High throughput estimation of functional cell activities reveals disease mechanisms and predicts relevant clinical outcomes

This work is supported by grants BIO2014- 57291-R from the Spanish Ministry of Economy and Competitiveness and “Plataforma de Recursos Biomoleculares y Bioinformáticos” PT13/0001/0007 from the ISCIII, both co-funded with European Regional Development Funds (ERDF); PROMETEOII/2014/025 from the Generalitat Valenciana (GVA-FEDER); Fundació la Marató TV3 (ref. 20133134); and EU H2020- INFRADEV-1-2015-1 ELIXIR-EXCELERATE (ref. 676559) and EU FP7-People ITN Marie Curie Project (ref 316861)

High throughput estimation of functional cell activities reveals disease mechanisms and predicts relevant clinical outcomes

This work is supported by grants BIO2014- 57291-R from the Spanish Ministry of Economy and Competitiveness and “Plataforma de Recursos Biomoleculares y Bioinformáticos” PT13/0001/0007 from the ISCIII, both co-funded with European Regional Development Funds (ERDF); PROMETEOII/2014/025 from the Generalitat Valenciana (GVA-FEDER); Fundació la Marató TV3 (ref. 20133134); and EU H2020- INFRADEV-1-2015-1 ELIXIR-EXCELERATE (ref. 676559) and EU FP7-People ITN Marie Curie Project (ref 316861)

Characterisation of chromatin modifiers in endometrial cancer

Chromatin organization is a critical regulator of gene expression and cell phenotype, and is frequently dysregulated in cancer. Endometrial cancer (EC) is the most common gynecological malignancy, and casues significant morbidity and mortality. EC is notable for recurrent alterations in chromatin including the ARID1A gene – a key component of the SWI/SNF remodeling complex – has emerged as a prevalent driver in EC, along with other remodelers such as CHD4 and BCOR. However, a systematic analysis of chromatin modifier alterations and their functional consequences in EC has not been done. This thesis presents a comprehensive investigation of genomic alterations in chromatin modifiers using whole genome sequencing (WGS) data from the Genomics England(GEL) 100,000 Genomes Project, the largest EC cohort to date. I demonstrate that while mutational processes vary across molecular subtypes, numerous chromatin modifiers are consistently altered across all subtypes. These genomic alterations frequently occur in different subunits of the same complex, such as alterations in CHD3, CHD4 and MBD3, subunits of the ATP-dependent chromatin remodeling complex NuRD. Additionally, I examine the correlation between driver mutations and patient survival, revealing that mutations in PBRM1 and CHD4 are associated with an increased risk of death after accounting for age, molecular subtype, and tumor mutation and copy number alteration burden. To complement the correlative analysis, I employ CRISPR-Cas9 gene editing to study the functional consequences of perturbations in selected chromatin modifiers (ARID1A, ARID1B, ARID5B, EP300, KMT2C, and SETD1B) in normal and malignant endometrial cells using transcriptomic and chromatin accessibility data. Furthermore, I explore the implications of the N1459S BCOR mutation, a hotspot mutation near-unique to EC. Considering the frequent occurrence of ARID1A mutations in malignant tissues and their absence in normal endometrium, I investigate tumor heterogeneity in endometrial cancer. I discuss the limitations of current methodologies and propose a deep learning approach to uncover the hidden evolutionary trajectories. With additional research, this approach could potentially facilitate understanding the sequence in which driver alterations occur. In summary, this work presents a resource for investigating chromatin organization in EC. The functional analyses using gene editing techniques confirm that EC-associated drivers disrupt essential cellular processes involved in oncogenesis. By providing the first systematic correlative and functional analyses of chromatin modifiers in EC, this thesis offers novel insights into EC biology

Multi-omics Integration for Gene Fusion Discovery and Somatic Mutation Haplotyping in Cancer

Cancer is a disease caused by changes to the genome and dysregulation of gene expression. Among many types of mutations, including point mutations, small insertions and deletions, large scale structural variants, and copy number changes, gene fusions are another category of genomic and transcriptomic alteration that can lead to cancer and which can serve as therapeutic targets. We studied gene fusion events using data from The Cancer Genome Atlas, including over 9,000 patients from 33 cancer types, finding patterns of gene fusion events and dysregulation of gene expression within and across cancer types. With data from the CoMMpass study (Multiple Myeloma Research Foundation), we generated the largest gene fusion study in multiple myeloma (742 patients), which is the second most common type of blood cancer, and which is driven by recurrent translocations. We then developed a novel tool for analyzing the haplotype context of somatic mutations. Linked-read whole genome sequencing enables haplotype resolution for analyzing somatic mutation patterns, which is lost during typical short-read sequencing and alignment. We analyzed a cohort of 14 multiple myeloma patients across disease stages, phasing three-quarters of high confidence somatic mutations and enabling us to interpret clonal evolution models at higher resolution. Finally, we also studied the co-evolution of the multiple myeloma tumor and microenvironment using single-cell RNA-sequencing, finding distinct patterns of tumor subclone evolution between disease stages in 14 patients. Our methods and results demonstrate the power of integrating data types to study complex and dynamic evolutionary pressures in cancer and point to future directions of research that aim to bridge gaps in research and clinical applications

MicroRNA and Cancer

MicroRNAs (miRs) are small noncoding RNAs that function as post-transcriptional regulators of gene expression and have important roles in almost all biological pathways. Deregulated miR expression has been detected in numerous cancers, where miRs act as both oncogene and tumor suppressors. Due to their important roles in tumorigenesis, miRs have been investigated as prognostic and diagnostic biomarkers and as useful targets for therapeutic intervention. From a therapeutic point of view, two modalities can serve to rectify gene networks in cancer cells. For oncomiRs, a rational means is downregulation through antagomirs. Moreover, observations of the pathological reductions in tumor-suppressive miRs have inspired the concept of “miR replacement therapy” to enhance the amount of these miRs, thereby restoring them to normal levels. However, the clinical applicability of miR-based therapies is severely limited by the lack of effective delivery systems. Therefore, to understand the role of this new class of regulators, we need to identify the mRNA targets regulated by individual miRs as well as to develop specific, efficient, and safe delivery systems for therapeutic miRs

Molecular Profiling of Prevention Strategies Using Rodent Models of Colorectal Cancer

Colorectal cancer (CRC) is the third most lethal cancer worldwide, caused by both genetic and environmental exposures, with underlying mechanisms that dovetail genetic, epigenetic, metabolomic, and gut microbiome influences. Adenomatous polyposis coli (Apc) is a tumor suppressor and a negative regulator of Wnt/b-catenin signaling, found mutated in over 70% of CRC cases. The current dissertation used two rodent models, one genetic and one environmental, in the context of cancer prevention to study the etiology of CRC and provide preclinical mechanistic insights. The environmental model incorporated a cooked meat derived mutagen, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), to mediate multi-organ carcinogenesis in the rat. The study focused on microRNA (miRNA) profiling in the PhIP model. miRNAs are stable negative regulators of gene targets, and play important roles in cancer and pluripotency. Rat tumor profiles and human pan-cancer datasets defined a miRNA signature of PhIP-induced multi-organ tumorigenesis. The miR-21^high/miR-126^low/miR-29c^low/miR-215^low/miR-145^low signature was associated with poor survival and reduced Klf4 levels, being predictive of possible environmental exposure and paradigm-shifting from ‘genotoxic’ to epigenetic regulation of dietary heterocyclic amines. A genetic angle was provided by the Apc-mutant polyposis in rat colon (Pirc) model, which harbors a large number of polyps in the colon, and recapitulates human familial adenomatous polyposis (FAP). The second study employed sequencing technology for mRNA, miRNA and 16S rRNA to assess the crosstalk between host, gut microbiota, and a cancer preventive diet in the Pirc model. Dietary spinach reduced tumor outcomes significantly in the Pirc model, and reversed host genetic effects on microbiota. mRNA and miRNA analyses revealed the importance of an inflammatory response in Pirc tumorigenesis, and implicated specific miRNA-mRNA associations, such as miR-145/Serpine1 and miR-34a/Klf4. The third study utilized untargeted metabolomics to investigate metabolic changes in the Pirc model along with the prevention effects of dietary spinach. The preliminary results indicated purine and lipid metabolism are important for tumorigenesis and prevention of colorectal cancer. Current technologies are providing new insights at the molecular level, incorporating ‘big data’ with genetic and phenotypic read-outs, to identify underlying leads associated with a designed dietary prevention strategy, which might help to mitigate the worldwide burden of CRC

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

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

Distinct functional roles of microRNA-23b and microRNA-26a in breast cancer pathogenesis

Tumour formation and metastasis are distinct processes that arise from cumulative alterations of genomic and epigenetic regulation. Uncontrolled modulation of cell cycle-related genes is crucial to tumour growth and additional genetic modifications provide cancer cells with motile and invasive phenotypes, leading to metastatic dissemination. The cytoskeleton constitutes the structural support to cell motility, invasion and adhesion. Among the best-characterised cytoskeletal modulators are the p21-activated kinases (PAKs). In breast cancer (BC), the HER2 pathway controls the cytoskeletal dynamics and cell motility via PAK activation, through distinct downstream signaling mechanisms. MicroRNAs (miRNAs) are small, non-coding RNAs that modulate gene expression post-transcriptionally. MiRNAs dysregulation can contribute to tumorigenicity, cell motility and metastasis by affecting relevant signaling pathways. We identified PAK2 as target of both miR-23b and miR-26a, implicating a direct role for these miRNAs in cytoskeletal remodeling. Experimentally, expression of miR-23b and miR-26a in BC cells promotes focal adhesions and cell spreading on substrates, but miR-23b alone controls cell-cell junctions and lamellipodia formation. Despite sharing the same target, the two miRNAs show additional distinct functions. MiR-26a overexpression in BC leads to formation of aneuploid cells associated with higher tumorigenicity. On the other hand, miR-23b inhibition enhances BC cell migration, invasion and metastasis in vivo. Clinically, low miR-23b levels correlate with metastatic development in BC patients. Mechanistically, growth factor-mediated signal transductions activate the transcription factor AP-1 and we show that this transcriptionally reduces miR-23b expression thus releasing PAK2 from its translational inhibition. The distinct cellular phenotypes described by the two miRNAs indicate that their global functions depend upon all the genes they regulate. Using RNA-sequencing and luciferase reporter assays, we validated a subset of genes as direct targets of either the two miRNAs. These genes are crucial to distinct molecular pathways and contribute to elucidate the observed phenotypes induced by miR-23b and miR-26a modulation.Open Acces

Unraveling the intricacies of spatial organization of the ErbB receptors and downstream signaling pathways

Faced with the complexity of diseases such as cancer which has 1012 mutations, altering gene expression, and disrupting regulatory networks, there has been a paradigm shift in the biological sciences and what has emerged is a much more quantitative field of biology. Mathematical modeling can aid in biological discovery with the development of predictive models that provide future direction for experimentalist. In this work, I have contributed to the development of novel computational approaches which explore mechanisms of receptor aggregation and predict the effects of downstream signaling. The coupled spatial non-spatial simulation algorithm, CSNSA is a tool that I took part in developing, which implements a spatial kinetic Monte Carlo for capturing receptor interactions on the cell membrane with Gillespies stochastic simulation algorithm, SSA, for temporal cytosolic interactions. Using this framework we determine that receptor clustering significantly enhances downstream signaling. In the next study the goal was to understand mechanisms of clustering. Cytoskeletal interactions with mobile proteins are known to hinder diffusion. Using a Monte Carlo approach we simulate these interactions, determining at what cytoskeletal distribution and receptor concentration optimal clustering occurs and when it is inhibited. We investigate oligomerization induced trapping to determine mechanisms of clustering, and our results show that the cytoskeletal interactions lead to receptor clustering. After exploring the mechanisms of clustering we determine how receptor aggregation effects downstream signaling. We further proceed by implementing the adaptively coarse grained Monte Carlo, ACGMC to determine if \u27receptor-sharing\u27 occurs when receptors are clustered. In our proposed \u27receptor-sharing\u27 mechanism a cytosolic species binds with a receptor then disassociates and rebinds a neighboring receptor. We tested our hypothesis using a novel computational approach, the ACGMC, an algorithm which enables the spatial temporal evolution of the system in three dimensions by using a coarse graining approach. In this framework we are modeling EGFR reaction-diffusion events on the plasma membrane while capturing the spatial-temporal dynamics of proteins in the cytosol. From this framework we observe \u27receptor-sharing\u27 which may be an important mechanism in the regulation and overall efficiency of signal transduction. In summary, I have helped to develop predictive computational tools that take systems biology in a new direction.\u2