Germ line mutations of the gene encoding the tricarboxylic acid (TCA) cycle enzyme fumarate hydratase (FH) cause a hereditary cancer syndrome known as hereditary leiomyomatosis and renal cell cancer (HLRCC). HLRCC-associated tumors harbor biallelic FH inactivation that results in the accumulation of the TCA cycle metabolite fumarate. Although it is known that fumarate accumulation can alter cellular signaling, if and how fumarate confers a growth advantage remain unclear. Here we show that fumarate accumulation confers a chronic proliferative signal by disrupting cellular iron signaling. Specifically, fumarate covalently modifies cysteine residues on iron regulatory protein 2 (IRP2), rendering it unable to repress ferritin mRNA translation. Simultaneously, fumarate increases ferritin gene transcription by activating the NRF2 (nuclear factor [erythroid-derived 2]-like 2) transcription factor. In turn, increased ferritin protein levels promote the expression of the promitotic transcription factor FOXM1 (Forkhead box protein M1). Consistently, clinical HLRCC tissues showed increased expression levels of both FOXM1 and its proliferation-associated target genes. This finding demonstrates how FH inactivation can endow cells with a growth advantage.

<p>In eukaryotic cells, DNA is packaged into a structure named chromatin which contains DNA and proteins. Nucleosomes are building blocks of chromatin and contain DNA wrapped around a histone octamer. Chromatin modifications (histone post-translational modifications and histone variants) play central roles in various cellular processes including gene expression and DNA damage response. Chromatin modifying enzymes use metabolites as co-substrates and co-factors, and changes in metabolic pathways and metabolite availability affects chromatin modifications and chromatin-associated functions. Moreover, recent studies have uncovered direct roles of metabolic enzymes in chromatin-associated functions. Fumarase, a TCA cycle enzyme that catalyzes the reversible conversion of fumarate to malate in mitochondria (a hydration reaction), is an example of an enzyme with dual functions in metabolism and genome integrity. Cytoplasmic fraction of yeast fumarase, Fum1p, localizes to the nucleus and promotes growth upon DNA damage. Fum1p promotes homologous recombination by enhancing DNA end resection. Human fumarase is involved in DNA repair by non-homologous end joining. Here, we provide evidence that yeast Fum1p and the histone variant Htz1p are also involved in DNA replication stress response and DNA repair by non-homologous end joining (NHEJ). Using mutants lacking the histone variant <i>HTZ1</i>, we show that high cellular levels of fumarate, by deletion of <i>FUM1</i> or addition of exogenous fumarate, suppressed the sensitivity to DNA replication stress by modulation of activity of Jhd2p. This suppression required sensors and mediators of the intra-S phase checkpoint, but not factors involved in the processing of replication intermediates. These results imply that high cellular levels of fumarate can confer resistance to DNA replication stress by bypassing or complementing the defects caused by loss of <i>HTZ1</i> and replication fork processing factors. We also show that upon induction of DSBs, exogenous fumarate conferred resistance to mutants with defects in NHEJ, early steps of homologous recombination (DNA end resection pathway) or late steps of homologous recombination (strand invasion and exchange). Taken together, these results link the metabolic enzyme fumarase and the metabolite fumarate to DNA damage response and show that modulation of DNA damage response by regulating activity of chromatin modifying enzymes is a plausible pathway linking metabolism and nutrient availability to chromatin-associated functions like genome integrity.<br><a></a></p>

Le cancer du rein papillaire de type 2 (PRCC2) est un cancer très agressif avec un potentiel métastatique élevé et pour lequel il n’y a pas de traitement efficace. La forme héréditaire de PRCC2 est associée au syndrome rare de la léiomyomatose cutanéo-utérine héréditaire (HLRCC). HLRCC est due à une mutation germinale hétérozygote du gène Fumarate Hydratase (FH) qui code l'enzyme du cycle de Krebs, la Fumarase. Le déficit en fumarase induit l’accumulation de fumarate et active les voies de signalisation du facteur de transcription inductible par l’hypoxie (HIF) et des espèces réactives de l’oxygène (ROS). Néanmoins, aucune mutation du gène FH n’a été rapportée dans les cas de PRCC2 sporadiques. Le projet de recherche porte sur la caractérisation moléculaire des PRCC2 héréditaires et sporadiques. Notre analyse du transcriptome a identifié des différences entre les signatures moléculaires des PRCC2 héréditaires et sporadiques. Cependant, l’étude d’immunohistochimie n'a pas révélé de biomarqueurs potentiels. Les analyses bio-informatiques de profils d’expression génique ont révélé que les tumeurs PRCC2 héréditaires et sporadiques partagent une dérégulation de la voie principale NRF2/KEAP1. Il a été montré que la surexpression de AKR1B10 (Aldo-Keto Reductase Family 1 Membre B10) est la conséquence directe de l’activation de l'élément de réponse antioxydant (ARE). Finalement, nous avons établi un nouveau modèle in vitro de lignée cellulaire, NCCFH1 (FH-/-), issue d’un patient HLRCC. NCCFH1 représente une plateforme idéale pour les études fonctionnelles, métaboliques et thérapeutiques. Bortézomib pourrait être la meilleure alternative thérapeutique pour les patients avec PRCC2. / Papillary Renal Cell Carcinoma type 2 (PRCC2) is known to be a very aggressive type of kidney cancer with a high metastatic potential, poor outcome and absence of effective therapy. Hereditary form of PRCC2 is associated with rare hereditary leiomyomatosis and renal cell carcinoma (HLRCC). HLRCC is characterized by germline heterozygous mutations in the Fumarate Hydratase (FH) gene that encodes an enzyme of the Krebs cycle, Fumarase. It has been shown that the accumulation of fumarate induces activation of Hypoxia Inducible Factor (HIF) and ROS (Reactive Oxygen Species) pathways. Nevertheless, no FH gene mutation has been reported in sporadic PRCC2 tumors. The goal of this study is to better characterize hereditary and sporadic PRCC2. Our transcriptome analysis identified the set of genes that are differentially expressed between the two types of PRCC2. Subsequent immunohistochemistry screening did not reveal any potential diagnostics biomarkers. Further, the comprehensive computational analysis of gene profiling data revealed that hereditary and sporadic PRCC2 share the similar molecular signature with NRF2-KEAP1 axis deregulation as one of the major pathway in both forms. We demonstrated that over expression of Aldo-keto reductase family 1 member B10 (AKR1B10) is the direct consequence of the antioxidant response element (ARE) activation shared in hereditary and sporadic tumors. Finally, we have established FH-deficient cell line (NCCFH1) a new preclinical model of hereditary PRCC2. It presents the perfect platform for studying the metabolic features and testing new therapies for hereditary PRCC2, while bortezomib appears to be a potential efficient therapeutic option.

Abstract Uterine leiomyomas are the most common benign tumours in females. They are myometrial neoplasms, may present single or multiple, and may be located in various sites of the uterus. Leiomyomas distort the uterine cavity and the uterus itself, causing abnormal vaginal bleeding, reduced fertility and also pelvic pressure and pain symptoms. The aim of this study was to elaborate current knowledge on familial uterine leiomyomas and to explore the possible association between uterine leiomyoma and cardiovascular disease risk factors, and also the association between leiomyomas and endometriosis. The natural history of familial uterine leiomyoma study showed significant differences between familial and non-familial leiomyoma cases, familial cases having more severe clinical characteristics. They presented with multiple uterine leiomyomas and were more often symptomatic. They were also diagnosed at a younger age. The prevalence study on uterine leiomyomas and endometriosis offered confirmation of an association between the diseases. Uterine leiomyomas and endometriosis seem to decrease female fertility independently of each other. Uterine leiomyomas related to the hereditary leiomyomatosis and renal cell cancer (HLRCC) tumour syndrome were studied in regard to their clinical characteristics and immunophenotype. The study provided evidence that women with HLRCC may be identified through distinct leiomyoma clinical characteristics, and routine-use IHC of CD34 and Bcl-2. Distinguishing these leiomyoma cases from sporadic ones may identify families affected by fumarate hydratase (fumarase, FH) mutation. Uterine leiomyoma and cardiovascular disease risk factors were studied in The Northern Finland Birth Cohort 1966 (NFBC1966). The study showed an association between leiomyomas and raised cardiovascular disease risk factors, serum lipids and metabolic syndrome in particular. These findings may suggest that there are shared predisposing factors underlying both uterine leiomyomas and adverse metabolic and cardiac disease risks, or that metabolic factors have a role in biological mechanisms underlying leiomyoma development. This study provides novel information on clinical characteristics of familial uterine leiomyomas and on the immunophenotype of HLRCC-related leiomyomas. The study also offers significant confirmation of associations between uterine leiomyomas and both endometriosis and several CVD risk factors. / Tiivistelmä Kohdun leiomyoomat ovat naisten yleisin hyvänlaatuinen kasvain. Ne ovat myometriumin neoplastisia muutoksia ja ne ilmenevät joko yksittäisinä tai monilukuisina, ja ne voivat sijaita missä tahansa kohdun myometriumia. Leiomyoomat muuttavat kohdun ja kohtuontelon säännöllistä muotoa. Lisäksi ne aiheuttavat vuotohäiriöitä, alentunutta hedelmällisyyttä, ja lantion alueen painetta ja kipua. Tämän tutkimuksen tavoitteena oli laajentaa nykyistä tietämystä suvuittain esiintyvistä kohdun leiomyoomista ja selvittää mahdollista leiomyoomien ja kardiovaskulaaritautiriskin assosiaatiota, ja lisäksi selvittää leiomyoomien ja endometrioosin assosiaatiota. Suvuittain esiintyvien kohdun leiomyoomien taudinkulkua selvittävässä tutkimuksessa osoitettiin merkittäviä eroja suvuittain ja ei-suvuittain esiintyvien leiomyoomien välillä. Suvuittain esiintyvien leiomyoomien kliininen taudinkuva oli vaikeampi, leiomyoomia oli kohdussa useampia ja ne aiheuttivat useammin oireita ja lisäksi ne diagnosoitiin nuoremmalla iällä. Kohdun leiomyoomien ja endometrioosin yleisyyttä selvittävä tutkimus antoi lisävahvistusta sille havainnolle, että nämä taudit assosioivat keskenään. Tutkimustuloksen mukaan leiomyoomat ja endometrioosi vähentävät naisen hedelmällisyyttä toisistaan riippumatta. Perinnöllinen kohdun leiomyomatoosi ja munuaissyöpä (hereditary leiomyomatosis and renal cell cancer, HLRCC) -kasvainoireyhtymään liittyvän kohdun leiomyoomia selvittävän tutkimuksen tuloksien mukaan HLRCC-naisten kohdun leiomyoomien kliiniset ominaisuudet poikkeavat satunnaisesti esiintyvien leiomyoomien ominaisuuksista. Naisella HLRCC voitaisiinkin tunnistaa näiden poikkeavien ominaisuuksien perusteella, sekä immunohistokemiallisilla värjäyksillä CD34 ja Bcl-2. Fumaraattihydrataasi (fumaraasi, FH) -geenin mutaatiota kantava suku voitaisiin siten tunnistaa yksittäisen HLRCC leiomyoomatapauksen avulla. Pohjois-Suomen syntymäkohortti 1966 (Northern Finland Birth Cohort 1966, NFBC1966) tutkittiin kohdun leiomyoomia ja kardiovaskulaarisairauden riskitekijöitä. Tutkimustuloksien perusteella kohdun leiomyoomat assosioivat koholla olevien kardiovaskulaarisairauden riskien kanssa, erityisesti seerumin lipidien ja metabolisen syndrooman suhteen. Näiden tutkimustulosten perusteella voidaan esittää, että leiomyoomien ja terveydelle epäedullisen metabolian ja kardiovaskulaaritaudin riskien taustalla on mahdollisesti joitain yhteisiä altistavia tekijöitä, tai että metabolisilla tekijöillä on rooli kohdun leiomyoomien tautimekanismissa. Tämä tutkimus on tuottanut uutta tietoa suvuittain esiintyvien kohdun leiomyoomien kliinisestä taudinkuvasta ja HLRCC:n liittyvien leiomyoomien immunofenotyypistä. Lisäksi tämä tutkimus esittää lisävahvistusta kohdun leiomyoomien ja endometrioosin assosiaatiolle sekä useille kardiovaskulaaririskitekijöille.

<p dir="ltr">The eukaryotic genome is organized and packaged into DNA-protein complexes called chromatin. Nucleosomes, the repeating unit of chromatin, are composed of DNA in complex with histone octamers containing two of each of the four core histones, the H2A, H2B, H3, and H4. Accessibility of nucleosomal DNA by other DNA-binding proteins can be regulated, in-part, by the type and level of nucleosome-associated post-translational modifications (PTMs). PTMs, such as acetylation of lysine residues, including H3K9, H3K14, and H4K16, can neutralize their positive charge, thereby reducing the affinity of acetylated histones for the negatively charged DNA sugar phosphate backbone. This, in turn, promotes the formation of relaxed open chromatin which is crucial for the accessibility of DNA by the transcription machinery. Consequently, histone acetylation is associated with active gene expression, while deacetylation, which restores positive charges of lysine residues, is linked with condensed chromatin structure, and the associated decrease in gene expression. Other PTMs such as histone methylation on H3K9, which prevents H3K9 acetylation, promotes gene silencing via serving as a binding site for HP1, a structural component of silenced heterochromatin. Interestingly, this epigenetic control of gene expression, typically used to regulate endogenous genes, also influences ectopic gene expression in host cells. Transgenes delivered into target cells undergo host cell-mediated assembly into chromatin. As a result, transgenes assembled into hypoacetylated condensed chromatin experience a dramatic loss of expression shortly after delivery into target cells. As part of this thesis, we assessed the impact of "priming" plasmid-based transgenes to adopt accessible chromatin states on transgene expression. Nucleosome positioning elements were introduced at promoters of transgenes, or vectors were pre-assembled into nucleosomes containing either unmodified histones or histone mutants mimicking constitutively acetylated states at residues H3K9 and H3K14, or H4K16 prior to their introduction into human cells. Transgene expression was then monitored by epifluorescence microscopy and flow cytometry over time. We found that DNA sequences capable of positioning nucleosomes influenced the expression of adjacent transgenes in a distance-dependent manner, even in the absence of pre-assembly into chromatin. Intriguingly, pre-assembly of plasmids into chromatin facilitated prolonged transgene expression compared to plasmids that were not pre-packaged into chromatin. Interactions between pre-assembled chromatin states and nucleosome positioning effects on reporter gene expression were also assessed. Overall, nucleosome positioning played a more significant role in influencing gene expression than priming with hyperacetylated chromatin states. These findings have direct relevance to ongoing efforts to develop durable plasmid-based gene therapies for genetically-derived disorders such as cancer.</p><p dir="ltr">In this thesis, we also explored the roles of the tumor suppressor enzyme fumarate hydratase (FH) in cellular responses to DNA replication stress. In humans, biallelic loss of function mutations in FH predisposes individuals to hereditary leiomyomatosis and renal cell carcinoma (HLRCC). However, the role of fumarase in HLRCC is not fully understood. The eukaryotic genome experiences thousands of lesions per cell each day, with DNA double-strand breaks (DSBs) being among the most deleterious. If unrepaired or repaired incorrectly, DSBs can lead to various disorders, including cell death and cancer. Cellular processes such as metabolism, transcription, and DNA replication are among the major endogenous sources of DSBs. Impediments to DNA replication that result in persistent stalling of replication forks can lead to fork collapse, thereby exposing unprotected single-stranded DNA (ssDNA) to endonuclease attack and the formation of double-strand breaks (DSBs). DNA-protein crosslinks that form irreversible complexes, an imbalance or exhaustion of deoxynucleotides (dNTPs), as well as the presence of inherently difficult-to-replicate genomic regions such as common fragile sites (CFSs), are some of the many obstacles that can halt fork progression. Intrinsically, however, all organisms have evolved response mechanisms designed to sense, prevent, detect, and resolve the different sources of DNA replication stress. These response mechanisms are governed by the highly conserved DNA replication checkpoint (DRC), a part of the intra-S phase checkpoint. The main function of the DRC is to halt cell cycle progression and activate DNA damage responses, such as DNA repair and subsequent replication fork restart. The highly conserved tricarboxylic acid (TCA) cycle enzyme fumarase (FH in humans, Fum1p in yeast), which functions to convert fumarate to malate in the mitochondrial TCA cycle, has been implicated in DRC responses in the cell’s nucleus. Upon exposure of yeast model organism to hydroxyurea (HU), an inhibitor of ribonucleotide reductase (RNR) that results in the exhaustion of cellular dNTPs, the expression of Fum1p is upregulated and Fum1p is translocated to the nucleus. Fum1p’s metabolite fumarate suppresses sensitivity to HU in yeast in a manner independent of modulating cellular dNTP levels. Notwithstanding, our understanding of these extra-mitochondrial functions of fumarase remains incomplete. For example, the nature of the impediments to DNA replication that are affected by fumarase is presently unclear. In addition, across all eukaryotes, fumarase lacks a canonical nuclear localization signal, and the means of its nuclear translocation upon DNA replication stress remain a mystery. In this study, our immunofluorescence experiments revealed that, similar to yeast, exposure of human cells to HU promoted the nuclear translocation of fumarase. We also performed Liquid Chromatography with tandem mass spectrometry (LC-MS/MS) using human cells exposed to DNA replication stress by HU to identify replication stress-dependent protein-protein interactions of FH. We observed that FH co-precipitated MUS81, a structure-specific endonuclease and a crucial component of cellular responses to DNA replication stress. MUS81 is localized to stalled replication forks during the S phase of the cell cycle, where it cleaves the three-way junctions created by stalled forks. MUS81 also functions to resolve recombination intermediates and Holliday junctions (HJs) during the S phase. MUS81 also localizes at common fragile sites (CFSs) during the G2/M phase. CFSs are genomic regions that are difficult-to-replicate, late-replicating, and tend to exit the S phase with under-replicated DNA. These CFSs are targeted by MUS81, cleaved, and replicated via Break-Induced Replication (BIR) that is dependent on POLD3 in a process called mitotic DNA synthesis (MiDAS). In our immunofluorescence studies, we observed that HLRCC-derived cancer cells expressing low levels of catalytically inactive fumarase exhibited bulky anaphase bridges. These observations are especially intriguing because the loss of MUS81 or inhibition of POLD3 is known to increase the frequency of bulky anaphase bridges, a phenotype associated with defects in MiDAS. Additionally, we observed that exposure of fumarase-deficient cells to aphidicolin (APH), an inhibitor of POLD3, dramatically increased the frequency of anaphase bridges. Furthermore, we found that upon exposure of human cells to APH, fumarase translocated to the nucleus. Whether fumarase suppresses the MiDAS defects observed in HLRCC cells is still under investigation. Taken together, the work described herein uncovers previously unknown roles of fumarase in DNA damage responses and provides direct links toward understanding how fumarase may function to safeguard genomic integrity.</p>

This thesis focuses on gynecopathology. It consists of a collection of seven papers published in pathology journals with impact factor. Introduction section contains selection of examples showing scientific application of molecular genetic methods. Further on the aims of individual research projects are described. The first project comprises histomophologic study of skin endometriosis addressing "mullerian" differentiation. A case report of a rare tumor namely borderline papillary serous tumor of the fimbriated end of the fallopian tube follows with molecular genetic analysis of KRAS, BRAF and p53 gene mutation status. Prospective longitudinal study on high grade squamous dysplasia (HSIL) of the cervix in HPV vaccinated women, so called DAV (dysplasia after vaccination), aims to elucidate pathogenesis of this phenomenon. Two other studies focus on incidence of fumarate hydratase deficient leiomyomas of the uterus and hereditary leiomyomatosis and renal cell carcinoma syndrome (HLRCC). The aim of those studies is to improve our diagnostic capability and increase detection rate of the patients with HLRCC syndrome. Finally a new subtype of HSIL namely bizarre cell dysplasia is described in two separate studies. Conclusion remarks contemplate the role of molecular genetics in surgical pathology.

This thesis focuses on gynecopathology. It consists of a collection of seven papers published in pathology journals with impact factor. Introduction section contains selection of examples showing scientific application of molecular genetic methods. Further on the aims of individual research projects are described. The first project comprises histomophologic study of skin endometriosis addressing "mullerian" differentiation. A case report of a rare tumor namely borderline papillary serous tumor of the fimbriated end of the fallopian tube follows with molecular genetic analysis of KRAS, BRAF and p53 gene mutation status. Prospective longitudinal study on high grade squamous dysplasia (HSIL) of the cervix in HPV vaccinated women, so called DAV (dysplasia after vaccination), aims to elucidate pathogenesis of this phenomenon. Two other studies focus on incidence of fumarate hydratase deficient leiomyomas of the uterus and hereditary leiomyomatosis and renal cell carcinoma syndrome (HLRCC). The aim of those studies is to improve our diagnostic capability and increase detection rate of the patients with HLRCC syndrome. Finally a new subtype of HSIL namely bizarre cell dysplasia is described in two separate studies. Conclusion remarks contemplate the role of molecular genetics in surgical pathology.