Proteomics-Based Studies to Reveal the Influence of Profilin1 Expression in Breast Cancer Cells During Oxidative Stress

Abstract

Relevant studies are crucial to indicate or shed more insight into metastatic breast cancer and its impact on women on a global level. It is essential to note that metastatic cancer is currently the leading cause of cancer-related death among women worldwide. Such statistics warrant enough reason why extensive research should be done on this strain of cancer to understand its commencement, development, and adverse effects on human health. Profilin1 (PFN1), a ubiquitously expressed cytoskeleton regulatory protein, is down-regulated in invasive and metastatic breast cancer. This regulatory protein, which is known to increased expression, suppresses breast cancer proliferation. Cancer proliferation affects tumorigenicity in cultured cells, where malignant tissue growth is enhanced primarily through mitotically active cells. The suppression of breast cancer proliferation assists in reducing tumorigenicity in vitro.<br/>One should note that there is an abundance of information regarding the use of research to indicate that PFN1 expression is up-regulated upon hydrogen peroxide (H2O2)-induced oxidative stress in some breast cancer cells. Depending on the concentration, H2O2 either plays an important role in cancer development or induces apoptosis in cancer cells selectively. Although the concept of PFN1 functionality and the effect of oxidative stress in cancer cells is attractive, many details are yet to be explored, constituting a significant section of the research theses and hypotheses. In particular, information is lacking on the consequences of the modification of PFN1 and its binding to specific ligands for cell differentiation and proliferation in higher eukaryotes.<br/>To advance biomarker discovery and targeted therapies for breast cancer, a clear understanding of the intricate molecular mechanisms leading to the aforementioned observations is a requisite. Using MDA-MB-231 as a model breast cancer cell line combined with proteomic approaches, we provide evidence that PFN1 overexpression is associated with alterations in the expression of proteins that have been functionally linked to cell proliferation, survival and motility. Specifically, we used tandem mass spectrometry-based post-translational modification (PTM) and label-free quantification to quantify protein expression differences across different samples. Spectrum analysis of the raw proteomic data was accomplished using PEAKS Studio software (Bioinformatics Solutions, Canada). To further biologically characterise proteomics data, we used the Database for Annotation, Visualisation and Integrated Discovery (DAVID) and STRING.<br/>Additionally, total ion current (TIC) and Western blots were used for proteomic data normalisation, validation, and used to check the quality of our data. Little, if anything, is presently known about the biological networks regulated by PFN1 that can explain its pleiotropic actions on cancer cells in vivo. It is hypothesised that the structure of PFN1 influences its cellular activities in the presence of cellular-induced oxidative stress. Therefore, the first specific aim is to determine the protein expression profile of metastatic cancer cells exposed to oxidative stress. The second aim of this thesis is to determine the PTM (glutathione modification) footprint of PFN1 in vitro and in vivo. The third is to determine how abolishing specific ligand interactions of PFN1 affects the proteomic profile of breast cancer cells. The fourth aim of the thesis is to determine how over-expressed specific ligands of PFN1 in cancer cells affect the proteomic profile of cells as a result of oxidative stress. Finally, this thesis seeks to observe as well as compare the proteomic profiles (also identified as protein expression profiles) that are situated between parental and transfected cell lines.<br/><b>Breakdown of the First Study</b><br/>In the first study, aimed to decipher breast cancer global proteomics data of cellular changes and molecular avenues at lower and higher concentrations of H2O2 exposure. Specifically, our label-free quantification and cluster analysis revealed significant modifications in ribosomal biogenesis and translational reprogramming, which might expand our understanding of the mechanism of chemotherapy resistance in tumours. Further, PTM analysis indicated that PFN1 undergoes glutathionylation at C71 under low concentration of H2O2, which might induce aggregation and likely modify its function.<br/><b>Breakdown of the Second Study</b><br/>The second study we investigated the PTM of glutathionylated PFN1 both in vitro and in breast cancer cells under oxidative effect and showed that all cysteine(C) can be glutathionylated but only PFN1 C71 glutathione modification results in protein precipitations and formation of stable oligomers. These first findings suggested a probable novel role of stabilisation (PTM) of PFN1 at C71, with an emphasis on how it contributes to the pathogenesis of amyotrophic lateral sclerosis (ALS).<br/><b>Breakdown of the third and fourth Studies</b><br/>In the last two studies, we monitored transfected MDA-MB-231 cells with overexpression of wt.PFN1 and its mutants (W3A, H119E, R88A and R88E-R136D) impaired in binding for three major ligands (actin, PI(4,5)P2 and poly-rich proline) in the presence and absence of oxidative stress (H2O2). We investigated, through labelfree quantification, alterations in global protein expression profiles of transfected breast cancer cells with expression of PFN1 mutants. We provide evidence that expression of mutant PFN1 is associated with alterations in the protein expressions that are functionally linked to translational initiation, cell-cell adhesion, canonical glycolysis, gene expression, movement of subcellular or cell components, response to the stress of the endoplasmic reticulum and regulating the apoptotic process. In addition, upon exposure of these PFN1 mutant transfected cells to oxidative stress, the data suggested differences in the protein expressions associated with cell death and Rho activation regulation between these mutants.<br/>The data resulting from all these studies, allowed us to contribute to a better understanding data for protein alterations and PFN1 modifications in vitro, and in breast cancer cells, in the absence and the presence of oxidative stress. This study identifies PFN1 signalling biological partners that govern cell survival and cell death in breast cancer cells and its role in the pathogenesis of breast cancer.