Use Model Organisms to Understand Molecular Mechanisms Underlying TSC

Tian Xu, Ph.D.
Howard Hughes Medical Institute/Yale University School of Medicine

The Xu lab is interested in utilizing model organisms to understand mechanisms of human diseases. We are developing and utilizing genetic approaches in model organisms to identify genes involved in disease processes and are exploring the genetic, biochemical, and developmental properties of the genes. Understanding molecular mechanisms underlying Tuberous Sclerosis Complex (TSC) and the involvement of the TSC tumor suppressors in mechanisms of size control is a major focus of the Xu lab.

To understand the developmental functions of tumor suppressors, we have been performing genetic screens to identify overgrowth mutations in mosaic flies (Xu et al., 1995; Potter et al., 2000). The cellular composition of these mosaic animals resembles those of cancer patients who are chimeric individuals carrying a small number of mutated somatic cells. Interestingly, all the identified mutations also deregulate organ size, suggesting that tumorigenesis might reflect an impairment of organ size control (Potter and Xu, 2001). Three classes of mutations have been isolated in our screens. Mutations in genes, such as lats, cause dramatic overproliferation, resulting in tumorous growth of mutant cells in mosaic animals and enlarged organs in homozygous mutants (Xu et al., 1995; Tao et al., 1999). Mice deficient for Lats1 develop soft-tissue sarcomas and ovarian stromal cell tumors, indicating a critical role for Lats in mammalian tumorigenesis tumorigenesis (St John et al., 1999). Lats proteins are a novel family of negative CDK regulators, which affect either G1/S or G2/M transition (Tao et al., 1999; Yang et al., 2001). Mutations in the second class, such as slimb, cause patterned outgrowths in mosaic animals and alter organ size by affecting signals that regulate pattern formation (Theodosiou et al., 1998). Mutations in the third class, such as the Drosophila homologs of human tumor suppressors, PTEN, TSC1, and TSC2, cause overgrowth of mutant clones in mosaic animals largely by increasing cell size (Huang et al., 1999; Potter et al., 2001).

Tuberous sclerosis complex (TSC) is a dominant disorder occurring in approximately 1/6000 births and is characterized by the presence of hamartomas in many organs, such as the brain, skin, heart, lung, and kidney (Cheadle et al., 2000). Two genes, TSC1 and TSC2, have been identified to contribute to inherited and sporadic TSC (Consortium, 1993; van Slegtenhorst et al., 1997). However, the mechanism of TSC action remains unknown.

Mutations in the Drosophila homolog of TSC1 (Tsc1) have been isolated from mosaic screens, and cells mutant for Tsc1 are dramatically increased in size (Potter et al., 2001; Tapon et al., 2001; Gao and Pan et al., 2001). Organ size is also increased in tissues that contain a majority of mutant cells. Mutations in the Drosophila Tsc2 gene have been previously shown to cause similar phenotypes, and it was suggested that the increase in cell size is due to polyploidy (Ito and Rubin, 1999). However, clones of Tsc1 mutant cells in the imaginal discs undergo additional divisions but retain normal ploidy (Potter et al., 2001; Tapon et al., 2001; Gao and Pan et al., 2001). The Tsc1 protein binds to Tsc2 in vitro. Overexpression of Tsc1 or Tsc2 alone has no effect, but co-overexpression leads to a decrease in cell size, cell number, and organ size (Potter et al., 2001; Tapon et al., 2001; Gao and Pan et al., 2001). This provided the first in vivo evidence showing that the two Tsc proteins function together as a unit.

We have previously shown that mutations in the Drosophila PTEN gene also cause phenotypes similar to mutations of Tsc1 or Tsc2 and, as in other organisms, Drosophila PTEN functions in the insulin pathway (Huang et al., 1999). Our genetic epistasis data showed that Drosophila Tsc1 and Tsc2 function together in the insulin signaling pathway at a position between Akt and S6K (Potter et al., 2001). Furthermore, Tsc phenotypes can be alleviated by alteration of S6K activities. Given that the insulin pathway and the TSC proteins are conserved from flies to humans, these results provided a mechanism for TSC action, and suggested that downstream components such as S6K could be potential drug targets for developing tuberous sclerosis therapeutics. Furthermore, the demonstration that the TSC proteins are potent negative regulators of insulin signaling suggested that they could be novel targets for anti-diabetic drugs.

Size control mechanisms could be critical targets for evolutionary events to alter the organism sizes, and for disease processes such as tumorigenesis that require increases in tissue size. We are continuing our efforts in analyzing the insulin/TSC pathway and its involvement in development and tumorigenesis. We will attempt to identify new components for the pathway by continuing our mosaic screens to identify new mutations with similar phenotypes, and by genetic modifier screens looking for Tsc1/Tsc2-interacting genes. We will also combine genetics with biochemical experiments to define the molecular mechanisms for the action of Tsc1 and Tsc2. For example, we are in the process of determining whether Akt regulates growth by directly phosphorylating Tsc2 and if Akt phosphorylates Tsc2, whether this affects the activity of the Tsc1/Tsc2 complex. Hopefully, identifying new components and defining their molecular relationships will not only help us to address how TSC genes and their pathway participate in mechanisms of size-control and how mutations in these genes lead to tumor development, but also provide potential assays for developing therapeutic drugs for TSC patients.

References:

Cheadle, J. P., Reeve, M. P., Sampson, J. R. and Kwiatkowski, D. J. (2000). Molecular genetic advances in tuberous sclerosis. Hum. Genet., 107:97-114.

Consortium, T. E. C. T. S. (1993). Identification and characterization of the tuberous sclerosis gene on chromosome 16. Cell, 75:1305-15.

Gao, X. and Pan, D. (2001). TSC1 and TSC2 tumor suppressors antagonize insulin signaling in cell growth. Genes Dev., 15:1383-92.

Huang, H., Potter, C. J., Tao, W., Li, D.-M., Brogiolo, W., Hafen, E., Sun, H. and Xu, T. (1999). PTEN affects cell size, cell proliferation and apoptosis during Drosophila eye development. Development, 126:5365-5372.

Ito, N., and Rubin, G. M. (1999). gigas, a Drosophila homolog of tuberous sclerosis gene product-2, regulates the cell cycle. Cell, 96:529-39.

Potter, C. J. and Xu, T. (2001). Mechanisms of Size Control. Current Opinions in Genetics & Development, 11:279-286.

Potter, C. J., Huang, H. and Xu, T. (2001). Drosophila Tsc1 functions with Tsc2 to antagonize insulin signaling in regulating cell growth, cell proliferation, and organ size. Cell, 105:357-368.

Potter, C.J., Turenchalk, G.S., and Xu, T. (2000). Drosophila in cancer research, an expanding role. Trends in Genetics, 16:33-39.

St. John, M.A.R., Tao, W., Fei, X., Fukumoto, R., Carcangiu, M.L., Brownstein, D.G., A.F. Parlow, McGrath, J. and Xu, T. (1999). Mice deficient for Lats1 develop soft tissue sarcomas, ovarian tumors and pituitary dysfunction. Nature Genetics, 21:182-186.

Tao, W., Zhang, S., Turenchalk, G.S., Stewart, R.A., St. John, M.A.R., Chen, W. and Xu, T. (1999). Human homologue of the Drosophila melanogaster lats tumor suppressor modulates CDC2 activity. Nature Genetics, 21:177-181.

Tapon, N., Ito, N., Dickson, B.J., Treisman, J.E. and Hariharan, I.K. (2001). The Drosophila tuberous sclerosis complex gene homologs restrict cell growth and cell proliferation. Cell, 105:345-55.

Theodosiou, N. A., Zhang, S., Wang, W. Y. and Xu, T. (1998). slimb coordinates wg and dpp expression in the dorsal-ventral and anterior-posterior axes during limb development. Development,125:3411-3416.

van Slegtenhorst, M., de Hoogt, R., Hermans, C., Nellist, M., Janssen, B., Verhoef, S., Lindhout, D., van den Ouweland, A., Halley, D., Young, J., Burley, M., Jeremiah, S., Woodward, K., Nahmias, J., Fox, M., Ekong, R., Osborne, J., Wolfe, J., Povey, S., Snell, R. G., Cheadle, J. P., Jones, A. C., Tachataki, M., Ravine, D., Kwiatkowski, D. J. and et al. (1997). Identification of the tuberous sclerosis gene TSC1 on chromosome 9q34. Science, 277:805-8.

Yang, X.L.. Li, D.M., Chen, W.L. and Xu, T. (2001). Human Homologue of Drosophila lats, LATS1, Negatively Regulate Growth by Inducing G2/M Arrest or Apoptosis. Oncogene, 20:6516-6523.

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