RASSF1A is a tumour suppressor, the silencing of which occurs through promoter methylation in a variety of human cancers. Loss of RASSF1A is associated with decreased sensitivity to DNA damaging agents and worse prognosis in breast, colon and lung cancers amongst others. RASSF1A functions in a number of cellular processes, promoting apoptosis in response to DNA damage or death receptor signalling, or cell cycle arrest at both G1/S and pro-metaphase checkpoints. As a scaffold protein, RASSF1A imparts these functions through direct interaction with target proteins. We have identified a novel interaction between RASSF1A and the SRC activator, OSSA. Further studies identify a role for RASSF1 in SRC signalling. We find that a second isoform of RASSF1, RASSF1C, the expression of which is maintained in cancers, is able to activate SRC. We also identify a novel tumour suppressor role for RASSF1A inhibiting SRC activation through binding of RASSF1C. SRC activation by RASSF1C expression promotes internalisation of adherens junctions leading to subsequent loss of tight junctions and cell polarity markers from sites of cell-cell contact. -catenin is also found to be re-localised throughout the cells from where it is hypothesised to be able to upregulate pro-proliferative genes. In addition, we find that RASSF1C expression promotes cell motility in both scratch wound and transwell assays. Finally, we show that RASSF1C expression enhances tumour cell aggressiveness using a mammosphere growth assay. We conclude that RASSF1C is an oncogene that can promote EMT through the activation of SRC family kinases. This function is inhibited by the tumour suppressor RASSF1A. This work highlights why RASSF1A is lost through epigenetic mechanisms and not mutation and why loss of RASSF1A is associated with more aggressive, metastatic cancers.

In addition to contributing genes at fertilization, the sperm cell induces the oocyte to leave its arrested state and resume metabolism in the process of activation. A hallmark of oocyte activation is a release of intracellular calcium (Ca2+i) from the endoplasmic reticulum. The mediators of oocyte activation have been studied in many animal models, while little is known in the bovine model. Both Src Family Kinase (SFK) and Phospholipase C (PLC) enzymes have been reported to be involved in oocyte activation in other animal models. In this dissertation are described experiments that define the role of SFK and PLC enzymes in the pathway leading to Ca2+i and calcium induced calcium release in bovine oocyte activation. Western blotting was used to discover that SFKs Src, Hck, and Lck are present in matured bovine oocytes, and Src, Blk, and Yes are present in acrosome reacted bovine spermatozoa. The PLC δ1 and δ3 are present in both matured bovine oocytes and spermatozoa. PLC δ4, γ2, and η2 are present in matured bovine oocytes. Microinjecting a known general SFK inhibitor, PP2, significantly decreases both Ca2+i and cleavage rates. Microinjecting a 13 amino acid peptide that mimics the phosphorylated carboxyl terminal region of pp60c-src decreases both Ca2+i and cleavage rates. Microinjecting a downstream substrate of pp60c-src sequestered any signal produced by Src and decreased Ca2+i and cleavage rates. Microinjecting primary antibodies raised against PLC isotypes blocked both Ca2+i and cleavage rates, giving insight to the mechanism of calcium induced calcium release in the bovine model. The PLC isotypes δ3, δ4, and γ2 decreased Ca2+i oscillations and cleavage rates, indicating they are involved in both IP3R and RyR activation. PLC δ4 and η2 did not impact Ca2+i but did significantly decrease cleavage rates. The data presented in this dissertation increase the understanding of the pathway leading to bovine oocyte activation and further confirm that the detailed pathway differs among animal models.

Acute myeloid leukemia (AML) is a clonal hematopoietic stem cell malignancy, marked by suppressed production of normal terminally differentiated and progenitor hematopoietic cells, and increased cellular proliferation, survival, invasion, and migration of poorly differentiated hematopoietic precursor cells called leukemic blasts. Clinical outcomes vary from good to very poor, and standard therapeutic regiments are only successful in inducing remission for approximately one half of patients. Through the use of phospho tyrosine mass spectrometry, we have identified putative candidate proteins which may be implicated in disease pathogenesis. Our in vitro data suggest a complex within the AML cell lines MOLM-14 and MV4-11 involving tyrosine phosphorylated DAP12, FCER1G, SYK, LYN, and CBL. In addition, we show the ability of high concentrations (µM) of SB203580, a p38α catalytic site inhibitor, to paradoxically sensitize cells to cytarabine while providing a modest proliferative advantage to cells treated with daunorubicin.

Acute myeloid leukemia (AML) is a clonal hematopoietic stem cell malignancy, marked by suppressed production of normal terminally differentiated and progenitor hematopoietic cells, and increased cellular proliferation, survival, invasion, and migration of poorly differentiated hematopoietic precursor cells called leukemic blasts. Clinical outcomes vary from good to very poor, and standard therapeutic regiments are only successful in inducing remission for approximately one half of patients. Through the use of phospho tyrosine mass spectrometry, we have identified putative candidate proteins which may be implicated in disease pathogenesis. Our in vitro data suggest a complex within the AML cell lines MOLM-14 and MV4-11 involving tyrosine phosphorylated DAP12, FCER1G, SYK, LYN, and CBL. In addition, we show the ability of high concentrations (µM) of SB203580, a p38α catalytic site inhibitor, to paradoxically sensitize cells to cytarabine while providing a modest proliferative advantage to cells treated with daunorubicin.

PURPOSE. Na, K-ATPase activity in lens epithelium is subject to control by Src family tyrosine kinases (SFKs). Previously we showed hyposmotic solution causes an SFK-dependent increase in Na, K-ATPase activity in the epithelium. Here we explored the role of cAMP in the signaling mechanism responsible for the SFK and Na, K-ATPase response. METHODS. Intact porcine lenses were exposed to hyposmotic Krebs solution (200 mOsm) then the epithelium was assayed for cAMP, SFK phosphorylation (activation) or Na, K-ATPase activity. RESULTS. An increase of cAMP was observed in the epithelium of lenses exposed to hyposmotic solution. In lenses exposed to hyposmotic solution SFK phosphorylation in the epithelium approximately doubled as did Na, K-ATPase activity and both responses were prevented by H89, a protein kinase A inhibitor. The magnitude of the SFK response to hyposmotic solution was reduced by a TRPV4 antagonist HC067047 added to prevent TRPV4-mediated calcium entry, and by a cytoplasmic Ca2+ chelator BAPTA-AM. The Na, K-ATPase activity response in the epithelium of lenses exposed to hyposmotic solution was abolished by BAPTA-AM. As a direct test of cAMP-dependent SFK activation, intact lenses were exposed to 8-pCPT-cAMP, a cell-permeable cAMP analog. 8-pCPT-cAMP caused robust SFK activation. Using Western blot, two calcium-activated adenylyl cyclases, ADCY3 and ADCY8, were detected in lens epithelium. CONCLUSIONS. Calcium-activated adenylyl cyclases are expressed in the lens epithelium and SFK activation is linked to a rise of cAMP that occurs upon hyposmotic challenge. The findings point to cAMP as a link between TRPV4 channel-mediated calcium entry, SFK activation, and a subsequent increase of Na, K-ATPase activity.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21447号 / 医博第4414号 / 新制||医||1032(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 武藤 学, 教授 平井 豊博, 教授 岩田 想 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

La famille des kinases Src, dont Lyn fait partie, joue un rôle clé dans le contrôle de nombreux processus biologiques. Lyn a une fonction bien établie dans les cellules hématopoïétiques et est notamment impliqué dans le maintien de différentes leucémies et son expression protéique est altérée dans les tumeurs solides. Plusieurs études ont mis en évidence qu’elle avait un rôle anti-apoptotique. Lyn peut être clivée par les caspases en donnant une protéine tronquée cytosolique. Nous avons ainsi montré que Lyn cytosolique (cLyn) régulait Bim, un membre pro-apoptotique de la famille Bcl-2, en le phosphorylant sur les tyrosines 92 et 161 en inhibant la fonction pro-apoptotique de Bim, en augmentant son interaction avec Bcl-XL, limitant ainsi la perméabilisation de la membrane externe mitochondriale et l’apoptose. Lyn possède également un rôle pro-inflammatoire. Nous avions préalablement montré que la surexpression de cLyn, chez la souris, conduit à un syndrome inflammatoire de la peau, ressemblant au psoriasis. Sur la base de ce résultat, nous avons voulu savoir si Lyn jouait un rôle dans cette maladie chronique de la peau. L’analyse de l’expression de Lyn chez des patients souffrant de psoriasis a montré que Lyn était surexprimée dans la peau lésionnelle par rapport à la peau non lésionnelle ou saine, résultats confirmés dans deux modèles de psoriasis chez la souris. De façon intéressante, nous avons montré que l’augmentation de l’expression de Lyn se situe à la fois dans le derme et dans l’épiderme chez l’homme et chez la souris, indiquant que le recrutement de cellules immunitaires dans la peau lésionnelle mais aussi la modulation de Lyn dans les kératinocytes sont impliqués. Par ailleurs, une augmentation de l’expression de Lyn a été observée dans les kératinocytes humains stimulés par le TNF-α et l'IL-17A. Afin de déterminer le rôle de Lyn dans cette maladie cutanée nous l’avons induit chez des souris déficientes pour Lyn. Une réduction significative du phénotype cutanée a été observée dans les souris LynKO, identifiant Lyn comme un nouvel acteur dans la pathogénie du psoriasis. De plus, nos résultats ont établi que l’expression de Lyn dans les kératinocytes semblait suffisante pour le maintien du phénotype psoriasique, indiquant un nouveau rôle de Lyn dans les kératinocytes. Au cours de ce travail, nous avions observé que les caspases inflammatoires étaient activées dans la peau lésionnelle de patients atteints du psoriasis. Les caspases inflammatoires, suite à leur activation, vont cliver l’IL-1β et l’IL-18, ce qui conduit à leur maturation. Nous avons alors voulu savoir si les caspases participaient au développement du psoriasis. Nous avons pu montrer que lorsque nous induisions du psoriasis chez des souris, l’invalidation des caspases inflammatoires ou son inhibition pharmacologique réduisait de façon significative le développement de la maladie. Bien que les cellules immunitaires et les kératinocytes soient capables de secréter de l’IL-1β via l’activation de l’inflammasome, nos données ont établi que seule l’activation des caspases inflammatoires dans le système immunitaire semblait nécessaire pour une réponse inflammatoire complète. En résumé, l’ensemble de mon travail de thèse a permis de montrer un mécanisme moléculaire par lequel la kinase Lyn régule négativement la voie apoptotique mitochondriale, ce qui peut contribuer à la transformation et/ou la résistance des cellules cancéreuses. D’autre part, nos résultats montrent que Lyn pourrait être un régulateur important du psoriasis, et notre étude indique que les caspases inflammatoires activées dans les cellules immunitaires sont impliquées dans la pathogenèse du psoriasis. A ce jour, bien que plusieurs traitements aient été développés pour le psoriasis, la maladie reste non résolue, donc le développement de cibles thérapeutiques contre Lyn et les caspases inflammatoires pourraient être intéressant pour le traitement de la maladie. / The Src family kinase, of which Lyn is one member, plays a key role in controlling many biological processes. Lyn has a well-established function in hematopoietic cells, presenting an important role in the regulation of hematopoietic abnormalities. In fact, Lyn plays a key role in maintaining several kind of leukemia, and furthermore it expression is altered in solid tumors. Different studies have shown that Lyn has an anti-apoptotic role. Lyn can be cleaved by caspases, cysteine proteases involved in apoptosis and inflammation, giving a new protein with a cytosolic location, different from the WT and membrane-anchored form. We have shown that the cytosolic form of Lyn (cLyn) regulated Bim, a pro-apoptotic member of the Bcl-2 family, involved in the control of mitochondrial apoptosis. We have identified that Bim is phosphorylated on tyrosine 92 and 161 by Lyn, resulting in an inhibition of its pro-apoptotic function, increasing its interaction with anti-apoptotic members such as Bcl-XL, thus limiting the permeabilization of mitochondrial outer membrane and impairing cell apoptosis. Lyn also has a pro-inflammatory role. We have previously shown that overexpression of the caspase-cleaved form of Lyn, in mice, leads to an inflammatory skin syndrome, resembling human psoriasis. Based on this result, we wanted to know if Lyn played a role in this chronic skin disease. Analysis of Lyn's expression in psoriasis patients showed that Lyn was overexpressed in lesional skin compared to non-lesional or healthy skin, which was subsequently confirmed in two mouse models of psoriasis disease. Interestingly, we have shown that the increase in Lyn expression is in both the dermis and the epidermis in humans and in mice, indicating that recruitment of immune cells into lesional skin but also the modulation of Lyn in keratinocytes are involved. To determine the role of Lyn in this skin disease we induced a psoriasis-like phenotype in Lyn deficient mice. A significant reduction in cutaneous phenotype was observed in LynKO mice compared to WT mice, identifying Lyn as a new player in the pathogenesis of psoriasis. In addition, our results established that Lyn expression in keratinocytes seemed crucial and sufficient for the maintenance of the psoriasis phenotype, indicating a new role of Lyn in the regulation of keratinocytes. During this work, we observed that inflammatory caspases were activated in lesional skin from psoriasis patients. Inflammatory caspases, following their activation within the inflammasome, will cleave IL-1β and IL-18, leading to their maturation. We then wanted to know if inflammatory caspases participated in the development of psoriasis. We were able to show that when we induced a psoriasis-like disease in mice, the invalidation of inflammatory caspases or its pharmacological inhibition significantly reduced the development of the disease compared to WT mice. Although immune cells and keratinocytes were able to secrete IL-1β via activation of the inflammasome, our data established that only activation of inflammatory caspases in the immune system seemed necessary for a complete inflammatory response. In summary, my thesis shows a molecular mechanism by which Lyn tyrosine kinase negatively regulates the mitochondrial apoptotic pathway, which may contribute to the transformation and/or chemotherapeutic resistance of cancer cells. On the other hand, our results show that Lyn could be an important regulator of psoriasis and our study indicates that inflammatory caspases activated in immune cells are involved in the pathogenesis of psoriasis. To date, although several treatments have been developed for psoriasis, the disease remains unresolved, so the development of therapeutic targets against Lyn and inflammatory caspases could be of interest for the treatment of the disease.