Single muscle fiber proteomics reveals unexpected mitochondrial specialization

Mammalian skeletal muscles are composed of multinucleated cells termed slow or fast fibers according to their contractile and metabolic properties. Here, we developed a high-sensitivity workflow to characterize the proteome of single fibers. Analysis of segments of the same fiber by traditional and unbiased proteomics methods yielded the same subtype assignment. We discovered novel subtype-specific features, most prominently mitochondrial specialization of fiber types in substrate utilization. The fiber type-resolved proteomes can be applied to a variety of physiological and pathological conditions and illustrate the utility of single cell type analysis for dissecting proteomic heterogeneity

Investigating the potential of metformin as an anti-cancer therapeutic in a model of breast disease

Metformin has been one of the most widely prescribed oral medications for type II diabetes for over six decades. It has recently received considerable attention because there is now evidence to show that metformin has a potential role in reducing the risk of cancer development and progression. However, the mechanisms behind the growth-inhibitory effect of metformin on breast cancer cells are not fully understood with little consensus on which tumour subtypes and/or patient populations will benefit from metformin treatment. Furthermore, it should be noted that much of the in vitro work published to date has used drug concentrations greatly exceeding the recommended clinical dose and most preclinical studies have given little attention to the cellular pharmacology of metformin uptake including the expression of metformin transporter molecules and intratumoral accumulation. As a result these studies may not translate directly into clinical practice. This project therefore tests the hypothesis that the anti-tumour effect of clinically relevant doses (0.03-0.3 mM) of metformin depends on breast cancer subtype and the presence of metformin transporters on breast cancer cells. Using immunohistochemistry on patient-derived tissues and various in vitro cell-based assays in a panel of increasingly transformed breast cell lines representing an in vitro model of breast disease progression, the expression of metformin transporters and the potential anti-proliferative effects of the clinical (0.03-0.3 mM) and potential tissue accumulation (1-5 mM) doses of metformin were evaluated. In parallel, global proteomic profiling was performed on three metastatic breast cancer cell lines to identify new potential molecular targets for metformin treatment. The data in this thesis show that metformin transporters are present on breast epithelial cells, pre-neoplastic, pre-invasive, invasive and metastatic breast cancer cells and that metformin has a cytostatic effect on the proliferation of these cells, causing cell cycle arrest, but not apoptosis at clinically relevant doses. The proteomic data suggest that metformin inhibits the expression of proteins within key cellular pathways in both triple negative breast cancer and the bone and lung-homed variants, with the lung-homed cells showing a greater response to metformin treatment. Taken together these data provide important novel insight into the useful role of metformin in breast cancer treatment, but further research is certainly required to identify biomarkers of response and mechanisms of action in breast cancer before metformin can be recommended in clinical practice

Exploiting myocardial ischemia/reperfusion models and advanced proteomic

"Acute Myocardial Infarction (AMI) remains a leading cause of death worldwide. After AMI, clinical restauration of blood flow aggravates tissue damage (Ischemia/Reperfusion, I/R injury), critically decreasing the number of viable cardiomyocytes (CMs). Besides CMs, other myocardial cell populations such as cardiac stem/progenitor cells (CSCs) and cardiac fibroblasts (CFs) play key roles in tissue pathology and regeneration upon AMI. Several studies have demonstrated the relevant role of endogenous CSCs in myocardial repair after I/R injury, supported by the establishment of a paracrine cross talk between CSCs and the injured tissue(...)"

Role of adipose tissue in the pathogenesis and treatment of metabolic syndrome

© Springer International Publishing Switzerland 2014. Adipocytes are highly specialized cells that play a major role in energy homeostasis in vertebrate organisms. Excess adipocyte size or number is a hallmark of obesity, which is currently a global epidemic. Obesity is not only the primary disease of fat cells, but also a major risk factor for the development of Type 2 diabetes, cardiovascular disease, hypertension, and metabolic syndrome (MetS). Today, adipocytes and adipose tissue are no longer considered passive participants in metabolic pathways. In addition to storing lipid, adipocytes are highly insulin sensitive cells that have important endocrine functions. Altering any one of these functions of fat cells can result in a metabolic disease state and dysregulation of adipose tissue can profoundly contribute to MetS. For example, adiponectin is a fat specific hormone that has cardio-protective and anti-diabetic properties. Inhibition of adiponectin expression and secretion are associated with several risk factors for MetS. For this purpose, and several other reasons documented in this chapter, we propose that adipose tissue should be considered as a viable target for a variety of treatment approaches to combat MetS

Mass Spectrometry-Based Protein Profiling And Investigations of TGF-ß1-Induced Epithelial-Mesenchymal Transition Signatures In Namru Murine Mammary Gland Epithelial Cells

Breast cancer is the second-most common cancer and the second-leading cause of cancer-related deaths in women. Despite advances in cancer early detection, prevention and treatment, breast cancer is still a major health challenge due to low survival caused by breast cancer metastasis. This warrants critical attention and intervention. From the proteomic standpoint, a protein-based multiplex system that provides large array of informative signals for cancer identification and prognosis is still limited. In this dissertation work, we developed two mass spectrometry-based strategies involving chemical biology tools for rapid protein fingerprinting of breast cancer cell lines, and for probing the O-linked N-acetylglucosamine (O-GlcNAc) proteome in transforming growth factor-beta (TGF-ß) induced epithelial-mesenchymal transition (EMT), a process that initiates metastasis. Investigation of O-GlcNAc EMT proteomics is critical in understanding how aberrant O-GlcNAc post-translational modification (PTM) promotes cancer invasion and metastasis, as well as in the identification of early stage therapeutic targets. Until now the role of O-GlcNAc PTM in TGF-ß-induced EMT is unknown. In Chapter 2, a novel ‘one-step cell processing’ method was developed as a prerequisite to rapid spectral profiling of mammalian cells using Matrix-Assisted Laser Desorption Ionization Time-of-Flight mass spectrometry (MALDI-TOF MS). Upon analysis of the mass spectral data of breast cancer cell lines with pattern recognition methods, discrimination between metastatic and non-metastatic cell lines was accomplished, demonstrating the potential of MALDI-MS profiling in breast cancer diagnosis. Chapter 3 reports a cleavable azide-reactive dibenzocyclooctyne-disulphide agarose-based beaded resin in Copper-free Click chemistry-based affinity enrichment of O-GlcNAc proteome from azido-GlcNAc labeled cellular extracts, that enabled the global O-GlcNAc proteomic profiling by shortgun proteomics with liquid chromatography-tandem mass spectrometry identification and label-free quantification. From TGF-ß-induced EMT in MNuMG cells 196 proteins were identified. 125 of these were putative O-GlcNAc proteins, 75% of which have been previously identified among O-GlcNAc affinity enrichment samples. Downstream bioinformatics analyses of the O-GlcNAc proteome data were performed using Ingenuity Pathway Analysis (IPA) software. In silico protein-protein interactions revealed a regulatory network for metastasis, while the most significantly represented metabolic and signaling pathways included glycolysis and several TGF-ß non-canonical pathways, respectively. A metastatic regulatory network that features core regulators β-catenin and cyclin-D1 both of which are regulated by O-GlcNAc transferase supports published study that shows that “O-GlcNAcylation Plays Essential Role in Breast Cancer Metastasis,” has led us to hypothesize that TGF-ß signaling cooperates with O-GlcNAc signaling in promoting EMT, invasion and metastasis, pending O-GlcNAc site-mapping and validation of the proteomic data

Exploring the role of metabolism in cancer and cardiac settings

Metabolism supports all aspects of cellular function. As such, dysregulated control of the enzymes and signalling pathways coordinating metabolism is common in diseases such as cancer and heart disease. Despite the critical role of metabolism in these diseases and their global burden, their complexity has limited the development of therapeutics that directly target their metabolism. Therefore, this thesis explored the role of extrinsic and intrinsic metabolic dysfunction and characterised examples of metabolism’s potential role in future therapeutics. This work utilised examples of diabetic cardiomyopathy (DCM) to illustrate pathology downstream of extrinsic metabolic dysfunction occurring with insulin resistance in type II diabetes mellitus (T2DM), which remodels the heart independent of adjacent vascular disease. Given that the pathological processes underlying DCM are poorly characterised, and no therapeutics are currently available, this work explored the utility of 2D monolayer, and 3D engineered heart tissue human-induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) models to characterise human-relevant DCM pathology in vitro and explore the trade-offs between model complexity and practicality. A multi-omics approach showed that both models demonstrated the expected shift to fatty acid metabolism and blunting of insulin signalling. The 3D model exhibited a more mature phenotype but showed increased variability. Moreover, phenotypes emerging in animal models of DCM were identified, such as deregulated CD36 trafficking and blunted hypoxia signalling. Examples were given for the intrinsic metabolic dysregulation that supports growth and survival in cancer. Transcriptomics from tumour biopsies was integrated with positron emission tomography (PET) measures for metabolite radiotracer uptake to infer this dysregulation in patients. We examined the utility of dynamic modelling of PET measures using the glucose radiotracer 2-deoxy-2 [18F]-fluoro-D-glucose (FDG). A robust correlation-based pathway approach established that simple dynamic models, which incorporate tracer perfusion and uptake across biological compartments, outperform static measures such as the standardised uptake value (SUV) and complex multi-compartmental models that overfit the data. They associated with the glycolysis term, absent from static findings, identified the most significant pathways, and showed robust detection of inflammatory signals. Dynamic approaches also more robustly established the pathways, such as oxidative phosphorylation, associated with metformin treatment, a metabolic drug under investigation for repurposing in cancer therapy. We used this established methodology to investigate an emerging radiotracer for glutamine uptake, 18F-Fluciclovine, evaluating its ability to characterise glutamine metabolism and its utility in the clinical setting for breast cancer. Immunohistochemistry and metabolomics confirmed that 18F-Fluciclovine was taken up by the glutamine transporter ASCT2 as a proxy for glutamine. 18F-Fluciclovine uptake was associated with pyrimidine metabolism, a biosynthetic pathway incorporating glutamine to supply proliferating cells with nucleotides. Clustering patients by pyrimidine metabolism expression captured the differences in oncogenic signalling, proliferation, and survival, highlighting pyrimidine metabolism’s role in pathology. Our radiogenomics analysis shows potential for metabolic drugs, like metformin, to treat cancer and cardiovascular disease concurrently. However, current cancer therapies like doxorubicin (DOX) lead to cardiotoxicity. Therefore, we used a rodent model of DOX cardiotoxicity to investigate the metabolic drug 5-aminoimidazole-4-carboxamide riboside (AICAR), previously shown to restore cardiac function. Transcriptomics explored a potential mechanism for this protection, confirming that DOX remodels the heart through substrate metabolism deregulation and fibrosis. AICAR, which activates AMP-activated protein kinase (AMPK), restored glycolytic gene expression and heme metabolism, indicative of reduced mitochondrial ferroptosis. A set of ‘rescue genes’ altered by DOX and restored by AICAR were identified, including evidence for alleviating DOX-induced MYC signalling that may mediate heart remodelling. This thesis used omics-based approaches to highlight metabolism’s role in human health and disease, characterising examples of intrinsic metabolic dysfunction, as seen in cancer, and extrinsic metabolic dysfunction, as in diabetic cardiomyopathy. The research employed in vitro, in vivo, and in situ measures to evaluate metabolic changes and their therapeutic implications, highlighting the potential of radiogenomics and hiPSC-CM models in advancing our understanding and treatment of metabolic diseases and the repurposing of metabolic drugs to target cancer and mitigate the cardiotoxicity that follows current treatment strategies