Cellular differentiation is a complex but fascinating process in all multicellular organisms. Differentiation can involve changes in numerous aspects of cell physiology; size, shape, polarity, metabolic activity, responsiveness to signals, and changes in gene expression profiles. These changes form the basis for differentiation to occur. The human hemochorial placenta is an intricate apposition of fetal and maternal tissues that is strategically juxtaposed at the interface, with its widespread ‘villous’ or tree-like projections, directly in contact with maternal blood. It is therefore, ideally suited to perform life-sustaining functions such as exchange of nutrients, respiratory gases and metabolic wastes, with the maternal supply. It also plays a central role in the maintenance of the immunologically privileged status of the fetal semi-allograft. Placental development is directed towards the establishment of a continuous nutrient supply to the developing fetus. This requires efficient access of maternal blood to a transporting surface, the multinucleate syncytiotrophoblast layer. This is made possible by the rapid proliferation and ensuing invasion of mononuclear trophoblasts into the maternal uterus and remodeling of the spiral arteries therein. It is interesting to note that in early pregnancy, it is the placenta that first engages its active growth and proliferation and only then, permits the logarithmic growth phase of the embryo. As a developing organ, the placenta undergoes constant tissue remodeling, which is characterized by the functional loss of trophoblast cells by apoptosis. Most of these changes occur at the trophoblast layer of the placental villous that is composed of two cell types: cytotrophoblasts (CT) and syncytiotrophoblasts (ST). The mononuclear cytotrophoblast cells, which are located between the syncytiotrophoblast layer and its basement membrane, proliferate and fuse during trophoblast differentiation to form the overlying multinucleated syncytium. CT are highly proliferating and invasive cells, in contrast to the ST which are non proliferative less invasive and functionally very active. Syncytiotrophoblast cells form the continuous, uninterrupted, multinucleated, epithelium-like surface of the placental villous that separates maternal blood from the villous interior. ST performs a crucial role in feto-maternal exchanges and serves as an endocrine tissue by its ability to synthesize and secrete a variety of hormones such as GnRH, chorionic gonadotrophin (CG), placental lactogen (PL) and steroid hormones involved in the homeostasis during pregnancy. Thus, differentiation of CT into ST serves as an ideal model to study cellular differentiation as morphologically and functionally these cells exhibit highly contrasting features. The molecular basis of cytotrophoblast differentiation has been studied using primary cultures of human trophoblast cells as a model system. Highly purified preparations of mononucleated cytotrophoblast cells can be isolated from preterm and term placental tissue by enzymatic dispersion. The isolated cells from term placental tissue aggregate spontaneously in culture and fuse to form a multinucleated syncytiotrophoblast which synthesizes and secretes placental lactogen (hPL), chorionic gonadotropin (hCG) and other syncytiotrophoblast-specific protein and steroid hormones . These in vitro changes, which recapitulate important activities accomplished by normal cytotrophoblast cells during in vivo maturation, implicate a critical relationship between the differentiation of cytotrophoblast cells into syncytiotrophoblast cells. Though primary cell culture is an ideal model to study these changes, it comes inherently associated with various problems like health risk of handling human tissues, time involved, variability in each placental samples depending on health status of the subject and quite often lack of history of the subject which makes the results from these experiments difficult to reproduce and assess. One way to overcome this is the cell culture model which is a reproducible experimental system and permits the direct observation of time-dependent processes and their experimental manipulation. BeWo cells, the cells which we have used in our study, were derived from human gestational choriocarcinoma. These cells are the highly invasive malignant counterparts of the normal human trophoblast wherein, the limited capacity for cell proliferation is far exceeded. However, they still retain important features of their normal counterpart, like the potential of hormone production and induced differentiation. Differentiation of CT to ST is precisely controlled by different agents such as transcription factors, hormones, growth factors, cytokines and oxygen levels. BeWo cells have been used by other investigators as well as by us and it has been shown that these cells can be induced to differentiate with the agents mentioned above and terminally differentiate into cells which express typical characteristics of the normal differentiating trophoblast; like morphological transition from cytotrophoblast to syncytiotrophoblast-like cells, increased production of protein and steroid hormones (hCG, hPL, estrogens, progesterone); increased activity of cellular alkaline phosphatase and arrested cell proliferation. Since these cells can be triggered by external agents to differentiate, they serve as a useful model for the study of changes that occur during differentiation. Using primary cells and various cell lines including BeWo cells, various groups have attempted to study trophoblast differentiation and the regulators that control this process. The results of such study have only come out with a list of genes or proteins which might be having a role in this process and no functional correlation has been drawn so far from these studies. The members of the syncytin protein family, ADAM (a disintegrin and metalloprotease) proteins may well be some of the main players in the process of trophoblast fusion; some of the requisites of trophoblast fusion being redistribution of phosphatidylserine to the outer leaflet of the plasma membrane and activity of certain intracellular proteases. Clearly, further studies on trophoblast differentiation are needed to answer the question of the precise identity of regulatory proteins and role of these proteins during differentiation. The present study is aimed at gaining insights into the process of trophoblast differentiation and the molecular events which occur during this process. Our aim is also to study the regulated process of differentiation using BeWo cell model and identify the differentially expressed genes and relate the known function of these gene products to changes seen during differentiation process. We have employed the Differential Display Reverse Transcriptase Polymerase Chain Reaction (DD-RTPCR) and Microarray analysis to monitor the changes in gene expression. In CHAPTER 1, a brief account of morphological, biochemical and physiological changes which occur during placentation and trophoblast differentiation is discussed. Various aspects of placental function are discussed in brief, with special reference to the many unique abilities of trophoblast cells that contribute to a successful pregnancy. Detailed accounts of molecular mechanism of cellular differentiation, the models used in these studies and the advantages and drawbacks have been highlighted. The results of the previous studies from our laboratory using different model system and the outcome of the study are also outlined in this chapter. The advantages and disadvantages of the primary cell lines and the ease of handling of continuous cell culture model, BeWo is also presented in this chapter. The aim and objective of our study is to understand the molecular mechanisms underlying the trophoblast differentiation and the literature available is reviewed in the light of the objective and the aims and scope of the present study. The details regarding the materials used and the techniques employed during the entire study are outlined in CHAPTER 2-‘Materials and Methods’. The conditions for culture of BeWo human choriocarcinoma cell line are described and details of procedures employed for the validation of BeWo cells as a model system for monitoring the process of cellular differentiation are mentioned in this chapter. The details of the procedures employed for isolation of RNA, Reverse Transcriptase Polymerase Chain Reaction (RTPCR), Differential Display RT-PCR (DD-RT-PCR), Microarray analysis, Northern Blot analysis and Western Blot analysis are also described. The principle of the MTT assay used for verifying the viability of cells following various treatments is provided along with the working protocol. This chapter also includes protocols of the in vivo studies in rat, the methods employed for rat uterine mince cultures and isolation of rat uterine epithelial cells and dose and duration of the various treatments with steroid hormones and their inhibitors, treatment with protein kinase inhibitors in cell culture system are also described. In addition, this chapter also describes the procedures for transfection of hTERT, silencing of SLPI gene using SiRNA approach, gelatin zymography, MAP Kinase assay, FACS, cloning and expression of SLPI protein and procedure employed for raising antibodies to SLPI in rabbit. Finally, details of statistical tests employed fro anlaysis of data are presented. The results obtained in the present study are presented in 4 chapters(Chapters 3-6), CHAPTER 3 describes the characterization and validation of model system employed- BeWo cells to study human trophoblastic differentiation. BeWo cells under normal culture conditions resemble cytotrophoblasts like cells and when treated with various effectors of differentiation can be induced ot differentiate into syncytiotrophoblasts. We used 10 µM Forskolin to induce differentiation in BeWo cells. Forskollin is known to induce characteristic changes associated with human trophoblast differentiation in these cells. Incubation of BeWo cultures in the presence of 10 µM Forskolin resulted in dramatic morphological biochemical changes intheir cytotrophoblast-like phenotype. Mononuclear cells were seen to fuse to form multinucleate syncytial structures over a period of 72-96 hours in culture. This process was also associated with an increased production of β-hCG, Endoglin and hTERT thereby validating this model system for study of human trophoblastic differentiation. Analysis of cell cycle genes in this system established the arrest of proliferation thus further validating the system. The viability of these cells, during the entire period of culture, was verified using the MTT assay. This chapter discusses the importance of in vitro cell culture systems in the study of human placental development, and also addresses the suitability of these model systems for the study of human trophoblast proliferation and differentiation. One of the important finding of our earlier studies was that arrest of proliferation was a prerequisite for trophoblast differentiation to occur. This conclusion was based on the fact that telomerase expression which is a hallmark of all proliferating cells was down regulated in BeWo cells by 48h as assessed by TRAP (Telomere Repeat Amplification Protocol) assay or RT-PCR analysis for hTERT which is the catalytic subunit of telomerase. Telomerase activity was undetectable by about 96th by which time syncytium formation is normally completed after the addition of differentiation inducing agents like Forskolin, TGF β etc. Although the telomeric holo enzyme consists of many components the subunits which are critical for enzyme action are hTERT and hTR; hTR; hTR which is the RNA component of telomerase is ubiquitously expressed in most cell types including telomerase negative cells such as differentiated somatic cells. Since the BeWo cells can be induced to differentiate into multinucleated ST by addition of Forskolin and periodically the aged ST are eliminated by apoptosis. It is very well documented that the life span of ST is very limited and the ST have to be replaced by the freshly formed ST out of fusion of CT. Considering this, it was of interest to test whether differentiation can be prevented or delayed by extending the expression of telomerase activity. This would further validate our system that one of the requisites for cells to differentiate is down regulation of hTERT in BeWo cells. This was achieved by transfection of BeWo cells with hTERT expression vector. The results of the study clearly established that we were able to over express hTERT in BeWo cells; we also noticed an increase in the proliferation of BeWo cells as assessed by BrdU incorporation. In agreement with this observation is the fact that, in contrast to the empty vector transfected cells, in hTERT transfected group, the cell density appeared to be clearly more at 72 h. That the decrease in the hTERT expression in the control (empty vector transfected) is not due to cell death was established by MTT assay, which indicated that there was no difference in the viability between control and hTERT transfected cells. Further more, results of analysis for a variety of cell proliferation and differentiation markers by RT-PCR and Western blot analysis clearly supports the conclusion that hTERT over expression delays syncytium formation. Although reports are available on the differential expression of genes during differentiation of CT to ST with both primary cell lines as well as BeWo cell line, relatively less is known about the functional importance of differentially expressed genes. In CHAPTER 4, results of our studies to profile the differentially expressed genes during Forskolin induced differentiation in BeWo cells by two approaches DD-RTPCR and microarray analysis and relate the known functions of these genes to changes that occur during the differentiation of CT to ST are presented. We identified several genes that had robust change during differentiation by DD RTPCR and the differential expression of ten transcripts was confirmed by Northern blot analysis. The genes which we identified were SLPI, Elongation factor-1 alpha -1, Prolyl hydroxylase beta, LIMO-4 etc. These genes were either shown to have a role during differentiation of cells or have functional role in the syncytiotrophoblasts. Secretory Leucocyte Protease Inhibitor was one of the differentially expressed transcripts which were significantly up regulated during Forskolin induced differentiation of BeWo cells. SLPI which is a 12 KDa protein reported to exhibit a variety of activities which include inhibition of proteases and elastase, in addition to antibacterial and anti inflammatory activities. It was chosen for our further studies because of its multifunctional role in placenta and also during implantation. Micro array analysis revealed the up-regulation of hCG, hCS, and Endoglin thus validating the experimental system. Several candidate genes that could influence trophoblast differentiation, cell adhesion and cellular proliferation were identified. Genes involved in cellular proliferation include cyclin M3, replication factor 3, signal-induced proliferation-associated gene 1, osteonectin, clusterin, etc clearly indicating a growth-arrested phenotype for the differentiating BeWo cells. Trophoblastic differentiation associated genes included adipose differentiation-related protein, GADD45A, PPAR binding protein, galectin 3, tubulins, collagen, stathmin, etc. The p53 tumor suppressor protein plays a major role in cellular response to DNA damage and other genomic aberrations. Activation of p53 can lead to either cell cycle arrest or DNA repair or apoptosis. Although we did not observe any change in the p53 mRNA levels, the total protein level as well the phosphorylation status of p53 was up regulated upon differentiation. We confirmed the down regulation of Cyclin D1, D2 and PCNA in differentiated cells and up regulation of CDK inhibitors, P27kip1, P21cip1which are p53 induced genes by RT-PCR and Western blot analysis. Phosphorylation of ser 20 leads to reduced interaction of p53 with its negative regulator MDM2. MDM2 inhibits the accumulation of p53 by targeting it for ubiquitination and proteosomal degradation. Analysis for the phosphorylated status of p53 revealed that specifically the ser 20 phosphorylated p53, was increased upon differentiation. Phosphorylation of ser-392 has been reported to influence the growth repressor function, DNA binding and transcriptional activation of p53 and in agreement with this, western blot analysis revealed an increase in the ser-392 phosphorylated p53. These results suggest that p53, a nuclear protein regulating several genes involved in proliferation and differentiation is playing a pivotal role in growth arrest during trophoblast differentiation. We also noticed that several components of the apoptotic cascade are differentially expressed in cytotrophoblast and the syncytiotrophoblasts layer, and these changes appear to be associated with the stage of apoptosis. Apoptosis is involved in the removal of aging syncytiotrophoblasts and it also promotes cytotrophoblast fusion and formation of the syncytial layer. We found that various apoptosis related genes are up regulated and anti apoptotic genes suppressed following differentiation in our micro array analysis. We identified the involvement of p53 in this process also and chapter 4 deals with this aspect. Genes which regulate the invasive behaviour of trophoblasts which include MMP2, cathepsin K, cystatin N, SLPI and cysterine-rich angiogenic inducer 61, etc. were found to be up regulated following differentiation in our micro array analysis, which establishes these differences in gene expression reflects the physiological changes that occur during placentation. The Co-ordinated regulation of ptoteases and protease inhibitors I (for example SLPI, cystatin B and MMP2) suggests that these genes play an important role in the regulation trophoblast invasion at the uterine-placenta interface in vivo. Our studies revealed that one of the transcripts,namely, SLPI(Secretory leukocyte protease inhibitor) was robustly up regulated as assessed in DDRT-PCR, micro-array, Northern blot and RT-PCR analysis. Considering its importance in implantation, placentation and maintenance of pregnancy several aspects of this multifunctional protein were studied in detail and the results are presented in CHAPTER 5. Studies on the regulation of this transcript in Be-Wo cells revealed that SLPI mRNA is regulated by progesterone in Be-Wo cells. The up regulation of SLPI mRNA by progesterone was specifically inhibited by Progesterone receptor antagonist, RU 486 and estradiol 17β did not have any effect on the expression of SLPI mRNA expression in BeWo cells. The absence of regulation of SLPI by estradiol in BeWo cells was also established by the fact that simultaneous addition of progesterone and aromatase inhibitor, fadrazole did not block the increase in SLPI expression. Interestingly in vivo and in vitro studies using rat uterine minces and rat epithelial cells revealed that SLPI mRNA is regulated by Estradiol 17β and the effect is specifically inhibited by estrogen receptor antagonists such as ICI 182780, Tamoxifen, and Centchorman. Promoter analysis of rat and human SLPI revealed the absence of a consensus progesterone responsive element (PRE) in human and estrogen responsive element (ERE) in rat, suggesting the possibility of a non-genomic action of progesterone or estrogen in the induction of SLPI mRNA. This was confirmed by the observation that induction of SLPI mRNA could be effectively blocked by the addition of Staurosporine, an inhibitor of protein kinase C along with progesterone and estrogen to either BeWo cells or rat uterine epithelial cells. These results suggest that the non-genomic action of steroid hormones may be involved in the induction of SLPI. In the present study, we have also identified the intracellular signaling pathway that regulates SLPI gene expression by using various protein kinase inhibitors. We have also shown that activation of MAP kinase pathway upon progesterone treatment and the involvement of protein kinases in this activation, permitting us to conclude the non genomic action of progesterone in induction of SLPI mRNA in BeWo cells. The results of these studies are presented in detail in Chapter 5. The observation that SLPI expression is markedly increased during differentiation and differentially regulated by progesterone and estradiol, and induction by non genomic pathway prompted us to undertake studies to investigate its role during differentiation. This was accomplished by using SiRNA to silence the expression of SLPI in Forskolin induced differentiating BeWo cells and the results of this study are presented in CHAPTER 6. Different concentrations and combinations of oligos were used to silence the SLPI gene and we found that effective knockdown (>80%) was achieved with SiRNA concentrations ranging from 5-25nM. A combination of oligos also increased the knockdown from 50% to 90% as assessed by RT-PCR and western blot analysis for mRNA and protein levels of SLPI respectively. We found that inhibition of SLPI expression by SiRNA also inhibited the morphological differentiation of BeWo cells. Functionally this was reflected, by increase in the protease activity as assessed by gelatin zymography. It should be noted that SLPI is a protease inhibitor; it inhibits a variety of proteases, including proteases from neutrophils, pancreatic acinar cells and mast cells and SLPI present in the syncytiotrophoblast may have a crucial role in controlling protease activity associated with invasiveness and differentiation. Inhibition of differentiation by silencing the expression of SLPI provides an opportunity to monitor the changes in gene expression where in a single gene has been silenced in contrast to the model employed in chapter 4. We carried out microarray analysis using control (Forskolin treated) and SLPI silenced (Forskolin treated) samples. The results revealed that proliferation and differentiation, apoptosis and inflammatory pathways genes are affected due to SLPI silencing and the results of this study are presented in CHAPTER 7. We confirmed the changes in gene expression by semi quantitative RT-PCR analysis of the some important genes in each pathway. A comparison of the results obtained with that of our earlier microarray analysis which is described in chapter 4 revealed that the changes in levels of expression of the genes involved in cell proliferation, differentiation, apoptosis and inflammation were completely reversed after silencing the expression of SLPI. We have presented in chapter 5 the importance of MAP kinase pathway in Forskolin induced differentiation and the activation of this pathway when SLPI expression is increased following progesterone treatment. Interestingly after silencing the expression of SLPI we found that MAP kinase pathway is affected. It was observed that silencing of SLPI expression resulted in inhibition of activation of MAP kinase as assessed by the phosphorylation status by ELISA and no activation of MAP kinase was observed in SLPI silenced Forskolin treated cells. CHAPTER 8 provides a general discussion of the results obtained in the present study in the light of current understanding the type of genes involved, changes during human trophoblastic proliferation and differentiation and the key players during this process. This chapter also brings out the importance of SLPI during trophoblastic differentiation, placentation, implantation and its regulation by steroid hormones. The highlights and salient features of the present study are summarized in this chapter. In CONCLUSION, the present investigation has led to the identification of specific genes involved in trophoblast differentiation, human placental growth and development. Also evident from this study is the usefulness of the trophoblastic cell culture system for the study of cellular differentiation. We have attempted to relate the gene expression changes to physiological changes that occur during placentation, implantation and pregnancy. Many of the regulatory events that we have described during human trophoblastic differentiation, may not only be restricted to these cells, but may represent common principles/features of cellular differentiation in general. Loss of differentiation is a wide-spread feature of tumor progression, and frequently accompanies aggressive neoplastic behavior. Our studies provide unequivocal evidence to support cellular differentiation as a natural barrier to malignant transformation. Most importantly we have shown that silencing of a single gene can disrupt this differentiation process and the importance of SLPI during differentiation process perse.

Les môles hydatiformes complètes (MHC) sont des lésions bénignes précancéreuses du placenta qui dans 5% des cas évoluent vers un cancer très prolifératif dénommé, choriocarcinome (CC). Différents travaux ont rapporté une corrélation entre le développement des MHC récurrentes et les mutations du gène NLRP7. Ce gène code pour la protéine NLRP7 de l’inflammasome dont l’activation contribue à la production d’IL-1 et IL-18. La majorité des travaux publiés à ce jour se sont intéressés à l’étude des mutations de NLRP7. En revanche, aucune étude n’a caractérisé son rôle dans la placentation normale et tumorale. L’objectif de mon projet de thèse était de caractériser le rôle de cette protéine dans le placenta normal et tumoral. Trois approches ont été utilisées, i) Clinique, en collaboration avec le CHU de Casablanca ; le centre de référence Français des Maladies Gestationnelles Trophoblastiques (MGT) et l’Université Mc Gill pour l’accès aux tissus et sera collectés chez des patientes MHC ou CC; ii) In Vitro/Ex Vivo, pour la caractérisation du rôle du NLRP7 dans les processus clés du développement placentaire normal et tumoral dans des systèmes de cultures en 2D et 3D. Deux lignées trophoblastiques ont été utilisées, les cellules non tumorales HTR-8 Sv/Neo et la lignée JEG3 issue d’un CC humain ; iii) In Vivo, par l’injection orthotopique de cellules JEG3, invalidées ou non pour l’expression du gène NLRP7 (stratégie ShRNA), dans le placenta de souris gestantes. L’impact tumoral des JEG3 suite à leur injection dans la corne utérine et dans la veine de la queue de souris non gestantes a également été étudié.Mes travaux ont démontré que la protéine NLRP7 est abondamment exprimée dans le placenta normal au cours du premier trimestre de la grossesse, que son expression est régulée positivement par l’hypoxie, paramètre clé du développement du placenta et que cette protéine contrôle les processus clés du développement placentaire comme la prolifération et la différentiation trophoblastique. Par ailleurs, j’ai aussi démontré que NLRP7 joue un rôle compensatoire important dans la pathologie du retard de croissance intra-utérin. En relation avec le développement tumoral placentaire, mes travaux ont démontré que, i) la protéine NLRP7 est augmentée dans le placenta de patientes MHC et CC, et que les protéines de la machinerie des inflammasomes sont aussi dérégulées, ii) les cellules tumorales JEG3 surexpriment le NLRP7 comparé au HTR-8 Sv/Neo, iii) l’invalidation de NLRP7 dans les JEG3 induit une baisse de leur prolifération et augmentation de leur migration et invasion dans les systèmes de culture 2D et 3D. L’étude in vivo a démontré que l’invalidation de NLRP7 diminue le développement et la métastase du CC humain dans les trois modèles étudiés. Les analyses immunohistologiques, RNAseq et anticorps-array ont permis la caractérisation des mécanismes régulés par la protéine NLRP7 dans les JEG3. L’ensemble de mes travaux ont mis en évidence le rôle critique de la protéine NLRP7 dans le développement placentaire normal et tumoral et proposent la machinerie NLRP7 comme cible potentielle pour le traitement du CC. / Complete hydatidiform moles (CHM) are benign precancerous lesions of the placenta that evolve in 5% of cases into a highly proliferative cancer called choriocarcinoma (CC). Numerous studies have reported correlations between the development of recurrent CHM and mutations in the NLRP7 gene. NLRP7 protein belongs the NLRP7-inflammasome, whose activation contributes to the production of mature IL-1 and IL-18. Most of the work published on NLRP7 was focused on the study of NLRP7 mutations in CHM. Though, no study has characterized its role in normal and tumor placental development. The aim of my thesis project was to characterize the role of this protein in the normal and tumor placenta. Three approaches were used, i) A clinical approach, in collaboration with Casablanca University Hospital; the French reference center of Trophoblastic Gestational Disease and with McGill University. These collaborations allowed for tissue access from MHC and CC patients; ii) An in Vitro / Ex Vivo approach for the characterization of the role of NLRP7 in key processes of normal and tumor placental development using 2D and 3D culture systems. Two trophoblastic cell types were used, a non-tumor cell line, the HTR-8 Sv/Neo and the JEG3 cells, derived from human CC; iii) An In Vivo approach through orthotopic injection of JEG3 cells, invalidated or not for the expression of the NLRP7 gene (ShRNA strategy), in the placenta of gravid mice. The tumor impact of JEG3 following their injection into the uterine horn and the vein of the tail of non-pregnant mice was also examined.The first part of my work showed that NLRP7 protein is abundantly expressed in the normal placenta during the first trimester of pregnancy, that its expression is upregulated by hypoxia, a key parameter in placental development, and that this protein controls key processes of placental development such as proliferation and differentiation. Importantly, I have also demonstrated that NLRP7 plays an important compensatory role in the pathology of intrauterine growth retardation. The second part of my work concerning the role of NLRP7 protein in the placental tumor development demonstrated that i) NLRP7 protein levels are increased in the placenta of MHC and CC patients, and that components of the inflammasome machinery are also deregulated, ii) the JEG3 cells overexpress NLRP7 compared to HTR-8 Sv/Neo, iii) NLRP7 knock-down in JEG3 induced a significant decrease in their proliferation and an increase in their migration and invasion both in the 2D and 3D culture systems. The in vivo study demonstrated that the knock-down of NLRP7 decreased the development and metastasis of human CC in the three tested routes. Immunohistological, RNAseq and antibody-array analyses allowed the characterization of the pathways regulated by the NLRP7 protein in JEG3 cells.Altogether my PhD project characterized the critical role of the NLRP7 protein in normal and tumor placental development and proposes the NLRP7 machinery as a potential target for CC treatment.

Les môles hydatiformes sont une prolifération placentaire prétumorale pouvant évolueren tumeur alors traitée par chimiothérapie. Afin de réduire la mortalité et d’optimiser laprise en charge thérapeutique, l’objectif de cette thèse est d’identifier les gènespermettant de prédire la transformation en tumeur post môlaire et la chimiorésistance.Concernant la prédiction de la transformation, l’analyse de l’expression de gènescandidatssur tissu molaire décrit la relocalisation apicale de la Syncytine-1 en cas detransformation maligne, sans modification de transcription de ses récepteurs ni de deuxautres enveloppes rétrovirales placentaires. L’analyse sans à priori du transcriptome par3 méthodes différentes n’a pas permis d’identifier de gène différentiellement expriméselon la transformation. Cela suggère que la variabilité interindividuelle et les diverscritères utilisés pour le diagnostic de tumeur nuisent à l’identification de biomarqueursrobustes.Concernant la prédiction de la chimiorésistance, une approche transcriptomique largespectre sur tissu tumoral de choriocarcinome identifie une réduction de transcriptiond’HLA-G en cas de monochimiorésistance, confirmée au niveau protéique par immunohistochimie. L’analyse en réseaux de l’ensemble des gènes différentiellementexprimés suggère que la monochimiorésistance est associée à une altération de ladifférenciation des lymphocytes T alors que la polychimiorésistance est associée à unealtération de la prolifération des cellules sanguines.In fine, l’objectivation de l’expression trophoblastique du point de contrôle PD-L1 aconduit à évaluer l’efficacité d’un anti PD-L1 chez les patientes chimiorésistantes. Lesrésultats encourageants de cet essai et la possibilité de stratifier les patientes à l’aidedes marqueurs HLA-G et Syncytine-1 incitent à évaluer la place de l’anti PD-L1 associéà une monochimiothérapie en première ligne de traitement des tumeurstrophoblastiques. / Hydatidiform moles are a pretumoral placental proliferation which can turn into a tumorrequiring chemotherapy. In order to reduce mortality and propose an optimal therapeuticmanagement, the aim of this thesis is to identify genes which are predictive of postmolartumor transformation and chemoresistance.Concerning the prediction of transformation, the expression analysis of candidate-geneson molar tissue shows a relocalization of Syncytin-1 at the syncytiotrophoblast apicalborder in moles followed by malignant transformation, without modification oftranscription of its receptors and two other retroviral placental envelopes. A wholetranscriptomeapproach using 3 different microarrays-based methods did not identify anydifferentially expressed gene according to the post molar evolution. This may reflect thatinter-individual variability and the different criteria used for tumor diagnosis impede theidentification of robust biomarkers.Concerning the prediction of chemoresistance, a broad-spectrum transcriptomicapproach on choriocarcinoma tumor tissue identifies a down regulation of HLA-G in case of monochemoresistance, confirmed at the protein level by immunohistochemistry.Pathway analysis of the differentially expressed genes suggests thatmonochemoresistance is associated with impaired T-cell differentiation, whereaspolychemoresistance is associated with impaired proliferation of blood cells.Ultimately, the evidence of trophoblastic ubiquitous expression of the PD-L1 immunecheckpoint led us to the evaluation of the efficacy of PD-L1 blockade in chemoresistantpatients. The encouraging results of this trial and the possibility of stratifying patientswith HLA-G and Syncytin-1 markers encourages the assessment of PD-L1 blockadecombined with monochemotherapy as a first line treatment for trophoblastic tumors.

Orientadores: Liliana Aparecida Lucci de Angelo Andrade, Fatima Aparecida Böttcher-Luiz / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-24T02:59:06Z (GMT). No. of bitstreams: 1 Pietro_Luciana_D.pdf: 3492137 bytes, checksum: 723d823e8ddb16925da7aa8f48f22ea1 (MD5) Previous issue date: 2013 / Resumo: A invasão do endométrio pelo trofoblasto extraviloso é fundamental no desenvolvimento do feto e da placenta, processo este controlado por fatores ligados à atividade imunológica e hormonal que, quando alterada, pode resultar em interrupção da gestação e/ou geração das chamadas doenças trofoblásticas gestacionais. Em algumas situações, pode haver evolução para o coriocarcinoma, neoplasia maligna do trofoblasto, em que há evidências da atuação das moléculas ligadas ao processo de fusão celular e inflamação. Porém, os estudos neste tema são incipientes e inconclusivos. Considerando essas informações, o objetivo deste trabalho é estudar de forma comparativa a expressão das citocinas LIF, IL-6 e do ativador de transcrição STAT-3, além da telomerase, em material de aborto, de placenta normal a termo e de coriocarcinoma. Métodos: a expressão destas moléculas foi avaliada pelos métodos: imunoistoquímica (IHQ), imunofluorescência (IF), Western Blotting (WB) e Real-Time PCR (RT-PCR), em amostras de material de aborto, placenta normal a termo e coriocarcinoma (N=12 cada um). Os ensaios de WB e Real-Time PCR empregaram material a fresco de placenta normal a termo e seu cultivo celular e cultura da linhagem BeWo. Resultados: no material de aborto, as reações de IHQs evidenciaram expressão moderada de IL-6 em 58,4% dos casos e intensa de STAT-3 em 33,3%. Na placenta normal, observou-se intensa marcação de IL-6 em 50% e de STAT- 3 em 16,7% dos casos, enquanto que, no coriocarcinoma, houve expressão intensa de IL-6 em 50% e de STAT-3 em 75% dos casos. Por outro lado, as reações para LIF tiveram expressão nula em todos os três grupos. Pelo WB houve expressão proteica de IL-6 apenas no material fresco de placenta normal e ausência de expressão na sua cultura primária e na linhagem BeWo; LIF não foi expresso em todos os grupos estudados. STAT-3 foi detectado no citoplasma em todos os grupos, entretanto, a expressão nuclear da STAT-3 fosforilada (pSTAT-3) não foi observada na IF e nem pelo WB. Na análise gênica pelo RTPCR houve forte expressão de IL-6 e STAT-3 no material fresco de placenta normal e expressão muito fraca na cultura primária de placenta normal e na linhagem BeWo; a expressão de LIF foi muito fraca em todos os grupos. Apenas a linhagem BeWo demonstrou forte expressão gênica da telomerase, contrastando com a completa falta de expressão no material fresco de placenta normal e em sua cultura primária. Conclusão: A intensa expressão IHQ de IL-6 e STAT-3 no coriocarcinoma indica a atuação de ambas na carcinogênese. A expressão proteica de IL-6 no material fresco de placenta normal e sua ausência no material de cultura primária e na linhagem BeWo pode ser ocasionado pelo contato célula-a-célula nas culturas aderentes, inibindo o crescimento celular e, consequentemente, as vias de sinalização. A falta de expressão da pSTAT-3 tanto na IF como por WB demonstra que a via JAK-STAT está sendo desativada. A ausência de expressão de LIF, em todos os métodos estudados, sugere que esta citocina poderia estar sendo inibida por meio de proteínas SOCS3 ou, atuando, de modo indireto, na proliferação celular do coriocarcinoma. O aumento da atividade da telomerase nas células BeWo reforça sua relação com o fenótipo maligno e a aponta como um bom marcador para progressão da doença / Abstract: The invasion of the endometrium by extravillous trophoblast is a fundamental process in the growth of the fetus and placenta. The process is controlled by factors related to the immune and hormonal activity that, when changed, may result in termination of pregnancy and development of so-called gestational trophoblastic diseases. In some cases, changes can result in malignancy, in which some molecules play a role in cell fusion process and inflammation, although studies in this area are inconclusive. Considering this information, the study had the aim of investigating the expression of cytokines LIF, IL-6, STAT- 3 and the function of telomerase to understand their participation in abortion, in normal at term placenta and choriocarcinoma. Methods: The expression of the molecules was assessed by immunohistochemical assay (IHC), immunofluorescence (IF), Western Blotting (WB) and Real-Time PCR (RT - PCR) using fixed material from biopsies of abortions, normal at term placentas and choriocarcinoma along with fresh tissue of normal at term placenta and their primary culture and BeWo cell line. Paraffin embedded material used in IHC and IF assays were obtained from the Department of Pathology files. Tests of WB and Real-Time PCR employed fresh material, obtained from cell cultures of normal at term placenta and the BeWo line. Results: IHC reactions to abortion biopsies showed moderate staining for IL-6 in 58.4% of cases and intense for STAT-3 in 33.3 % of cases. In biopsies of normal placenta, there was intense reaction for IL-6 in 50% of cases, intense for STAT-3 in 16.7%; choriocarcinoma showed intense staining for IL- 6 in 50% of cases and also for STAT-3 in 75% of cases. On the other hand, LIF expression was missing in all three groups. WB analyses showed IL-6 protein in fresh material from normal placentas, but no expression in placenta primary cultures and BeWo line. LIF was absent in all groups. Cytoplasmic STAT-3 was observed in all groups, while the nuclear expression of phosphorylated STAT-3 was absent. On gene analyses a strong expression of IL-6 and STAT- 3 was observed from fresh normal placenta, but very weak expression in primary cultures of normal placenta and BeWo cell line. LIF expression was very weak in all groups. In regard to the gene expression of telomerase, it was strong in the BeWo line which contrasted with its complete lack of expression in fresh normal placenta and its primary culture. Conclusion: The high expression of IL-6 and STAT-3 in biopsies of choriocarcinoma indicates the role of both in tumor progression. Regarding protein expression, the presence of IL-6 in the material from fresh normal placenta, and its absence in primary culture and BeWo line may be caused by the cell-to-cell contact cultures by inhibiting cell growth and thus signaling pathways. However, the lack of expression of phosphorylated STAT-3 whether through IF or WB shows that its JAK-STAT pathway is inhibited. Lack of expression of the LIF suggests that it might be involved indirectly in choriocarcinoma cell proliferation or be inhibited by SOCS3 protein. Moreover, the increased telomerase activity of BeWo cells enhances their relation to the malignant phenotype and indicates a good marker for disease progression / Doutorado / Ciencias Biomedicas / Doutora em Ciências Médicas