Suppression and activation of lipid signalling in cancer : tumour suppressor PTEN and oncogenic sphingosine kinase

Haddadi, Nahal

Suppression and activation of lipid signalling in cancer : tumour suppressor PTEN and oncogenic sphingosine kinase

Haddadi, Nahal

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Publication Type:: Thesis
Issue Date:: 2019

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Field	Value	Language
dc.contributor.author	Haddadi, Nahal
dc.date.accessioned	2019-09-17T04:50:21Z
dc.date.available	2019-09-17T04:50:21Z
dc.date.issued	2019
dc.identifier.uri	http://hdl.handle.net/10453/135943
dc.description	University of Technology Sydney. Faculty of Science.	en_AU
dc.description.abstract	The PTEN tumour suppressor is the second most frequently mutated tumour suppressor gene in cancer. It is a lipid and protein phosphatase that negatively regulates the well-known pro-proliferative and anti-apoptotic phosphatidylinositol 3-kinase (PI3K)/AKT signalling pathway to modulate cell proliferation, cell cycle progression and cell survival. Sphingosine kinase 1 (SphK1), a known tumour promoter/oncogene, has been shown to activate the PI3K/Akt pathway to enhance resistance to apoptosis. The loss of function of PTEN and/or the overexpression of SphK1 contribute to tumorigenesis. This thesis describes the analysis of novel, cancer associated mutations of PTEN, to determine their effect(s) on wild type (WT) PTEN function, and also explores differential SphK1 isoform expression in cancers. Previous work in our laboratory described 10 novel somatic mutations of PTEN in primary colorectal tumours. To determine the functional consequences of these novel cancer-associated PTEN mutations, the WT PTEN and the series of PTEN mutants (K62R, Y65C, K125E, K125X, E150Q, D153N, D153Y, N323K and the C124S and G129E controls) were transiently transfected into: (A) PTEN null U87MG glioblastoma cells, and WT PTEN expressing (A) HCT116 colon cancer and (C) MCF7 breast cancer cells. Transfected cells were then assayed for cell proliferation, cell cycle phase distribution and AKT phosphorylation. In U87MG cells, 50% of the PTEN mutants (Y65C, K125E, E150Q and D153Y) exhibited statistically significant reductions in cell proliferation, but not to the level of that of WT PTEN. In both the HCT116 and MCF7 cell lines, 80% of the PTEN mutants (K62R, Y65C, K125E, K125X, E150Q, D153Y and N323K) displayed reduced cell proliferation rates but none produced reductions comparable with WT PTEN. Further, relative to WT PTEN, 75% of PTEN mutants (K62R, K125X, E150Q, D153N and N323K) possessed functional deficiency in cell cycle inhibitory capacity in the G2 phase in U87MG cells. In contrast, 90% of PTEN mutants (K62R, K125E, K125X, E150Q, D153N, D153Y and N323K) possessed functional deficiency in the cell cycle inhibitory capacity in either the G1 or G2 phase in HCT116 cells. In MCF7 cells, 100% and 60% (K62R, Y65C, D153N, D153Y and N323K) of the PTEN mutants had functional deficiency in cell cycle inhibitory capacity in either the G1 or G2 phase, respectively. The analyses of endogenous suppression of phosphorylation of AKT revealed that 40% of PTEN mutants (K125E, K125X and D153N) show deficiency in pAKT suppression in the U87MG cell line while 60% of the mutants showed such deficiency in the HCT116 cell line (K125X, E150Q, D153N, D153Y and N323K). All but one (K62R) of the PTEN mutants showed a deficiency in the ability to suppress the level of endogenous phosphorylated AKT in the MCF7 cells. Overall, the results of the functional assays showed that the somatic mutations of the PTEN gene alter PTEN tumour suppressor function. Expression of SphK1, a positive upstream regulator of the Akt pathway, is expressed as 2 major isoforms, SphK1-43kDa (SphK1a) and SphK1-51kDa (SphK1b), with similar SphK1 activity. However, to date, there is no literature on the expression of the two SphK1 isoforms in cell lines or human tissues. Profiling the expression of the two SphK isoforms in various cancer cell lines (n=24), primary cancer tissues (n=28) and paired adjacent tissues (n=28), demonstrated that the SphK1a isoform is expressed in all cell lines and tissues (both normal and cancer) studied, however, expression of SphK1b is cell and tissue specific, including breast, prostate and lung. Balancing signalling pathways and maintaining cellular homeostasis, as observed through the PTEN/SphK1 swinging pendulum is important and the study of these pathways is crucial in gaining further understanding the opposing regulatory mechanisms, which may be exploited for the future prevention and treatment of cancers.	en_AU
dc.format	Thesis (PhD)
dc.language.iso	en_AU	en_AU
dc.relation	https://opus.lib.uts.edu.au/bitstream/10453/135943/2/02whole.pdf
dc.rights	The author owns the copyright in this thesis including all reproduction and reuse rights for the work. The work may not be altered without the permission of the copyright owner. Attribution is essential when quoting or paraphrasing from this thesis.
dc.rights	au.edu.uts.lib/ppc
dc.rights	info:eu-repo/semantics/openAccess
dc.title	Suppression and activation of lipid signalling in cancer : tumour suppressor PTEN and oncogenic sphingosine kinase	en_AU
dc.type	Thesis	en_AU
utslib.copyright.status	open_access

Abstract:

The PTEN tumour suppressor is the second most frequently mutated tumour suppressor gene in cancer. It is a lipid and protein phosphatase that negatively regulates the well-known pro-proliferative and anti-apoptotic phosphatidylinositol 3-kinase (PI3K)/AKT signalling pathway to modulate cell proliferation, cell cycle progression and cell survival. Sphingosine kinase 1 (SphK1), a known tumour promoter/oncogene, has been shown to activate the PI3K/Akt pathway to enhance resistance to apoptosis. The loss of function of PTEN and/or the overexpression of SphK1 contribute to tumorigenesis. This thesis describes the analysis of novel, cancer associated mutations of PTEN, to determine their effect(s) on wild type (WT) PTEN function, and also explores differential SphK1 isoform expression in cancers. Previous work in our laboratory described 10 novel somatic mutations of PTEN in primary colorectal tumours. To determine the functional consequences of these novel cancer-associated PTEN mutations, the WT PTEN and the series of PTEN mutants (K62R, Y65C, K125E, K125X, E150Q, D153N, D153Y, N323K and the C124S and G129E controls) were transiently transfected into: (A) PTEN null U87MG glioblastoma cells, and WT PTEN expressing (A) HCT116 colon cancer and (C) MCF7 breast cancer cells. Transfected cells were then assayed for cell proliferation, cell cycle phase distribution and AKT phosphorylation. In U87MG cells, 50% of the PTEN mutants (Y65C, K125E, E150Q and D153Y) exhibited statistically significant reductions in cell proliferation, but not to the level of that of WT PTEN. In both the HCT116 and MCF7 cell lines, 80% of the PTEN mutants (K62R, Y65C, K125E, K125X, E150Q, D153Y and N323K) displayed reduced cell proliferation rates but none produced reductions comparable with WT PTEN. Further, relative to WT PTEN, 75% of PTEN mutants (K62R, K125X, E150Q, D153N and N323K) possessed functional deficiency in cell cycle inhibitory capacity in the G2 phase in U87MG cells. In contrast, 90% of PTEN mutants (K62R, K125E, K125X, E150Q, D153N, D153Y and N323K) possessed functional deficiency in the cell cycle inhibitory capacity in either the G1 or G2 phase in HCT116 cells. In MCF7 cells, 100% and 60% (K62R, Y65C, D153N, D153Y and N323K) of the PTEN mutants had functional deficiency in cell cycle inhibitory capacity in either the G1 or G2 phase, respectively. The analyses of endogenous suppression of phosphorylation of AKT revealed that 40% of PTEN mutants (K125E, K125X and D153N) show deficiency in pAKT suppression in the U87MG cell line while 60% of the mutants showed such deficiency in the HCT116 cell line (K125X, E150Q, D153N, D153Y and N323K). All but one (K62R) of the PTEN mutants showed a deficiency in the ability to suppress the level of endogenous phosphorylated AKT in the MCF7 cells. Overall, the results of the functional assays showed that the somatic mutations of the PTEN gene alter PTEN tumour suppressor function. Expression of SphK1, a positive upstream regulator of the Akt pathway, is expressed as 2 major isoforms, SphK1-43kDa (SphK1a) and SphK1-51kDa (SphK1b), with similar SphK1 activity. However, to date, there is no literature on the expression of the two SphK1 isoforms in cell lines or human tissues. Profiling the expression of the two SphK isoforms in various cancer cell lines (n=24), primary cancer tissues (n=28) and paired adjacent tissues (n=28), demonstrated that the SphK1a isoform is expressed in all cell lines and tissues (both normal and cancer) studied, however, expression of SphK1b is cell and tissue specific, including breast, prostate and lung. Balancing signalling pathways and maintaining cellular homeostasis, as observed through the PTEN/SphK1 swinging pendulum is important and the study of these pathways is crucial in gaining further understanding the opposing regulatory mechanisms, which may be exploited for the future prevention and treatment of cancers.

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http://hdl.handle.net/10453/135943