Ectopic Expression of Germline Genes Drives Malignant Brain Tumor Growth in Drosophila

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Science  24 Dec 2010:
Vol. 330, Issue 6012, pp. 1824-1827
DOI: 10.1126/science.1195481


Model organisms such as the fruit fly Drosophila melanogaster can help to elucidate the molecular basis of complex diseases such as cancer. Mutations in the Drosophila gene lethal (3) malignant brain tumor cause malignant growth in the larval brain. Here we show that l(3)mbt tumors exhibited a soma-to-germline transformation through the ectopic expression of genes normally required for germline stemness, fitness, or longevity. Orthologs of some of these genes were also expressed in human somatic tumors. In addition, inactivation of any of the germline genes nanos, vasa, piwi, or aubergine suppressed l(3)mbt malignant growth. Our results demonstrate that germline traits are necessary for tumor growth in this Drosophila model and suggest that inactivation of germline genes might have tumor-suppressing effects in other species.

The Drosophila tumor-suppressor gene lethal (3) malignant brain tumor [l(3)mbt] was identified as a temperature-sensitive mutation that caused malignant growth in the larval brain (1). Other l(3)mbt mutant alleles obtained later show the same temperature-sensitive phenotype (2). L(3)mbt’s closest homologs, Drosophila Scm (Sex comb on midleg) and Sfmbt (Scm-related gene containing four mbt domains), encode Polycomb Group proteins (3). L3MBTL1, the human homolog of Drosophila L(3)MBT (3), is a transcriptional repressor (4) that is found in a complex with core histones, heterochromatin protein 1γ (HP1γ), and RB (Retinoblastoma protein) and can compact nucleosomes (5). Drosophila L(3)MBT is a substoichiometric component of the dREAM-MMB complex, which includes the two Drosophila Retinoblastoma-family proteins and the Myb-MuvB (MMB) complex (6). Depletion of components of the dREAM/MMB complex in Drosophila Kc cells by RNA interference results in genome-wide changes in gene expression (7). These data strongly suggest that l(3)mbt function might contribute to establishing and maintaining certain differentiated states through the stable silencing of specific genes (3, 7).

To identify the genes whose misexpression might account for the growth of l(3)mbt tumors (henceforth referred to as mbt tumors), we carried out genome-wide gene expression profiling of l(3)mbtE2 and l(3)mbtts1 homozygous and transheterozygous larval brains raised at restrictive temperature (29°C). We also analyzed l(3)mbtts1 tumors at the 1st, 5th, and 10th rounds of allograft culture in adult flies (T1, T5, and T10, respectively). Brains from homozygous white1118 (w1118), l(3)mbtE2, or l(3)mbtts1 larvae raised at permissive temperature (17°C) were used as controls. For comparison, we also profiled larval brain malignant neoplasms caused by mutation in brain tumor (brat) as well as allograft cultures at T1,T5, and T10 of tumors caused by mutants in brat, lethal giant larvae (lgl), miranda (mira), prospero (pros), and partner of inscuteable (pins) (8).

Hierarchical clustering plots of these data (table S1) reveal three distinct clusters that include control larval brains, mbt larval brain tumors, and cultured l(3)mbtts1 tumors, respectively (fig. S1). From these data, we identified 151 genes that were either overexpressed (n = 125) or underexpressed (n = 26) in all three larval mbt tumor types compared to all three controls (table S2). From this list, we removed those genes that were also up- (n = 23) or down-regulated (n = 14) in larval brat neoplasms and, hence, likely to encode functions generally required for larval brain tumor growth. We refer to the expression levels of the remaining 102 up-regulated genes as the mbt signature (MBTS) (table S3). MBTS is notably enhanced in cultured mbt tumors and can be used unequivocally to distinguish mbt tumors from other cultured malignant brain neoplasms like lgl, mira, pros, pins, or brat (Fig. 1A and table S3). Individual MBTS genes, however, are also up-regulated in some of these tumors.

Fig. 1

Gene expression profile of mbt tumors. (A) Heatmap of expression levels of the genes that are most significantly up-regulated in larval brain mbt tumors (mbt tumor signature, MBTS). Samples include wild-type larval brains, larval mbt tumors, and different types of larval brain malignant neoplasms in the 1st, 5th, and 10th rounds of allograft culture (T1, T5, and T10, respectively). (B) MBTS genes with known germline functions.

The function of most MBTS genes remains unknown. However, a quarter of them (26 of 102) are genes required in the germ line (Fig. 1B and table S4A). For instance, nanos (nos), female sterile(1)Yb (fs(1)Yb), and zero population growth (zpg) function in the establishment of the pole plasm in the egg and cystoblasts differentiation (9). The gonad-specific thioredoxins ThioredoxinT (TrxT) and deadhead (dhd), giant nuclei (gnu), corona (cona), hold'em (hdm), matotopetli (topi), and the female germline-specific γTUB37C isoform function during oocyte differentiation, meiosis, and syncytial embryo development (1015). Also piwi, aubergine (aub), krimper (krimp), and tejas (tej) are involved in the biogenesis of Piwi-interacting RNAs (piRNAs) that protect germline cells against transposable elements and viruses (16, 17). Some of these genes also have functions that are not germline related. For instance, some piwi alleles display synthetic lethality (18), and nos is required during nervous system development (19).

Driven by the high percentage of MBTS genes that have germline functions, we looked for other germline-related genes that do not meet the stringent criteria applied to select the 102 MBTS genes, but are overexpressed in mbt tumors (table S4B). Among these we found the genes that encode the synaptonemal complex protein Crossover suppressor on 3 of Gowen [C(3)G] and the cell cycle kinase Pan gu (PNG), which interact with the proteins encoded by the MBTS genes cona and gnu, respectively (11, 13). The same applies to Squash (SQU), Spindle-E (SPN-E), Maelstrom (MAEL), and AGO3, components of the piRNA machinery, which colocalize with other MBTS proteins in nuage (16, 17).

To determine whether the mRNAs that we found ectopically expressed in mbt tumors are translated, we checked for protein expression with a selected number of currently available antibodies. Given the key role of VASA in the assembly of the pole plasm and germline development (20), we included it in this study, even though vasa mRNA levels are not significantly increased in mbt tumors. By Western blot, we confirmed that PIWI, AUB, and VASA are ectopically expressed in mbt tumors (Fig. 2A). Immunofluorescence studies also revealed the ectopic expression in l(3)mbtts1 brains raised at 29°C of C(3)G, SQU, and VASA (Fig. 2B). These results show that some of the germline genes ectopically expressed in mbt tumors are translated. However, we have not been able to confirm the expression of other proteins, including MAEL, ORB, BAM, GNU, and TOPI, which suggests that, possible technical problems aside, either the corresponding mRNAs are not translated or these proteins might be unstable in such an ectopic environment. The expression of VASA, by contrast, suggests that other mRNAs whose levels are not appreciably increased in mbt tumors might actually be ectopically translated.

Fig. 2

Ectopic expression of germline proteins in l(3)mbtts1 larval brain tumors. (A) Western blot. PIWI, AUB, and VASA are ectopically expressed in l(3)mbtts1 brain tumors. αTUB is used as a loading control. (B) Immunofluorescence. VASA, SQU, and C(3)G are overexpressed in l(3)mbtts1 brains raised at 29°C. Low-magnification views (left) reveal VASA staining concentrated in the outer proliferative center (OPC) and in undifferentiated cells of the central brain (CB) (scale bar, 50 μm). High-magnification views (middle and right) show that SQU and C(3)G localize in the cytoplasm and on condensed chromatin, respectively (scale bar, 10 μm). Brains were counterstained with DAPI (4′,6′-diamidino-2-phenylindole) (DNA) and antibodies against the neuroblast marker MIRA.

Prompted by the expression in l(3)mbtts1 brains of several genes involved in the biogenesis and regulation of piRNAs, we sequenced 23- to 30-nucleotide RNAs from l(3)mbtts1 larval brain tumors and from wild-type brains and ovaries. We found 117 known piRNAs and microRNAs (miRNAs) in l(3)mbtts1 larval brain tumor samples (table S5). Of these, 31 are either not expressed in wild-type brains or are expressed there at less than 10% their level in larval brain tumors. Most of them are highly expressed in wild-type ovaries, thus substantiating further the ectopic acquisition of germline traits that characterizes mbt tumors.

We do not know which, if any, of the germline genes that are up-regulated in mbt tumors are direct targets of l(3)mbt or if their ectopic expression is a downstream consequence of intermediate events. The putative direct targets of l(3)mbt are many. The dREAM-MMB complex, of which L(3)MBT is a substoichiometric component (6), has been found to be promoter-proximal to 32% of Drosophila genes, and MMB factors are known to regulate transcription of a wide range of genes in Drosophila Kc cells (7). In addition, we do not have an estimate for the number of proteins like VASA that, despite their low mRNA expression levels, might be up-regulated in mbt tumors. Indeed, many of these genes, as well as the piRNAs and miRNAs expressed in mbt tumors, might themselves regulate the basal transcription and translation machineries, adding a further layer of gene expression modulation (2123).

We then determined the extent to which ectopic expression of germline genes contributes to mbt tumor growth. To this end, we first quantified larval brain growth in individuals that were mutant for l(3)mbtts1 alone, or double mutant for l(3)mbtts1 and one of several of the germline genes that are ectopically expressed in mbt tumors (Fig. 3). Measured as the total amount of protein, the average brain size in l(3)mbtts1 (21 ± 6 μg of protein per brain, n = 5) is about seven times as large (P < 0.0001) as that in control w1118 larvae, a difference that is not significantly reduced by the additional loss of zpg, Pxt, or AGO3. However, brain overgrowth is reduced to a size similar to that of the control in l(3)mbtts1 larvae that are also mutant for either piwi (P < 0.0001), vasa (P < 0.0001), aub (P = 0.0003), or nos (P = 0.001) (Fig. 3). The loss of piwi does not prevent brain overgrowth in brat k06028 mutant larvae (P = 0.72). We then quantified tumor growth after allograft in adult flies (Fig. 3). The frequency with which l(3)mbtts1 homozygous larval brain tissue develops tumors in this assay (70%, n = 67) is not significantly reduced by the additional loss of zpg or AGO3 and is only moderately reduced by the loss of Pxt (P = 0.03), but it is markedly reduced by the additional loss of piwi (P < 0.0001), vasa (P < 0.0001), aub (P = 0.0002), or nos (P < 0.0001). The frequency of brat k06028 tumor formation (80%, n = 10) is not affected by the loss of piwi (73%, n = 15) or nos (86%, n = 7, P = 1). These results demonstrate that the ectopic expression of germline genes, particularly piwi, vasa, nos, and aub, significantly contributes to mbt tumor growth.

Fig. 3

The role of ectopically expressed germline genes in mbt tumor growth. Brain micrographs were taken from larvae of the corresponding genotypes raised at 29°C (scale bars, 100 μm). Larval brain size is shown as mean ± SD (in micrograms) of protein per brain (n = number of brains). Adult fly micrographs were taken 10 days after implantation of green fluorescent protein (GFP)–labeled larval brain tissue. In the absence of tumor growth, GFP signal is either undetectable or is localized to a very small piece of green tissue that is about the size of the implant (arrows). Tumor growth was quantified as the percentage (%) of n hosts in which the implanted tissue (green) grew over the entire abdomen of the host. P-values refer to the difference between each double-mutant combination and l(3)mbtts1, or between piwi1 bratK06028 and bratk06028.

A closely reminiscent soma-to-germline transformation observed in mutants in the Caenorhabditis elegans Rb homolog LIN-35, as well as in long-lived C. elegans strains (20, 24, 25), has led some to propose that the acquisition of germline characteristics by somatic cells might contribute to increased fitness and survival, a mechanism that could contribute to the transformation of mammalian cells (24, 25). Also in humans, some genes that are predominantly expressed in germline cells and have little or no expression in somatic adult tissues become aberrantly activated in various malignancies, including melanoma and several types of carcinomas (26, 27). These are known as cancer-testis (CT) genes or cancer-germline (CG) genes (28). A subset of these CG genes encode antigens that are immunogenic in cancer patients and are being pursued as biomarkers and as targets for therapeutic cancer vaccines (29, 30).

Human CG genes are suspected to contribute to oncogenesis germline traits like immortality, invasiveness, and hypomethylation (28), but their actual role in cancer remains unknown. Our results demonstrate that ectopic germline traits are necessary for tumor growth in Drosophila mbt tumors, suggesting that their inactivation might have tumor-suppressing effects in other species. Some germline genes up-regulated in mbt tumors are orthologs of human CG genes like PIWIL1/piwi (31, 32), NANOS1/nanos (33), and SYCP1 /c(3)G (34). The list of genes up-regulated in mbt tumors includes many other germline genes that might also be relevant in human cancer.

Supporting Online Material

Material and Methods

Fig. S1

Tables S1 to S5


References and Notes

  1. We thank E. Gateff, R. Lehmann, P. Zamore, A. Spradling, F. Azorin, P. Lasko, S. Hawley, M. Siomi, T. Kai, T. Orr-Weaver, H. White-Cooper, D. McKearin, T. Schupbach, Hybridoma Bank, and Bloomington and Tübingen Drosophila Stock centers for antibodies and fly stocks; H. Auer, the IRB Functional Genomics Facility, and the European Molecular Biology Laboratory Genomics Core Facility for invaluable technical guidance; J. Januschke and members of our laboratory for discussions; and M. Llamazares for proofreading. Work in our laboratory is supported by ONCASYM-037398, BFU2006-05813, BFU2009-07975, SGR2005, SRG200, CEN-20091016, and Consolider-Ingenio CSD2006-23. A.J. is a recipient of a Ministerio de Ciencia e Innovacion Formacion de Personal Investigador fellowship. Array data sets are deposited at Gene Expression Omnibus (accession no. GSE24917).
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