The retinoblastoma protein (pRB) negatively regulates cell proliferation by limiting the activity of the family of E2F transcription factors. In Drosophila, mutation of the DEAD-box helicase belle (bel) relieves an E2F/pRB induced G1 cell cycle arrest; however, the mechanism of this rescue is unknown. Here, we show that the level of the cyclin-dependent kinase inhibitor Dacapo (Dap), homolog of mammalian p21/p27, is strongly reduced both in bel mutant cells in vivo and in tissue culture cells depleted of Bel by RNA interference. Interestingly, the loss of bel also partially alleviates an ectopically induced G1 cell cycle arrest. Additionally, we show that Bel undergoes nucleocytoplasmic shuttling. Thus, inactivation of bel renders cells less sensitive to several anti-proliferative signals inducing G1 arrest.

RNA interference (RNAi) is a regulatory gene silencing system found in nearly all eukaryotic organisms that employs small RNAs, typically 20–25 nucleotides long, to target complementary sequences found in mRNAs. RNA helicases use ATP to unwind double-stranded RNA (dsRNA), and are known to participate at nearly every level of RNA metabolism. A multitude of RNA helicases have been isolated from screens for essential RNAi factors, and even the earliest models of the RNAi pathway have presumed an RNA helicase to function at the level of small RNA duplex unwinding. However, while many components that function in RNAi have been uncovered and characterized, the exact placement in the pathway and ascription of a specific biochemical function of an RNA helicase in RNAi remains elusive. Recent studies have delved deeper into the precise role of some RNA helicases. Surprisingly, these studies have revealed nontraditional roles, which may not even require the helicase activity. Such findings suggest that RNA helicases regulate gene silencing at nearly every level of the RNAi pathways.

Summary

Background

The Drosophila archipelago gene (ago) encodes the specificity component of a ubiquitin-ligase that targets the Cyclin E and dMyc proteins for degradation. Its human ortholog Fbw7 is commonly lost in many cancers, suggesting that failure to degrade ago/Fbw7 targets leads to excess tissue growth.

Results

Here we show that although loss of ago induces hyperplasia of some organs, it paradoxically shrinks the size of the adult eye. We find that this reflects a requirement for ago to restrict apoptotic activity of the rbf1/e2f1 pathway adjacent to the eye-specific morphogenetic furrow: ago mutant cells display elevated de2f1 activity, express the pro-death dE2f1 targets hid and rpr, and undergo high rates of apoptosis. This death and the resulting small-eye phenotype are dependent on rbf1, de2f1, hid, and the rbf1/de2f1 regulators cyclin E and dacapo, but are independent of dp53. A transactivation-deficient de2f1 allele blocks MF-associated apoptosis of ago mutant cells but does not retard their clonal overgrowth, indicating that intact de2f1 function is required for the death but not overproliferation of ago cells. Alleles of EGFR and wg pathway components further modulate the ago apoptotic and eye size phenotypes, suggesting these pathways control rates of de2f1-driven apoptosis among ago mutant cells.

Conclusions

These data show that ago loss requires a collaborating block in cell death to efficiently drive tissue overgrowth and that this conditional growth-suppressor phenotype reflects a role for the gene in restricting apoptotic output of the rbf1/de2f1 pathway. Moreover, the susceptibility of ago mutant cells to succumb to this apoptotic program appears to depend on local variations in extracellular signaling that could thus determine tissue-specific fates of ago mutant cells.

Previous studies in Drosophila melanogaster have demonstrated that many tumor suppressor pathways impinge on Rb/E2F to regulate proliferation and survival. Here, we report that Tuberous Sclerosis Complex 1 (TSC1), a well-established tumor suppressor that regulates cell size, is an important regulator of dE2F1 during development. In eye imaginal discs, the loss of tsc1 cooperates with rbf1 mutations to promote ectopic S-phase and cell death. This cooperative effect between tsc1 and rbf1 mutations can be explained, at least in part, by the observation that TSC1 post-transcriptionally regulates dE2F1 expression. Clonal analysis revealed that the protein level of dE2F1 is increased in tsc1 or tsc2 mutant cells and conversely decreased in rheb or dTor mutant cells. Interestingly, while s6k mutations have no effect on dE2F1 expression in the wild-type background, S6k is absolutely required for the increase of dE2F1 expression in tsc2 mutant cells. The canonical TSC/Rheb/Tor/S6k pathway is also an important determinant of dE2F1-dependent cell death, since rheb or s6k mutations suppress the developmentally regulated cell death observed in rbf1 mutant eye discs. Our results provide evidence to suggest that dE2F1 is an important cell cycle regulator that translates the growth-promoting signal downstream of the TSC/Rheb/Tor/S6k pathway.

Author Summary

Tuberous Sclerosis Complex genes 1 (TSC1) is a downstream component of the Insulin Receptor signaling pathway that is often deregulated in many tumors. In this study, we discovered that the fruit fly homolog of TSC1 regulates E2F transcription factor by controlling protein expression. E2F family proteins are key regulators of cellular division, and other tumor promoting events are previously shown to regulate E2F activity. Our findings demonstrate the importance of altering the E2F activity during tumorigenesis and provide new insights into the crosstalk between tumor promoting events.

Functional inactivation of the Retinoblastoma (pRB) pathway is an early and obligatory event in tumorigenesis. The importance of pRB is usually explained by its ability to promote cell cycle exit. Here, we demonstrate that, independently of cell cycle exit control, in cooperation with the Hippo tumor suppressor pathway, pRB functions to maintain the terminally differentiated state. We show that mutations in the Hippo signaling pathway, wts or hpo, trigger widespread dedifferentiation of rbf mutant cells in the Drosophila eye. Initially, rbf wts or rbf hpo double mutant cells are morphologically indistinguishable from their wild-type counterparts as they properly differentiate into photoreceptors, form axonal projections, and express late neuronal markers. However, the double mutant cells cannot maintain their neuronal identity, dedifferentiate, and thus become uncommitted eye specific cells. Surprisingly, this dedifferentiation is fully independent of cell cycle exit defects and occurs even when inappropriate proliferation is fully blocked by a de2f1 mutation. Thus, our results reveal the novel involvement of the pRB pathway during the maintenance of a differentiated state and suggest that terminally differentiated Rb mutant cells are intrinsically prone to dedifferentiation, can be converted to progenitor cells, and thus contribute to cancer advancement.

Author Summary

The inability to respond to growth inhibitory cues is one acquired trait of a cancer cell. Almost all such signals are eventually routed through the Retinoblastoma (pRB) tumor suppressor pathway. Therefore, inactivation of the pRB pathway is considered to be an early and obligatory event during transformation of a normal cell into a malignant cancer cell. In this study, we found that inactivation of the Hippo pathway makes Rb mutant cells prone to undergo morphological changes and to become less differentiated, progenitor-like cells. Furthermore, we show that this was independent of the failure of Rb mutant cells to properly respond to cell cycle exit cues. These results are significant since, in general, tumors containing progenitor-like cells have a higher potential to progress through later stages of tumorigenesis and to become more aggressive and more deadly. Thus, the inactivation of Rb not only renders cells insensitive to growth inhibitory signals, but also sensitizes cells to revert to a progenitor-like state.

The Hippo pathway negatively regulates the cell number in epithelial tissue. Upon its inactivation, an excess of cells is produced. These additional cells are generated from an increased rate of cell division, followed by inappropriate proliferation of cells that have failed to exit the cell cycle. We analyzed the consequence of inactivation of the entire E2F family of transcription factors in these two settings. In Drosophila, there is a single activator, dE2F1, and a single repressor, dE2F2, which act antagonistically to each other during development. While the loss of the activator dE2F1 results in a severe impairment in cell proliferation, this defect is rescued by the simultaneous loss of the repressor dE2F2, as cell proliferation occurs relatively normally in the absence of both dE2F proteins. We found that the combined inactivation of dE2F1 and dE2F2 had no significant effect on the increased rate of cell division of Hippo pathway mutant cells. In striking contrast, inappropriate proliferation of cells that failed to exit the cell cycle was efficiently blocked. Furthermore, our data suggest that such inappropriate proliferation was primarily dependent on the activator, de2f1, as loss of de2f2 was inconsequential. Consistently, Hippo pathway mutant cells had elevated E2F activity and induced dE2F1 expression at a point when wild-type cells normally exit the cell cycle. Thus, we uncovered a critical requirement for the dE2F family during inappropriate proliferation of Hippo pathway mutant cells.

Author Summary

The E2F transcription factor family is considered to be the best-characterized downstream target of the retinoblastoma protein (pRB). The pRB pathway is functionally inactivated in most tumor cells, and it is thought that unrestrained activity of E2F drives inappropriate proliferation in tumors. We utilized the relative simplicity of the Drosophila model to determine the role of the dE2F family in proliferation of cells following inactivation of the recently identified Hippo tumor suppressor pathway. We found that Hippo pathway mutant cells require the dE2F family to delay the cell cycle exit and to proliferate inappropriately when wild-type cells enter quiescence. This is significant since the loss of the entire dE2F family exerts almost no effect on the ability of Hippo pathway mutations to accelerate proliferation of actively dividing cells. Thus, the importance of the dE2F family in cells with an inactivated tumor suppressor pathway varies in different contexts. This discovery may have implications in designing anti-cancer therapies that inhibit E2F activity.

In Drosophila melanogaster, the loss of activator de2f1 leads to a severe reduction in cell proliferation and repression of E2F targets. To date, the only known way to rescue the proliferation block in de2f1 mutants was through the inactivation of dE2F2. This suggests that dE2F2 provides a major contribution to the de2f1 mutant phenotype. Here, we report that in mosaic animals, in addition to de2f2, the loss of a DEAD box protein Belle (Bel) also rescues proliferation of de2f1 mutant cells. Surprisingly, the rescue occurs in a dE2F2-independent manner since the loss of Bel does not relieve dE2F2-mediated repression. In the eye disc, bel mutant cells fail to undergo a G1 arrest in the morphogenetic furrow, delay photoreceptor recruitment and differentiation, and show a reduction of the transcription factor Ci155. The down-regulation of Ci155 is important since it is sufficient to partially rescue proliferation of de2f1 mutant cells. Thus, mutation of bel relieves the dE2F2-mediated cell cycle arrest in de2f1 mutant cells through a novel Ci155-dependent mechanism without functional inactivation of the dE2F2 repressor.

E2F and retinoblastoma tumor suppressor protein pRB are important regulators of cell proliferation; however, the regulation of these proteins in vivo is not well understood. In Drosophila there are two E2F genes, an activator, de2f1, and a repressor, de2f2. The loss of de2f1 gives rise to the G1/S block accompanied by the repression of E2F-dependent transcription. These defects can be suppressed by mutation of de2f2. In this work, we show that the de2f1 mutant phenotype is rescued by the loss of the pre-mRNA splicing factor SR protein B52. Mutations in B52 restore S phase in clones of de2f1 mutant cells and phenocopy the loss of the de2f2 function. B52 acts upstream of de2f2 and plays a specific role in regulation of de2f2 pre-mRNA splicing. In B52-deficient cells, the level of dE2F2 protein is severely reduced and the expression of dE2F2-dependent genes is deregulated. Reexpression of the intronless copy of dE2F2 in B52-deficient cells restores the dE2F2-mediated repression. These results uncover a previously unrecognized role of the splicing factor in maintaining the G1/S block in vivo by specific regulation of the dE2F2 repressor function.

To gain insight into the essential functions of E2F, we have examined the phenotypes caused by complete inactivation of E2F and DP family members in Drosophila. Our results show that dDP requires dE2F1 and dE2F2 for DNA-binding activity in vitro and in vivo. In tissue culture cells and in mutant animals, the levels of dE2F and dDP proteins are strongly interdependent. In the absence of dDP, the levels of dE2F1 and dE2F2 decline dramatically, and vice versa. Accordingly, the cell cycle and transcriptional phenotypes caused by targeting dDP mimic the effects of targeting both dE2F1 and dE2F2 and are indistinguishable from the effects of inactivating all three proteins. Although trans-heterozygous dDP mutant animals develop to late pupal stages, the analysis of somatic mutant clones shows that dDP mutant cells are at a severe proliferative disadvantage when compared directly with wild-type neighbors. Strikingly, the timing of S-phase entry or exit is not delayed in dDP mutant clones, nor is the accumulation of cyclin A or cyclin B. However, the maximal level of bromodeoxyuridine incorporation is reduced in dDP mutant clones, and RNA interference experiments show that dDP-depleted cells are prone to stall in S phase. In addition, dDP mutant clones contain reduced numbers of mitotic cells, indicating that dDP mutant cells have a defect in G2/M-phase progression. Thus, dDP is not essential for developmental control of the G1-to-S transition, but it is required for normal cell proliferation, for optimal DNA synthesis, and for efficient G2/M progression.