epidermal-growth-factor and Tuberous-Sclerosis

Topics: Adolescent; Adult; Astrocytes; Cell Communication; Cell Differentiation; Cell Proliferation; Cells, Cultured; Epidermal Growth Factor; Female; Humans; Induced Pluripotent Stem Cells; Infant; Male; Neuroglia; Neurons; Organoids; Signal Transduction; Synapses; Tuberous Sclerosis; Young Adult

Tuberous sclerosis complex (TSC) is caused by mutations in TSC1 or TSC2 genes. Lymphangioleiomyomatosis (LAM) can be sporadic or associated with TSC and is characterized by widespread pulmonary proliferation of abnormal α-smooth muscle (ASM)-like cells. We investigated the features of ASM cells isolated from chylous thorax of a patient affected by LAM associated with TSC, named LAM/TSC cells, bearing a germline TSC2 mutation and an epigenetic defect causing the absence of tuberin. 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. LAM/TSC cells spontaneously detach probably for the inactivation of the focal adhesion kinase (FAK)/Akt/mTOR pathway and display the ability to survive independently from adhesion. Non-adherent LAM/TSC cells show an extremely low proliferation rate consistent with tumour stem-cell characteristics. Moreover, LAM/TSC cells bear characteristics of stemness and secrete high amount of interleukin (IL)-6 and IL-8. Anti-EGF receptor antibodies and rapamycin affect proliferation and viability of non-adherent cells. In conclusion, the understanding of LAM/TSC cell features is important in the assessment of cell invasiveness in LAM and TSC and should provide a useful model to test therapeutic approaches aimed at controlling their migratory ability.

Topics: Antibodies; Base Sequence; Cell Adhesion; Cell Death; Cell Proliferation; Cell Survival; Cells, Cultured; Epidermal Growth Factor; Epigenesis, Genetic; Epithelial-Mesenchymal Transition; ErbB Receptors; Female; Humans; Interleukin-6; Interleukin-8; Lymphangioleiomyomatosis; Molecular Sequence Data; Sirolimus; Tuberous Sclerosis; Tuberous Sclerosis Complex 2 Protein; Tumor Suppressor Proteins

Epidermal growth factor (EGF), hepatocyte growth factor (HGF), and vascular endothelial growth factor (VEGF) regulate angiogenesis and cell growth in the developing brain. EGF, HGF, and VEGF modulate the activity of the mammalian target of rapamycin (mTOR) cascade, a pathway regulating cell growth that is aberrantly activated in tuberous sclerosis complex (TSC). We hypothesized that expression of EGF, HGF, VEGF, and their receptors EGFR, c-Met, and Flt-1, respectively, would be altered in TSC. We show by cDNA array and immunohistochemical analysis that EGF, EGFR, HGF, c-Met, and VEGF, but not Flt-1, mRNA, and protein expression was up-regulated in Tsc1 conditional knockout (Tsc1(GFAP)CKO) mouse cortex. Importantly, these alterations closely predicted enhanced expression of these proteins in tuber and subependymal giant cell astrocytoma (SEGA) specimens in TSC. Expression of EGF, EGFR, HGF, c-Met, and VEGF protein, as well as hypoxia inducible factor-1α, a transcription factor that regulates VEGF levels and is also modulated by mTOR cascade activity, was enhanced in SEGAs (n = 6) and tubers (n = 10) from 15 TSC patients. Enhanced expression of these growth factors and growth factor receptors in human SEGAs and tubers and in the Tsc1(GFAP)CKO mouse may account for enhanced cellular growth and proliferation in tubers and SEGAs and provides potential target molecules for therapeutic development in TSC.

Topics: Animals; Brain; Cerebral Cortex; Child; Epidermal Growth Factor; Female; Hepatocyte Growth Factor; Humans; Male; Mice; Mice, Knockout; Oligonucleotide Array Sequence Analysis; Phosphorylation; Ribosomal Protein S6 Kinases; RNA, Messenger; Tuberous Sclerosis; Tuberous Sclerosis Complex 1 Protein; Tumor Suppressor Proteins; Vascular Endothelial Growth Factor A

Patients with tuberous sclerosis complex (TSC) develop hamartomas containing biallelic inactivating mutations in either TSC1 or TSC2, resulting in mammalian target of rapamycin (mTOR) activation. Hamartomas overgrow epithelial and mesenchymal cells in TSC skin. The pathogenetic mechanisms for these changes had not been investigated, and the existence or location of cells with biallelic mutations ("two-hit" cells) was unclear. We compared TSC skin hamartomas (angiofibromas and periungual fibromas) with normal-appearing skin of the same patient, and we observed more proliferation and mTOR activation in hamartoma epidermis. Two-hit cells were not detected in the epidermis. Fibroblast-like cells in the dermis, however, exhibited allelic deletion of TSC2, in both touch preparations of fresh tumor samples and cells grown from TSC skin tumors, suggesting that increased epidermal proliferation and mTOR activation were not caused by second-hit mutations in the keratinocytes but by mesenchymal-epithelial interactions. Gene expression arrays, used to identify potential paracrine factors released by mesenchymal cells, revealed more epiregulin mRNA in fibroblast-like angiofibroma and periungual fibroma cells than in fibroblasts from normal-appearing skin of the same patient. Elevation of epiregulin mRNA was confirmed with real-time PCR, and increased amounts of epiregulin protein were demonstrated with immunoprecipitation. Epiregulin stimulated keratinocyte proliferation and phosphorylation of ribosomal protein S6 in vitro. These results suggest that hamartomatous TSC skin tumors are induced by paracrine factors released by two-hit cells in the dermis and that proliferation with mTOR activation of the overlying epidermis is an effect of epiregulin.

Topics: Cell Proliferation; Epidermal Growth Factor; Epiregulin; Epithelium; Gene Expression Profiling; Hamartoma; Humans; Mesoderm; Paracrine Communication; Protein Kinases; RNA, Messenger; TOR Serine-Threonine Kinases; Tuberous Sclerosis; Tuberous Sclerosis Complex 2 Protein; Tumor Suppressor Proteins

Reversible protein phosphorylation is an essential cellular regulatory mechanism. Many proteins integrate and are modulated by multiple phosphorylation events derived from complex signaling cues. Simultaneous detection and quantification of temporal changes in all of a protein's phosphorylation sites could provide not only an immediate assessment of a known biochemical activity but also important insights into molecular signaling mechanisms. Here we show the use of stable isotope-based quantitative MS to globally monitor the kinetics of complex, ordered phosphorylation events on protein players in the canonical mitogen-activated protein kinase signaling pathway. In excellent agreement with activity assays and phosphospecific immunoblotting with the same samples, we quantified epidermal growth factor-induced changes in nine phosphorylation sites in the extracellular signal-regulated kinase (ERK)/p90 ribosomal S6 kinase-signaling cassette. Additionally, we monitored 14 previously uncharacterized and six known phosphorylation events after phorbol ester stimulation in the ERK/p90 ribosomal S6 kinase-signaling targets, the tuberous sclerosis complex (TSC) tumor suppressors TSC1 and TSC2. By using quantitative phosphorylation profiling in conjunction with pharmacological kinase inhibitors we uncovered a ERK-independent, protein kinase C-dependent pathway to TSC2 phosphorylation. These results establish quantitative phosphorylation profiling as a means to simultaneously identify, quantify, and delineate the kinetic changes of ordered phosphorylation events on a given protein and defines parameters for the rapid discovery of important in vivo phosphoregulatory mechanisms.

Topics: Binding Sites; Cell Line; Epidermal Growth Factor; Humans; MAP Kinase Signaling System; Mass Spectrometry; Phorbol Esters; Phosphorus Isotopes; Phosphorylation; Protein Kinase C; Repressor Proteins; Ribosomal Protein S6 Kinases, 90-kDa; Tuberous Sclerosis; Tuberous Sclerosis Complex 1 Protein; Tuberous Sclerosis Complex 2 Protein; Tumor Suppressor Proteins

Tuberous sclerosis complex (TSC) is a tumor suppressor gene disorder characterized by mutations in the TSC1 or TSC2 genes. These mutations lead to the development of benign tumors involving smooth muscle cells, causing life-threatening lymphangioleiomyomatosis. We isolated and characterized two types of cells bearing a mutation in TSC2 exon 18 from a renal angiomyolipoma of a TSC patient: one population of alpha-actin-positive smooth muscle-like cells with loss of heterozygosity for the TSC2 gene (A(+) cells) and another of nonloss of heterozygosity keratin 8/18-positive epithelial-like cells (R(+) cells). Unlike control aortic vascular smooth muscle cells, A(+) cells required epidermal growth factor (EGF) to grow and substituting EGF with insulin-like growth factor (IGF)-1 failed to increase the cell number; however, omission of EGF did not cause cell loss. The A(+) cells constantly released IGF-1 into the culture medium and constitutively showed a high degree of S6K phosphorylation even when grown in serum-free medium. Exposure to antibodies against EGF and IGF-1 receptors caused a rapid loss of A(+) cells: 50% by 5 days and 100% by 12 days. Signal transduction mediated by EGF and IGF-I receptors is therefore involved in A(+) cell survival. These results may offer a novel therapeutic perspective for the treatment of TSC complications and lymphangioleiomyomatosis.

Topics: Actins; Adult; Angiomyolipoma; Aorta; Cell Culture Techniques; Cell Proliferation; Cell Survival; DNA Mutational Analysis; Epidermal Growth Factor; Exons; Female; Fluorescein-5-isothiocyanate; Fluorescent Antibody Technique, Indirect; Fluorescent Dyes; Genes, Tumor Suppressor; Genetic Markers; Humans; Immunohistochemistry; Insulin-Like Growth Factor I; Keratins; Loss of Heterozygosity; Microsatellite Repeats; Muscle, Smooth; Muscle, Smooth, Vascular; Mutation; Phosphorylation; Rhodamines; Ribosomal Protein S6 Kinases, 70-kDa; Tuberous Sclerosis; Tuberous Sclerosis Complex 1 Protein; Tuberous Sclerosis Complex 2 Protein; Tumor Suppressor Proteins