Regulation of cell growth by Notch signaling and its differential requirement in normal vs. tumor-forming stem cells in Drosophila

Abstract

Cancer stem cells (CSCs) are postulated to be a small subset of tumor cells with tumor-initiating ability that shares features with normal tissue-specific stem cells. The origin of CSCs and the mechanisms underlying their genesis are poorly understood, and it is uncertain whether it is possible to obliterate CSCs without inadvertently damaging normal stem cells. Here we show that a functional reduction of eukaryotic translation initiation factor 4E (eIF4E) in Drosophila specifically eliminates CSC-like cells in the brain and ovary without having discernable effects on normal stem cells. Brain CSC-like cells can arise from dedifferentiation of transit-amplifying progenitors upon Notch hyperactivation. eIF4E is up-regulated in these dedifferentiating progenitors, where it forms a feedback regulatory loop with the growth regulator dMyc to promote cell growth, particularly nucleolar growth, and subsequent ectopic neural stem cell (NSC) formation. Cell growth regulation is also a critical component of the mechanism by which Notch signaling regulates the self-renewal of normal NSCs. Our findings highlight the importance of Notch-regulated cell growth in stem cell maintenance and reveal a stronger dependence on eIF4E function and cell growth by CSCs, which might be exploited therapeutically.

Figure 1.

N-dependent cell growth is required for type II NB maintenance. (A) A schematic drawing of Drosophila late larval CNS showing type I and type II NB lineages within the central brain area. (CB) Central brain; (OL) optical lobe; (VNC) ventral nerve cord. (B) A diagram of a type II NB lineage. Distinct cell types within the hierarchy can be identified by using combinations of cell fate markers: type II NB: Dpn (Deadpan, red)⁺, Pros (Prospero, blue)⁻; immature IPs: Dpn⁻, Pros⁻; mature IPs: Dpn⁺, cytoplasmic Pros; GMC or neurons: Dpn⁻, nuclear Pros. (C) N reporter E(spl)mγ-GFP (green) expression in type II NB lineage (mature IPs, closed arrowheads) showing differential N activity in different cell types. Dlg staining (purple) outlines the cell cortex. (White closed arrowheads) Mature IPs. From this panel on, NBs are marked with brackets and immature IPs are marked with white open arrowheads. (D–G) Clonal analysis of type II NBs of spdo (D) or aph-1 (E) mutants at various times ACI. Newly born daughter cells are marked with closed arrowheads. (F) Wild-type NBs served as control. (H,I) Analysis of the nucleoli (red; anti-fibrillarin) of the NBs and their daughter cells within spdo mutant (I) or wild-type (H) clones. (J) Quantification of nucleolar/cellular volume ratio of wild-type or spdo mutant NBs. (*) P < 0.002 versus control in Student's t-test; n = 6–8. (K) A working model depicting N regulation of NB fate through control of their cellular and nucleolar sizes. (Orange dot) Nucleolus; (red) NB; (green) immature IP; (purple) mature IP; (blue) GMCs or neurons. Bar, 10 μm.

Figure 2.

N signaling promotes cell growth and dedifferentiation of IPs into ectopic NBs. (A) Ectopic NBs (Dpn⁺, Pros⁻; yellow arrowheads) in ada mutant clones. From this panel on, yellow arrowheads mark ectopic NBs. (B) Ectopic NBs in ada mutant clones contain enlarged nucleoli compared with IPs in wild-type clones (white arrowheads). (C) Quantification of nucleolar/cellular volume ratio in normal or ectopic NBs within ada mutant or N overactivation clones. (*) P < 0.0001; n = 8–10. (D) CldU pulse and chase revealed a cell cycle delay of ectopic NBs in ada mutant clones. After a 20-h chase, CldU was undetectable in the primary NBs (bracket) within wild-type or ada mutant clones. The only cells retaining the CldU label (white arrowheads) in wild-type clones were terminally differentiated neurons furthest from the primary NB. In contrast, CldU was detectable in some ectopic NBs within ada mutant clones (bigger nucleoli; yellow arrowheads). (Green) CldU; (red) fibrillarin; (blue) GFP. (E) Cell lineage tracing showing that mature IPs of ada mutants could dedifferentiate back into type II NBs (yellow bracket; identified by the expression of NB marker Mira and the absence of mature IP and GMC marker Ase), while wild-type mature IPs only generate GMCs or neurons (closed arrowhead; Mira⁻). Note that Ase is specifically expressed in type I but not type II NBs. A type I NB (Ase⁺Mira⁺) is shown in the left panel. (F) A schematic model summarizing cell lineage tracing data shown in E. (Green) Mira labeling NBs, immature IPs, and mature IPs; (red) lacZ; (blue) Ase. In ada mutants, immature and mature IPs can dedifferentiate into type II NBs (purple arrows). (G) Ectopic NBs induced by N overactivation specifically in mature IPs (driven by Erm-GAL4) contain larger nucleoli (yellow arrowheads) than control IPs (white arrowhead). (H) A working model proposing that when N signaling is overactivated (N↑), immature IPs (or mature IPs, not shown) gradually increase cellular and nucleolar sizes and dedifferentiate into a stem cell. Bar, 10 μm.

Figure 3.

eIF4E knockdown efficiently and specifically inhibits brain tumor formation. (A) Effects of NB-specific knockdown of eIF4E (driven by 1407-GAL4) on ectopic NB formation in ada, brat, or lgl mutants or aPKC^CAAX overexpression backgrounds. (Green) NBs marked by Dpn; (red) neurons marked by Pros. Posterior views of a single brain lobe are shown. (B) eIF4E knockdown has no discernable effects on normal NB development or on ectopic NB formation resulting from symmetric division of type I NBs in cnn mutants. From this panel on, the yellow dotted line marks the boundary between the optic lobe (left) and the central brain (right) areas. Central brain NBs can be distinguished from optic lobe NBs based on their medial/superficial location in the brain and larger size. (C) Quantification of data from A and B. (*) P < 0.0001; n = 15–20. (D) Clonal analysis of type II NBs in wild-type, eIF4E mutant, N^act, or N^act; eIF4E backgrounds. (E) eIF4E expression (red) in wild-type, ada, or spdo mutant type II NBs. (F,G) Quantification of data from D and E. Bars: A,B, 100 μm; D, 20 μm; E, 10 μm.

Figure 4.

dMyc and eIF4E constitute a regulatory loop in NB regulation. (A) dMyc (red) expression in wild-type, ada, or spdo mutant type II NBs. The MARCM clones were analyzed at 48 h ACI. (B) dMyc-lacZ is highly expressed in the NB but not its daughter cells in a wild-type type II NB clone. In ada mutant clones, however, ectopic NBs (yellow arrowheads) also show up-regulated dMyc transcription. (Green) GFP; (red) dMyc-lacZ; (blue) Pros; (brackets) NBs. (C) Ectopic NB formation in brat mutants is suppressed by dMyc RNAi, and the tumor suppression effect of eIF4E knockdown is partially relieved when dMyc is overexpressed. (*) P < 0.0001; n = 15–20. (D) An increase in IP nucleolar size (green, open arrowheads) promoted by dMyc overexpression is strongly suppressed by eIF4E RNAi. (*) P < 0.0001; n = 30–40. (E) Up-regulation of eIF4E-lacZ expression in NBs and IPs by dMyc. (*) P < 0.015; n = 20–30. (F) Positive regulation of eIF4E protein levels by dMyc in IPs (open arrowheads). (*) P < 0.01; n = 20–30. (G) High eIF4E-lacZ expression in both wild-type NBs and ectopic NBs of ada mutant. (Green) eIF4E-lacZ; (red) Dpn. (H) eIF4E-lacZ expression in wild-type or ada mutant type II NB clones. (Green) GFP; (red) eIF4E-lacZ; (blue) Pros; (brackets) NBs. (I) A working model depicting the eIF4E–dMyc feedback regulatory loop in promoting cell growth within NBs. Bars: A,B,D–H, 10 μm; C, 100 μm.

Figure 5.

Biochemical characterization of the N-dMyc–eIF4E molecular circuitry. (A–C) ChIP with the anti-Su(H) antibody or a control IgG on wild-type third instar larval brain chromatin. (A) Schematic representation of the dmyc locus. (Orange rectangles) Coding regions; (gray rectangles) noncoding regions; (lines) introns; (blue bars) two putative Su(H)-binding sites matching the consensus sequence (C/T)GTGGGAA(A/C). Enrichment of Su(H) at the dmyc-A but not dmyc-B amplicon is determined by both standard PCR (B) and real-time quantitative PCR (C). A region in E(spl) containing Su(H)-binding sites (Bailey and Posakony 1995) and a region in the rp49 promoter without such sites serve as positive and negative controls, respectively. (**) P < 0.0001. (D,E) Coimmunoprecipitation between transfected human eIF4E and Drosophila dMyc (D) or human eIF4E and endogenous c-Myc (E) in HEK293T nuclear extracts. GFP serves as a negative control. (IP) Immunoprecipitation; (IB) immunoblotting. Input represents 4% of total. (F–H) ChIP with anti-dMyc antibody or a control IgG on wild-type third instar larval brain chromatin. (F) Schematic representation of the eIF4E locus. Blue bars represent a cluster of adjacent noncanonical E boxes. Enrichment of dMyc at the eIF4E locus is shown by standard PCR (G) and real-time quantitative PCR (H). A region in Nnp-1 harboring canonical E boxes CACGTG and a region within the rp49 promoter serve as positive and negative controls, respectively. (*) P < 0.01. Error bars indicate standard deviation of three independent experiments.

Figure 6.

The N-dMyc–eIF4E module operates in normal or ectopic NBs. (A) dMyc or eIF4E knockdown alone has a mild or no effect on NB cell growth, respectively, as assayed by their cell size, nucleolar size, or ratio of nucleolar/cell size, whereas knocking down both leads to a drastic reduction in NB cell growth. Overexpression of a wild-type form of the eIF4E transgene shows no discernible effect on NB cellular or nucleolar sizes. NB nucleoli are indicated by arrowheads. (Green) Dlg; (red) fibrillarin. (**) P < 0.0001; (*) P < 0.05; n = 30–40. (B) Knocking down either dMyc or eIF4E alone has no noticeable effect on type II NB maintenance, but knocking down both leads to premature differentiation and NB loss. (Green) Dpn; (red) Pros. (*) P < 0.001; n = 15–20. Magnified images are shown on the right, with individual NBs encircled with white dotted lines. (Arrowheads) Pros⁺ Dpn⁺ cell. (C) Type II NB lineages in wild-type, 1407>N RNAi; Rheb, or1407>N-IR; dMyc backgrounds are delineated by white dashed lines, and the NB in each lineage is indicated by a star. The yellow dotted line indicates the boundary between the optical lobe (left) and the central brain (right) region. Amplified images of a representative type II NB lineage in each genotype are shown at the bottom. Larvae were processed at 96 h ALH. (D) Quantification of type II NB number in wild-type, 1407>dMyc, 1407>N-IR, 1407>N-IR; dMyc, 1407>N-IR; GFP, or 1407>N-IR; Rheb brain lobes. (**) P < 0.0005; n = 15–20. Only dorsal–medial (DM) type II NBs generating surface layer progeny were counted. (E) A working model of dMyc overexpression preventing the stem cell fate loss in N signaling-defective NBs by boosting cell growth. (Orange dot) Nucleolus; (red) NB; (green) immature IP; (purple) mature IP; (blue) GMCs or neurons. (F) Effects of eIF4E inhibitor treatment on wild-type NB maintenance and ectopic NB formation induced by N overactivation or Ada inactivation. (*) P < 0.0001; n = 15–20. Bars: A,C, bottom panel, 10 μm; C, top panel, 50 μm; B,F, 100 μm.

Figure 7.

The dMyc–eIF4E regulatory loop also operates in Drosophila GSCs. (A) A schematic drawing of Drosophila early oogenesis. GSCs (pink) reside at the anterior tip of the germarium, directly contacting the stem cell niche (purple). The GSCs continuously self-renew and produce a daughter cell that moves away from the niche and becomes the differentiating CB (light pink), which gives rise to 16 post-mitotic cystocytes (CC; green). The GSCs and CBs can be identified by the presence of the membranous spectrosome (red spot), which develops into a branched fusome in differentiating cysts (Lin et al. 1994). (OO) Oocyte; (NC) nurse cells. (B) eIF4E (red) expression is enriched in the GSCs (open arrowhead, containing large nucleoli) compared with post-mitotic cysts (closed arrowhead, containing small nucleoli) in wild-type ovaries. In mei-P26^mfs1 mutants, eIF4E is highly expressed in all CSCs that contained enlarged nucleoli. (C) mei-P26^mfs1/1 mutant egg chambers often carry ectopic GSC-like cells (white arrowheads) marked by the spectrosomes. Such intermediate ovarian tumor phenotypes of mei-P26 can be effectively suppressed by removing one copy of eIF4E but not rheb. Note that the defect of irregular nurse cell number in mei-P26^mfs1/1 mutants is not suppressed by reduction of eIF4E function (yellow arrowhead). (Green) Vasa (germ cell-specific marker); (red) Hts (marker for the spectrosome and the fusome). (D) Statistical analysis of data shown in C; n = 450–500 ovarioles examined for each genotype. (E,F) mei-P26^fs1 mutants show a strong ovarian tumor phenotype (88.3% tumorous ovarioles [open arrowhead], 6.25% ovarioles containing nurse cells, 1.25% ovarioles containing eggs, and 4.2% empty ovarioles), which can be significantly suppressed by removing one copy of eIF4E (63.9% tumorous ovarioles, 26.0% ovarioles containing nurse cells [white arrowheads], 5.3% ovarioles containing eggs [yellow arrowhead], and 4.8% empty ovarioles) or one copy of dMyc (72.4% tumorous ovarioles, 18.6% ovarioles containing nurse cells [white arrowheads], 4.8% ovarioles containing eggs, and 4.2% empty ovarioles). Images in these panels are assembled from individual images acquired with a 40× objective. (F) eIF4E or dMyc heterozygosity has no effect on the maintenance of GSCs (open arrowheads) in an otherwise wild-type background (open arrowheads). (G) Statistical analysis of data shown in E; n = 480–520 ovarioles examined for each genotype. Bars: B,F, 10 μm; C, 50 μm; E, 100 μm.