Tuberous sclerosis complex (TSC), an autosomal dominant disease, is characterized by the formation of hamartomas in various organs such as brain, kidney, skin, retina and heart, and is caused by mutations in TSC1 e TSC2 tumor suppressor genes, encoding hamartin and tuberin, respectively. Lymphangioleiomyomatosis (LAM) is a rare lung disease characterized by cystic lung destruction leading to progressive respiratory failure and formation of abdominal tumors. LAM can be sporadic or associated to TSC and LAM lung alterations resulting from proliferation of neoplastic cells bearing mutations in either TSC1 or TSC2 genes. A metastatic process has been proposed in dissemination of LAM and TSC cells to explain the identical TSC2 mutations and loss of heterozigosity (LOH) patterns in LAM cells of lung nodules, angiomyolipomas and lymph nodes of the same sporadic LAM patients, that is consistent with metastatic spread among organs. The aim of this project is to study the role of cytokines and matrix metalloproteinases in LAM and TSC. LAM/TSC cells, isolated from chylous of a patient affected by LAM associated to TSC, bear a TSC2 germline mutation and do not express tuberin for an epigenetic silencing of the TSC2 second allele that confirms our previously published evidence that an epigenetic alterations of TSC2 can cause the loss of tuberin in TSC cells. Treatment of LAM/TSC cells with 5-azacytidine that inhibits CpG DNA methylation led to the expression of tuberin as consequence of the chromatin remodeling. Proliferation of LAM/TSC cells is epidermal growth factor (EGF)-dependent and blockade of EGF receptor causes cell death, as we previously showed in cells lacking tuberin, while rapamycin, an mTOR inhibitor, significantly reduced proliferation. LAM/TSC cells have the ability to grow independently from adhesion and survive in adherent and nonadherent condition. For these features these cells are a good model to study the mechanisms of motility in LAM and TSC and the relation to tuberin expression. We studied the effect of anti-EGFR Ab and rapamycin on motility, cytokines and MMPs (Matrix metalloproteinases) expression. LAM/TSC cells spontaneously detach and undergo spontaneous cycles of adhesion and nonadhesion conditions likely for the inactivation of the focal adhesion kinase (FAK)/Akt/mTOR pathway. In LAM/TSC cells FAK inhibition caused the reduction of AKT phosphorylation which was followed by inhibition of mTOR phosphorylation and mTOR autophosphorylation and, consequently, by a strong reduction of S6 phosphorilation as it occurs in nonadherent cells. Anti-EGFR antibody and rapamycin reduced the viability of adherent and nonadherent cells while 5-azacytidine slightly increased the percentage of detaching cells. Nonadherent LAM/TSC cells underwent an extremely low proliferation rate consistent with tumour stem cell characteristics. One of the step driving EMT is the repression of E-cadherin, resulting in the loss of cell-cell adhesion. LAM/TSC cells did not express E-cadherin, but they express vimentin, marker of mesenchymal cells. EMT process controls the migration of cancer cells from primary tumors depending on an inflammatory microenvironment. LAM/TSC cells secreted high amount of IL-6, IL-8 and IL-1α, cytokines crucial for a variety of cancer cells. The levels of IL-6, IL-8 and IL-1α were not affected by rapamycin or anti-EGFR antibody but were regulated by tuberin expression since their levels were reduced by 5-azacytidine incubation. MMPs degrade and modify the extracellular matrix (ECM), facilitating detachment of cells from the tissue. Levels of MMP-2 and MMP-9 in urine are predictive of disease status in a variety of cancers and also in LAM. MMP-2 and MMP-9 levels are high in urine of LAM patients and MMP-2 is substantially up-regulated in their lung tissue. Matrilisin (MMP-7) contributes to tumor progression, invasion and is overexpressed in several types of invasive cancers. In adherent status LAM/TSC cells expressed higher levels of MMP-2 mRna and lower of MMP-7 than in nonadherent condition. Consistent with ECM degration and invasive features respectively,. MMP-2 and MMP-7 mRna expression appeared to be related to tuberin since they were significantly reduced by 5-azacytidine incubation. Anti-EGFR Ab and rapamycin significantly decreased MMP-2 mRna expression while their effect was the opposite on MMP-7. Extracellular matrix metalloproteinase inducer (EMMPRIN), is thought to affect tumor progression through its ability to stimulate MMPs expression. EMMPRIN expression, quantified by cytometric analysis, was reduced by 5-azacytidine and was much higher in LAM/TSC cells than in MCF7, breast cancer cells. Anti-EGFR antibody and rapamycin did not change EMMPRIN levels. For the mesenchymal feature of LAM/TSC cells and their ability to survive in adherent and nonadherent conditions, we evaluated LAM/TSC motility in wound healing assay providing an indication of cell migration rate. LAM/TSC cells closed wounds in about 11h, much faster than tumoral cells such as MCF7. Cell motility of LAM/TSC cells is related to tuberin expression since 5-azacytidine treatment, and the consequent induced-tuberin expression, increased the time of wound closure to 19h. However, 5-azacytidine treatment did not have any effect on MMP-2 and MMP-7 mRna expression in wound healing assay. Rapamycin and anti-EGFR Ab decreased the migration rate of LAM/TSC cells leading to the closure of wound in about 25h and causing an induced an increase of MMP-2 and MMP-7 levels. However during wound closure (50%) MMP-2 secretion was increased by anti-EGFR and rapamycin treatments and normalized at complete wound closure. In conclusion, LAM/TSC cells express invasive and migratory features which appear to be related to tuberin expression . Phosphorylation of FAK and MMP-2 expression was reduced in nonadherent LAM/TSC cells compared to adherent cells suggesting that FAK signaling was required for the activation of MMP-2 expression. MMP-2 and MMP-7 are related to the adherent and nonadherent conditions and are involved in the metastatic process proposed in dissemination of LAM cells. LAM/TSC cells have the ability to migrate and their motility is related to tuberin expression and reduced by anti-EGFR antibody and rapamycin. Rapamycin and anti-EGFR Ab control MMP-2 and MMP-7 expression and secretion during cell migration. These data suggest a specific role for MMP-2 and MMP-7 in LAM/TSC cell motility and thereby in the pathogenesis of LAM and TSC indicating their involvement in the metastatic process proposed in dissemination of LAM cells. The understanding of LAM/TSC cell features in motility is important for the assessment of cell invasiveness in LAM and TSC and provide highlights to sustain MMPs pathways as potential target for LAM. TSC and TSC related disorders.

STUDIO DELLE CITOCHINE E DELLE METALLOPROTEASI NELLA LINFANGIOLEIOMIOMATOSI E NELLA SCLEROSI TUBEROSA / E. Orpianesi ; tutor: A. Gorio ; coordinatore: A. Gorio. - : . Università degli Studi di Milano, 2014 Jan 30. ((26. ciclo, Anno Accademico 2013. [10.13130/orpianesi-emanuela_phd2014-01-30].

STUDIO DELLE CITOCHINE E DELLE METALLOPROTEASI NELLA LINFANGIOLEIOMIOMATOSI E NELLA SCLEROSI TUBEROSA

E. Orpianesi
2014-01-30

Abstract

Tuberous sclerosis complex (TSC), an autosomal dominant disease, is characterized by the formation of hamartomas in various organs such as brain, kidney, skin, retina and heart, and is caused by mutations in TSC1 e TSC2 tumor suppressor genes, encoding hamartin and tuberin, respectively. Lymphangioleiomyomatosis (LAM) is a rare lung disease characterized by cystic lung destruction leading to progressive respiratory failure and formation of abdominal tumors. LAM can be sporadic or associated to TSC and LAM lung alterations resulting from proliferation of neoplastic cells bearing mutations in either TSC1 or TSC2 genes. A metastatic process has been proposed in dissemination of LAM and TSC cells to explain the identical TSC2 mutations and loss of heterozigosity (LOH) patterns in LAM cells of lung nodules, angiomyolipomas and lymph nodes of the same sporadic LAM patients, that is consistent with metastatic spread among organs. The aim of this project is to study the role of cytokines and matrix metalloproteinases in LAM and TSC. LAM/TSC cells, isolated from chylous of a patient affected by LAM associated to TSC, bear a TSC2 germline mutation and do not express tuberin for an epigenetic silencing of the TSC2 second allele that confirms our previously published evidence that an epigenetic alterations of TSC2 can cause the loss of tuberin in TSC cells. Treatment of LAM/TSC cells with 5-azacytidine that inhibits CpG DNA methylation led to the expression of tuberin as consequence of the chromatin remodeling. Proliferation of LAM/TSC cells is epidermal growth factor (EGF)-dependent and blockade of EGF receptor causes cell death, as we previously showed in cells lacking tuberin, while rapamycin, an mTOR inhibitor, significantly reduced proliferation. LAM/TSC cells have the ability to grow independently from adhesion and survive in adherent and nonadherent condition. For these features these cells are a good model to study the mechanisms of motility in LAM and TSC and the relation to tuberin expression. We studied the effect of anti-EGFR Ab and rapamycin on motility, cytokines and MMPs (Matrix metalloproteinases) expression. LAM/TSC cells spontaneously detach and undergo spontaneous cycles of adhesion and nonadhesion conditions likely for the inactivation of the focal adhesion kinase (FAK)/Akt/mTOR pathway. In LAM/TSC cells FAK inhibition caused the reduction of AKT phosphorylation which was followed by inhibition of mTOR phosphorylation and mTOR autophosphorylation and, consequently, by a strong reduction of S6 phosphorilation as it occurs in nonadherent cells. Anti-EGFR antibody and rapamycin reduced the viability of adherent and nonadherent cells while 5-azacytidine slightly increased the percentage of detaching cells. Nonadherent LAM/TSC cells underwent an extremely low proliferation rate consistent with tumour stem cell characteristics. One of the step driving EMT is the repression of E-cadherin, resulting in the loss of cell-cell adhesion. LAM/TSC cells did not express E-cadherin, but they express vimentin, marker of mesenchymal cells. EMT process controls the migration of cancer cells from primary tumors depending on an inflammatory microenvironment. LAM/TSC cells secreted high amount of IL-6, IL-8 and IL-1α, cytokines crucial for a variety of cancer cells. The levels of IL-6, IL-8 and IL-1α were not affected by rapamycin or anti-EGFR antibody but were regulated by tuberin expression since their levels were reduced by 5-azacytidine incubation. MMPs degrade and modify the extracellular matrix (ECM), facilitating detachment of cells from the tissue. Levels of MMP-2 and MMP-9 in urine are predictive of disease status in a variety of cancers and also in LAM. MMP-2 and MMP-9 levels are high in urine of LAM patients and MMP-2 is substantially up-regulated in their lung tissue. Matrilisin (MMP-7) contributes to tumor progression, invasion and is overexpressed in several types of invasive cancers. In adherent status LAM/TSC cells expressed higher levels of MMP-2 mRna and lower of MMP-7 than in nonadherent condition. Consistent with ECM degration and invasive features respectively,. MMP-2 and MMP-7 mRna expression appeared to be related to tuberin since they were significantly reduced by 5-azacytidine incubation. Anti-EGFR Ab and rapamycin significantly decreased MMP-2 mRna expression while their effect was the opposite on MMP-7. Extracellular matrix metalloproteinase inducer (EMMPRIN), is thought to affect tumor progression through its ability to stimulate MMPs expression. EMMPRIN expression, quantified by cytometric analysis, was reduced by 5-azacytidine and was much higher in LAM/TSC cells than in MCF7, breast cancer cells. Anti-EGFR antibody and rapamycin did not change EMMPRIN levels. For the mesenchymal feature of LAM/TSC cells and their ability to survive in adherent and nonadherent conditions, we evaluated LAM/TSC motility in wound healing assay providing an indication of cell migration rate. LAM/TSC cells closed wounds in about 11h, much faster than tumoral cells such as MCF7. Cell motility of LAM/TSC cells is related to tuberin expression since 5-azacytidine treatment, and the consequent induced-tuberin expression, increased the time of wound closure to 19h. However, 5-azacytidine treatment did not have any effect on MMP-2 and MMP-7 mRna expression in wound healing assay. Rapamycin and anti-EGFR Ab decreased the migration rate of LAM/TSC cells leading to the closure of wound in about 25h and causing an induced an increase of MMP-2 and MMP-7 levels. However during wound closure (50%) MMP-2 secretion was increased by anti-EGFR and rapamycin treatments and normalized at complete wound closure. In conclusion, LAM/TSC cells express invasive and migratory features which appear to be related to tuberin expression . Phosphorylation of FAK and MMP-2 expression was reduced in nonadherent LAM/TSC cells compared to adherent cells suggesting that FAK signaling was required for the activation of MMP-2 expression. MMP-2 and MMP-7 are related to the adherent and nonadherent conditions and are involved in the metastatic process proposed in dissemination of LAM cells. LAM/TSC cells have the ability to migrate and their motility is related to tuberin expression and reduced by anti-EGFR antibody and rapamycin. Rapamycin and anti-EGFR Ab control MMP-2 and MMP-7 expression and secretion during cell migration. These data suggest a specific role for MMP-2 and MMP-7 in LAM/TSC cell motility and thereby in the pathogenesis of LAM and TSC indicating their involvement in the metastatic process proposed in dissemination of LAM cells. The understanding of LAM/TSC cell features in motility is important for the assessment of cell invasiveness in LAM and TSC and provide highlights to sustain MMPs pathways as potential target for LAM. TSC and TSC related disorders.
GORIO, ALFREDO
GORIO, ALFREDO
Tuberous sclerosis complex ; Lymphangioleiomyomatosis ; Matrix metalloproteinases ; Motility ; Rapamycin; anti-EGFR antibody
Settore BIO/14 - Farmacologia
STUDIO DELLE CITOCHINE E DELLE METALLOPROTEASI NELLA LINFANGIOLEIOMIOMATOSI E NELLA SCLEROSI TUBEROSA / E. Orpianesi ; tutor: A. Gorio ; coordinatore: A. Gorio. - : . Università degli Studi di Milano, 2014 Jan 30. ((26. ciclo, Anno Accademico 2013. [10.13130/orpianesi-emanuela_phd2014-01-30].
Doctoral Thesis
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/2434/230542
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