PSA 2016

These preprints were automatically compiled into a PDF from the collection of papers deposited in PhilSci-Archive in conjunction with the PSA 2016

Use of multiple platform “omics” datasets to define new biomarkers in oral cancer and to determine biological processes underpinning heterogeneity of the disease

Oral cancer in early stages (I and II) may be curable by surgery or radiation therapy alone but advanced stage disease (III and IV) has a relatively low survival rate. The pathogenic pathways that contribute to Oral Squamous Cell Carcinoma (OSCC) remain poorly characterised and the critical factor in the lack of prognostic improvement is that a significant proportion of cancers initially are asymptomatic lesions and are not diagnosed or treated until they reach an advanced stage. Hence, a clinically applicable gene expression signature is in high demand and improved characterization of the OSCC gene expression profile would constitute substantial progress. For OSCC, possible themes that might be addressed using microarray data include distinguishing the disease from normal at the molecular level; determining whether specific biomarkers or profiles are predictive for tumour behaviour; and identifying biologic pathways necessarily altered in tumourigenesis, potentially illuminating novel therapeutic targets. However, OSCC is a heterogeneous disease, making diagnostic biomarker development difficult. Although this phenotypic variation is striking when one compares OSCC from different geographic locales, little is known about the underpinning biological mechanisms. Cancer may be accompanied by the production and release of a substantial number of proteins, metabolites and/or hormones into the blood, saliva, and other body fluids that could also serve as useful markers for assessing prognosis, metastasis, monitoring treatment, and detecting malignant disease at an early stage. The primary aim of this thesis is to investigate metabolomic and transcriptomic profiles using multiple bioinformatics approaches and biological annotation tools in an attempt to identify specific biomarkers and prediction models for OSCC from each profile as well as from the interface outcomes of integrating the two platforms. Additional aims of the thesis go further to identify the mechanisms underlying the biological changes during tumorigenic transformation of OSCC, as well as to determine biological processes underpinning the heterogeneity of the disease among populations. Two review studies were carried out in this thesis. The review study of published transcriptomic profiles of OSCC specified several genes and pathways exhibiting substantially altered expression in cancerous versus noncancerous states across studies. However, the result of the review suggests not relying on the final set of genes published by the individual studies, but to access the raw data of each study and start subsequent analysis from that stage using unified bioinformatics approaches to acquire useful and complete understanding of the systems biology. The other review study focused on the metabolic profiles of OSCC and revealed a systemic metabolic response to cancer, which bears great potential for biomarker development and diagnosis of oral cancer. However, the metabolic signature still needs to improve specificity for OSCC from other types of cancer. In an attempt to detect a robust gene signature of OSCC overcoming the limitation of the transcriptomic review in accessing the raw data from the previous works, four public microarray raw datasets (comprising 365 tumour and normal samples) of OSCC were successfully integrated using ComBat data integration method in R software, determining the common set of genes, biomarkers, and the relative regulatory pathways possibly accountable for tumour transformation and growth in OSCC. Examination of the meta-analysis datasets showed several discriminating gene expression signatures for OSCC relative to normal oral mucosa; with a signature of 8 genes (MMP1, LAMC2, PTHLH, TPBG, GPD1L, MAL, TMPRSS11B, and SLC27A6) exhibiting the best discriminating performance and show potential as a diagnostic biomarker set. In addition, 32 biomarkers specific to OSCC and HNSCC were identified with the majority involved in extracellular matrix (ECM), interleukins, and peptidase activity where around 2/3 of them are located in the extracellular space and plasma membrane. Additionally, investigation of the interactive network created by merging metabolic and transcriptomic profiles highlighted the significant molecular and cellular biofunctions, pathways, and biomarkers distinguishing OSCC from normal oral mucosa. The results highlighted interactions of significantly altered expression of Dglucose, ethanol, glutathione, GABA, taurine, choline, creatinine, and pyruvate metabolites with the expressed PTGS2, IL1B, IL8, IL6, MMP1, MMP3, MMP9, SERPINE1, COL1A1, COL4A1, LAMC2, POSTN, ADAM12, CDKN2A, PDPN, TGM3, SPINK5, TIMP4, KRT19, and CRYAB biomarkers of OSCC. Such a pattern may represent a clinically useful surrogate for the presence of OSCC which might help in deciphering some of the obscure multifaceted mechanisms underlying carcinogenesis of OSCC which emerged from dysregulated genetic and metabolic system of the body. In an attempt to define pathways of importance in two phenotypically different forms of OSCC, transcriptomic analysis of OSCC from UK and Sri Lankan patients was undertaken. The development of OSCCs in UK and Sri Lankan populations appears largely mediated by similar biological pathways despite the differences related to race, ethnicity, lifestyle, and/or exposure to environmental carcinogens. However, results revealed a highly activated “Cell-mediated Immune Response” in Sri Lankan tumour and normal samples relative to UK cohorts which may reflects a role in resistance of patients to invasiveness, metastasis, and mortality observed in Sri Lankan relative to UK patients. In conclusion, multiple molecular profiles were able to identify a unique transcriptomic profile for OSCC and could further discriminate the tumour from normal oral mucosa on the basis of 8 genes. Altered expression of several metabolic and transcriptomic biomarkers specific for OSCC were identified, along with several dysregulated pathways and molecular processes found common in patient with oral cancer. Integrating both metabolomic and transcriptomic signatures revealed a promising strategy in analysing the concurrent perturbation in both genetic and metabolic systems of the body. Additional results revealed possible impact of specific supplementary dietary components in boosting the immune system of the body against invasion, progression, and metastasis of the disease. Further clinical studies are required to confirm and validate the current results

Psr1p interacts with SUN/sad1p and EB1/mal3p to establish the bipolar spindle

Regular Abstracts - Sunday Poster Presentations: no. 382During mitosis, interpolar microtubules from two spindle pole bodies (SPBs) interdigitate to create an antiparallel microtubule array for accommodating numerous regulatory proteins. Among these proteins, the kinesin-5 cut7p/Eg5 is the key player responsible for sliding apart antiparallel microtubules and thus helps in establishing the bipolar spindle. At the onset of mitosis, two SPBs are adjacent to one another with most microtubules running nearly parallel toward the nuclear envelope, creating an unfavorable microtubule configuration for the kinesin-5 kinesins. Therefore, how the cell organizes the antiparallel microtubule array in the first place at mitotic onset remains enigmatic. Here, we show that a novel protein psrp1p localizes to the SPB and plays a key role in organizing the antiparallel microtubule array. The absence of psr1+ leads to a transient monopolar spindle and massive chromosome loss. Further functional characterization demonstrates that psr1p is recruited to the SPB through interaction with the conserved SUN protein sad1p and that psr1p physically interacts with the conserved microtubule plus tip protein mal3p/EB1. These results suggest a model that psr1p serves as a linking protein between sad1p/SUN and mal3p/EB1 to allow microtubule plus ends to be coupled to the SPBs for organization of an antiparallel microtubule array. Thus, we conclude that psr1p is involved in organizing the antiparallel microtubule array in the first place at mitosis onset by interaction with SUN/sad1p and EB1/mal3p, thereby establishing the bipolar spindle.postprin

Removal of antagonistic spindle forces can rescue metaphase spindle length and reduce chromosome segregation defects

Regular Abstracts - Tuesday Poster Presentations: no. 1925Metaphase describes a phase of mitosis where chromosomes are attached and oriented on the bipolar spindle for subsequent segregation at anaphase. In diverse cell types, the metaphase spindle is maintained at a relatively constant length. Metaphase spindle length is proposed to be regulated by a balance of pushing and pulling forces generated by distinct sets of spindle microtubules and their interactions with motors and microtubule-associated proteins (MAPs). Spindle length appears important for chromosome segregation fidelity, as cells with shorter or longer than normal metaphase spindles, generated through deletion or inhibition of individual mitotic motors or MAPs, showed chromosome segregation defects. To test the force balance model of spindle length control and its effect on chromosome segregation, we applied fast microfluidic temperature-control with live-cell imaging to monitor the effect of switching off different combinations of antagonistic forces in the fission yeast metaphase spindle. We show that spindle midzone proteins kinesin-5 cut7p and microtubule bundler ase1p contribute to outward pushing forces, and spindle kinetochore proteins kinesin-8 klp5/6p and dam1p contribute to inward pulling forces. Removing these proteins individually led to aberrant metaphase spindle length and chromosome segregation defects. Removing these proteins in antagonistic combination rescued the defective spindle length and, in some combinations, also partially rescued chromosome segregation defects. Our results stress the importance of proper chromosome-to-microtubule attachment over spindle length regulation for proper chromosome segregation.postprin

The impact of the environment on DNA methylation in humans and zebrafish

DNA methylation is a chemical modification to the DNA strand, which can control gene expression. DNA methylation can be modified by the environment. For example, tobacco use substantially alters DNA methylation, and hence DNA methylation therefore provides a route through which the environment can lead to alterations in gene expression. Consequently, alterations to DNA methylation patterns have been associated with disease phenotypes in humans and other mammals. However, the precise role of environmentally-induced DNA methylation changes in the onset of pathological phenotypes is not often clearly defined. Here, we investigate the response of DNA methylation to two different environmental exposures – adulthood cannabis and in utero tobacco exposure. These environmental exposures are important because they are associated with adverse phenotypes – long-term cannabis use, particularly through adolescence, is associated with adverse psychosocial wellbeing. The development of conduct problem (CP, including autism and antisocial behaviour disorder) in childhood and adolescence is associated with exposure to tobacco during development (in utero). However, as yet, no studies have explored the role of DNA methylation in the link between these exposures and their associated phenotypic effects. Therefore, here we first asked whether DNA methylation in a longitudinal human cohort, the Christchurch Health and Development Study (CHDS), was altered in response to long term cannabis exposure, with and without tobacco. Using the Illumina EPIC array, we detected nominal differential DNA methylation in response to cannabis specifically, in genes associated with the following pathways; Cholinergic synapse, glutamatergic synapse and dopaminergic synapse. These observations show a potential mediation between DNA methylation in the observed phenotypic effects of cannabis use. In order to develop a tool to investigate this association further, we assessed the efficacy of a targeted, high throughput amplicon-based approach, bisulfite - based amplicon sequencing (BSAS), to replicate differential methylation at loci identified via EPIC array. We found that the ability of BSAS to detect equivalent differential methylation was locus-specific, meaning that it has value as a validation and replication tool, but that each locus for validation must be tested before being applied to a large study. Cannabis use is a contentious issue, mainly because of the debate around its therapeutic but also its psychoactive properties. In order to quantify the impact of both of its main cannabinoids, (-)-trans-∆9-tetrahydrocannabinol (THC) and cannabidiol (CBD) were exposed to zebrafish embryos. Following exposure reduced representation bisulfite sequencing (RRBS) was used to quantify their impact of each cannabinoid on DNA methylation. Differential methylation was found in each of the exposure groups, findings demonstrated the greatest number of methylation differences was in the CBD exposure group. CBD DNA methylation differences were found in genes that have roles in neurodevelopment, neurotransmission and behaviour. THC DNA methylati on differences on the other hand were found to alter genes with roles in the axon guidance and retinal ganglion pathways, supporting the role of DNA methylati on in the biological response to THC. Furthermore, our data revealed a role for both THC and CBD in brain related pathways, indicating that further research is needed to understand the full biological impacts of the two compounds. Next, to determine if tobacco-induced DNA methylation alterations are important in the link between in utero tobacco exposure and the development of CP, here, we applied BSAS to a subset of CHDS participants to assess DNA methylation in in utero-exposed individuals compared to non-exposed individuals, with and without CP. We selected a panel of genes with known roles in in utero neurodevelopment, and identified differential methylation that was specific to individuals exposed to tobacco during development, who had high CP scores. We imply that developmentally-induced DNA methylati on alterations may be playing a role in the development of CP in exposed individuals. To investigate this further, we applied a genome-wide approach (EPIC array) to a larger cohort and identified nominal significance at genes involved in global developmental delay and neurological disorders, indicating that, in addition to CP, visual impairment may be a phenotypic response to in utero tobacco exposure. Lastly, we discuss whether DNA methylation analysis in whole blood samples is able to predict DNA methylation changes in brain tissue. To answer this question, we used publicly available data of the top lists of differentially methylated CpG sites in blood and brain tissue from individuals with schizophrenia. We found that, the methylation of individual CpG sites did not replicate between tissues, the genes and pathways that have biological relevance to schizophrenia (e.g. mTOR signalling pathway and the mRNA surveillance pathway) were identified in both tissue types, demonstrating the value and applicability of whole blood as a proxy tissue. Overall, here we demonstrate a role for DNA methylation in the biological response to cannabis, and a link between in utero tobacco exposure and development of CP. Further research is required to understand the mechanism through which these changes can contribute to disease

Applying the Free-Energy Principle to Complex Adaptive Systems

The free energy principle is a mathematical theory of the behaviour of self-organising systems that originally gained prominence as a unified model of the brain. Since then, the theory has been applied to a plethora of biological phenomena, extending from single-celled and multicellular organisms through to niche construction and human culture, and even the emergence of life itself. The free energy principle tells us that perception and action operate synergistically to minimize an organism’s exposure to surprising biological states, which are more likely to lead to decay. A key corollary of this hypothesis is active inference—the idea that all behavior involves the selective sampling of sensory data so that we experience what we expect to (in order to avoid surprises). Simply put, we act upon the world to fulfill our expectations. It is now widely recognized that the implications of the free energy principle for our understanding of the human mind and behavior are far-reaching and profound. To date, however, its capacity to extend beyond our brain—to more generally explain living and other complex adaptive systems—has only just begun to be explored. The aim of this collection is to showcase the breadth of the free energy principle as a unified theory of complex adaptive systems—conscious, social, living, or not