| 1. |
- Bertran, Esther, et al.
(författare)
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Overactivation of the TGF-β pathway confers a mesenchymal-like phenotype and CXCR4-dependent migratory properties to liver tumor cells
- 2013
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Ingår i: Hepatology. - : Ovid Technologies (Wolters Kluwer Health). - 0270-9139 .- 1527-3350. ; 58:6, s. 2032-44
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Tidskriftsartikel (refereegranskat)abstract
- UNLABELLED: Transforming growth factor-beta (TGF-β) is an important regulatory suppressor factor in hepatocytes. However, liver tumor cells develop mechanisms to overcome its suppressor effects and respond to this cytokine by inducing other processes, such as the epithelial-mesenchymal transition (EMT), which contributes to tumor progression and dissemination. Recent studies have placed chemokines and their receptors at the center not only of physiological cell migration but also of pathological processes, such as metastasis in cancer. In particular, CXCR4 and its ligand, stromal cell-derived factor 1α (SDF-1α) / chemokine (C-X-C motif) ligand 12 (CXCL12) have been revealed as regulatory molecules involved in the spreading and progression of a variety of tumors. Here we show that autocrine stimulation of TGF-β in human liver tumor cells correlates with a mesenchymal-like phenotype, resistance to TGF-β-induced suppressor effects, and high expression of CXCR4, which is required for TGF-β-induced cell migration. Silencing of the TGF-β receptor1 (TGFBR1), or its specific inhibition, recovered the epithelial phenotype and attenuated CXCR4 expression, inhibiting cell migratory capacity. In an experimental mouse model of hepatocarcinogenesis (diethylnitrosamine-induced), tumors showed increased activation of the TGF-β pathway and enhanced CXCR4 levels. In human hepatocellular carcinoma tumors, high levels of CXCR4 always correlated with activation of the TGF-β pathway, a less differentiated phenotype, and a cirrhotic background. CXCR4 concentrated at the tumor border and perivascular areas, suggesting its potential involvement in tumor cell dissemination.CONCLUSION: A crosstalk exists among the TGF-β and CXCR4 pathways in liver tumors, reflecting a novel molecular mechanism that explains the protumorigenic effects of TGF-β and opens new perspectives for tumor therapy.
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| 2. |
- Bertran, Esther, et al.
(författare)
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Role of CXCR4/SDF-1 alpha in the migratory phenotype of hepatoma cells that have undergone epithelial-mesenchymal transition in response to the transforming growth factor-beta.
- 2009
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Ingår i: Cellular Signalling. - : Elsevier BV. - 0898-6568 .- 1873-3913. ; 21:11, s. 1595-1606
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Tidskriftsartikel (refereegranskat)abstract
- Treatment of FaO rat hepatoma cells with TGF-beta selects cells that survive to its apoptotic effect and undergo epithelial-mesenchymal transitions (EMT). We have established a cell line (T beta T-FaO, from TGF-beta-treated FaO) that shows a mesenchymal, de-differentiated, phenotype in the presence of TGF-beta and is refractory to its suppressor effects. In the absence of this cytokine, cells revert to an epithelial phenotype in 3-4 weeks and recover the response to TGF-beta. T beta T-FaO show higher capacity to migrate than that observed in the parental FaO cells. We found that FaO cells express low levels of CXCR4 and do not respond to SDF-1 alpha. However, TGF-beta up-regulates CXCR4, through a NF kappaB-dependent mechanism, and T beta T-FaO cells show elevated levels of CXCR4, which is located in the presumptive migration front. A specific CXCR4 antagonist (AMD3100) attenuates the migratory capacity of T beta T-FaO cells on collagen gels. Extracellular SDF-1 alpha activates the ERKs pathway in T beta T-FaO, but not in FaO cells, increasing cell scattering and protecting cells from apoptosis induced by serum deprivation. Targeted knock-down of CXCR4 with specific siRNA blocks the T beta T-FaO response to SDF-1 alpha. Thus, the SDF-1/CXCR4 axis might play an important role in mediating cell migration and survival after a TGF-beta-induced EMT in hepatoma cells.
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| 3. |
- Caja, Laia, et al.
(författare)
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Differential intracellular signalling induced by TGF-beta in rat adult hepatocytes and hepatoma cells : implications in liver carcinogenesis.
- 2007
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Ingår i: Cellular Signalling. - : Elsevier BV. - 0898-6568 .- 1873-3913. ; 19:4, s. 683-94
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Tidskriftsartikel (refereegranskat)abstract
- The transforming growth factor-beta (TGF-beta) regulates hepatocyte growth, inhibiting proliferation and inducing apoptosis. Indeed, escaping from the TGF-beta suppressor actions might be a prerequisite for liver tumour progression. In this work we show that TGF-beta plays a dual role in regulating apoptosis in FaO rat hepatoma cells, since, in addition to its pro-apoptotic effect, TGF-beta also activates survival signals, such as AKT, the epidermal growth factor receptor (EGFR) being required for its activation. TGF-beta induces the expression of the EGFR ligands transforming growth factor-alpha (TGF-alpha) and heparin-binding EGF-like growth factor (HB-EGF) and induces intracellular re-localization of the EGFR. Cells that overcome the apoptotic effects of TGF-beta undergo morphological changes reminiscent of an epithelial-mesenchymal transition (EMT) process. In contrast, TGF-beta does not activate AKT in adult hepatocytes, which correlates with lack of EGFR transactivation and no response to EGFR inhibitors. Although TGF-beta induces TGF-alpha and HB-EGF in adult hepatocytes, these cells show very low expression of TACE/ADAM 17 (TNF-alpha converting enzyme), which is required for EGFR ligand proteolysis and activation. Furthermore, adult hepatocytes do not undergo EMT processes in response to TGF-beta, which might be due, at least in part, to the fact that F-actin re-organization induced by TGF-beta in FaO cells require the EGFR pathway. Finally, results indicate that EGFR transactivation does not block TGF-beta-induced cell cycle arrest in FaO cells, but must be interfering with the pro-apoptotic signalling. In conclusion, TGF-beta is a suppressor factor for adult quiescent hepatocytes, but not for hepatoma cells, where it plays a dual role, both suppressing and promoting carcinogenesis.
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| 4. |
- Caja, Laia, et al.
(författare)
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Dissecting the effect of targeting the epidermal growth factor receptor on TGF-β-induced-apoptosis in human hepatocellular carcinoma cells.
- 2011
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Ingår i: Journal of Hepatology. - : Elsevier BV. - 0168-8278 .- 1600-0641. ; 55:2, s. 351-358
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND & AIMS: Transforming growth factor-beta (TGF-β) induces apoptosis in hepatocytes, a process that is inhibited by the epidermal growth factor receptor (EGFR) pathway. The aim of this work was to ablate EGFR in hepatocellular carcinoma (HCC) cells to understand its role in impairing TGF-β-induced cell death.METHODS: Response to TGF-β in terms of apoptosis was analyzed in different HCC cell lines and the effect of canceling EGFR expression was evaluated.RESULTS: TGF-β induces apoptosis in some HCC cells (such as Hep3B, PLC/PRF/5, Huh7, or SNU449), but it also mediates survival signals, coincident with the up-regulation of EGFR ligands. Inhibition of the EGFR, either by targeted knock-down with specific siRNA or by pharmacological inhibition, significantly enhances apoptotic response. TGF-β treatment in EGFR targeted knock-down cells correlates with higher levels of the NADPH oxidase NOX4 and changes in the expression profile of BCL-2 and IAP families. However, other HCC cells, such as HepG2, which show over activation of the Ras/ERKs pathway, SK-Hep1, with an endothelial phenotype, or SNU398, where the TGF-β-Smad signaling is altered, show apoptosis resistance that is not restored through EGFR blockade.CONCLUSIONS: The inhibition of EGFR in HCC may enhance TGF-β-induced pro-apoptotic signaling. However, this effect may only concern those tumors with an epithelial phenotype which do not bear alterations in TGF-β signaling nor exhibit an over-activation of the survival pathways downstream of the EGFR.
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| 5. |
- Caja, Laia, et al.
(författare)
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Overactivation of the MEK/ERK pathway in liver tumor cells confers resistance to TGF-{beta}-induced cell death through impairing up-regulation of the NADPH oxidase NOX4.
- 2009
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Ingår i: Cancer Research. - 0008-5472 .- 1538-7445. ; 69:19, s. 7595-7602
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Tidskriftsartikel (refereegranskat)abstract
- Transforming growth factor-beta (TGF-beta) induces apoptosis in hepatocytes, being considered a liver tumor suppressor. However, many human hepatocellular carcinoma (HCC) cells escape from its proapoptotic effects, gaining response to this cytokine in terms of malignancy. We have recently reported that the apoptosis induced by TGF-beta in hepatocytes requires up-regulation of the NADPH oxidase NOX4, which mediates reactive oxygen species (ROS) production. TGF-beta-induced NOX4 expression is inhibited by antiapoptotic signals, such as the phosphatydilinositol-3-phosphate kinase or the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) pathways. The aim of the present work was to analyze whether resistance to TGF-beta-induced apoptosis in HCC cells is related to the impairment of NOX4 up-regulation due to overactivation of survival signals. Results indicate that inhibition of the MAPK/ERK kinase (MEK)/ERK pathway in HepG2 cells, which are refractory to the proapoptotic effects of TGF-beta, sensitizes them to cell death through a mitochondrial-dependent mechanism, coincident with increased levels of BIM and BMF, decreased levels of BCL-XL and MCL1, and BAX/BAK activation. Regulation of BMF, BCL-XL, and MCL1 occurs at the mRNA level, whereas BIM regulation occurs post-transcriptionally. ROS production and glutathione depletion are only observed in cells treated with TGF-beta and PD98059, which correlates with NOX4 up-regulation. Targeting knockdown of NOX4 impairs ROS increase and all the mitochondrial-dependent apoptotic features by a mechanism that is upstream from the regulation of BIM, BMF, BCL-XL, and MCL1 levels. In conclusion, overactivation of the MEK/ERK pathway in liver tumor cells confers resistance to TGF-beta-induced cell death through impairing NOX4 up-regulation, which is required for an efficient mitochondrial-dependent apoptosis.
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| 6. |
- Caja, Laia, et al.
(författare)
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The transforming growth factor-beta (TGF-β) mediates acquisition of a mesenchymal stem cell-like phenotype in human liver cells.
- 2011
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Ingår i: Journal of Cellular Physiology. - : Wiley. - 0021-9541 .- 1097-4652. ; 226:5, s. 1214-1223
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Tidskriftsartikel (refereegranskat)abstract
- Transforming growth factor-beta (TGF-β) mediates several and sometime opposite effects in epithelial cells, inducing growth inhibition, and apoptosis but also promoting an epithelial to mesenchymal transition (EMT) process, which enhances cell migration and invasion. TGF-β plays relevant roles in different liver pathologies; however, very few is known about its specific signaling and cellular effects in human primary hepatocytes. Here we show that TGF-β inhibits proliferation and induces pro-apoptotic genes (such as BMF or BIM) in primary cultures of human fetal hepatocytes (HFH), but also up-regulates anti-apoptotic genes, such as BCL-XL and XIAP. Inhibition of the epidermal growth factor receptor (EGFR), using gefitinib, abrogates the increase in the expression of the anti-apoptotic genes and significantly enhances cell death. Simultaneously, TGF-β is able to induce an EMT process in HFH, coincident with Snail up-regulation and a decrease in E-cadherin levels, cells showing mesenchymal proteins and reorganization of the actin cytoskeleton in stress fibers. Interestingly, these cells show loss of expression of specific hepatic genes and increased expression of stem cell markers. Chronic treatment with TGF-β allows selection of a population of mesenchymal cells with a de-differentiated phenotype, reminiscent of progenitor-like cells. Process is reversible and the mesenchymal stem-like cells re-differentiate to hepatocytes under controlled experimental conditions. In summary, we show for the first time that human hepatocytes may respond to TGF-β inducing different signals, some of them might contribute to tumor suppression (growth inhibition and apoptosis), but others should mediate liver tumor progression and invasion (EMT and acquisition of a stem-like phenotype).
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| 7. |
- Caja, Laia, et al.
(författare)
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The tyrphostin AG1478 inhibits proliferation and induces death of liver tumor cells through EGF receptor-dependent and independent mechanisms.
- 2011
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Ingår i: Biochemical pharmacology. - : Elsevier BV. - 1873-2968 .- 0006-2952. ; 82:11, s. 1583-1592
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Tidskriftsartikel (refereegranskat)abstract
- Hepatocellular carcinoma (HCC) is one of the most common causes of cancer-related death. Different signaling pathways are de-regulated in this pathogenesis, among them the epidermal growth factor receptor one (EGFR/Erb1). Here we show that blockage of this pathway by the tyrphostin 4-(3-chloroanilino)-6,7-dimethoxyquinazoline (AG1478) in different liver tumor cell lines promotes both inhibition of cell proliferation and induction of cell death, which are coincident with arrest in the G1 phase of the cell cycle, caspase-3 activation and DNA fragmentation. AG1478 up-regulates the expression of the pro-apoptotic member of the BCL-2 family BIM and down-regulates the expression of the anti-apoptotic BCL-XL and MCL1. Furthermore, it also decreases the levels of the caspase inhibitors HIAP2 and XIAP. The treatment of HCC cells with AG1478 enhanced the apoptosis induced by other pro-apoptotic stimuli, such as the physiological cytokine, TGF-β, highly expressed in liver tumors, or the chemotherapeutic drug doxorubicin. The effects observed by AG1478 were broader than the ones seen by silencing of the EGFR with siRNA, which indicates that this drug might act on other targets different from the EGFR. In this same line of evidence, AG1478 retained some cytotoxic effects in cells where EGFR has been targeted knock-down with shRNA. Interestingly, AG1478 preferentially acts on liver tumor cells, being untransformed cells much less responsive to its cytotoxic effects. In conclusion, AG1478 could be a potential therapeutic drug to be used in HCC.
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| 8. |
- Chisari, Andrea N, et al.
(författare)
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Lack of amino acids in mouse hepatocytes in culture induces the selection of preneoplastic cells.
- 2012
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Ingår i: Cellular Signalling. - : Elsevier BV. - 0898-6568 .- 1873-3913. ; 24:1, s. 325-32
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Tidskriftsartikel (refereegranskat)abstract
- Protein malnutrition occurs when there is insufficient protein to meet metabolic demands. Previous works have indicated that cycles of protein fasting/refeeding enhance the incidence of early lesions during chemical carcinogenesis in rat liver. The general objective of this work was to study the effect of aminoacids (Aa) deprivation on the proliferation and survival of hepatocytes, to understand its possible involvement in the generation of pre-neoplastic stages in the liver. Lack of Aa in the culture medium of an immortalized mice hepatocyte cell line induced loss in cell viability, correlating with apoptosis. However, a subpopulation of cells was able to survive, which showed a more proliferative phenotype and resistance to apoptotic stimuli. Escaping to Aa deprivation-induced death is coincident with an activated mTOR signaling and higher levels of phospho-AKT and phospho-ERKs, which correlated with increased activation of EGFR/SRC pathway and overexpression of EGFR ligands, such as TGF-α and HB-EGF. Lack of Aa induced a rapid increase in reactive oxygen species (ROS) production. However, cells that survived showed an enhancement in the levels of reduced glutathione and a higher expression of γ-GCS, the regulatory enzyme of glutathione synthesis, which can be interpreted as an adaptation of the cells to counteract the oxidative stress. In conclusion, results presented in this paper indicate that it is possible to isolate a subpopulation of hepatocytes that are able to grow in the absence of Aa, showing higher capacity to proliferate and survive, reminiscent of a preneoplastic phenotype.
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| 9. |
- Crosas-Molist, Eva, et al.
(författare)
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Vascular smooth muscle cell phenotypic changes in patients with Marfan syndrome
- 2015
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Ingår i: Arteriosclerosis, Thrombosis and Vascular Biology. - 1079-5642 .- 1524-4636. ; 35:4, s. 960-972
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Tidskriftsartikel (refereegranskat)abstract
- OBJECTIVE: Marfan's syndrome is characterized by the formation of ascending aortic aneurysms resulting from altered assembly of extracellular matrix microfibrils and chronic tissue growth factor (TGF)-β signaling. TGF-β is a potent regulator of the vascular smooth muscle cell (VSMC) phenotype. We hypothesized that as a result of the chronic TGF-β signaling, VSMC would alter their basal differentiation phenotype, which could facilitate the formation of aneurysms. This study explores whether Marfan's syndrome entails phenotypic alterations of VSMC and possible mechanisms at the subcellular level.APPROACH AND RESULTS: Immunohistochemical and Western blotting analyses of dilated aortas from Marfan patients showed overexpression of contractile protein markers (α-smooth muscle actin, smoothelin, smooth muscle protein 22 alpha, and calponin-1) and collagen I in comparison with healthy aortas. VSMC explanted from Marfan aortic aneurysms showed increased in vitro expression of these phenotypic markers and also of myocardin, a transcription factor essential for VSMC-specific differentiation. These alterations were generally reduced after pharmacological inhibition of the TGF-β pathway. Marfan VSMC in culture showed more robust actin stress fibers and enhanced RhoA-GTP levels, which was accompanied by increased focal adhesion components and higher nuclear localization of myosin-related transcription factor A. Marfan VSMC and extracellular matrix measured by atomic force microscopy were both stiffer than their respective controls.CONCLUSIONS: In Marfan VSMC, both in tissue and in culture, there are variable TGF-β-dependent phenotypic changes affecting contractile proteins and collagen I, leading to greater cellular and extracellular matrix stiffness. Altogether, these alterations may contribute to the known aortic rigidity that precedes or accompanies Marfan's syndrome aneurysm formation.
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| 10. |
- Lopez-Luque, Judit, et al.
(författare)
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Downregulation of Epidermal Growth Factor Receptor in hepatocellular carcinoma facilitates Transforming Growth Factor-beta-induced epithelial to amoeboid transition
- 2019
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Ingår i: Cancer Letters. - : ELSEVIER IRELAND LTD. - 0304-3835 .- 1872-7980. ; 464:Nov, s. 15-24
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Tidskriftsartikel (refereegranskat)abstract
- The Epidermal Growth Factor Receptor (EGFR) and the Transforming Growth Factor-beta (TGF-beta) are key regulators of hepatocarcinogenesis. Targeting EGFR was proposed as a promising therapy; however, poor success was obtained in human hepatocellular carcinoma (HCC) clinical trials. Here, we describe how EGFR is frequently downregulated in HCC patients while TGF-beta is upregulated. Using 2D/3D cellular models, we show that after EGFR loss, TGF-beta is more efficient in its pro-migratory and invasive effects, inducing epithelial to amoeboid transition. EGFR knock-down promotes loss of cell-cell and cell-to-matrix adhesion, favouring TGF-beta-induced actomyosin contractility and acquisition of an amoeboid migratory phenotype. Moreover, TGF-beta upregulates RHOC and CDC42 after EGFR silencing, promoting Myosin II in amoeboid cells. Importantly, low EGFR combined with high TGFBI or RHOC/CDC42 levels confer poor patient prognosis. In conclusion, this work reveals a new tumour suppressor function for EGFR counteracting TGF-beta-mediated epithelial to amoeboid transitions in HCC, supporting a rational for targeting the TGF-beta pathway in patients with low EGFR expression. Our work also highlights the relevance of epithelial to amoeboid transition in human tumours and the need to better target this process in the clinic.
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