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The tuberous sclerosis complex (TSC) pathway and mechanism of size control C.J. Potter, L.G. Pedraza, H. Huang and T. Xu1 Howard Hughes Medical Institute, Department of Genetics, Yale University School of Medicine, New Haven, CT 06510, U.S.A.
Abstract We have identified three groups of growth-constraint genes using mosaic genetic screens in Drosophila melanogaster, including PTEN (phosphatase and tensin homologue deleted on chromosome 10), and the tuberous sclerosis complex (TSC) genes, Tsc1 and Tsc2. Our studies show that all three groups of genes participate in mechanisms that regulate organ and organism size in animals. We propose that mechanisms of organ size control are critical targets for diseases, such as tumorigenesis, which require an increase in tissue size and total mass, and for evolutionary events that alter the size of organisms. Using genetic and biochemical methods, we have shown that Tsc1 and Tsc2 function in the insulin/phosphoinositide 3-kinase (PI3K)/Akt pathway. We have shown that Akt regulates the Tsc1–Tsc2 complex by directly phosphorylating Tsc2. We have shown further that S6 kinase (S6K) is a downstream component of the PI3K/Akt/TSC pathway and reduction of S6K activity can block TSC defects. Recent studies from many laboratories have now confirmed our findings in mice, rats and human patients, and have shown that drugs that antagonize S6K activities, such as rapamycin, diminish tumours in TSC-deficient mice and rats. Clinical trials based on these findings have begun. Given that other components of the pathway, such as PTEN, are also mutated in a large number of cancer patients and that these components regulate intracellular insulin signalling, therapeutics based on the knowledge of the pathway could have effects beyond the TSC patient population.
Our laboratory 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 tumour suppressors in mechanisms of size control is a major focus of our laboratory. To understand the developmental functions of tumour suppressors, we have been performing genetic screens to identify overgrowth mutations in mosaic flies [1,2]. The cellular composition of these mosaic animals resembles those of cancer patients who are chimaeric 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 [3]. Three classes of mutation have been isolated in our screens. Mutations in genes such as lats cause dramatic overproliferation, resulting in tumourous growth of mutant cells in mosaic animals and enlarged organs in homozygous mutants [1,4]. Mice deficient for Lats1 develop soft-tissue sarcomas and ovarian stromal cell tumours, indicating a critical role for Lats protein in mammalian tumorigenesis [5]. Key words: Drosophila genetics, phosphoinositide 3-kinase (PI3K)/PTEN/Akt/tuberous sclerosis complex (TSC) pathway, size-control mechanisms, tuberous sclerosis gene function, tumourigenesis. Abbreviations used: PI3K, phosphoinositide 3-kinase; TSC, tuberous sclerosis complex; PKB, protein kinase B; S6K, S6 kinase. 1
To whom correspondence should be addressed (e-mail
[email protected]).
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Lats proteins are a novel family of negative cyclin-dependent kinase (‘CDK’) regulators, which affect either G1 /S or G2 /M transition [4,6]. In the second class, mutations of genes such as slimb lead to increased cell proliferation as a consequence of duplicated structures [7]. In the third class, mutant cells acquire a growth advantage and exhibit an increase in cell size. This class includes mutant alleles of Drosophila Tsc1 [8–10] and Drosophila Tsc2 [11], as well as mutant alleles of the Drosophila homologue of the tumour suppressor PTEN (phosphatase and tensin homologue deleted on chromosome 10) [12–14]. TSC is a dominant disorder occurring in approx. 1/6000 births and is characterized by the presence of hamartomas in many organs, such as the brain, skin, heart, lung and kidney [15]. Two genes, TSC1 and TSC2, have been identified to contribute to inherited and sporadic TSC [16,17]. However, the mechanism of TSC action remains unknown. Mutations in the Drosophila homologue of TSC1 (Tsc1) have been isolated from mosaic screens, and cells mutant for Tsc1 are dramatically increased in size [8–10]. 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 [11]. However, clones of Tsc1 mutant cells in the imaginal discs undergo additional divisions but retain normal ploidy [8–10]. Furthermore, ectopic overexpression of Tsc1 or Tsc2 alone in Drosophila tissues has no effect, but cooverexpression of Tsc1 and Tsc2 leads to dramatic decreases in cell growth and cell proliferation [8–10]. Since Tsc1 and
TSC Genes – Novel Players in the Growth Regulation Network
Tsc2 form a complex in vitro [8] and in vivo [10], these results indicate that Tsc1 and Tsc2 function as a complex in vivo to regulate cellular growth. The similarity in phenotypes caused by mutation of Drosophila PTEN and Tsc1/Tsc2 suggested that they might share a common biological function [8]. Indeed, patients with hereditary mutations of human PTEN, like TSC1 and TSC2 patients, also develop hamartomas in multiple organs. PTEN functions to antagonize the conserved insulin/phosphoinositide 3-kinase (PI3K) signalling pathway in humans and flies [12,18]. PTEN is also one of the most frequently mutated genes involved in the development of human cancers [19], indicating that the insulin signalling pathway plays a pivotal role in tumorigenesis. Since TSC1 and TSC2 also function as tumour suppressors, these results suggested that TSC1/TSC2 might also function as antagonists of the insulin signalling pathway. In Drosophila, mutations of the positive components of the insulin signalling pathway – insulin receptor, PI3K, Akt/protein kinase B (PKB) and S6 kinase (S6K) – lead to marked decreases in cell size [20–22]. To determine if, and where, Tsc1 and Tsc2 function within the insulin signalling pathway, we have performed classic genetic epistasis experiments with components of this pathway, and have showed that the Tsc1–Tsc2 complex functions between Akt and S6K in the insulin signalling pathway [8]. However, the molecular mechanism by which the insulin/PI3K/Akt pathway transduces signals through Tsc1–Tsc2, and Akt regulates Tsc1–Tsc2 activity, is unknown. The serine/threonine kinase Akt plays a pivotal role in multiple processes. Akt regulates cell growth via the indirect activation of TOR and S6K. Indeed, expression of Akt in Drosophila and mammalian tissues leads to dramatic increases in cell growth/size. Akt also plays a pivotal role in tumorigenesis, since the ability of PTEN to function as a tumour suppressor results from its role in negatively regulating the activity of Akt [23]. However, the direct mechanism by which Akt stimulates growth in vivo is unclear. We have now shown that Drosophila Akt/PKB stimulates growth by phosphorylating the Tsc2 tumour suppressor and inhibiting formation of the Tsc1–Tsc2 complex [24]. We show that Akt/PKB directly phosphorylates Drosophila Tsc2 in vitro on conserved residues Ser-924 and Thr-1518. Mutation of these sites renders Tsc2 insensitive to Akt/PKB signalling, making the Tsc1–Tsc2 complex more stable within the cell. Increasing Akt/PKB signalling in vivo leads to a marked increase in cell growth and size, disrupts Tsc1/Tsc2 association, and disturbs the distinct subcellular localization of Tsc1 and Tsc2. Furthermore, all Akt/PKB growth signals are blocked by expression of a Tsc2 mutant lacking its Akt phosphorylation sites. Thus Tsc2 appears to be the critical target of Akt in mediating growth for the insulin signalling pathway. Since the Akt, Tsc1 and Tsc2 molecules, as well as the Akt phosphorylation sites in Drosophila Tsc2, are conserved from insects to humans, phosphorylation of Tsc2 by Akt might be a conserved mechanism of growth control. Indeed, recent studies from the Guan [25] and Cantley [26]
laboratories have shown that Akt does phosphorylate TSC2 in mammalian cells. It was suggested previously that Akt and S6K are activated independently and are components of parallel pathways [27]. Our genetic epistasis experiments showed previously that Akt and S6K are in the same pathway and Akt acts upstream of Tsc1/Tsc2 while S6K acts downstream of Tsc1/Tsc2 [8]. Consistent with this pathway epistasis result, we have shown that expression of either fly or human S6K can suppress the effect of overexpression Tsc1 and Tsc2, including overexpression of Tsc2 with mutated Aktphosphorylation sites [24]. Our studies have shown that Drosophila Tsc1 and Tsc2 function in the insulin/PI3K/PTEN/Akt pathway. We have shown that Akt regulates the Tsc1–Tsc2 complex by directly phosphorylating Tsc2 and these Akt-phosphorylation sites are conserved in all TSC2 proteins including human TSC2. We have further shown that S6K is a downstream component of the PI3K/PTEN/Akt/TSC pathway and reduction of S6K activity can block TSC defects. Recent studies from multiple laboratories have now confirmed our findings in mice, rats and human patients, and have shown that drugs antagonizing S6K activities, such as rapamycin, diminish tumours in TSC-deficient mice and rats. Given that other components of the pathway, such as PTEN, are also mutated in large numbers of cancer patients and that TSC1 and TSC2 negatively regulate intracellular insulin signalling, therapeutics developed based on the knowledge of the pathway information could have effects beyond the TSC patient population. We propose that mechanisms of organ size control are critical targets for diseases such as tumorigenesis, which requires an increase in tissue size and total mass, and for evolutionary events that alter sizes of organisms. The studies of size-control mechanisms are important for our understanding of cancer biology and for developing potential therapeutic agents.
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Received 24 January 2003
