Author: Maressa Anne Bruhn
Bruhn, Maressa Anne, 2012 The role of serum and glucocorticoid inducible kinase 3 in the regulation of cell growth and malignant transformation, Flinders University, School of Biological Sciences
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The most well established downstream effector of phosphatidylinositol 3-kinase (PI3-K) signalling is the v-akt murine thymoma viral oncogene homolog/protein kinase B (AKT/PKB) kinase family, with many studies highlighting the critical importance of this family in normal cell physiology, including cell growth, proliferation and survival, in addition to disease states such as cancer. However, more recently AKT-independent PI3-K signalling pathways have been reported, including signalling via serum and glucocorticoid inducible kinase (SGK) 3, a member of the SGK family of serine/threonine kinases. The three SGK kinases (SGK1, -2, -3) share 54% structural homology with the AKT kinases in the catalytic domain, and have shown to be similarly activated in a PI3-K-dependent manner via 3-phosphoinositide-dependent kinase 1 (PDK1). Moreover, the SGK and AKT families share many of the same downstream targets including glycogen synthase kinase 3 beta (GSK3β), forkhead transcription factor 3a (FOXO3a), and Bcl-2 associated death promoter (BAD). The level of similarity existing between these two kinase families suggests possible functional redundancy, however studies using single isoform AKT and SGK knockout mice suggests isoform specific signalling. Furthermore distinct differences in cellular localisation between these kinases make it more likely that they each have important and specific roles. In addition to the recent studies demonstrating that SGK3 is likely to be an important factor involved in AKT-independent malignant cell transformation, earlier studies in our laboratory demonstrated that the SGK3 isoform revealed increased transcript expression in a panel of ovarian tumour cells compared with SGK1 and SGK2, making SGK3 an interesting candidate to further characterise in both normal cell physiology and tumourigenesis. The AKT family has been widely reported as a key downstream effector of PI3-K signalling to cell growth, thus this thesis firstly aimed to examine a possible role for SGK3 in cell growth and proliferation. Using multiple SGK3 gain-of-function epithelial and fibroblast cell lines, studies presented here revealed a strong role for SGK3 in signalling to components of the cell growth pathway, regulating macromolecular (RNA and protein) content, cell size, and regulating ribosomal-DNA (rDNA) transcription. Furthermore, using the mammalian target of rapamycin complex 1 (mTORC1) inhibitor rapamycin, these studies were also able to demonstrate that SGK3 is able to regulate cell growth in a largely mTORC1-dependent manner. Moreover, studies presented herein were also able to explore the importance of SGK3s reported endosomal localisation, demonstrating SGK3 functions optimally to influence cell growth when localised at the endosomal compartment. Following the identification of SGK3 as an important modulator of cell growth, the second aim was to investigate the influence of SGK3 on malignant cell transformation and tumourigenesis. These studies utilised multiple pre-tumourigenic genetically defined SGK3 gain-of-function cell lines to obtain functional readouts for a variety of well-established hallmarks of tumourigenesis, including anchorage-independent growth, cell migration and chemoresistance. Moreover, these studies incorporated the use of AKT gain-of-function cell lines in order to make comparisons between the ability of SGK3 and AKT to promote tumourigenic hallmarks. Results from these studies revealed both SGK3 and the AKT isoforms to promote anchorage-independent growth, in addition to a role for AKT in promoting cell migration, suggesting that in addition to the AKT family, SGK3 is also an important effector of malignant transformation. Finally, the third aim of this project was to extend functional studies to global gene expression analysis, in an attempt to further define mechanisms associated with SGK3 function, and identify possible novel associations existing between SGK3 and other factors. Global gene analysis was conducted using Affymetrix genechip microarrays of all SGK3 and AKT gain-of-function cell lines used for functional studies. Through the use of two different bioinformatic approaches, including the MetaCore (TM) platform by GeneGo, along with Gene Set Enrichment Analysis (GSEA) from the Broad Institute, all data was interrogated to determine novel associations with SGK3, in addition to determining possible mechanisms associated with readouts observed in earlier functional analyses. These studies revealed not only known associations with SGK3, but also novel associations including demonstrating a possible role for SGK3 in the immune response, in addition to possible involvement in lysophosphatidic acid (LPA) pathway signalling. Moreover, these gene expression studies enabled comparison of differential gene regulation between both SGK3 and the AKT isoforms. In summary, studies presented in this thesis demonstrate for the first time an important role for SGK3 in regulating cell growth via regulation of rDNA transcription, which is likely to be largely dependent on mTORC1. Furthermore, SGK3 also appears to play a critical role in the regulation of malignant transformation, which is consistent with recent reports demonstrating a role for SGK3 in AKT-independent PI3-K oncogenic signalling. Additionally, global gene expression studies allowed for the detection of novel connections with SGK3 including a role for SGK3 in the immune response. In summary this thesis furthers our understanding of the role of SGK3 in health and disease and provides an important platform that can be used as a basis for future investigations into SGK3 function.
Keywords: SGK3,cell growth,PI3K,cell transformation
Subject: Biological Sciences thesis
Thesis type: Doctor of Philosophy
School: School of Biological Sciences
Supervisor: Karen Sheppard, Ross Hannan, Catherine Abbott