The Ste20‐like kinase (SLK) has been shown to be expressed in all cell lines and tissues. Previous studies using siRNA and dominant negative approaches have established that SLK plays important roles in cell growth, cytoskeletal dynamics and cell migration. However, the SLK-dependent signaling mechanisms have yet to be elucidated. To further investigate the role of SLK in those processes, we have assessed the effect of a genetic deletion of SLK on cell growth, apoptosis and cell motility. Conditional SLK-floxed fibroblasts were derived and the SLK gene was inactivated in established cell lines. Using cell counts and flow cytometry, we show that SLK deletion does not affect cell growth or progression through the cell cycle. Similarly, Boyden chamber migration assay showed that SLK deletion did not affect cell motility. Using a scratch wound assays and immunofluorescence, we assessed the localization of cytoskeletal protein during migration. Our data show that Paxillin and FAK are still recruited to the leading edge of migrating cells in the absence of SLK. However, nocodazole release studies show that SLK-deficient cells have a faster focal adhesion turnover rate. As SLK has been shown to play a role in apoptosis, we tested the effect of SLK deletion on cell death. Using multiple apoptotic triggers, we show that SLK deletion does not affect apoptosis in fibroblasts and that signaling downstream of those triggers is unchanged. Overall, in contrast to siRNA studies, our data show that the genetic deletion of SLK doesn’t affect cell growth, apoptosis or cell migration. This is likely due to the activation of compensatory mechanisms, bypassing the requirements for SLK.

Duchenne Muscular Dystrophy (DMD) is a fatal X-linked disorder affecting 1 out of every 3500 male births. The underlying cause of DMD is mutations within the dystrophin gene resulting in loss of protein expression, which leads to myofiber instability and damage. The constant damage of skeletal muscle causes sustained immune infiltration, marked by increased levels of cytokines, such as TGF-beta. Interestingly, TGF-beta can decrease the myogenic potential of satellite cells, thus preventing muscle regeneration. Previously, our lab has shown that knockdown of the Ste20 Like Kinase, SLK, in normal mammary epithelial cells was sufficient to delay TGF-beta induced epithelial to mesenchymal transition. Therefore, we speculated that decreasing SLK levels would be sufficient to decrease the anti-myogenic effects of TGF-beta both in cultured myoblasts and in a mouse model of muscular dystrophy. In the first section of this study, we explored the effect of muscle specific deletion of SLK on muscle development and regeneration. Skeletal muscle specific deletion of SLK did not impair muscle development, but caused a myopathy in older mice. Additionally, muscle regeneration was delayed, but not inhibited by SLK deletion. These findings indicated that SLK has beneficial roles in skeletal muscle, but was not absolutely required for optimal muscle development and regeneration. In the second section, we investigated the potential for SLK knockdown to mitigate the anti-myogenic effects of TGF-beta in vitro. Decreasing levels of SLK restored myoblast differentiation in the presence of TGF-beta in a p38 dependent manner. In the final section, we determined that SLK levels are elevated in dystrophic muscle and that subsequent deletion of SLK in the mdx mouse enhances terminal differentiation of myoblasts without further exacerbating the pathology of the disease. Collectively, this work demonstrates that SLK inhibition can provide a protective effect against the anti-myogenic effects of TGF-beta via upregulation of p38 activity.

Over the past two decades, the mammalian Ste20-like kinase (SLK) has been characterized for its role in regulating cellular migration, proliferation and apoptosis in fibroblasts and myoblasts. In mammary epithelial cells, SLK has been shown to be required for efficient epithelial-to-mesenchymal transition and to be activated downstream of the HER2/Neu-oncogene to control chemotactic cellular migration. Here, we assessed the role of SLK in HER2/Neu-induced mammary tumorigenesis in vivo. As SLK is activated downstream of HER2/Neu, we hypothesized that the loss of SLK would significantly delay tumor progression in a mouse model of HER2-positive breast cancer. As we have shown that global attenuation of SLK kinase activity results in embryonic lethality, a conditional SLK knockout mouse model was generated. To study the role of SLK in HER2-positive breast cancer, we crossed these conditional SLK knockout mice with mice expressing HER2/Neu linked to Cre recombinase in the mammary luminal epithelium. Unexpectedly, we have demonstrated that conditional deletion of SLK significantly accelerates Neu-induced mammary tumor onset and decreases overall survival. SLK deletion results in the induction of Sox10 which drives mammary stem/progenitor activity and cooperates with HER2/Neu to drive tumor growth. Using the Cancer Genome Atlas, we have supported previous findings and validated Sox10 as a potential biomarker of the Triple-negative Breast Cancer subtype. Furthermore, we have uncovered that SLK deletion results in enhanced activation of both PDK1 and AKT. We provide evidence that Sox10 induction requires signaling through a novel AKT/Sox9-dependent pathway following SLK deletion. Taken together, our data suggests that SLK may be required to maintain cells in a fully differentiated state and that loss of SLK in HER2/Neu-induced breast cancer drives a more basal/stem-like phenotype through the induction of Sox10.

The precise mechanism regulating focal adhesion disassembly has yet to be elucidated. Recently, we have implicated the Ste20-like kinase SLK in mediating efficient focal adhesion turnover and cell migration in a Rac-1 and FAK-dependent manner. Although an indirect association of this kinase with the microtubule network has been determined, the exact involvement of SLK in the disassembly of the adhesion complex remains unclear. With the identification of the focal adhesion protein paxillin as a substrate of SLK, we show that SLK regulates adhesion turnover through its phosphorylation at S250. Mutation of S250 to a threonine residue ablates SLK phosphorylation of paxillin in vitro and results in reduced adhesion turnover and migration in vivo. Additionally, our studies demonstrate that overexpression of the paxillin S250T mutation prevents the redistribution of paxillin to the membrane ruffle in migrating cells. The complete loss of polyubiquitylation in the S250T mutant, combined with no observed reduction in S250T protein expression, suggests that S250 phosphorylation is required for a ubiquitin-mediated modification that regulates paxillin redistribution within the cell. Moreover, we show that phosphorylation of S250 is required for paxillin to interact with FAK. An observed accumulation of phospho-FAKY397 in cells overexpressing the paxillin S250T mutant suggests that phosphorylation of S250 is involved in regulating FAK-dependent focal adhesion dynamics. Consequently, our data suggests that SLK regulates adhesion turnover through the phosphorylation of paxillin at S250.

The precise mechanism regulating focal adhesion disassembly has yet to be elucidated. Recently, we have implicated the Ste20-like kinase SLK in mediating efficient focal adhesion turnover and cell migration in a Rac-1 and FAK-dependent manner. Although an indirect association of this kinase with the microtubule network has been determined, the exact involvement of SLK in the disassembly of the adhesion complex remains unclear. With the identification of the focal adhesion protein paxillin as a substrate of SLK, we show that SLK regulates adhesion turnover through its phosphorylation at S250. Mutation of S250 to a threonine residue ablates SLK phosphorylation of paxillin in vitro and results in reduced adhesion turnover and migration in vivo. Additionally, our studies demonstrate that overexpression of the paxillin S250T mutation prevents the redistribution of paxillin to the membrane ruffle in migrating cells. The complete loss of polyubiquitylation in the S250T mutant, combined with no observed reduction in S250T protein expression, suggests that S250 phosphorylation is required for a ubiquitin-mediated modification that regulates paxillin redistribution within the cell. Moreover, we show that phosphorylation of S250 is required for paxillin to interact with FAK. An observed accumulation of phospho-FAKY397 in cells overexpressing the paxillin S250T mutant suggests that phosphorylation of S250 is involved in regulating FAK-dependent focal adhesion dynamics. Consequently, our data suggests that SLK regulates adhesion turnover through the phosphorylation of paxillin at S250.

The precise mechanism regulating focal adhesion disassembly has yet to be elucidated. Recently, we have implicated the Ste20-like kinase SLK in mediating efficient focal adhesion turnover and cell migration in a Rac-1 and FAK-dependent manner. Although an indirect association of this kinase with the microtubule network has been determined, the exact involvement of SLK in the disassembly of the adhesion complex remains unclear. With the identification of the focal adhesion protein paxillin as a substrate of SLK, we show that SLK regulates adhesion turnover through its phosphorylation at S250. Mutation of S250 to a threonine residue ablates SLK phosphorylation of paxillin in vitro and results in reduced adhesion turnover and migration in vivo. Additionally, our studies demonstrate that overexpression of the paxillin S250T mutation prevents the redistribution of paxillin to the membrane ruffle in migrating cells. The complete loss of polyubiquitylation in the S250T mutant, combined with no observed reduction in S250T protein expression, suggests that S250 phosphorylation is required for a ubiquitin-mediated modification that regulates paxillin redistribution within the cell. Moreover, we show that phosphorylation of S250 is required for paxillin to interact with FAK. An observed accumulation of phospho-FAKY397 in cells overexpressing the paxillin S250T mutant suggests that phosphorylation of S250 is involved in regulating FAK-dependent focal adhesion dynamics. Consequently, our data suggests that SLK regulates adhesion turnover through the phosphorylation of paxillin at S250.

The precise mechanism regulating focal adhesion disassembly has yet to be elucidated. Recently, we have implicated the Ste20-like kinase SLK in mediating efficient focal adhesion turnover and cell migration in a Rac-1 and FAK-dependent manner. Although an indirect association of this kinase with the microtubule network has been determined, the exact involvement of SLK in the disassembly of the adhesion complex remains unclear. With the identification of the focal adhesion protein paxillin as a substrate of SLK, we show that SLK regulates adhesion turnover through its phosphorylation at S250. Mutation of S250 to a threonine residue ablates SLK phosphorylation of paxillin in vitro and results in reduced adhesion turnover and migration in vivo. Additionally, our studies demonstrate that overexpression of the paxillin S250T mutation prevents the redistribution of paxillin to the membrane ruffle in migrating cells. The complete loss of polyubiquitylation in the S250T mutant, combined with no observed reduction in S250T protein expression, suggests that S250 phosphorylation is required for a ubiquitin-mediated modification that regulates paxillin redistribution within the cell. Moreover, we show that phosphorylation of S250 is required for paxillin to interact with FAK. An observed accumulation of phospho-FAKY397 in cells overexpressing the paxillin S250T mutant suggests that phosphorylation of S250 is involved in regulating FAK-dependent focal adhesion dynamics. Consequently, our data suggests that SLK regulates adhesion turnover through the phosphorylation of paxillin at S250.

This thesis on the Swedish voluntary women’s defence organisation Riksförbundet Sveriges lottakårer (SLK), studies how the board and the members of SLK discussed the problems and the meaning of voluntary defence work during the 1960s and 1970s. Furthermore the thesis aims to engage with previous research and give an explanation to why people may be interested in voluntary defence. Views varied greatly within SLK: some meant that the voluntary grounds of participation gave a sense of elite status to the organisation; however others saw voluntary work as untenable, since more women at the time had both work and family obligations. Another issue was professionalisation versus socialisation. While some members wanted to see a centralisation and professionalisation of the organisation, others found it more important to safeguard the social functions and independence of the local corps unit. The results suggest that gender structures as well as individual agency create multiple reasons and meanings behind voluntary defence work.

La migration cellulaire est un mécanisme important pour divers processus cellulaires tels que l’embryogenèse et la cicatrisation. De même, elle participe à des processus pathologiques notamment l’invasion des cellules malignes et la formation des métastases cancéreux. La dissémination métastatique est un processus très compliqué. L’acquisition du pouvoir migratoire invasif par la cellule maligne ainsi que son potentiel métastatique est gérée par le cytosquelette qui est dynamiquement modifié et contrôlé par des voies de signalisation intracellulaires. Cependant, la physiologie des cellules métastatiques et les cascades de signalisation qui les poussent à métastaser ne sont toujours pas comprises. Les protéines Ezrine, Radixine et Moésine (ERMs) jouent un rôle important dans l’organisation du cytosquelette au cortex cellulaire et elles sont des déterminantes clés de la migration cellulaire. Ainsi, une dérégulation à ce niveau peut conduire à une migration cellulaire aberrante. D’où l’implication des ERMs dans différents cancers agressifs et invasifs. Les ERMs sont régulées en aval de plusieurs acteurs cellulaires notamment les récepteurs membranaires. Plusieurs études ont rapporté que le récepteur au thromboxane A2 (RTXA2), un récepteur couplé à la protéine G (RCPG) favorise les métastases. Il a été décrit surtout dans le cadre de cancer du sein triple négatif (CSTN), l’un des cancers les plus mortels chez la femme. Les RCPG possèdent un rôle central dans presque toutes les fonctions physiologiques et constituent la plus grande famille des cibles médicamenteuses. D’une manière intéressante, les deux laboratoires de Dr Sébastien Carréno et Dr Michel Bouvier, ont découvert que le RTXA2 active les protéines ERMs à travers la GTPase RhoA. Dans ce projet de recherche on a identifié une nouvelle voie de signalisation liant le RTXA2 aux ERMs à travers la GTPase RhoA et la kinase SLK. Cette voie est impliquée dans la migration des cellules de cancer du sein triple négatif. Ainsi, on a pu démontrer que la moésine et la kinase SLK agissaient en aval du récepteur étudié pour favoriser la vitesse et la directionnalité de la migration des cellules de CSTN. 6 On a montré que la migration cellulaire dirigée en aval du RTXA2 est due à une polarité de la moésine au front de la migration. On a constaté aussi que la moésine est responsable d’une polarité des filaments d’actine au front de la migration suite à une activation du récepteur. Ce travail a mis en évidence une nouvelle cascade de signalisation importante pour la migration des cellules cancéreuses agressives triples négatives du sein ce que pourrait être une nouvelle cible des thérapies anti-métastatiques. / Cell migration is an important mechanism for various cellular processes such as embryogenesis and cicatrization. Likewise, it controls pathological processes including the invasion of malignant cells and the formation of metastases. Metastasis is a very complicated process. The acquisition of invasive migratory power by a malignant cell as well as its metastatic potential is regulated by the cytoskeleton which is dynamically modified and controlled by intracellular signaling pathways. However, metastatic cells physiology and the cascades causing their metastases are not clear yet. Ezrin, Radixin and Moesin (ERMs) proteins have an important role in organizing the cytoskeleton at the cell cortex and they are key determinants of cell motility. Thus, a deregulation at this point may lead to an aberrant cell migration. Hence, the involvement of ERMs in various aggressive and invasive cancers. ERMs are regulated downstream of several cellular actors in particular membrane receptors. Several studies have reported that the thromboxane A2 receptor (TXA2R), a G protein coupled receptor (GPCR) promotes metastasis. It has been described especially in the context of triple negative breast cancer (TNBC), one of the deadliest cancers in women. GPCR have a central role in almost all physiological functions and constitute the largest family of drug targets. Interestingly, the two laboratories of Dr Sébastien Carréno and Dr Michel Bouvier, have discovered that the TXA2R activates ERM proteins through the GTPase RhoA. In this research project, we have identified a new signaling pathway linking the TXA2 receptor to ERMs via RhoA and the kinase SLK. This pathway is involved in the migration of triple negative breast cancer cells. Thus, we demonstrated that moesin and SLK acted downstream of the receptor to promote the speed and directionality of TNBC cells migration. We discovered that the directed cell migration downstream of TXA2R is due to a polarization of moesin at the leading edge. We also observed that moesin is responsible for actin filaments polarity at the leading edge following an activation of the receptor. So, this work has revealed a new signaling cascade important for the migration of aggressive triple negative breast cancer cells which could be a new target for anti-metastatic therapies.