Dissertations / Theses on the topic 'HSC70; Cancer'

The Hsp70 family of molecular chaperones is essential for protein folding, re-folding misfolded client proteins, clearance of aberrant client proteins, and can also inhibit programmed cell death. There are two major cytosolic members of this family: the constitutive Hsc70, and the inducible Hsp72. Under stress conditions the Hsp70 family protects the cell from protein related damage by the induction of Hsp72. Hsc70 and Hsp72 are highly homologous with minor differences in substrate binding. In cancers, Hsp72 is commonly induced and this induction is thought to aid in cancer cell survival. In these studies we demonstrate the differential regulation of the prosurvival kinase Akt by Hsc70 and Hsp72. We demonstrate that of the two cytosolic forms, Hsp72 is the primary Akt regulator. Using a phenothiazine class inhibitor of Hsp70-family activity, methylene blue, we demonstrate dose dependent decreases in the levels of Akt; produced breast cancer specific cell death. This cell death could be rescued by the use of an Hsp70 family ATPase stimulating compound, SW02. We also demonstrate a similar phenotype with a rhodacyanine class Hsp70 family inhibitor, YM-1, also capable of reducing Akt and causing cancer specific cytotoxicity. The resulting Akt decreases were sufficient to block a tamoxifen-resistance pathway, allowing previously resistant cells to regain sensitivity to tamoxifen. These results demonstrate the capabilities of Hsp70 family inhibitors as potent compounds for the treatment of breast cancer.

Les HSP ou protéines de stress ont été découvertes chez la drosophile par Ritossa en 1962. Hautement conservées entre les espèces, elles sont essentielles à l’homéostasie cellulaire et plus encore à la survie lors de stress d’origine diverse (chimique, physique, métabolique etc…). Actuellement, chez les mammifères, il existe cinq principales familles d'HSPs en fonction de leur poids moléculaires : HSP110, HSP90, HSP70 et HSP60 et les petites HSPs à laquelle appartient HSP27 (Kampinga et al., 2009). Parmi les HSPs, HSP70 est la plus fortement induite que ce soit par des agressions telles que le stress oxydatif, les agents anticancéreux ou les radiations ionisantes. HSP70 est, contrairement aux cellules « normales », fortement exprimée dans les cellules cancéreuses et confère à ces cellules une résistance aux drogues anticancéreuses (Goloudina, Demidov & Garrido, 2012) favorisant ainsi le développement tumoral. Les propriétés cyto-protectrices de HSP70 ont été jusqu’à présent attribuées par ces fonctions intracellulaires, principalement en inhibant le processus apoptotique à différentes étapes clés de la signalisation cellulaire (Ravagnan et al., 2001). Cependant une forme membranaire de HSP70 a été détectée à la surface des exosomes dérivant des cellules tumorales (Kuppner et al., 2001). HSP70 membranaire participerait au processus de tumorigenèse en inhibant l’activation des cellules myéloïdes suppressives (MDSCs) et en conséquence, la réponse immune anti-tumorale (Pfister et al., 2007; Schmitt, Gehrmann, Brunet, Multhoff, & Garrido, 2007). Par cette double facette, HSP70 représente donc une cible thérapeutique de choix pour la thérapie anticancéreuse. Compte tenu du rôle clé de HSP70, à la fois par ses fonctions intracellulaires et extracellulaires dans le développement tumoral, l’un des projets phare de notre groupe a été de développer des inhibiteurs spécifiques de HSP70, notamment des aptamères peptidiques et des peptides. Dans un premier travail, nous avons démontré in vitro que deux aptamères A8 et A17 (et son dérivé P17), interagissent avec HSP70 sur des domaines différents et sensibilisent les cellules cancéreuses à la mort induite par des agents chimiothérapeutiques. Des études in vivo réalisées chez la souris et le rat confirment ces résultats et mettent en évidence une réduction significative de la croissance tumorale par ces aptamères peptidiques. Dans un deuxième travail, nous avons généré un dérivé de l’aptamère A8, le peptide P8.1. Nous avons démontré que ce peptide est capable de neutraliser la forme membranaire de HSP70, présente à la surface des exosomes, bloquant ainsi l’activation des MDSCs et restaurant la réponse immunitaire anti-tumorale. A plus long terme, ce travail vise à mettre au point et à valider en clinique une thérapie anticancéreuse plus efficace, en associant aux traitements anticancéreux actuels, les inhibiteurs de HSP70
Heat shock proteins (HSPs) were first discovered in Drosophila by Ritossa in 1962. As stress proteins, HSPs are induced in response to a wide variety of physiological and environmental insults. HSPs have a cyto-protective function and act as molecular chaperones by assisting the folding of nascent or misfolded proteins and by preventing their aggregation. Mammalian HSPs have been classified into 5 families according to their molecular weight: HSP110, HSP90, HSP70, HSP60 and the family of small HSPs such as HSP27 (Kampinga et al., 2009). The most well-known inducible stress chaperone HSP70 is hardly detectable at basal level in normal “non-stressed” cells, but in cancer cells HSP70 is constitutively highly expressed. In that respect, this HSP play a key role in oncogenesis and in resistance to chemotherapeutic drugs (Goloudina et al., 2012).Until now, the cytoprotective properties of HSP70 were attributed to its intracellular functions mainly via its ability to block the apoptotic process at key points of the signal (Ravagnan et al., 2001). More recently, a membrane bound form of HSP70 was detected but also at the surface of exosomes derived from tumor cells but not non-cancerous cells (Kuppner et al., 2001). Moreover, growing evidence support the critical role of this membrane-bound HSP70 in the process of tumorigenesis (Pfister et al., 2007; Schmitt et al., 2007) via the activation of myeloid suppressor cells (MDSCs), which inhibit the anti-tumor immune response (Chalmin et al., 2010). Thereby, HSP70 by this dual action represents an attractive target for new anti-cancer therapy.In that aim, we developed specific inhibitors of HSP70, including peptide aptamers and peptides. In this work, we demonstrated that two aptamers A8, A17 (and the peptide P17), interact with different domains of HSP70 and, significantly sensitized cancer cells to apoptosis induced by chemotherapeutic drugs. Accordingly, in vivo studies in mice and rats showed a significant reduction of tumor growth by these inhibitors. Finally, we generate an A8 derived peptide called P8.1 that specifically neutralized the extracellular region of HSP70 at the surface of exosomes. Our results demonstrated that this peptide P8.1 inhibits MDSC activation and restored the antitumor immune response in vitro and in vivo, respectively.Overall, our work will help to develop and validate more effective cancer therapy based on the association of conventional chemotherapy with HSP70 inhibitors

La mise au point d'un vaccin anti-tumoral efficace requiert l'identification de nombreux antigènes associés aux tumeurs, dont l'expression est fréquente dans des tumeurs d'origines histologiques variées. La protéine de choc thermique Hsp70 inductible (ou Hsp72) est fréquemment sur-exprimée dans les tumeurs humaines malignes d'origines variées, telles que le carcinome du sein, du poumon, du colon, du col de l'utérus et les ostéosarcomes. Afin d' établir l'intérêt de Hsp70 comme antigène associé aux tumeurs, trois peptides de Hsp70 ont été sélectionnés pour leur haute affinité pour HLA-A*0201. Ces peptides induisent des lymphocytes T cytotoxiques anti-tumoraux "in vivo" dans des souris transgéniques pour HLA-A*0201 et "in vitro" à partir de PBMC des donneurs sains HLA-A0201+. Ces épitopes sont la cible d'une réponse immunitaire. .
The design of a broad application tumor vaccine requires the identification of tumor antigens expressed in a majority of tumors of various origins. The major stress-induccible heat shock protein Hsp70 (a. K. A Hsp72) is frequently overexpressed in human tumors of various histological origin, such as breast, lung, colorectal, cervical carcinoma and osteosarcoma. To assess the value of Hsp70 as a tumor associated antigen, three peptides from Hsp70 were selected for their high affinity for HLA-A*0201. These peptides were able to trigger anti-tumor cytotoxic T lymphocytes "in vivo" in HLA-A*0201-transgenic HHD mice and "in vitro" in HLA-A*0201+ healthy donors. These epitopes are tagets of an immune reponse in many HLA-A0201+ breast cancer patients. Hsp70 is thus a valuable tumor antigen for broad application tumor immunotherapy

Le cancer est aujourd’hui une maladie qui fait malheureusement partie de notre quotidien, et bien qu’il soit de mieux en mieux traité, de plus en plus de gens en sont atteints. La recherche s’oriente sur deux pans : la thérapie et le diagnostic. Les nouvelles thérapies présentent des résultats spectaculaires jamais atteints à ce jour, mais l’amélioration du diagnostic peut aussi sauver des vies. Il est établi que plus un cancer est diagnostiqué tôt, plus le patient a de chances de guérir. Idem pour le suivi de la maladie. Dans ce contexte de précision et de précocité, la biopsie liquide émerge et possède un avenir très prometteur. Elle consiste en l’étude d’analytes présents dans les fluides corporels, particulièrement la circulation sanguine. On y retrouve principalement, mais pas exclusivement, l’ADN tumoral circulant, les cellules tumorales circulantes (CTCs) et les exosomes.Ce manuscrit a pour objectif de replacer mon travail de recherche, qui porte sur les exosomes, dans le contexte de la biopsie liquide, afin de laisser libre court aux comparaisons et à la compréhension de leur potentiel diagnostic. Les exosomes sont des nanovésicules libérées par les cellules dans le sang. Elles contiennent du matériel génétique, des lipides et des protéines. Une étape clé pour leur utilisation en tant que biomarqueur est de différencier les exosomes dérivés de tumeur de ceux dérivés d’autres cellules de l’organisme.La protéine de stress Heat shock protein-70 (HSP70) a été décrite comme étant surexprimée dans les cellules cancéreuses et associée à un mauvais pronostic. Nous avons précédemment démontré que seuls les exosomes dérivés de cellules cancéreuses portaient HSP70 à la membrane. Dans ce travail, nous avons ouvert une étude clinique pilote prospective incluant des patients atteints d’un cancer du sein et du poumon afin de déterminer s’il était possible de détecter et quantifier les HSP70-exosomes dans le sang de patients atteints de tumeurs solides malignes.Nous avons montré que le taux de HSP70 dans les exosomes, contrairement à la forme soluble, reflétait le contenu en HSP70 dans la biopsie tumorale. Le taux de HSP70-exosomes circulants est augmenté chez les patients atteints d’un cancer à un stade métastatique comparé aux non-métastatique et aux donneurs sains. Nous avons ensuite démontré que les niveaux de HSP70-exosomes étaient corrélés au statut de la maladie, et étaient potentiellement de meilleurs marqueurs que les CTCs. Enfin, nous avons indiqué que le taux de HSP70-exosomes étaient inversement corrélés à la réponse au traitement, et, par conséquent, que le suivi du taux de HSP70 dans les exosomes pourrait être utile dans la prédiction de la réponse au traitement
Actually, cancer is unfortunately part of our daily life. Although it is better treated, more and more people are affected. Cancer research is working on two side: therapy and diagnosis. The next-generation therapies show wonderful results that were never reached so far, but diagnosis improvement can also save lives. It is well established that the earlier the cancer is diagnosed, the better the survival rate. Idem for the follow-up of the disease. In this context of precision and earliness, liquid biopsy spark interest and seems to have a bright future. It consists in the investigation of liquid analytes, particularly within the bloodstream, such as circulating tumor DNA, circulating tumor cells (CTCs) and exosomes.This manuscript aims to put my research work on exosomes in the context of liquid biopsy, in order to let you compare with other analytes and understand their diagnosis potential. Exosomes are nanovesicles released by all cells that can be found in the blood. They carry genetic material, proteins end lipids. A key point for their use as potential biomarkers in cancer is to differentiate tumor-derived exosomes from other circulating nanovesicles.Heat shock protein-70 (HSP70) has been shown to be abundantly expressed by cancer cells and to be associated with bad prognosis. We previously showed that exosomes derived from cancer cells carried HSP70 in the membrane while those from non- cancerous cells did not. In this work, we opened a prospective clinical pilot study including breast and lung cancer patients to determine whether it was possible to detect and quantify HSP70 exosomes in the blood of patients with solid cancers.We found that circulating exosomal HSP70 levels, but not soluble HSP70, reflected HSP70 content within the tumour biopsies. Circulating HSP70 exosomes increased in metastatic patients compared to non-metastatic patients or healthy volunteers. Further, we demonstrated that HSP70-exosome levels correlated with the disease status and, when compared with circulating tumor cells, were more sensitive tumor dissemination predictors. Finally, our case studies indicated that HSP70-exosome levels inversely correlated with response to the therapy and that, therefore, monitoring changes in circulating exosomal HSP70 might be useful to predict tumor response and clinical outcome

HSP70 est une protéine de choc thermique surexprimée dans les cellules cancéreuses. Elle inhibe le processus apoptotique permettant la survie des cellules exposées à divers stress. Cette fonction protectrice d’HSP70 implique l’interaction et la neutralisation des protéines Apaf-1 et AIF. Nous avons montré qu’ADD70, un peptide contenant la séquence d’AIF nécessaire à son interaction avec HSP70, se lie et neutralise HSP70 dans le cytosol. Il sensibilise les cellules cancéreuses à la mort induite par une grande variété de stimuli apoptotique. In vivo, l’expression, de ce peptide par les cellules tumorales diminue leur tumorigénicité chez des animaux syngéniques immunocompétents sans affecter leur croissance chez des animaux immunodéficients. De plus, ADD70 sensibilise les cellules cancéreuses coliques de rat et les cellules de mélanome de souris au cisplatine, un agent anti-cancéreux. Ces données nous montrent l’intérêt de cibler l’interaction AIF/HSP70 dans la thérapie anti-cancéreuse
HSP70 is a heat shock protein overexpressed in several cancer cells. It prevents apoptosis, thereby increasing the survival of cells exposed to a wide range of lethal stimuli. These protective functions of HSP70 involve its interaction with and neutralization of Apaf-1, implicated in caspase activation, and AIF, involved in caspase-independent cell death. We have shown that a peptide containing the AIF sequence involved in its interaction with HSP70, binds to and neutralizes HSP70 in the cytosol, thereby sensitizing cancer cells to apoptosis induced by a variety of death stimuli. The expression of ADD70 in tumor cells decreases their tumorigenicity in syngeneic animals without affecting their growth in immunodeficient animals. In addition, ADD70 sensitizes rat colon cancer cells and mouse melanoma cells to the chemotherapeutic agent cisplatin. Altogether, these data indicate the potential interest of targeting the HSP70 interaction with AIF for cancer therapy

Hop (the Hsp90/Hsp70 organising protein) is a co-chaperone that acts as an adapter between the major molecular chaperones Hsp90 and Hsp70 during the cellular assembly of the Hsp90 complex. The Hsp90 complex regulates the stability and conformational maturation of a range of important cellular proteins, many of which are deregulated in cancer. In this study, we hypothesised that Hop knockdown inhibits proliferation and migration of cancer cells. We characterised the expression of Hop in cell models of different cancerous status, and provided evidence that Hop was upregulated in tumour cells compared to normal cell counterparts. Using an RNA interference approach, a 60-90% knockdown of Hop was achieved for up to 144 hours in the MDA-MB-231 and Hs578T breast cancer cell lines. Hop knockdown resulted in downregulation of the Hsp90 client proteins, Akt and Stat3, as well as a change in the expression of other Hsp90 co-chaperones, p23, Cdc37 and Aha1, while no change in the levels of Hsp90 or Hsp70 was observed. Silencing of Hop impaired cell proliferation in Hs578T cells but an increase in proliferation in MDA-MB-231, suggesting that the role of Hop in cancer cell proliferation was dependent on type of cancer cell. Hop knockdown in Hs578T and MDA-MB- 231 cells did not lead to any significant changes in the half maximal inhibitory concentrations (IC50) of selected small molecule inhibitors (paclitaxel, geldanamycin and novobiocin) in these cell lines after 72 hours. Hop knockdown cells were however, more sensitive than control cells to the Hsp90 inhibitors geldanamycin and novobiocin at earlier time points and in the presence of the drug transporter inhibitor, verapamil. Hop knockdown caused a decrease in cell migration as measured by the wound healing assay in both Hs578T and MDA-MB-231 cells. Hop was present in purified pseudopodia fractions of migrating cells, and immunofluorescence analysis showed that Hop colocalised with actin at the leading edges of pseudopodia, points of adhesion and at intercellular junctions of cells that have been stimulated to migrate with the chemokine stromal derived factor-1. Hop was able to bind to actin in vitro using actin cosedimentation assays, and silencing of Hop dramatically reduced the capacity of Hs578T cells to form pseudopodia. These results establish a correlation between Hop and actin dynamics, pseudopodia formation and migration in the context of Hop silencing, and collectively suggest that Hop plays a role in cancer cell migration. This study presents experimental evidence for a promising alternative to targeting Hsp90 and Hsp70 chaperones, a novel drug target in cancer therapy.

Mitosis is an important process for the generation of two genetically identical daughter cells. Formation of a bipolar mitotic spindle requires the presence of two centrosomes as these are required to form each pole of the spindle. However, cancer cells, frequently possess extra centrosomes. Therefore, they have developed mechanisms to cluster them into two poles to enable mitotic pseudo-spindle formation and cell survival. Inhibiting centrosome clustering offers a unique and attractive therapeutic approach to selectively kill cells with amplified centrosomes by promoting formation of multipolar spindle poles and mitotic catastrophe. Centrosome clustering mechanisms include proteins with specific roles in microtubule dynamics, microtubule attachments to centrosomes, kinetochores and the cell cortex, as well as the spindle assembly checkpoint. Here, we identify a new pathway that contributes to centrosome clustering and involves the Nek6 kinase and Hsp72 chaperone. Nek6, as well as its upstream activators Plk1 and Aurora-A, targets Hsp72 to the poles of cells with amplified centrosomes. Blocking Hsp72 or Nek6 activity leads to formation of multipolar spindles with poles that always contain centrosomes, whereas other centrosome de-clustering agents trigger formation of acentrosomal poles. Indeed, inhibition of Hsp72 in ALL cells led to an increase of multipolar spindle frequency that correlated with centrosome amplification. Dynein/dynactin and phospho-Hsp72 colocalise to kinetochores and we suggest that they are required for proper attachment of microtubules to kinetochores to facilitate a stable bipolar mitotic spindle and potentially centrosome clustering. Additionally, loss of Hsp72 or Nek6 function did not disrupt either mitotic spindle formation or mitotic progression in non-cancer derived cells versus cancer cells. Hence, the Nek6-Hsp72 pathway, and its potential downstream target dynein, may act as a novel pathway of centrosome clustering that reveals a new opportunity for targeting centrosome clustering and mitotic progression in cancer cells.

HOP (Heat shock protein 70/ Heat shock protein 90 organising protein) is a co-chaperone essential for client protein transfer from HSP70 to HSP90 within the HSP90 chaperone machine and has been found to be up-regulated in various cancers. However, minimal in vitro information can be found on the regulation of HOP expression. The aim of this study was to analyse the HOP gene structure across known orthologues, identify and characterise the HOP promoter, and identify the regulatory mechanisms influencing the expression of HOP in cancer. We hypothesized that the expression of HOP in cancer cells is likely regulated by oncogenic signalling pathways linked to cis-elements within the HOP promoter. An initial study of the evolution of the HOP gene speciation was performed across identified orthologues using Mega5.2. The evolutionary pathway of the HOP gene was traced from the unicellular organisms to fish, to amphibian and then to land mammal. The synteny across the orthologues was identified and the co-expression profile of HOP analysed. We identified the putative promoter region for HOP in silico and in vitro. Luciferase reporter assays were utilized to demonstrate promoter activity of the upstream region in vitro. Bioinformatic analysis of the active promoter region identified a large CpG island and a range of putative cis-elements. Many of the cis-elements interact with transcription factors which are activated by oncogenic pathways. We therefore tested the regulation of HOP levels by rat sarcoma viral oncogene homologue (RAS). Cancer cell lines were transfected with mutated RAS to observe the effect of constitutively active RAS expression on the production of HOP using qRT-PCR and Western Blot analyses. Additionally, inhibitors of the RAS signalling pathway were utilised to confirm the regulatory effect of mutated RAS on HOP expression. In cancer cell lines containing mutated RAS (Hs578T), HOP was up-regulated via a mechanism involving the MAPK signalling pathway and the ETS-1 and C/EBPβ cis-elements within the HOP promoter. These findings suggest for the first time that Hop expression in cancer may be regulated by RAS activation of the HOP promoter. Additionally, this study allowed us to determine the murine system to be the most suited genetic model organism with which to study the function of human HOP.

L’utilisation de modèles animaux a permis la découverte de mécanismes importants du développement en général, et du développement tumoral en particulier afin d’établir et mettre au point de nouveaux traitements. Le poisson zèbre (Danio rerio), est de plus en plus utilisé dans le cadre de ces recherches du fait de ses nombreux avantages comme par exemple la transparence de ses larves ou une forte homologie avec l’homme. Plusieurs approches ont été développées chez ce poisson comme l’invalidation transitoire d’un gène afin d’identifier le rôle d’une protéine dans le développement, ou alors la transplantation de cellules tumorales de mammifères et étudier les réponses aux traitements anti-tumoraux.C’est dans l’un de ces deux contextes que nous avons étudié le rôle du monoxyde d’azote dans le développement tumoral. Pour cela nous avons utilisé une sonde fluorescente et avons pu détecter in vivo une production de monoxyde d’azote associée aux cellules tumorales xénogreffées, dont l’utilisation d’un capteur du NO, le cPTIO s’est traduit par une perte de cellules tumorales et une baisse de l’expression d’un facteur angiogénique le VEGF, démontrant une utilisation potentielle dans du cPTIO comme molécule anti-tumorale.L’autre volet d’étude a été l’identification du rôle de HSF1 dans le développement et la différenciation des globules rouges chez le poisson zèbre comme modèle expérimental, dans des conditions de non stress thermique. Pour cela, nous avons inactivé transitoirement le gène hsf1 grâce aux morpholinos, et avons constaté des défauts dans le développement, mais aussi une altération de la différenciation des érythrocytes
The use of animal models has led to the discovery of important mechanisms of biology development in general, and tumor development in particular, to establish and develop new treatments. The Zebrafish (Danio rerio) is increasingly used nowadays as part of this research because of its many advantages such as the transparency of the larvae, and the high homology with human. Several approaches have been developed in this fish, as the gene-knockdown in order to identify the role of a protein in the development, or the tumor transplantation of mammalian cells to study anti-tumor treatments response.It is in one of these two contexts that we studied the role of nitric oxide in tumor development in zebrafish. We used a fluorescent probe and were able to detect in vivo the production of nitric oxide associated with xenograft tumor cells. The use of an NO scanvenger, the cPTIO resulted in a loss of tumor cells and a decrease in the expression of an angiogenic factor VEGF, showing a potential in the use of cPTIO as antitumor molecule.We used also the transitory invalidation of hsf1 gene, in order to explore a potential new role in the development and red blood cells differentiation in zebrafish as an experimental model, in non heat-shocked conditions. We found that HSF1 was important for the differentiation of erythrocytes, and its inactivation also reflected defects in development

Un certain nombre d’études épidémiologiques font état d’une augmentation de l’infertilité masculine durant ces cinquante dernières années, en particulier dans les pays industrialisés, mais aussi d’une augmentation des malformations de l’appareil reproducteur masculin telles que la cryptorchidie (absence de migration des testicules dans les bourses) ou l’hypospadias (malformation du pénis), et des cancers testiculaires. Des données expérimentales suggèrent que ces anomalies du tractus génital mâle sont liées. Ces symptômes forment le syndrome de dysgénésie testiculaire. Les causes d’apparition ce syndrome semblent être d’origine environnementale. En effet, les évolutions relativement rapides de ce syndrome suggèrent des facteurs dynamiques, en lien avec le mode de vie ou l’environnement. Une des hypothèses est que, l’exposition durant la vie fœtale ou néonatale à des composés présents dans l’environnement capables d’interférer avec le système hormonal (perturbateurs endocriniens environnementaux, PEEs), serait responsable de l’augmentation de l’incidence de ces pathologies. Au banc des principaux accusés, les molécules qui possèdent des activités de type estrogénique ou antiandrogénique. A ce jour, les mécanismes d’action à l’origine du syndrome de dysgénésie testiculaire sont encore mal connus. Certaines études suggèrent des mécanismes de type épigenétique dans les effets à long terme des PEEs. L’objectif de notre travail était d’identifier et caractériser les mécanismes d’action de type épigenétique impliqué dans l’infertilité mâle. Pour cela, nous avons utilisé un modèle expérimental (rats nouveau-nés) reposant sur une exposition développementale à un estrogène (estradiol benzoate). Ce modèle induit chez le rat adulte un phénotype d’hypospermatogenèse liée à une à apoptose chronique des cellules germinales testiculaires. Nous montrons que ce phénotype est lié à l’altération de deux voies, impliquant en amont des effecteurs épigénétiques. La première voie implique la famille des miR-29s. Ainsi, nous observons une augmentation de l’expression des miR-29a, b, c qui provoque une diminution de deux de ses cibles: la protéine antiapoptotique MCL-1 et les enzymes de méthylation de l’ADN DNMTs. La chute des DNMTs entraine une hypométhylation globale (estimée à travers le gène Line-1) et spécifique du facteur de choc thermique HSF1. Ceci provoque une réexpression de ces facteurs entrainant l’apoptose des cellules germinales adultes. La deuxième voie implique le miR-18a. L’augmentation de son expression provoque une chute de l’expression de sa cible HSF2 qui régule la protéine de choc thermique HSP70/HSPA2. Le faible taux d’HSPA2 est une autre explication de l’apoptose des cellules germinales dans notre modèle. Nous montrons aussi que ce phénotype est irréversible lorsque l’exposition à lieu chez le nouveau-né alors qu’il est réversible quand l’exposition à lieu à l’âge adulte. Ces données suggèrent que l’exposition néonatale à l’estradiol benzoate induit une programmation développementale de l’hypospermatogenèse.Enfin, les anomalies tissulaires d’expression des miRNAs se retrouvent au niveau sanguin, suggérant leur utilisation potentielle comme biomarqueurs. Nous avons validé cet aspect chez l’homme en montrant que l’expression des miR29s et du miR-18a était plus élevée chez les patients oligo- ou azoospermiques que les chez patients normospermiques.En conclusion, nos résultats indiquent que l’hypospermatogenèse due à une apoptose chronique des cellules germinales observée chez l’animal adulte après exposition néonatale à l’EB met en jeu une modification d’expression de plusieurs effecteurs épigénétiques clés: miR-29s, miR-18a et DNMTs. De plus, les miR-29s et miR-18a pourraient être de nouveaux biomarqueurs circulants non invasifs de la stérilité masculine dans le contexte d’une oligo ou azoospermie chez l'homme
Epidemiological studies have reported an increase in male infertility over the past fifty years, especially in industrialized countries, but also an increase in malformations of the male reproductive tract such as cryptorchidism (no migration of the testes into the scrotum) and hypospadias (malformation of the penis), and testicular cancers. Experimental data suggest that these abnormalities of the male genital tract are related. These symptoms form the testicular dysgenesis syndrome. The causes of the occurrence of this syndrome appear to be environmental in origin. Indeed, the relatively rapid evolution of this syndrome suggests dynamic factors related to lifestyle or environment. One hypothesis is that exposure during fetal or neonatal life to compounds present in the environment can interfere with the hormonal system (environmental endocrine disruptors), would be responsible for the increased incidence of these pathologies. Bench of the main accused, molecules that have estrogenic or anti-androgenic activity types. To date, the mechanisms of action behind the testicular dysgenesis syndrome are poorly understood. Some studies suggest that epigenetic mechanisms are at playThe objective of our work was to identify and characterize the epigenetic mechanisms of action involved in male infertility induced by neonatal exposure to xenoestrogen. For this, we used an experimental model based on a developmental exposure to estrogen (estradiol benzoate). This model induced in adult rats a hypospermatogenesis phenotype due to chronic apoptosis of germ cells.We show that this phenotype is related to an alteration of two pathways, involving upstream effectors epigenetic. The first pathway involves the family of miR- 29s. Thus, we observe an up-regulation of miR -29a, b, c, which causes a decrease in two of his targets: the anti-apoptotic protein MCL- 1 and the enzymes of DNA methylation DNMTs. Falling DNMTs leads to a global hypomethylation (estimated through the Line -1 gene) and to specific hypomethylation of the heat shock factor, HSF1. This causes a re-expression of factors that induce apoptosis in adult germ cells. The second pathway involves up-regulation of miR -18a that causes a down-regulation of its target HSF2 which regulates the heat shock protein HSP70/HSPA2. The down-regulation of HSPA2 is another explanation of germ cell apoptosis in our model. We also show that this phenotype is irreversible when the estrogen exposure takes place in the newborn whereas it is reversible when exposure takes place in adulthood, suggesting that neonatal exposure to estradiol benzoate induced a developmental programming of hypospermatogenesis.Finally, abnormal tissue expressions of miRNAs are found in the blood, suggesting their potential use as biomarkers. We validated this aspect in humans showing that the expression of miR29s and miR-18a was higher in patients with decrease or no sperm counts compared to normal sperm count. In conclusion, our results indicate that hypospermatogenesis due to chronic germ cell apoptosis observed in adult animals after neonatal exposure to EB involves a change in expression of several key epigenetic effectors: miR-29, miR-18a and DNMTs. In addition, miR-29 and miR-18a could be new non invasive circulating biomarkers of men infertility

Un certain nombre d'études épidémiologiques font état d'une augmentation de l'infertilité masculine durant ces cinquante dernières années, en particulier dans les pays industrialisés, mais aussi d'une augmentation des malformations de l'appareil reproducteur masculin telles que la cryptorchidie (absence de migration des testicules dans les bourses) ou l'hypospadias (malformation du pénis), et des cancers testiculaires. Des données expérimentales suggèrent que ces anomalies du tractus génital mâle sont liées. Ces symptômes forment le syndrome de dysgénésie testiculaire. Les causes d'apparition ce syndrome semblent être d'origine environnementale. En effet, les évolutions relativement rapides de ce syndrome suggèrent des facteurs dynamiques, en lien avec le mode de vie ou l'environnement. Une des hypothèses est que, l'exposition durant la vie fœtale ou néonatale à des composés présents dans l'environnement capables d'interférer avec le système hormonal (perturbateurs endocriniens environnementaux, PEEs), serait responsable de l'augmentation de l'incidence de ces pathologies. Au banc des principaux accusés, les molécules qui possèdent des activités de type estrogénique ou antiandrogénique. A ce jour, les mécanismes d'action à l'origine du syndrome de dysgénésie testiculaire sont encore mal connus. Certaines études suggèrent des mécanismes de type épigenétique dans les effets à long terme des PEEs. L'objectif de notre travail était d'identifier et caractériser les mécanismes d'action de type épigenétique impliqué dans l'infertilité mâle. Pour cela, nous avons utilisé un modèle expérimental (rats nouveau-nés) reposant sur une exposition développementale à un estrogène (estradiol benzoate). Ce modèle induit chez le rat adulte un phénotype d'hypospermatogenèse liée à une à apoptose chronique des cellules germinales testiculaires. Nous montrons que ce phénotype est lié à l'altération de deux voies, impliquant en amont des effecteurs épigénétiques. La première voie implique la famille des miR-29s. Ainsi, nous observons une augmentation de l'expression des miR-29a, b, c qui provoque une diminution de deux de ses cibles: la protéine antiapoptotique MCL-1 et les enzymes de méthylation de l'ADN DNMTs. La chute des DNMTs entraine une hypométhylation globale (estimée à travers le gène Line-1) et spécifique du facteur de choc thermique HSF1. Ceci provoque une réexpression de ces facteurs entrainant l'apoptose des cellules germinales adultes. La deuxième voie implique le miR-18a. L'augmentation de son expression provoque une chute de l'expression de sa cible HSF2 qui régule la protéine de choc thermique HSP70/HSPA2. Le faible taux d'HSPA2 est une autre explication de l'apoptose des cellules germinales dans notre modèle. Nous montrons aussi que ce phénotype est irréversible lorsque l'exposition à lieu chez le nouveau-né alors qu'il est réversible quand l'exposition à lieu à l'âge adulte. Ces données suggèrent que l'exposition néonatale à l'estradiol benzoate induit une programmation développementale de l'hypospermatogenèse.Enfin, les anomalies tissulaires d'expression des miRNAs se retrouvent au niveau sanguin, suggérant leur utilisation potentielle comme biomarqueurs. Nous avons validé cet aspect chez l'homme en montrant que l'expression des miR29s et du miR-18a était plus élevée chez les patients oligo- ou azoospermiques que les chez patients normospermiques.En conclusion, nos résultats indiquent que l'hypospermatogenèse due à une apoptose chronique des cellules germinales observée chez l'animal adulte après exposition néonatale à l'EB met en jeu une modification d'expression de plusieurs effecteurs épigénétiques clés: miR-29s, miR-18a et DNMTs. De plus, les miR-29s et miR-18a pourraient être de nouveaux biomarqueurs circulants non invasifs de la stérilité masculine dans le contexte d'une oligo ou azoospermie chez l'homme.

M.Sc.
Breast cancer is the most commonly diagnosed cancer and cause of death in women world wide as well as in South Africa. Cancer is characterized by over-proliferation of cells or the inhibition of programmed cell death known as apoptosis, a well coordinated process that results in the activation of several proteases and other hydrolytic enzymes. Apoptosis is regulated by enhancers and inhibitors, such as heat shock proteins (Hsps) that modulate the apoptotic process according to the demands of specific cells. Hsps can regulate the release of pro-apoptotic factors from the mitochondria as well as inhibit key steps in the apoptotic cascade such as activation of caspases. The Hsp70 family constitutes the most conserved and best studied class of Hsps and the stress-induced Hsp70 also blocks the apoptotic pathway at different levels. Hsp70 is furthermore overexpressed in several tumor cells and can effectively inhibit cell death induced by a wide range of stimuli including several cancer related stresses such as hyperthermia, chemotherapeutic agents and nonsteroidal anti-inflammatory drugs (NSAIDs) i.a. aspirin (acetylated salicylic acid) In addition to their potent analgesic, antipyretic and anti-inflammatory activity, NSAIDs can inhibit cell proliferation and induce apoptosis in many cancer cell lines. However, NSAIDs can also lower the temperature threshold for Hsp70 induction and induce a transcriptionally inert intermediate of Hsp70 that can be converted to a transcriptionally active state by a subsequent exposure to heat shock. This suggests that NSAIDs act differently under heat stress, possibly increasing cellular protection through the heat shock response in cancer cells with already elevated levels of Hsps. It is therefore hypothesized that the synergistic use of heat shock with salicylic acid (SA) treatment will increase Hsp70 expression and protein accumulation and further enhance the resistance of breast cancer cells to apoptosis. The effects of SA on its own or in combination with HS on the viability of MCF-7 breast cancer cells as well as Hsp70 protein levels and gene expression were therefore investigated. SA treatments were found to induce cell death in a dose-dependent manner with the most significant decrease in viability observed after treatment with 20 mM SA.

M.Sc.
Heat shock (HS) proteins and HS transcription factors (HSFs) have been coined as the ‘Achilles Heel’ for cancer therapy, since they have been found to be overexpressed in cancer cells and are required for cell survival during tumour progression and metastasis. Hsp70 and other members of the Hsp family have been shown to inhibit apoptosis at several different stages, contributing to resistance to chemotherapy. NSAIDs, like salicylates and aspirin, are used for the treatment and prevention of cancers such as breast cancer. SA has been shown to enhance HSF-DNA binding and results in the increased expression of heat-induced Hsp70 which is antiapoptotic. We hypothesise that SA treatment can result in the potentiation of Hsp70 in MCF-7 cells further increasing their resistance to apoptosis and thus the aim of this study was to investigate the dose-responsive effects of salicylic acid (SA) in the presence and absence of heat shock on components of the pro and antiapoptotic components of the apoptotic pathway. MCF-7 cells, which naturally overexpress Hsp70, were treated with several doses of SA in the presence and absence of a mild heat shock, followed by analysis of Hsp70 and several pro and antiapoptotic members of intrinsic and extrinsic apoptotic pathways, including Bcl-2, Bax, caspase 6 and 8, JNK, AIF and APAF-1. Induced Hsp70 accumulation by the SA treatments in the presence and absence of heat shock enhanced apoptosis in cells exposed to SA whereas higher concentrations of SA combination with heat shock induced necrosis and a decrease in Hsp70 accumulation in MCF-7 cells. Identification of the effects which specific concentrations of SA in the presence and absence of heat shock had on the apoptotic pathway constituents helped highlight potential pathways by which cell death could occur in MCF-7 cells through the downregulation of Hsp70. It is most likely that MCF-7 cell death is occurring due to the release of reactive oxygen species (ROS) which in turn lead to necrosis or death may be achieved via a cathepsin-B-mediated cell death pathway where both of these possibilities need to be further investigated.

HSP70 plays an important role in cancer development. However, the molecular role of HSP70 in cancer is poorly understood. Previous work from our laboratory demonstrated that HSP70 is essential for initiation of Her2-positive breast cancer by controlling oncogene-induced senescence. Here we demonstrate that HSP70 is critical for both initiation and progression of mammary cancer. Interestingly, the role of HSP70 in cancer development did not involve its canonical function as a molecular chaperone. Instead, HSP70 had multiple effects on signaling pathway components related to tumor initiation, growth, and metastasis, such as FOXM1, HIF1, NF-𝜅B, and SRC. HSP70 regulated signaling networks via association with the co-chaperone, BAG3, a scaffold protein with capacity to interact with multiple key regulators of cell signaling. Using SRC as a model, we demonstrated that association with HSP70 attenuates BAG3's interaction with the SH3 domain of SRC. We also show that an HSP70-interacting small molecule, YM-1, can specifically inhibit the HSP70-BAG3 signaling axis, leading to selective inhibition of tumor growth in vivo and in vitro. This compound mimicked the effects seen with depletion of HSP70 in a dose dependent manner, providing a proof of principle that the association of HSP70 and BAG3 is needed for regulation of these pathways. Additionally, a second generation of YM-1 analogs, JG-98 and JG-84, were shown to be more potent than YM-1 while acting in a similar fashion on signaling pathways. A less potent analog, JG-36, was not able to modulate these pathways as effectively. These studies demonstrate that the HSP70-BAG3 axis is a major regulator of cancer signaling and suggest that targeting the interface between HSP70 and BAG3 is a novel therapeutic approach.

碩士
國立臺灣大學
獸醫學研究所
98
Immunization with xenogeneic DNA is a promising cancer treatment, as it generates autoantibodies and cytotoxic T cells to break the tumor tolerance against self-antigens. Heat shock protein 70 (HSP70) is overexpressed in many kinds of tumors and is believed to be heavily involved in tumor progression. This study employed a xenogeneic chicken HSP70 (chHSP70) DNA vaccine in a canine transmissible venereal tumor (CTVT) model in beagles to break the tumor tolerance by inducing immune responses towards canine HSP70 self-antigens. In this study, three vaccination groups were created: the first (G1) was designed to evaluate the prophylactic efficiency of the chHSP70 DNA vaccine by delivering the vaccine prior to tumor inoculation; the second (G2) was designed to evaluate the therapeutic efficacy in developed tumors by vaccinating the dogs after tumor inoculation; and the third (G3) consisted of the same vaccination schedule as that of G1, with the exception that the intramuscular injection/electroporation method used to administer the third vaccination in G1 was replaced with a transdermal injection. Four CTVT-bearing dogs that received no treatment (NT) served as controls, and one dog in each vaccination group immunized with empty vector served as a vector control. Tumor growth was notably inhibited only in the G1 dogs, in which the vaccination program triggered tumor regression much sooner (beginning in week 9) than in the G2 (week 18), G3 (week 12) and NT (week 14) dogs. The CD4+ subpopulation of tumor-infiltrating lymphocytes was significantly increased during tumor regression in the G1 dogs as compared with the G2 and NT dogs (56.77% vs. 23.56% and 22.73%, respectively) and was similar to that of G3. The tumor-specific cytotoxicity of peripheral blood mononuclear cells (PBMCs) in all dogs in the three vaccination groups was dramatically enhanced, and ELISpot assay indicated that canine HSP70-specific IFN-

碩士
國立清華大學
生命科學系
88
ABSTRACT Geldanamycin (GA) specifically binds to HSP90 and disrupts the interaction of HSP90 and its target proteins. The binding will lead to substrate protein dissociation from HSP90 and may affect their structures and functions. Herein, we showed that exposure of non-small lung cancer H460 cells to 0.5 mM GA leads to enhancement of the synthesis of the 70 kDa heat shock proteins (HSP70s). The induction of HSP70s by GA is concentration- and time-dependent and this process coincides to the accumulation of its mRNA. By using the specific probes for hsp70-1 and hsp70b, we found that HSP70-1 is abundantly involved in GA-induced HSP70s. Furthermore importantly, we demonstrated for the first time that the PKC pathway is significant in this process since it is inhibited by H-7 and H-8 but not affected by HA1043. Similar results can be found in the protein and mRNA level. These data lead us to conclude that PKC pathway plays a major role in the GA-induced HSP70-1 expression in H460 cells. By using the electrophoretic mobility shift assay (EMSA), we showed that proximal Heat shock element (HSE1) was responsible for the inducible expression of HSP70. We found that the nuclear extracts prepared from GA-treated cells exhibited a significant increase in binding activity toward Heat shock element. Moreover, this increase in binding activity toward the Heat shock element is reduced by H-7 and H-8 but not affected by HA1004. We concluded that the PKC pathway is the major pathway involved in GA-induced expression of HSP70-1 in H460 cells.

Proteínas de Choque Térmico são um grupo de proteínas que é induzido em resposta ao stress e situações lesivas. As várias proteínas que pertencem a este grupo estão divididas em famílias, de acordo com o seu peso molecular. Podem ser distinguidas a HSP72, HSP40, HSP60, HSP70, HSP90 e uma família adicional de grandes HSPs. Este é um grupo de proteínas que é constitutivamente expresso em células normais sendo responsável pela homeostase celular. É de facto o seu nível de expressão basal que sustenta os processos que ocorrem num ambiente intracelular saudável. Uma vez que as HSPs interagem com muitas outras proteínas, elas são referidas como “ajudantes moleculares”, enquanto as proteínas que elas ajudam recebem o nome de “proteínas cliente”. Contudo, também existem HSPs que ajudam outras HSPs. Nesta situação elas são chamadas de “co-ajudantes”, como no caso da HSP40 com a HSP70 ou HSP90. Apesar das HSPs terem sido primeiramente descobertas após um choque térmico, atualmente sabe-se que podem ser induzidas por diversos outros estímulos. O seu desempenho em situações de stress é ajudar as células a restaurar o seu ambiente fisiológico, sendo fundamentais para a viabilidade e sobrevivência dos organismos. Células cancerígenas também tiram vantagem destes mecanismos. Um processo oncológico é uma situação lesiva que culmina na activação das HSPs, que contribuem para os piores prognósticos nas doenças neoplásicas. Uma abordagem terapêutica que surgiu com o estudo das HSPs no desenvolvimento tumoral foi a criação de moléculas que inibem as HSPs e as suas funções. Desta forma, a Oncologia Translacional tornou-se um campo muito importante, permitindo que nanoproteínas, como as HSPs, sejam usadas como potenciais alvos anti-cancerígenos. HSP90 é considerada o alvo terapêutico perfeito, entre todas as HSPs, visto que os seus inibidores demonstram os melhores resultados. Uma vez que estes fármacos estão ainda numa fase de experimentação, espécies de roedores e canídeos de laboratório têm sido usados em diversos ensaios clínicos de inibidores de HSPs. Geralmente as HSPs encontram-se excessivamente expressas na maioria dos tumores, tanto em humanos como em animais. Considerando as semelhanças epidemiológicas, morfológicas e biológicas que os seres humanos partilham com os animais de companhia, espera-se que os tumores espontâneos destas espécies sejam o melhor modelo animal para estudar a doença em pacientes humanos.
Heat Shock Proteins are a group of proteins that is induced in response to stress and harmful situations. The several proteins that belong to this group are further divided into families, based on their molecular weight, distinguished by the terms HSP27, HSP40, HSP60, HSP70, HSP90 and an additional family of large HSPs. This is a group of proteins that is constitutively expressed in normal cells, being responsible for the maintenance of the celular homeostasis. It is indeed their basal level of expression that sustains the pathways that occur in healthy intracellular environments. Since HSPs interact with multiple other proteins, they are referred to as “molecular chaperones”, while the proteins that they help receive the name of “client proteins”. However, there are some HSPs that assist other HSPs. In this situation they are called “co-chaperones”, as in the case of HSP40 with both HSP70 and HSP90. Although HSPs were first discovered after a heat shock, nowadays it is known that they can be induced by a variety of other stimuli. The work they do in stressful situations is to help the cells to restore their physiological environments, being fundamental for viability and survival of organisms. Cancer cells also take advantage of these mechanisms. An oncologic process is a harmful situation, which culminates with the activation of HSPs, allowing cancer cells to survive to otherwise lethal conditions. They also contribute to the worst prognosis of the neoplastic disease. A therapeutic approach that was invented by studying the role of HSPs in tumorigenesis was the creation of molecules that inhibit HSPs and their functions. This way, Translational Oncology has become a very important field, allowing nanoproteins, like HSPs, to be used as potencial anticancer targets. HSP90 is considered the perfect drug target among all HSPs, with its inhibitors being the ones with the most promising results. Because these drugs are still in the experimental phase, laboratory rodent and canine species have been used in several clinical trials of HSPs inhibitors. Generally, HSPs are overexpressed in the majority of cancers, in both humans and animals. Given the epidemiological, morphologic and biological similarities that human beings share with companian animals, it was concluded that spontaneous tumors in this species provide the best model to study the disease in humans.

碩士
國立清華大學
生命科學系
89
The heat-shock proteins (HSPs) or chaperones can protect and stabilize the folding and functions of normal proteins in cells. Chaperones are generally composed of large, evolutionarily conserved families within which the members are highly related, yet might differ in subcellular localization or gene regulation. We used semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) to analyze the expression profile of HSP70 isoforms in human non-small cell lung cancer H460 cells under CdCl2 stress. The expressions of hsp70-1, hsp70-2, hsp70-hom, hsp70-3, grp78, hsp70B, and hsp70B’ were monitored by amplifying from a conserved, common primer set and then differentiated by isoform-specific restriction enzymes. It is known that CdCl2 could elicit different cellular responses and gene expressions in a concentration-dependent and treatment time-dependent manner. Consistent with which, in our system, the expression level or pattern of chaperones varied low concentration (5 uM) and high concentration (40 uM to 60 uM) of CdCl2. We compared the changes of the newly synthesized chaperones by metabolic labeling with [35S]methionine and of the total chaperones by Western blotting analysis in CdCl2 treatment. We further analyzed changes of gene expression of individual isoforms by RT-PCR. From our study, this novel method can be performed to differentiate the members of HSP70 family under stress. Here, it has been found that the main members of HSP70 family in CdCl2-treated cells include hsp70-1, hsp70-2, and grp78. Furthermore, other four members of HSP70 family (hsp70-hom, hsp70-3, hsp70B, and hsp70B’) can be analyzed by this novel method. These four members don’t take part in stress conditions with CdCl2-treated. 二、英文附錄 1. ABSTRACT....................................2 2. INTRODUCTION................................3 3. EXPERIMENTAL PROCEDURES.....................7 4. RESULTS....................................11 5. DISCUSSION.................................18 6. REFERENCES.................................23 7. FIGURE LEGENDS.............................30 8. FIGURES....................................34 9. TABLES.....................................45

Understanding the cellular response thermal stress is important for improving thermoablative treatments of cancer. Cells generally respond to thermal stress by expressing heat shock proteins, or undergoing cell death by apoptosis or necrosis. Most of our detailed knowledge regarding these cellular phenomena has been gathered in vitro in two dimensional (2D) environments. Yet, little is known about how prostate cancer cells respond to thermal stress in a more physiologically relevant three dimensional (3D) environment. Several approaches were used to investigate this question, all of which focused on controlled heating of cells in both two dimensional (2D) and 3D culture. Tools and assays were developed to investigate cellular response to thermal stress in 2D and 3D environments. A computer-controlled heating apparatus was constructed to heat cell cultures to precise temperatures and durations. Three dimensional growth environments were produced using Matrigel, a commercially available extracellular matrix (ecm) mixture. Transcriptional expression of heat shock protein 70 (HSP70) was measured using a green fluorescent protein (GFP) reporter gene under the control of an HSP promoter. Apoptosis, necrosis and HSP70 transcription was measured using flow cytometry analysis. Quantitative polymerase chain reaction (qPCR) and microscopy revealed that transmembrane targets may be involved in the mechanism of the effect which 3D culture has on the cellular response to heat shock. The results herein demonstrate that the 3D growth environment, may be protective to the cell in that the percentage of cells that undergo apoptosis or necrosis when exposed to heat shock are reduced. Furthermore, HSP70 expression is enhanced in 3D culture at a specific thermal dose and integrins and heat shock proteins may be part of the mechanism by which the ecm exerts its protective effect against thermal stress.
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