A role for heat shock protein 90 (Hsp90) in fibronectin matrix dynamics
- Authors: O'Hagan, Kyle Leonard
- Date: 2013
- Subjects: Molecular chaperones , Heat shock proteins , Metastasis , Cancer -- Treatment
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4157 , http://hdl.handle.net/10962/d1018260
- Description: To date, a significant portion of research has been devoted to understanding the biological role of the molecular chaperone, heat shock protein 90 (Hsp90), in cancer development and metastasis. Studies have alluded to over 300 clients for intracellular Hsp90, many of which are involved in oncogenic signaling pathways, making Hsp90 a bone fide drug target with several inhibitors already in clinical trials. In recent years, a limited number of extracellular Hsp90 clients have been elucidated with roles in cancer cell migration and invasion. Examples of such clients include matrix metalloproteinase-2 (MMP-2), LRP-1/CD91 and HER-2. Inhibition of extracellular Hsp90 using cellimpermeable inhibitors has been shown to reduce cancer cell migration and metastasis by a hitherto undefined mechanism. Using surface biotinylation and an enzyme linked immunosorbent assay, we provided evidence to support that Hsp90 was found extracellularly in cancers of different origin, cell type and malignancy. Next, we isolated extracellular Hsp90-containing complexes from MDA-MB-231 breast cancer cells using a cell impermeable crosslinker followed by immunoprecipitation and identified by mass spectrometry that the extracellular matrix protein, fibronectin, co-precipitated with Hsp90β. This interaction between Hsp90β and fibronectin was confirmed using pull down assays and surface plasmon resonance spectroscopy with the purified proteins. The ability of exogenous Hsp90β to increase the insoluble fibronectin matrix in Hs578T breast cancer cells indicated a role for Hsp90 in fibronectin matrix stability or fibrillogenesis. Hsp90 knockdown by RNA interference or inhibition with the small molecule inhibitor, novobiocin, resulted in a dose and time-dependent reduction of the extracellular fibronectin matrix. Furthermore, novobiocin was shown to cause the internalization of a fluorescently-labeled exogenous fibronectin matrix incorporated into the extracellular matrix by Hs578T cells. This suggested endocytosis as a possible mechanism for fibronectin turnover. This was supported by the colocalization of fibronectin with key vesicular trafficking markers (Rab-5 and LAMP-1) in small, intracellular vesicles. Furthermore, treatment with the vesicular trafficking inhibitor, methyl-β-cyclodextrin, resulted in a dose-dependent recovery in the extracellular fibronectin matrix following treatment with novobiocin. Taken together, these data provided the first evidence to suggest fibronectin as a new client of Hsp90 and that Hsp90 was involved in regulating extracellular fibronectin matrix dynamics.
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- Authors: O'Hagan, Kyle Leonard
- Date: 2013
- Subjects: Molecular chaperones , Heat shock proteins , Metastasis , Cancer -- Treatment
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4157 , http://hdl.handle.net/10962/d1018260
- Description: To date, a significant portion of research has been devoted to understanding the biological role of the molecular chaperone, heat shock protein 90 (Hsp90), in cancer development and metastasis. Studies have alluded to over 300 clients for intracellular Hsp90, many of which are involved in oncogenic signaling pathways, making Hsp90 a bone fide drug target with several inhibitors already in clinical trials. In recent years, a limited number of extracellular Hsp90 clients have been elucidated with roles in cancer cell migration and invasion. Examples of such clients include matrix metalloproteinase-2 (MMP-2), LRP-1/CD91 and HER-2. Inhibition of extracellular Hsp90 using cellimpermeable inhibitors has been shown to reduce cancer cell migration and metastasis by a hitherto undefined mechanism. Using surface biotinylation and an enzyme linked immunosorbent assay, we provided evidence to support that Hsp90 was found extracellularly in cancers of different origin, cell type and malignancy. Next, we isolated extracellular Hsp90-containing complexes from MDA-MB-231 breast cancer cells using a cell impermeable crosslinker followed by immunoprecipitation and identified by mass spectrometry that the extracellular matrix protein, fibronectin, co-precipitated with Hsp90β. This interaction between Hsp90β and fibronectin was confirmed using pull down assays and surface plasmon resonance spectroscopy with the purified proteins. The ability of exogenous Hsp90β to increase the insoluble fibronectin matrix in Hs578T breast cancer cells indicated a role for Hsp90 in fibronectin matrix stability or fibrillogenesis. Hsp90 knockdown by RNA interference or inhibition with the small molecule inhibitor, novobiocin, resulted in a dose and time-dependent reduction of the extracellular fibronectin matrix. Furthermore, novobiocin was shown to cause the internalization of a fluorescently-labeled exogenous fibronectin matrix incorporated into the extracellular matrix by Hs578T cells. This suggested endocytosis as a possible mechanism for fibronectin turnover. This was supported by the colocalization of fibronectin with key vesicular trafficking markers (Rab-5 and LAMP-1) in small, intracellular vesicles. Furthermore, treatment with the vesicular trafficking inhibitor, methyl-β-cyclodextrin, resulted in a dose-dependent recovery in the extracellular fibronectin matrix following treatment with novobiocin. Taken together, these data provided the first evidence to suggest fibronectin as a new client of Hsp90 and that Hsp90 was involved in regulating extracellular fibronectin matrix dynamics.
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The screening and characterisation of compounds for modulators of heat shock protein (Hsp90) in a breast cancer cell model
- Authors: Moyo, Buhle
- Date: 2013 , 2013-07-18
- Subjects: Heat shock proteins Breast -- Cancer Breast -- Cancer -- Chemotherapy Breast -- Cancer -- Treatment Cancer cells Naphthoquinone PQQ (Biochemistry)
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4060 , http://hdl.handle.net/10962/d1004129
- Description: Breast cancer is a leading cause of cancer death in Africa. Hsp90 has been identified as a target for anti-cancer treatments as its inhibition results in the disruption and ubiquitin–proteasome degradation of activated oncoproteins. Currently, there are no US Food and Drug Administration approved Hsp90 inhibitor drugs and existing Hsp90 inhibitors such as geldanamycin and novobiocin are hepatotoxic and display a low affinity for Hsp90, respectively. Therefore, there is a need for the development of Hsp90 inhibitors with improved inhibitory properties. In this study twelve natural compounds bearing a quinone nucleus were screened and characterised for the modulation of Hsp90. The compounds analysed formed three series; the sargaquinoic acid (SQA), naphthoquinone, and pyrroloiminoquinone alkaloid series. Certain compounds exhibited half maximal inhibitory concentrations of between 3.32 μM and 12.4 μM, while others showed no antiproliferative activity at concentrations of up to 500 μM in the MDA-MB-231 breast adenocarcinoma cell line. Immunofluorescence and Western analyses indicated that the modulation of Hsp90 and partner proteins by SQA was more similar to that of novobiocin. Isothermal titration calorimetry analyses suggested that SQA interacted with Hsp90β with a low affinity, and saturation-transfer difference nuclear magnetic resonance confirmed that this interaction with Hsp90β occurred through the methyl moiety bound to 1, 4 benzoquinone of SQA. Pulldown assays indicated SQA disrupted the association between Hsp90 and Hop dose-dependently, more similarly to novobiocin. Immunofluorescence and Western analyses performed on naphthoquinone and pyrroloiminoquinone alkaloid compounds indicated modulation of Hsp90 and Hsp90 partner proteins by the compounds. Naphthoquinone compounds were prioritised for analysis for binding to Hsp90β over the pyrroloiminoquinone alkaloid compounds. Lapachol interacted with Hsp90β with a low affinity however; this interaction was thought to be too weak to disrupt the association of Hsp90 and Hop. The remaining naphthoquinone compounds showed no interaction with Hsp90β, thus allowing the determination of a preliminary structure-activity relationship for these compounds. To the best of our knowledge, this is the first study to describe a systematic subcellular analysis of the effects of geldanamycin and novobiocin in comparison to sargaquinoic acid and compounds of the naphthoquinone and pyrroloquinoline scaffold on Hsp90 and its partner proteins. , Microsoft� Word 2010 , Adobe Acrobat 9.54 Paper Capture Plug-in
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- Authors: Moyo, Buhle
- Date: 2013 , 2013-07-18
- Subjects: Heat shock proteins Breast -- Cancer Breast -- Cancer -- Chemotherapy Breast -- Cancer -- Treatment Cancer cells Naphthoquinone PQQ (Biochemistry)
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4060 , http://hdl.handle.net/10962/d1004129
- Description: Breast cancer is a leading cause of cancer death in Africa. Hsp90 has been identified as a target for anti-cancer treatments as its inhibition results in the disruption and ubiquitin–proteasome degradation of activated oncoproteins. Currently, there are no US Food and Drug Administration approved Hsp90 inhibitor drugs and existing Hsp90 inhibitors such as geldanamycin and novobiocin are hepatotoxic and display a low affinity for Hsp90, respectively. Therefore, there is a need for the development of Hsp90 inhibitors with improved inhibitory properties. In this study twelve natural compounds bearing a quinone nucleus were screened and characterised for the modulation of Hsp90. The compounds analysed formed three series; the sargaquinoic acid (SQA), naphthoquinone, and pyrroloiminoquinone alkaloid series. Certain compounds exhibited half maximal inhibitory concentrations of between 3.32 μM and 12.4 μM, while others showed no antiproliferative activity at concentrations of up to 500 μM in the MDA-MB-231 breast adenocarcinoma cell line. Immunofluorescence and Western analyses indicated that the modulation of Hsp90 and partner proteins by SQA was more similar to that of novobiocin. Isothermal titration calorimetry analyses suggested that SQA interacted with Hsp90β with a low affinity, and saturation-transfer difference nuclear magnetic resonance confirmed that this interaction with Hsp90β occurred through the methyl moiety bound to 1, 4 benzoquinone of SQA. Pulldown assays indicated SQA disrupted the association between Hsp90 and Hop dose-dependently, more similarly to novobiocin. Immunofluorescence and Western analyses performed on naphthoquinone and pyrroloiminoquinone alkaloid compounds indicated modulation of Hsp90 and Hsp90 partner proteins by the compounds. Naphthoquinone compounds were prioritised for analysis for binding to Hsp90β over the pyrroloiminoquinone alkaloid compounds. Lapachol interacted with Hsp90β with a low affinity however; this interaction was thought to be too weak to disrupt the association of Hsp90 and Hop. The remaining naphthoquinone compounds showed no interaction with Hsp90β, thus allowing the determination of a preliminary structure-activity relationship for these compounds. To the best of our knowledge, this is the first study to describe a systematic subcellular analysis of the effects of geldanamycin and novobiocin in comparison to sargaquinoic acid and compounds of the naphthoquinone and pyrroloquinoline scaffold on Hsp90 and its partner proteins. , Microsoft� Word 2010 , Adobe Acrobat 9.54 Paper Capture Plug-in
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