| 1. |
- Herdenberg, Carl, 1982-
(författare)
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Molecular and physiological functions of LRIG proteins and netrin-1 in health and disease
- 2021
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The leucine-rich repeats and immunoglobulin-like domains (LRIG) gene family has three members, LRIG1, LRIG2, and LRIG3, that encode three structurally similar transmembrane proteins. LRIG1 is a receptor tyrosine kinase regulator, tumor suppressor, and stem cell marker in the skin, intestine, and brain. LRIG2 and LRIG3 have been less studied but shown to interact with LRIG1. The different roles and mechanisms of action of LRIG proteins have not yet been fully elucidated. In Caenorhabditis elegans (C. elegans), the LRIG homolog SMA-10 regulates bone morphogenetic protein (BMP) signaling; however, this function has not been demonstrated for mammalian LRIG proteins. In mice, the gene encoding the neurodevelopmental guidance cue netrin-1, Ntn1, interacts with Lrig3 in inner ear development. The physical interactions between LRIG proteins and other proteins are mostly unknown. Here, we describe an LRIG1-centered protein interaction network that regulates growth factor receptor levels. The LRIG1 interactome comprised LRIG2 and LRIG3 as well as many unanticipated proteins. An unbiased pathological examination of female mice with different Lrig3 genotypes (homozygous, heterozygous, or knockout) revealed a reduced incidence of spontaneous fatty liver and lymphocytic hyperplasia of the spleen in Lrig3-null mice. Female Lrig3-null mice also had a lower incidence of microvesicular cytoplasm in the liver after eight weeks on a high-fat diet. To further explore the molecular and physiological functions of LRIG proteins, we generated Lrig-null (Lrig1-/-;Lrig2-/-;Lrig3-/-) mouse embryonic fibroblasts (MEFs), which displayed a deficiency in adipogenesis caused by impaired BMP signaling. LRIG1 and LRIG3, but not LRIG2, sensitized cells to BMP and rescued the adipogenesis deficiency in Lrig-null MEFs. In C. elegans, the LRIG homolog sma-10 was needed for proper lipid accumulation. By analyzing data from the UK Biobank and GENiAL cohort, we found that certain LRIG1 gene variants were associated with a higher body mass index (BMI) yet protected against type 2 diabetes. This effect was probably mediated by altered adipocyte morphology. CRISPR/Cas9-mediated ablation of Ntn1 revealed that the BMP-promoting function of LRIG1 and LRIG3 was opposed by netrin-1, which functioned as an inhibitor of BMP signaling via its receptor neogenin.In summary, the present thesis describes a novel LRIG protein interaction network, the regulation of BMP signaling by LRIG proteins and netrin-1, and an important function of LRIG proteins in regulating fat metabolism with implications for human metabolic health.
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| 2. |
- Tzavlaki, Kalliopi
(författare)
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Regulation of cell differentiation and invasion by members of the TGFß family
- 2021
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Transforming growth factor β (TGFβ) and bone morphogenetic protein (BMP) signaling pathways are important in embryonic development and tissue homeostasis, but also have complex roles in the context of cancer. TGFβ promotes epithelial to mesenchymal transition (EMT) a physiological developmental process, often hijacked in different types of cancer, eventually leading to cancer cell invasion and metastasis. BMP signaling is involved in bone formation, angiogenesis and neural cell differentiation, but also regulates cancer by inducing EMT and its reversion. Liver kinase B1 (LKB1) is a tumor suppressor protein kinase involved in the regulation of cell metabolism, proliferation and polarity. First, we investigated how LKB1 negatively regulates BMP signaling and we demonstrated that LKB1 interacts with one of the BMP type I receptors and mediates its degradation, leading to the inhibition of BMP-induced cell differentiation.We then focused on the role of LKB1 in the establishment of mammary epithelial polarity. Upon LKB1 depletion, normal mammary epithelial cells lost the ability to form polarized acini, and displayed enhanced TGFβ responses. The use of a chemical inhibitor targeting TGFβ type I receptor restored the formation of acini, therefore we concluded that the contribution of LKB1 to mammary epithelial polarity is dependent on the regulation of autogenous TGFβ signaling.Glioblastoma (GBM) is a brain malignancy, that is highly invasive and heterogeneous in terms of cell differentiation. TGFβ enhances the self-renewal potential of glioblastoma stem cells (GSCs), while BMP promotes their differentiation towards the astrocytic lineage. In the second part of this thesis, we investigated the role of different effectors downstream of TGFβ/BMP signaling in GBM. Snail is a well-established inducer of EMT in carcinomas but in the context of GBM, we demonstrated that Snail was induced by BMP7, and via its interaction with Smad signaling effectors, enhanced BMP while it suppressed TGFβ signaling, thus promoting the astrocytic differentiation of GSCs and suppressing stemness.Finally, the role of the TGFβ/BMP target gene, CXXC5, was investigated in GBM. CXXC5 expression was enriched in GSCs that express high levels of stem cell markers, and depletion of CXXC5 led to reduced self-renewal capacity of GBM cells. Further analysis indicated that CXXC5 epigenetically regulates stemness-related genes by counteracting the activity of the polycomb repressor complex 2 (PRC2), thus affecting the histone modification pattern on the regulatory elements of these genes. Collectively, the thesis provides evidence on mechanisms that regulate cell differentiation by interfering with TGFβ/BMP signaling.
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| 3. |
- Kolliopoulos, Constantinos
(författare)
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Role of TGFβ-induced hyaluronan-CD44 signaling in cancer progression
- 2021
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Hyaluronan, a prevalent glycosaminoglycan of the extracellular space often accumulates in pathological conditions, such as chronic inflammation, infection, and cancer. Hyaluronan synthase HAS2 has been responsible for the synthesis and deposition of hyaluronan in a variety of tumors. We have shown that HAS2 was required for efficient transforming growth factor β (TGFβ)-induced epithelial to mesenchymal transition (EMT), a developmental program which is commandeered by cancer cells to increase their migratory and invasive capacity. In study I, our findings show that long non-coding RNA Has2as has a key role in TGFβ- and Has2-induced breast cancer EMT, migration and acquisition of stemness.Hyaluronan conveys its signaling properties via binding to its cell surface receptor CD44, a well-established stem cell marker in a plethora of tumors. CD44 exerts its signaling properties by interacting with components of the actin cytoskeleton machinery, and by acting as a co-receptor for other receptor tyrosine or threonine kinases impacting their signaling properties. Furthermore, CD44 is subjected to proteolytic cleavage, which eventually liberates the cytoplasmic tail (CD44-ICD). CD44-ICD translocates to the nucleus and alters gene expression. In study II, our findings support that TRAF4/6 mediates pro-tumorigenic effects of CD44, and suggests that inhibitors of CD44 signaling via TRAF4/6 and RAC1 may be beneficial in the treatment of tumor patients.Glioblastoma (GBM) multiforme remains one of the most aggressive and lethal types of brain tumors worldwide with a poor prognosis. In study III, we have initiated studies to elucidate the CD44-dependent molecular mechanisms in GBM progression by knocking out (KO) CD44 by employing CRISPR/Cas9 gene editing in glioma U251MG cells.Aberrant hyaluronan levels are also found during infectious diseases. In study V, we show that in a cohort study of dengue patients, high levels of circulating Dengue Nonstructural Protein 1 (NS1) correlate with high levels of serum hyaluronan. Moreover, we propose that hyaluronan can serve as a prognostic marker for the onset of warning signs during the course of dengue viral infection. Mechanistically, NS1 treatment-induced hyaluronan production contributing to increased vascular permeability.In study IV, we have identified a bifurcating loop during TGFβ signaling, whereby transcriptional induction of NUAK1 serves as a negative checkpoint and NUAK2 induction positively contributes to signaling and terminal differentiation responses to TGFβ activity.In summary, the current thesis provides mechanistic insights into the roles of TGFβ-induced hyaluronan-CD44 interactions in cancer progression.
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| 4. |
- Tan, E-Jean, 1979-
(författare)
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Transcriptional and Epigenetic Regulation of Epithelial-Mesenchymal Transition
- 2013
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The transforming growth factor beta (TGFβ) is a cytokine that regulates a plethora of cellular processes such as cell proliferation, differentiation, migration and apoptosis. TGFβ signals via serine/threonine kinase receptors and activates the Smads to regulate gene expression. Enigmatically, TGFβ has a dichotomous role as a tumor suppressor and a tumor promoter in cancer. At early stages of tumorigenesis, TGFβ acts as a tumor suppressor by exerting growth inhibitory effects and inducing apoptosis. However, at advanced stages, TGFβ contributes to tumor malignancy by promoting invasion and metastasis.The pro-tumorigenic TGFβ potently triggers an embryonic program known as epithelial-mesenchymal transition (EMT). EMT is a dynamic process whereby polarized epithelial cells adapt a mesenchymal morphology, thereby facilitating migration and invasion. Downregulation of cell-cell adhesion molecules, such as E-cadherin and ZO-1, is an eminent feature of EMT. TGFβ induces EMT by upregulating a non-histone chromatin factor, high mobility group A2 (HMGA2). This thesis focuses on elucidating the molecular mechanisms by which HMGA2 elicits EMT.We found that HMGA2 regulates a network of EMT transcription factors (EMT-TFs), such as members of the Snail, ZEB and Twist families, during TGFβ-induced EMT. HMGA2 can interact with Smad complexes to synergistically induce Snail expression. HMGA2 also directly binds and activates the Twist promoter. We used mouse mammary epithelial cells overexpressing HMGA2, which are mesenchymal in morphology and highly invasive, as a constitutive EMT model. Snail and Twist have complementary roles in HMGA2-mesenchymal cells during EMT, and tight junctions were restored upon silencing of both Snail and Twist in these cells. Finally, we also demonstrate that HMGA2 can epigenetically silence the E-cadherin gene. In summary, HMGA2 modulates multiple reprogramming events to promote EMT and invasion.
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| 5. |
- Batool, Tahira
(författare)
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Heparan sulfate dependent cell signaling and associated pathophysiology : Implications in tumorigenesis and embryogenesis
- 2018
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Heparan sulfate proteoglycans (HSPGs) consist of a protein core to which several linear, negatively charged heparan sulfate (HS) chains are covalently attached. HSPGs are expressed on the cell surface and in the extra-cellular matrix (ECM) where they have diverse biological functions, for example co-receptor functions. The diverse functions of HS are linked to structural variability of the polysaccharide. In this thesis, I investigated HS structure-function relationship by using different cell and animal models of one HS-biosynthetic enzyme, glucuronyl C5-epimerase (Hsepi) and one enzyme responsible for post synthetic modification, heparanase.Deletion of Hsepi in mice resulted in neonatal lethality, with multiple organ defects, indicating the importance of HS in embryogenesis. Up-regulated expression of heparanase is found in most human tumor tissues, correlating with increased metastatic potential and decreased survival of cancer patients.In the first project, I focused on the effects of HS on cancer associated cell signaling and found that heparanase overexpression attenuated TGF-β1 stimulated Smad phosphorylation in tumor cells because of increased sulfation degree and turnover rate of HS.Heparanase role in cancer progression has led to clinical trials where inhibition of heparanase activity is currently being evaluated as a potential cancer treatment. Heparin, a HS-related polysaccharide, is being used to inhibit heparanase activity. In my second project, we studied the effect of low molecular weight heparin (LMWH) on cisplatin resistance of ovarian cancer cells (A2780cis). LMWH treatment of A2780cis cells reduced Wnt-activity in these cells and consequently reduce the drug resistance.In paper III, we continued exploring the HS/heparanase role in cancer by using heparanase overexpressing mice (Hpa-tg). We found Lewis Lung Carcinoma (LLC2) cells showed faster growth, bigger tumors and more metastasis in the Hpa-tg mice as compared to wild-type (WT) mice, because of suppressed antitumor immunity in the Hpa-tg mice.In paper IV and V, we studied the structure-function relationship of HS by using Hsepi-/- mice model. Hsepi-/- results in HS-chains lacking IdoA, which makes the chain rigid and consequently affects its co-receptor function. Skeletal malformation in Hsepi-/- mice, led us in paper IV to investigate bone morphogenic protein (BMP), an important signal molecule during embryogenesis and known to interact with HS. We found upregulation of a number of BMPs and expression of P-smad1/5/8, but reduced expression of inhibitory Smads and Gremlin1 in the Hsepi-/- MEF cells. The study indicated that the developmental defects in Hsepi mice could be contributed by a higher BMP signaling. In paper V we investigated the lung of the Hsepi-/- mice. The distal lung of 17.5 days old embryos remained populated by epithelial tubules, because of impaired differentiation of type I cells of the lungs. Potential mechanisms behind the failure of type I cell formation was identified to be reduced vascularization and a sustained signaling of Smad pathways.
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| 6. |
- Bellomo, Claudia
(författare)
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TGFβ and LXR signaling in hepatocellular carcinoma
- 2018
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Hepatocellular carcinoma (HCC) is one of the most prevalent cancer types in the Western world and in the Asia-pacific regions, with its incidence expected to rise up to 22 million cases till 2020. Hepatocellular carcinoma etiology is mainly due to hepatitis B (HBV) and hepatitis C (HCV) infections, and to a lesser extent it is determined by the development of alcohol-driven cirrhosis and non-alcoholic steatohepatitis (NASH). Furthermore, HCC is characterized by a high mortality rate, with poor prognostic expectance and limited therapeutic options currently available in the clinics.Transforming growth factor beta (TGFβ) is a pleiotropic cytokine with a janus-role in HCC and in other malignancies. TGFβ can in fact elicit either tumor-suppressive and tumor- promoting effects depending on tumor stage, microenvironmental and immunological cues. In HCC specifically, TGFβ determines cytostasis and cellular senescence during the first stages of tumor development, while it enhances HCC malignancy and progression in the later stages due to increased invasiveness, acquired resistance to cytostatic actions and tumor immunotolerance.Liver X receptors (LXRα/NR1H3 and LXRβ/NR1H2) are transcription factors of the nuclear hormone receptor family, which play an important role in oxysterol metabolism and reverse- cholesterol transport to the liver. Their involvement in malignancies has been studied so far to a limited extend, with evidence of both tumor-suppressive -via cytostatic mechanisms- and tumor- immunotolerance activities. Moreover, the potential crosstalk of LXR and TGFβ pathways has not been yet unraveled in the context of hepatocellular carcinoma.We have described (Paper I) a high-content imaging platform for the screening of small molecules able to revert the TGFβ-induced epithelial to mesenchymal transition (EMT) in human keratinocytes. This screening allowed us to identify LXR agonists as epithelial plasticity modulators in established terminally differentiated and mouse embryonic fibroblast, as well as in epithelial and mesenchymal HCC cell lines.We have identified (Paper II) the transcription factor SNAI1 (Snail) as the mediator of the crosstalk between TGFβ and LXRα pathways in epithelial and mesenchymal HCC cell models. LXRα activation diminishes the transcriptional induction of SNAI1 by TGFβ, thus antagonizing the induction of mesenchymal features and the production of reactive oxygen species by TGFβ. However, we have unraveled that LXRα and TGFβ signaling still positively interact in increasing cytostasis in HCC, in order to preserve liver epithelial features.We have described (Paper III) that LXRα activation counteracts the transcriptional induction of α smooth muscle actin (αSMA), a major hallmark of fibroblast activation, elicited by TGFβ in patient-derived primary liver fibroblasts.In conclusion, we herein report that the signaling crosstalk between TGFβ and LXRα pathways results in antagonistic effects either on parenchymal and fibroblast cell lines representative of the HCC disease, suggesting the potential future application of LXR agonists as clinical therapeutic options.
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| 7. |
- Dahl, Markus
(författare)
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Mechanisms for Quantitative Regulation of TGF-ß Signaling
- 2012
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Cancer is a widely spread disease, and many cancer variants are today still difficult to treat. Efforts are being made to understand the complexity of cancer, both at a clinical level but also at a pre-clinical level. The aim is of course to merge the research from both disciplines, as an example, find out how to treat a tumour in a patient and what molecular mechanisms are behind the origin of the tumour. Basic research provides a platform that in the long run will help to create treatments for many cancer variants that exist today. Transforming Growth Factor Beta (TGF-ß) is a cytokine that regulates many cellular events such as cell differentiation, cell proliferation and migration. TGF-ß signaling is important to study since many studies show that patients with cancer actually have accumulated mutations in proteins connected to the pathway. In this thesis I try to enhance the knowledge of the TGF-ß signaling pathway, looking in more detail how the signaling output is regulated by the response to the ligand, explained in paper four. Furthermore I try to reveal the protein network that control transmission of the signal from the cell surface to the nucleus. We found that PARP-1 (paper one and two) and PARP-2 (paper three) associates with the signaling pathway to regulate the Smad proteins and to negatively regulate the transcription of Smad target genes.
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| 8. |
- Gélabert, Caroline
(författare)
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Micro-environmental control of tumor differentiation and invasiveness
- 2023
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The transforming growth factor β (TGFβ) family participates in embryonic development and adult tissue homeostasis. In early stages of tumorigenesis, TGFβ promotes cell cycle arrest and apoptosis; however, in advanced malignancies, TGFβ promotes tumor cell migration and metastasis via the induction of epithelial-tomesenchymal transition (EMT). A new aspect of the regulation of TGFβ signalling is the participation of non-coding RNAs, molecules that are not translated into proteins but are nevertheless important regulators of gene expression. The expression of the long non-coding RNA LINC00707 was identified being down-regulated by TGFβ by engaging the transcription factor KLF6. LINC00707 resides in the cytoplasm where it interacts with and sequesters the Smad proteins, which mediate TGFβ signals. Thus, when TGFβ signaling downregulates LINC00707, the Smad complex is free to enter the nucleus and regulate its target genes implicated in the EMT process. It is also important to consider the biology of cells in their microenvironment. The growth of solid tumors leads to regional deprivation of nutrients within a tumor. Glutamine deprivation in mesenchymal and epithelial hepatocellular carcinoma cell lines showed a large change in gene expression related to TGFβ signaling in cells adapted to glutamine deprivation, suggesting a dependence of TGFβ signaling on glutamine metabolism. In mesenchymal cells, we observed a mesenchymal-to-epithelial transition associated with reduced metabolic activity and a reduced generation of reactive oxygen species. Mitochondrial reactive oxygen species are known for their capacity to regulate various signalling pathways associated with diverse cellular responses. This work also identified the polarity protein Par3 as a negative regulator of mitochondrial activity in glioblastoma cells. Moreover, Par3 leads to suppressed invasiveness and sustained clonogenicity of glioblastoma cells.In summary, this work describes novel regulatory mechanisms that affect different aspects of cancer biology in both epithelial (carcinoma) and nonepithelial (glial) tumor cells. A central component that unifies these new mechanisms of cancer cell regulation is the TGFβ signaling pathway. In addition to its novel findings, this work opens several questions whose investigation can provide deeper mechanistic understanding of the action of the key RNAs or proteins analysed in this thesis.
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| 9. |
- Lönn, Peter, 1980-
(författare)
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Regulation of TGF-β Signaling by Post-Translational Modifications
- 2010
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Transforming growth factor-β (TGF-β) signaling is initiated when the ligand binds to type II and type I serine/threonine kinase receptors at the cell surface. Activated TGF-β type I receptors phosphorylate R-Smads which relocate, together with co-Smads, to the cell nucleus and regulate transcription. Enhancement or repression of Smad-specific gene targets leads to intracellular protein compositions which organize functional complexes and thus govern cellular processes such as proliferation, migration and differentiation. TGF-β/Smad signaling relays are regulated by various post-translational modifications. From receptors to gene promoters, intricate interplays between phosphorylation, acetylation, ubiquitination and numerous other modifications, control Smad signaling initiation and duration. However, many steps in the cascade, including receptor internalization, Smad nuclear shuttling and transcriptional termination, still remain elusive. The open gaps in our understanding of these mechanisms most likely involve additional post-translational regulations. Thus, the aim of the present investigation was to identify novel modulators of TGF-β/Smad signaling. In the first part of this thesis, we show the importance of ADP-ribosylation in Smad-mediated transcription. We identified poly(ADP-ribose) polymerase 1 (PARP-1) as a Smad interacting protein. Our work revealed that PARP-1 forms direct interactions with Smad3/4, and PARylates residues in their MH1 domains. This modification restricts Smads from binding to DNA and attenuates Smad-activated transcription. PARylation is reversed by the glycohydrolase PARG. We provide evidence that PARG can de-ADP-ribosylate Smads, which enhances Smad-promoted gene regulation. In the second part, we examine a Smad-dependent gene target of TGF-β signaling, salt inducible kinase 1 (SIK). After induction, SIK cooperates with Smad7 and Smurf2 to downregulate the TGF-β type I receptor. The mechanism relies on both the kinase and UBA domain of SIK as well as the E3-ligase activity of Smurf2. In summary, we have unveiled two enzyme-dependent TGF-β/Smad modulatory mechanisms; SIK promoted receptor turnover and PARP-1/PARG-regulated Smad signaling.
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| 10. |
- Shahidi Dadras, Mahsa, 1984-
(författare)
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Regulation of cell polarity and invasion by TGF-β and BMP signaling
- 2017
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Transforming growth factor β (TGF-β) and bone morphogenetic protein (BMP) signaling pathways are involved in many physiological processes during embryonic and adult life. TGF-β promotes epithelial to mesenchymal transition (EMT). We identified a gene target of TGF-β signaling, encoding the salt-inducible kinase 1 (SIK1). A potential substrate of this kinase, the polarity protein Par3, is an established regulator of tight junction assembly. SIK1 associates with Par3, can potentially phosphorylate Par3 and leads to its degradation, contributing to tight junction disassembly.Glioblastoma multiforme (GBM) is a common malignancy in the central nervous system, characterized by high heterogeneity, invasiveness, and resistance to therapy. One of the causes of heterogeneity and therapy-resistance is the existence of glioblastoma stem cells (GSCs). TGF-β signaling promotes self-renewal while BMP signaling induces differentiation of GSCs. Snail is a potent inducer of the EMT in carcinomas. However, in the context of GBM, Snail induces BMP signaling and represses TGF-β signaling through interaction with SMADs, the signaling mediators of TGF-β and BMP. In conclusion, Snail differentially regulates the activity of the opposing BMP and TGF-β pathways, thus promoting an astrocytic fate switch and repressing stemness in GSCs.Although profound changes in cell polarity is a hallmark of invasive malignancies, little is known about the role of the polarity machinery in tumor suppression. Patient transcriptomic data suggested low Par3 expression, correlating with poor survival of the GBM patients. Par3 silencing decreased the GSC self-renewal capacity and enhanced their invasiveness. Transcriptomic analysis indicates that loss of Par3 leads to downregulation of genes encoding mitochondrial enzymes that generate ATP. These results support a novel role of Par3 in GBM, beyond its contribution to junctional contacts between cells.Another regulator of TGF-β and BMP signaling is the liver kinase B1 (LKB1). According to GBM patient mRNA analysis, high levels of LKB1 correlate with poor prognosis. Silencing of LKB1 in GSCs impairs invasion and self-renewal capacity due to downregulation of genes involved in these processes. Moreover, loss of LKB1 induces mitochondrial dysfunction, leading to decreased ATP levels. Collectively, this thesis has delivered a group of novel regulatory pathways that control critical aspects of cancer cell polarity, invasion and stemness.
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