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)
Need to Define Hamartomas

5. Hamartomas Defined as a Common Benign Tumor Result from:
abnormal formation of normal tissue
excessive cellular growth
Growths are called “Tubers”
Define Tubers
Describe how they are normally asymptomatic, undetected, and only cause problems if they invade on another organs resources (mainly space)

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
Define Positional Cloning
41kb = 4.1 Million Basepair

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
RHEB GAP Instead of RAS-GAP
State functions of GAP- Deactivating Active RAS/RHEB Complex
More Clues to Overall function- Deactivates an Active Protein (RHEB)

10. Tuberin Pathway

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

15. Loss of Function Mutation in either TSC1 or TSC2
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 is now constitutively Active
mTOR
Protein Synthesis/
Cell Growth
Rheb is now constitutively Active
Rheb excessively activates mTOR
mTOR activation yields excessive Protein Synthesis and 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 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 Rapamyacin binds mTOR, deactivating it
Rheb
mTOR
Rapamyacin
Protein Synthesis/
Cell Growth
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

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:
Kobayashi T, Hirayama Y, Kobayashi E, Kubo Y, Hino O 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 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 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. (
Yeung RS: Multiple roles of the tuberous sclerosis complex genes. Genes Chromosomes Cancer 38: , 2003