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Although the understanding of genetic predisposition to prostate cancer (PCa) has been improved through genome-wide association studies (GWAS), little is known about the biological implication of germline variants residing in coding or non-coding regions in cancer development and progression. Our hypothesis is that inherited variants may predispose to specific early recurrent genomic events observed in PCa adenocarcinomas, possibly in the context of variable androgen receptor (AR) signaling that changes during a man’s lifetime. Recent in silico analysis by our group on potential association between germline variants and PCa specific somatic lesions identified a non-coding polymorphic regulatory element at the 7p14.3 locus associated with DNA repair and hormone regulated transcript levels and with an early recurrent prostate cancer specific somatic mutation in the Speckle-Type POZ protein (SPOP) gene (OR=5.54, P=1.22e-08) in human prostate tissue data. In order to functionally characterize the polymorphic 7p14.3 locus (rs1376350, single nucleotide polymorphism, G>A), we set up to establish isogenic cell lines harboring the minor allele by using the CRISPR/Cas9 system. In parallel, CRISPR/Cas9 system was used to knock out different portion of the region encompassing the 7p14.3 variant and to eliminate transcription factors (TFs) binding sites that were identified from previous in silico analysis (i.e. AR and CCAAT/Enhancer Binding Protein (C/EBP) beta (CEBPβ)). The transcriptomes of edited pools and edited single clones from macrodeletion (731 bp), microdeletion (50 bp) and alterations of TFs binding sites were analyzed and compared to the transcriptomes of isogenic cells heterozygous (A/G) and homozygous (A/A) for the minor allele A of the risk variant rs1376350 (with or without AR overexpression). These data identified a set of genes scattered throughout the genome with the same pattern of deregulation suggesting the implication of the variant on the regulation of genes residing in different chromosomes. Additionally, ChIP-qPCR experiments for histone modification supported the identification of the 7p14.3 locus with enhancer activity. Furthermore, ChIP-qPCR of histone mark associated with transcriptional activation or repression in isogenic cells harboring the minor allele A upon AR overexpression showed that the activity of the locus is higher for the minor allele A compared to G, independently from AR activation. Despite the limitations of our model and the current lack of validation in other cells, we confirmed that some of the differentially expressed genes that emerged from the comparative analysis of edited cells are deregulated in human normal and tumor prostate samples as well. This work is a proof of concept of germline predisposition to molecularly distinct cancer subclasses and has the potential to nominate new mechanisms of cancer development. Future work aims to elucidate the mechanisms implicated in the deregulation of the transcriptome by combining the information obtained until now with potential new players that we expect to identify by Mass Spectrometry experiments. To clarify the link between the 7p14.3 variant and the somatic mutations in SPOP, we plan to express mutant SPOP in isogenic cells harboring the minor allele and to asses DNA damage response upon overexpression or silencing of TFs binding at and around the rs1376350 variant. My work is an example of how the CRISPR/Cas9 system can be used to develop a technical framework with convergent approaches to functionally characterize polymorphic regulatory regions including but not limited to the establishment of isogenic cells upon single nucleotide editing.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第24089号 / 医博第4865号 / 新制||医||1059(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 武藤 学, 教授 万代 昌紀, 教授 松田 文彦 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 新制・課程博士 / 博士(生命科学) / 甲第25448号 / 生博第519号 / 新制||生||69(附属図書館) / 京都大学大学院生命科学研究科統合生命科学専攻 / (主査)教授 河内 孝之, 教授 荒木 崇, 教授 中野 雄司 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

Cancer is a complex disease shaped by a heterogeneous landscape of inherited genetic variants and acquired somatic aberrations. Although specific patterns of somatic aberrations within key pathways are recognized as hallmarks of many cancers, and mounting evidence suggests a significant interplay between germline and somatic variants, the intricate relationship between germline predisposition and the disruption of these pathways remains poorly understood. Here, I present an integrative approach using multi-omics data to functionally characterize germline variants and explore the heterogeneous landscape of somatic mutations, with the aim of establish mechanistic links between functional variants and the disruption of cancer-related biological processes. To enable the identification of functional variants, I initially performed a comprehensive characterization of functionally annotated transcriptional regulatory elements, establishing a hierarchy of ‘consensus’ elements across multiple levels of abstraction. This analysis generated a vast collection of consensus promoters, enhancers, and active enhancers, spanning 198 cell lines and 38 tissue types, with aggregate data providing global consensus definitions for each element type. Additionally, ‘total binding affinity’ method was employed, integrating 1000 Genomes Project genotype data and thousands of transcription factor binding motifs, to further characterize and functionally annotate these regulatory elements. The results generated from this analysis can be interactively explored and visualized through the CONREL web application. To allow effective annotation of individual’s ancestry, I developed and successfully employed an improved version of EthSEQ (version 3), an R package that provides a rapid and reliable pipeline for ancestry annotation. Accurate stratification of individual ancestry is essential for correctly interpreting the impact of genomic variations in associations studies. EthSEQ version 3 was successfully utilized to determine the genetic ancestry of over 500 pediatric patients diagnosed with 11 different tumor types, enabling further investigation into the genetic landscape of patients confidently identified as of European ancestry.To further investigate into the interplay between germline and somatic variants, I conducted genome-wide association studies across 33 cancer types characterized by The Cancer Genome Atlas, using binary traits defined by somatic aberration profiles in ten oncogenic signaling pathways. Functional links between associated variants and somatic profiles were investigated through cis-eQTL data to identify regulatory interactions with pathway-related genes. Additionally, using GWAS summary statistics I employed polygenic scores to examine the contribution of germline genetic variation to somatic molecular profiles, tumor subtypes, and clinical outcomes such as patient survival and tumor aggressiveness. Polygenic scores were validated using external data from PCAWG and CCLE datasets. Lastly, to explore the heterogeneity of somatic mutational profiles, I employed a network-based approach to propagate somatic alterations through a molecular interaction network, aiming to reveal novel patterns of somatic alteration with potential significance in cancer. I then conducted a series of GWAS analyses, utilizing traits defined by combinations of these propagated somatic scores across genes involved in well-defined DNA repair pathways. Overall, I demonstrate that germline genetics can describe patients’ genetic liability to develop specific cancer molecular and clinical profiles. Understanding the functional roles of genetic variants can provide valuable insights into the biological mechanisms underlying a disease or trait.

I used the C. elegans genetic model to examine the role of ceramide biosynthesis (sphingolipid pathway) and iron regulation and found that each process impacts germline development and function. Using a sphingolipid specific antibody mAb15B4, I found that sphingolipids are associated with germ granules (P granules) within C. elegans and zebrafish; thus, suggesting conservation of macromolecules associated with germ granules. Phenotype analysis of ceramide biosynthesis mutants in C. elegans revealed that this pathway is essential for normal germline function in the aging adult hermaphrodite; specifically, precocious germline senescence was observed. Furthermore, I found that disruption of ceramide biosynthesis, via the hyl-2 deletion mutation, negatively impacts mAb15B4 localization at the P granules. Through genetic suppression analysis, I determined that insulin signaling and lipid biosynthesis can modulate the mAb15B4 localization to P granules. Additional, phenotype analysis showed that ceramide biosynthesis dysfunction decreased fecundity, and led to germline structure defects and uterine tumors. Through suppression analysis, I determined that modulation of the insulin signaling pathway suppressed the precocious germline senescence due to ceramide biosynthesis dysfunction. Since the presence of uterine tumors is associated with reproductive senescence I concluded that ceramide biosynthesis has a role in germline maintenance in the aging of the germline (germline senescence). The other important fate of a germ cell is programmed cell death. Apoptosis, which occurs through a highly conserved molecular pathway, is a normal component of growth and homeostatic processes. I used C. elegans to gain a greater understanding of the cisd gene function. The C. elegans genome has three previously uncharacterized cisd genes which code for CISD-1 (homology to vertebrate mitoNEET/CISD1 and NAF-1/CISD2) and CISD-3.1 and CISD-3.2 (homology to vertebrate Miner2/CISD3). I determined that independent disruption of the cisd genes resulted in a significant increase in the number of cell corpses within the adult hermaphrodite germline. Genetic analysis was used to examine the dysfunction of cisd-1 relative to the cell death canonical pathway genes. The increased gamete cell death in the cisd-1 hermaphrodite is suppressed by the ced-9 (Bcl-2 homolog) gain-of-function and requires functional CED-3 (caspase) and CED-4 (APAF). Additionally, the increased germ cell programmed cell death is facilitated by the pro-apoptotic, CED-9-binding protein, CED-13. Further analysis of the cisd gene family members show that cisd-3.2 dysfunction leads to germline defects and reproductive dysfunction, suggesting defects in germline stem cell proliferation. Expression analysis using the cisd promoters to drive fluorescent protein reporters showed that the cisd gene family is expressed in various tissues including the germline; fusion protein analysis showed that CISD-3 is mitochondrial localized. I propose that cisd-3.2 germline defects are a result of abnormal mitochondrial function. Combined, this work is significant because it identifies sphingolipids as a new component of embryonic P granules, a role for ceramide biosynthesis in reproductive senescence, and places the cisd gene family members as regulators of physiological germline programmed cell death acting through CED-13 and the core apoptotic machinery. Furthermore, it is the first study to show that a CISD3 protein family member is required for normal germline function. These findings support the idea that ceramide biosynthesis and iron regulation are core components in germline development and function.

NEET proteins belong to a unique class of [2Fe-2S] cluster proteins that have been shown to participate in various biological processes such as regulating iron, reactive oxygen species and apoptosis within the cell and are localized to the mitochondria. Disruption of the mitochondrial NEET proteins are associated with different human diseases such as obesity, neurodegeneration, cancer and diabetes. In humans, a missense mutation in the CISD2 gene results in a heritable multisystem disorder termed Wolfram syndrome 2 (WFS2), a disease which displays an early onset of juvenile diabetes and various neuropsychiatric disorders. The C. elegans genome contains three previously uncharacterized cisd genes: cisd-1, which has homology to the human CISD1 and CISD2, and cisd-3.1 and cisd-3.2, both of which have homology to the human CISD3. Disruption of the cisd-3 gene(s) function results in mis-regulation of proteostasis in the mitochondria, whereas cisd-1 and cisd-3.1 disruption impacts proteostasis in the endoplasmic reticulum. Reduction of cisd-3.2 gene function also leads to a developmental delay in C. elegans. A knockout mutation of the cisd-3.2(pn68) gene function results in various germline defects including delayed development progression and morphological defects. Furthermore, I show the cisd gene(s) and protein expression profiles is present relative to sex, tissue type and developmental stages. This work is significant because it provides further insight of the essential role of CISD-3 relative to C. elegans. Furthermore, my studies can contribute to new genetic discoveries that will widen the scientific research relative to NEET protein family studies.