Establishment of human OCT4 as a putative HSP90 client protein: a case for HSP90 chaperoning pluripotency
- Authors: Sterrenberg, Jason Neville
- Date: 2015
- Subjects: Induced pluripotent stem cells , Heat shock proteins , Stem cells , Transcription factors , Molecular chaperones
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/194010 , vital:45415 , 10.21504/10962/194010
- Description: The therapeutic potential of stem cells is already being harnessed in clinical trails. Of even greater therapeutic potential has been the discovery of mechanisms to reprogram differentiated cells into a pluripotent stem cell-like state known as induced pluripotent stem cells (iPSCs). Stem cell nature is governed and maintained by a hierarchy of transcription factors, the apex of which is OCT4. Although much research has elucidated the transcriptional regulation of OCT4, OCT4 regulated gene expression profiles and OCT4 transcriptional activation mechanisms in both stem cell biology and cellular reprogramming to iPSCs, the fundamental biochemistry surrounding the OCT4 transcription factor remains largely unknown. In order to analyze the biochemical relationship between HSP90 and human OCT4 we developed an exogenous active human OCT4 expression model with human OCT4 under transcriptional control of a constitutive promoter. We identified the direct interaction between HSP90 and human OCT4 despite the fact that the proteins predominantly display differential subcellular localizations. We show that HSP90 inhibition resulted in degradation of human OCT4 via the ubiquitin proteasome degradation pathway. As human OCT4 and HSP90 did not interact in the nucleus, we suggest that HSP90 functions in the cytoplasmic stabilization of human OCT4. Our analysis suggests HSP90 inhibition inhibits the transcriptional activity of human OCT4 dimers without affecting monomeric OCT4 activity. Additionally our data suggests that the HSP90 and human OCT4 complex is modulated by phosphorylation events either promoting or abrogating the interaction between HSP90 and human OCT4. Our data suggest that human OCT4 displays the characteristics describing HSP90 client proteins, therefore we identify human OCT4 as a putative HSP90 client protein. The regulation of the transcription factor OCT4 by HSP90 provides fundamental insights into the complex biochemistry of stem cell biology. This may also be suggestive that HSP90 not only regulates stem cell biology by maintaining routine cellular homeostasis but additionally through the direct regulation of pluripotency factors. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2015
- Full Text:
- Date Issued: 2015
- Authors: Sterrenberg, Jason Neville
- Date: 2015
- Subjects: Induced pluripotent stem cells , Heat shock proteins , Stem cells , Transcription factors , Molecular chaperones
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/194010 , vital:45415 , 10.21504/10962/194010
- Description: The therapeutic potential of stem cells is already being harnessed in clinical trails. Of even greater therapeutic potential has been the discovery of mechanisms to reprogram differentiated cells into a pluripotent stem cell-like state known as induced pluripotent stem cells (iPSCs). Stem cell nature is governed and maintained by a hierarchy of transcription factors, the apex of which is OCT4. Although much research has elucidated the transcriptional regulation of OCT4, OCT4 regulated gene expression profiles and OCT4 transcriptional activation mechanisms in both stem cell biology and cellular reprogramming to iPSCs, the fundamental biochemistry surrounding the OCT4 transcription factor remains largely unknown. In order to analyze the biochemical relationship between HSP90 and human OCT4 we developed an exogenous active human OCT4 expression model with human OCT4 under transcriptional control of a constitutive promoter. We identified the direct interaction between HSP90 and human OCT4 despite the fact that the proteins predominantly display differential subcellular localizations. We show that HSP90 inhibition resulted in degradation of human OCT4 via the ubiquitin proteasome degradation pathway. As human OCT4 and HSP90 did not interact in the nucleus, we suggest that HSP90 functions in the cytoplasmic stabilization of human OCT4. Our analysis suggests HSP90 inhibition inhibits the transcriptional activity of human OCT4 dimers without affecting monomeric OCT4 activity. Additionally our data suggests that the HSP90 and human OCT4 complex is modulated by phosphorylation events either promoting or abrogating the interaction between HSP90 and human OCT4. Our data suggest that human OCT4 displays the characteristics describing HSP90 client proteins, therefore we identify human OCT4 as a putative HSP90 client protein. The regulation of the transcription factor OCT4 by HSP90 provides fundamental insights into the complex biochemistry of stem cell biology. This may also be suggestive that HSP90 not only regulates stem cell biology by maintaining routine cellular homeostasis but additionally through the direct regulation of pluripotency factors. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2015
- Full Text:
- Date Issued: 2015
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:
- Date Issued: 2012
- 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:
- Date Issued: 2012
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