The role of NEDD9 in TGFβ mediated tumour initiating cell dynamics in the Claudin-low breast cancer subtype

Breast cancer is an extremely heterogeneous disease comprising at least ten different subtypes, each exhibiting different characteristics in progression, prognosis and response to treatment. Transforming growth factor β (TGFβ) a member of the TGF superfamily of cytokines differentially regulates breast tumour-initiating cells (BTICs). In the claudin-low breast cancer subtype, TGFβ increases the tumour initiating capacity through the ability of the scaffolding protein NEDD9 to unite the TGFβ/SMAD and Rho-Actin-SRF pathways. Previously, oncogenicity has been ascribed to the level of NEDD9 protein expression. However, my analysis indicates this mediation by NEDD9 is irrespective of NEDD9 protein expression, which is ubiquitously overexpressed in the majority of cancer types including breast cancer. In this thesis, I demonstrate how in the Claudin-low breast cancer subtype NEDD9 protein expression and post-translational modification are influenced by TGFβ pathway activation. I identify significant NEDD9 interacting proteins and their downstream effectors which contribute towards oncogenic TGFβ signalling pathways. A key TGFβ specific NEDD9 interactor identified in this subtype is the metabolic isoenzyme PKM2. Through a variety of techniques, I explore the known roles of PKM2 in the regulation of oncogenic metabolic reprogramming and demonstrate how these processes are influenced by its association with NEDD9. Finally, I investigate potential translational applications and biomarkers of TGFβ mediated, NEDD9/PKM2 dependent downstream signalling pathways, using large clinical breast cancer datasets and patient-derived tumour xenograft (PDTX) models. These data suggest a novel mechanism by which oncogenic TGFβ signalling regulates cellular proliferation and self-renewal via β-Catenin/c-Myc regulation of altered metabolism in the Claudin-low breast cancer subtype, a process which is dependent upon the scaffolding protein NEDD9 and the metabolic enzyme PKM2. Together, these data suggest that a combined biomarker of TGFβ signalling and c-Myc expression may be useful in identifying a subset of Claudin-low breast cancer patients who would be sensitive to inhibition of Wnt/β-Catenin signalling. Additionally, due to the dependence of these tumours on c-Myc driven glutamine dependent metabolic processes, metabolic magnetic resonance imaging may be useful for monitoring response in these patients

Extracellular matrix proteins in hypoxia, 3D cancer spheroids as a model

The extracellular matrix (ECM) is a non-cellular network of cross-linked macromolecules, such as collagens, glycoproteins (fibronectin and laminins), and proteoglycans. The ECM plays a critical role in mediating cell adhesion, migration, differentiation, and proliferation. Thus, abnormal ECM remodeling can lead to pathological states and cancer metastasis. Fibroblasts, the most abundant cell type in the tumor stroma, produce several ECM components and proteins. Hypoxia (low oxygen level) is a critical factor in cancer growth. The disruption in oxygen homeostasis leads to ECM remodeling. Therefore, many ECM proteins, such as collagens and laminins, are expressed differently. Increased deposition of these proteins can lead to ECM stiffness alterations, which can lead to cancer progression. 3D cell culture methods (spheroids) have gained increasing interest since they provide a more tissue-like environment compared to traditional 2D cell culture methods. We used 3D spheroids containing cancer cells and fibroblasts to mimic cutaneous squamous cell carcinoma (cSCC) tumors. In this study, western blot results showed that in hypoxia, collagen prolyl hydroxylases (P4HA1 and P4HA2) and collagen lysyl hydroxylase (PLOD2) expression increased in both transformed keratinocytes and metastatic cSCC cells when they were cocultured with human primary skin fibroblasts. Laminin-332 expression was, however, downregulated. Immunofluorescence staining confirmed that P4HA1 expression was upregulated in 3D spheroids in hypoxic conditions. Proliferation assay showed that cell proliferation increased in hypoxia in mono- and cocultured spheroids. Mass spectrometry experiments also revealed different ECM protein expressions in hypoxia compared to normoxia. These results show that 3D spheroids containing cancer cells and fibroblasts are an indispensable tool for detecting ECM alterations in hypoxic conditions

Semi-Synthesis of the Transcription Factor SMAD2 Containing Caging Groups and Phosphoaminoacid Analogues

Post-translational modification (PTM) of a protein refers to any chemical change that occurs to the protein after its ribosomal synthesis. The seemingly endless number of PTMs can endow proteins with new functionalities that are not present in the unmodified proteins. In order to study the functions of PTMs on a given protein, it is often necessary to have access to pure preparations of the modified protein and its analogues. Traditional biological methods frequently do not allow for the isolation of significant amounts of pure modified proteins, therefore chemical methods are often employed in this regard. Protein semi-synthesis is a chemical method that entails the melding of at least two protein fragments in which at least one fragment is isolated from a biotic source while another fragment is synthesized by chemical methods. This framework enables the installation of PTMs into the complete protein through chemical control over the synthesized fragment without the need to synthesize the entire protein molecule, which is often a practically impossible task. This thesis describes efforts employing the protein semi-synthesis technique known as expressed protein ligation (EPL) to the study of the cellular signaling protein Smad2. Smad2 is activated by phosphorylation, which is the best characterized reversible PTM. Once phosphorylated, Smad2 accumulates in the nucleus of cells where it helps to direct transcriptional changes that affect cell behavior. Techniques were developed to cage Smad2 by directly blocking the activating phosphates on Smad2 with bulky photoremovable groups. This affords the investigator control over the timing and localization of Smad2 activity by judicious application of light of the appropriate wavelength to remove the photocaging group. This approach can be employed to generate caged analogues of any phosphorylated protein. A parallel caging approach was developed that relies upon indirect blockade of Smad2 phospho-dependent activity through the installation of a caging group on the C-terminus of the protein. This approach was compatible with a fluorescence reporter that is fluorescent only when the photocaging group is removed, thus allowing for selective monitoring of the activated form of the protein. This protein was introduced into live cells and upon activation allowed for real time visualization of the active protein through fluorescence microscopy. The activity of Smad2 is dependent upon differential protein-protein interactions that the phosphorylated protein is able to participate in while the non-phosphorylated protein is not. In an effort to identify new binding partners that are sensitive to the phosphorylated state of Smad2, methods were developed to install stable phosphoanalogues (phosphonates) into Smad2. These analogues were used to identify a candidate Smad2-binding protein, PRMT5, that preferentially binds non-phosphorylated Smad2. Studies are ongoing to determine if this interaction has physiological relevance

Proteomic Characterization of Ovarian and Breast Cancer Microenvironments for Improved Diagnostics and Therapeutic Targeting

Cancers exist within complex microenvironments formed by heterogeneous cell types. This diversity creates significant challenges for detection, diagnosis and treatment. Mass spectrometry-based proteomics is a powerful approach capable of characterizing complex biological systems which are characteristic of cancer biology. In this thesis, proteomics was utilized to answer several questions related to ovarian cancer diagnosis and detection, and the effects of NODAL, an embryonic morphogen, on the breast cancer secretome and stromal cell recruitment. First, I compared multiple sample preparation techniques and found high-pH/low-pH fractionation to yield the greatest proteome coverage over commonly used approaches. Second, I compared the proteomes from two ovarian cancer subtypes (high-grade serous and endometrioid) for which histological discrimination remains difficult in a proportion of cases. I documented several unknown proteins, including KIAA1324, which were validated and confirmed to improve the differential diagnosis of endometrial ovarian cancer. Third, I extensively characterized extracellular vesicle proteomes from biological fluids (conditioned media, plasma and ascites) to catalogue potential biomarkers associated with malignant ovarian cancer. I detected many factors associated with advanced stage, high-grade serous ovarian cancer including CFHR4, MUC1, APCS and PZP that may be useful for early detection. Last, I characterized the global effects of the Transforming Growth Factor-β superfamily member NODAL on the breast cancer secretome and stromal cell recruitment in vitro. I found a previously unknown role for NODAL in modulating pro-inflammatory factors, including CXCL1 and IL6 that were correlated with multipotent stromal cell recruitment. In summary, this work represents a significant contribution to the histological assessment and detection of ovarian cancer and our understanding of the malignant properties of NODAL within the breast cancer microenvironment

Proteomics and phosphoproteomics applied to cell signaling and cancer

Signaling networks control and regulate outcomes in cells and organisms in both normal physiology and pathophysiological states. Signaling is traditionally represented and studied as a series of stepwise enzymatic events constituting a cascade. However, it is increasingly apparent that such representations limit understanding of signal transduction since these linear cascades function in an interconnected network that includes extensive cross talk among receptors and pathways. Mass spectrometry (MS)-based proteomics is a useful tool that allows a system-wide investigation of signaling events at the levels of post-translational modifications (PTMs), protein-protein interactions and changes in protein expression on a large scale. This technology now allows accurate quantification of thousands of proteins and their modifications in response to any perturbation. This thesis work is dedicated to the optimization and employment of quantitative mass spectrometry to cellular signaling and an application to segregate two lymphoma subtypes at the levels of protein expression and phosphorylation, employing state of the art liquid chromatography (LC)-MS/MS technologies coupled with improved sample preparation techniques and data analysis algorithms. In the first project I investigated the feasibility of a new, high accuracy fragmentation method called higher energy collisional dissociation (HCD) for the analysis of phospho-peptides. Using this method we were able to measure the phospho-proteome of a single cell line in 24h of measurement time which was a great improvement to previous capabilities. This fragmentation method that was originally thought to be slower and less sensitive than the standard method of low resolution collision induced dissociation (CID) fragmentation. However, our work proves this not to be the case and we showed that HCD outperformed the existing low resolution strategy [1]. In the second project I employed this HCD fragmentation technique on the LTQ-Orbitrap Velos for addressing the clinical question of segregating two subtypes of diffuse B-cell lymphoma (DLBCL). These subtypes are histologically indistinguishable but had been segregated on the basis of a gene expression signature. I employed the recently developed ‘super-SILAC’ approach with a ‘super-SILAC mix’ of multiple labeled cell lines. This heavy reference mix was spiked into several cell lines derived from the two DLBCL subtypes and analyzed LC-MS, resulting in successful segregation based on a distinct proteomic signature [2]. The third project deals with the in-depth analysis of the phospho-proteome of a human cancer cell line on a quadrupole-Orbitrap mass spectrometer using a label-free quantification approach. Our analysis uncovered about 50,000 distinct phosphorylated peptides in a single cell type across a number of cellular conditions allowing assessment of global properties of this large dataset. Strikingly, we found that at least three-quarters of the proteome can be phosphorylated which is much higher than current estimates. We also analyzed phosphotyrosine events using enrichment with anti-phospho-tyrosine antibodies to identify more than 1,500 site specific phosphorylation events. Unexpectedly tyrosine phosphorylated proteins were enriched among higher abundance proteins. The observed difference in phospho-protein abundance correlated with the substrate Km values of tyrosine kinases. For the first time we calculated site specific occupancies using label- free quantification and observed widespread full phosphorylation site occupancy during mitosis. In the final and main project, I applied proteomics and phospho-proteomics to the study of signal transduction in response to transforming growth factor-beta (TGF-β), a multifunctional cytokine. TGF-β signaling regulates many biological outcomes including cell growth, differentiation, morphogenesis, tissue homeostasis and regeneration. The cellular responses to this multifunctional ligand are diverse and can even be opposed to each other, depending on the cell type and the conditions. To shed light on the reasons for the different outcomes, we analyzed the early phospho-proteome and ensuing proteome alterations in response to TGF-β treatment in a keratinocyte cell line. The early SILAC based phospho-proteome analysis uncovered over 20,000 phosphorylation events across five time points (0 to 20 min) of TGF-β treatment. Building on our recent advances in instrumentation, sample preparation, and data analysis algorithms we measured a deep TGF-β responsive proteome at six late time points (6h to 48h) with corresponding controls in only eight days of measurement time. Our label-free approach identified about 8,000 proteins and quantified more than 6,000 of them. This deep proteome covered well established pathways involved in TGF-β signaling, allowing global evaluation at the level of individual pathway members. Combining the TGF-β responsive proteome with an in-silico upstream regulator analysis, we correctly retrieved several known and predicted novel transcription factors driving TGF-β induced cytostasis, de-differentiation and epithelial to mesenchymal transition (EMT). The combined analysis of transcription factor regulation with early phosphorylation changes and proteome changes enabled visualization of the intricate interplay of key transcription factors, kinases and various pathways driving cytostatis, EMT and other processes induced by TGF-β. In summary, my thesis developed a highly efficient phospho-proteomic workflow, which was applied to the measurement of a very deep phospho-proteome of a single cancer cell line allowing analysis of its global features. The main achievement was the first in-depth and combined study of the phospho-proteome and resulting proteome changes following a defined signaling event, in this case leading to a time-resolved view of TGF- β signaling events relevant in cancer

Search for markers and molecular mechanisms of aggressive endometrial cancer : profiling of aggressive vs non-aggressive endometrial cancers

Endometrial cancer is the seventh frequent type of cancer among women. Identification of new prognostic markers is important to optimize treatment and follow-up of all EC patients. Understanding the molecular mechanisms of carcinogenesis may pave through discovery of further EC molecular markers. Cohort studies often disregard the individual features of tumors. Since such features may represent an opportunity to individualize cancer treatment, an innovative approach for their assessment should be developed. In this study (paper I), we addresses the previously overlooked individual characteristics of endometrial cancers, which could serve as a wellspring of information regarding specific molecular processes; regulators of such processes could potentially be useful for predicting aggression in individual endometrial tumors. Systemic analysis of individual proteome profiles represented that different proteins may be impacted in the individual endometrial tumors of different patients, but the impact of these proteins on basic cell functions may still be similar. The correlation between publically available gene expression data sets of profiling of endometrial tumors and our proteome profiling supports the conclusion that individual tumor features are doubtlessly crucial in endometrial tumorigenesis and are not inconsistent individual variations. IHC validation using tissue microarray analysis of MST1 and PKN1 proteins suggested their potential to serve as predictive biomarkers for endometrial cancer as well as efficacy of this approach. Transforming growth factor-β (TGFβ) and epidermal growth factor (EGF) are two potent regulators of tumorigenesis. Signaling cross-talk between TGFβ and EGF involves a number of regulators which define the impact on cell physiology (Paper II and III). In paper II, we discuss mammalian sterile-like 1 kinase (MST1) as a negative regulator of combined TGFβ and EGF signaling. We observed that enhanced expression of MST1 inhibited the combined action of TGFβ1 and EGF on cell invasiveness, migration and proliferation. Monitoring of the intracellular regulatory proteins showed that the MST1 contribution to TGFβ-EGF cross-talk may involve focal adhesion kinase and E-cadherin, but not activation of Smad2. Our data elucidated the negative feedback role of MST1 on TGFβ1‑ and EGF-regulated cell invasiveness, migration and proliferation. Our results from paper III demonstrated that protein kinase N1 (PKN1) modulated responses of HEC-A-1 endometrial cancer cells to TGFβ1 and EGF. PKN1 had an inhibitory effect on stimulation of cell migration, and PKN1 kinase activity was required for the inhibitory effect of TGFβ and EGF on cell proliferation and invasiveness. We observed that phosphorylation of Smad2, FAK and Erk1/2 correlated with cellular response to TGFβ1 and EGF. PKN1 modulates TGFβ and EGF-dependent regulation of cell proliferation, migration and invasiveness, and is therefore a component of the signaling network downstream of TGFβ and EGF. Thus, our findings provided insights into different mechanisms of tumorigenesis and on the impact of cross-talk between signaling pathways on tumor developmen

Site-Specific Incorporation of Biochemical and Biophysical Probes into Proteins Using Expressed Protein Ligation

Protein engineering can be made far more powerful if a protein is not only expressed recombinantly but also altered covalently using synthetic chemistry. These two methods are brought together in the protein semi-synthesis technique Expressed Protein Ligation (EPL). In EPL, recombinant and synthetic polypeptides are joined together via a chemoselective ligation reaction. EPL was originally used to attach synthetic constructs to the C-terminus of recombinant proteins, but is now used to attach recombinant or synthetic polypeptides either at the N- or C-terminus of a protein or into the core of a protein. This thesis illustrates, with three distinct applications, the development of EPL from its original definition to its current understanding. In the first application, a general strategy was developed for the site-specific incorporation of fluorophores into proteins using Abl-SH3 as a model system. In the second application, chemistries were developed that allowed the site-specific introduction of phospho-amino acids into proteins, in this case using the transforming growth factor β receptor I as the model system. In the final application, EPL was used to synthesize several modified versions of the E. coli sigma factor σ70, demonstrating that this method can be used to probe extremely large macromolecules. These studies revealed that EPL works under a variety of reaction conditions and provided paradigms for using this technique to site-specifically insert fluorophores and phosphate groups into proteins. The chemical manipulation of proteins by EPL will be an important tool as researchers strive to characterize the proteomes of organisms

The characterisation and role of mighty during myogenesis

Myogenesis, or skeletal muscle formation, begins during embryogenesis and involves the proliferation of myoblasts followed by their exit from the cell-cycle to differentiate and form myotubes. This formation of skeletal muscle is a complex process involving many genes and various signalling pathways. Mighty is a novel myogenic gene discovered at AgResearch by the Functional Muscle Genomics (FMG) group in a genetic screen performed on the muscle of myostatin null and wild-type mice. It was found that heavily muscled mice, lacking myostatin, had increased expression of the mighty gene. This gene was found to be conserved, with cognates found in mammals, amphibians, teleosts, and arthropods. Mighty was found to be expressed in a variety of tissues, but only skeletal muscle showed increased mighty mRNA expression in myostatin null mice, indicating the specific regulation of mighty by myostatin in skeletal muscle (Marshall, 2005). The aim of this study was to characterise the mighty protein and examine its role in myogenesis to elucidate mighty's function. To undertake this study, antibodies specific for the full-length mighty protein and antibodies specific for a peptide region of mighty were characterised. Results using these antibodies, showed endogenous mighty, from myoblasts, to be a low-abundant, nuclear protein which shows a mobility of ~52 kDa in SDS gels, different to that of recombinant mighty protein. The mobility difference of endogenous mighty compared to recombinant mighty appears to be due to phosphorylation and may involve other post-translational modifications. In agreement, the determined isoelectric point (~5.7) of endogenous mighty also appears to be the result of phosphorylation. Interestingly, 52 kDa mighty was not detected in muscle extracts, but a ~30 kDa protein was specifically detected, indicating multiple forms, and subsequent roles, for mighty protein. Mass spectrometry (MS) was also performed for further characterisation of the mighty protein and possible post-translational modifications. Although hits were achieved with both recombinant mighty proteins, endogenous mighty MS analysis was not accomplished due to its low-abundance. The function of the mighty protein in myoblasts was investigated during proliferation and differentiation. The results indicate that proliferating myoblasts have low levels of mighty in G0 and increased levels in G1/S during the cell cycle. This differential expression of mighty may involve cell cycle exit at the G1/S phase. Differentiation results showed mighty to be upregulated before MyoD during differentiation, placing mighty very early in the differentiation hierarchy. This agrees with previous results by Marshall (2005) which showed mighty to upregulate MyoD through IGF-II expression. Enhanced differentiation was also seen in double muscle bovine myoblasts concomitantly with increased mighty expression. In conclusion, mighty appears to be a post-translationally modified protein that plays an early role in myogenic differentiation. This role in differentiation appears to be upstream of MyoD through the upregulation of IGF-II and may be linked to cell cycle exit in the G1 phase of the cell cycle