeostasis to remove themselves receptor-dependent after extrinsic signaling or after internal stress by BCL-2 proteins on the outer mitochondrial membrane (OMM). Mitochondrial BCL-2 proteins are also often needed for receptor-mediated signaling in apoptosis. Then, the truncated BH3-only protein BID (tBID) blocks retrotranslocation of the pro-apoptotic BCL-2 proteins BAX and BAK from the mitochondria into the cytosol. BAX and BAK in turn permeabilize the OMM. Although the BCL-2 proteins are controlled by a complex regulatory network, a specific mechanism for the inhibition of tBID remained unknown. Curiously, it was suggested that hexokinases, which channel glucose into the metabolism, have an intriguing function in the regulation of apoptosis. Recent analysis of transient hexokinase interactions with BAX revealed its participation in the inhibition of BAX and also BAK by retrotranslocation from mitochondria to the cytosol. In contrast to general apoptosis inhibition by anti-apoptotic BCL-2 proteins, hexokinase I and hexokinase 2 specifically inhibit tBID and thus the mitochondrial apoptosis pathway in response to death receptor signaling. Mitochondrial hexokinase localization and BH3 binding of cytosolic hexokinase domains are prerequisites for protection against receptor-mediated cell death, whereas glucose metabolism is not. This mechanism protects cells from apoptosis induced by cytotoxic T cells.

Ziel der vorliegenden Arbeit war es, die Rolle des pro-apoptotischen Proteins Bim am endoplasmatischen Retikulum (ER) und an den Mitochondrien zu untersuchen. Für diese Untersuchungen wurden zwei Isoformen von Bim verwendet, zum einen BimL, welches an den Motor Dynein Komplex gebunden ist, zum anderen BimS, welches im Zytosol lokalisiert ist. Um eine konditionale Expression von Bim zu erreichen, wurde Myc-markierte humane cDNA unter der Kontrolle des Tet-Off Systems in einen adenoviralen Vector kloniert. Eine Überexpression von BimL und BimS induzierte in der Prostatakarzinomzelllinie DU145 Bax- und Bak-abhängigen apoptotischen Zelltod. Eine Überexpression des anti-apoptotischen Proteins Bcl-2 lokalisiert am ER zeigte eine vollständige Hemmung der Bim-induzierten Apoptose, was die Wichtigkeit des ER unterstreicht. Überexpression von Bcl-2 an den Mitochondrien führte eine partielle Hemmung herbei. Bim Expression induzierte Bax- und Bak-abhängig den Zusammenbruch des mitochondrialen Membranpotentials. Dieses wurde ebenso in mit am ER lokalisiertem Bcl-2 Zellen beobachtet. Bcl-2 lokalisiert an den Mitochondrien verminderte dagegen mitochondriale Permeabilisation. Proteinanalysen zeigten eine Hochregulierung von ER-Stress Proteinen nach Bim Überexpression. Zusätzlich wurde Cytochrom c Freisetzung aus den Mitochondrien und Aktivierung von Caspase-9, -3 und -8 beobachtet. Mit einem Breitband-Caspase Hemmer konnte der Bim-induzierte Zelltod vollständig gehemmt werden, was zeigt, dass Caspasen essentiell sind. Zusammenfassend kann gesagt werden, dass Bim, parallel zum mitochondrialen Signalweg, ER-Stress auslöst und, dass Bim eine effektive Apoptose durch die Interaktion des ER und der Mitochondrien induziert. / The aim of this thesis was to investigate the role of the pro-apoptotic BH3-only protein Bim, at the endoplasmic reticulum (ER) and the mitochondria. For this purpose, a full length human myc-tagged Bim cDNA was cloned into an adenoviral vector, which allows for the conditional expression of the transgene under the control of a Tet-Off-system. Two different Bim isoforms were used for these investigations. One was BimL, which is bound to the motor dynein complex of the microtubule and the other one was BimS, which is localized in the cytosol. The enforced expression of each of these two isoforms in the prostate cancer cell line DU145, showed the capability of BimL and BimS to induce apoptosis via either Bak or Bax. Also, Bax- and Bak-dependent breakdown of the mitochondrial membrane potential upon overexpression of either Bim isoforms was measured. This effect was also observed in cells overexpression the anti-apoptotic protein Bcl-2 at the ER. However, targeting Bcl-2 to the mitochondria partially inhibited Bim-induced mitochondrial permeabilization. These findings indicated the execution of the intrinsic apoptotic pathway upon Bim signalling. Nevertheless, expression of Bcl-2 at the mitochondria partially suppressed Bim-induced apoptosis whereas ER-targeted Bcl-2 entirely prevented cell death induction by Bim underlining the importance of the ER. Further, an upregulation of ER stress proteins upon Bim expression was seen. Cytochrome c release form the mitochondria and activation of caspase-9, -3 and -8 was observed. In addition, the complete inhibition of Bim-induced cell death by a pan caspase inhibitor revealed that caspases are crucial. In conclusion, Bim induces the mitochondrial apoptotic pathway and, in parallel, triggers ER stress. It seems that Bim mediates cell death through the interaction of the mitochondria and the ER. The ER-mitochondria cross-talk leads to the amplification of the apoptotic death signal.

Bcl-2 family members are important regulators of apoptosis, and their tampered expression is often involved in oncogenesis. Of particular importance are the levels of Bcl-2 family members in forming lymphomas. We studied two groups of murine thymic T cell lymphomas derived from either Bcl-2 or Bax overexpression in order to predict their sensitivity and resistance to treatments. While the growth rate and histological characteristics were similar for both lymphoma groups, Bax-derived lymphomas failed to undergo cell cycle arrest following radiation treatment and had frequent p53 mutations. In contrast, Bcl-2-derived lymphomas often halted proliferation following radiation delivery and rarely had p53 mutations. Bax-derived lymphomas were uniformly sensitive to treatment with 2-deoxy-D-glucose (2DG) while all Bcl-2-derived lymphomas were resistant. This led us to hypothesize that the Bcl-2 family is involved in 2DG-induced cell death. Focusing on the mechanism of 2DG toxicity in Bax-derived lymphomas, our studies demonstrate the following: cell death involved the activation of proapoptotic Bax, was effectively blocked by anti-apoptotic Bcl-2, and was mediated, at least in part, by the BH3-only family member Bim. Based on these results, we explored whether a BH3 mimetic (ABT-737) could sensitize lymphomas to 2DG killing. Indeed, a combination of ABT-737 with 2DG enhanced killing in Bax-derived lymphomas and resensitized Bcl-2-overexpressing lymphomas to 2DG. Since both 2DG and BH3 mimetics are currently in clinical trials, understanding their killing mechanisms and optimal combinations are of potential clinical significance. The work in this dissertation demonstrates a novel role of Bcl-2 family member proteins in regulating 2DG toxicity and may predict response to 2DG treatment. The information found presents a new strategy of combining 2DG with BH3 mimetics to improve existing lymphoma therapies.