Anticancer therapeutics are often limited to suboptimal doses due to their lack of selectivity for tumor cells and resultant damage to healthy tissue. These limitations motivated researchers to develop tumor-specific delivery systems for improved therapeutic efficacy and reduced unintended cytotoxicity. Polyamidoamine dendrimers offer an ideal platform for designing targeted therapeutics with tunable characteristics that optimize pharmacokinetic behavior and targeting specificity. Ligand conjugation to dendrimer provides the biochemical interaction necessary to activate tumor-specific receptors for receptor-mediated endocytosis and effective internalization of polyplexes. Tumor-specific receptors overexpressed in carcinomas, like folate receptor-alpha (FOLRα), are targeted by ligand-conjugated dendrimer to allow enhanced internalization of dendrimer and its therapeutic cargo. We examined the cellular trafficking dynamics and potential of folate-conjugated dendrimer for nucleic acid delivery in vitro. Results show folate-conjugation to G4 PAMAM dendrimer (G4FA) confers enhanced uptake in FOLRα-positive tumor cells. Cells internalize G4FA in a receptor-dependent manner with specificity for FOLRα-positive tumor cells.

Apoptosis (Type I) and autophagy (Type II) represent two major forms of programmed cell death. Numerous anticancer agents employed in standard chemotherapy or novel targeted therapy induce both apoptosis and autophagy. Of note, a cytoprotective autophagic response often counteracts apoptosis triggered by such agents, potentially contributing to drug-resistance. Mechanistically, autophagy and apoptosis share molecular regulatory mechanisms primarily governed by the Bcl-2 family proteins. However, since autophagy acts as the double-edge sword in cancer, whether autophagy should be inhibited or activated in cancer treatment remains the subject of debate. Here we report a) a novel autophagy-targeted strategy that targeting the adaptor SQSTM1/p62 induces “inefficient” autophagy due to cargo-loading failure and converts cytoprotective autophagic response to apoptosis via the BH3-only protein NBK/Bik (Part 1); and b) a new mechanism for acquired drug-resistance in which the BH3-only protein Bim acts as a dual-agent regulating both autophagy and apoptosis (Part 2).

The cancers of liver, colon and breast are amongst the top five most prevalent and most fatal worldwide. As the Raf/MEK/ERK pathway is frequently deregulated in hepatocellular carcinoma (HCC), sorafenib, a Raf kinase inhibitor, became the first systemic therapy approved for the treatment of patients with HCC. However, sorafenib only produced modest effects with low response rates in the clinic. Similarly, regorafenib, which was approved for the treatment of metastatic colorectal cancer (CRC), has had a poor response rate in the clinic. Since phosphodiesterase type 5 has been reported to be overexpressed in HCC and CRC, we hypothesized that sildenafil, a phosphodiesterase type 5 inhibitor, could enhance the toxicities of sorafenib and regorafenib in HCC and CRC cells, respectively. Our in vitro data indicated that the drugs interacted strongly to kill cancer cells via induction of ER stress, autophagy and apoptosis. In accordance with these findings, our in vivo data demonstrated a significant reduction in tumor growth. The second study in this manuscript was conducted based on the growing body of evidence about the significant contribution of EGFR and JAK/STAT signaling to the breast tumorigenesis. Our preliminary in vitro data demonstrated that the concurrent inhibition of these two pathways by lapatinib, a dual ERBB1/2 inhibitor, and ruxolitinib, a JAK1/2 inhibitor, synergistically killed breast cancer cells of all types, including the resistant triple negative subtype. Our mechanistic studies showed that the combination of ruxolitinib and lapatinib triggered cytotoxic mitophagy, and autophagy-dependent activation of BAX and BAK leading to the mitochondrial dysfunction.

La croissance d’une tumeur requiert la formation de son propre réseau vasculaire par angiogenèse. Ce processus est orchestré par une multitude de médiateurs dont le VEGF (Vascular Endothelial Growth Factor) qui interagit avec des récepteurs surexprimés à la surface des cellules endothéliales des vaisseaux avoisinant la tumeur. Les thérapies ciblées anticancéreuses jouent sur cette surexpression pour concevoir de nouvelles molécules spécifiques de la zone tumorale. Un nouvel agent de ciblage a ainsi vu le jour au sein du laboratoire. Il s’agit d’un cyclopeptide de 17 acides aminés, nommé CBO-P11 qui présente une haute affinité pour les récepteurs du VEGF. Afin de prolonger le temps de circulation du peptide dans le sang, des nanoparticules de poly(fluorure de vinylidène) (PVDF), polymère biocompatible, ont été utilisées comme vecteur. Les nanoparticules ont tout d’abord été synthétisées par une polymérisation radicalaire en émulsion dans l’eau, puis radio-greffées avec de l’acide acrylique (AA) pour obtenir du PVDF-g-PAA, ce qui a permis par la suite de les fonctionnaliser avec le CBO-P11 et un agent d’imagerie par « click » chemistry via un bras espaceur de type oligo(éthylène glycol). Différents marqueurs ont été étudiés à savoir deux sondes fluorescentes pour l’imagerie optique et un agent de contraste pour l’imagerie par résonance magnétique à bas champ. Chaque étape de synthèse a été caractérisée par de nombreuses techniques telles que la diffusion statique et dynamique de la lumière, la diffusion de neutrons aux petits angles, la spectrométrie photoélectronique de rayons X, la spectrométrie infrarouge, la spectrométrie de fluorescence et la polarisation dynamique nucléaire. Des études in vitro sur des cellules endothéliales ont permis de montrer que les nanoparticules n’étaient pas cytotoxiques et que le CBO-P11 lié au vecteur conservait sa spécificité pour les récepteurs du VEGF. Ces outils multifonctionnels présentent un grand potentiel pour cibler et imager les zones tumorales. / Tumor growth requires the formation of its own vascular network by angiogenesis. This process is mediated through numerous factors including VEGF (Vascular Endothelial Growth Factor) which interacts with receptors overexpressed on endothelial cells of blood vessels neighboring tumor. Targeted anticancer therapies take advantage of this overexpression to design tumor-specific molecules. A new targeting agent, CBO-P11 was synthesized in the laboratory. It is a 17-mer cyclopeptide with a high affinity for VEGF receptors. In order to increase the blood circulation time of this peptide, nanoparticles of poly(vinylidene fluoride) (PVDF), biocompatible polymer, were used as carrier. Nanoparticles were synthesized by radical polymerization in aqueous emulsion, then radio-grafted with acrylic acid (AA) to obtain PVDF-g-PAA. Nanoparticles were functionalized with CBO-P11 and an imaging agent by “click” chemistry via an oligo(ethylene glycol)-like spacer arm. Different dyes were used: two fluorescent probes for optical imaging and a contrast agent for low-field magnetic resonance imaging. Each step of the nanoparticles preparation was monitored and successfully characterized by numerous techniques such as dynamic and static light scattering, small-angle neutron scattering, X-ray photoelectron spectroscopy, infrared spectroscopy, fluorescence spectroscopy and dynamic nuclear polarization. In vitro studies were performed on endothelial cells and have shown that nanoparticles were not cytotoxic and that CBO-P11 conjugated to nanoparticles kept its specificity for VEGF receptors. Those multifunctional tools offer a great potential to target and image tumor.

Les sarcomes sont des cancers rares touchant toutes les zones du corps humain, caractérisés par une grande diversité de nature, de comportement clinique et de réponse aux thérapies existantes, certains étant de bon pronostic, d’autres très difficilement curables.Leur traitement de référence est la chirurgie ; la radiothérapie et les protocoles de chimiothérapie n’ayant que peu évolué lors des 30 dernières années.Récemment des caractéristiques génétiques leur étant propres ont été découvertes, prédictives de leur agressivité et contre lesquelles il est possible de diriger des drogues spécifiques pouvant améliorer le pronostic et diminuer les effets secondaires des thérapies conventionnelles.C’est sur l’inhibition d’Aurora Kinase A et B et CHK1 que s’est focalisé ce travail avec le test des effets de deux nouvelles drogues sur 9 types de cellules cancéreuses sarcomateuses avec des résultats très prometteurs, qu’il conviendra de conforter par d’autres expériences, notamment sur l’animal. / Sarcomas are rare cancers, which may arise in all parts of human body. They are characterized by great diversity in their nature, clinical behavior and response to existing therapeutics. Some are of good prognosis and others hard to cure.Their treatment essentially relies on surgery and radiotherapy or chemotherapy haven’t know major breakthrough over the last 3 decades.Recently new genetics abnormalities linked to sarcomas have been discovered. Their analysis can predict their aggressiveness and it is now possible to develop targeted therapies against them. This could help improving cancer prognosis and/or limiting conventional drugs adverse effects.Our work focused on Aurora Kinase A and B and CHK1 inhibition, testing the effects of 2 new drugs on 9 types of sarcoma cells with promising results, which we will confort by other experiments, including on the animal.

En France, le cancer colorectal (CCR) est la 2ème cause de décès par cancer. Chez les patients, lorsque la tumeur est localisée, elle est réséquée par chirurgie. Toutefois, 50% des patients sont diagnostiqués à un stade métastatique, ces patients sont alors traités par chimiothérapie (FOLFOX/FOLFIRI), souvent en combinaison avec des thérapies ciblées incluant des anticorps tel que le Cetuximab (anti-EGFR) ou le Bevacizumab (anti-VEGF). Cependant, il reste encore environ 40% des patients qui ne répondent pas au traitement et l’une des causes les mieux établies est l'influence des mutations de la voie RAS en aval du récepteur à l'EGF sur la réponse au Cetuximab. C’est pourquoi l’identification de nouvelles cibles moléculaires accessibles aux anticorps permettrait le développement de nouveaux modèles de thérapie ciblée. Depuis peu, les Claudines ont suscité un intérêt pour le ciblage tumoral comme la claudin-4 dans les cancers de l’endomètre ou de la prostate. Très récemment, les résultats d'une étude clinique ont démontré que la combinaison d'un anticorps anti-Claudin-18.2, l'Acm IMAB362, avec la chimiothérapie prolongeait nettement la survie chez des patients atteints de cancer gastrique avancé.C’est dans cette optique que nous nous sommes focalisés sur les claudines dans le CCR. Tout d’abord en étudiant leur expression dans des échantillons de patients atteints de CCR métastatique. En analysant l’expression des claudines dans les nouveaux sous-types moléculaires, nous avons montré que certaines pouvaient avoir une valeur pronostique. Nous avons également identifié des claudines comme cibles thérapeutiques potentielles dans le CCR, parmi elles la CLDN1. En effet, nous avons montré que la forme membranaire de la CLDN1 était surexprimée dans les tumeurs primaires et métastases du CCR. Nous avons développé un anticorps monoclonal (Acm) ciblant les parties extracellulaires de la CLDN1. Nous avons montré que le ciblage thérapeutique de la CLDN1 par Acm provoquait un ralentissement significatif de la croissance, la migration et l'invasion des cellules tumorales aussi bien in vitro que in vivo. Afin d’améliorer l’efficacité du ciblage de la CLDN1 par Acm, nous avons couplé ce dernier avec une toxine, générant ainsi un Antibody Drug conjugated (ADC). Nous avons montré que le ciblage de la CLDN1 par un ADC diminuait la survie cellulaire in vitro en culture cellulaire 2D, mais également celle des sphéroïdes via un effet cytotoxique. Ce travail a permis d'établir la preuve de concept du ciblage de la CLDN1 aussi bien par Acm que par ADC. Afin de finaliser ce travail, ces résultats doivent être confirmés in vivo. A terme, nous espérons que ce ciblage puisse trouver sa place au sein de l’arsenal thérapeutique du CCR métastatique, en particulier chez les patients résistants aux traitements. / Colorectal cancer (CRC) is one of the major causes of cancer-related deaths in the Western world. When localized, CRC is often curable by surgery. However, 50% of patients are diagnosed at a metastatic stage, these patients are then treated with chemotherapy (FOLFOX / FOLFIRI), often in combination with targeted therapies including antibodies such as Cetuximab (anti-EGFR) or Bevacizumab (anti -VEGF). Despite these treatment, almost 40% of patients develop resistance. One of the most known resistance mechanisms of resistance is due to the RAS pathway downstream of the EGF receptor in response to Cetuximab. Therefore, more therapeutic options are required particularly by identifying new molecular targets that can be reached by antibodies. Recently, Claudines have generated interest as targets in cancer, such as claudin-4 in endometrial or prostate cancer. More recently, the results of a clinical study demonstrated that the combination of an anti-Claudin-18.2 antibody, IMAB362 mAb, with chemotherapy significantly prolonged survival in patients with advanced gastric cancer.Therefore, one of our aims was to focus on claudins in CCR. First, by studying their expression in metastatic CRC patient samples. We demonstrated the prognostic value of some claudins, after analyzing their gene expression in the new molecular subtype of CRC. Beside, we identified some claudins as potential therapeutic targets in CCR, among them claudin-1 (CLDN1). Indeed, we showed that the membrane form of CLDN1 is overexpressed in primary tumors and metastases of CRC. Therefore, we developed a monoclonal antibody (mAb) targeting the extracellular parts of CLDN1. We showed that therapeutic targeting of CLDN1 by mAb significantly decreased tumor cell growth, migration and invasion both in vitro and in vivo. In order to improve the efficiency of targeting CLDN1 by mAb, we conjugated it with a toxin, thus generating an Antibody Drug Conjugate (ADC). We showed that CLDN1 targeting by an ADC decreased cell survival in vitro in 2D cell culture, but also spheroids growth via a cytotoxic effect.This work demonstrated the proof of concept of CLDN1 targeting by both mAb and ADC. In order to achieve this work, the next steps remains on testing ADC affect in vivo models. CLDN1 is a good therapeutic target in the CCR. In the long term, we hope that its targeting can find its place within the therapeutic arsenal of metastatic CRC, particularly in treatment of resistant patients.

Object: The aim of the study was to evaluate the early changes in pituitary hormone levels after severe traumatic brain injury (sTBI) and compare hormone levels to basic neuro-intensive care data, a systematic scoring of the CT-findings and to evaluate whether hormone changes are related to outcome. Methods: Prospective study, including consecutive patients, 15-70 years, with sTBI, Glasgow Coma Scale (GCS) score <= 8, initial cerebral perfusion pressure > 10 mm Hg, and arrival to our level one trauma university hospital within 24 hours after head trauma (n = 48). Serum samples were collected in the morning (08-10 am) day 1 and day 4 after sTBI for analysis of cortisol, growth hormone (GH), prolactin, insulin-like growth factor 1 (IGF-1), thyroid-stimulating hormone (TSH), free triiodothyronine (fT3), free thyroxine (fT4), follicular stimulating hormone (FSH), luteinizing hormone (LH), testosterone and sex hormone-binding globulin (SHBG) (men). Serum for cortisol and GH was also obtained in the evening (17-19 pm) at day 1 and day 4. The first CT of the brain was classified according to Marshall. Independent staff evaluated outcome at 3 months using GOS-E. Results: Profound changes were found for most pituitary-dependent hormones in the acute phase after sTBI, i.e. low levels of thyroid hormones, strong suppression of the pituitary-gonadal axis and increased levels of prolactin. The main findings of this study were: 1) A large proportion (54% day 1 and 70% day 4) of the patients showed morning s-cortisol levels below the proposed cut-off levels for critical illness related corticosteroid insufficiency (CIRCI), i.e. < 276 nmol/L (= 10 ug/dL), 2) Low s-cortisol was not associated with higher mortality or worse outcome at 3 months, 3) There was a significant association between early (day 1) and strong suppression of the pituitary-gonadal axis and improved survival and favorable functional outcome 3 months after sTBI, 4) Significantly lower levels of fT3 and TSH at day 4 in patients with a poor outcome at 3 months. 5) A higher Marshall CT score was associated with higher day 1 LH/FSH-and lower day 4 TSH levels 6) In general no significant correlation between GCS, ICP or CPP and hormone levels were detected. Only ICPmax and LH day 1 in men was significantly correlated. Conclusion: Profound dynamic changes in hormone levels are found in the acute phase of sTBI. This is consistent with previous findings in different groups of critically ill patients, most of which are likely to be attributed to physiological adaptation to acute illness. Low cortisol levels were a common finding, and not associated with unfavorable outcome. A retained ability to a dynamic hormonal response, i.e. fast and strong suppression of the pituitary-gonadal axis (day 1) and ability to restore activity in the pituitary-thyroid axis (day 4) was associated with less severe injury according to CT-findings and favorable outcome.

Cancer cells display aberrant receptors on their surface that can serve as targets for the development of directed drug therapies. As such, our group has utilized two parallel approaches to redirect the cytotoxic properties of a ribosome-inactivating protein (RIP), Shiga-Like Toxin 1 (SLT 1), by altering its receptor specificity to target and kill cancer cells. The first combinatorial protein library was constructed such that a randomized 7 AA long peptide was inserted within the cytotoxic domain (A chain) of SLT-1. A high-throughput protein-based screening campaign identified a novel A chain toxin variant (named SLT 1AIYSNKLM) capable of targeting and killing human melanoma cells. This variant harbours a peptide insert (IYSNKLM) that directs the A chain to kill human melanoma cell lines. Equilibrium binding studies using 125I-radiolabeled SLT-1AIYSNKLM were conducted to determine the equilibrium binding constant and receptor density on 518-A2 human melanoma cells. When injected into SCID mice bearing a human melanoma xenograft, nanoSPECT/CT imaging as well as the biodistribution profile showed marked tumour uptake and retention of the radiolabeled toxin variant. Furthermore, preliminary experiments have shown that the SLT-1AIYSNKLM receptor is a protein, highlighting the potential for this method to be used in the discovery of novel biomarkers. A second approach was employed to demonstrate that our toxin-based combinatorial library system can be adapted to target known cancer biomarkers. Specifically, SLT-1 A chain variants harbouring 12-residue inserts were expressed in a phage display library. The library was screened against cell lines expressing the human colon cancer marker carcinoembryonic antigen (CEA; CD66e; CEACAM-5) to identify candidates that not only targeted, but internalized into cancer cells within a 1 h period. Variant, CSTA-10, was found to kill CEA-expressing BxPC-3 cells. Overall, the directed evolution of an RIP template such as SLT-1 represents a novel and powerful strategy for the identification of tumour-targeted toxin variants.

Cancer is a set of complex genetic diseases driven by diverse genomic alterations. The genomic study of cancer has enabled the discovery of novel, targetable events in almost all cancer types and in turn, has led to the development of new, targeted cancer therapies benefiting patients; however, the recent explosion of genomic datasets has also resulted in huge lists of new oncogenic factors of unknown biological relevance, and uncertainty over how best to use the data appropriately to influence patient care. Some of the most pressing questions surround the use of statistical methods to identify actionable genomic alterations in cancer and the identification of driving oncogenes in the context of the genomic evolution of cancer cells, undergone before, during, and after prolonged treatment regimens.

Cancer cells display aberrant receptors on their surface that can serve as targets for the development of directed drug therapies. As such, our group has utilized two parallel approaches to redirect the cytotoxic properties of a ribosome-inactivating protein (RIP), Shiga-Like Toxin 1 (SLT 1), by altering its receptor specificity to target and kill cancer cells. The first combinatorial protein library was constructed such that a randomized 7 AA long peptide was inserted within the cytotoxic domain (A chain) of SLT-1. A high-throughput protein-based screening campaign identified a novel A chain toxin variant (named SLT 1AIYSNKLM) capable of targeting and killing human melanoma cells. This variant harbours a peptide insert (IYSNKLM) that directs the A chain to kill human melanoma cell lines. Equilibrium binding studies using 125I-radiolabeled SLT-1AIYSNKLM were conducted to determine the equilibrium binding constant and receptor density on 518-A2 human melanoma cells. When injected into SCID mice bearing a human melanoma xenograft, nanoSPECT/CT imaging as well as the biodistribution profile showed marked tumour uptake and retention of the radiolabeled toxin variant. Furthermore, preliminary experiments have shown that the SLT-1AIYSNKLM receptor is a protein, highlighting the potential for this method to be used in the discovery of novel biomarkers. A second approach was employed to demonstrate that our toxin-based combinatorial library system can be adapted to target known cancer biomarkers. Specifically, SLT-1 A chain variants harbouring 12-residue inserts were expressed in a phage display library. The library was screened against cell lines expressing the human colon cancer marker carcinoembryonic antigen (CEA; CD66e; CEACAM-5) to identify candidates that not only targeted, but internalized into cancer cells within a 1 h period. Variant, CSTA-10, was found to kill CEA-expressing BxPC-3 cells. Overall, the directed evolution of an RIP template such as SLT-1 represents a novel and powerful strategy for the identification of tumour-targeted toxin variants.