Title

 

Authors

E. Orpianesi
 
Doctorate School in Biochemical, Nutritional and Metabolic Science
Dept. of Medicine, Surgery and Dentistry
Università degli Studi di Milano, Italy

 

Abstract

Tuberous sclerosis complex (TSC), an autosomal dominant disease characterized by hamartoma formation in various organs, is caused by mutations in TSC1 e TSC2  tumor suppressor genes, encoding hamartin and tuberin respectively. They form a complex that acts downstream of PI3K and Akt, and upstream of Rheb, inhibiting mammalian target of rapamycin(mTOR) and p70S6K1.Tuberin regulates and is, itself, regulated by p42/44 mitogen-activated protein kinase (MAPK). Cells with TSC2 mutation have been found in angiomyolipomas and lung lesions of lymphangioleiomyomatosis (LAM) patients (1). LAM is a rare disease characterized by widespread proliferation of abnormal smooth muscle-like cells, that leads to cystic lung destruction. We isolated and characterized tuberin-deficient cells from the chylous of LAM/TSC patient with germline TSC2 mutation. LAM/TSC cells do not express tuberin likely for the methylation of the TSC2 promoter since tuberin expression can be induced by chromatin remodelling agent such as 5-azacytidine and trichostatin A. It has been shown that an epigenetic event can cause loss of heterozigosity in TSC2 cells (2). LAM/TSC cells are smooth muscle (positive to alpha-actin antibody), express CD44V6 and HMB45 antibodies, markers of LAM and TSC cells, and required epidermal growth factor (EGF) for proliferation such as other TSC2 cell population isolated in our laboratory. The blockade of EGF receptor with monoclonal antibodies gradually reduces proliferation and causes cell death. The acquisition of mesenchymal characteristics is a transient event, important for migration and tissue invasion of cancer cells. LAM/TSC cells bear mesenchymal characteristics such as positivity for vimentin, while E-cadherin, usually not expressed in invasive cancer cells, is not detectable. It is well known that interleukin-8 (IL-8) and -6 (IL-6) production is regulated by PI3K and MAPK pathways. LAM/TSC cells secreted high amount of IL-8 and IL-6, and both rapamycin or anti-EGFR antibody exposure did not affect their levels. IL-6 and IL-8 release was blocked by 5-azacytidine exposure and consequent tuberin expression. Metylprednisolone, an antinflammatory drug, and repertaxin, an IL-8 receptor antagonist, significantly inhibited any cytokine expression.Chemokine, which induce migration of leukocytes and other cells, are considered to be synthesized largely by cells of the immune system, resulting in cell recruitment to a site of inflammation as part of an inflammatory response (3). To evaluate the factor driving migration of LAM/TSC cells some growth factor were tested. LAM/TSC cells derived from chylous secreted GM-CSF and MCP-1 since following overnight incubation with cycloheximide (CHX) their levels dropped to zero in ELISA test. Anti-EGFR antibody incubation for 48 hours significantly increased the levels of MCP-1 and slightly of GM-CSF. Rapamycin caused a not statistically significant increase. MCP-1 is known to induce directional migration of macrophages, T cells and NK cells that representing an important factor for chemotactic modulation of leukocyte infiltration.It has been suggested that the increase in leukocyte infiltration depends on interference with EGFR activation since exposure to anti-EGFR antibody upregulated MCP-1 (4).
In conclusion, LAM/TSC cells secrete high amount of IL-6, IL-8, MCP-1 and GM-CSF. Rapamycin and anti-EGFR antibody treatments did not affect IL-6 and IL-8 release, while metylprednisoloneand repertaxin cause a significant decrease. The elevated production of MCP-1 and GM-CSF and the effect of anti-EGFR antibody might be very important to understand the relationship between LAM/TSC cells and recruitment of cytokine in the lung.                                                                    

1. Lesma E.  et al. (2005). Am J Pathol vol 167(4):1093-103
2. Lesma E. et al. (2009). Am J Pathol vol 174(6):2150-9
3. Balkwill F. et al. (2004). Nat Rev Cancer vol 4:540-550
4. Hoffmann T.K. et al. (2009). Int.J.Cancer vol 124,2589-2596