Tuberous sclerosis gene products in proliferation control

Mutation Research 488 (2001) 233–239

Review

Tuberous sclerosis gene products in proliferation control Markus Hengstschläger a,∗ , David M. Rodman b , Angelina Miloloza a , Elke Hengstschläger-Ottnad a , Margit Rosner a , Marion Kubista a a

Obstetrics and Gynecology, Prenatal Diagnosis and Therapy, University of Vienna, Währinger Gürtel 18-20, A-1090 Vienna, Austria b Cardiovascular Pulmonary Research Laboratory, Center for Genetic Lung Disease, University of Colorado Health Sciences Center, Denver, CO 80262, USA Received 22 January 2001; received in revised form 5 March 2001; accepted 5 March 2001

Abstract Two genes, TSC1 and TSC2, have been shown to be responsible for tuberous sclerosis (TSC). The detection of loss of heterozygosity of TSC1 or TSC2 in hamartomas, the growths characteristically occurring in TSC patients, suggested a tumor suppressor function for their gene products hamartin and tuberin. Studies analyzing ectopically modulated expression of TSC2 in human and rodent cells together with the finding that a homolog of TSC2 regulates the Drosophila cell cycle suggest that TSC is a disease of proliferation/cell cycle control. We discuss this question including very recent data obtained from analyzing mice expressing a modulated TSC2 transgene, and from studying the effects of deregulated TSC1 expression. Elucidation of the cellular functions of these proteins will form the basis of a better understanding of how mutations in these genes cause the disease and for the development of new therapeutic strategies. © 2001 Elsevier Science B.V. All rights reserved. Keywords: Tuberous sclerosis; TSC1; TSC2; Proliferation; Cell cycle

1. Introduction TSC is an autosomal dominant condition affecting about 1 in 6000 individuals. The severity of this disease and its impact on the quality of life can be extremely variable. It is characterized by mental retardation and epilepsy associated with benign cortical tubers (hamartomas) as well as by other lesions, also named hamartomas, affecting kidneys, heart, lung and skin. Primary diagnostic criteria are, e.g. facial angiofibromas, peringual fibromas, calcified ∗ Corresponding author. Tel.: +43-1-40400-7847; fax: +43-1-40400-7848. E-mail address: [email protected] (M. Hengstschläger).

retinal hamartomas, cortical tubers or renal angiomyolipomas. Especially the cortical tubers and the complications from the kidney hamartomas often result in significant morbidity and mortality [1]. Since the cloning and characterization of the two disease-causing genes, TSC1 on chromosome 9q34 [2] and TSC2 on chromosome 16p13.3 [3], a substantial effort has been made to understand the spectrum of mutations in these genes as well as the possible functions of the two resultant gene products, hamartin and tuberin. Linkage analysis suggested that about half of large families were linked to TSC1 and half to TSC2, whereas in sporadic cases more often TSC2 mutations are detected. The mutation spectra of TSC1 and TSC2 are very heterogeneous and published TSC mutations include large deletions/rearrangements for

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TSC2 and insertions, deletions, nonsense, point splicing and mis-sense mutations in both genes. Although TSC is a dominant disorder, mutations in the TSC genes are recessive at the level of the affected cell, demonstrating that their gene products act as tumor suppressors. Loss of heterozygosity can be detected in the hamartomas of TSC patients. So far no other gene defect is known that would mimic the phenotypical effects of TSC1 or TSC2 mutations (reviewed in [4,5]). Data from investigating in vitro and in vivo models of deregulated expression of mammalian TSC2, together with functional analyses of Drosophila TSC2 triggered the discussion whether TSC is a disease of proliferation/cell cycle control. Very recent results from studying a TSC2 transgene in mice and modulated TSC1 expression in cell lines provide further insights into the role of TSC gene products in the regulation of proliferation. Here we present a summary and interpretation of data in the literature on the role of TSC gene products in proliferation control with the aim to get a clearer picture on whether deregulated proliferation/cell cycle control could be the molecular mechanism of the development of this disease.

2. Deregulated expression of tuberin and hamartin affects the regulation of proliferation A common approach to analyze a putative function of tumor suppressor genes in proliferation control is to analyze their ability, when ectopically overexpressed in cells, to inhibit cell proliferation. For TSC2 this experiment was performed for the first time by Orimoto et al. [6] and Jin et al. [7]. The Eker rat is predisposed to the development of multiple neoplasias in the kidney, uterus, and spleen due to a germ-line mutation in the rat homolog of the human TSC2 gene [8,9]. The homozygous Eker mutant condition is lethal in mid-gestation characterized by disrupted neuroepithelial growth and development [10,11] and TSC2 null mice also die at mid-gestation [12,13]. Tetracyclin-dependent conditional overexpression of TSC2 in an Eker-rat derived kidney tumor cell line suppressed its proliferation rate [6]. Jin et al. [7] demonstrated that re-introduction of stably-expressed wild-type TSC2 into two independently-derived renal carcinoma Eker rat cell lines downregulated colony formation capacity, proliferation rate measured by

cell counting and anchorage-independent cell growth. Ectopic expression of high levels of TSC2 in cells derived from Eker rat embryos homozygous for the Eker-mutant TSC2 gene has been shown to increase the proportion of cells in G0/G1 [14]. All the results described above have been obtained by analyzing overexpression of TSC2 in TSC2-deficient cells. So the question remained whether high ectopic TSC2 can negatively regulate proliferation of TSC2-positive cells. To prove that, full-length rat TSC2 has been placed under the control of a regulatable tetracycline promoter and has been introduced into Rat1 fibroblasts stably expressing the tetracycline repressor/VP16 transactivator fusion protein. Conditional overexpression of tuberin suppressed colony formation and cell proliferation [7]. Overexpression of TSC2 in Rat1 fibroblasts and in SKNSH human neuroblastoma cells (both TSC2-positive) triggered an increase in the amount of G0/G1 cells [14,15]. Cell counting experiments analyzing TSC2-positive HeLa cells revealed that proliferation decreases under selection for high ectopically expressed TSC2 levels (Miloloza and Hengstschläger, in preparation). Very recently published data further show that high levels of wild-type TSC2 transfected into NIH-3T3 cells have negative effects on the cellular proliferation rate [16]. Studies of the functional consequences of modulated TSC1 expression are less complete. Very recently, it was demonstrated that TSC1, like TSC2, when overexpressed attenuates proliferation [17,18]. Functional loss of some tumor suppressor molecules can induce quiescent (differentiated) cells to proliferate and thereby these cells become susceptible to the transformation process. Several of these tumor suppressors have activities that are high in G0/G1 cells and become downregulated when cells (re-)enter the cell cycle. Different mechanisms are known to regulate these stage-specific activities, including cell cycle-dependent synthesis and degradation. Such mechanisms very likely do not regulate the functions of tuberin and hamartin, since it has been shown that these proteins are expressed in all phases of the cell cycle [17,19]. A variety of different mechanisms can be hypothesized as cell cycle regulating functions of these proteins and further investigation in this direction are required. Knudson’s “two-hit” hypothesis postulates that a germ-line alteration in a tumor suppressor gene

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inherited from an affected parent, is complemented by a second somatic mutation leading to a complete loss of function of this gene resulting in tumor development [20]. Consistent with a tumor suppressor function for TSC1 and TSC2, loss of heterozygosity (LOH) for these gene loci has been demonstrated in hamartomas of TSC patients (reviewed in [4,5]). It is tempting to speculate that at least in specific cell types loss of functional hamartin or tuberin can induce uncontrolled proliferation. Experimental confirmation of this hypothesis was provided using antisense TSC2 oligonucleotides. Antisense inhibition of TSC2 expression induced quiescent Rat1 fibroblasts to enter the cell cycle, shortened G1 phase of the ongoing cell cycle of Rat1 cells and human SKNSH neuroblastoma cells and suppressed cell cycle withdrawal during serum arrest [19]. In perfect agreement with these observations, analyses of cardiomyocytes from rats homozygous for the Eker-mutation (TSC2-deficient cells) demonstrated that loss of TSC2 inhibits cell-cycle withdrawal [21]. That TSC2-negative cells exhibit an increased proliferation rate compared to their TSC2-positive counterparts has further been shown analyzing fibroblasts homozygous for a TSC2-mutation derived from Eker rat embryos [14] and in ELT3 smooth muscle cells (these cells are described in [22]) derived from an Eker rat uterine leiomyoma (Rodman, in preparation). Furthermore, although disruption of the TSC2 gene results in embryonic death in mice, histological analyses suggest the presence of enhanced ventricular myocardial proliferation in homozygous mutant fetuses [13]. Another approach to analyze the cellular consequences of loss of functional TSC2 has been used by generating TSC2 mutants, with the hope that expression of these modified cDNAs would override the cellular functions of the endogenous gene product. It has been found that whereas transfection of wild-type TSC2 into NIH-3T3 cells had negative effects on the cellular proliferation rate, overexpression of several of the modified cDNAs increased proliferation in vitro. Furthermore, cardiomyocyte DNA synthesis in adult mice carrying one of these modified TSC2 cDNAs was elevated 35-fold during isoproterenol-induced hypertrophy [16]. Evidence that TSC2 plays a role in controlling Drosophila cell cycle was provided by studying the effects of mutations in gigas, a Drosophila homolog of TSC2. Although the

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direct molecular mechanism remains elusive, gigas mutant cells endoreplicate their DNA, suggesting that gigas is required for the decision whether to enter M phase or S phase in Drosophila [23]. So far, experiments investigating the cellular consequences of downregulated TSC1 expression with respect to cell proliferation have not been performed in mammalian cells. The Drosophila homolog of TSC1, rocky, has been described [23]. Recent data show that TSC1 (rocky) and TSC2 (gigas) mutants have the same (or indistinguishable) phenotypes. It is now believed that both mutants induce cell enlargement, not cell cycle arrest, suggesting that TSC1/TSC2 restrict cell size (cell growth) in Drosophila (Tapon, Ito and Hariharan in preparation).

3. The current knowledge about the molecular mechanism of proliferation control in TSC The effects of a tumor suppressor on cell proliferation can either be direct or indirect. Some, such as the retinoblastoma protein, are direct cell cycle regulators and others are part of the regulation of another specific biochemical pathway (e.g. endocytosis), what at the very end triggers effects on cell proliferation. For the TSC gene products it is not clarified at the moment whether they have direct or only indirect effects on the control of cell proliferation (see below). It must be a major aim to elucidate the mechanism through which a tumor gene product affects proliferation. This approach provides an opportunity to elucidate the molecular function of the gene of interest. Taken together, all the data described above leave no doubt that deregulated expression of hamartin or tuberin can have effects visible via loss of proliferation control. However, neither of the two TSC gene products have been demonstrated to have a direct role in cell cycle control. Even the data on Drosophila’s gigas, described above, although demonstrating a clear cell cycle deregulation upon mutation in this TSC2 homolog, do not as yet provide a direct molecular mechanism for this phenotype. From the biochemical functions of hamartin and tuberin postulated in the literature one would currently favor a model, in which the observed proliferation effects are indirect. Tuberin has been shown to function as a GTPase-activating protein for Rap1a, but not for rap2, ras, rho or rac [24]. Since

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Rap1 has been shown to induce DNA synthesis when microinjected into 3T3 cells [25], one could imagine that activation of Rap1 via loss of tuberin is an indirect way to regulate proliferation. It has been reported that tuberin also functions as a GTPase-activating protein for Rab5 and influences endocytosis [26]. Roles for tuberin in regulating transcription [27,28] and neuronal differentiation [15] have also been suggested. Although all of those functions could be responsible for indirect effects on proliferation, further investigations are required to understand their impact on regulation of the development of the uncontrolled growths characteristic for TSC. Hamartin has been shown to bind to ezrin/radixin/moesin proteins, which form crosslinks between cortical actin filaments and the plasma membrane, and to regulate Rho GTPases, the actin-based cytoskeleton and cell adhesion [29]. One could speculate that TSC gene products may also transmit information about changes in cell adhesion to the nucleus, and are probably involved in normal cell death that accompanies cell detachment. In support for this, hamartin is suggested to be a nuclear shuttling protein, which accumulates in the nucleus upon inhibition of nuclear export with leptomycin B (R. Lamb, personal communication). More detailed analyses of the (direct or indirect) effects of altered hamartin or tuberin expression on the mammalian cell cycle machinery revealed that overexpression of both induces the expression of the cyclin-dependent kinase inhibitor p27 [14,17]. Since upregulation of p27 is a well known consequence of decreased cell proliferation [30,31], these findings should so far only be interpreted as a consequence of the effects of TSC gene product on proliferation rather than to be the mechanism. It has been reported that the TSC2-negative immortalized Eker rat cell line EEF8 exhibits more p27 in the cytoplasm than the TSC2-positive immortalized Eker rat cell line EEF4, although both were found to express p27 in the nucleus [14]. No direct correlation between TSC2 expression and p27 delocalization was established, so that these data could be interpreted as a difference between two immortalized cell lines rather than a consequence of TSC2 expression. However, these data heve been confirmed in other cellular systems. Inactivation of tuberin via overexpression of specific “dominant negative” TSC2 mutants induced a more or less complete transition of p27 from the nucleus to

the cytoplasm of serum synchronized 3T3 cells [16]. Furthermore, p27 was shown to be completely cytosolic in TSC2-negative ELT3 cells (cells are described in [22]), whereas p27 was largely nuclear in wild type smooth muscle cells (Rodman, in preparation). A major question is whether the effects of TSC gene products on p27 might depend on the cellular background and cannot be found in every cellular system analyzed, what would be in agreement with the finding that in vivo TSC protein expression is restricted to specific organs and tissues (recently reviewed in [32]). High levels of p27 do not affect cell cycle control equally in TSC2-positive and in TSC2-deficient cells, as demonstrated by analysis of EEF4 and EEF8 cells [14] and ELT3 cells (Rodman, in preparation). Still, additional work in different cellular systems is required to prove that this is directly connected to the function of TSC gene products rather than simply a late consequence of a transformation process occurring upon deregulated TSC gene expression. It is well known that deregulation of p27, including its delocalization, is associated with the development of a wide variety of tumors [33]. On the other hand, delocalization of M-phase cyclins in TSC2-deficient Drosophila embryos [23] suggest that anomalous protein trafficking might indeed underlie the altered cell cycle regulation observed in TSC2-negative cells. Another interesting aspect for a better understanding of the relation of TSC gene products and proliferation control is the intracellular localization of hamartin and tuberin. Probably due to cell type specific differences, to the low abundance or to antibody limitations these proteins have differently been localized in previous investigations [29,34–38]. It was demonstrated that tuberin and hamartin associate physically in vivo [35,36]. All these reports provide evidence for a cytosolic tuberin–hamartin complex. Very recently, nuclear localization of hamartin in pancreatic acinar tissues and some cells in the granular layer of cerebellum was observed [32]. Together with the suggestion that hamartin is a nuclear shuttling protein (R. Lamb, personal communication, see above) this adds an additional potential indirect mechanism through which cell cycle may be affected. Accordingly, additional work is required studying the precise localization of these proteins before one of the two models, indirect or direct involvement of hamartin or tuberin in cellular proliferation control, can be favored.

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4. The role of deregulation of proliferation control in the development of tuberous sclerosis — conclusions and questions for the future All the data summarized above clearly show that TSC gene products, when ectopically overexpressed, share the capacity to downregulate (but not to shut off) proliferation of a variety of human, rat and mouse cells. In addition, data obtained by different experimental approaches in different cellular systems provide strong evidence that at least in specific cell types loss of functional tuberin (or hamartin) can induce uncontrolled proliferation. Accordingly, it would be of great importance to investigate in detail the naturally occurring models for TSC1- or TSC2-negative cells, namely hamartomas proven to harbor LOH. It would be most important to elucidate whether or not all these growths result from cells exhibiting deregulated proliferation. If hamartomas could develop without deregulated proliferation this would implicate that the effects on proliferation cannot be the unique causative mechanism. If proliferation control would be the major mechanism of the development of the disease one could postulate a model in which the activities of wild-type TSC gene products keep cells in the non-proliferating, differentiated status. Homozygous mutations in TSC genes would trigger loss of this gatekeeper activity and would initiate cells to proliferate. This would imply that every natural occurring disease-causing mutation in a TSC gene would affect its property to arrest cells. The proof of the existence of natural-occurring mutations, which do not influence TSC activities on cell cycle control, would ultimately demonstrate that TSC can also develop independently of deregulated cell proliferation. An explanation would be that mutations in TSC genes in the first step lead to deregulation of a so far unknown biochemical cascade. And in a second step then triggers (even probably overlapping) deregulations of different controls, such as for cell migration, differentiation, endocytosis, proliferation depend on the affected cell type and all involved in the development of hamartomas. Anyhow, this would then prove that deregulation of proliferation is not the first step primary mechanism, but rather one of the possible second step effects downstream of a so far unknown biochemical control. Do high levels of tuberin or hamartin downregulate the proliferation rate of only a specific set of cells,

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whereas other cells do not react? Does the loss of TSC gene products in one cell type induce proliferation, whereas in another cell type the presence of these proteins is essential for cell cycling? These questions are of particular interest since in terms of cell type specificity the not identical expression pattern between hamartin and tuberin suggests that these two proteins do not always associate in all organs and tissues and might differently be involved in intracellular cascades of different cells [32,38,39]. Recently, it was demonstrated that tuberin and hamartin associate physically in vivo, suggesting that these two proteins function in the same complex [35,36]. On the other hand, it has been demonstrated that hamartin and tuberin exist in co-localizing and independent forms in endogenous expressions. These results indicate that tuberin and hamartin, besides having shared functions, may also possess unique functions in different cell compartments [32,38]. Can TSC1 arrest TSC2-negative cells and vice versa? These and similar questions need to be answered. In Drosophila, mutated TSC2 (gigas) has been shown to induce endoreplication [23]. Until now it has not been investigated whether endoreplication occurs upon loss of TSC2 in mammalian cells. Tumor suppressors often mediate their cell cycle effects by inducing quiescent cells to proliferate via deregulating the biochemical cascade responsible for the transition from G0/G1 phase to S phase. However, until now a potential role of hamartin or tuberin has not been mapped to any phase of the normal mammalian cell cycle. Overexpression of these molecules increases the amount of G0/G1 cells [14,17] but at least hamartin appears to also affect the proportion of cells in G2/M phase to some extent [17]. Overexpression to superphysiologic levels, although widely used to prove the capacity of tumor suppressors to downregulate proliferation, might not be the right approach to answer such questions in detail. Furthermore, is any of the postulated biochemical activities of tuberin or hamartin described above (GAP-activity, binding of ezrin, etc.) necessary for their effects on proliferation control? In conclusion, we believe it to be of great importance to further investigate how hamartin and tuberin affect the control of proliferation in different cell types. Although these effects might occur only indirectly and might even be unrelated to the development of disease, analyzing the causative mechanisms will without doubt provide important insights into the

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functions of the genes responsible for TSC. Recently, it has been suggested that most if not all cancers acquire the same set of functional capabilities during their development, albeit through various mechanistic strategies. Six essential alterations in cell physiology that collectively dictate malignant growth have been suggested: self-sufficiency in growth signals (no normal cell can proliferate in the absence of growth stimulatory signals), insensitivity to antigrowth signals (such as, e.g. TGFß), evasion of programmed cell death (apoptosis), limitless replicative potential (downregulation/loss of the intrinsic, cell-autonomous program that limits multiplication), sustained angiogenesis (in order to progress to a larger size neoplasias must develop angiogenic ability), and tissue invasion and metastasis [40]. TSC hamartomas very rarely progress to malignancy, and therefore, alterations inducing metastasis might probably not occur in these tumors. Today, the knowledge about the development of TSC hamartomas is only rudimentary. It has been shown that downregulation of tuberin expression allows proliferation under serum arrested conditions [19]. To which extent TSC proteins are involved in the regulation of, e.g. apoptosis, telomere maintenance, insensitivity to antigrowth signals or angiogenesis has not been clarified. These aspects can be investigated either by studying the effects of modulated TSC gene expressions in different cells or by analyzing the physiological status of hamartoma cells. Acknowledgements The authors wish to thank R. Lamb and N. Ito for sharing data prior to publication and D.S. Hunter for critically reading the manuscript. We apologize to those whose data have been cited indirectly because of space limitations. Work in the laboratory of M.H. is funded by the Austrian Federal Ministry of Science and Transport (GZ 70.037/2-Pr/4/98). M.K. is supported by the Austrian Ludwig Boltzmann Institute for Clinical and Experimental Oncology. D.M.R. is supported by funding from the LAM Foundation. References [1] M.R. Gomez, J.R. Sampson, V. Holets-Whittemore, Tuberous Slerosis Complex, 3rd Edition, Oxford University Press, New York, 1999.

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