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p110α Proteomics and PTEN Localization in Breast Cancer Models

Don-Doncow, Nicholas (2012) MOBT19 20112
Degree Projects in Molecular Biology
Abstract
Breast cancer is the most common cancer type among women. The 5-year survival rate for invasive breast cancer is generally good at approximately 80-85%, however it is not uncommon for late recurrences to develop – the relative survival at 15 years is approximately 60%, in contrast to for example uterine cancer where the 5-year is about 80% and very few late deaths occur (80% survival at 15-years). Therefore, greater understanding of tumor biology is necessary to develop better therapies and improve long-term survival.
Cancer is driven by different genetic and epigenetic aberrations including mutations, deletions, insertions and chromosomal rearrangements that affect normal cellular function and gene expression and give the cells survival... (More)
Breast cancer is the most common cancer type among women. The 5-year survival rate for invasive breast cancer is generally good at approximately 80-85%, however it is not uncommon for late recurrences to develop – the relative survival at 15 years is approximately 60%, in contrast to for example uterine cancer where the 5-year is about 80% and very few late deaths occur (80% survival at 15-years). Therefore, greater understanding of tumor biology is necessary to develop better therapies and improve long-term survival.
Cancer is driven by different genetic and epigenetic aberrations including mutations, deletions, insertions and chromosomal rearrangements that affect normal cellular function and gene expression and give the cells survival and growth advantages. One of the most commonly mutated pathways in cancer is the phosphatidylinositol 3-kinase (PI3K) pathway. This pathway is known to regulate a wealth of cellular processes including cell survival, proliferation, apoptosis and growth. PI3Ks, such as the oncogene p110α (encoded by PIK3CA), are responsible for phosphorylating phosphatidylinositols and phosphoinositides, forming potent lipid second messengers that activate downstream PI3K signaling. The main suppressor of this pathway is Phosphatase and TENsin homolog (PTEN), a tumor suppressor gene which acts in direct opposition to PI3Ks, dephosphorylating the active lipid second messengers and thereby turning off downstream signaling. PIK3CA and PTEN are frequently mutated in cancer, and mutational activation of p110α or loss of PTEN function appear to be important in sensitivity and resistance to certain therapies such as anti-HER2 agents, which are in clinical use today, or PARP inhibitors, that are in clinical trials. Recently, evidence for a role for PTEN in the nucleus has emerged, but it's exact function in the nucleus is not well understood.
The work presented in this thesis focuses on the PI3K/PTEN pathway and can be split into two parts. The first project focused on a method to purify p110α protein from breast cancer cell lines for detection of these mutations using multiple reaction monitoring (MRM) mass spectroscopy. Unlike conventional mass spectroscopy, MRM allows one to specify target peptide ions including wildtype and mutant forms to search for, detect, and quantitate. A viable immunoprecipitation method has been optimized in order to obtain purified samples of p110α protein for the fine-tuning of the MRM analysis. The establishment of an MRM analysis may provide a new tool for detection and quantification of mutant peptides. In the second project, we established a nuclear extraction protocol and in situ immunofluorescence method that can detect PTEN’s localization into the nucleus. We present data here that PTEN can localize to the nucleus under both oxidative stress and DNA damage. PTEN increased in concentration in the nucleus when treated with either hydrogen peroxide, or gamma-radiation or UV irradiation-mediated DNA damage. An identification of PTEN response to DNA damage can prove to be important in understanding what other tumor-suppressive roles PTEN may have as help inform more effective treatments, such as combinations with PARP inhibitors. (Less)
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author
Don-Doncow, Nicholas
supervisor
organization
course
MOBT19 20112
year
type
H2 - Master's Degree (Two Years)
subject
language
English
id
3799710
date added to LUP
2013-05-23 13:51:27
date last changed
2013-05-23 13:51:27
@misc{3799710,
  abstract     = {Breast cancer is the most common cancer type among women. The 5-year survival rate for invasive breast cancer is generally good at approximately 80-85%, however it is not uncommon for late recurrences to develop – the relative survival at 15 years is approximately 60%, in contrast to for example uterine cancer where the 5-year is about 80% and very few late deaths occur (80% survival at 15-years). Therefore, greater understanding of tumor biology is necessary to develop better therapies and improve long-term survival. 
Cancer is driven by different genetic and epigenetic aberrations including mutations, deletions, insertions and chromosomal rearrangements that affect normal cellular function and gene expression and give the cells survival and growth advantages. One of the most commonly mutated pathways in cancer is the phosphatidylinositol 3-kinase (PI3K) pathway. This pathway is known to regulate a wealth of cellular processes including cell survival, proliferation, apoptosis and growth. PI3Ks, such as the oncogene p110α (encoded by PIK3CA), are responsible for phosphorylating phosphatidylinositols and phosphoinositides, forming potent lipid second messengers that activate downstream PI3K signaling. The main suppressor of this pathway is Phosphatase and TENsin homolog (PTEN), a tumor suppressor gene which acts in direct opposition to PI3Ks, dephosphorylating the active lipid second messengers and thereby turning off downstream signaling. PIK3CA and PTEN are frequently mutated in cancer, and mutational activation of p110α or loss of PTEN function appear to be important in sensitivity and resistance to certain therapies such as anti-HER2 agents, which are in clinical use today, or PARP inhibitors, that are in clinical trials. Recently, evidence for a role for PTEN in the nucleus has emerged, but it's exact function in the nucleus is not well understood.
The work presented in this thesis focuses on the PI3K/PTEN pathway and can be split into two parts. The first project focused on a method to purify p110α protein from breast cancer cell lines for detection of these mutations using multiple reaction monitoring (MRM) mass spectroscopy. Unlike conventional mass spectroscopy, MRM allows one to specify target peptide ions including wildtype and mutant forms to search for, detect, and quantitate. A viable immunoprecipitation method has been optimized in order to obtain purified samples of p110α protein for the fine-tuning of the MRM analysis. The establishment of an MRM analysis may provide a new tool for detection and quantification of mutant peptides. In the second project, we established a nuclear extraction protocol and in situ immunofluorescence method that can detect PTEN’s localization into the nucleus. We present data here that PTEN can localize to the nucleus under both oxidative stress and DNA damage. PTEN increased in concentration in the nucleus when treated with either hydrogen peroxide, or gamma-radiation or UV irradiation-mediated DNA damage. An identification of PTEN response to DNA damage can prove to be important in understanding what other tumor-suppressive roles PTEN may have as help inform more effective treatments, such as combinations with PARP inhibitors.},
  author       = {Don-Doncow, Nicholas},
  language     = {eng},
  note         = {Student Paper},
  title        = {p110α Proteomics and PTEN Localization in Breast Cancer Models},
  year         = {2012},
}