Breast cancer stem cells and epithelial mesenchymal plasticity : implications for chemoresistance

Tumour heterogeneity is a key characteristic of cancer and has significant implications relating to tumour response to chemotherapy as well as patient prognosis and potential relapse. It is being increasingly accepted that tumours are clonal in origin, suggestive of a tumour arising from a deregulated or mutated cell. Cancer stem cells (CSC) possess these capabilities, and with appropriate intracellular triggers and/or signalling from extracellular environments, can purportedly differentiate to initiate tumour formation. Additionally through epithelial mesenchymal plasticity (EMP), where cells gain and maintain characteristics of both epithelial and mesenchymal cell types, epithelial-derived tumour cells have been shown to de-differentiate to acquire cancer stem attributes, which also impart chemotherapy resistance. This new paradigm places EMP centrally in the process of tumour progression and metastasis, as well as modulating drug response to current forms of chemotherapy. Furthermore, EMP and CSCs have been identified in cancers arising from different tissue types making it a possible generic therapeutic target in cancer biology. Using breast cancer (BrCa) as an example, we summarise here the current understanding of CSCs, the role of EMP in cancer biology - especially in CSCs and different molecular subtypes, and the implications this has for current and future cancer treatment strategies.

Converse Bergmann cline in a eucalyptus herbivore, paropsis atomaria olivier (coleoptera: chrysomelidae) : phenotypic plasticity or local adaptation?

Aim To measure latitude-related body size variation in field-collected Paropsis atomaria Olivier (Coleoptera: Chrysomelidae) individuals and to conduct common-garden experiments to determine whether such variation is due to phenotypic plasticity or local adaptation. Location Four collection sites from the east coast of Australia were selected for our present field collections: Canberra (latitude 35°19' S), Bangalow (latitude 28°43' S), Beerburrum (latitude 26°58' S) and Lowmead (latitude 24°29' S). Museum specimens collected over the past 100 years and covering the same geographical area as the present field collections came from one state, one national and one private collection. Methods Body size (pronotum width) was measured for 118 field-collected beetles and 302 specimens from collections. We then reared larvae from the latitudinal extremes (Canberra and Lowmead) to determine whether the size cline was the result of phenotypic plasticity or evolved differences (= local adaptation) between sites. Results Beetles decreased in size with increasing latitude, representing a converse Bergmann cline. A decrease in developmental temperature produced larger adults for both Lowmead (low latitude) and Canberra (high latitude) individuals, and those from Lowmead were larger than those from Canberra when reared under identical conditions. Main conclusions The converse Bergmann cline in P. atomaria is likely to be the result of local adaptation to season length.

Stem cell–related gene expression in clonal populations of mesenchymal stromal cells from bone marrow

Decline in the frequency of potent mesenchymal stem cells (MSCs) has been implicated in ageing and degenerative diseases. Increasing the circulating stem cell population can lead to renewed recruitment of these potent cells at sites of damage. Therefore, identifying the ideal cells for ex vivo expansion will form a major pursuit of clinical applications. This study is a follow-up of previous work that demonstrated the occurrence of fast-growing multipotential cells from the bone marrow samples. To investigate the molecular processes involved in the existence of such varying populations, gene expression studies were performed between fast- and slow-growing clonal populations to identify potential genetic markers associated with stemness using the quantitative real-time polymerase chain reaction comprising a series of 84 genes related to stem cell pathways. A group of 10 genes were commonly overrepresented in the fast-growing stem cell clones. These included genes that encode proteins involved in the maintenance of embryonic and neural stem cell renewal (sex-determining region Y-box 2, notch homolog 1, and delta-like 3), proteins associated with chondrogenesis (aggrecan and collagen 2 A1), growth factors (bone morphogenetic protein 2 and insulin-like growth factor 1), an endodermal organogenesis protein (forkhead box a2), and proteins associated with cell-fate specification (fibroblast growth factor 2 and cell division cycle 2). Expression of diverse differentiation genes in MSC clones suggests that these commonly expressed genes may confer the maintenance of multipotentiality and self-renewal of MSCs.

Morphologic and genomic characterisation of the koala Chlamydia pneumoniae strain

Chlamydia pneumoniae is a common human and animal pathogen associated with a wide range of upper and lower respiratory tract infections. In more recent years there has been increasing evidence to suggest a link between C. pneumoniae and chronic diseases in humans, including atherosclerosis, stroke and Alzheimer’s disease. C. pneumoniae human strains show little genetic variation, indicating that the human-derived strain originated from a common ancestor in the recent past. Despite extensive information on the genetics and morphology processes of the human strain, knowledge concerning many other hosts (including marsupials, amphibians, reptiles and equines) remains virtually unexplored. The koala (Phascolarctos cinereus) is a native Australian marsupial under threat due to habitat loss, predation and disease. Koalas are very susceptible to chlamydial infections, most commonly affecting the conjunctiva, urogenital tract and/or respiratory tract. To address this gap in the literature, the present study (i) provides a detailed description of the morphologic and genomic architecture of the C. pneumoniae koala (and human) strain, and shows that the koala strain is microscopically, developmentally and genetically distinct from the C. pneumoniae human strain, and (ii) examines the genetic relationship of geographically diverse C. pneumoniae isolates from human, marsupial, amphibian, reptilian and equine hosts, and identifies two distinct lineages that have arisen from animal-to-human cross species transmissions. Chapter One of this thesis explores the scientific problem and aims of this study, while Chapter Two provides a detailed literature review of the background in this field of work. Chapter Three, the first results chapter, describes the morphology and developmental stages of C. pneumoniae koala isolate LPCoLN, as revealed by fluorescence and transmission electron microscopy. The profile of this isolate, when cultured in HEp-2 human epithelial cells, was quite different to the human AR39 isolate. Koala LPCoLN inclusions were larger; the elementary bodies did not have the characteristic pear-shaped appearance, and the developmental cycle was completed within a shorter period of time (as confirmed by quantitative real-time PCR). These in vitro findings might reflect biological differences between koala LPCoLN and human AR39 in vivo. Chapter Four describes the complete genome sequence of the koala respiratory pathogen, C. pneumoniae LPCoLN. This is the first animal isolate of C. pneumoniae to be fully-sequenced. The genome sequence provides new insights into genomic ‘plasticity’ (organisation), evolution and biology of koala LPCoLN, relative to four complete C. pneumoniae human genomes (AR39, CWL029, J138 and TW183). Koala LPCoLN contains a plasmid that is not shared with any of the human isolates, there is evidence of gene loss in nucleotide salvage pathways, and there are 10 hot spot genomic regions of variation that were previously not identified in the C. pneumoniae human genomes. Sequence (partial-length) from a second, independent, wild koala isolate (EBB) at several gene loci confirmed that the koala LPCoLN isolate was representative of a koala C. pneumoniae strain. The combined sequence data provides evidence that the C. pneumoniae animal (koala LPCoLN) genome is ancestral to the C. pneumoniae human genomes and that human infections may have originated from zoonotic infections. Chapter Five examines key genome components of the five C. pneumoniae genomes in more detail. This analysis reveals genomic features that are shared by and/or contribute to the broad ecological adaptability and evolution of C. pneumoniae. This analysis resulted in the identification of 65 gene sequences for further analysis of intraspecific variation, and revealed some interesting differences, including fragmentation, truncation and gene decay (loss of redundant ancestral traits). This study provides valuable insights into metabolic diversity, adaptation and evolution of C. pneumoniae. Chapter Six utilises a subset of 23 target genes identified from the previous genomic comparisons and makes a significant contribution to our understanding of genetic variability among C. pneumoniae human (11) and animal (6 amphibian, 5 reptilian, 1 equine and 7 marsupial hosts) isolates. It has been shown that the animal isolates are genetically diverse, unlike the human isolates that are virtually clonal. More convincing evidence that C. pneumoniae originated in animals and recently (in the last few hundred thousand years) crossed host species to infect humans is provided in this study. It is proposed that two animal-to-human cross species events have occurred in the context of the results, one evident by the nearly clonal human genotype circulating in the world today, and the other by a more animal-like genotype apparent in Indigenous Australians. Taken together, these data indicate that the C. pneumoniae koala LPCoLN isolate has morphologic and genomic characteristics that are distinct from the human isolates. These differences may affect the survival and activity of the C. pneumoniae koala pathogen in its natural host, in vivo. This study, by utilising the genetic diversity of C. pneumoniae, identified new genetic markers for distinguishing human and animal isolates. However, not all C. pneumoniae isolates were genetically diverse; in fact, several isolates were highly conserved, if not identical in sequence (i.e. Australian marsupials) emphasising that at some stage in the evolution of this pathogen, there has been an adaptation/s to a particular host, providing some stability in the genome. The outcomes of this study by experimental and bioinformatic approaches have significantly enhanced our knowledge of the biology of this pathogen and will advance opportunities for the investigation of novel vaccine targets, antimicrobial therapy, or blocking of pathogenic pathways.

Clonal characterization of bone marrow derived stem cells and their application for bone regeneration

Tissue engineering allows the design of functionally active cells within supportive bio-scaffolds to promote the development of new tissues such as cartilage and bone for the restoration of pathologically altered tissues. However, all bone tissue engineering applications are limited by a shortage of stem cells. The adult bone marrow stroma contains a subset of nonhematopoietic cells referred to as bone marrow mesenchymal stem cells (BMSCs). BMSCs are of interest because they are easily isolated from a small aspirate of bone marrow and readily generate single- cell-derived colonies. These cells have the capacity to undergo extensive replication in an undifferentiated state ex vivo. In addition, BMSCs have the potential to develop either in vitro or in vivo into distinct mesenchymal tissues, including bone, cartilage, fat, tendon, muscle, and marrow stroma. Thus, BMSCs are an attractive cell source for tissue engineering approaches. However, BMSCs are not homo- geneous and the quantity of stem cells decreases in the bone marrow in aged population. A sequential loss of lineage differentiation potential has been found in the mixed culture of bone marrow stromal cells due to a heterogenous popu- lation. Therefore, a number of studies have proposed that homogenous bone marrow stem cells can be generated from clonal culture of bone marrow cells and that BMSC clones have the greatest potential for the application of bone regeneration in vivo

Investigation into the introduction of clonal strains of Pseudomonas aeruginosa to the cystic fibrosis population by consumption of raw salad vegetables

Most salad vegetables are eaten fresh by consumers. However, raw vegetables may pose a risk of transmitting opportunistic bacteria to immunocompromised people, including cystic fibrosis (CF) patients. In particular, CF patients are vulnerable to chronic Pseudomonas aeruginosa lung infections and this organism is the primary cause of morbidity and mortality in this group. Clonal variants of P. aeruginosa have been identified as emerging threats to people afflicted with CF; however it has not yet been proven from where these clones originate or how they are transmitted. Due to the organisms‟ aquatic environmental niche, it was hypothesised that vegetables may be a source of these clones. To test this hypothesis, lettuce, tomatoes, mushrooms and bean sprout packages (n = 150) were analysed from a green grocer, supermarket and farmers‟ market within the Brisbane region, availability permitting. The internal and external surfaces of the vegetables were separately analysed for the presence of clonal strains of P. aeruginosa using washings and homogenisation techniques, respectively. This separation was in an attempt to establish which surface was contaminated, so that recommendations could be made to decrease or eliminate P. aeruginosa from these foods prior to consumption. Soil and water samples (n = 17) from local farms were also analysed for the presence of P. aeruginosa. Presumptive identification of isolates recovered from these environmental samples was made based on growth on Cetrimide agar at 42°C, presence of the cytochrome-oxidase enzyme and inability to ferment lactose. P. aeruginosa duplex real-time polymerase chain reaction assay (PAduplex) was performed on all bacterial isolates presumptively identified as P. aeruginosa. Enterobacterial repetitive intergenic consensus strain typing PCR (ERIC-PCR) was subsequently performed on confirmed bacterial isolates. Although 72 P. aeruginosa were isolated, none of these proved to be clonal strains. The significance of these findings is that vegetables may pose a risk of transmitting sporadic strains of P. aeruginosa to people afflicted with CF and possibly, other immunocompromised people.

Distributed plasticity analysis of steel frame structures comprising non-compact sections

Application of 'advanced analysis' methods suitable for non-linear analysis and design of steel frame structures permits direct and accurate determination of ultimate system strengths, without resort to simplified elastic methods of analysis and semi-empirical specification equations. However, the application of advanced analysis methods has previously been restricted to steel frames comprising only compact sections that are not influenced by the effects of local buckling. A research project has been conducted with the aim of developing concentrated plasticity methods suitable for practical advanced analysis of steel frame structures comprising non-compact sections. A primary objective was to produce a comprehensive range of new distributed plasticity analytical benchmark solutions for verification of the concentrated plasticity methods. A distributed plasticity model was developed using shell finite elements to explicitly account for the effects of gradual yielding and spread of plasticity, initial geometric imperfections, residual stresses and local buckling deformations. The model was verified by comparison with large-scale steel frame test results and a variety of existing analytical benchmark solutions. This paper presents a description of the distributed plasticity model and details of the verification study.

Myosin light chain kinase regulates synaptic plasticity and fear learning in the lateral amygdala

Learning and memory depend on signaling mole- cules that affect synaptic efficacy. The cytoskeleton has been implicated in regulating synaptic transmission but its role in learning and memory is poorly understood. Fear learning depends on plasticity in the lateral nucleus of the amygdala. We therefore examined whether the cytoskeletal-regulatory protein, myosin light chain kinase, might contribute to fear learning in the rat lateral amygdala. Microinjection of ML-7, a specific inhibitor of myosin light chain kinase, into the lateral nucleus of the amygdala before fear conditioning, but not immediately afterward, enhanced both short-term memory and long-term memory, suggesting that myosin light chain kinase is involved specifically in memory acquisition rather than in posttraining consolidation of memory. Myosin light chain kinase inhibitor had no effect on memory retrieval. Furthermore, ML-7 had no effect on behavior when the train- ing stimuli were presented in a non-associative manner. An- atomical studies showed that myosin light chain kinase is present in cells throughout lateral nucleus of the amygdala and is localized to dendritic shafts and spines that are postsynaptic to the projections from the auditory thalamus to lateral nucleus of the amygdala, a pathway specifically impli- cated in fear learning. Inhibition of myosin light chain kinase enhanced long-term potentiation, a physiological model of learning, in the auditory thalamic pathway to the lateral nu- cleus of the amygdala. When ML-7 was applied without as- sociative tetanic stimulation it had no effect on synaptic responses in lateral nucleus of the amygdala. Thus, myosin light chain kinase activity in lateral nucleus of the amygdala appears to normally suppress synaptic plasticity in the cir- cuits underlying fear learning, suggesting that myosin light chain kinase may help prevent the acquisition of irrelevant fears. Impairment of this mechanism could contribute to pathological fear learning.

Soiling the seed : microenvironment and epithelial mesenchymal plasticity

In the past decade we have come to appreciate that the microenvironment has the potential for major influence on the cancer cell. An extreme case for this occurs when the cancer cell changes its environment in the context of metastasis, where this may in part underpin the altered biology of cells in metasases. Increasing evidence suggests that changes in the cellular microenvironment contribute to tumourigenesis and metastasis, but the molecular basis of these alterations is not well understood. Reactive stroma provides oncogenic signals to facilitate tumourigenesis and metastasis—co-implantation of normal human epithelial cells in vivo with irradiated, carcinogen treated, or cancer derived fibroblasts leads to the enhancement or formation of malignant tumours.

Role of intratumoural heterogeneity in cancer drug resistance : molecular and clinical perspectives

Drug resistance continues to be a major barrier to the delivery of curative therapies in cancer. Historically, drug resistance has been associated with over-expression of drug transporters, changes in drug kinetics or amplification of drug targets. However, the emergence of resistance in patients treated with new-targeted therapies has provided new insight into the complexities underlying cancer drug resistance. Recent data now implicate intratumoural heterogeneity as a major driver of drug resistance. Single cell sequencing studies that identified multiple genetically distinct variants within human tumours clearly demonstrate the heterogeneous nature of human tumours. The major contributors to intratumoural heterogeneity are (i) genetic variation, (ii) stochastic processes, (iii) the microenvironment and (iv) cell and tissue plasticity. Each of these factors impacts on drug sensitivity. To deliver curative therapies to patients, modification of current therapeutic strategies to include methods that estimate intratumoural heterogeneity and plasticity will be essential.

Paradoxical enhancement of fear extinction memory and synaptic plasticity by inhibition of the histone acetyltransferase p300

It is well established that the coordinated regulation of activity-dependent gene expression by the histone acetyltransferase (HAT) family of transcriptional coactivators is crucial for the formation of contextual fear and spatial memory, and for hippocampal synaptic plasticity. However, no studies have examined the role of this epigenetic mechanism within the infralimbic prefrontal cortex (ILPFC), an area of the brain that is essential for the formation and consolidation of fear extinction memory. Here we report that a postextinction training infusion of a combined p300/CBP inhibitor (Lys-CoA-Tat), directly into the ILPFC, enhances fear extinction memory in mice. Our results also demonstrate that the HAT p300 is highly expressed within pyramidal neurons of the ILPFC and that the small-molecule p300-specific inhibitor (C646) infused into the ILPFC immediately after weak extinction training enhances the consolidation of fear extinction memory. C646 infused 6 h after extinction had no effect on fear extinction memory, nor did an immediate postextinction training infusion into the prelimbic prefrontal cortex. Consistent with the behavioral findings, inhibition of p300 activity within the ILPFC facilitated long-term potentiation (LTP) under stimulation conditions that do not evoke long-lasting LTP. These data suggest that one function of p300 activity within the ILPFC is to constrain synaptic plasticity, and that a reduction in the function of this HAT is required for the formation of fear extinction memory.