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

Therapy for word finding difficulties using phonological and orthographic cues: a clinical application in progress

Semantic therapy for anomia is well established both in research and in clinical practise. Research into phonological therapy is more limited and the results more equivocal. This paper will describe an ongoing clinical study which uses combined phonological and orthographic cues to treat anomia in a case series design. The study includes a single cue and a choice of cue condition, and also assesses the effect of repeated presentation of a picture for naming without cues on word retrieval. The results of therapy for picture naming and for word retrieval in natural conversation will be presented for two participants

Influence of Oligomerization State on the Structural Properties of Invasion Plasmid Antigen B (IpaB) from Shigella flexneri in the Presence and Absence of Phospholipid Membranes

Shigella flexneri causes bacillary dysentery, an important cause of mortality among children in the developing world. Shigella secretes effector proteins via its type III secretion system (T3SS) to promote bacterial uptake into human colonic epithelial cells. The T3SS basal body spans the bacterial cell envelope anchoring a surface-exposed needle. A pentamer of invasion plasmid antigen D (IpaD) lies at the nascent needle tip and IpaB is recruited into the needle tip complex upon exposure to bile salts. From here, IpaB forms a translocon pore in the host cell membrane. Although the mechanism by which IpaB inserts into the membrane is unknown, it was recently shown that recombinant IpaB can exist as either a monomer or tetramer. Both of these forms of IpaB associate with membranes, however, only the tetramer forms pores in liposomes. To reveal differences between these membrane-binding events, Cys mutations were introduced throughout IpaB, allowing site-specific fluorescence labeling. Fluorescence quenching was used to determine the influence of oligomerization and/or membrane association on the accessibility of different IpaB regions to small solutes. The data show that the hydrophobic region of tetrameric IpaB is more accessible to solvent relative to the monomer. The hydrophobic region appears to promote membrane interaction for both forms of IpaB, however, more of the hydrophobic region is protected from solvent for the tetramer after membrane association. Limited proteolysis demonstrated that changes in IpaB’s oligomeric state may determine the manner by which it associates with phospholipid membranes and the subsequent outcome of this association

Potential zoonotic sources of SARS‐CoV‐2 infections

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) causing coronavirus disease-2019 (COVID-19) likely has evolutionary origins in other animals than humans based on genetically related viruses existing in rhinolophid bats and pangolins. Similar to other animal coronaviruses, SARS-CoV-2 contains a functional furin cleavage site in its spike protein, which may broaden the SARS-CoV-2 host range and affect pathogenesis. Whether ongoing zoonotic infections are possible in addition to efficient human-to-human transmission remains unclear. In contrast, human-to-animal transmission can occur based on evidence provided from natural and experimental settings. Carnivores, including domestic cats, ferrets and minks, appear to be particularly susceptible to SARS-CoV-2 in contrast to poultry and other animals reared as livestock such as cattle and swine. Epidemiologic evidence supported by genomic sequencing corroborated mink-to-human transmission events in farm settings. Airborne transmission of SARS-CoV-2 between experimentally infected cats additionally substantiates the possibility of cat-to-human transmission. To evaluate the COVID-19 risk represented by domestic and farmed carnivores, experimental assessments should include surveillance and health assessment of domestic and farmed carnivores, characterization of the immune interplay between SARS-CoV-2 and carnivore coronaviruses, determination of the SARS-CoV-2 host range beyond carnivores and identification of human risk groups such as veterinarians and farm workers. Strategies to mitigate the risk of zoonotic SARS-CoV-2 infections may have to be developed in a One Health framework and non-pharmaceutical interventions may have to consider free-roaming animals and the animal farming industry

Challenges in accessing fieldwork in rural Himalayas: an emerging researcher’s experiences

This article is the reflection of an emerging researcher who has experienced different nudges from the beginning of accessing the research field to the final stage of collating data in rural Nepal. My experiences and reflections may provide provocations to researchers to consider before they undertake field research. Areas that researchers should be aware of before entering the field include strategies of approaching the resources, socio-cultural and language diversities, topography and travel complication. The article offers those considering field research in remote areas, such as rural Nepal, an opportunity for rethinking a case study research approach, choosing appropriate methodological tools, sampling strategies and accessing resources

Generalisation to untreated items in anomia therapy: links to individuals’ language processing

The aim of therapy for word-retrieval problems with people with aphasia is for carry-over to everyday communication. This may be most likely if the therapy generalises to untreated words . However, Howard (2000) claims there is little convincing evidence for generalisation. He suggests that both ‘semantic’ and ‘phonological’ therapies work by strengthening the links (‘mapping’) between semantic and phonological representations. This paper investigates generalisation to untreated items in anomia therapy by combining data over two recent studies and from the literature

Context-Specific Effects of TGF-β/SMAD3 in Cancer Are Modulated by the Epigenome.

The transforming growth factor beta (TGF-β) signaling pathway exerts opposing effects on cancer cells, acting as either a tumor promoter or a tumor suppressor. Here, we show that these opposing effects are a result of the synergy between SMAD3, a downstream effector of TGF-β signaling, and the distinct epigenomes of breast-tumor-initiating cells (BTICs). These effects of TGF-β are associated with distinct gene expression programs, but genomic SMAD3 binding patterns are highly similar in the BTIC-promoting and BTIC-suppressing contexts. Our data show cell-type-specific patterns of DNA and histone modifications provide a modulatory layer by determining accessibility of genes to regulation by TGF-β/SMAD3. LBH, one such context-specific target gene, is regulated according to its DNA methylation status and is crucial for TGF-β-dependent promotion of BTICs. Overall, these results reveal that the epigenome plays a central and previously overlooked role in shaping the context-specific effects of TGF-β in cancer.S.J.V. was supported by a grant from the Dutch Cancer Foundation (KWF).This is the final version of the article. It was first available from Elsevier via http://dx.doi.org/10.1016/j.celrep.2015.11.04

Computational approach to discriminate human and mouse sequences in patient-derived tumour xenografts.

Background - Patient-Derived Tumour Xenografts (PDTXs) have emerged as the pre-clinical models that best represent clinical tumour diversity and intra-tumour heterogeneity. The molecular characterization of PDTXs using High-Throughput Sequencing (HTS) is essential; however, the presence of mouse stroma is challenging for HTS data analysis. Indeed, the high homology between the two genomes results in a proportion of mouse reads being mapped as human. Results - In this study we generated Whole Exome Sequencing (WES), Reduced Representation Bisulfite Sequencing (RRBS) and RNA sequencing (RNA-seq) data from samples with known mixtures of mouse and human DNA or RNA and from a cohort of human breast cancers and their derived PDTXs. We show that using an In silico Combined human-mouse Reference Genome (ICRG) for alignment discriminates between human and mouse reads with up to 99.9% accuracy and decreases the number of false positive somatic mutations caused by misalignment by >99.9%. We also derived a model to estimate the human DNA content in independent PDTX samples. For RNA-seq and RRBS data analysis, the use of the ICRG allows dissecting computationally the transcriptome and methylome of human tumour cells and mouse stroma. In a direct comparison with previously reported approaches, our method showed similar or higher accuracy while requiring significantly less computing time. Conclusions - The computational pipeline we describe here is a valuable tool for the molecular analysis of PDTXs as well as any other mixture of DNA or RNA species