TSC2 GENE ENCODES FOR TUBERIN

1. TSC2 GENE ENCODES FOR TUBERIN By May Doan

2. Content • Disease: Tuberous Sclerosis • TSC2 Gene Discovered • General Roles within the Organism • Molecular Roles within the Cell • Mutations and Cancer • Possible Treatments • Summary

3. Disease: Tuberous Sclerosis • Autosomal dominant • Rare, multisystem disorder • Loci assigned at chromosomes 9 and 16 • Characterized by: • Cortical lesions called tubers • Benign tumors called hamartomas arising from the brain, heart, lungs, and kidneys • Intractable epilepsy • Mental retardation • Autism What causes tuberous sclerosis?

4. TSC2 Gene Discovered • PFGE: 5 TSC-related deletions were IDed at Chromosome 16. • Positional Cloning: These deletions were mapped to a 120kb “candidate region” in which four genes were isolated. • One gene, now TSC2, was interrupted in all five deletions. • Northen blot analysis identified either shortened transcript or reduced expression in TSC patients. • This confirmed that TSC2 (5.5 kb) is the chromosome 16 TSC gene. So what is TSC2?

5. TSC2 is a Tumor Supressor Gene • Follows Knudson’s “two-hit” hypothesis • Loss of Heterozygosity through various mutations: • Germline insertions • De novo deletions • TSC2 is NOT TSC1 • Similarities • Differences What does TSC2 do within the organism?

6. General Roles within the Organism How does TSC-2 function molecularly? What are the mechanisms in which it executes all these roles?

7. TSC2 is a Kinase and Molecular Switch • Regulate many biological processes=Participates in many signaling pathways • mTOR • WNT/B-catenin • TSC2tuberin (kinase domain and GAP domain)

8. mTOR Pathway • TSC2 • a Rheb-GTPase that suppresses mTOR signaling • suspends the phosphorylation of p70S6K and 4E-BP1 • thereby regulates cell proliferation

9. TSC2 MutationsCancer

10. Possible Treatment: Rapamycin WEEK 2: extensive tumor cell death DAY 4: reduction in tumor size

11. Additional Comments • Have not documented a consistent complete pathologic response to long-term rapamycin treatment. • In fact, after two months, there was evidence of active kidney disease. • Only short-term clinical efficacy with minimal toxocity. • Still issues to be worked out • optimal dosing schedule • duration of responses • potential resistance

12. WNT/B-Catenin Pathway • Observations/Correlations • CyclinD gene promoter is responsive to the effects of WNT/B-catenin stimulation. • CyclinD mRNA is elevated in cortical tumors like hamartomas. • inhibition of TSC2 expression by anti-sense oligos was accompanied by increased cyclin D1 protein • Conclusion • May be a link between the TSC2 and the B-catenin signaling pathway as a possible mechanism of cyclin D1 up-regulation in TSC pathology.

13. WNT/Beta-Catenin Pathway • Hypothesis: The level of B-catenin is correspondingly up-regulated by increased protein stability • Aka: if B-catenin is not degraded, it is overly stable, producing more than normal CyclinD • Known • TSC1/TSC2 act downstream of DSH and upstream of B-catenin. • Conclusion • This places the function of the tuberin at the level of the B-catenin degradation complex. • Experiment • Co-immunoprecipitation analyses showed interaction between TSC2 and GSK3/Akin • amount of endogenous GSK3 associated with tuberin in 293 cells was reduced by Wnt stimulation. • Conclusion • TSC2 binds the GSK3/Axin complex and promotes B-catenin degradation. Upon Wnt stimulation, DSH destabilizes the degradation complex and increases the free B-catenin pool.

14. Summary • Tuberous Sclerosis is an autosomal dominant disease characterized by skin lesions and hamartomas. • TSC2 was discovered in 1993 through positional cloning. • TSC2 regulates many biological processes. • TSC2’s protein product, tuberin, is a kinase and molecular switch that participates in many pathways • Pathway related to cell proliferation: mTOR • Rapamycin has had positive clinical results to treat TSC in rats. • Pathway related to cell fate determination and migration: WNT/B-catenin