BIOLOGICAL OVERVIEW

So far, relatively few mechanisms have been shown to be capable of regulating both cell proliferation and cell death in a coordinated manner. In a screen for Drosophila mutations that result in tissue overgrowth, the gene salvador (sav) that promotes both cell cycle exit and cell death was identified. Elevated Cyclin E and Thread/DIAP1 levels are found in mutant cells, resulting in delayed cell cycle exit and impaired apoptosis. Salvador contains two WW domains and binds to the Warts protein kinase. The human ortholog of salvador (hWW45) is mutated in several cancer cell lines. Thus, salvador restricts cell numbers in vivo by functioning as a dual regulator of cell proliferation and apoptosis (Tapon, 2002).

To identify genes that restrict cell growth or cell numbers in vivo, a screen was conducted in the Drosophila eye for mutations that increase the relative representation of mutant tissue compared to wild-type tissue. Using FLP/FRT-induced mitotic recombination, clones of mutant tissue (marked white) were compared in size to sister clones of wild-type tissue (marked red). Those flies were retained whose eyes contained an excess of mutant over wild-type tissue. So far, mutations have been identified in at least 23 distinct loci that elicit this phenotype. These included negative regulators of cell proliferation such as archipelago (ago) as well as homologs of human tumor-suppressor genes including PTEN, TSC1, and TSC2 (Tapon, 2002).

Three alleles of sav were identified. A fourth allele, sav⁴, was isolated by Jessica Treisman. sav¹ and sav² generate eyes that have an increased representation of mutant tissue (white colored: marked by the expression of a mutant allele of the gene white) over wild-type tissue (red) when compared to the parent chromosome. sav³ elicits a more severe phenotype; in addition to a further increase in the representation of mutant tissue, the mutant tissue protrudes from the eye in folds. sav⁴ exhibits an intermediate phenotype. Clones of sav³ mutant tissue generated in other parts of the fly including the notum and haltere also display outgrowths. All four alleles are lethal when homozygous, in trans to each other or in trans to the deletion Df(3R)hh that spans the sav locus (Tapon, 2002).

In the eye disc, sav clones contain cells that continue to proliferate for 12–24 hr after their normal counterparts stop dividing. Studies of cycling cells show almost no differences between wild-type and mutant populations. However, given that mutant clones contain more ommatidia than wild-type twin spots, accelerated growth must have occurred in mutant tissue anterior to the furrow. Even a relatively minor growth advantage exhibited by mutant cells at every cell cycle can eventually result in increased clone size when amplified by the approximately nine rounds of cell division that occur in the eye primordium prior to the passage of the MF. A subtle change in cell cycle parameters may not easily be detected (Tapon, 2002).

In sav clones, elevated Cyclin E protein levels are observed in the basal nuclei posterior to the MF in the eye imaginal disc. These cells normally stop dividing when they downregulate Cyclin E protein levels. In discs containing many sav clones, the stripe of cyclin E RNA expression is broader and more intense. Thus, the increased level of Cyclin E protein is, at least in part, a result of elevated cyclin E RNA levels. Thus, an inability to downregulate Cyclin E/cdk activity may be the result of increased levels of cyclin E RNA as occurs in sav clones as well as in conditions of impaired protein degradation, or reduced levels of the cdk inhibitor Dacapo. In each case, cell cycle exit is delayed (Tapon, 2002).

Elevated DIAP1 levels are likely to underlie the absence of the developmentally regulated apoptosis in sav clones in the pupal retina as well as the resistance to hid-induced and rpr-induced apoptosis in the larval imaginal disc. The elevated DIAP1 levels appear to result from alterations in posttranscriptional regulation of DIAP1 expression. Recent work has shown that both Rpr and Hid can downregulate DIAP1 levels either by promoting the autoubiquitination of DIAP1 or by causing a generalized inhibition of translation that especially impacts proteins with a short half-life such as DIAP1. Either of these mechanisms is likely to be less efficient in cells that already have elevated levels of DIAP1 (Tapon, 2002).

Sav normally functions to downregulate the basal level of DIAP1 protein. In the absence of Sav, higher levels of DIAP1 accumulate. This increases the level of Hid or Rpr activity that is required to overcome DIAP1-mediated inhibition of caspase activation. Consistent with this model, the more potent form of Hid, Hid-Ala5, is able to partially overcome the increased levels of DIAP1 in sav clones and induce a low level of caspase activity (Tapon, 2002).

Sav appears capable of regulating both cell cycle exit and apoptosis by virtue of its ability to modulate the levels of two key regulators -- Cyclin E and DIAP1. Loss of sav appears to increase cyclin E levels transcriptionally and DIAP1 levels by a posttranscriptional mechansim. Since cell number is determined by both the extent of cell proliferation as well as apoptosis, sav could function as a key regulator of cell number by virtue of its ability to regulate both processes (Tapon, 2002).

One of few pathways that can directly regulate both cell proliferation and cell death is the Ras/MAPK pathway. Ras can promote cell proliferation by promoting growth, and MAP kinase can phosphorylate and inactivate Hid and also reduce Hid transcription. The results indicate that sav might function in a distinct pathway. (1) No change in diphospho-ERK level is observed in sav mutant clones. (2) Cell death induced by the MAP kinase-resistant Hid-Ala5 protein (where five putative MAPK phosphorylation sites have been mutated to alanines) is also reduced by a loss of sav function. However, it is still possible that sav might function downstream of the MAPK family proteins (Tapon, 2002).

Clones of cells mutant for wts generate large tumor-like growths in Drosophila. Wts' human ortholog LATS1 binds to the cdc2 protein kinase in a cell cycle-dependent manner and inhibits its activity. Thus, it has been suggested that excessive Cyclin A/cdc2 may cause excessive cell proliferation by promoting both the G1/S and G2/M transitions. The interaction between wts and cdc2, however, does not explain the excessive and inappropriate growth (mass accumulation) that appears to drive the cell proliferation in clones of wts mutant cells. The defect in cell death in wts cells is also not easily accounted for by the interaction of Wts with cdc2. The data raise the possibility that sav and wts might interact (via a WW domain-PY motif-dependent interaction) and function to promote cell cycle exit and apoptosis during development. However, wts is likely to have sav-independent functions as well. While sav mutations appear to result in a subtle increase in growth rate, the very strong overrepresentation of wts mutant tissue in third instar larval discs indicates that wts mutations must cause a much greater increase in growth rate (Tapon, 2002).

Mice lacking the warts ortholog LATS1 display pituitary hyperplasia and develop slow-growing tumors. This contrasts with the dramatic overgowth phenotype observed in wts mutants in Drosophila. These differences may be due to the presence of other wts homologs (e.g., LATS2) in mammals that can partially compensate for LATS1 inactivation. It is concluded that the strategy of conducting phenotype-based screens in model organisms followed by a search for mutations in cancer cell lines may help identify new tumor suppressor genes (Tapon, 2002).

Differential requirement of Salvador-Warts-Hippo pathway members for organ size control in Drosophila melanogaster

The Salvador-Warts-Hippo (SWH) pathway contains multiple growth-inhibitory proteins that control organ size during development by limiting activity of the Yorkie oncoprotein. Increasing evidence indicates that these growth inhibitors act in a complex network upstream of Yorkie. This complexity is emphasised by the distinct phenotypes of tissue lacking different SWH pathway genes. For example, eye tissue lacking the core SWH pathway components salvador, warts or hippo is highly overgrown and resistant to developmental apoptosis, whereas tissue lacking fat or expanded is not. This study explores the relative contribution of SWH pathway proteins to organ size control by determining their temporal activity profile throughout Drosophila eye development. Eye tissue lacking fat, expanded or discs overgrown displays elevated Yorkie activity during the larval growth phase of development, but not in the pupal eye when apoptosis ensues. Fat and Expanded do possess Yorkie-repressive activity in the pupal eye, but loss of fat or expanded at this stage of development can be compensated for by Merlin. Fat appears to repress Yorkie independently of Dachs in the pupal eye, which would contrast with the mode of action of Fat during larval development. Fat is more likely to restrict Yorkie activity in the pupal eye together with Expanded, given that pupal eye tissue lacking both these genes resembles that of tissue lacking either gene. This study highlights the complexity employed by different SWH pathway proteins to control organ size at different stages of development (Milton, 2010).

The SWH pathway controls Drosophila eye size by limiting growth during the larval stage of development and by restricting proliferation and promoting apoptosis during pupal development. Eyes lacking core SWH pathway components (e.g. sav, wts or hpo) are significantly larger than eyes lacking the non-core components ft, ex, dco or Mer. Owing to this disparity, it has been hypothesized that ft and ex only partially affect SWH pathway activity, whereas sav, wts and hpo have stronger effects, or, alternatively, that non-core components affect pathway activity in a temporally restricted fashion. Analysis of tissue recessive for ft, ex or dco³ revealed that Yki activity was elevated during larval eye development when tissues are actively growing and proliferating, but not during pupal development when apoptosis ensues, supporting the idea that Ft, Ex and Dco influence SWH pathway activity in a temporally restricted fashion. However, when tissue lacking both Mer and ft, or Mer and ex, was analysed, Yki activity was found to be elevated during both larval and pupal development, similar to the Yki activity profile observed in tissue lacking core SWH pathway proteins. This is consistent with previous reports showing that Mer acts in parallel to both Ft and Ex, and that these proteins can compensate for each other to control SWH pathway activity. Therefore, Ft and Ex do contribute to SWH pathway regulation in the pupal eye to ensure appropriate exit from the cell cycle and developmental apoptosis, but these functions can be executed by Mer in their absence, suggesting a degree of plasticity in the regulation of Yki activity by non-core SWH pathway proteins. The ability of Mer to compensate for Ft or Ex cannot simply be explained by compensatory increases in Mer protein in pupal eye tissues lacking ft or ex, since Mer expression levels were found to be unaltered in these tissues (Milton, 2010).

Previous analyses of tissue lacking both ft and ex showed that these proteins function, at least in part, in parallel to control growth of larval imaginal discs. The current analysis of ft,ex double-mutant tissue suggests that these proteins are likely to function together to control Yki activity in the pupal eye. Yki activity was not elevated in tissue lacking ft, ex or both genes, showing that these genes cannot compensate for each other in the pupal eye. This is consistent with the notion that Ft influences the activity of downstream SWH pathway proteins by multiple mechanisms, an idea that is supported by THE analysis of the requirement of the atypical myosin, Dachs, for Ft signalling in the pupal eye. During larval imaginal disc development, Ft can influence Yki activity by repressing Dachs activity, which in turn can repress the core SWH pathway protein Wts. Analysis of pupal eye tissue that lacks both Mer and ft, or Mer, ft and dachs, showed that Yki activity was elevated in each scenario. This shows that in the pupal eye, the ability of Ft to compensate for Mer is not reliant on Dachs, and implies that Ft can employ different modes of signal transduction throughout eye development. However, because Ft and Mer can compensate for each other it is not possible to formally conclude that normal signal transduction by Ft in the pupal eye occurs independently of Dachs (Milton, 2010).

Expression of Ex is tightly controlled in response to alterations in SWH pathway activity at both the transcriptional and post-transcriptional levels. Interestingly, it was also found that Ex expression is controlled in a temporal fashion throughout eye development; Ex is expressed at relatively high levels in the larval eye, but at very low levels in the pupal eye. Despite the fact that Ex expression is very low in the pupal eye, it clearly retains function at this stage of development because it can compensate for loss of Mer to restrict Yki activity. The dynamic expression profile of Ex suggests that factors that influence its expression play an important role in defining overall eye size in Drosophila. At present, only two transcriptional regulatory proteins have been shown to influence the expression of ex: Yki and Sd. There are conflicting reports on whether Yki and Sd control basal expression of ex in larval imaginal discs. It is clear, however, that Yki and Sd collaborate to drive ex expression when the activity of the SWH pathway is suppressed, presumably as part of a negative-feedback loop. Despite the fact that basal ex expression is low in the pupal eye, the ex promoter is still responsive to Yki, as Ex expression is substantially elevated in pupal eye clones lacking hpo or Mer and ex. Future investigation of the ex promoter will help to clarify understanding of the complex fashion by which expression of the ex gene is controlled, and should aid understanding of eye size specification in Drosophila (Milton, 2010).

This study emphasises the complexity of the means by which the activity of core SWH pathway proteins is regulated by non-core proteins such as Ft, Ex, Mer and Dco. The signalling mechanisms employed by non-core proteins appear to differ at discrete stages of development in order to achieve appropriate organ size during the larval growth period of eye development, and to subsequently sculpt the eye by regulating apoptosis during pupal development (Milton, 2010).

PROTEIN STRUCTURE

Amino Acids - 608

Structural Domains

The sav mutations were localized to the interval 93F11-13 to 94D10-13. High-resolution meiotic mapping localized sav to a 20 kb region that contained five ORFs. All five ORFs were sequenced completely and it was found that all four sav chromosomes had truncating mutations in CG13831. The other four ORFs did not have any amino acid changes. Five independent cDNA clones of CG13831 were examined by restriction mapping, and two independent clones were sequenced completely. The longest clone is 2.2 kb long, which is in agreement with the approximate size of the RNA determined by Northern blotting. The predicted ORF, encoding a protein of 608 amino acids, includes the entire coding region since there is a stop codon upstream and in-frame with the ATG codon. The predicted Sav protein has two WW domains, and its C-terminal portion includes a domain that is likely to adopt the conformation of a coiled-coil. Sav is most similar to the human protein hWW45 (Valverde, 2000) and to the protein encoded by the C. elegans ORF T10H10.3. WW domains are known to mediate protein-protein interactions with various proline-containing motifs. The more C-terminal WW domain lacks the second conserved tryptophan residue that is required for substrate binding and is unlikely to be a functional WW domain. The N-terminal WW domain contains all of the appropriate conserved residues. This putative WW domain is predicted to belong to the Group I family of WW domains that is predicted to interact with the PPXY ('PY') motif (Tapon, 2002).

EVOLUTIONARY HOMOLOGS

Since mutations in sav lead to excessive cell proliferation and reduced cell death, tests were performed to see whether hWW45 might be a mutational target in cancer. hWW45 maps to the chromosomal region 14q13–14q23 (Valverde, 2000), a locus that is subject to allelic loss in a variety of cancers, including renal cancers, ovarian cancers, and malignant mesothelioma. The entire coding region of hWW45 was sequenced in a panel of 52 tumor-derived cell lines, representing a broad range of tissue types. One colon cancer cell line, HCT15, had a heterozygous C to A mutation at nucleotide 554, resulting in a substitution of aspartic acid for alanine at codon 185. This mutation was not present in 185 population-based controls (370 chromosomes), indicating that it is not a common polymorphism. HCT15 carries a mutation in the mismatch repair gene MSH6, which appears to enhance the frequency of point mutations in other genes. More significantly, two renal cancer cell lines, ACHN and 786-O, were found to have deletions involving hWW45. The normal allele was not present in either cell line, indicating that these cell lines are either homozygous or hemizygous for the deletion. The hWW45 transcript was undetectable by RT-PCR in both cell lines, and a Southern blot using a probe derived from the 3' portion of the gene demonstrated that this part of the gene was absent in both cell lines. In cell line 786-O, PCR analysis of genomic DNA indicated that there is a deletion of ~157 kb with the 5' breakpoint between exons 2 and 3 of hWW45. The deletion in ACHN of ~138 kb encompassed the entire gene. The common region of overlap between these two deletions is only 21 kb, containing exons 3–5 of hWW45. No other transcription units were identified within this 21 kb interval, using the GENSCAN exon prediction program. Thus, deletions have been identified that would inactivate the human ortholog of sav in at least two cancer cell lines (Tapon, 2002).

Originally identified in Drosophila, the Warts(Wts)/Lats protein kinase has been proposed to function with two other Drosophila proteins, Hippo (Hpo) and Salvador (Sav), in the regulation of cell cycle exit and apoptosis. In mammals, two candidate Warts/Lats homologs, termed Lats1 and Lats2, have been described, and the targeted disruption of LATS1 in mice increases tumor formation. Little, however, is known about the function and regulation of human Lats kinases. Human Mst2, a STE20-family member and purported Hpo ortholog, phosphorylates and activates both Lats1 and Lats2. Deletion analysis reveals that regulation of Lats1 occurs through the C-terminal, catalytic domain. Within this domain, two regulatory phosphorylation sites were identified by mass spectrometry. These sites, S909 in the activation loop and T1079 within a hydrophobic motif, have been highly conserved during evolution. Moreover, a direct interaction is observed between Mst2 and hWW45, a putative ortholog of Drosophila Sav. These results indicate that Mst2-like kinases regulate Lats kinase activities in an evolutionarily conserved regulatory pathway. Although the function of this pathway remains poorly understood in mammals, it is intriguing that, in Drosophila, it has been linked to development and tissue homeostasis ( Chan, 2005 ).

date revised: 1 September 2002

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