THE UNIVERSITY of NORTH CAROLINA at CHAPEL HILL

TSC2 and Tuberous Sclerosis

Charles G. Sproule IVApril 4, 2006Biology 169

THE UNIVERSITY of NORTH CAROLINA at CHAPEL HILL

Tuberous Sclerosis● A Rare Genetic Disorder affecting 1

million people worldwide● Abbreviated TSC● Discovered in the

late 1800’s

THE UNIVERSITY of NORTH CAROLINA at CHAPEL HILL

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

THE UNIVERSITY of NORTH CAROLINA at CHAPEL HILL

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)

THE UNIVERSITY of NORTH CAROLINA at CHAPEL HILL

Hamartomas

● Defined as a Common Benign Tumor● Result from:

– abnormal formation of normal tissue

– excessive cellular growth

● Growths are called “Tubers”

THE UNIVERSITY of NORTH CAROLINA at CHAPEL HILL

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

THE UNIVERSITY of NORTH CAROLINA at CHAPEL HILL

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

THE UNIVERSITY of NORTH CAROLINA at CHAPEL HILL

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

THE UNIVERSITY of NORTH CAROLINA at CHAPEL HILL

GTPase Activating Protein

THE UNIVERSITY of NORTH CAROLINA at CHAPEL HILL

Tuberin Pathway

THE UNIVERSITY of NORTH CAROLINA at CHAPEL HILL

Tuberin Pathway: Simplified

● Components Downstream of Hamartin/Tuberin Complex:– Rheb: Rheb-GTP activates mTOR– mTOR: Directly Phosphorylates Transcription Factors– Transcription Factors Cause:

● Protein Synthesis ● Cell Growth

Growth Factors

Akt/Erk1

Hamartin

Tuberin

Rheb

mTOR

THE UNIVERSITY of NORTH CAROLINA at CHAPEL HILL

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

THE UNIVERSITY of NORTH CAROLINA at CHAPEL HILL

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

THE UNIVERSITY of NORTH CAROLINA at CHAPEL HILL

Loss of FunctionGrowth Factors

Akt/Erk1

Hamartin

Tuberin

Rheb

mTOR

Normal Pathway

THE UNIVERSITY of NORTH CAROLINA at CHAPEL HILL

Loss of Function

● Mutation in either TSC1 or TSC2● Hamartin/Tuberin Complex is not functional

Growth Factors

Akt/Erk1

Hamartin

Tuberin

Rheb

mTOR

THE UNIVERSITY of NORTH CAROLINA at CHAPEL HILL

Loss of Function

Rheb

mTOR

● Rheb is now constitutively Active● Rheb excessively activates mTOR● mTOR activation yields excessive Protein

Synthesis and Cell Growth

THE UNIVERSITY of NORTH CAROLINA at CHAPEL HILL

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

THE UNIVERSITY of NORTH CAROLINA at CHAPEL HILL

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

THE UNIVERSITY of NORTH CAROLINA at CHAPEL HILL

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

THE UNIVERSITY of NORTH CAROLINA at CHAPEL HILL

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

THE UNIVERSITY of NORTH CAROLINA at CHAPEL HILL

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

THE UNIVERSITY of NORTH CAROLINA at CHAPEL HILL

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

THE UNIVERSITY of NORTH CAROLINA at CHAPEL HILL

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.

THE UNIVERSITY of NORTH CAROLINA at CHAPEL HILL

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

THE UNIVERSITY of NORTH CAROLINA at CHAPEL HILL

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

THE UNIVERSITY of NORTH CAROLINA at CHAPEL HILL

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

Rheb

mTOR Rapamyacin

THE UNIVERSITY of NORTH CAROLINA at CHAPEL HILL

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

THE UNIVERSITY of NORTH CAROLINA at CHAPEL HILL

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