No description available.

No description available.

Background HER2-positive breast cancer is a poor prognostic subgroup, even if treated with anti-HER2 directed therapy. Trastuzumab is an important HER2-targeting antibody but only limited patients respond to this drug, and acquired resistance is a common problem. HER3 has been shown to be a key candidate in mediating resistance to trastuzumab and other ErbB inhibitors. The aims of the project are to investigate the resistance mechanisms and the relevant biomarkers in relation to trastuzumab treatment and resistance in HER2-positive breast cancer, in particular, HER3 subcellular localisation and HER3 phosphorylation. Methods Effects of trastuzumab on HER3 subcellular localisation and HER3 phosphorylation in relation to MET receptor were studied using western blots, nuclear fractionation, confocal microscopy, and immunoprecipitation in a panel of HER2-positive cell lines, including SKBr3 and BT474 breast cancer cells in which trastuzumab resistance was induced by long-term drug exposure. Effects of drug and knockdown experiments were tested by cell viability and proliferation assays. HER3 and MET expression was assessed by immunohistochemistry in xenograft tumours and human tissue samples, and clinical impact was assessed in different cohorts of HER2-positive breast cancer patients. Results Acquired trastuzumab resistant SKBr3 cells showed an increase of nuclear HER3_100kD, which was derived from C-terminus of HER3. Nuclear HER3_100kD could be due to the proteolytic cleavage of HER3 since it was reduced by ADAM17 or gamma-secretase inhibitor. In a panel of HER2-positive cell lines and xenograft samples, nuclear HER3 was observed only in the resistant cells. In addition, nuclear HER3 was associated with poor progression-free and overall survivals in HER2-positive breast cancer patients. It was also found that HER3 phosphorylation was maintained in acquired trastuzumab resistant cells, which was contributed by the ligand independent interaction of MET and HER3. Higher MET expression was associated with better overall survival in HER2-positive, breast cancer patients who were not treated with trastuzumab. Conclusions Nuclear HER3 was found in trastuzumab resistant cells and appeared to result from HER3 proteolytic cleavage mediated by ADAM17 and gamma-secretase. Further studies are required to investigate its mechanism and to identify the HER3 cleavage sites. MET was a key factor in maintaining HER3 phosphorylation during trastuzumab resistance. Lastly, nuclear HER3 and MET could be two potential biomarkers in HER2-positive breast cancer.

De part, leur implication dans la prolifération cellulaire, l'invasion et leur surexpression dans de nombreux cancers, les récepteurs à tyrosine kinase de la famille HER constituent des cibles de choix en oncologie. Parmi ces récepteurs, le récepteur HER3 semble pertinent car il est impliqué dans la tumorigenèse de nombreux cancers (sein, ovaire, pancréas, mélanome…) et il est associé à un mauvais pronostic. De plus, la surexpression du récepteur HER3 est souvent associée à l'apparition de résistance aux thérapies ciblées. Nous avons sélectionné plusieurs anticorps anti-HER3 humains et murins respectivement par « phage display » et fusion cellulaire. Ces derniers reconnaissent spécifiqument le récepteur HER3. Parmi les nombreux anticorps découvert, nous avons sélectionné un anticorps anti-HER3 humain H4B-121 et 2 anticorps anti-HER3 murins 9F7-F11 et 16D3-C1. Ces derniers ont la capacité à faire régresser des tumeurs épidermoide, pancréatique et triple négative chez la souris, en présence ou en abscence de neuréguline et indépendamment du status HER2 et P53/PTEN. Cette inhibition est possible grâce à un blocage du cycle cellulaire en phase G1, une inhibition de la prolifération ainsi qu'une induction de l'apoptose. Ces trois anticorps sont capables de bloquer l'hétérodimérisation des récepteurs HER2/HER3 ainsi que la phosphorylation du récepteur HER3. Ils sont aussi capables d'inhiber la phosphorylation de la protéine AKT ainsi que de ses cibles (MDM2, FOXO et XIAP). De plus, nous avons montré que nos anticorps sont capables de lyser les cellules tumorales par ADCC (Antibody-dependent cell-mediated cytotoxicity). Cette étude démontre que ces anticorps anti-HER3 représentent une nouvelle thérapie pour les cancers du pancréas et du sein triple négatif. / Due to their implication in the cellular proliferation, the invasion and their surexpression in numerous cancers, the tyrosine kinase receptors of HER family constitute one of the best targets in oncology. Within this family, the human epidermal growth factor receptor 3 (HER3) plays a role in tumorigenesis of different cancers (Breast, melanoma, pancreas and ovary). This receptor is implicated in drug resistance and he is over expressed in cancers that are not eligible for the currently approved targeted therapies. To this end, we generated specific antibodies (Abs) against domain 1 (D1) and domain 3 (D3) of HER3 that recognize epitopes that do not overlap with the neuregulin-binding site. The fully human H4B-121 Ab and the mouse monoclonal Abs 16D3-C1 and 9F7-F11 inhibited tumor growth in nude mice xenografted with epidermoid, pancreatic, or triple-negative breast cancer cells independently of NRG addiction, HER2 status and p53/PTEN mutations. The combination of one anti-HER3 Ab and trastuzumab improved tumor growth inhibition in mice xenografted with HER2(low) cancer cell lines, for which trastuzumab alone shows no or moderate efficiency. Ab-induced disruption of tumor growth was associated with G1 cell cycle arrest, proliferation inhibition, and apoptosis of cancer cells. Anti-HER3 Abs blocked HER2/HER3 heterodimerization and HER3 phosphorylation at the cell membrane, leading to inhibition of phosphorylation of the downstream AKT targets murine double minute 2, X-linked inhibitor of apoptosis, and forkhead box O1. Anti-HER3 Abs can also induice antibody dependant cell-mediated cytotoxicity. This study demonstrates that anti-HER3 D1 and D3 Abs could represent a new option for immunotherapy of pancreatic and triple-negative breast cancers.

Ce travail s'est intéresser aux difficultés de la prise en charge de la dépression en explorant plusieurs niveaux de représentations : celui des soignants, des patients, de leur famille. La dépression est une pathologie fréquente en oncologie avec une prévalence d’environ 15% qui augmente avec l'aggravation de la maladie cancéreuse. Elle aggrave les autres symptômes tels que la douleur, la fatigue, l'anxiété et retentit sur la qualité de vie et la survie des patients. La prise en charge spécifique du cancer est plus difficile chez les patients déprimés. Les différents objectifs de cette recherche sont de déterminer les freins de la prise en charge de la pathologie dépressive en oncologie et de sensibiliser les soignants à cette problématique en proposant une réflexion sur les modalités de prise en charge. Après une avoir effectué une revue de la littérature, nous avons débuté un travail de traduction et validation d’une échelle de dépistage de la dépression qui semblait adapté à notre population et aux modalités de prise en charge. Cette échelle a ensuite été proposée et utilisée en routine dans différents lieux de prise en charge. Elle a permis de confirmer les résultats retrouvés dans la littérature internationale quant à la fréquence de la dépression et son association avec d’autres symptômes. Ce travail a été complété par une évaluation de la concordance entre l’évaluation par le patient, le médecin et l’infirmier de la symptomatologie dépressive. Les niveaux de concordance retrouvés étaient faibles. Des résultats similaires ont été retrouvés lorsque l’on s’est intéressé à l’utilisation de différentes méthodes pour évaluer la dépression chez la personne âgée atteinte d’un cancer en phase avancée. Nous avons complété ce travail par une étude rétrospective ayant pour objectif d’évaluer l’impact de l’intervention d’une équipe de soins de support sur la sévérité de la dépression des patients atteints d’un cancer en phase avancée avec des résultats positifs, ainsi qu’une synthèse de la littérature quant aux traitements pharmacologiques existants pour la dépression.La dernière partie a utilisé une autre approche de la dépression avec pour objectif d’évaluer les représentations sociales de la dépression dans plusieurs groupes (soignants et famille de patients. Après une discussion générale de ce travail ainsi que la présentation des limites, nous conclurons par les perspectives quant à la prise en charge de la dépression en oncologie. / This work is exploring the associated issues with the management of depression among cancer patients by exploring several levels of representations: health care professionals, patients and patients’ families. Depression is a common disease in oncology with a prevalence of 15%, which increases with the worsening of the cancer disease. It worsens other symptoms such as pain, fatigue, anxiety and impacts patients’ quality of life and survival. Furthermore, cancer specific management is more difficult among depressed patients. The different objectives of this research were to explore the barriers to depression management in oncology and to make health care porfessional aware of this disease by proposing a reflection on the modalities of care.After having conducted a literature review, we started a study to translate and validate a depression screening scale that seemed suited to our population and modalities of care. This scale was then proposed and used routinely in different places of care. Our results were consistent with the international literature regarding the prevalence of depression and its association with other symptoms. We have then explored correlation between depression assessment by the patient, the physician and the nurse. The levels of agreement were found weak. Similar results were found in another study exploring the use of different methods to assess depression in elderly patients with advanced cancer.After these studies, we conducted a retrospective study aiming to evaluate the impact of the intervention of a supportive care team on the severity of depression in advanced cancer patients with positive results and a summary of the existing literature on depression pharmacological treatments.The last part used a different approach to depression study with the goal to assess social representations of depression in several groups (health care professionals, patients and their family). After a general discussion of this work and the presentation of limitations, we conclude with the prospects for the management of depression in oncology.

Telomeres are nucleoprotein structures that contain non-coding (TTAGGG) tandem repeats and associated telomere binding proteins at the end of chromosomes. As a consequence of end-replication losses, telomeres undergo gradual erosion with ongoing cell division. It is hypothesised that in addition to the end-replication problem, mutational mechanisms may contribute to telomere erosion by generating large-scale telomeric deletion events. As short dysfunctional telomeres are capable of fusion to other chromosome ends, large-scale telomeric deletions can lead to genomic instability which in turn may drive tumour progression. The primary aim of this thesis was to investigate putative mutational mechanisms that could lead to large-scale telomeric deletion. The role of oxidative stress and it potential contribution to telomere dynamics was assessed. The induction of fragility and replication inhibition at telomeres was also examined. Furthermore, the role that G-quadruplex structure within telomere repeat sequences and the possible induction of replication fork stalling and resolution as single or double stranded breaks was also considered as a mutational mechanism that could lead to telomere deletion. High-resolution analysis of telomere dynamics using Single Telomere Length Analysis (STELA), following the induction of oxidative stress in IMR90 fibroblasts, revealed that oxidative damage does not appear to affect the rate of telomere erosion, or the frequency of large-scale telomeric deletion. The data are more consistent with the view that premature senescence does not arise as a consequence of accelerated telomere erosion, but instead more likely results from stochastic DNA damage across the rest of the genome. The analysis of telomere dynamics following the induction of chromosome fragility, showed that telomere length in Seckel cell (SCK) fibroblasts were significantly different from those of untreated cells following treatments with aphidicolin with an increase in stochastic telomeric deletion. Whilst in MRC5 fibroblasts, the induction of the telomere fragility impacted on the upper to lower allele ratio, with a loss of the longer telomere length distributions. The stabilisation of G-quadruplex structures using the G-quadruplex ligand (RHPS4), together with ATRX knockdown, showed that an absence of ATRX sensitised cells to the ligand, but that the stabilisation of G-quadruplexes, did not significantly affect the telomere dynamics as determined using STELA. Taken together, the data presented in this thesis are not consistent with a role for oxidative stress, or the formation of G-quadruplex structures, in generating large-scale telomeric deletion; however telomeric mutational events may occur following the induction of chromosome fragile sites, specifically in the context of an ATR deficiency.

Antigens (Ags) expressed by cancer cells can be specifically recognised by T cells contributing to anti-tumour immunity. For this reason, tumour Ag-specific T helper type 1 (Th1) cells can be detected in cancers and their infiltration is predictive of a prolonged disease-free survival. However, their activity is suppressed by T cells with inhibitory functions i.e. regulatory T cells (Tregs). High infiltration of tumours by these cells often correlates with poor survival. It is therefore crucial to understand their activity. Since Th1 and Treg cells are activated after Ag recognition, it is plausible that tumour Ag-specific Tregs inhibit Th1-mediated tumour immunity. Furthermore, vaccination, rather than inducing effector T cells, may be detrimental by activating Tregs. Increased numbers of CD25hi Treg cells are found in cancer patients, including CRC. However, in contrast to many cancers, the phenotype of these cells has not been studied in CRC patients. In this thesis, phenotypes of circulating, colon and tumour infiltrating Tregs are described. A high percentage of intratumoural CD4+CD25hi Tregs was found to express immunosuppressive molecules and these cells were enriched in late-stage CRC tumours. In addition, this thesis describes the measurement of Th1 and Treg cells recognising the oncofetal Ag, 5T4. Four immunogenic regions within the oncofoetal tumour Ag 5T4 and 14 peptides able to bind to most commonly found human HLA-DR alleles were identified. CD4+CD25hi cells mediated the control of tumour Ag 5T4-specific Th1 responses in CRC patients and their suppressive activity was not restricted to Th1 cells of the same Ag specificity. Also, responses to some, but importantly not all, immunogenic helper peptides discovered were influenced by Tregs. Thus Tregs may contribute to CRC progression by influencing tumour Ag- specific responses of Th1 cells. This body of work brings more information regarding the tumour Ag-specificity of Tregs and offers a future rationale to design peptide-based vaccines which exclude Treg peptides.

Mammalian/mechanistic target of mTOR complex 1 (mTORC1) regulates multiple cellular processes, including de novo protein synthesis, autophagy and apoptosis. mTORC1 overactivation occurs in a range of cancers and benign tumour dispositions as a result of mutations which increase mitogenic stimulus or cause malfunction of the tuberous sclerosis complex, the prime regulator of mTORC1 activity. mTORC1 overactivation results in elevated endoplasmic reticulum (ER) stress which, at low levels, elicits a pro-survival response. However, prolonged or excessive ER stress causes cell death. The present study utilised clinically relevant drug combinations to simultaneously enhance levels of ER stress and inhibit compensatory survival pathways in in vitro models of mTORC1 overactivity in order to cause non-genotoxic cell death. The main drugs used in this study were nelfinavir, an ER stress-inducer, chloroquine, an autophagy inhibitor, and bortezomib, a proteasome inhibitor. The key findings of this study include identification of drug combinations nelfinavir and chloroquine, nelfinavir and mefloquine, or nelfinavir and bortezomib to induce significant and selective cell death in mTORC1-driven cells, as measured by flow cytometry with DRAQ7 staining and western blot analysis for cleavage of apoptotic markers. Cell death is likely mediated through ER stress signalling, as shown by increased ER stress markers at both the level of mRNA and protein. Of interest, this study found cell death as a result of combined treatment with nelfinavir was not dependent on proteasome inhibition by nelfinavir, or autophagy inhibition by chloroquine. Additionally, nelfinavir-chloroquine-mediated cell death was completely rescued by inhibition of the vacuolar ATPase by bafilomycin-A1. In conclusion, mTORC1 overactive cells have higher basal levels of ER stress which can be manipulated with drug treatment beyond a survivable threshold, whereas cells capable of reducing mTORC1 signalling are able to survive. This study ascertained a combination of nelfinavir and chloroquine, nelfinavir and mefloquine, or nelfinavir and bortezomib, to cause effective cytotoxicity in mTORC1-driven cells.

The reactive stroma in prostate cancer is predominantly composed of myofibroblasts, which are thought to be required for tumour progression. Bone-marrow derived mesenchymal stem cells (BM-MSCs) are known to migrate into the tumour and are one of the many potential precursors of myofibroblasts. The factors secreted by cancer cells which may drive myofibroblastic differentiation of MSC, however are poorly understood. The aim of this thesis was to explore for the first time, the impact of TGF-β1 expressing exosomes (nano-sized vesicles) secreted by prostate cancer cells in directing the differentiation of BM-MSCs and subsequently the functions of exosome differentiated BM-MSCs. Exosomes isolated from prostate cancer cells skewed BM-MSCs away from differentiating into adipocytes, and instead towards alpha-smooth muscle actin (α-SMA) positive myofibroblasts. BM-MSCs treated with exosomes exhibited enhanced secretion of VEGF-A, HGF and had an altered transcript profile with heightened matrix metalloproteinases (MMP-1, -3 and -13). Impairing the secretion of exosomes by Rab27a knockdown or depleting exosomes from prostate cancer cells culture media by high speed ultracentrifugation, attenuated myofibroblastic differentiation of BM-MSCs, demonstrating exosomes as the key driving factor for this. Furthermore, differentiation of BM-MSCs into myofibroblasts was dependent on exosomally tethered TGF-β1, however BM-MSCs treated with soluble TGF-β1 at the same dose, failed to obtain the same myofibroblastic phenotype. The exosome-differentiated MSCs enhanced endothelial and cancer cell proliferation and migration, supported endothelial vessel formation and promoted tumour cell invasion into peri-tumoural matrix in vitro. In conclusion, this study reports prostate cancer exosomes expressing TGF-β1, to dominantly modulate the fate of BM-MSCs, generating cells with tumour promoting myofibroblastic traits.

MicroRNAs (miRNAs) are small RNAs of ~22 nucleotides in length that regulate gene expression post-transcriptionally. MicroRNAs regulate many biological processes, and may contribute to cancer initiation and progression. In the normal gastrointestinal mucosa, myofibroblasts are involved in wound healing, and in tumours, they play a role in cancer progression. The present study aimed to determine the miRNA expression profiles in different populations of myofibroblasts from gastric and oesophageal tissues, namely, cancer-associated myofibroblasts (CAMs), adjacent non-tumour tissue myofibroblasts (ATMs) and normal tissue myofibroblasts (NTMs), and to investigate the processes that are influenced by differential miRNA abundance in different myofibroblast populations. In addition, miRNA profiling of a putative myofibroblast precursor cell type, mesenchymal stromal cells (MSCs), was studied. Global miRNA expression profiling of a panel of previously characterised gastric and oesophageal myofibroblasts was determined using locked nucleic acid (LNA) miRNA arrays. The microarray data for hsa-miR-29b, hsa-miR-181d, hsa-miR-214 and hsa-miR-424 were validated by quantitative RT-PCR. Using unsupervised miRNA datasets, principal component analysis (PCA) segregated gastric and oesophageal NTMs, and CAMs from gastric and oesophageal carcinomas. Analysis of global miRNA expression showed distinct profile of MSCs in comparison to gastric and oesophageal NTMs. Hierarchical clustering analysis of miRNAs exhibiting significantly different abundance revealed distinct clusters between CAMs and ATMs, CAMs and NTMs and between ATMs and NTMs. In 12 paired samples of gastric CAMs and their corresponding ATMs, 12 miRNAs were significantly different with hsa-miR-181d being the most up-regulated miRNA in CAMs. Using already validated targets of 10 of these miRNAs, an analysis using MetaCore® indicated that the regulation of cell cycle progression and Wnt signalling were differentially affected. In a comparison of each CAM and its corresponding ATM, Wnt signalling was the only process that was significantly targeted by miRNAs that were different in abundance, in all 12 pairs. Using previously determined gene expression data, pairwise analysis of 20 transcripts associated with Wnt signalling showed a significant increase in WNT5A and TGFB2 mRNAs in CAMs compared to their ATMs. Additionally, Western blot analysis showed increased expression of Wnt-5a in CAMs. The results of migration and EdU incorporation assays indicated that Wnt-5a induced the migration and proliferation of both CAMs and ATMs. The abundance of hsa-miR-181d was significantly increased in CAMs by Wnt-5a. Migration of AGS (gastric cancer) cells was stimulated by Wnt-5a and was inhibited in the presence of TIMP-3, a validated target of the miR-181 family. After hsa-miR-181d knockdown, the migration and proliferation of CAMs were significantly decreased, and stimulation with Wnt-5a reversed the migratory inhibition of hsa-miR-181d knockdown CAMs. Conditioned medium from hsa-miR-181d knockdown CAMs inhibited the migration of AGS cells. The findings suggest that differential miRNA abundance is a feature of normal myofibroblasts from the stomach and oesophagus, and myofibroblasts from normal and cancer tissues. One consequence is increased Wnt-5a in gastric CAMs that is implicated in the regulation of cell migration, in health and disease, at least partly through modulation of hsa-miR-181d.