Analysis of the interaction of Hsp90 with the extracellular matrix protein fibronectin (FN)
- Authors: Hunter, Morgan Campbell
- Date: 2014
- Subjects: Heat shock proteins , Fibronectins , Extracellular matrix proteins , Breast -- Cancer
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4170 , http://hdl.handle.net/10962/d1020960
- Description: Mounting evidence suggests that Hsp90 is present and functionally active in the extracellular space. The biological function of extracellular Hsp90 (eHsp90) remains relatively uncharacterized compared to that of intracellular Hsp90. eHsp90 has been shown to interact with a finite number of extracellular proteins, however, despite the identification of eHsp90 interacting proteins, the function of eHsp90 in these complexes is unknown. Several reports suggest a role for eHsp90α in cell migration and invasion. Reported targets for eHsp90 stimulated cell migration include MMPs, LRP-1, tyrosine kinase receptors and possible others unidentified. Limited studies report a role for eHsp90β. Recently, Hsp90α and Hsp90β were isolated in a complex containing fibronectin (FN) on the surface of MDA-MB-231 breast cancer cells. Herein, we report direct binding of Hsp90α and Hsp90β to FN using a solid phase binding assay and surface plasmon resonance (SPR) spectroscopy. SPR spectroscopy showed that Hsp90β bound the 70 kDa amino-terminal fragment of FN (FN70), but that binding of FN to Hsp90β was not limited to FN70. Confocal microscopy showed regions of colocalization of Hsp90 with extracellular FN matrix fibrils in Hs578T breast cancer cell lines. Treatment of Hs578T breast cancer cells with novobiocin (an Hsp90 inhibitor) and an LRP-1 blocking antibody resulted in a loss of FN matrix and FN endocytosis (novobiocin treated). Addition of exogenous Hsp90β was able to recover such effect after both treatments. FN was shown to colocalize with intracellular LRP-1 in novobiocin treated Hs578T cells. Immunoprecipitation of an LRP-1 containing complex showed the presence of Hsp90 and 70 and 120+ kDa FN fragments. Treatment of Hs578T cells with novobiocin increased the level of FN120+ bound in LRP-1 immunoprecipitate. Exogenous Hsp90β decreased the level of low and high molecular weight FN fragments in a complex with LRP-1, despite the fact that higher levels of lower molecular weight FN fragments were detected in this cell lysate compared to the other treatments. We report FN as a novel interacting protein of eHsp90. Taken together, we provide evidence for a direct role of eHsp90β in FN matrix remodeling. We suggest that Hsp90 plays a direct role in FN matrix dynamics through interaction with FN and LRP-1. The identification of FN as a novel interacting protein of eHsp90 suggests a role for Hsp90 in FN matrix remodeling, which is important for a number of fundamental cellular processes including cell migration and metastasis.
- Full Text:
- Authors: Hunter, Morgan Campbell
- Date: 2014
- Subjects: Heat shock proteins , Fibronectins , Extracellular matrix proteins , Breast -- Cancer
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4170 , http://hdl.handle.net/10962/d1020960
- Description: Mounting evidence suggests that Hsp90 is present and functionally active in the extracellular space. The biological function of extracellular Hsp90 (eHsp90) remains relatively uncharacterized compared to that of intracellular Hsp90. eHsp90 has been shown to interact with a finite number of extracellular proteins, however, despite the identification of eHsp90 interacting proteins, the function of eHsp90 in these complexes is unknown. Several reports suggest a role for eHsp90α in cell migration and invasion. Reported targets for eHsp90 stimulated cell migration include MMPs, LRP-1, tyrosine kinase receptors and possible others unidentified. Limited studies report a role for eHsp90β. Recently, Hsp90α and Hsp90β were isolated in a complex containing fibronectin (FN) on the surface of MDA-MB-231 breast cancer cells. Herein, we report direct binding of Hsp90α and Hsp90β to FN using a solid phase binding assay and surface plasmon resonance (SPR) spectroscopy. SPR spectroscopy showed that Hsp90β bound the 70 kDa amino-terminal fragment of FN (FN70), but that binding of FN to Hsp90β was not limited to FN70. Confocal microscopy showed regions of colocalization of Hsp90 with extracellular FN matrix fibrils in Hs578T breast cancer cell lines. Treatment of Hs578T breast cancer cells with novobiocin (an Hsp90 inhibitor) and an LRP-1 blocking antibody resulted in a loss of FN matrix and FN endocytosis (novobiocin treated). Addition of exogenous Hsp90β was able to recover such effect after both treatments. FN was shown to colocalize with intracellular LRP-1 in novobiocin treated Hs578T cells. Immunoprecipitation of an LRP-1 containing complex showed the presence of Hsp90 and 70 and 120+ kDa FN fragments. Treatment of Hs578T cells with novobiocin increased the level of FN120+ bound in LRP-1 immunoprecipitate. Exogenous Hsp90β decreased the level of low and high molecular weight FN fragments in a complex with LRP-1, despite the fact that higher levels of lower molecular weight FN fragments were detected in this cell lysate compared to the other treatments. We report FN as a novel interacting protein of eHsp90. Taken together, we provide evidence for a direct role of eHsp90β in FN matrix remodeling. We suggest that Hsp90 plays a direct role in FN matrix dynamics through interaction with FN and LRP-1. The identification of FN as a novel interacting protein of eHsp90 suggests a role for Hsp90 in FN matrix remodeling, which is important for a number of fundamental cellular processes including cell migration and metastasis.
- Full Text:
Molecular chaperone expression and function in breast cancer and breast cancer stem cells
- Authors: Sterrenberg, Jason Neville
- Date: 2012
- Subjects: Breast -- Cancer , Stem cells , Cancer cells
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4141 , http://hdl.handle.net/10962/d1016238
- Description: The Cancer Stem Cell (CSC) theory suggests that cancers arise from and are maintained by a subpopulation of cancer cells with stem cell properties. Molecular chaperones are key components of cellular regulation. The overexpression of chaperones has become synonymous with cancer cells with chaperones being recognized as bona fide anti-cancer drug targets. Although chaperone activity has been characterized in cancer cells, very little is known about the cellular functions of chaperones in cancer stem cells. We set out to compare the expression of selected molecular chaperones in non-stem cancer cell and cancer stem cell enriched populations isolated from breast cancer lines, in order to identify chaperones differentially expressed between the two populations for further biological characterization. In order to isolate breast cancer stem cells from the MCF-7 and MDA-MB-231 breast cancer cell lines, three cancer stem cell isolation and identification techniques were utilized based on (1) cell surface marker expression (CD44+/CD24- and CD44+/CD24-/EpCAM+ phenotypes), (2) aldehyde dehydrogenase enzyme activity (ALDHHi) and (3) ability to grow in anchorage-independent conditions. The MDA-MB-231 and MCF-7 breast cancer cell lines displayed CD44+/CD24- cell populations with the MCF-7 cell line additionally displaying a large CD44+/CD24-/EpCAM+ population. Although both cell lines showed similar ALDHHi populations, they differed substantially with respect to anchorage-independent growth. MCF-7 cells were able to form anchorage-independent colonies while the MDA-MB-231 cell line was not. Anchorage-independent MCF-7 cells showed enrichment in CD44+/CD24- and CD44+/CD24-/EpCAM+ cells compared to adherent MCF-7 cells, and were selected for gene expression studies. Gene expression studies identified 22 genes as being down-regulated at the mRNA level in the anchorage-independent MCF-7 cells, while only 2 genes (BAG1 and DNAJC12) were up-regulated. The down-regulation of selected chaperones in anchorage independent MCF-7 cells was confirmed at the protein level for selected chaperones, including DNAJB6, a type II DNAJ protein shown to be involved in the regulation of Wnt signaling. In order to characterize the effect of DNAJB6 expression on BCSCs we developed a pCMV mammalian expression plasmid for both DNAJB6 isoforms (DNAJB6L and DNAJB6S). We successfully constructed mutants of the conserved histidine-proline-aspartic acid (HPD) motif of the J domain of DNAJB6S and DNAJB6L. These constructs will allow the analysis of the role of DNAJB6 in cancer stem cell function. To the best of our knowledge, this is the first report to focus on the comparative expression of molecular chaperones in normal and cancer stem cell enriched breast cancer populations.
- Full Text:
- Authors: Sterrenberg, Jason Neville
- Date: 2012
- Subjects: Breast -- Cancer , Stem cells , Cancer cells
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4141 , http://hdl.handle.net/10962/d1016238
- Description: The Cancer Stem Cell (CSC) theory suggests that cancers arise from and are maintained by a subpopulation of cancer cells with stem cell properties. Molecular chaperones are key components of cellular regulation. The overexpression of chaperones has become synonymous with cancer cells with chaperones being recognized as bona fide anti-cancer drug targets. Although chaperone activity has been characterized in cancer cells, very little is known about the cellular functions of chaperones in cancer stem cells. We set out to compare the expression of selected molecular chaperones in non-stem cancer cell and cancer stem cell enriched populations isolated from breast cancer lines, in order to identify chaperones differentially expressed between the two populations for further biological characterization. In order to isolate breast cancer stem cells from the MCF-7 and MDA-MB-231 breast cancer cell lines, three cancer stem cell isolation and identification techniques were utilized based on (1) cell surface marker expression (CD44+/CD24- and CD44+/CD24-/EpCAM+ phenotypes), (2) aldehyde dehydrogenase enzyme activity (ALDHHi) and (3) ability to grow in anchorage-independent conditions. The MDA-MB-231 and MCF-7 breast cancer cell lines displayed CD44+/CD24- cell populations with the MCF-7 cell line additionally displaying a large CD44+/CD24-/EpCAM+ population. Although both cell lines showed similar ALDHHi populations, they differed substantially with respect to anchorage-independent growth. MCF-7 cells were able to form anchorage-independent colonies while the MDA-MB-231 cell line was not. Anchorage-independent MCF-7 cells showed enrichment in CD44+/CD24- and CD44+/CD24-/EpCAM+ cells compared to adherent MCF-7 cells, and were selected for gene expression studies. Gene expression studies identified 22 genes as being down-regulated at the mRNA level in the anchorage-independent MCF-7 cells, while only 2 genes (BAG1 and DNAJC12) were up-regulated. The down-regulation of selected chaperones in anchorage independent MCF-7 cells was confirmed at the protein level for selected chaperones, including DNAJB6, a type II DNAJ protein shown to be involved in the regulation of Wnt signaling. In order to characterize the effect of DNAJB6 expression on BCSCs we developed a pCMV mammalian expression plasmid for both DNAJB6 isoforms (DNAJB6L and DNAJB6S). We successfully constructed mutants of the conserved histidine-proline-aspartic acid (HPD) motif of the J domain of DNAJB6S and DNAJB6L. These constructs will allow the analysis of the role of DNAJB6 in cancer stem cell function. To the best of our knowledge, this is the first report to focus on the comparative expression of molecular chaperones in normal and cancer stem cell enriched breast cancer populations.
- Full Text:
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