2. TSC1/Hamartin and Facial Angiofibromas Biology 169 Ann Hau

3. Overview Tuberous Sclerosis Complex (TSC) & facial angiofibromas Gene: TSC1 Protein: Hamartin TSC1 mutation and cancer TSC1 Hamartin Mutation X Truncated hamartin TUMOR GROWTH

4. Tuberous Sclerosis Complex (TSC) Bourneville’s Disease Development of multiple hamartomas Tumor growth in skin, kidneys, brain, heart, eyes, lungs, teeth Seizures, autism, mental retardation

5. Tuberous Sclerosis Complex (TSC) Variable phenotypic manifestations 1 in 6,000 live births; 1 million people worldwide; 50,000 Americans

6. Facial Angiofibromas Manifestation of TSC in skin Benign tumors of the face Treat with laser treatment or dermabrasion www.schoolscience.co.uk/.../ mrc/15/page4.html

7. Mild facial angiofibroma Severe facial angiofibroma www.schoolscience.co.uk/.../ mrc/15/page4.html

8. Genetics of TSC Autosomal dominant disorder Mutation via LOH in one of two known tumor suppressor genes –TSC2 on chromosome 16 –TSC1 on chromosome 9 Transmittance –Genetic inheritance (1/3 of cases) –Spontaneous genetic mutation (2/3 of cases)

9. TSC1 and RB: Similar LOH at Locus Hereditary TSC: 1/3 of cases Sporadic TSC: 2/3 of cases Lodish, et al. Molecular Cell Biology, 4 th ed.

10. TSC1 Gene Identification by positional cloning Location at 9q34 23 exons Encodes hamartin Homolog of TSC1 transcript in yeast S. pombe van Slegtenhorst, M., et al. Identification of the tuberous sclerosis gene TSC1 on chromosome 9q34. Science 277: 805-808, 1997.

11. Hamartin Protein 1,164 amino acids; 130 kD Hydrophilic Transmembrane domain Coiled-coil region

12. TSC1/Hamartin: Normal function Tumor suppressor Forms a functional complex with TSC2/tuberin in multiple signaling cascades Regulates growth, proliferation, migration, and differentiation

13. TSC1/Hamartin: Normal function Hamartin-tubulin interaction –Hamartin binds to tuberin via coiled-coil domain, inhibits tuberin ubiquitination (Benvenuto et al., 2000) –Hamartin protein expression and hamartin- tubulin interaction detected throughout entire cell cycle (Miloloza, et al. 2000) –Phosphorylation may be key regulatory mechanism controlling TSC1-TSC2 function (Aicher, et al. 2001)

14. Tee, et al. 2002. Tuberin-hamartin complex and mTOR pathway

15. TSC: GENES GONE WILD!

16. Mutations of TSC1 32 distinct mutations identified –30 truncating, 1 missense, 1 splice site mutation –Small deletions, insertions, point mutations –No genomic deletions or genomic rearrangements Mutated in sporadic bladder tumors (Hornigold, et al. 1999)

17. TSC Lesions Shared characteristics –Abnormalities in cell size, number, morphology, and location –Suggests role of TSC genes in proliferation, growth, migration, differentiation

18. EXPERIMENTAL EVIDENCE

19. What happens when there is no TSC1? Mutants of TSC1 homolog in S. pombe –Defective uptake of nutrients from environment (van Slegtenhorst, et al. 2004) Mutants of TSC1 homolog in Drosophila –“Gigas” phenotype: increased cell and organ size; relatively normal differentiation and morphology (Ito, et al. 1999)

20. Ito, et al. gigas, a Drosophila Homolog of Tuberous Sclerosis Gene Product-2, Regulates the Cell Cycle Cell. Vol 96, 529-539, 19 February 1999

21. Mouse Models TSC1 knockout mice via gene targeting (Kobayashi et al. 2001) –TSC1 -/- mutants died around embryonic days 10.5-11.5; growth failure, anaemia –TSC1 +/- mutants developed renal and extrarenal tumors that have a loss of wildtype TSC1 allele

22. Kobayashi, 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. Proc. Nat. Acad. Sci. 98: 8762-8767, 2001.

23. Mouse Models TSC1 and TSC2 knockouts –Similar phenotypes suggest that a common pathway for TSC1/2 products exists –Heterozygous TSC1 and TSC2 mice have increased astrocyte proliferation: hamartin and tuberin are important growth regulators for astrocytes Uhlmann, E., et al. Heterozygosity for the tuberous sclerosis complex (TSC) gene products results in increased astrocyte numbers and decreased p27-Kip1 expression in TSC2 +/- cells. Oncogene 21: 4050-4059, 2002.

24. Summary TSC pathology implies role of TSC1 in regulating growth, proliferation, differentiation, and migration TSC1 -/- mice phenotype indicates role in embryonic development of brain TSC1-TSC2 complex acts as a tumor suppression in the mTOR-S6K pathway

25. Summary TSC1 is a negative regulator of growth TSC1 mutation causes cells to grow faster: increased translation through constitutively active mTOR pathway Overgrowth of cells leads to growths of TSC; cells are growing when they shouldn’t be!