An unregulated regulator: Vasa expression in the development of somatic cells and in tumorigenesis

Summary

Growing evidence in diverse organisms shows that genes originally thought to function uniquely in the germ line may also function in somatic cells, and in some cases even contribute to tumorigenesis. Here we review the somatic functions of Vasa, one of the most conserved “germ line” factors among metazoans. Vasa expression in somatic cells is tightly regulated and often transient during normal development, and appears to play essential roles in regulation of embryonic cells and regenerative tissues. Its dysregulation, however, is believed to be an important element of tumorigenic cell regulation. In this perspectives paper, we propose how some conserved functions of Vasa may be selected for somatic cell regulation, including its potential impact on efficient and localized translational activities and in some cases on cellular malfunctioning and tumorigenesis.

Introduction

More than 40 genes that are normally expressed selectively in the germ line of many model organisms are also expressed in various tumor types in humans (Simpson et al., 2005). Recapitulating functions of a germ line program thus has been hypothesized to contribute to the characteristic features of neoplastic diseases, including immortality, hypomethylation of DNA, and metastatic migrations (Hanahan and Weinberg, 2011). Indeed, in C. elegans, mutations of the retinoblastoma (Rb) tumor suppressor complex induce somatic cells to express germ line genes, leading cells to revert to patterns of gene expression normally restricted to germ cells (Wang et al., 2005). Further, C. elegans mutants with decreased insulin-like signaling (e.g. daf-2, the insulin-like receptor, and age-1, the downstream PI3K mutant strains) cause mis-expression of the germ line factors pie-1 and pgl in the somatic cells. These altered somatic cells are then more resistant to stress-induced damage and have increased cellular lifespans (or negligible senescence) (Curran et al., 2009). This result is particularly noteworthy considering C. elegans somatic cells are genetically programmed to cease cell divisions after the animal reaches adulthood. These observations suggest that the acquisition of germ line elements may contribute to somatic phenotypes, and even of tumorgenic cells.

Vasa was first identified in Drosophila (Hay et al., 1988; Lasko and Ashuburner, 1988) as a polar granule component consisting of cytoplasmic granules inherited by posterior pole cells, which directs the fate of the cell to the germ line (Illmensee and Mahowald, 1974). It is a member of the DEAD-box RNA helicase family (for the molecular classification and characterization of Vasa, see reviews of Raz, 2001; Linder, 2006; Gustafson and Wessel, 2010; Lasko, 2013), similar in sequence to eukaryotic initiation factor 4A (eIF4A) (Hay et al., 1988; Lasko and Ashuburner, 1988). Vasa is considered to function as a translational regulator in the germ line (Hay et al., 1988; Lasko and Ashburner, 1988; Linder et al., 1989; Sengoku et al., 2006). Follow-up reports in Drosophila suggest that Vasa associates with eIF5B, an essential translation initiation factor required for joining of ribosomal subunits (Carrera et al. 2000; Pestova et al. 2000), and is involved in translation of gurken in oocytes and mei-P26 mRNA in germ line stem cells (Johnstone and Lasko 2004; Liu et al., 2010). A recent report in insect ovarian cells also suggests that Vasa binds to mRNAs with its DEAD-box domain and functions in the generation of germ line-specific, Piwi-interacting RNAs (piRNAs) that protect animal genomes against transposons and are essential for fertility (Xiol et al., 2014). Vasa thus appears to have broad functions, including translational regulation of specific mRNAs and piRNA generation, which contribute to cell fate determination within the germ line.

Vasa is one of the most conserved germ line factors in various metazoan organisms, based on sequence, expression patterns, and functions in the germ line. Its knockdown in major model organisms compromises the animal's reproductive capabilities with varying severity between different organisms, or even between sexes of the same species. For instance, Vasa knockdown in Drosophila results in sterility in females but not in males (Styhler et al., 1998; Lasko and Ashburner, 1990; Renault, 2012), whereas in mice it causes sterility in males but not in females (Tanaka et al., 2000). In C. elegans, GLH1, one of four Vasa homologs in this organism, is important both for germ cell development and for gamete maturation (Roussell and Bennett, 1993; Gruidl et al., 1996; Kuznicki et al., 2000; Spike et al., 2008). In zebrafish, maternal Vasa is stable throughout early development and is necessary for initial germ cell formation, and Vasa produced embryonically is required for germ cell maintenance (Braat et al., 2001; Hartung et al., 2014). In Medaka, on the other hand, Vasa protein synthesized in the embryo is important for germ cell migration but not for survival (Li et al., 2009. The mechanism of these variable Vasa inputs among different species and sexes is unclear. It was recently proposed that Vasa might have multiple different functions in each cell type or organism by using different partner proteins (Dehghani and Lasko, 2015). The identity of such distinct interactors though, remains unclear.

Recent reports in more diverse organisms suggest that Vasa might also function outside of the germ line. These somatic functions of Vasa include various types of embryonic cells (Yajima and Wessel, 2011a; Schwager et al, 2014), regenerative tissues (Wagner et al., 2012; Yajima and Wessel, 2015), and even in tumorigenic cells (Janic et al., 2010). These reports cast potential additional functions of Vasa and/or of Vasa's interacting proteins in the soma. In this Perspectives paper, we speculate on the functional impact of Vasa in somatic cells as well as on potential contributions during tumorigenesis.

Vasa's contributions as a mitotic factor during embryogenesis

Although expression and activities of Vasa are most prominent in the germ line, Vasa protein and/or mRNA is often widely expressed in somatic lineages during embryogenesis of many organisms (summarized in Table 1). Proteins and mRNAs are often stored maternally in the oocyte and embryo, and thus Vasa presence in the resultant somatic cells does not necessary indicate a functional role. Initial observations in Drosophila showed that maternal Vasa contributes to the formation of both the germ cells and the embryonic abdominal segments (Schupbach and Wieschaus, 1986; Hay et al., 1990), implying a broad impact on embryogenesis. Flies mutant for Vasa embryonically, however, are viable as homozygotes, suggesting no essential zygotic roles in somatic development of Drosophila. Recent studies in the sea urchin and spider, on the other hand, demonstrated that Vasa does function in somatic cells during embryogenesis. In the sea urchin, Vasa was found to localize to the nuclear periphery at S-phase of the cell cycle in blastomeres and accumulate on the mitotic apparatus at M-phase of every blastomere during early embryogenesis. The knockdown of Vasa in these embryos resulted in defects of proper spindle organization and chromosome segregation, cell cycle delays and often death of the embryo (Yajima and Wessel, 2011a & b). In spider embryogenesis, Vasa is localized to small puncta ubiquitously distributed throughout the cortex before PGCs emerged at the late germ band stage and its knockdown by RNA interference resulted in defects of mitotic progression in the embryo (Schwager et al, 2014). Importantly, this function of Vasa as a mitotic regulator was also reported in Drosophila germ line stem cells (Pek and Kai, 2010). In those cells, Vasa concentrated in the perinuclear region during S-phase and to the spindle and chromosome region during M-phase. Its functionality was highlighted in mitotic progression by regulating the localization of chromosome-associated proteins, which in turn mediated chromosome condensation and segregation (Pek and Kai, 2011). These reports suggest a conserved function of Vasa as a mitotic regulator both in the germ line and in the soma (Fig. 1).

Table 1.

Summary of Vasa expression and functional contributions in the soma

Organism	mRNA	Protein	Functional contribution	References
Fly (Drosophila melanogaster)	Uniformly distributed during syncytial blastoderm stage.	Uniformly distributed during syncytial blastoderm stage.	Abdominal segment formation.	Schupbach and Wieschaus, 1986; Lasko and Ashburner, 1988 & 1990; Hay et al., 1988
Spider (Parasteatoda tepidariorum)	Uniformly distributed in the early embryo, and becomes highly expressed in mesoderm and ectoderm after germ band formation.	Uniformly distributed in the early embryo, and becomes undetectable after germ band formation.	Early embryonic mitotic divisions and spindle integrity.	Schwager et al, 2014
Planarians (Dugesia japonica and Schmidtea mediterranea)	Expressed at the lower level in the neoblasts and highly expressed in the growing blastema during regeneration.	ND	Neoblast proliferation and differentiation during regeneration.	Shibata et al., 1999 Wagner et al. 2012
Hydra (Hydra magnipapillata)	Expressed in multipotent stem cells (i-cells) and ectodermal epithelial cells.	ND	ND	Mochizuki et al., 2001
C. elegans	Expressed in all cells of young embryos and reduced after the 8- to 10-cell stage.	Background level	ND	Gruidl et al., 1996; Kuznicki et al., 2000
Polychaete (Platynereis dumerilii and Capitella sp. I)	Uniformly distributed until the four cell stage, and becomes confined to the ventral plate and the mesodermal bands at 15pf and to mesodermal posterior growth zone (MPGZ) during larval and adult stages.	Distributed in the yolk free cytoplasm of all blastomeres at 4-cell stage, and becomes enriched to secondary mesoblast cells and MPGZ during larval and adult stages.	ND	Rebscher et al., 2007; Dill and Seaver, 2008
Sea urchin (Strongylocentrotus purpuratus)	Uniformly distributed in every cell until gastrula stage, and transiently expressed in the cells of the adult precursor (rudiment).	Enriched on the mitotic apparatus of every early embryonic cell, and transiently expressed in the adult precursor cells and wound-healing tissues.	Early embryonic mitotic cell cycle progression, wound-healing, and developmental reprograming.	Yajima and Wessel, 2011b & 2015
Ascidians (Ciona intestinalis)	Uniformly distributed in each cell until 16-cell stage and widely expressed in the postplasm and the trunk region.	Concentrated at the postplasm at 64-cell and enriched in the posterior-most cells (the B7.6 cells) and the trunk region.	ND	Shirae-Kurabayashi et al, 2006
Zebrafish	Localized to the distal ends of the first and second cleavage planes at 4-cell stage.	Distributed throughout the embryo during embryogenesis.	ND	Yoon et al., 1997; Braat et al, 1999 & 2000; Kosaka et al., 2007
Frog (Xenopus laevis)	Expressed during early embryogenesis by RT-PCR analysis.	Uniformly expressed until tailbud stage, and becomes enriched in the mesoderm during early tadpole stage.	ND	Ikenishi et al., 1996; Li et al., 2010
Chick	ND	Localized in the basal cleavage furrow in the early embryo, and becomes scattered in the hypoblast layer after stage X.	ND	Tsunekawa et al., 2000

*ND, no data

Fig 1.

Vasa is involved in mitotic progression. A. Vasa localizes on the mitotic apparatus and is essential for cell cycle progression of sea urchin embryos (Adapted from Yajima and Wessel, 2011a) and Drosophila germ line stem cells (Adapted from Pek and Kai, 2010). Vasa is enriched in peri-nuclear regions prior to mitotic entry (Interphase), localizes on the mitotic apparatus during M-phase, and disappears at the end of mitosis, followed by peri-nuclear localization at the beginning of next mitotic entry. Vasa knockdown (right panels) in those cells result in defects of spindle organization and chromosome segregation. The knockdown image of sea urchin is an embryo at 16-cell stage, and that of Drosophila is a germ line stem cell at Anaphase. B. A cartoon diagram of Vasa protein (red) localization in the sea urchin embryonic cells (left panel) and Drosophila germline stem cells (middle panel). Both in sea urchin embryonic cells and Drosophila germ line stem cells, Vasa is evenly distributed over the spindle (green) and DNA (blue) during mitosis, and its knockdown (right panel) results in cell division defects, including abnormal chromosomal segregation. Further, in the sea urchin, Vasa becomes more enriched toward the vegetal blastomere (a future germ line) of the spindle at 16-cell stage by an unknown mechanism.

Detailed functional contributions of Vasa in mitotic regulation of somatic cells are yet to be identified but it may “simply” function to increase translation locally. A recent report in the sea urchin embryo suggests that Vasa interacts with most mRNAs in the cell and its knockdown results in inhibition of overall protein synthesis down to approximately 20% that of the normal embryo (Yajima and Wessel, 2015). Vasa may thus function by contributing to translation of a large number of mRNAs, which allows cells to produce sufficient amounts of proteins for rapid cell replication and differentiation during embryogenesis, especially in a large cell with rapid cell fate transitions.

Vasa's function in the soma during regeneration and tumorigenesis

Vasa also appears to function within the soma during regeneration and somatic tumorigenesis. In planarian regenerative tissues, Vasa expression is upregulated (Wagner et al., 2012), as it is in Drosophila brain tumor cells (Janic et al., 2010), and even in human cancer cells (Hashimoto et al., 2008; Nguyen et al., 2011; Kim et al., 2014). In planarians, transcriptional profiling identified that expression of several germ line genes are upregulated in adult proliferating neoblasts during regeneration. Among those genes, the vasa-like gene, vasa-1 was identified to be essential for proliferative expansion to promote the process of differentiation in neoblast descendants, suggesting its functional role in stem cell regulation (Wagner et al., 2012).

When a malignant brain tumor was induced in Drosophila (by inactivation of a polycomb repressor gene, lethal (3) malignant brain tumor [l(3)mbt]; Gateff et al., 1993), 102 genes were found to increase transcript levels specific to this mutant (Janic et al., 2010). Among these genes, a quarter of them (26 of 102) are known factors required for the Drosophila germ line. In addition to vasa, these genes included, for instance, nanos, necessary for pole plasm development (Wong et al., 2005) and piwi and aubergine, involved in the biogenesis of Piwi interacting RNAs (piRNAs) that protect germ line cells against transposable elements and viruses (Klattenhoff and Therurkauf, 2008; Patil and Kai, 2010). Importantly, not only were these genes expressed in the tumor cells, but inhibition of vasa, piwi, aubergine, or nanos halted tumor growth, suggesting that the expression of these factors in the somatic tumor had an essential function. In this process of tumorigenesis regulation, interestingly, vasa was not upregulated at the mRNA level but its protein expression was increased. This is distinct from other germ line factors such as piwi and aubergine, which were up-regulated both at the mRNA and protein levels during tumorigenesis. This finding suggests that vasa mRNA (and potentially Vasa protein as well) is expressed at low levels during normal soma development, and can be differentially regulated and utilized upon appropriate stimulation. This post-transcriptional regulation of Vasa is a common theme (e.g. Wagner et al., 2012; Yajima and Wessel, 2015) likely important for this gene function.

DDX4 (the Vasa ortholog in humans) expression was also found in various types of cancer tissues in humans. In ovarian cancer cells, DDX4 expression was significantly increased together with CD133, a cancer stem cell marker, whereas almost all CD133-negative cells had no DDX4 expression, implying that DDX4 may play an important role in cancer stem cells (Kim et al., 2014). Further, in the SKOV-3 (epithelial ovarian cancer) cell line, artificially forced-expression of DDX4 resulted in up-regulation of 10 proteins and down-regulation of 6 proteins. Among these proteins altered by DDX4, 14-3-3σ protein was down-regulated but its mRNA level was not changed, suggesting that DDX4 may be involved in post-transcriptional regulation of 14-3-3σ (Hashimoto et al., 2008). Importantly, 14-3-3σ is frequently suppressed in human breast, gastric, lung and ovarian cancers (Ferguson et al., 2000; Iwata et al., 2000; Osada et al., 2002; Akahira et al., 2004). 14-3-3σ is known to sequester the mitotic initiation complex (cdk1-cyclin B1) to the cytoplasm in response to the DNA damage, and induces cell cycle arrest for the G2 checkpoint (Chan et al., 1999). With the forced expression of DDX4 in which 14-3-3σ is down-regulated, the SKOV-3 ovarian cancer cells appeared to ignore the G2 checkpoint and underwent rapid cell cycle progression even in the presence of DNA damage (Hashimoto et al., 2008). In these cells, DDX4 may have precociously nucleated the mitotic initiation complex and induced pre-mature mitotic entry by down-regulating 14-3-3σ activities. If this speculation is correct, DDX4 overexpressing cells could then continue to proliferate with damaged genetic materials, which can eventually result in tumor formation. These speculations need to be tested through further experiments. One could test, for instance, if forced expression of 14-3-3σ would rescue DDX4-overexpression phenotypes, slow down cell cycle progression and enable repair of DNA damage in the genome. The SKOV cell line used here is derived from ovarian carcinomas whose molecular status and cellular dynamics may be quite different from normal cells or even non-ovarian cancer cells. Another important experiment is to test the protein content of cells with or without DDX4 present to determine if the proteome varies by DDX4's functionality in non-ovarian cells. Since Vasa protein level in the cell is thought to be regulated by the Ubiquitin proteasome-dependent protein degradation mechanism (Liu et al., 2003; Styhler et al., 1998 and 2002; Kugler et al., 2010; Woo et al., 2006 a & b), introducing constitutively active forms of DDX4 into the cell would be important to identify its functional roles. For these experiments, one could mutate putative protein degradation recognition sites, or may target DDX4 expression to an ectopic sub-cellular region where ‘Vasa-regulators’ may be absent. These experiments both in non-oncogenic and in non-ovarian-cancer cells will potentially reveal actual and conserved contributions of DDX4 to cancer cell regulation.

These recent results in tumorgenic and cancerous cells suggest that somatic cells that have been considered Vasa-null in their normal state could potentially induce Vasa expression through post-transcriptional regulation under certain stimuli. Indeed, in the sea urchin embryo, Vasa mRNA and protein appears to be consistently expressed at low levels in various types of somatic cells during normal development, but the protein can be up-regulated ‘transiently’ from the same amount of mRNA during the formation of adult precursor tissues, wound-healing, or developmental re-programming, processes which require cells to undergo active proliferation and differentiation (Voronina et al., 2008; Yajima and Wessel, 2015). During these regulatory processes, importantly, the number of cells that entered into M-phase was increased 2-fold (Yajima and Wessel, 2015). Vasa might thus assist cells to temporarily up-regulate translation of molecules essential for cell proliferation. In somatic lineages, therefore, Vasa might be essential for ‘a transient induction’ of active cell proliferation through its translational activities that normally occur in the germ line.

In the germ line and gonads, on the other hand, Vasa protein is enriched and functional, yet these cells typically divide slowly. This may be because of different binding partners of Vasa or other more dominating factors that have inhibitory effects on the Vasa's function or cell cycle control in these cells. For instance, Nanos, another germ line factor, is known to inhibit cyclin-B mRNA translation and functions as a cell cycle repressor, preventing the germ cells from cell cycle progression (Barker et al., 1992; Asaoka et al., 1999; Kadyrova et al., 2007; Juliano et al., 2010). Whether the somatic cells described above also recover this regulatory mechanism ‘transiently’ to manipulate the level of Vasa activities for proper embryogenesis or regeneration has yet to be tested. Another possibility is that Vasa needs to be modified in order to be functional in some somatic cells (as described in the next section). In tumorigenic cells, however, this regulatory mechanism may be inactivated. Vasa over-expression will then not adequately limit the continual translation of Vasa's interactors such as cyclin-B and other embryonic mRNAs (Liu et al., 2009; Yajima and Wessel, 2015). This may result in over expression of embryonic proteins and a recapitulation of the embryonic molecular status in the cells, but only in an “unregulated” manner. This could lead to uncontrolled cellular activities, which may in some cases enable the retention of continuously proliferating tumorgenic cells.

Vasa function is tightly regulated by post-transcriptional mechanisms

Knockdown or overexpression of genes of interest has been a gold standard to allow researchers to identify the gene's functional contributions as well as its surrounding molecular network during development. Vasa over-expression, however, often results in no dramatic phenotype in an organism's normal development (Ikenishi and Yamakita, 2003; Orsborn et al., 2007; Kugler et al., 2010; Gustafson et al., 2011), suggesting again that the presence of Vasa mRNA and/or protein in the soma does not immediately indicate its function in those cells, and that Vasa function is tightly regulated by post-transcriptional mechanisms. In Drosophila embryos, it is thought that Vasa protein is actively degraded in the soma through E3 Ubiquitin ligase pathways, resulting in embryos with Vasa enriched only in the germ line (Liu et al., 2003; Styhler et al., 1998 and 2002; Kugler et al., 2010; Woo et al., 2006 a & b). The knockdown of these Ubiquitin pathways in the embryo, however, resulted only in higher expression of Vasa in the germ line but did not appear to induce Vasa accumulation in the soma even when its mRNA was present. These facts suggest that a differential mechanism of Vasa regulation in the germ line and the soma. In the sea urchin embryo, similarly, Vasa protein over-expression did not have a significant phenotype (Gustafson et al., 2011). Therefore, introducing excessive amounts of Vasa protein usually “simply” results in extra degradation of Vasa protein at each cell cycle, and no developmental defects in these embryos. It should be noted that Vasa does not appear to be sufficient for translational activity on its own; it may be rate limiting in some cells, and/or be part of a complex of translational regulators that are upregulated upon stimuli that leads to increased translation of cellular mRNAs. Further, a recent report in Drosophila with a series of deletions /mutations in the vasa gene demonstrated that multiple domains in the N- and C-terminal regions of Vasa protein are important for its localization in the germ line and for its functions in transposon repression, posterior patterning, and pole cell specification. Notably, some of these engineered mutant -Vasa proteins resulted in the increase or decrease of Vasa protein accumulation in the germ line but not in the somatic cells, suggesting again a simple mutation of the gene or inhibition of degradation pathway is insufficient to induce Vasa accumulation in the soma (Dehghani and Lasko, 2015). Embryos thus appear to have a high fidelity system to keep titrated levels of Vasa protein, especially in the somatic cells. This strong post-transcriptional and -translational regulation of Vasa makes it difficult to artificially over-express Vasa proteins to the level where a phenotype may be apparent.

Further, modifications of Vasa also appear critical for its differential activities in some cases. In Drosophila oogenesis, it has been reported that phosphorylated-Vasa activity is required for translation of Gurken (Grk), which is responsible for axis specification of oocytes (Ghabrial and Schupbach, 1999; Klattenhoff et al., 2007). In the DNA-repair mutants (e.g. spindle-B/DMC1/RAD51-like, spindle-C and okra/Rad54) or the repeat-associated small interfering RNA (rasiRNA) pathway mutants (e.g. armitage, spindle-E, aubergine), Vasa protein appears to migrate distinctively on a poly-acrylamide gel relative to wild-type ovary samples. This retardation was reversed after phosphatase treatment, indicating that the retarded mobility resulted from a phosphorylated form of Vasa. This retardation was also restored by an additional mutation in the double-strand-break signaling (e.g. mei-41, mnk), which is activated in the presence of DNA-damage or -double-strand breaks. Therefore, in the DNA-repair and rasiRNA mutants, persistence of double-strand break activates the DNA-damage checkpoint pathway, which results in phosphorylation of Vasa and then blocks efficient translation of Grk, leading to axial patterning defects. These results suggest that Vasa may be differentially modified in each lineage or developmental stage for its differential activities. In support of this idea, recent efforts in Drosophila to identify functional regions of Vasa in vivo (Dehghani and Lasko, 2015) demonstrated that many of the Vasa mutations that would be expected to abrogate DEAD-box helicase function localized and functioned appropriately in oogenesis, but mis-functioned in embryonic patterning, pole cell specification, grk activation, or transposon repression. Therefore, Vasa uses different domains and modifications for each of its differential activities during development. This fact implies that post-transcriptional modifications of Vasa are essential for its differential activities in each lineage and/or role. Vasa (DDX4) protein also appears phosphorylated in sea urchins (Guo et al., 2015), and potentially in mice and humans based on the search in the PhosphoSite proteomic database (http://www.phosphosite.org; Gustafson and Wessel, 2010), yet no detailed analysis has been provided especially in somatic cells. In the future, accomplishing the phosphor-proteomics analysis in the germ line, embryonic cells, regenerative tissues, and in tumor cells will likely reveal if Vasa undergoes differential modifications for each cellular process and for each development stage. The identified Vasa phosphorylation sites could then be mutated to test if any induce differential functions in each cell type. These detailed functional assays will be essential to reveal differential functional mechanisms of Vasa.

Hypothesis: potential functions of Vasa in the soma

Both in germ line and somatic lineages, Vasa is sometimes found in granule-like aggregations. Recent work in the insect ovarian cell line suggests that Vasa functions as a transient amplifier complex that mediates biogenesis of secondary piRNAs with Piwi proteins (Xiol et al., 2014). Vasa forms a tight clamp (interaction) on the RNA to anchor the amplifier onto transposon transcripts to inhibit their activities in the germ line at the perinuclear region. Although this RNA binding activity of Vasa has not been tested in the soma, members of this amplifier complex, Piwi proteins, have been also recently proposed to work in somatic stem cells (Ross et al., 2014). This casts an interesting insight that Vasa and Piwi may function in a similar manner for quality control of the genomes in stem cells. In the sea urchin embryo, similarly, it was found that Vasa is essential for the clustering of various mRNAs to the nuclear envelope and to the mitotic apparatus. Further, Vasa knockdown results in ~80% reduction of total protein synthesis in these embryos (Yajima and Wessel, 2015). These results suggest again that Vasa's primary function may be in clustering of various mRNAs to a proper sub-cellular localization (e.g. mitotic apparatus) for precise and efficient translation. This is likely essential for the quality control of embryonic development in which each blastomere in a single embryo undergoes robust but different translation for differential cell fate determination. Indeed, temporally and spatially controlled mRNA translation on the mitotic apparatus has been proposed as an important biological event for proper embryonic cell regulation (Raff et al., 1990; Gross and Fry, 1966; Blower et al., 2007).

In Xenopus and sea urchin embryos, ribosomes and translation factors are reported to be tightly associated to the mitotic apparatus (Suprenant, 1993; Liska et al., 2004; Mitchison et al., 2004; Blower et al., 2007), suggesting potential translational activities on the mitotic apparatus during M-phases. The large cells of 100-1000 microns such as eggs require distinct mechanisms of molecular regulation; simple expression and diffusion of molecules may not accomplish sufficient distribution of molecules to each daughter cell during rapid cell divisions of a large-sized cell (e.g. every 30~40 minutes per cell division for Xenopus and sea urchin embryos). Localized translation on the mitotic apparatus is therefore considered to provide a mechanism important for efficient and precise protein production necessary for rapid cell divisions and differential cell fate determinations during embryogenesis. In these embryonic cells, Vasa may recruit various mRNAs to the mitotic apparatus and help their interactions with translational regulators during M-phase. By doing so, Vasa could regulate translation in a temporally and spatially controlled-manner during embryogenesis. This hypothesis has to be tested by analyzing the subcellular function of Vasa on the mitotic apparatus in developing embryos. This can be accomplished by recruiting Vasa localization to the ectopic sub-cellular region to sequester Vasa from the mitotic apparatus. One could then analyze if the sequestered Vasa induces ectopic localized translation, and how that could result in changes of overall translation efficiency, cell divisions, and embryonic development. This sub-cellular level of analysis will be essential in the future to delineate the potential functions of Vasa as a localized translational regulator during embryogenesis.

These actively dividing embryonic cells as well as tumorgenic cells are also known to use distinct mechanisms of energy metabolism. Differentiated cells with slower cell cycling typically use oxidative phosphorylation for energy generation, which produces 32~38 mol ATP/ mol glucose. Rapidly dividing embryonic cells, on the other hand, have altered metabolisms that rely more on anaerobic glycolysis (Warburg-like metabolism; Warburg, 1930, 1956a, 1956b; Kondoh, 2007; Hsu and Sabatini, 2008), which produces only 2 mol ATP/ mol glucose. This altered pathway is hypothesized to allow the diversion of glycolytic intermediates into various biosynthetic pathways, including those generating nucleotides and amino acids, facilitating the biosynthesis of the macromolecules and organelles required for assembly of new cell proliferation (DeBerardinis et al., 2008; Vander Heiden et al., 2009). Perhaps, in the environment of anaerobic glycolysis with low-ATP production, Vasa may be essential for efficient translation using minimal amounts of ATP to meet the required level of protein synthesis during rapid embryonic cell divisions. A recent report in Drosophila demonstrated a functional involvement of germ line factors in the glycolytic pathway; glycolytic enzymes (e.g. Aldolase, GAPDH, Phosphoglycerate Kinase, Enolase, and Pyruvate Kinase) were found to be components of germ plasm. Importantly, GAPDH was enriched with a germ line specific Tudor/Vasa complex, and Pyruvate Kinase directly interacted with Tudor in germ cells. Further, knockdown of Enolase or Pyruvate Kinase resulted in reduction of piRNAs, and compromised germ cell formation. Through the interaction with germ line factors, therefore, glycolytic pathway components appear to function in protecting the genome from transposable elements and in germ cell formation (Gao et al., 2015). Since Tudor and Vasa are reported to function together in the germ cells (Arkov et al., 2006), it will be intriguing to test if Vasa is involved in this process of metabolic pathway. Further, it is important to identify if Vasa is in any way associated with a particular metabolic state of the cell (e.g. glucose consumption rate, and generation of lactate and ATP) during regulation of embryonic and tumorgenic cells. This can be measured by conventional HPLC analysis and enzymatic assays, or by Mass spectrometry-based metabolomics that can provide broad information on the chemical composition of complex (metabolomes) in cell extracts. The outcomes of these experiments may reflect Vasa's “employment” during minimal ATP-generation, perhaps for more efficient translational contributions during embryogenesis and tumorigenesis.

Additional future questions

Many cells and organisms appear to have a tightly regulated system for Vasa expression, especially in the soma, implying a broad biological importance. In tumorgenic cells where cellular dynamics are often altered, on the other hand, this tight control over Vasa may be lacking, allowing cells to accumulate Vasa and its target factors (e.g cell cycle materials) more effectively to contribute to a part of tumorigenic processes. Investigating the mechanisms of Vasa regulation in naturally Vasa-expressing tumorgenic cells may be thus helpful to understand the nature of this molecule function in the cells. Further, identifying “upstream regulator(s)” of Vasa in these tumorigenic cells could also decipher how this molecule may be tightly regulated for its transient function during normal development, especially in the soma. DDX4 (Vasa ortholog in humans) mutations in the cancer cell genome have been reported so far in 55 cancer cell types, which include prostate, ovarian, breast, glioma, melanoma, uterine, and many other cancer cell lines or tissues (Fig. 2, Identified by the cBioPortal search; Gao et al., 2013; Cerami et al., 2012). Through this genomic mutation-based search, however, the cause or consequence of these DDX4 mutations in cancer cells appears rather random; some cancer cell lines such as prostate cancer cells lack ddx4, whereas other cells such as breast cancer cell lines duplicate ddx4 genes in the genome. It is thus unlikely that DDX4 is a cause of cancer initiation nor is consistently involved in a major step of cancer cell development, yet rather might contribute to tumorigenesis as an important translation factor when expressed in the context of other translation factors of the cancer cell phenotype. Considering that Vasa's post-transcriptional modifications are critical to development of various somatic cells, it is important to rely on a protein level database for analyzing Vasa function in cancer cells. Other germ line factors may also be resolved with such an approach. In concert with metabolomics studies, one could then potentially predict even more close associations with tumorigenesis.

Fig 2.

The cBioPortal (http://www.cbioportal.org/) database search result. Cancer cell types that contain mutations in the DDX4 (Vasa) gene in the genome are listed. X-axis lists cancer type, and Y-axis indicates the frequency of the alterations in the cancers. Mutations of DDX4 in the genome is indicated in green, deletion in blue, amplification in red, and multiple alterations in gray colored balls indicate cancer cell types. The text in parenthesis indicates the original data source of each cancer cell.

In conclusion, most cells of the body do not have detectable level of Vasa, but when Vasa protein is expressed in somatic and/or tumorgenic cells, Vasa is essential for cellular regulation. We think of this as a Vasa addiction that perhaps alters the cellular metabolism, including translational regulation. Were Vasa protein altered in those addicted cells, the cell then crashes and burns. Such theoretical compensatory regulation within these cells will inform us as to how Vasa fundamentally functions in the cell. Further study of the determinants of Vasa localization and activity in various somatic cells including cancerous cells should help elucidate why ectopic Vasa expression can be detrimental.

Highlights.

• Vasa function in somatic cell regulation.

• Vasa in some cases contribute to tumorigenesis.

• Post-transcriptional regulation of Vasa is a key for this gene function.