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Hormone receptor positive (HR+) breast cancer represent 70% of all breast tumours. Its oncogenesis is multiple step process thought to be driven by the presence of two transcription factors, the oestrogen receptor (ER) and/or the progesterone receptor (PR). Therefore, the endocrine-targeted therapeutic approach has been focused on both receptors as prognostic markers and therapeutic targets, aiming to alter the oestrogen signalling for patients with ERα-positive disease. The benefits gained from the treatment appear to be limited by developing either de novo or acquired resistance following a period of response to tamoxifen. In the present work, global quantitative proteomics was combined with the analysis of fractions enriched in target subcellular locations (nuclear and cytoplasmic fractions), this has allowed measurement of the changes in total abundance and in the compartmental abundance/distribution between the nucleus and cytoplasm for several thousand proteins differentially expressed in MCF-7cells in response to oestrogen and tamoxifen stimulation in MCF-7 cells. In the first part of the thesis, we have used a proteomics subcellular spatial razor approach to look at changes in total protein abundance and in protein distribution between the nucleus and cytoplasm following exposure of MCF7 breast cancer cells to oestradiol. The dominant response of MCF7 cells to oestrogen stimulation involves dynamic changes in protein subcellular spatial distribution rather than changes in total protein abundance. Of the 3604 quantitatively monitored proteins, only about 2% show substantial changes in total abundance (>2-fold), whereas about 20% of the proteins show substantial changes in local abundance and/or redistribution of their subcellular location, with up to 16-fold changes in their local concentration in the nucleus or the cytoplasm. The second part of the thesis focuses on the spatial distribution of proteins in the three sub-proteome fractions (total lysate, nuclear and cytoplasmic fractions) in 4-OHT stimulated MCF-7 cells. Of the 3493 quantitatively monitored proteins, 97 of quantified proteins were detected as a core data set with differential abundance (DA) that was significantly changed in total abundance and/or subcellular location (P < 0.05). More than 50% of the proteins show significant changes in local abundance and/or redistribution of their subcellular location, only 19 proteins show substantial changes in the total lysate. Following the rigorous downstream analysis of the pathways significantly overrepresented and GO terms significantly enriched, this analysis showed that protein changes in abundance with 4-OHT across multiple sample types (CNT) were involved in pathways related to metabolism, signal transduction, growth and proliferation, and development. The nuclear response involved upregulation of the proteins participating in the GPCR downstream signalling pathway leading to accumulated response in the cancer pathway by moderately modulating the cell survival (PI3K/AKT) pathway in a circadian rhythm. Furthermore, this was accompanied by substantial changes in the local abundance and /or the redistribution of the metastasis mediating proteins and apoptotic cleavages of cell adhesion proteins. We propose that dynamic redistribution of the subcellular location of multiple proteins in response to stimuli is a fundamental characteristic of cells and suggest that perturbation of cellular spatial control may be an important feature of cancer.

Oxidative stress is a biological state that occurs due to imbalance between reactive oxygen species (ROS) and the antioxidant system leading to subcellular disturbance. The purpose of this thesis was to determine changes in protein abundance/distribution between nuclear and cytoplasmic cell compartments in the normal human IMR90 fibroblasts subjected to mild oxidative stress. The experimental design was to exert tert- butyl hydrogen peroxide upon the IMR90 cell lines to induce mild oxidative stress followed by fractionation of the cells into nucleus and cytoplasm. Cellular response was measured by the use of subcellular spatial razor approach based on quantitative shotgun LC-MS/MS-based proteomics with SILAC isotope labeling. It has been found that, in response to the treatment, proteins were redistributed between nucleus and cytoplasm including numerous proteins that were not previously known to associate with oxidative stress. We found 121 most significant proteins with known function at unexpected subcellular location. These proteins were known to contribute to different cellular processes such as, transcription, iron/haem metabolism, TCA cycle, glycolysis, autophagy, signal transduction and ATP synthesis, and are consistent with functional networks that are spatially distributed across the cell. Specific metabolic pathways of NRF2 and proline regulatory axis were found to play an important part on the cellular response to mild oxidative stress. Iron metabolism with iron/haem as a cofactor and mitochondrial proteins were prominent into the response as well. By initial comparison of the oxidative stress fibroblasts with Cdc7 depleted fibroblasts after induction of origin activation checkpoint, it was found that both responses affect proteins related to glycolysis, TCA cycle and proline regulatory axis. Evidence suggested nuclear import/export of proteins induced by the treatment. Subcellular spatial razor results for response of fibroblasts to oxidative stress clearly suggested that, to obtain comprehensive pictures of cellular function, measurements of global changes in total protein abundance need to be combined with measurement of the dynamic subcellular spatial redistribution of proteins.