Snail regulates BMP and TGFβ pathways to control the differentiation status of glioma-initiating cells

Glioblastoma multiforme is a brain malignancy characterized by high heterogeneity, invasiveness, and resistance to current therapies, attributes related to the occurrence of glioma stem cells (GSCs). Transforming growth factor β (TGFβ) promotes self-renewal and bone morphogenetic protein (BMP) induces differentiation of GSCs. BMP7 induces the transcription factor Snail to promote astrocytic differentiation in GSCs and suppress tumor growth in vivo. We demonstrate that Snail represses stemness in GSCs. Snail interacts with SMAD signaling mediators, generates a positive feedback loop of BMP signaling and transcriptionally represses the TGFB1 gene, decreasing TGFβ1 signaling activity. Exogenous TGFβ1 counteracts Snail function in vitro, and in vivo promotes proliferation and re-expression of Nestin, confirming the importance of TGFB1 gene repression by Snail. In conclusion, novel insight highlights mechanisms whereby Snail differentially regulates the activity of the opposing BMP and TGFβ pathways, thus promoting an astrocytic fate switch and repressing stemness in GSCs

Regulation of cell differentiation and invasion by members of the TGFß family

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

Regulation of cell differentiation and invasion by members of the TGFß family

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

TGF-β Signaling

Transforming growth factor-beta (TGF-beta) represents an evolutionarily conserved family of secreted polypeptide factors that regulate many aspects of physiological embryogenesis and adult tissue homeostasis. The TGF-beta family members are also involved in pathophysiological mechanisms that underlie many diseases. Although the family comprises many factors, which exhibit cell type-specific and developmental stage-dependent biological actions, they all signal via conserved signaling pathways. The signaling mechanisms of the TGF-beta family are controlled at the extracellular level, where ligand secretion, deposition to the extracellular matrix and activation prior to signaling play important roles. At the plasma membrane level, TGF-beta s associate with receptor kinases that mediate phosphorylation-dependent signaling to downstream mediators, mainly the SMAD proteins, and mediate oligomerization-dependent signaling to ubiquitin ligases and intracellular protein kinases. The interplay between SMADs and other signaling proteins mediate regulatory signals that control expression of target genes, RNA processing at multiple levels, mRNA translation and nuclear or cytoplasmic protein regulation. This article emphasizes signaling mechanisms and the importance of biochemical control in executing biological functions by the prototype member of the family, TGF-beta

NOX4 regulates TGF beta-induced proliferation and self-renewal in glioblastoma stem cells

Y Glioblastoma (GBM) is the most aggressive and common glioma subtype, with a median survival of 15 months after diagnosis. Current treatments have limited therapeutic efficacy; thus, more effective approaches are needed. The glioblastoma tumoural mass is characterised by a small cellular subpopulation - glioblastoma stem cells (GSCs) - that has been held responsible for glioblastoma initiation, cell invasion, proliferation, relapse and resistance to chemo- and radiotherapy. Targeted therapies against GSCs are crucial, as is understanding the molecular mechanisms that govern the GSCs. Transforming growth factor beta (TGF beta) signalling and reactive oxygen species (ROS) production are known to govern and regulate cancer stem cell biology. Among the differentially expressed genes regulated by TGF beta in a transcriptomic analysis of two different patient-derived GSCs, we found NADPH oxidase 4 (NOX4) as one of the top upregulated genes. Interestingly, when patient tissues were analysed, NOX4 expression was found to be higher in GSCs versus differentiated cells. A functional analysis of the role of NOX4 downstream of TGF beta in several patient-derived GSCs showed that TGF beta does indeed induce NOX4 expression and increases ROS production in a NOX4-dependent manner. NOX4 downstream of TGF beta regulates GSC proliferation, and NOX4 expression is necessary for TGF beta-induced expression of stem cell markers and of the transcription factor nuclear factor erythroid 2-related factor 2 (NRF2), which in turn controls the cell's antioxidant and metabolic responses. Interestingly, overexpression of NOX4 recapitulates the effects induced by TGF beta in GSCs: enhanced proliferation, stemness and NRF2 expression. In conclusion, this work functionally establishes NOX4 as a key mediator of GSC biology

The liver kinase B1 supports mammary epithelial morphogenesis by inhibiting critical factors that mediate epithelial-mesenchymal transition

The liver kinase B1 (LKB1) controls cellular metabolism and cell polarity across species. We previously established a mechanism for negative regulation of transforming growth factor β (TGFβ) signaling by LKB1. The impact of this mechanism in the context of epithelial polarity and morphogenesis remains unknown. After demonstrating that human mammary tissue expresses robust LKB1 protein levels, whereas invasive breast cancer exhibits significantly reduced LKB1 levels, we focused on mammary morphogenesis studies in three dimensional (3D) acinar organoids. CRISPR/Cas9-introduced loss-of-function mutations of STK11 (LKB1) led to profound defects in the formation of 3D organoids, resulting in amorphous outgrowth and loss of rotation of young organoids embedded in matrigel. This defect was associated with an enhanced signaling by TGFβ, including TGFβ auto-induction and induction of transcription factors that mediate epithelial-mesenchymal transition (EMT). Protein marker analysis confirmed a more efficient EMT response to TGFβ signaling in LKB1 knockout cells. Accordingly, chemical inhibition of the TGFβ type I receptor kinase largely restored the morphogenetic defect of LKB1 knockout cells. Similarly, chemical inhibition of the bone morphogenetic protein pathway or the TANK-binding kinase 1, or genetic silencing of the EMT factor SNAI1, partially restored the LKB1 knockout defect. Thus, LKB1 sustains mammary epithelial morphogenesis by limiting pathways that promote EMT. The observed downregulation of LKB1 expression in breast cancer is therefore predicted to associate with enhanced EMT induced by SNAI1 and TGFβ family members

The protein kinase LKB1 negatively regulates bone morphogenetic protein receptor signaling

The protein kinase LKB1 regulates cell metabolism and growth and is implicated in intestinal and lung cancer. Bone morphogenetic protein (BMP) signaling regulates cell differentiation during development and tissue homeostasis. We demonstrate that LKB1 physically interacts with BMP type I receptors and requires Smad7 to promote downregulation of the receptor. Accordingly, LKB1 suppresses BMP-induced osteoblast differentiation and affects BMP signaling in Drosophila wing longitudinal vein morphogenesis. LKB1 protein expression and Smad1 phosphorylation analysis in a cohort of non-small cell lung cancer patients demonstrated a negative correlation predominantly in a subset enriched in adenocarcinomas. Lung cancer patient data analysis indicated strong correlation between LKB1 loss-of-function mutations and high BMP2 expression, and these two events further correlated with expression of a gene subset functionally linked to apoptosis and migration. This new mechanism of BMP receptor regulation by LKB1 has ramifications in physiological organogenesis and disease

The protein kinase LKB1 negatively regulates bone morphogenetic protein receptor signaling

The protein kinase LKB1 regulates cell metabolism and growth and is implicated in intestinal and lung cancer. Bone morphogenetic protein (BMP) signaling regulates cell differentiation during development and tissue homeostasis. We demonstrate that LKB1 physically interacts with BMP type I receptors and requires Smad7 to promote downregulation of the receptor. Accordingly, LKB1 suppresses BMP-induced osteoblast differentiation and affects BMP signaling in Drosophila wing longitudinal vein morphogenesis. LKB1 protein expression and Smad1 phosphorylation analysis in a cohort of non-small cell lung cancer patients demonstrated a negative correlation predominantly in a subset enriched in adenocarcinomas. Lung cancer patient data analysis indicated strong correlation between LKB1 loss-of-function mutations and high BMP2 expression, and these two events further correlated with expression of a gene subset functionally linked to apoptosis and migration. This new mechanism of BMP receptor regulation by LKB1 has ramifications in physiological organogenesis and disease

The protein kinase LKB1 negatively regulates bone morphogenetic protein receptor signaling

The protein kinase LKB1 regulates cell metabolism and growth and is implicated in intestinal and lung cancer. Bone morphogenetic protein (BMP) signaling regulates cell differentiation during development and tissue homeostasis. We demonstrate that LKB1 physically interacts with BMP type I receptors and requires Smad7 to promote downregulation of the receptor. Accordingly, LKB1 suppresses BMP-induced osteoblast differentiation and affects BMP signaling in Drosophila wing longitudinal vein morphogenesis. LKB1 protein expression and Smad1 phosphorylation analysis in a cohort of non-small cell lung cancer patients demonstrated a negative correlation predominantly in a subset enriched in adenocarcinomas. Lung cancer patient data analysis indicated strong correlation between LKB1 loss-of-function mutations and high BMP2 expression, and these two events further correlated with expression of a gene subset functionally linked to apoptosis and migration. This new mechanism of BMP receptor regulation by LKB1 has ramifications in physiological organogenesis and disease