1. TSC1 and Facial Angiofibromas
Mathilde Sabourin
Biology 169
March 30, 2006

2. Tuberous Sclerosis Complex (TSC)
Autosomal dominant inheritance of germline mutation in TSC1 or TSC2
Affects 1 in 6,000 people
65% of cases caused by sporadic mutations
Almost 100% penetrance
Widespread development of hamartomas
Clinical triad for diagnosis
Other common symptoms
Susceptibility of organs to benign, rare tumors: brain, skin, heart, lungs and kidneys.
Hamartomas=tumor-like growths; rarely progress to malignancy
Clinical triad=mental retardation, seizures (caused by brain lesions) and adenoma sebaceum (facial angiofibromas)
Other symptoms=autism and ADHD
Besides facial angiofibromas, all of these other symptoms are infantile manifestations.
Pulmonary lymphangiomyomatosis (LAM) is seen only in female TSC patients: benign smooth muscle cell proliferation in the lungs; often results in lung collapse.
The highest morbidity and mortality is caused by brain and renal lesions.

3. Facial Angiofibromas Major cutaneous disfigurement
Used in clinical triad to diagnose TSC

4. The TSC1 Gene Found on human chromosome 9q34 Encodes for Hamartin
Tumor suppressor gene with TSC2
Expressed in all cells and tissues, but especially skeletal muscle cells; during all cell cycle phases, including G0
Proteins from TSC1 and TSC2 form a complex to function as a tumor suppressor.

5. Hamartin 130kDa hydrophilic protein No homology to tuberin (TSC2)
Important coiled-coil domain
Co-localizes with tuberin in cytoplasm
Regulates cell growth and proliferation
Or homology to other vertebrate proteins.
Coiled-coil domain has been shown to be the only domain necessary for binding to tuberin and for the protein function
Hamartin phosphorylation by CDK1 decreases activity of the protein complex; phosphorylation by GSK3B increases the stability of the complex.
Hamartin could be responsible for the proper localization of the complex; or it could be needed to activate tuberin’s GAP domain.

6. Pathway for Hamartin
Acts in insulin pathway for cell growth regulation
Hamartin/tuberin complex suppresses Rheb
Rheb activates mTOR pathway
mTOR is a serine/threonine kinase that increases growth and proliferation by phosphorylating p70S6K and 4E-BP1
p70S6K phosphorylates ribosomal protein S6, which leads to increased ribosome biogenesis
4E-BP1 binds eIF4E and inhibits 5’ cap-dependent mRNA translation; inactive when phosphorylated, so protein synthesis can occur
Rheb needs to be GTP-bound in order to activate mTOR. Hamartin/tuberin complex is a Rheb GAP, so it makes Rheb be GDP-bound and inactive. Growth factors and insulin cause the inactivating phosphorylation of hamarin and tuberin by several kinases; this then allows synthesis of proteins and growth.
AKT/PKB/PI3K phosphorylate tuberin and suppress its GAP activity; CDK1 phosphorylates hamartin.

7. Pathway recap Insulin PI3K TSC1/2 Rheb 4E-BP1 mTOR S6K
Protein synthesis

8. Two-hit model Tumor suppressor gene LOH seen in tumor tissues
Most cases (65%) are sporadic
So they have to be hit twice in their lifetime

9. Mouse model TSC1-/- is lethal Many problems with development
Development of renal and extra-renal tumors
Homozygous mutant embryos dies between 10.5 and 11.5 days
Null mutants were pale and edematous, had pericardial effusions, hypoplastic liver, enlarged heart.
10% of heterozygous males died before 18mths, whereas 45% of heterozygous females died early. Death was usually caused by excessive amounts of blood in the peritoneal cavity (as a result of vascular tumors in the liver).
Figure: A) macroscopic view of heterozygous and homozygous embryos at day 12. See unclosed neural tube and exencephaly.
B) Histology of head region. Homozygous enlarged to twice the size. Arrows point to the unclosed region of the neural fold.
C) Immunostaining of neural tube
D) Histological analysis of the heart.
Murine Embryo Fibroblasts with null TSC1 mutation had slower growth rate, less F-actin and focal adhesion formation, constitutive phosphorylation of S6 and S6K

10. TSC1 and Cancer Common mutations Up-regulated mTOR pathway
LAM and “benign metastasis”
Drosophila gigas mutant cells
All TSC1 mutations are nonsense or frameshift mutations resulting in protein truncation.
Loss of TSC1 causes constitutive activation of mTOR pathway, which leads to increased protein synthesis and the formation of clones of cells.
LAM: Astrinidis et. al suggest a “benign metastasis” model for LAM pathogenesis, where benign TSC1 or 2 mutated cells can metastasize to the lungs from other organs; same TSC2 mutation was found in native LAM and recurrent LAM after lung transplant.
This could be explained by the fact that cells without hamartin or tuberin can migrate aberrantly. They activate the GTPases that regulate the actin cytoskeleton, cell morphology and migration.
Mutant in Drosophila homolog of TSC1; B shows enlarged eye size of mutant fly; C shows section of fly retina, with enlarged cells lacking rhabdomeres; D shows enlarged bristles on the anterior wing margin

11. Treatments Surgical removal of benign tumors
CO2 laserbrasion for disfiguring facial angiofibromas
Lung transplant for LAM
Rapamycin reduces tumor size
Rapamycin works downstream of TSC1/2 complex and inhibits mTOR. This has been shown to reduce the tumor size in mutants; it’s in clinical trials for treatment of LAM.

12. References
Astrinidis A et. al. “Tuberous sclerosis complex: linking growth and energy signaling pathways with human disease.” Oncogene ; 24;
Garami A et. al. “Insulin Activation of Rheb, a Mediator of mTOR/S6K/4E-BP Signaling, Is Inhibited by TSC1 and 2.” Molecular Cell. June 2003; 11;
Kobayashi T et. al. “A germ-line Tsc1 mutation causes tumor development and embryonic lethality that are similar, but not identical to, those caused by Tsc2 mutation in mice.” PNAS. 17 July 2001; 98;
Kwiatkowski DJ et. al. “A mouse model of TSC1 reveals sex-dependent lethality from liver hemangiomas, and up-regulation of p70S6 kinase activity in Tsc1 null cells.” Human Molecular Genetics ; 11;
Lamb RF et. al. “The TSC1 tumour suppressor hamartin regulates cell adhesion through ERM proteins and the GTPase Rho.” Nature Cell Biology. May 2000; 2;
Milolosa A et. al. “The TSC1 gene product, hamartin, negatively regulates cell proliferation.” Human Molecular Genetics ; 9;
Tapon N et. al. “The Drosophila Tuberous Sclerosis Complex Gene Homologs Restrict Cell Growth and Cell Proliferation.” Cell. May 4, 2001; 105;
Van Slegtenhorst M et. al. “Identification of the Tuberous Sclerosis Gene TSC1 on Chromosome 9q34.” Science. 8 August 1997; 277;
Van Slegtenhorst M et. al. “Interaction between hamartin and tuberin, the TSC1 and TSC2 gene products.” Human Molecular Genetics ; 7;