<p>Despite its ubiquitous abundance, Hsp90 inhibitors have shown promise in anti-cancer clinical trials, suggesting that Hsp90 inhibitors selectively target tumor cells while exhibiting minimal effects in normal cells. Extracellular expression of heat shock protein 90 (eHsp90) by tumor cells is strongly correlated with malignancy. Development of small molecule probes that can specifically detect eHsp90 in vivo may therefore have utility in the early detection of malignancy. We synthesized a fluorescent cell impermeable Hsp90 inhibitor, HS-131, to target eHsp90 in vivo. HS-131 was characterized biochemically to ensure specificity for eHsp90, and an inactive analog was also synthesized to be used as an in vivo control. </p><p>Through confocal microscopy, eHsp90 can be visualized with cell impermeable, fluorophore-tagged Hsp90 inhibitors. High resolution confocal and real time lattice light sheet microscopy showed that probe-bound eHsp90 accumulates in punctate structures on the plasma membrane of breast tumor cells and is subsequently actively internalized. This internalization occurs in the presence and absence of inhibitors. The extent of internalization correlates with tumor cell aggressiveness, and this process can be induced in benign cells by over-expressing p110HER2, leading to malignant transformation of these cells. Internalization of eHsp90 is also increased after inhibition of Hsp70, suggesting that overcompensation of the heat shock response can also upregulate the eHsp90 trafficking mechanism. Whole body 3D cryo fluorescence imaging and histology of flank and spontaneous tumor-bearing mice strongly suggests that eHsp90 expression is a unique phenomenon in vivo. </p><p>Taken together, these results suggest that active and differential internalization of eHsp90 in aggressive cancer cells contributes to the selectivity observed upon Hsp90 inhibitor treatment and may provide a novel metastatic biomarker for solid tumors and may lead to the development of a tumor-specific drug delivery system.</p> / Dissertation

Doctor Pharmaceuticae - DPharm / Natural products have been an important source of drugs and novel lead compounds in drug discovery. Their unique scaffolds have led to the synthesis of derivatives that continue to give rise to medicinally relevant agents. Thus, natural product-inspired drugs represent a significant proportion of drugs in the market and with several more in development. Cancer is among the leading public health problems and a prominent cause of death globally. Chemotherapy has been important in the management of this disease even though side effects that arise due to lack of selectivity is still an issue.

No description available.

No description available.

No description available.

No description available.

The heat shock response (HSR) is a robust cellular reaction to mitigate protein damage from heat and other challenges to the proteome. This protective molecular program in humans is controlled by heat shock transcription factor 1 (HSF1). Activation of HSF1 leads to the induction of an array of cytoprotective genes, many of which code for chaperones. These chaperones, known as heat shock proteins (HSPs), are responsible for maintaining the functional integrity of the proteome. HSPs achieve this by promoting proper folding and assembly of nascent proteins, refolding denatured proteins, and processing for degradation proteins and aggregates which cannot be returned to a functional conformation. The powerful ability of the heat shock response to promote cell survival makes its master regulator, HSF1, an important point of research. To garner a better understanding of HSF1, we reviewed the role of the highly dynamic HSF1 protein structure and investigated how HSF1 affects cancer cell behavior and drug response. Cancers can be characterized in part by abhorrent replication, self-sufficient growth signaling, invasion, and evasion of apoptosis. HSF1 has been found to promote proliferation, invasion, and drug resistance in several types of cancer; including lung and ovarian cancer. Ovarian cancer has elevated levels of HSF1, but the role of HSF1 in ovarian cancer behavior had not been previously examined. Researching the role of HSF1 in ovarian cancer is merited, because treatment outcomes are poor due to the high frequency of late stage detection and drug resistance. We hypothesized that HSF1 is important in the malignant growth and drug resistance of ovarian cancer. We have created ovarian cancer cell lines with inducible knockdown of HSF1 to investigate how HSF1 contributes to the behavior of ovarian cancer. This allowed us to examine the behavior of cells in the absence HSF1. Both 2D and 3D spheroid tissue culture models were used to study how HSF1 contributes to the growth and invasion of ovarian cancer cells after treatment with the transforming growth factor β (TGFβ) cytokine. Additionally, we studied how HSF1 reduction modulates the response to multiple therapeutic drugs. Our research shows that HSF1 induces epithelial-mesenchymal transition (EMT) in a 3D growth model. Our work also demonstrates that reduction of HSF1 sensitizes ovarian cancer cells to multiple drugs.

HIF1α et HIF2α sont des protéines largement impliquées dans le développement de pathologies posant des problèmes majeurs de santé publique, comme le cancer. Leur activité, qui est régulée prioritairement par leur stabilité via le système ubiquitine-protéasome, coordonne de nombreux processus cellulaires susceptibles de favoriser le développement de ces maladies. Un enjeu récent de la recherche thérapeutique est d’identifier des partenaires protéiques pouvant réguler les protéines HIFα, afin de mettre au point des thérapies ciblées. Les protéines de choc thermique (HSPs) sont une classe de protéines dont une des fonctions essentielles est de réguler l’homéostasie protéique dans la cellule, en interagissant avec le protéasome. Certaines d’entre elles, HSP27 et HSP90, ont la faculté de pouvoir réguler spécifiquement la stabilité de nombreuses protéines souvent elles-mêmes impliquées dans l’apparition de ces pathologies. L’objectif de ce travail était de savoir si ces deux HSPs peuvent contrôler la stabilité de la protéine HIF2α. Nos résultats suggèrent qu’HSP27 pourrait stimuler la dégradation de HIF2α en favorisant son ubiquitination. Ce résultat est surprenant, en raison du rôle connu d’HSP27 dans la progression tumorale. Il est donc nécessaire de le confirmer et d’en préciser les processus biologiques sous-jacents. D’autre part, nos autres résultats semblent confirmer qu’HIF2α est une protéine cliente d’HSP90. De plus, nous montrons pour la première fois que l’inhibition d’HSP90 par le 17-DMAG diminue la production de VEGF dépendante de HIF2α. Des travaux récents suggèrent qu’HIF2α a un rôle prédominant dans la progression tumorale, et peut constituer une cible globale de choix dans plusieurs types de cancer. Il conviendrait d’évaluer la capacité des inhibiteurs d’HSP90 à supprimer des fonctions de HIF2α nouvellement décrites, comme son rôle dans la maintenance des cellules souches cancéreuses. / Both HIF1α and HIF2α proteins are highly involved in the development of pathologies, such as cancers, which are prime public health issues. These proteins are primarily controlled at the protein level by ubiquitin-dependant degradation, and regulate numerous cellular processes which are likely to favor the development of these diseases. A recent issue in therapeutic research is to identify partners that might regulate the expression and the activity of the HIFα proteins, with the aim to elaborate targeted therapies. Heat shock proteins (HSPs) form a family of proteins whose main function is to regulate protein homeostasis in cells, which they achieve through interaction with the ubiquitin-proteasome pathway. HSP27 and HSP90 are able to specifically control the stability of certain client proteins involved in those pathologies. In the present work, we sought to determine whether these HSPs could regulate the expression of the HIF2α protein. Our results suggest that HSP27 may induce ubiquitin and proteasome-dependant degradation of HIF2α, which is quite intriguing given the well-known role of HSP27 in tumor promotion. This needs to be confirmed and the underlying biological significance of such a regulation remains to be defined. Our results also may confirm that HIF2α is an HSP90 client protein. Moreover, we show for the first time that inhibition of HSP90 by 17-DMAG decreases the HIF2α-dependant VEGF production. Recent studies emphasize HIF2α as a major promoter of tumor progression, and suggest that HIF2α may constitute an attractive global target in several cancer types. Therefore, the ability of HSP90 inhibitors to disrupt newly described HIF2α functions, such as cancer stem cell maintenance, should be evaluated.