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.

Prostatacancer är en vanligt förekommande sjukdom i Sverige. För att få bukt med sjukdomen krävs att mer resurser sätts in för att effektivisera forskningen inom området. Ett samarbete mellan Karolinska Institutet, Kungliga Tekniska Högskolan och Janssen- Cilag AB har resulterat i LUPA, ett informationsvisualiseringssystem, med ambitionen att åstadkomma en effektivare process för hypotesgenerering gällande prostatacancer. Det här projektet handlar om att validera systemets funktion för den tilltänkta målgruppen; det vill säga att göra hypotesgenereringsprocessen enklare och mer effektiv för forskare som arbetar med prostatacancerdata i Stockholms län. Från resultatet kan man se att ett informationsvisualiseringsverktyg skulle vara användbart för att effektivisera dagens cancerforskningsprocess. Tidsåtgången för nystartade projekts första grundläggande steg skulle kunna reduceras drastiskt, särskilt stegen för införskaffning av data och datamanipulation. Dessutom upplevdes systemet främja hypotesgenereringsprocessen genom enkelheten att kvickt generera nya visuella strukturer för olika variabler. Slutligen visade det här projektet att organiserad och ren rådata är sällsynt. Testdeltagarna uppskattade informationsvisualiseringen men såg mest potential i dess möjlighet att städa och strukturera data. / Prostate cancer is a common disease in Sweden. In order to overcome the disease, more resources must be invested to streamline research in the field. A collaboration between Karolinska Institutet, the Royal Institute of Technology and Janssen-Cilag AB has resulted in LUPA, an information visualization system with the ambition of achieving a more efficient process for hypothesis generation regarding prostate cancer. This thesis project is about validating the system's function for the intended target group; that is, to make the hypothesis generation process simpler and more efficient for researchers working with prostate cancer data in Stockholm County. From the results, it can be seen that an information visualization tool would be useful to streamline the current cancer research process. The time required for the first basic steps of new projects could be drastically reduced, especially the steps for data acquisition and data manipulation. In addition, the system was perceived to promote the hypothesis generation process through the simple methods of quickly generating new visual structures for different variables. Finally, this project showed that organized and clean raw data is rare. The test participants appreciated the information visualization but saw the most potential in its ability to clean and structure data.

Current cancer research is beginning to address the psychosocial implications of a prostate cancer (PCa) diagnosis from not just the perspective of quality of life of the patient, but of his partner as well. Such inquiries have created novel intervention programs aiming to alleviate the adverse side effects that a PCa diagnosis may inflict on the couple. Assessing efficacy of couple-based interventions, however, has been a difficult task due to the lack of homogeneity between studies regarding the operationalization process of primary outcome variables, as well as the instruments being used to measure them. This thesis, in response, aims to provide a detailed assessment of how previous interventions operationalized their targeted variables, the reported psychometric analysis of the instruments of measurement, and which instruments yielded statistically significant results. A narrative review was conducted using a database search strategy to collect articles regarding couple-based interventions that focused on outcomes related to PCa diagnosis, treatment, and survivorship. Out of the ten articles that passed the screening method, forty-two outcomes were identified, ranging from physical, social, and mental well-being of the couple, to the impact PCa had on their relationship quality. The outcomes were grouped into eight categories: quality of life, appraisal of PCa outcomes, sexual/physical well-being, relationship assessment, coping, mental health, knowledge, and distress. Various scales were used to measure similar outcomes with some articles failing to report on the psychometric properties of their chosen instruments. This assessment aims to provide future researchers with an indication as to what outcomes have been previously targeted and their corresponding methods of operationalization, categorization, and analysis. The multitude of assessed outcomes, the lack of uniformity on best practices in PCa couple intervention research, and the general failure to report on reliability and validity of measures may serve as significant barriers to producing high-quality evidence that can inform the development of future research and practice. This review provides the research community an aid in the development of behavioral interventions, and potentially, practice, via offering recommendations on certain outcomes that remain underreported within interventions. It is the ultimate aim of this project to assist in fostering a true public health for all.

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