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Genetic interactions between the Drosophila tumor suppressor gene ept and the stat92E transcription factor.

Gilbert MM, Beam CK, Robinson BS, Moberg KH - PLoS ONE (2009)

Bottom Line: We find that this phenotype is very sensitive to the genetic dose of stat92E, the transcriptional effector of the Jak-Stat signaling pathway, and that this pathway undergoes strong activation in ept mutant cells.Genetic evidence indicates that stat92E contributes to cell cycle deregulation and excess cell size phenotypes that are observed among ept mutant cells.These findings identify ept as a cell-autonomous inhibitor of the Jak-Stat pathway and suggest that excess Jak-Stat signaling makes a significant contribution to proliferative and tissue architectural phenotypes that occur in ept mutant tissues.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Emory University School of Medicine, Atlanta, Georgia, United States of America.

ABSTRACT

Background: Tumor Susceptibility Gene-101 (TSG101) promotes the endocytic degradation of transmembrane proteins and is implicated as a mutational target in cancer, yet the effect of TSG101 loss on cell proliferation in vertebrates is uncertain. By contrast, Drosophila epithelial tissues lacking the TSG101 ortholog erupted (ept) develop as enlarged undifferentiated tumors, indicating that the gene can have anti-growth properties in a simple metazoan. A full understanding of pathways deregulated by loss of Drosophila ept will aid in understanding potential links between mammalian TSG101 and growth control.

Principal findings: We have taken a genetic approach to the identification of pathways required for excess growth of Drosophila eye-antennal imaginal discs lacking ept. We find that this phenotype is very sensitive to the genetic dose of stat92E, the transcriptional effector of the Jak-Stat signaling pathway, and that this pathway undergoes strong activation in ept mutant cells. Genetic evidence indicates that stat92E contributes to cell cycle deregulation and excess cell size phenotypes that are observed among ept mutant cells. In addition, autonomous Stat92E hyper-activation is associated with altered tissue architecture in ept tumors and an effect on expression of the apical polarity determinant crumbs.

Conclusions: These findings identify ept as a cell-autonomous inhibitor of the Jak-Stat pathway and suggest that excess Jak-Stat signaling makes a significant contribution to proliferative and tissue architectural phenotypes that occur in ept mutant tissues.

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Related in: MedlinePlus

stat92E promotes growth of ept tumors.Bright-field images of (A) control discs [FRT80B/M(3)], (B) ept mutant discs [ept2/M(3)], or ept mutant, stat92E heterozygous discs [ept2/M(3),stat92E06346/+] from wandering-stage larvae. Yellow arrows in (C) denote the two lobes of tissue resembling eye discs. (D) Flow cytometric analysis of control (grey fill), ept2/M(3) mutant (black line), and ept2/M(3),stat92E06346/+ (dotted line) eye-antennal discs shows that reducing stat92E gene dosage partially rescues of cell cycle and cell size (inset) defects in ept2 mutant tissues. Percentages of cells in each stage of the cell cycle are indicated. The FACS data are representative of multiple experiments.
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pone-0007083-g001: stat92E promotes growth of ept tumors.Bright-field images of (A) control discs [FRT80B/M(3)], (B) ept mutant discs [ept2/M(3)], or ept mutant, stat92E heterozygous discs [ept2/M(3),stat92E06346/+] from wandering-stage larvae. Yellow arrows in (C) denote the two lobes of tissue resembling eye discs. (D) Flow cytometric analysis of control (grey fill), ept2/M(3) mutant (black line), and ept2/M(3),stat92E06346/+ (dotted line) eye-antennal discs shows that reducing stat92E gene dosage partially rescues of cell cycle and cell size (inset) defects in ept2 mutant tissues. Percentages of cells in each stage of the cell cycle are indicated. The FACS data are representative of multiple experiments.

Mentions: Eye-antennal discs were engineered to lack ept function by the eyFLP,Minute (Min) technique. In this configuration, mitotic recombination produces Min/Min cells that die and ept mutant cells that populate the disc and structures derived from it. These discs (hereafter referred to as ‘ept/M(3)’) overgrow into large unstructured tumors [5] (Fig. 1, compare A vs. B), indicating that loss of the gene can bypasses normal limits on organ size. To test the genetic requirements of this tumor-like phenotype, we screened a small collection of alleles of signaling, polarity, and growth regulatory genes (stat92E, crb, lgl, Drosophila aPKC, yki, cyclinD, dMyc, s6k and others) for their ability to suppress size and/or architectural phenotypes associated with loss of ept/tsg101. A single copy of the stat92E06346 loss-of-function allele [32] was found to significantly reduce the size of ept eye-antennal tumors (Fig. 1C) and cause them to grow as two distinct tissue lobes (yellow arrows in Fig. 1C) rather than the large mass of tissue that otherwise characterizes ept mutant eye-antennal discs. This effect on organ size and architecture is completely penetrant, and is accompanied by a 15-fold increase in the frequency of pupation (0.4% of ept/(3) animals [n = 245] vs. 6% of ept/M(3),stat92E06346/+ animals [n = 186]). To determine the effect of stat92E heterozygosity on the proliferative properties of cells within ept tumors, it was necessary to first establish the effect of ept loss on larval cell division and growth. Flow cytometric analysis indicates that cells within ept mutant tumors are enlarged relative to control cells (inset in Fig. 1D, black line vs grey fill) and show an increase in the percentage of cells in the S- and G2/M-phases of the cell cycle compared to cells in control FRT80B/M(3) discs (Fig. 1D, black line vs. grey fill). At a cellular level, the stat92E06346 allele slightly reduces the size of cells within these tumors while increasing the fraction of G1 cells and decreasing the fraction of G2/M cells (Fig. 1D and inset; dotted line vs. black line). Thus a diploid dose of the stat92E gene is required for the effect of ept loss on G1/S cell cycle phasing and to a somewhat lesser degree for the enlarged size of ept mutant imaginal disc cells. As cells in ept tumors are highly proliferative [5], the former effect suggests that stat92E promotes S-phase entry in ept mutant cells. This conclusion agrees with published data showing that overexpression of the mitogen Upd in the eye disc increases the number of cells in S-phase [25].


Genetic interactions between the Drosophila tumor suppressor gene ept and the stat92E transcription factor.

Gilbert MM, Beam CK, Robinson BS, Moberg KH - PLoS ONE (2009)

stat92E promotes growth of ept tumors.Bright-field images of (A) control discs [FRT80B/M(3)], (B) ept mutant discs [ept2/M(3)], or ept mutant, stat92E heterozygous discs [ept2/M(3),stat92E06346/+] from wandering-stage larvae. Yellow arrows in (C) denote the two lobes of tissue resembling eye discs. (D) Flow cytometric analysis of control (grey fill), ept2/M(3) mutant (black line), and ept2/M(3),stat92E06346/+ (dotted line) eye-antennal discs shows that reducing stat92E gene dosage partially rescues of cell cycle and cell size (inset) defects in ept2 mutant tissues. Percentages of cells in each stage of the cell cycle are indicated. The FACS data are representative of multiple experiments.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2747001&req=5

pone-0007083-g001: stat92E promotes growth of ept tumors.Bright-field images of (A) control discs [FRT80B/M(3)], (B) ept mutant discs [ept2/M(3)], or ept mutant, stat92E heterozygous discs [ept2/M(3),stat92E06346/+] from wandering-stage larvae. Yellow arrows in (C) denote the two lobes of tissue resembling eye discs. (D) Flow cytometric analysis of control (grey fill), ept2/M(3) mutant (black line), and ept2/M(3),stat92E06346/+ (dotted line) eye-antennal discs shows that reducing stat92E gene dosage partially rescues of cell cycle and cell size (inset) defects in ept2 mutant tissues. Percentages of cells in each stage of the cell cycle are indicated. The FACS data are representative of multiple experiments.
Mentions: Eye-antennal discs were engineered to lack ept function by the eyFLP,Minute (Min) technique. In this configuration, mitotic recombination produces Min/Min cells that die and ept mutant cells that populate the disc and structures derived from it. These discs (hereafter referred to as ‘ept/M(3)’) overgrow into large unstructured tumors [5] (Fig. 1, compare A vs. B), indicating that loss of the gene can bypasses normal limits on organ size. To test the genetic requirements of this tumor-like phenotype, we screened a small collection of alleles of signaling, polarity, and growth regulatory genes (stat92E, crb, lgl, Drosophila aPKC, yki, cyclinD, dMyc, s6k and others) for their ability to suppress size and/or architectural phenotypes associated with loss of ept/tsg101. A single copy of the stat92E06346 loss-of-function allele [32] was found to significantly reduce the size of ept eye-antennal tumors (Fig. 1C) and cause them to grow as two distinct tissue lobes (yellow arrows in Fig. 1C) rather than the large mass of tissue that otherwise characterizes ept mutant eye-antennal discs. This effect on organ size and architecture is completely penetrant, and is accompanied by a 15-fold increase in the frequency of pupation (0.4% of ept/(3) animals [n = 245] vs. 6% of ept/M(3),stat92E06346/+ animals [n = 186]). To determine the effect of stat92E heterozygosity on the proliferative properties of cells within ept tumors, it was necessary to first establish the effect of ept loss on larval cell division and growth. Flow cytometric analysis indicates that cells within ept mutant tumors are enlarged relative to control cells (inset in Fig. 1D, black line vs grey fill) and show an increase in the percentage of cells in the S- and G2/M-phases of the cell cycle compared to cells in control FRT80B/M(3) discs (Fig. 1D, black line vs. grey fill). At a cellular level, the stat92E06346 allele slightly reduces the size of cells within these tumors while increasing the fraction of G1 cells and decreasing the fraction of G2/M cells (Fig. 1D and inset; dotted line vs. black line). Thus a diploid dose of the stat92E gene is required for the effect of ept loss on G1/S cell cycle phasing and to a somewhat lesser degree for the enlarged size of ept mutant imaginal disc cells. As cells in ept tumors are highly proliferative [5], the former effect suggests that stat92E promotes S-phase entry in ept mutant cells. This conclusion agrees with published data showing that overexpression of the mitogen Upd in the eye disc increases the number of cells in S-phase [25].

Bottom Line: We find that this phenotype is very sensitive to the genetic dose of stat92E, the transcriptional effector of the Jak-Stat signaling pathway, and that this pathway undergoes strong activation in ept mutant cells.Genetic evidence indicates that stat92E contributes to cell cycle deregulation and excess cell size phenotypes that are observed among ept mutant cells.These findings identify ept as a cell-autonomous inhibitor of the Jak-Stat pathway and suggest that excess Jak-Stat signaling makes a significant contribution to proliferative and tissue architectural phenotypes that occur in ept mutant tissues.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Emory University School of Medicine, Atlanta, Georgia, United States of America.

ABSTRACT

Background: Tumor Susceptibility Gene-101 (TSG101) promotes the endocytic degradation of transmembrane proteins and is implicated as a mutational target in cancer, yet the effect of TSG101 loss on cell proliferation in vertebrates is uncertain. By contrast, Drosophila epithelial tissues lacking the TSG101 ortholog erupted (ept) develop as enlarged undifferentiated tumors, indicating that the gene can have anti-growth properties in a simple metazoan. A full understanding of pathways deregulated by loss of Drosophila ept will aid in understanding potential links between mammalian TSG101 and growth control.

Principal findings: We have taken a genetic approach to the identification of pathways required for excess growth of Drosophila eye-antennal imaginal discs lacking ept. We find that this phenotype is very sensitive to the genetic dose of stat92E, the transcriptional effector of the Jak-Stat signaling pathway, and that this pathway undergoes strong activation in ept mutant cells. Genetic evidence indicates that stat92E contributes to cell cycle deregulation and excess cell size phenotypes that are observed among ept mutant cells. In addition, autonomous Stat92E hyper-activation is associated with altered tissue architecture in ept tumors and an effect on expression of the apical polarity determinant crumbs.

Conclusions: These findings identify ept as a cell-autonomous inhibitor of the Jak-Stat pathway and suggest that excess Jak-Stat signaling makes a significant contribution to proliferative and tissue architectural phenotypes that occur in ept mutant tissues.

Show MeSH
Related in: MedlinePlus