Crosstalk between cancer and stellate cells is pivotal in pancreatic cancer, resulting in differentiation of stellate cells into myofibroblasts that drives tumour progression. To assess... Show moreCrosstalk between cancer and stellate cells is pivotal in pancreatic cancer, resulting in differentiation of stellate cells into myofibroblasts that drives tumour progression. To assess cooperative mechanisms in a 3D context, we generated chimeric spheroids using human and mouse cancer and stellate cells. Species-specific deconvolution of bulk-RNA sequencing data revealed cell type-specific transcriptomes underpinning invasion. This dataset highlighted stellate-specific expression of transcripts encoding the collagen-processing enzymes ADAMTS2 and ADAMTS14. Strikingly, loss of ADAMTS2 reduced, while loss of ADAMTS14 promoted, myofibroblast differentiation and invasion independently of their primary role in collagen-processing. Functional and proteomic analysis demonstrated that these two enzymes regulate myofibroblast differentiation through opposing roles in the regulation of transforming growth factor β availability, acting on the protease-specific substrates, Serpin E2 and fibulin 2, for ADAMTS2 and ADAMTS14, respectively. Showcasing a broader complexity for these enzymes, we uncovered a novel regulatory axis governing malignant behaviour of the pancreatic cancer stroma. Show less
Aims Telomere attrition in cardiomyocytes is associated with decreased contractility, cellular senescence, and upregulation of proapoptotic transcription factors. Pim1 is a cardioprotective kinase... Show moreAims Telomere attrition in cardiomyocytes is associated with decreased contractility, cellular senescence, and upregulation of proapoptotic transcription factors. Pim1 is a cardioprotective kinase that antagonizes the aging phenotype of cardiomyocytes and delays cellular senescence by maintaining telomere length, but the mechanism remains unknown. Another pathway responsible for regulating tetomere length is the transforming growth factor beta (TGF beta) signalling pathway where inhibiting TGF beta signalling maintains telomere Length. The relationship between Pim1 and TGF beta has not been explored. This study delineates the mechanism of telomere length regulation by the interplay between Pim1 and components of TGF beta signalling pathways in proliferating A549 cells and post-mitotic cardiomyocytes.Methods and results Telomere length was maintained by lentiviral-mediated overexpression of PIM1 and inhibition of TGF beta signalling in re A549 cells. Telomere length maintenance was further demonstrated in isolated cardiomyocytes from mice with cardiac-specific overexpression of PIM1 and by pharmacological inhibition of TGF beta signalling. Mechanistically, Pim1 inhibited phosphorylation of Smad2, preventing its translocation into the nucleus and repressing expression of TGF beta pathway genes.Conclusion Pim1 maintains tetomere lengths in cardiomyocytes by inhibiting phosphorylation of the TGF beta pathway downstream effectors Smad2 and Smad3, which prevents repression of telomerase reverse transcriptase. Findings from this study demonstrate a novel mechanism of telomere length maintenance and provide a potential target for preserving cardiac function.[GRAPHICS]. Show less
TGF beta-SMAD3 signaling is a major driving force for cancer metastasis, while BMP-SMAD1/5 signaling can counteract this response. Analysis of gene expression profiles revealed that an increased... Show moreTGF beta-SMAD3 signaling is a major driving force for cancer metastasis, while BMP-SMAD1/5 signaling can counteract this response. Analysis of gene expression profiles revealed that an increased TGF beta-SMAD3 and a reduced BMP-SMAD1/5 targeted gene expression signature correlated with shortened distant metastasis free survival and overall survival of patients. At molecular levels, we discovered that TGF beta abolished BMP-induced SMAD1/5 activation in the highly-invasive breast cancer MDA-MB-231 cells, but to a less extent in the non-invasive cancer and normal breast cells. This suggests an inverse correlation between BMP signaling and invasiveness of tumor cells and TGF beta signaling acts in a double whammy fashion in driving cancer invasion and metastasis. Sustained ERK activation by TGF beta was specifically observed in MDA-MB-231 cells, and MEK inhibitor (MEKi) treatment restored BMP-SMAD1/5 signaling while not affecting SMAD2/3 activation. FK506 potently activated BMP, but not TGF beta signaling in breast cancer cells. MEKi or FK506 alone inhibited MDA-MB-231 extravasation in a zebrafish xenograft cancer model. Importantly, when administrated at suboptimal concentrations MEKi and FK506 strongly synergized in promoting BMP-SMAD1/5 signaling and inhibiting cancer cell extravasation. Furthermore, this combination of suboptimal concentrations treatment in a mouse tumor model resulted in real-time reduction of BMP-SMAD1/5 signaling in live tumors, and consequently potently inhibited tumor self-seeding, liver and bone metastasis, but not lung and brain metastasis. Mechanistically, it is the first time to identify BMP-SMAD1/5 signaling as an underlying molecular driver for organ-specific metastasis. Combining of MEKi and FK506, or their analogues, may be explored for clinical development of breast cancer. Show less
Hua, W.; Dijke, P. ten; Kostidis, S.; Giera, M.; Hornsveld, M. 2020
Metastasis is the most frequent cause of death in cancer patients. Epithelial-to-mesenchymal transition (EMT) is the process in which cells lose epithelial integrity and become motile, a critical... Show moreMetastasis is the most frequent cause of death in cancer patients. Epithelial-to-mesenchymal transition (EMT) is the process in which cells lose epithelial integrity and become motile, a critical step for cancer cell invasion, drug resistance and immune evasion. The transforming growth factor-beta (TGF beta) signaling pathway is a major driver of EMT. Increasing evidence demonstrates that metabolic reprogramming is a hallmark of cancer and extensive metabolic changes are observed during EMT. The aim of this review is to summarize and interconnect recent findings that illustrate how changes in glycolysis, mitochondrial, lipid and choline metabolism coincide and functionally contribute to TGF beta-induced EMT. We describe TGF beta signaling is involved in stimulating both glycolysis and mitochondrial respiration. Interestingly, the subsequent metabolic consequences for the redox state and lipid metabolism in cancer cells are found to be in favor of EMT as well. Combined we illustrate that a better understanding of the mechanistic links between TGF beta signaling, cancer metabolism and EMT holds promising strategies for cancer therapy, some of which are already actively being explored in the clinic. Show less
The epicardium, the outer layer of the heart, has been of interest in cardiac research due to its vital role in the developing and diseased heart. During development, epicardial cells are active... Show moreThe epicardium, the outer layer of the heart, has been of interest in cardiac research due to its vital role in the developing and diseased heart. During development, epicardial cells are active and supply cells and paracrine cues to the myocardium. In the injured adult heart, the epicardium is re-activated and recapitulates embryonic behavior that is essential for a proper repair response. Two indispensable processes for epicardial contribution to heart tissue formation are epithelial to mesenchymal transition (EMT), and tissue invasion. One of the key groups of cytokines regulating both EMT and invasion is the transforming growth factor beta (TGF beta) family, including TGF beta and Bone Morphogenetic Protein (BMP). Abundant research has been performed to understand the role of TGF beta family signaling in the developing epicardium. However, less is known about signaling in the adult epicardium. This review provides an overview of the current knowledge on the role of TGF beta in epicardial behavior both in the development and in the repair of the heart. We aim to describe the presence of involved ligands and receptors to establish if and when signaling can occur. Finally, we discuss potential targets to improve the epicardial contribution to cardiac repair as a starting point for future investigation. Show less
Johnson, B.V.; Kumar, R.; Oishi, S.; Alexander, S.; Kasherman, M.; Vega, M.S.; ... ; S 2020
BACKGROUND: The X-chromosome gene USP9X encodes a deubiquitylating enzyme that has been associated with neurodevelopmental disorders primarily in female subjects. USP9X escapes X inactivation, and... Show moreBACKGROUND: The X-chromosome gene USP9X encodes a deubiquitylating enzyme that has been associated with neurodevelopmental disorders primarily in female subjects. USP9X escapes X inactivation, and in female subjects de novo heterozygous copy number loss or truncating mutations cause haploinsufficiency culminating in a recognizable syndrome with intellectual disability and signature brain and congenital abnormalities. In contrast, the involvement of USP9X in male neurodevelopmental disorders remains tentative.METHODS: We used clinically recommended guidelines to collect and interrogate the pathogenicity of 44 USP9X variants associated with neurodevelopmental disorders in males. Functional studies in patient-derived cell lines and mice were used to determine mechanisms of pathology.RESULTS: Twelve missense variants showed strong evidence of pathogenicity. We define a characteristic phenotype of the central nervous system (white matter disturbances, thin corpus callosum, and widened ventricles); global delay with significant alteration of speech, language, and behavior; hypotonia; joint hypermobility; visual system defects; and other common congenital and dysmorphic features. Comparison of in silico and phenotypical features align additional variants of unknown significance with likely pathogenicity. In support of partial loss-of-function mechanisms, using patient-derived cell lines, we show loss of only specific USP9X substrates that regulate neurodevelopmental signaling pathways and a united defect in transforming growth factor signaling. In addition, we find correlates of the male phenotype in Usp9x brain-specific knockout mice, and further resolve loss of hippocannpal-dependent learning and memory.CONCLUSIONS: Our data demonstrate the involvement of USP9X variants in a distinctive neurodevelopmental and behavioral syndrome in male subjects and identify plausible mechanisms of pathogenesis centered on disrupted transforming growth factor beta signaling and hippocampal function. Show less
Sundqvist, A.; Voytyuk, O.; Hamdi, M.; Popeijus, H.E.; Bijlsma-van der Burgt, C.; Janssen, J.; ... ; Dam, H. van 2019
Transforming growth factor-beta (TGF beta) has both tumor-suppressive and tumor-promoting effects in breast cancer. These functions are partly mediated through Smads, intracellular transcriptional... Show moreTransforming growth factor-beta (TGF beta) has both tumor-suppressive and tumor-promoting effects in breast cancer. These functions are partly mediated through Smads, intracellular transcriptional effectors of TGF beta. Smads form complexes with other DNA-binding transcription factors to elicit cell-type-dependent responses. Previously, we found that the collagen invasion and migration of pre-malignant breast cancer cells in response to TGF beta and epidermal growth factor (EGF) critically depend on multiple Jun and Fos components of the activator protein (AP)-1 transcription factor complex. Here we report that the same process is negatively regulated by Jun N-terminal kinase (JNK)-dependent cJun phosphorylation. This was demonstrated by analysis of phospho-deficient, phospho-mimicking, and dimer-specific cJun mutants, and experiments employing a mutant version of the phosphatase MKP1 that specifically inhibits JNK. Hyper-phosphorylation of cJun by JNK strongly inhibited its ability to induce several Jun/Fos-regulated genes and to promote migration and invasion. These results show that MEK-AP-1 and JNK-phospho-cJun exhibit distinct pro- and anti-invasive functions, respectively, through differential regulation of Smad- and AP-1-dependent TGF beta target genes. Our findings are of importance for personalized cancer therapy, such as for patients suffering from specific types of breast tumors with activated EGF receptor-Ras or inactivated JNK pathways. Show less
Moursel, L.G.; Graaf, L.M. van der; Bulk, M.; Roon-Mom, W.M.C. van; Weerd, L. van der 2019
In severe forms of cerebral amyloid angiopathy (CAA) pathology, vascular calcification has been observed in the cerebral cortex, both in vivo on MRI and CT, and post-mortem using histopathology.... Show moreIn severe forms of cerebral amyloid angiopathy (CAA) pathology, vascular calcification has been observed in the cerebral cortex, both in vivo on MRI and CT, and post-mortem using histopathology. However, the pathomechanisms leading to calcification of CAA-laden arteries are unknown. Therefore, we investigated the correlation between calcification of cortical arterioles and several potential modulators of vascular calcification using immunohistochemistry in a unique collection of brain material of patients with a hereditary form of CAA, namely hereditary cerebral hemorrhage with amyloidosis-Dutch type (HCHWA-D or D-CAA). We show a topographical association of osteopontin (OPN) and TGF beta signaling factor phospho-SMAD2/3 (pSMAD2/3) in calcified CAA vessel walls. OPN and pSMAD2/3 gradually accumulate in vessels prior to calcification. Moreover, we found that the vascular accumulation of Collagen 1 (Col1), OPN and pSMAD2/3 immunomarkers correlated with the CAA severity. This was independently of the vessel size, including capillaries in the most severe cases. We propose that calcification of CAA vessels in the observed HCHWA-D cases may be induced by extracellular OPN trapped in the fibrotic Col1 vessel wall, independently of the presence of vascular amyloid. Show less
Hereditary cerebral hemorrhage with amyloidosis-Dutch type (HCHWA-D) is an early onset hereditary form of cerebral amyloid angiopathy (CAA) pathology, caused by the E22Q mutation in the amyloid... Show moreHereditary cerebral hemorrhage with amyloidosis-Dutch type (HCHWA-D) is an early onset hereditary form of cerebral amyloid angiopathy (CAA) pathology, caused by the E22Q mutation in the amyloid beta (A beta) peptide. Transforming growth factor beta 1 (TGF beta 1) is a key player in vascular fibrosis and in the formation of angiopathic vessels in transgenic mice. Therefore, we investigated whether the TGF beta pathway is involved in HCHWA-D pathogenesis in human postmortem brain tissue from frontal and occipital lobes. Components of the TGF pathway were analyzed with quantitative RT-PCR. TGF beta 1 and TGF beta Receptor 2 (TGFBR2) gene expression levels were significantly increased in HCHWA-D in comparison to the controls, in both frontal and occipital lobes. TGF beta-induced pro-fibrotic target genes were also upregulated. We further assessed pathway activation by detecting phospho-SMAD2/3 (pSMAD2/3), a direct TGF beta down-stream signaling mediator, using immunohistochemistry. We found abnormal pSMAD2/3 granular deposits specifically on HCHWA-D angiopathic frontal and occipital vessels. We graded pSMAD2/3 accumulation in angiopathic vessels and found a positive correlation with the CAA load independent of the brain area. We also observed pSMAD2/3 granules in a halo surrounding occipital vessels, which was specific for HCHWA-D. The result of this study indicates an upregulation of TGF beta 1 in HCHWA-D, as was found previously in AD with CAA pathology. We discuss the possible origins and implications of the TGF beta pathway deregulation in the microvasculature in HCHWA-D. These findings identify the TGF beta pathway as a potential biomarker of disease progression and a possible target of therapeutic intervention in HCHWA-D. Show less
