Le mélanome et les polymorphismes associés au dysfonctionnement du système immunitaire

La recherche biomédicale profite de plus en plus au développement des techniques de séquençage et d'analyse de l'ADN. Les coûts du séquençage ont drastiquement baissés au cours de ces dernières années et les genomes-wides associations studies (GWAS) ont révolutionné l'approche de la recherche génétique en mettant en évidence associations et single-nucleotide-polymorphisms (SNPs) qui pourraient être importantes pour la susceptibilité à développer des maladies dites communes. La majorité des cancers appartiennent à cette définition de maladie commune, ils sont généralement causés par une accumulation de lésions/mutations de l'ADN aboutissant à une perte de contrôle de la prolifération et du cycle cellulaire. Ces mutations peuvent être héréditaires, acquises ou une combinaison des deux. Dans la plupart des cancers communs (cancers qui n'ont pas une hérédité familiale importante) les mutations de l'ADN sont souvent amenées par des facteurs tels que inflammation chronique, tabac, virus, exposition aux radiations, aux agents chimiques. Ceci est le cas pour le mélanome également, un cancer de la peau qui est corrélé à l'exposition des rayons UV solaires ou artificiels. Une hypothèse largement acceptée aujourd'hui est que les tumeurs, à travers leur accumulation progressive de mutations somatiques et d'anomalies chromosomiques, finissent par échapper au contrôle exercé par le système immunitaire. Il est par conséquence imaginable que des polymorphismes naturels puissent renforcer ou affaiblir la capacité du système immunitaire à freiner voir arrêter la progression tumorale

Branch site haplotypes that control alternative splicing

We show that the allele-dependent expression of transcripts encoding soluble HLA-DQβ chains is determined by branchpoint sequence (BPS) haplotypes in DQB1 intron 3. BPS RNAs associated with low inclusion of the transmembrane exon in mature transcripts showed impaired binding to splicing factor 1 (SF1), indicating that alternative splicing of DQB1 is controlled by differential BPS recognition early during spliceosome assembly. We also demonstrate that naturally occurring human BPS point mutations that alter splicing and lead to recognizable phenotypes cluster in BP and in position −2 relative to BP, implicating impaired SF1-BPS interactions in disease-associated BPS substitutions. Coding DNA variants produced smaller fluctuations of exon inclusion levels than random exonic substitutions, consistent with a selection against coding mutations that alter their own exonization. Finally, proximal splicing in this multi-allelic reporter system was promoted by at least seven SR proteins and repressed by hnRNPs F, H and I, supporting an extensive antagonism of factors balancing the splice site selection. These results provide the molecular basis for the haplotype-specific expression of soluble DQβ, improve prediction of intronic point mutations and indicate how extraordinary, selection-driven DNA variability in HLA affects pre-mRNA splicin

Capturing the biological impact of CDKN2A and MC1R genes as an early predisposing event in melanoma and non melanoma skin cancer

Germline mutations in CDKN2A and/or red hair color variants in MC1R genes are associated with an increased susceptibility to develop cutaneous melanoma or non melanoma skin cancer. We studied the impact of the CDKN2A germinal mutation p.G101W and MC1R variants on gene expression and transcription profiles associated with skin cancer. To this end we set-up primary skin cell co-cultures from siblings of melanoma prone-families that were later analyzed using the expression array approach. As a result, we found that 1535 transcripts were deregulated in CDKN2A mutated cells, with over-expression of immunity-related genes (HLA-DPB1, CLEC2B, IFI44, IFI44L, IFI27, IFIT1, IFIT2, SP110 and IFNK) and down-regulation of genes playing a role in the Notch signaling pathway. 3570 transcripts were deregulated in MC1R variant carriers. In particular, genes related to oxidative stress and DNA damage pathways were up-regulated as well as genes associated with neurodegenerative diseases such as Parkinson's, Alzheimer and Huntington. Finally, we observed that the expression signatures indentified in phenotypically normal cells carrying CDKN2A mutations or MC1R variants are maintained in skin cancer tumors (melanoma and squamous cell carcinoma). These results indicate that transcriptome deregulation represents an early event critical for skin cancer development

Primary Immunodeficiency and Cancer Predisposition Revisited: Embedding Two Closely Related Concepts Into an Integrative Conceptual Framework

Common understanding suggests that the normal function of a “healthy” immune system safe-guards and protects against the development of malignancies, whereas a genetically impaired one might increase the likelihood of their manifestation. This view is primarily based on and apparently supported by an increased incidence of such diseases in patients with specific forms of immunodeficiencies that are caused by high penetrant gene defects. As I will review and discuss herein, such constellations merely represent the tip of an iceberg. The overall situation is by far more varied and complex, especially if one takes into account the growing difficulties to define what actually constitutes an immunodeficiency and what defines a cancer predisposition. The enormous advances in genome sequencing, in bioinformatic analyses and in the functional in vitro and in vivo assessment of novel findings together with the availability of large databases provide us with a wealth of information that steadily increases the number of sequence variants that concur with clinically more or less recognizable immunological problems and their consequences. Since many of the newly identified hard-core defects are exceedingly rare, their tumor predisposing effect is difficult to ascertain. The analyses of large data sets, on the other hand, continuously supply us with low penetrant variants that, at least in statistical terms, are clearly tumor predisposing, although their specific relevance for the respective carriers still needs to be carefully assessed on an individual basis. Finally, defects and variants that affect the same gene families and pathways in both a constitutional and somatic setting underscore the fact that immunodeficiencies and cancer predisposition can be viewed as two closely related errors of development. Depending on the particular genetic and/or environmental context as well as the respective stage of development, the same changes can have either a neutral, predisposing and, in some instances, even a protective effect. To understand the interaction between the immune system, be it “normal” or “deficient” and tumor predisposition and development on a systemic level, one therefore needs to focus on the structure and dynamic functional organization of the entire immune system rather than on its isolated individual components alone

The Risk of Cancer Associated with Immunosuppressive Therapy for Skin Diseases

The possible carcinogenic risk of immunosuppressive therapies is an important issue in everyday clinical practise. Carcinogenesis is a slow multi step procedure, thus a long latency period is needed before cancer develops. PUVA therapy is used for many skin diseases including psoriasis, early stage cutaneous T cell lymphoma, atopic dermatitis, palmoplantar pustulosis and chronic eczema. There has been concern about the increased melanoma risk associated to PUVA therapy, which has previously been associated with an increased risk on non-melanoma skin cancer, especially squamous cell carcinoma. The increased risk of basal cell carcinoma (BCC) is also documented but it is modest compared to squamous cell carcinoma (SCC). This thesis evaluated melanoma and noncutaneous cancer risk associated to PUVA, and the persistence of nonmelanoma cancer risk after the cessation of PUVA treatment. Also, the influence of photochemotherapy to the development of secondary cancers in cutaneous T cell lymphoma and the role of short term cyclosporine in later cancer development in inflammatory skin diseases were evaluated. The first three studies were performed on psoriasis patients. The risk of melanoma started to increase 15 years after the first treatment with PUVA. The risk was highest among persons who had received over 250 treatments compared to those under 250 treatments. In noncutaneous cancer, the overall risk was not increased (RR=1.08,95% CI=0.93-1.24), but significant increases in risk were found in thyroid cancer, breast cancer and in central nervous system neoplasms. These cancers were not associated to PUVA. The increased risk of SCC was associated to high cumulative UVA exposure in the PUVA regimen. The patients with high risk had no substantial exposure to other carcinogens. In BCC there was a similar but more modest tendency. In the two other studies, the risk of all secondary cancers (SIR) in CTCL patients was 1.4 (95% CI=1.0-1.9). In separate sites, the risk of lung cancer, Hodgkin and non-Hodgkin lymphomas were increased. PUVA seemed not to contribute to any extent to the appearance of these cancers. The carcinogenity of short-term cyclosporine was evaluated in inflammatory skin diseases. No increased risk for any type of cancer including the skin cancers was detected. To conclude, our studies confirm the increased skin cancer risk related to PUVA treatment in psoriasis patients. In clinical practice, this has led to a close and permanent follow-up of patients treated with PUVA. In CTCL patients, PUVA treatment did not contribute to the development of secondary cancers. We could not detect any increase in the risk of cancer in patients treated with short term cyclosporine, unlike in organ transplant patients under such long-term therapy.Vaikeiden tulehduksellisten ihosairauksien hoidossa joudutaan käyttämään elimistön puolustusjärjestelmään vaikuttavia hoitoja ja lääkkeitä. Näitä hoitomuotoja ovat mm. PUVA-hoito sekä siklosporiinilääkitys. Edellämainitut hoidot ovat tehokkaita, mutta puolustusjärjestelmään vaikuttavina hoitoina niihin voi pitkällä aikavälillä liittyä lisääntynyt syöpäriski. Syövän kehittyminen on monimutkainen, useita vuosia kestävä tapahtumasarja. Hoitojen mahdolliset pitkäaikaissivuvaikutukset eivät ole olleet tiedossa niitä aloitettaessa, ja epidemiologiset pitkäaikaistutkimukset ovat ainoa tapa saada uutta tietoa eri hoitomuotojen mahdollisesta syöpäriskistä. PUVA-hoito toteutetaan herkistämällä iho ensin psoraleenilla ja sen jälkeen valottamalla UVA-säteillä. Yleisiä PUVA:lla hoidettavia ihosairauksia ovat mm. psoriaasi, lichen planus, palmoplantaarinen pustuloosi, prurigo nodularis, ihon T-solulymfooma ja erilaiset ekseemat. Immuunivastetta hillitsevällä siklo-sporiinilla hoidetaan nykyisin esim. atooppista ekseemaa, psoriaasia, palmoplantaarista pustuloosia ja hankalaa käsiekseemaa. Molempiin edellä mainittuihin hoitoihin tiedetään liittyvän lisääntynyt syöpäriski. PUVA-hoito lisää erityisesti ihon okasolusyöpien riskiä, ja myös siklosporiinia saavilla elinsiirtopotilailla näiden syöpien riski on lisääntynyt. Väitöskirjatyössä tutkittiin sisäisen PUVA-hoidon yhteyttä melanooman ja sisäelinsyöpien kehittymiseen sekä sisäisen PUVA-hoidon yhteyttä ihosyöpien ilmaantumiseen PUVA-hoidon päättymisen jälkeen. Lisäksi tutkittiin ihon T-solulymfoomaa sairastavien potilaiden muiden syöpien riskiä ja niiden mahdollista yhteyttä PUVA-hoitoon. Tutkimme myös lyhytaikaisen, ihotauteihin käytetyn siklosporiinihoidon vaikutusta myöhemmin ilmaantuviin syöpiin. Tuloksissa pitkäaikaiseen sisäiseen PUVA-hoitoon liittyi lisääntynyt melanoomariski, joka oli suurimmillaan yli 250 PUVA-hoitokertaa saaneilla ja/tai yli 15 vuoden kuluttua hoidon aloittamisesta. Sisäelinsyöpien kokonaisriski ei ollut kohonnut, mutta kilpirauhas-, rinta- ja keskushermostosyöpien osuus oli lisääntynyt. Nämä potilaat eivät kuitenkaan olleet saaneet suuria PUVA-hoitoannoksia (yli 300 kertaa). PUVA-hoidon ihosyöpiä (oka- ja tyvisolusyöpiä) lisäävä vaikutus on pysyvä, vaikka hoito lopetetaan. Ihon T-solulymfoomaa sairastavilla potilailla keuhkosyöpä- ja lymfoomariski olivat kohonneet, mutta PUVA-hoidolla ei ollut vaikutusta tähän. Potilailla, jotka olivat saaneet lyhytaikaisesti siklosporiinihoitoa, ei voitu todeta lisääntynyttä syöpäriskiä eikä ihosyöpien määrä ollut merkittävästi lisääntynyt. Yhteenvetona väitöskirjatyö osoitti, että PUVA-hoidon syöpää aiheuttava vaikutus rajoittuu pelkästään iholle. Pitkäaikainen PUVA-hoito lisää melanoomariskiä ja myös muiden ihosyöpien riskiä, vaikka hoito olisi jo lopetettu. Iholymfoomapotilailla todetut syövät eivät olleet yhteydessä PUVA-hoitoihin, ja lyhyt-aikaisen siklosporiinihoidon ei voitu osoittaa aiheuttavan syöpiä. PUVA-hoidon osalta löydös on merkittävä ja on johtanut runsaasti PUVA-hoitoja saaneiden potilaiden tarkkaan seurantaan

DNA repair protein complexes, functionality and significance for repair efficiency and cell survival

DNA‐reparasjons‐protein‐komplekser, funksjonalitet og signifikans for reparasjonseffektivitet og skadetoleranse All informasjon om en organisme er lagret i vår arvestoff, DNA. DNA er et relativt ustabilt makromolekyl som konstant blir utsatt for farer som truer dets integritet, både fra omgivelsene og fra kjemiske prosesser inne i selve cellen. I tillegg kan baser spontant bli mistet uten noe form for påvirkning. Selve kopieringen av DNA, den såkalte DNA-replikasjonen er svært rask og er en kritisk prosess i cellen hvor mye kan gå galt. I tillegg kan ureparerte DNA-skader ved replikasjonen foreviges i form av mutasjoner. Mutasjoner i gener som koder for proteiner som regulerer cellens vekst og død kan resultere i ukontrollert cellevekst og dermed kreft. En av cellens strategier for å sikre effektiv og trygg replikasjon og reparasjon av DNA’et er å samarbeide ved å danne proteinkomplekser, hvorav PCNA ofte spiller en sentral rolle. PCNA sitter som en homotrimerisk ring rundt DNA-tråden som replikeres, og fungerer som en plattform for binding av mange proteiner. I tillegg til binding av DNA-replikasjonsproteiner, bindes også mange DNA-reparasjonsproteiner til PCNA, og sørger for effektiv reparasjon av skadet DNA både før og etter selve replikasjonen. I tillegg er PCNA involvert i DNA-syntese ved reparasjon som ikke er assosiert med replikasjon. I 1998 ble det funnet et motiv (en peptid-sekvens) som er ansvarlig for at mange proteiner bindes til PCNA, kalt PCNA Interacting Peptide (PIP). I artikkel 1 fant vi ved hjelp av blant annet fluorescerende proteiner og konfokal mikroskopi et nytt motiv som er viktig for proteiners binding til PCNA. Dette motivet fant vi først i det direkte alkyleringsreparasjons-proteinet; human AlkB homologue 2 og derfor kalte vi motivet AlkB homologue 2 PCNA Interacting Motif (APIM). I denne artikkelen verifiserer vi et funksjonelt APIM motiv i fem proteiner og viser at over-uttrykk av dette motivet gjør celler mer sensitive for alkylerende skade. Dette tyder på at overuttykk av APIM hemmer bindingen mellom APIM-inneholdende DNA reparasjons-proteiner og PCNA slik at de ikke reparerer DNA-skadene optimalt. I samme artikkel viser vi også at APIM er konservert i mer enn 200 proteiner, blant annet i nukleotideeksisjonsreparasjons (NER) proteinet Xeroderma Pigmentosum group A (XPA), og i artikkel 2 verifiserer vi at APIM også er et funksjonelt PCNA bindende motiv i XPA. Vi viser og at overuttrykk av APIM-peptidet gjør celler mer sensitive for skade fra UV-lys, en type DNA-skade som hovedsakelig blir reparert av NER. I tillegg finner vi bevis som støtter at det er redusert funksjon av XPA som er årsak til at cellene er mer UV-sensitive ved overuttrykk av APIM, antagelig pga. svekket binding til PCNA. I artikkel 3 ser vi nærmere på baseeksisjonsreparasjons- og singeltrådbruddsreparasjons-proteinet XRCC1. Dette er i likhet med PCNA og XPA et protein uten enzymatisk funksjon, men med mange bindingspartnere, blant annet PCNA. Hvilken del av XRCC1 som er viktig for dens funksjon i cella er derimot ikke helt klarlagt, noe vi undersøker nærmere i denne artikkelen. Det viser seg at den delen av XRCC1 som har evnen til å binde PCNA og alkyleringsreparasjons-proteinet MPG er den eneste XRCC1 mutanten som kan stimulere reparasjon av alkyleringsskader, noe som igjen bekrefter viktigheten av å binde seg til PCNA. Oppsummert tar dette arbeidet for seg hvordan DNA-reparasjonsproteiner binder seg til hverandre og PCNA, og hvordan dette påvirker evnen til å reparere DNA og dermed tåle DNA-skade.PhD i molekylærmedisi

Molecular Genetic Mechanisms in Cancers of Keratinocytic Origin

Keratinocytic cancers (KC) comprise a group of diseases that have a broad spectrum clinically and pathologically. At one end of the spectrum are benign proliferations (acanthomas), and at the other end are malignant tumors with aggressive growth and metastatic potential. Traditionally, about 80% of KC cases have basal cell carcinoma (BCC) and 20% have cutaneous squamous cell carcinoma (cSCC). Both tumors have different phenotypic features due to different oncogenic pathways. cSCC is biologically different and requires a different approach due to the higher risk of local recurrence, metastasis and death. Genetic factors play an important role in the development of KC. Family and family history studies, the presence of KC as a feature of rare hereditary syndromes, and genetic association studies give us clues in this regard. More than 20 genetic syndromes associated with KC have been described. Some syndromes are associated with multiple BCC, some with multiple cSCC, and some with both BCC and cSCC. Environmental risk factors include exposure to ultraviolet light radiation and immunosuppression in both tumors. Exposure to ionizing radiation is most common in BCC, while smoking and photosensitive drug use are among the environmental risk factors for cSCC. Molecular, epidemiological, and clinical studies will help better understand the cellular processes involved in tumorigenesis, and develop new strategies for treating and preventing KCs