TSC2 and Tuberous Sclerosis

1. TSC2 and Tuberous Sclerosis Charles G. Sproule IV April 4, 2006 Biology 169

2. Tuberous Sclerosis • A Rare Genetic Disorder affecting 1 million people worldwide • Abbreviated TSC • Discovered in the late 1800’s

3. Tuberous Sclerosis • Incidence: 1 in every 6,000 Births • 1/3 of All Cases are Inherited • Inherited as Autosomal Dominant • 2/3 of All Cases are Sporadic • With proper treatment, most victims live a normal life

4. Clinical Manifestations • TSC is characterized in multiple ways: • Hamartomas in multiple organ systems • Most often occur in the Brain, Heart, Kidneys, Skin, and Lungs. • Behavioral Manifestations • Autism, Mental Retardation, Learning Disabilities • Seizure Disorders • Many patients are epileptic • Diagnosed by the “Clinical Triad” • Seizures, Behavioral Manifestations, and Presence of Benign Tumors (Mainly Hamartomas)

5. Hamartomas • Defined as a Common Benign Tumor • Result from: • abnormal formation of normal tissue • excessive cellular growth • Growths are called “Tubers”

6. The TSC2 Gene • Tuberous Sclerosis caused by a mutation in either the TSC1 or TSC2 Gene • TSC2 Gene • Discovered in 1993 via positional cloning • Maps to Chromosome 16 (16p13.3) • Evolutionarily Highly-Conserved Throughout Flies, Mice, and Humans • Encodes for the Protein “Tuberin” • Very Large Gene • Spans 41 Kilobases • Has 41 Exons

7. Tuberin Protein • Characteristics: • 1807 Amino Acids • 190 kilo-Daltons • Expressed in most embryonic and mammalian cells • Found in Cytoplasm of Cells • Has two main functional domains: • Hamartin Interaction Domain • GAP Domain

8. Tuberin: A Closer Look • Hamartin Interaction Domain: • Responsible for Interaction with TSC1 Protein Hamartin • Hamartin and Tuberin form a complex in a functional pathway • GAP Domain (GTPase Activating Protein) • Very important clue as to function of protein

9. GTPase Activating Protein

10. Tuberin Pathway

11. Tuberin Pathway: Simplified Growth Factors Akt/Erk1 • Components Downstream of Hamartin/Tuberin Complex: • Rheb: Rheb-GTP activates mTOR • mTOR: Directly Phosphorylates Transcription Factors • Transcription Factors Cause: • Protein Synthesis • Cell Growth Hamartin Tuberin Rheb mTOR Protein Synthesis/ Cell Growth

12. Mutations in TSC2 • Various Genomic Mutations can occur in TSC2 • 2/3 of all mutations found have truncating effect • Small amount (<10%) are large genomic deletions • Misense, Nonsense, and Insertions/Deletions usually found • Truncating Mutation would mean a loss of the GAP Domain Function

13. TSC2 Mutations and Tuberin • Mutation in TSC2 can result in: • Loss of Function of Tuberin Protein • Looking at Tuberin, we Know That: • Tuberin is a Rheb-GTPase • Healthy Tuberin suppresses Growth • Mutation could cause excessive cell growth • Therefore we know that Tuberin is a: • TUMOR SUPPRESSOR GENE

14. Loss of Function Growth Factors Akt/Erk1 Hamartin Tuberin Rheb mTOR Protein Synthesis/ Cell Growth Normal Pathway

15. Loss of Function Growth Factors Akt/Erk1 • Mutation in either TSC1 or TSC2 • Hamartin/Tuberin Complex is not functional Hamartin Tuberin Rheb mTOR Protein Synthesis/ Cell Growth

16. Loss of Function Rheb mTOR • Rheb is now constitutively Active • Rheb excessively activates mTOR • mTOR activation yields excessive Protein Synthesis and Cell Growth Protein Synthesis/ Cell Growth

17. TSC2 Mutations and Cancer • Excessive Cell Growth = Tumorigenesis! • TSC2 Mutations cause excessive cell growth • Hamartomas are caused by excessive cell growth • Thus, TSC2 mutations cause Hamartomas • Both TSC2 genes must be mutated • Follows Knudson’s 2-Hit Model for Tumor Suppressor Genes

18. Homozygous Mice Knockouts • Embryonic Lethal at age 10.5 Days • Tuberin is thus essential for embryologic development • Embryos exhibited: • Unclosed Neural Tubes • Thickened Myocardium • Thickened Myocardium is indicative of Tuberin function in suppressing cell growth

19. Homozygous Mice Knockouts • (Above) Neural Crest Malformation in Embryos at 10.5 and 11.5 Days • (Side) Failure of Neural Crest to Close at 11.5 Days

20. Homozygous Mice Knockouts • Embryologic Myocardial Defects at age 12.5 and age 11.5 Days • Note increased cell density • Figures show excessive cell growth early on in embryos • Excessive cell growth indicative of TSC2 Mutation

21. Heterozygous Mouse Mutations • Germ Line Heterozygous Mutations in TSC2 • Causes renal carcinomas in mice • Carcinomas are 100% penetrant • Unknown reason for phenotypic discrepancy among mice and humans • Additional cancers were also found in some mice • Cancers of Liver • Brain Tumors

22. Heterozygous Mouse Mutations • Kidneys from a 7-Month Heterozygous Mutant • Note renal carcinoma formation (White Spots) • Bottom Figure subjected to carcinogens • This induced earlier LOH • More severe renal carcinomas were found

23. TSC2 Mutations in Humans • TSC2 LOF Mutations cause Tuberous Sclerosis in Humans • Deficient cell-growth signaling pathway • Leads to benign tumors, or hamartomas, in multiple organ systems.

24. TSC Prognosis/Treatment • Severity of TSC varies on a case by case basis • TSC2 mutations may cause more severe cases of TSC • Most live a normal life with routine checkups • < 2% of TSC tumors become malignant • Treatment Includes • Anti-seizure Medications • Intervention and Pharmacology for Behavioral Problems • Removal Of Tumors

25. Rapamyacin • mTOR is a proto-oncogene downstream of the TSC complex • Once active, it promotes protein synthesis and thus cell growth • mTOR – mammalian target of Rapamyacin • Rapamyacin is an mTOR inhibitor • Rapamyacin is already widely used in preventing immune system dysfunction during surgeries

26. Rapamyacin Rheb mTOR Rapamyacin • Rapamyacin binds mTOR, deactivating it • All Downstream signaling is halted, and thus cell growth is contained • Currently in Phase II Trials • So far Results are good Protein Synthesis/ Cell Growth

27. What Lies Ahead • Continued Pursuit of Rapamyacin/ Pharmacology • Fully Understanding Tuberin Function • Main Function is suppressing mTOR and cell growth • Depending on Cell Type, Hamartin/Tuberin may play roles in: • mTOR Inhibition • WNT/β-Catenin Signaling • Intracellular Trafficking

28. References • Brendan D. Manning, M. Nicole Logsdon, Alex I. Lipovsky, Derek Abbott, David J. Kwiatkowski, and Lewis C. Cantley. Feedback inhibition of Akt signaling limits the growth of tumors lacking Tsc2. Genes & Dev., Aug 2005; 19: 1773 - 1778. • Kobayashi T, Hirayama Y, Kobayashi E, Kubo Y, Hino O. 1995. A germline insertion in the tuberous sclerosis (Tsc2) gene gives rise to the Eker rat model of dominantly inherited cancer [published erratum appears in Nat Genet 1995 Feb9(2):218]. Nat Genet 9:70 –74. • Kobayashi, T., Minowa, O., Kuno, J., Mitani, H., Hino, O., and Noda, T. (1999). Renal carcinogenesis, hepatic hemangiomatosis, and embryonic lethality caused by a germ-line Tsc2 mutation in mice. Cancer Res. 59, 1206–1211. • Manning BD, Tee AR, Logsdon MN, Blenis J, Cantley LC. 2002.Identification of the tuberous sclerosis complex-2 tumor suppressor gene product tuberin as a target of the phosphoinositide 3-kinase/akt pathway. Mol Cell 10:151–162. • Tuberous Sclerosis Alliance.(http://www.tsalliance.org) 2005. • Yeung RS: Multiple roles of the tuberous sclerosis complex genes. Genes Chromosomes Cancer 38: 368-375, 2003