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The role of lats in cell cycle regulation and tumorigenesis
Biochimica et Biophysica Acta 1424 (1999) M9^M16 www.elsevier.com/locate/bba
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The role of lats in cell cycle regulation and tumorigenesis Gregory S. Turenchalk a
a;b
, Maie A.R. St. John
a;c
, Wufan Tao
a;b
, Tian Xu
a;b;
*
Howard Hughes Medical Institute, Yale University School of Medicine, Boyer Center for Molecular Medicine, 295 Congress Avenue, New Haven, CT 06536-0812, USA b Department of Genetics, Yale University School of Medicine, Boyer Center for Molecular Medicine, 295 Congress Avenue, New Haven, CT 06536-0812, USA c Cell Biology, Yale University School of Medicine, Boyer Center for Molecular Medicine, 295 Congress Avenue, New Haven, CT 06536-0812, USA Received 1 March 1999; accepted 28 June 1999
Keywords: lats; Kinase; Tumor suppressor; Cancer; Cell cycle; Cyclin-dependent protein kinase inhibitor; Cyclin-dependent protein kinase; Cyclin; Drosophila
Contents 1.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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2.
Tumor suppressors and the cell cycle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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3.
The identi¢cation of Lats . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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4.
lats mutant cells proliferate faster than wild-type cells . . . . . . . . . . . . . . . . . . . . . . . . . . .
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5.
Lats is involved in a cell-cell communication mechanism . . . . . . . . . . . . . . . . . . . . . . . . .
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6.
Human Lats1, a functional homolog of the lats gene . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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7.
Human lats: a novel regulator of the cell cycle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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8.
Lats is a tumor suppressor in mammals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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9.
Understanding the lats mutant phenotype in light of the biochemical function of lats . . . .
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10. A model for the Lats function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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* Corresponding author. Tel.: +1 (203) 737-2623; Fax: +1 (203) 737-2630; E-mail : [email protected] 0304-419X / 99 / $ ^ see front matter ß 1999 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 4 1 9 X ( 9 9 ) 0 0 0 2 1 - 9
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1. Introduction The study of tumor suppressors and their role in carcinogenesis requires an understanding of the intricacies of the cell cycle. Mechanisms controlling the precise timing and sequence of cell cycle events as well as checkpoints insuring the ¢delity of those events are the key targets that can be disrupted to result in tumorigenesis. Transitions between each stage of the cell cycle are controlled by the activity of cyclin-dependent protein kinases (CDKs) [1,2]. Positive regulatory subunits, the cyclins, are required for activity of CDKs and it is the synthesis and destruction of these cyclins that drive the cell cycle. In addition to regulation by cyclin subunits, the CDKs are also positively and negatively regulated by reversible phosphorylation reactions and by the binding of CDK inhibitors (CDIs), which play a key role in the maintenance of cell cycle checkpoints [3,4]. The large tumor suppressor gene (lats) originally discovered in Drosophila [5,6] has now shown to be a tumor suppressor in mammals [7] and behaves as a new type of negative regulator of CDKs. 2. Tumor suppressors and the cell cycle Human tumor suppressors often function as negative regulators of the cell cycle. Mutations in tumor suppressor genes can lead to failure of cell cycle checkpoint controls, resulting in the accumulation of genetic changes contributing to a tumor phenotype [8]. Several tumor suppressors are known to modulate the activities of CDK/cyclin complexes either directly or indirectly. For example, p53 activates the transcription of the p21 (p21WAF1/CIP1) CDK inhibitor in response to DNA damage signals and p21 in turn binds and inactivates the CDK4,6/cyclin D complexes [9]. p16, another CDI, is a potent tumor suppressor that acts directly on the CDK4/cyclin D complex [10]. 3. The identi¢cation of Lats Many components of the cell cycle machinery in Drosophila are similar to those found in vertebrates
[11,12]. Fly homologs such as Rb and p21 have been identi¢ed and shown to be functionally conserved [13^15]. This strong evolutionary conservation of cell cycle components along with the strength of the fruit £y as a genetic research organism makes Drosophila an attractive model system for the study of cancer. Previous Drosophila screens required dissection of dead larvae and pupae to reveal over-proliferated tissues [16,17]. Mosaic screens in Drosophila which are designed to mimic the genetic make-up of cancer patients have allowed the isolation of overproliferation mutations which are lethal at earlier developmental stages [5,18]. One of the genes identi¢ed in the screen is the lats gene [5]. The latsX1 mutation was genetically mapped to the 100A1-5 polytene chromosome region and the lats locus was further de¢ned by a single complementation group of over 50 alleles. Somatic cells mutant for lats over-proliferate dramatically to form large tumors in a wide variety of tissues in mosaic adults (Fig. 1A). The degree of over-proliferation in clones varies among the lats alleles with the latsX1 mutation showing the most dramatic phenotype with clones as large as 1/5 of the body size. Homozygous mutants for the various lats alleles display a wide range of developmental defects. Strong alleles cause homozygous animals to exhibit embryonic lethality. Moderate alleles exhibit lethality in a range of larval and pupal stages and a subgroup of the moderate pupal lethal alleles causes giant animal phenotypes with dramatically over-proliferated neural and epithelial tissues. Weak alleles cause a variety of phenotypes including held out wings with broadened blades, rough eyes with ventral outgrowths and outgrowths on the dorsal-anterior region of the head. The lats transcriptional unit resides in a complicated genomic region where it is located within the intron of another gene and is £anked by transcripts from two other genes. Rescue experiments were required to de¢nitively determine that lats is responsible for the observed phenotypes. Expression of the lats cDNA under the control of the heat shockinducible promoter rescues the lethality of lats alleles and suppresses tumor formation in lats mosaic £ies [5]. lats encodes a putative Ser/Thr kinase with a large N-terminal domain (also known as wts, [5,6]).
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Fig. 1. Lats has a tumor suppressor function in £ies and mice. (A) This adult £y contains a clone of lats mutant cells which have over-proliferated to form a large tumor outgrowth. (B) This homozygous lats1 knockout mouse also developed a tumor in the form of a soft tissue sarcoma.
4. lats mutant cells proliferate faster than wild-type cells Sectioning of lats tumors in mosaic £ies revealed unpatterned over-proliferated tissues with many lobes and folds and the size and shape of the mutant cells are heterogeneous and irregular [5]. The behavior of lats mutant clones was in contrast to that of clones mutant for other Drosophila tumor supressors such as l(1)discs large-1 (dlg, [19,20]) and l(2)giant larvae (lgl, [21,22]). The mutant cells in these tumor suppressors proliferate slower than wild-type cells. Mutant clones for these genes induced during the ¢rst instar larval stage are competed away during growth and do not form detectable clones in the adults. lats mutant clones induced at similar developmental stages form dramatically over-proliferated
tissues in adults. This suggests that lats mutant cells proliferate faster than wild-type cells. Cells in overproliferated mutant clones on the body are capable of di¡erentiation and produce bristles and hairs, although the morphologies of these structures are not entirely wild-type. Careful examination of numerous marked mutant clones veri¢es that only lats mutant cells are over-proliferating and that wild-type cells are una¡ected. Thus, the lats over-proliferation phenotype is not caused by prevention of di¡erentiation and the phenotype is cell autonomous [5]. 5. Lats is involved in a cell-cell communication mechanism The lats mutant phenotypes indicate a role for lats
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in the size control mechanism that regulates cell proliferation. It has been shown that proliferating cells in a developing Drosophila imaginal disc communicate to maintain a constant disc size. Imaginal discs can undergo regeneration to form a normal-sized disc when a small region of the disc is surgically removed [23,24]. Consistent with the notion that proliferation is regulated by local cell-cell interaction, it has been shown that DNA replication and mitosis in growing discs occurs in small, non-clonal cell clusters throughout the disc [25^27]. The size control mechanism is an intrinsic property of the cells in each disc as demonstrated by transplantation experiments that show that young discs transplanted into adult hosts grow until they reach their normal size [28]. Such size control mechanisms also exist in invertebrates [23,24,29]. Mutations in lats dramatically disrupt the size and morphology of Drosophila imaginal discs. Mutant cells in lats mosaic disc clones can over-proliferate to form massive outgrowths that are sometimes larger than the size of a normal mature disc and £ies homozygous for many lats alleles have an enlarged body size and dramatically overgrown discs. This is in stark contrast to mosaic experiments with mutations in other genes that e¡ect cell division. Disc's mosaic for mutations in minute or dE2F contain cells of di¡erent genotypes and the number of progeny cells from a given parental cell can vary dramatically in a mature disc, but the overall size of the mature disc is una¡ected [30^32]. These data show that alterations in cell proliferation are not always su¤cient to disrupt the size control mechanism. Furthermore, the fact that in a minute mosaic disc one group of cells is over-proliferated while the rest are under-proliferated to achieve a constant disc size indicates that a cellcell communication component is involved in the size control mechanism. The cell autonomous over-proliferation phenotype of lats mutant cells suggests that they are defective in receiving signals to inhibit cell proliferation sent by neighboring wild-type cells. 6. Human Lats1, a functional homolog of the lats gene We have isolated a human homolog of the Drosophila lats gene (human Lats1). Expression of human
Lats1 cDNA under the control of the heat shockinducible promoter completely suppresses tumor formation in lats mosaic £ies and Lats1 rescues all of the developmental defects in homozygous lats mutants including embryonic lethality [33]. This demonstrates that human Lats1 is an authentic homolog of the Drosophila lats tumor suppressor. The functional conservation between both molecules provides the opportunity to use the strengths of each experimental organism to explore the biochemical and genetic properties of the Lats protein. We have also identi¢ed a mouse lats homolog (Lats1) [7] along with additional lats-like molecules in Caenorhabditis elegans (ce-Lats-l) and Saccharomyces cerevisiae (sc-Lats-l). In addition to highly conserved C-terminal kinase domains, these proteins also share homologous N-terminal regions. This suggests that Lats molecules may function in all eukaryotic organisms. Another group of previously identi¢ed putative kinases (e.g. Dbf2 and Dbf20 from budding yeast; Ndr, nuclear Dbf2-related proteins from humans, £ies and nematodes) share high levels of sequence similarity with Lats proteins (e.g. 44% identity between Lats1 and h-Ndr [34^36]) but lack sequences corresponding to the Lats N-terminal region indicating that they are not genuine homologs of Lats. Indeed, neither £y Ndr nor C-terminal Lats alone can rescue lats mutants (S.Z., W.C. and T.X., unpublished results). It has previously also been reported that lats is a £y homolog of the human myotonic dystrophy kinase [6,37], but the myotonic dystrophy kinase lacks sequences corresponding to the Lats Nterminal region and its kinase domain also lacks unique sequences which are present within the Lats and Dbf kinases [5]. Thus, it is highly unlikely that it is a human homolog of Drosophila Lats. 7. Human lats: a novel regulator of the cell cycle Biochemical investigation of the human Lats1 protein using immunoprecipitation from Hela cells [33] reveals that Lats1 is phosphorylated in a cell cycledependent manner. The Lats1 protein exists in a phosphorylated form during the late prophase and remains phosphorylated during the metaphase. Dephosphorylated Lats1 becomes detectable during en-
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trance into the anaphase and by the beginning of telophase, most of the Lats1 protein is dephosphorylated. Lats1 remains dephosphorylated during the G1, S, G2 and G0 phases. Further immunoprecipitation experiments were used to investigate whether Lats1 is directly involved in the regulation of the cell cycle. Cdc2 co-immunoprecipitates with Lats1 in mitotic cells. The amount of Cdc2 co-precipitated was most abundant during early mitosis (equivalent to about 25% of the amount of Cdc2 complexed with cyclinB), but the amount progressively decreases in subsequent stages of the cell cycle and is absent in quiescent cells. The variation in co-precipitated Cdc2 levels is not due to £uctuations in overall Cdc2 levels during the cell cycle, since the total Cdc2 levels remain at a near constant level in cycling cells [38]. This suggests that Lats1 preferentially associates with Cdc2 during early mitosis, which is a period when Lats1 is in its phosphorylated form. Experiments utilizing the baculovirus expression system revealed that Cdc2 and Lats1 proteins could be coimmunoprecipitated using either anti-human Lats1 or anti-Cdc2 antibodies, suggesting that the in vivo Lats1/Cdc2 complex may result from direct binding of the two proteins. The yeast two-hybrid assay [39,40] was used to examine the binding between Lats1 and Cdc2 and the results corroborated the co-immunoprecipitation data. Full length Lats1 and the N-terminal region of Lats1 interacted with Cdc2 in the assay. The C-terminal kinase domain, however, did not interact with Cdc2, indicating that Lats1 associates with Cdc2 through its N-terminal domain. Neither full length Lats1 nor the N-terminal region of Lats1 revealed any interaction with the two G1 cell cycle kinases, CDK2 and CDK4, indicating that the association between Lats1 and Cdc2 is speci¢c. The speci¢c association of Lats1 and Cdc2 suggests that Lats1 may act as a tumor suppressor through negative regulation of Cdc2 activity. Histone H1 kinase assays using immunoprecipitated Lats1/Cdc2 and Cdc2/cyclin B complexes show that in contrast to the Cdc2/cyclin B complex, the Lats1/Cdc2 complex exhibits no detectable kinase activity for histone H1. Neither cyclin A nor cyclin B proteins could be detected in the Lats1/Cdc2 immunocomplex when tested using anti-cyclin A and B antibodies, which indicates that Lats1 may modulate Cdc2 activity in
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a di¡erent manner than that of most known CDIs [41,42]. This biochemical data were further supported by genetic interactions between lats, cdc2 and cyclin A, in Drosophila. Reducing the dose of cdc2 resulted in suppression of the lats late pupal lethal alleles leading to a rescue of the lethality and other morphological phenotypes. Reduction of cdc2 activity is also able to suppress the over-proliferation phenotypes of moderate lats alleles. In agreement with the results of the yeast two-hybrid assay for human Lats1 and CDK2, reduction of Cdc2c, the Drosophila homolog of CDK2 [43,44], does not modify the lats mutant phenotypes. cyclin A and cyclin B were tested in a similar manner and while cyclin B failed to show a genetic interaction with lats, cyclin A was able to suppress the lats mutant phenotype in a manner similar to cdc2. The speci¢c genetic interactions between lats, cdc2 and cyclin A support the biochemical data suggesting that Lats regulates cell proliferation by negatively regulating Cdc2/cyclin A activity. 8. Lats is a tumor suppressor in mammals To investigate the function of Lats in mammals, we generated Lats1 knockout mice (Lats13=3 ). These Lats13=3 mice exhibit several developmental and hormonal defects including a lack of mammary gland development, infertility and growth retardation, but more importantly, these mice develop soft tissue sarcomas (Fig. 1B) and ovarian stromal cell tumors and are highly susceptible to carcinogenic treatments [7]. 9. Understanding the lats mutant phenotype in light of the biochemical function of lats The role of Lats as a negative regulator of Cdc2/ cyclin A is consistent with the Drosophila Lats phenotype as a tumor suppressor. Ectopic activation of Cdc2/cyclin A in G1-arrested cells by over-expression of cyclin A can drive the G1/S transition in cells that lack cyclin E [45,46] and this G1/S activity is greatly enhanced when both cyclin A and an activated form of Cdc2 are over-expressed. Evidence from two other Drosophila mutants, roughex (rux) and ¢zzy-related (fzr), support the notion that increased Cdc2/cyclin
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A activity in Drosophila can result in over-proliferation. rux mutants cause cells to accumulate cyclin A in early G1 and the cells progress into the S phase precociously [47,48]. Loss of fzr, a Cdc20-related gene, results in the accumulation of mitotic cyclins in G1 cells and drives progression through an extra division cycle in the embryonic epidermis [49]. Consistent with these data, cyclin A has shown to accumulate abnormally in lats mutant cells and lats mutant phenotypes can be suppressed by cdc2 and cyclin A mutations [33]. The lats phenotype is unique in several respects. lats mutants deregulate Cdc2/cyclin A activity which impacts both the G1/S and G2/M checkpoints. This deregulation of Cdc2/cyclin A by lats mutants appears to be speci¢c, whereas other mutations such as rux, ¢zzy (fzy) and fzr accumulate multiple mitotic cyclins which a¡ect the activities of several Cdc2/cyclin complexes [47,49,50]. The cyclin A in lats mutant cells is degraded during late mitosis, which indicates that many aspects of the cell cycle are still normal in lats mutants. Lats a¡ects a speci¢c CDK/cyclin complex and the particular CDK/cyclin complex that Lats abnormally activates participates at two di¡erent cell cycle stages as opposed to other mutations that either activate a CDK/cyclin complex during a single cell cycle stage or throughout the entire cell cycle. The combination of these properties
provides an explanation for the severity of the overproliferation phenotype in Drosophila lats mutants. Although inactivation of lats causes tumors in both Drosophila and mice, the correlation between genotype and phenotype varies. Every lats3=3 cell over-proliferates in mosaic £ies [5] while only restricted tissues in Lats3=3 mice develop tumors [7]. There is an important di¡erence between the cell cycle in mammals and in Drosophila. In mammals, the Cdc2/cyclin A complex is involved in the regulation of G2/M [8,51], but in Drosophila, Cdc2/cyclin A functions at G1/S in addition to its function at G2/ M. In mammals, Cdc2 is not responsible for G1/S regulation and this function is instead provided by a di¡erent CDK, CDK2, which complexes with cyclin A for its G1/S activity [8]. It is likely that such complexities and redundancies of the mammalian genome are responsible for the phenotypic di¡erences between mice and £ies. 10. A model for the Lats function Although it has not yet been demonstrated that Lats is an active kinase, an additional function for the Lats C-terminal kinase domain has been suggested by yeast two-hybrid experiments which have
Fig. 2. A model for the Lats function. Phosphorylation of Lats at early mitosis could change its conformation to free the Lats N-terminal region from the Lats C-terminal kinase domain. This could then allow the Lats N-terminal region to bind to Cdc2, which leads to inactivation of the mitotic kinase activity of Cdc2. Cdc2/cyclin A is involved in the regulation of G2/M in both £ies and mammals, but the Cdc2/cyclin A complex is only involved in the regulation of G1/S in the £y.
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shown that the N-terminal region of Lats1 interacts much stronger with Cdc2 than full length Lats1 does. This inmplies that the C-terminal kinase domain of Lats1 has a negative e¡ect on the binding between the Lats1 N-terminal region and Cdc2. One possible explanation for this phenomenon is that the Lats1 Cterminal kinase region exerts this e¡ect by binding intramolecularly to its own N-terminal region. This explanation is supported by the positive interaction between the Lats1 C-terminal kinase domain and the N-terminal region of Lats1 in the two-hybrid assay. Since the phosphorylation of Lats1 seems to be a prerequisite for binding to Cdc2, an interesting model for the Lats1 function can be proposed. Phosphorylation of Lats1 may change its conformation and disrupt the intramolecular association between the N-terminus and C-terminus of Lats1, freeing the Nterminal domain of Lats1 for Cdc2 binding. If this model (Fig. 2) is correct, it will be necessary to identify any protein kinases and phosphatases which modulate the phosphorylation state of Lats1 during the cell cycle to gain a full understanding of the regulation of Lats1 activity. In both £ies and mammals, cyclin A and cyclin B are degraded over di¡erent time courses during mitosis [52,53], although the precise mechanism of such di¡erential inactivation of Cdc2/cyclins is unknown. Our biochemical and genetic data indicate that Lats speci¢cally modulates Cdc2/cyclin A activity, but not Cdc2/cyclin B activity. cyclin A but not cyclin B interacts with lats genetically and lats mutant cells abnormally accumulate cyclin A but not cyclin B ([33]). Lats may, therefore, be an important player in the di¡erential inactivation of Cdc2/cyclin complexes during mitosis. Yeast two-hybrid assays demonstrate that Lats speci¢cally interacts with Cdc2 but not other CDKs and this ¢nding is supported by genetic data in Drosophila which show that Lats interacts with cdc2 but not the £y CDK2 homolog, cdc2c. Given that p16and p21-like CDK inhibitors have not been found for Cdc2, it is possible that Cdc2 and the rest of the CDKs are negatively regulated by di¡erent families of proteins. Alternatively, the activity of each CDK could be modulated by both types of negative regulators. Over-expression of Cdc2 and cyclin A has been reported in multiple types of human tumors [54^56]
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and negative regulators of CDK/cyclins (e.g. p16) have shown to be tumor suppressors in mammals [57]. Our ¢nding that Lats negatively regulates the Cdc2/cyclin A complex and the fact that Lats1 behaves as a tumor suppressor in the mouse suggest that inactivation of Lats1 will be involved in tumor development in humans as well. This demonstrates that studies using Drosophila as a model organism can produce results that are directly relevant to human cancer despite the vast evolutionary distance between £ies and humans. We are currently using Drosophila genetics to identify genes in the lats pathway. Given the conservation of the lats gene, many components of the lats pathway are likely to be conserved from £ies to humans as well. Characterization of these lats interacting genes will not only provide mechanistic insight into how lats functions, but it will also provide candidate genes that may be involved in human tumorigenesis. Acknowledgements We thank N.A. Theodosiou and other members of the Xu lab for comments and K. Sepanek for assistance. G.S.T is a NSF graduate fellow. M.A.R.S.J. is a MSTP student. W.T. was an Anna Fuller fellow. This work was supported in part by the National Cancer Institute and Grants from NIH (R01CA69408). References [1] [2] [3] [4] [5] [6] [7]
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The role of lats in cell cycle regulation and tumorigenesis Gregory S. Turenchalk a
a;b
, Maie A.R. St. John
a;c
, Wufan Tao
a;b
, Tian Xu
a;b;
*
Howard Hughes Medical Institute, Yale University School of Medicine, Boyer Center for Molecular Medicine, 295 Congress Avenue, New Haven, CT 06536-0812, USA b Department of Genetics, Yale University School of Medicine, Boyer Center for Molecular Medicine, 295 Congress Avenue, New Haven, CT 06536-0812, USA c Cell Biology, Yale University School of Medicine, Boyer Center for Molecular Medicine, 295 Congress Avenue, New Haven, CT 06536-0812, USA Received 1 March 1999; accepted 28 June 1999
Keywords: lats; Kinase; Tumor suppressor; Cancer; Cell cycle; Cyclin-dependent protein kinase inhibitor; Cyclin-dependent protein kinase; Cyclin; Drosophila
Contents 1.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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2.
Tumor suppressors and the cell cycle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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3.
The identi¢cation of Lats . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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4.
lats mutant cells proliferate faster than wild-type cells . . . . . . . . . . . . . . . . . . . . . . . . . . .
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5.
Lats is involved in a cell-cell communication mechanism . . . . . . . . . . . . . . . . . . . . . . . . .
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6.
Human Lats1, a functional homolog of the lats gene . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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7.
Human lats: a novel regulator of the cell cycle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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8.
Lats is a tumor suppressor in mammals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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9.
Understanding the lats mutant phenotype in light of the biochemical function of lats . . . .
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10. A model for the Lats function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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* Corresponding author. Tel.: +1 (203) 737-2623; Fax: +1 (203) 737-2630; E-mail : [email protected] 0304-419X / 99 / $ ^ see front matter ß 1999 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 4 1 9 X ( 9 9 ) 0 0 0 2 1 - 9
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1. Introduction The study of tumor suppressors and their role in carcinogenesis requires an understanding of the intricacies of the cell cycle. Mechanisms controlling the precise timing and sequence of cell cycle events as well as checkpoints insuring the ¢delity of those events are the key targets that can be disrupted to result in tumorigenesis. Transitions between each stage of the cell cycle are controlled by the activity of cyclin-dependent protein kinases (CDKs) [1,2]. Positive regulatory subunits, the cyclins, are required for activity of CDKs and it is the synthesis and destruction of these cyclins that drive the cell cycle. In addition to regulation by cyclin subunits, the CDKs are also positively and negatively regulated by reversible phosphorylation reactions and by the binding of CDK inhibitors (CDIs), which play a key role in the maintenance of cell cycle checkpoints [3,4]. The large tumor suppressor gene (lats) originally discovered in Drosophila [5,6] has now shown to be a tumor suppressor in mammals [7] and behaves as a new type of negative regulator of CDKs. 2. Tumor suppressors and the cell cycle Human tumor suppressors often function as negative regulators of the cell cycle. Mutations in tumor suppressor genes can lead to failure of cell cycle checkpoint controls, resulting in the accumulation of genetic changes contributing to a tumor phenotype [8]. Several tumor suppressors are known to modulate the activities of CDK/cyclin complexes either directly or indirectly. For example, p53 activates the transcription of the p21 (p21WAF1/CIP1) CDK inhibitor in response to DNA damage signals and p21 in turn binds and inactivates the CDK4,6/cyclin D complexes [9]. p16, another CDI, is a potent tumor suppressor that acts directly on the CDK4/cyclin D complex [10]. 3. The identi¢cation of Lats Many components of the cell cycle machinery in Drosophila are similar to those found in vertebrates
[11,12]. Fly homologs such as Rb and p21 have been identi¢ed and shown to be functionally conserved [13^15]. This strong evolutionary conservation of cell cycle components along with the strength of the fruit £y as a genetic research organism makes Drosophila an attractive model system for the study of cancer. Previous Drosophila screens required dissection of dead larvae and pupae to reveal over-proliferated tissues [16,17]. Mosaic screens in Drosophila which are designed to mimic the genetic make-up of cancer patients have allowed the isolation of overproliferation mutations which are lethal at earlier developmental stages [5,18]. One of the genes identi¢ed in the screen is the lats gene [5]. The latsX1 mutation was genetically mapped to the 100A1-5 polytene chromosome region and the lats locus was further de¢ned by a single complementation group of over 50 alleles. Somatic cells mutant for lats over-proliferate dramatically to form large tumors in a wide variety of tissues in mosaic adults (Fig. 1A). The degree of over-proliferation in clones varies among the lats alleles with the latsX1 mutation showing the most dramatic phenotype with clones as large as 1/5 of the body size. Homozygous mutants for the various lats alleles display a wide range of developmental defects. Strong alleles cause homozygous animals to exhibit embryonic lethality. Moderate alleles exhibit lethality in a range of larval and pupal stages and a subgroup of the moderate pupal lethal alleles causes giant animal phenotypes with dramatically over-proliferated neural and epithelial tissues. Weak alleles cause a variety of phenotypes including held out wings with broadened blades, rough eyes with ventral outgrowths and outgrowths on the dorsal-anterior region of the head. The lats transcriptional unit resides in a complicated genomic region where it is located within the intron of another gene and is £anked by transcripts from two other genes. Rescue experiments were required to de¢nitively determine that lats is responsible for the observed phenotypes. Expression of the lats cDNA under the control of the heat shockinducible promoter rescues the lethality of lats alleles and suppresses tumor formation in lats mosaic £ies [5]. lats encodes a putative Ser/Thr kinase with a large N-terminal domain (also known as wts, [5,6]).
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Fig. 1. Lats has a tumor suppressor function in £ies and mice. (A) This adult £y contains a clone of lats mutant cells which have over-proliferated to form a large tumor outgrowth. (B) This homozygous lats1 knockout mouse also developed a tumor in the form of a soft tissue sarcoma.
4. lats mutant cells proliferate faster than wild-type cells Sectioning of lats tumors in mosaic £ies revealed unpatterned over-proliferated tissues with many lobes and folds and the size and shape of the mutant cells are heterogeneous and irregular [5]. The behavior of lats mutant clones was in contrast to that of clones mutant for other Drosophila tumor supressors such as l(1)discs large-1 (dlg, [19,20]) and l(2)giant larvae (lgl, [21,22]). The mutant cells in these tumor suppressors proliferate slower than wild-type cells. Mutant clones for these genes induced during the ¢rst instar larval stage are competed away during growth and do not form detectable clones in the adults. lats mutant clones induced at similar developmental stages form dramatically over-proliferated
tissues in adults. This suggests that lats mutant cells proliferate faster than wild-type cells. Cells in overproliferated mutant clones on the body are capable of di¡erentiation and produce bristles and hairs, although the morphologies of these structures are not entirely wild-type. Careful examination of numerous marked mutant clones veri¢es that only lats mutant cells are over-proliferating and that wild-type cells are una¡ected. Thus, the lats over-proliferation phenotype is not caused by prevention of di¡erentiation and the phenotype is cell autonomous [5]. 5. Lats is involved in a cell-cell communication mechanism The lats mutant phenotypes indicate a role for lats
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in the size control mechanism that regulates cell proliferation. It has been shown that proliferating cells in a developing Drosophila imaginal disc communicate to maintain a constant disc size. Imaginal discs can undergo regeneration to form a normal-sized disc when a small region of the disc is surgically removed [23,24]. Consistent with the notion that proliferation is regulated by local cell-cell interaction, it has been shown that DNA replication and mitosis in growing discs occurs in small, non-clonal cell clusters throughout the disc [25^27]. The size control mechanism is an intrinsic property of the cells in each disc as demonstrated by transplantation experiments that show that young discs transplanted into adult hosts grow until they reach their normal size [28]. Such size control mechanisms also exist in invertebrates [23,24,29]. Mutations in lats dramatically disrupt the size and morphology of Drosophila imaginal discs. Mutant cells in lats mosaic disc clones can over-proliferate to form massive outgrowths that are sometimes larger than the size of a normal mature disc and £ies homozygous for many lats alleles have an enlarged body size and dramatically overgrown discs. This is in stark contrast to mosaic experiments with mutations in other genes that e¡ect cell division. Disc's mosaic for mutations in minute or dE2F contain cells of di¡erent genotypes and the number of progeny cells from a given parental cell can vary dramatically in a mature disc, but the overall size of the mature disc is una¡ected [30^32]. These data show that alterations in cell proliferation are not always su¤cient to disrupt the size control mechanism. Furthermore, the fact that in a minute mosaic disc one group of cells is over-proliferated while the rest are under-proliferated to achieve a constant disc size indicates that a cellcell communication component is involved in the size control mechanism. The cell autonomous over-proliferation phenotype of lats mutant cells suggests that they are defective in receiving signals to inhibit cell proliferation sent by neighboring wild-type cells. 6. Human Lats1, a functional homolog of the lats gene We have isolated a human homolog of the Drosophila lats gene (human Lats1). Expression of human
Lats1 cDNA under the control of the heat shockinducible promoter completely suppresses tumor formation in lats mosaic £ies and Lats1 rescues all of the developmental defects in homozygous lats mutants including embryonic lethality [33]. This demonstrates that human Lats1 is an authentic homolog of the Drosophila lats tumor suppressor. The functional conservation between both molecules provides the opportunity to use the strengths of each experimental organism to explore the biochemical and genetic properties of the Lats protein. We have also identi¢ed a mouse lats homolog (Lats1) [7] along with additional lats-like molecules in Caenorhabditis elegans (ce-Lats-l) and Saccharomyces cerevisiae (sc-Lats-l). In addition to highly conserved C-terminal kinase domains, these proteins also share homologous N-terminal regions. This suggests that Lats molecules may function in all eukaryotic organisms. Another group of previously identi¢ed putative kinases (e.g. Dbf2 and Dbf20 from budding yeast; Ndr, nuclear Dbf2-related proteins from humans, £ies and nematodes) share high levels of sequence similarity with Lats proteins (e.g. 44% identity between Lats1 and h-Ndr [34^36]) but lack sequences corresponding to the Lats N-terminal region indicating that they are not genuine homologs of Lats. Indeed, neither £y Ndr nor C-terminal Lats alone can rescue lats mutants (S.Z., W.C. and T.X., unpublished results). It has previously also been reported that lats is a £y homolog of the human myotonic dystrophy kinase [6,37], but the myotonic dystrophy kinase lacks sequences corresponding to the Lats Nterminal region and its kinase domain also lacks unique sequences which are present within the Lats and Dbf kinases [5]. Thus, it is highly unlikely that it is a human homolog of Drosophila Lats. 7. Human lats: a novel regulator of the cell cycle Biochemical investigation of the human Lats1 protein using immunoprecipitation from Hela cells [33] reveals that Lats1 is phosphorylated in a cell cycledependent manner. The Lats1 protein exists in a phosphorylated form during the late prophase and remains phosphorylated during the metaphase. Dephosphorylated Lats1 becomes detectable during en-
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trance into the anaphase and by the beginning of telophase, most of the Lats1 protein is dephosphorylated. Lats1 remains dephosphorylated during the G1, S, G2 and G0 phases. Further immunoprecipitation experiments were used to investigate whether Lats1 is directly involved in the regulation of the cell cycle. Cdc2 co-immunoprecipitates with Lats1 in mitotic cells. The amount of Cdc2 co-precipitated was most abundant during early mitosis (equivalent to about 25% of the amount of Cdc2 complexed with cyclinB), but the amount progressively decreases in subsequent stages of the cell cycle and is absent in quiescent cells. The variation in co-precipitated Cdc2 levels is not due to £uctuations in overall Cdc2 levels during the cell cycle, since the total Cdc2 levels remain at a near constant level in cycling cells [38]. This suggests that Lats1 preferentially associates with Cdc2 during early mitosis, which is a period when Lats1 is in its phosphorylated form. Experiments utilizing the baculovirus expression system revealed that Cdc2 and Lats1 proteins could be coimmunoprecipitated using either anti-human Lats1 or anti-Cdc2 antibodies, suggesting that the in vivo Lats1/Cdc2 complex may result from direct binding of the two proteins. The yeast two-hybrid assay [39,40] was used to examine the binding between Lats1 and Cdc2 and the results corroborated the co-immunoprecipitation data. Full length Lats1 and the N-terminal region of Lats1 interacted with Cdc2 in the assay. The C-terminal kinase domain, however, did not interact with Cdc2, indicating that Lats1 associates with Cdc2 through its N-terminal domain. Neither full length Lats1 nor the N-terminal region of Lats1 revealed any interaction with the two G1 cell cycle kinases, CDK2 and CDK4, indicating that the association between Lats1 and Cdc2 is speci¢c. The speci¢c association of Lats1 and Cdc2 suggests that Lats1 may act as a tumor suppressor through negative regulation of Cdc2 activity. Histone H1 kinase assays using immunoprecipitated Lats1/Cdc2 and Cdc2/cyclin B complexes show that in contrast to the Cdc2/cyclin B complex, the Lats1/Cdc2 complex exhibits no detectable kinase activity for histone H1. Neither cyclin A nor cyclin B proteins could be detected in the Lats1/Cdc2 immunocomplex when tested using anti-cyclin A and B antibodies, which indicates that Lats1 may modulate Cdc2 activity in
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a di¡erent manner than that of most known CDIs [41,42]. This biochemical data were further supported by genetic interactions between lats, cdc2 and cyclin A, in Drosophila. Reducing the dose of cdc2 resulted in suppression of the lats late pupal lethal alleles leading to a rescue of the lethality and other morphological phenotypes. Reduction of cdc2 activity is also able to suppress the over-proliferation phenotypes of moderate lats alleles. In agreement with the results of the yeast two-hybrid assay for human Lats1 and CDK2, reduction of Cdc2c, the Drosophila homolog of CDK2 [43,44], does not modify the lats mutant phenotypes. cyclin A and cyclin B were tested in a similar manner and while cyclin B failed to show a genetic interaction with lats, cyclin A was able to suppress the lats mutant phenotype in a manner similar to cdc2. The speci¢c genetic interactions between lats, cdc2 and cyclin A support the biochemical data suggesting that Lats regulates cell proliferation by negatively regulating Cdc2/cyclin A activity. 8. Lats is a tumor suppressor in mammals To investigate the function of Lats in mammals, we generated Lats1 knockout mice (Lats13=3 ). These Lats13=3 mice exhibit several developmental and hormonal defects including a lack of mammary gland development, infertility and growth retardation, but more importantly, these mice develop soft tissue sarcomas (Fig. 1B) and ovarian stromal cell tumors and are highly susceptible to carcinogenic treatments [7]. 9. Understanding the lats mutant phenotype in light of the biochemical function of lats The role of Lats as a negative regulator of Cdc2/ cyclin A is consistent with the Drosophila Lats phenotype as a tumor suppressor. Ectopic activation of Cdc2/cyclin A in G1-arrested cells by over-expression of cyclin A can drive the G1/S transition in cells that lack cyclin E [45,46] and this G1/S activity is greatly enhanced when both cyclin A and an activated form of Cdc2 are over-expressed. Evidence from two other Drosophila mutants, roughex (rux) and ¢zzy-related (fzr), support the notion that increased Cdc2/cyclin
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A activity in Drosophila can result in over-proliferation. rux mutants cause cells to accumulate cyclin A in early G1 and the cells progress into the S phase precociously [47,48]. Loss of fzr, a Cdc20-related gene, results in the accumulation of mitotic cyclins in G1 cells and drives progression through an extra division cycle in the embryonic epidermis [49]. Consistent with these data, cyclin A has shown to accumulate abnormally in lats mutant cells and lats mutant phenotypes can be suppressed by cdc2 and cyclin A mutations [33]. The lats phenotype is unique in several respects. lats mutants deregulate Cdc2/cyclin A activity which impacts both the G1/S and G2/M checkpoints. This deregulation of Cdc2/cyclin A by lats mutants appears to be speci¢c, whereas other mutations such as rux, ¢zzy (fzy) and fzr accumulate multiple mitotic cyclins which a¡ect the activities of several Cdc2/cyclin complexes [47,49,50]. The cyclin A in lats mutant cells is degraded during late mitosis, which indicates that many aspects of the cell cycle are still normal in lats mutants. Lats a¡ects a speci¢c CDK/cyclin complex and the particular CDK/cyclin complex that Lats abnormally activates participates at two di¡erent cell cycle stages as opposed to other mutations that either activate a CDK/cyclin complex during a single cell cycle stage or throughout the entire cell cycle. The combination of these properties
provides an explanation for the severity of the overproliferation phenotype in Drosophila lats mutants. Although inactivation of lats causes tumors in both Drosophila and mice, the correlation between genotype and phenotype varies. Every lats3=3 cell over-proliferates in mosaic £ies [5] while only restricted tissues in Lats3=3 mice develop tumors [7]. There is an important di¡erence between the cell cycle in mammals and in Drosophila. In mammals, the Cdc2/cyclin A complex is involved in the regulation of G2/M [8,51], but in Drosophila, Cdc2/cyclin A functions at G1/S in addition to its function at G2/ M. In mammals, Cdc2 is not responsible for G1/S regulation and this function is instead provided by a di¡erent CDK, CDK2, which complexes with cyclin A for its G1/S activity [8]. It is likely that such complexities and redundancies of the mammalian genome are responsible for the phenotypic di¡erences between mice and £ies. 10. A model for the Lats function Although it has not yet been demonstrated that Lats is an active kinase, an additional function for the Lats C-terminal kinase domain has been suggested by yeast two-hybrid experiments which have
Fig. 2. A model for the Lats function. Phosphorylation of Lats at early mitosis could change its conformation to free the Lats N-terminal region from the Lats C-terminal kinase domain. This could then allow the Lats N-terminal region to bind to Cdc2, which leads to inactivation of the mitotic kinase activity of Cdc2. Cdc2/cyclin A is involved in the regulation of G2/M in both £ies and mammals, but the Cdc2/cyclin A complex is only involved in the regulation of G1/S in the £y.
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shown that the N-terminal region of Lats1 interacts much stronger with Cdc2 than full length Lats1 does. This inmplies that the C-terminal kinase domain of Lats1 has a negative e¡ect on the binding between the Lats1 N-terminal region and Cdc2. One possible explanation for this phenomenon is that the Lats1 Cterminal kinase region exerts this e¡ect by binding intramolecularly to its own N-terminal region. This explanation is supported by the positive interaction between the Lats1 C-terminal kinase domain and the N-terminal region of Lats1 in the two-hybrid assay. Since the phosphorylation of Lats1 seems to be a prerequisite for binding to Cdc2, an interesting model for the Lats1 function can be proposed. Phosphorylation of Lats1 may change its conformation and disrupt the intramolecular association between the N-terminus and C-terminus of Lats1, freeing the Nterminal domain of Lats1 for Cdc2 binding. If this model (Fig. 2) is correct, it will be necessary to identify any protein kinases and phosphatases which modulate the phosphorylation state of Lats1 during the cell cycle to gain a full understanding of the regulation of Lats1 activity. In both £ies and mammals, cyclin A and cyclin B are degraded over di¡erent time courses during mitosis [52,53], although the precise mechanism of such di¡erential inactivation of Cdc2/cyclins is unknown. Our biochemical and genetic data indicate that Lats speci¢cally modulates Cdc2/cyclin A activity, but not Cdc2/cyclin B activity. cyclin A but not cyclin B interacts with lats genetically and lats mutant cells abnormally accumulate cyclin A but not cyclin B ([33]). Lats may, therefore, be an important player in the di¡erential inactivation of Cdc2/cyclin complexes during mitosis. Yeast two-hybrid assays demonstrate that Lats speci¢cally interacts with Cdc2 but not other CDKs and this ¢nding is supported by genetic data in Drosophila which show that Lats interacts with cdc2 but not the £y CDK2 homolog, cdc2c. Given that p16and p21-like CDK inhibitors have not been found for Cdc2, it is possible that Cdc2 and the rest of the CDKs are negatively regulated by di¡erent families of proteins. Alternatively, the activity of each CDK could be modulated by both types of negative regulators. Over-expression of Cdc2 and cyclin A has been reported in multiple types of human tumors [54^56]
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and negative regulators of CDK/cyclins (e.g. p16) have shown to be tumor suppressors in mammals [57]. Our ¢nding that Lats negatively regulates the Cdc2/cyclin A complex and the fact that Lats1 behaves as a tumor suppressor in the mouse suggest that inactivation of Lats1 will be involved in tumor development in humans as well. This demonstrates that studies using Drosophila as a model organism can produce results that are directly relevant to human cancer despite the vast evolutionary distance between £ies and humans. We are currently using Drosophila genetics to identify genes in the lats pathway. Given the conservation of the lats gene, many components of the lats pathway are likely to be conserved from £ies to humans as well. Characterization of these lats interacting genes will not only provide mechanistic insight into how lats functions, but it will also provide candidate genes that may be involved in human tumorigenesis. Acknowledgements We thank N.A. Theodosiou and other members of the Xu lab for comments and K. Sepanek for assistance. G.S.T is a NSF graduate fellow. M.A.R.S.J. is a MSTP student. W.T. was an Anna Fuller fellow. This work was supported in part by the National Cancer Institute and Grants from NIH (R01CA69408). References [1] [2] [3] [4] [5] [6] [7]
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