We have now characterized the tumor phenotype of all 28 zebrafish lines in our collection with heterozygous mutations in ribosomal protein genes and found that 17 of these are tumor-prone, displaying an incidence of zMPNSTs that is far higher than that of wild-type fish. As noted previously (Amsterdam et al., 2004
), these tumor-prone lines include mutants for proteins in both the large and small ribosomal subunits. We did not find a correlation between the severity of the rp
mRNA reduction in the homozygous rp
mutants and the tumor incidence of the adult heterozygotes. As noted, however, a large difference in the degree of rp
message reduction between homozygotes from two rp
lines may correspond to a much smaller difference between the respective heterozygotes. Since it has been shown that mRNAs encoding certain rps are normally produced in excess (Bortoluzzi et al., 2001
; Thorrez et al., 2008
), a reduction of the mRNA encoding such rps to just over half the wild-type level in heterozygous fish may have no adverse consequences.
Intriguingly, we found that the most highly tumor-prone lines have a significant impairment of growth. In one high-tumor line, hi258 (rpL35), the growth defect was apparent as early as the embryonic stages. Furthermore, this growth impairment is detected at the cellular level, as rp heterozygous cells from another high-tumor line, hi10 (rpL36a), are out-competed by wild-type cells in chimeric embryos. This defect in growth of the hi10 (rpL36a) heterozygous cells appeared to worsen over time, suggesting that the growth impairment observed in day 4 heterozygous embryos that persists into adulthood reflects an intrinsic defect at the cellular level. The observed defect in growth, coupled with the fact that a ribosomal protein gene is mutated, strongly suggests that the underlying defect is an impairment in protein translation.
A number of rp
flies and RpL24
heterozygous mice are smaller at the organismal level but do not have smaller cells (Oldham et al., 2000
; Oliver et al., 2004
). Similarly, an analysis of cell size in wild-type and rp
heterozygous zebrafish showed a greater variation within fish of the same genotype than between genotypes (data not shown). This suggests that rp
heterozygotes are smaller because they have fewer cells, not smaller cells, which is consistent with our finding that rp
heterozygous cells are out-competed by wild-type cells. One possible connection between the observed growth defect and tumorigenesis is that a mutation that would not normally confer a growth advantage might do so amongst a population of growth-impaired cells (Bilousova et al., 2005
). Thus, the growth defect might simply make these cells more susceptible to oncogenic mutations.
On the other hand, tumorigenesis might be a direct result of a translation defect. While we have thus far been unable to directly demonstrate experimentally that protein translation is reduced in the rp
heterozygotes [nor is overall protein synthesis reduced in the tumors (MacInnes et al., 2008
)], there is precedent to suggest that even subtle defects in global translation can have dramatic consequences. For example, tumor cells treated with the rapamycin analog RAD001 are impaired only slightly in global translation, but this is enough to reduce the translation of p21 such that its induction is inhibited after DNA damage (Beuvink et al., 2005
). In addition, it is possible that a translation defect could be manifested in the rp
heterozygotes by a selective translation of mRNAs; i.e.
, under conditions of impaired translational capacity, certain messages may be translated at the expense of others that would normally also be translated (Rajasekhar et al., 2003
). Interestingly, zMPNSTs from rp
heterozygous fish contain p53 mRNA but do not express detectable levels of p53 protein, even under conditions (DNA damage, proteasome inhibition) which should prevent its degradation (MacInnes et al., 2008
). It is possible that certain cell types, such as the cell of origin (presumably nerve sheath cells) that give rise to zMPNSTs, may be more susceptible to a translation defect than other cell types. Alternatively, it may be that translation in the cell of origin is less profoundly affected than in other cells. In this model, most of the cells in the organism could have a global decrease in protein synthesis, resulting in the organismal growth defect, while nerve sheath cells might only lose expression of select messages (such as p53) without compromising their growth properties.
Either of these models might also explain why the correlation between growth impairment and tumor susceptibility is not absolute. As noted, three lines displayed a growth impairment, but were not tumor-prone [hi2430
), and hi3893
)]. In addition, there were three other lines that were prone to develop zMPNSTs, but were not significantly growth-impaired [hi1026
), and hi1987
)] (). These data suggest the possibility that while a general growth impairment predisposes to tumor development, tumorigenesis also depends on some unknown modifying factors that may be cell-type specific. As different rp
s are expressed at different levels in different cell types (Bortoluzzi et al., 2001
; Thorrez et al., 2008
), it is possible that an alteration of a given rp
gene dosage that affects protein synthesis in most cells sufficiently to cause an organismal growth phenotype might not result in a tumor-promoting level of translation impairment in the nerve sheath cells.
Another possible explanation for the finding that three rp heterozygous lines had a moderate incidence of zMPNSTs but no observable growth impairment is that there may be stochastic variation in the severity of the translation defect among individual fish. It is important to realize that the average weight of a population of rp heterozygotes was used to determine whether the line had an overall growth impairment. As shown for the hi258 line, there certainly exists a large variation in size among the heterozygotes (). It is currently not known, and will be important to determine, if the fish that are smallest at a young age are the ones that will eventually develop tumors. It will also be interesting to determine whether the size of the fish correlates with the time to onset of cancer. Thus, there may exist an even finer correlation between growth and tumorigenicity than we have discovered thus far.
Regardless of the precise mechanism of tumor promotion, our data show that heterozygosity of numerous rp genes causes growth impairment at the organismal level reminiscent of the Minute phenotype of rp mutant flies and mice. This growth defect is predictive of a predisposition to develop zMPNSTs and clearly precedes tumorigenesis. In addition, these mutant lines may be useful to use as recipients in cell transplant experiments where it is desirable to have a greater expansion of the donor cell population, similar to the use of Minute strains in Drosophila.