Abstract The stem cell is a primitive cell that is capable of dividing to reproduce itself and can give rise to a selection of differentiated progeny. Stem cells are thought to be involved in or even main factors in many diseases. In postnatal humans, mesenchymal tissues have the capacity to regenerate from stem cells called mesenchymal stem cells (MSC). It is currently thought that these cells will become the basis of therapy for many diseases. In the present study, a novel in vitro method was developed to examine human bone marrow derived MSC differentiation into osteoblast lineage, and to study the role of MSC in a variety of mesenchymal diseases. The ability of MSCs to differentiate into osteoblasts was investigated during aging. In addition, the interindividual variability in the osteogenesis of MSCs and in the osteoblastic response of MSC to estrogen and testosterone was studied. Furthermore, an ex vivo model using a human aortic valve microenvironment was developed to explore whether the extracellular matrix influences the osteoblastic differentiation of the MSC. Finally, the role of MSC in neurofibromatosis type 1 (NF1) related congenital pseudarthrosis of the tibia (CPT) was studied. It was found that after menopause the osteogenic potential of MSCs does not decrease. It was also found that estrogen receptor (ER) alpha genotype confers interindividual variability of response to estrogen and testosterone in MSC derived osteoblasts. In addition, it was found that the non-calcified valves with living valve cells inhibited osteogenesis of co-cultured MSCs, whereas the calcified and devitalised valves promoted differentiation towards an osteoblastic lineage. Finally, MSCs from NF1-related pseudarthrosis showed altered NF1 gene expression, poor osteoblastic differentiation and bone formation. In conclusion, MSC can be easily isolated from the bone marrow and MSC has the capacity to regenerate tissue even at later stages of life. These results could help explain the contradictory effects of 17β-estradiol (E2) on osteoblasts in vitro and might also provide new insights into understanding the differences in responses to hormone replacement therapy. It seems that adult stem cells from bone marrow undergo milieu-dependent differentiation to express phenotypes that are similar to cells in the local microenvironment. Finally, the NF1 gene was shown to have a role in bone development and remodelling.

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Des études épidémiologiques ont démontré que l’exposition aux pesticides tels que le chlordécone augmente le risque du cancer, en particulier de la prostate. Il avait été précédemment montré au laboratoire que le chlordécone possède des propriétés pro-angiogéniques impliquant la libération de NO et la production de VEGF suite à l’activation du récepteur aux oestrogènes de type alpha (ERα). Les processus angiogéniques pouvant impliquer les espèces réactives de l’oxygène (EROs), produites notamment par la mitochondrie, l’objectif de ce travail a été d’évaluer la contribution de la biogenèse mitochondriale et du stress oxydatif dans l’angiogenèse induite par le chlordécone.Les résultats obtenus montrent que la biogenèse mitochondriale n’est pas essentielle pour la réponse angiogénique du chlordécone puisqu’elle n’est retrouvée que pour de forte concentration de chlordécone. Les mécanismes mis en jeu ont été identifiés au niveau des cellules endothéliales humaines. Ils impliquent une régulation spatio temporellede la production des EROs impliquant dans un premier temps la NADPH oxydase, elle même capable de stimuler la production mitochondriale d’EROs via la voie impliquant la NO synthase. Le chlordécone serait par ailleurs capable de favoriser la localisation périnucléaire des EROs afin de favoriser la production de VEGF. L’ensemble de ces effets implique le récepteur aux oestrogènes. Ce travail a donc permis d’identifier les mécanismes cellulaires impliqués dans la modulation de l’angiogenèse par le chlordécone. Ces mécanismes moléculaires pourraient contribuer à identifier de nouvelles cibles permettant de réguler les processus angiogéniques et la tumorigenèse induites par ce toxique. / Epidemiological studies report that exposure to pesticides like chlordecone increases risk of prostate cancer and tumorigenesis. We have reported recently that the pro-angiogenic effect of chlordecone involving NO release and VEGF production is mediated through activation of α isoform of the estrogen receptor (ERα). Since mitochondria and ROS have been implicated inthe process of angiogenesis, this study aims to determine the contribution of mitochondrial biogenesisand oxidative stress in chlordecone-induce dangiogenesis. Firstly, our results indicate that mitochondrial biogenesis is not essential for chlordecone angiogenic response. We also identified the molecular mechanism involved; chlordecone induces endothelial cells angiogenesis by a spatiotemporal regulation of ROS production involving NADPH oxidase then mitochondrial O2 -via a NO sensitive pathways through activation of ERα.These findings propose that a molecular mechanism may partly explain the epidemiological evidence implicating chlordecone as risk factor of prostatic cancer.

<p>Osteoporosis causes over two million skeletal fractures in the United States every year in people over 50 years of age. Age-related bone loss results from imbalanced bone turnover mainly caused by decreases in sex hormones and skeletal mechanobiology. Estrogen receptor β (ERβ) in osteocytes (Ot) has been proposed to mediate skeletal structural adaptations in response to estrogen and mechanical stimuli. However, direct <em>in vivo</em> studies on Ot-ERβ are lacking, and relevant <em>in vitro</em> studies are mostly made in two-dimensional (2D) culture models, whose cellular environment restricts Ot morphology and biology. To better understand the mechanisms of estrogen-ERs in age-related bone loss, it is important to investigate the role of Ot-ERβ in skeletal turnover in response to sex hormonal and mechanical cues and develop a novel 3D culture model that can reproduce Ot morphology for future <em>in vitro</em> ER studies. The role of Ot-ERβ in bone turnover and skeletal adaptive response to mechanical load were examined in male and female mice at 12wk and 30wk old. Ot-ERβ shows age- and sex-dependent effects on bone morphology. Young male mice with Ot-ERβ deletion (ERβ-dOT) showed increased vertebral cancellous bone, whereas decreased cortical and cancellous vertebral bone mass appeared in adult male ERβ-dOT mice. No difference in bone mass occurred in female mice between genotypes. Ot-ERβ mediates tibial mechanoadaptation in cortical but not cancellous in young and adult male mice but plays an inhibitory role in young female mice during cortical mechanoadaptation. Gonadectomy studies on young adult mice revealed that deletion of Ot-ERβ inhibits the sex hormone withdrawal-induced decreases in bone mass and skeletal strength for male mice but did not play a major role for female mice. Lastly, a novel 3D <em>in vitro</em> culture system was developed using collagen-mineral composites for investigating culture mineralization, osteocyte biology, and osteocyte-osteoblast interaction. Cell viability and cellular differentiation were validated after 3 days and 56 days of culture. Optimal PSC-HA culture conditions were determined based on osteocyte differentiation, gene expression analyses, and tissue mineralization. Overall, this work takes novel steps to demonstrate the <em>in vivo</em> role osteocyte-ERβ plays in skeletal morphology and mechanobiology and develops a novel <em>in vitro</em> 3D culture using PSC-HA composites. These advances will contribute to future mechanistic studies of sex hormone receptors in osteoblasts and osteocytes in age-related bone loss using controlled <em>in vitro</em> environments. </p>

Bröstcancer är en multifaktoriell sjukdom och är den vanligaste cancerformen hos kvinnor, cirka var tionde kvinna får någon gång diagnosen. År 2017 var det drygt 10 000 kvinnor som diagnostiserades med bröstcancer och 1400 som avled till följd av sjukdomen i Sverige. Den vanligaste typen är en icke-invasiv form kallad duktal cancer in situ. De flesta bröstcancrarna uttrycker receptorer för östrogenreceptorn (ER) och klassas som ER-positiva tumörer, många tumörer uttrycker också receptorer för progesteron (PgR). ER är en prediktiv biomarkör där positivitet idag används för att identifiera de patienter som svarar på antiöstrogenbehandling med exempelvis läkemedlet tamoxifen (TAM). TAM används för behandling av bröstcancer hos pre- och postmenopausala ER-positiva kvinnor. Syftet med denna litteraturstudie var att undersöka huruvida effekten av TAM kan förutsägas med hjälp av prediktiva biomarkörer vid adjuvant behandling av ER-positiv bröstcancer hos pre- och postmenopausala kvinnor. Arbetet innefattade artikelsökningar och baseras på 9 retrospektiva vetenskapliga studier hämtade från en avhandling och databasen PubMed. Analyserade markörer var proteinuttrycket av HOXB13, AIB1, mTOR, Akt, ER, PgR, EGFR, HER2, CXCL10, CXCR3 samt genuttryck av HOXB13, IL17BR, CYP2C19, CCND1 och RSF1. Studierna analyserade patientmaterial från studier som jämfört TAM vs kontroll eller olika behandlingsregimer av TAM. Ett lågt proteinuttryck av HOXB13 var prediktivt för fördelaktig TAM-effekt samt att ett högt uttryck förutsäger mindre fördelaktig effekt hos ER-positiva patienter. Vidare visades att fosforylerat (p) mTOR enskilt och i kombination med p-ER och/eller p-Akt var prediktivt för ett reducerat TAM-svar. Resultaten som presenteras visar att ett högt uttryck av AIB1 var prediktivt för ett fördelaktigt TAM-svar hos ER-positiva. CXCL10 och CXCR3 var båda prediktiva för ett förbättrat TAM-svar jämfört med kontroll. Vidare visar resultaten att genuttrycken för HOXB13 och förhållandet mellan HOXB13:IL17BR förutsäger effekten av en längre behandling på 5 år jämfört med 2 år. En variant allel av CYP2C19*2 genotypen identifierar patienter med allvarlig prognos men som fördelaktigt kan behandlas med TAM. Resultaten påvisar även att endast ER-status inte är prediktivt för en fördelaktig TAM-behandling och att PgR-analys kan användas som tillägg vid ER-negativitet för att identifiera en liten subgrupp av bröstcancerpatienter som med fördel kan behandlas med TAM. HER2-positivitet påvisar ingen nytta av TAM hos ER-positiva patienter. Resultaten avseende CCND1 påvisar att genamplifiering ej var lämpad som prediktiv markör men att RSF1-genamplifiering kan vara en potentiell markör för avsaknad av TAM-fördel. Sammanfattningsvis kan TAM-effekt förutsägas med hjälp av prediktiva markörer så som HOXB13, AIB1, CXCL10/CXCR3 m.fl., det krävs dock verifiering av resultaten i större kontrollerade studier i form av retrospektiva metaanalyser eller prospektiva studier för att dra en generell slutsats. / For women breast cancer is the most common cancer diagnosis in Sweden, approximately 10 000 patients were diagnosed in 2017. Breast cancer is the leading cause of death among women around the world and in Sweden it is the second most common cause of death. Breast cancer arise from the terminal duct lobular units of the breast, most tumour types are classified as non-invasive called cancer in situ, specifically as ductal cancer in situ. When diagnosing breast cancer there are different ways to characterize tumours. One example is the use of prognostic and predictive biomarkes, which provides information about prognosis and treatment effect. The most common analysis is that of estrogen receptor (ER) and progesterone receptor (PgR) expression. Estrogen and progesterone regulate proliferation of mammary glands and the development of breast tissue. Approximately 85 % of tumours express these two steroid receptors. The presence of ER in cancer cells is the most important marker for prediction of adjuvant tamoxifen treatment effect. Tamoxifen is a selective estrogen receptor modulator, which upon binding to the ER in breast tissue regulate the proliferative effects and acts as an anti-estrogen. Tamoxifen is used in the treatment of breast cancer in pre and postmenopausal women with ER-positive tumours. Some patients, however, will still not respond to treatment and experience a breast cancer relapse within 15 years. Because of this it’s important to aquire knowledge of and validate these prognostic and predictive biomarkes as well as futher study breast cancer biology to better individualise treatment according to patient needs.  The aim of this study was to evaluate potential predictive biomarkers in the treatment of ER-positive breast cancer with adjuvant tamoxifen. Specifically, if the effect of tamoxifen can be predicted using specific biomarkers. Potential future biomarkes evaluated were the expression of HOXB13, IL17BR, CYP2C19, CCND1 and RSF1 genes and the protein expression of mTOR, Akt, ER, PgR, EGFR, HER2, HOXB13, AIB1, CXCL10 and CXCR3. This litterature study is based on the analysis of 10 different retrospecitve studies of predictive markers based on patient materials from original randomized controlled trials evaluating the efficacy of tamoxifen. Seven of the studies were collected using the database PubMed, however two studies were collected from a thesis. The search was limited to articles which studied tumour material of pre- or postmenopausal female breast cancer patients randomized to adjuvant tamoxifen treatment or control. Based on the results presented, potential predicitive biomarkes of tamoxifen efficacy and benefit of breast cancer patients could be a high protein expression of AIB1, a low expression of the HOXB13 protein and a high expression of CXCL10 and CXCR3. Moreover, predicitve markers of tamoxifen benefit could also be a CYP2C19*2 variant allel. A reduced response to treatment could be predicted with the positive expression of p-mTOR-s2448, p-ER-s167/s305 and p-Akt. HER2-positivity could be a marker for identifying patients who will not respond to tamoxifen and may benefit greater from other treatments. These results, however, needs to be verified in bigger cohorts of breast cancer patients, e.g. in meta-analyses or prospective randomized studies.

Indiana University-Purdue University Indianapolis (IUPUI) / Breast cancer remains the most prevalent malignancy diagnosed in women. More than two thirds of all diagnosed breast cancers are estrogen receptor (ER)-positive and are dependent on estrogen signaling. Drugs for the treatment of ER-positive breast cancer can be divided into three classes: selective estrogen receptor modulators (SERMs), selective estrogen receptor down-regulators (SERDs) and aromatase inhibitors (AIs). However, the efficacy and safety of SERMs, SERDs and AIs are compromised by side effects or tumor resistance. One possible way of improving treatment efficacy and safety profiles is to develop agents with dual aromatase inhibitory and ER modulatory activity. Over the past 30 years, tamoxifen, a SERM, has become the most widely used drug for the adjuvant treatment of breast cancer. The metabolism of tamoxifen has a complex profile involving both active and inactive metabolites, among which endoxifen, 4-hydroxytamoxifen (4-HT) and norendoxifen (Nor) have been shown to have ER modulatory activity. Previous studies have also shown that norendoxifen is a potent AI in vitro. These preliminary studies support the utilization of tamoxifen metabolites as lead compounds for the development of dual AI/SERM(D) agents. Hydroxynorendoxifen (Hdn) was identified as a novel tamoxifen metabolite, with an average plasma concentration of 0.82 nM. Nor and Hdn were potent and relatively selective AIs, with Kis of 70 nM and 20 nM, respectively. Nor and Hdn have high binding affinity for ER-α and ER-β, with EC50 values less than 35 nM. Nor and Hdn can inhibit breast cancer cell proliferation with high potency, with IG50s of 25 nM and 9 nM, respectively. Nor and Hdn can suppress progesterone receptor (PGR) mRNA expression level by reducing it by 68% and 86%. Moreover, a series of Nor analogues were shown to have both potent aromatase inhibitory activity and high ERs binding affinity. Results from this dissertation will contribute to three aspects: 1) the identification of Hdn as a tamoxifen metabolite illustrated a more comprehensive metabolism profile of tamoxifen; 2) the data suggest Nor and Hdn possess dual aromatase inhibitory and ER antagonistic activity; 3) a series of Nor analogues were characterized as lead compounds for the development of dual AI/SERM(D) agents.

Cancers represent second the most common cause of death in the Czech Republic. The most common are breast and colorectal cancers. Identification of prognostic factors improving decision-making approaches for treatment optimization belongs to the key aims of clinical research in oncology. Carriers of mutation in cancer-susceptibility genes represent a small but clinically important group of high-risk patients. The implementation of NGS have accelerated predisposing genes analyses. The large extent of data about the presence of variants in predisposing genes is in striking contrast to only a very limited information available about clinico-pathological characteristics of mutation carriers. Determination of the risk of tumor development in carriers of rare mutations or variants of unclear significance in genes with incomplete penetrance represent substantial drawbacks of current NGS analyses. To address these issues, we have attempted i) to introduce a unified approach to NGS analysis in breast cancer patients, ii) to characterize importance of prognostic factors in BRCA1/BRCA2 mutation carriers, and iii) to identify the cancer risks in carriers of germline mutations in the CHEK2 gene. Colorectal cancer represents seemingly histologically homogeneous disease. However, at the molecular level it can be...

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