Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2011 Sep 29:11:57.
doi: 10.1186/1471-213X-11-57.

Loss of the Drosophila cell polarity regulator Scribbled promotes epithelial tissue overgrowth and cooperation with oncogenic Ras-Raf through impaired Hippo pathway signaling

Karen Doggett  1 , Felix A Grusche, Helena E Richardson, Anthony M Brumby
Affiliations

Loss of the Drosophila cell polarity regulator Scribbled promotes epithelial tissue overgrowth and cooperation with oncogenic Ras-Raf through impaired Hippo pathway signaling

Karen Doggett et al. BMC Dev Biol. .

Abstract

Background: Epithelial neoplasias are associated with alterations in cell polarity and excessive cell proliferation, yet how these neoplastic properties are related to one another is still poorly understood. The study of Drosophila genes that function as neoplastic tumor suppressors by regulating both of these properties has significant potential to clarify this relationship.

Results: Here we show in Drosophila that loss of Scribbled (Scrib), a cell polarity regulator and neoplastic tumor suppressor, results in impaired Hippo pathway signaling in the epithelial tissues of both the eye and wing imaginal disc. scrib mutant tissue overgrowth, but not the loss of cell polarity, is dependent upon defective Hippo signaling and can be rescued by knockdown of either the TEAD/TEF family transcription factor Scalloped or the transcriptional coactivator Yorkie in the eye disc, or reducing levels of Yorkie in the wing disc. Furthermore, loss of Scrib sensitizes tissue to transformation by oncogenic Ras-Raf signaling, and Yorkie-Scalloped activity is required to promote this cooperative tumor overgrowth. The inhibition of Hippo signaling in scrib mutant eye disc clones is not dependent upon JNK activity, but can be significantly rescued by reducing aPKC kinase activity, and ectopic aPKC activity is sufficient to impair Hippo signaling in the eye disc, even when JNK signaling is blocked. In contrast, warts mutant overgrowth does not require aPKC activity. Moreover, reducing endogenous levels of aPKC or increasing Scrib or Lethal giant larvae levels does not promote increased Hippo signaling, suggesting that aPKC activity is not normally rate limiting for Hippo pathway activity. Epistasis experiments suggest that Hippo pathway inhibition in scrib mutants occurs, at least in part, downstream or in parallel to both the Expanded and Fat arms of Hippo pathway regulation.

Conclusions: Loss of Scrib promotes Yorkie/Scalloped-dependent epithelial tissue overgrowth, and this is also important for driving cooperative tumor overgrowth with oncogenic Ras-Raf signaling. Whether this is also the case in human cancers now warrants investigation since the cell polarity function of Scrib and its capacity to restrain oncogene-mediated transformation, as well as the tissue growth control function of the Hippo pathway, are conserved in mammals.

PubMed Disclaimer

Figures

Figure 1
Figure 1
scrib mutant cells expressing bskDN in the eye disc have impaired Hippo pathway signaling. Confocal sections through 3rd instar larval eye/antennal discs (A-F), posterior to the left in this and all subsequent figures. bskDN-expressing clones (A, C, E), and scrib1 clones expressing bskDN (B, D, F) were generated with ey-FLP and are positively marked by GFP expression (green, or magenta in the merges). Grayscale is DIAP1 (A-B), β-GAL (C-F), and F-actin to show tissue morphology (A-F). A white bar indicates the location of the morphogenetic furrow (MF) where cells are G1-phase arrested. (A, B) DIAP1 is expressed throughout the larval eye/antennal disc, and levels of the protein increase posterior to the MF in the differentiating portion of the eye disc. bskDN-expressing clones exhibit normal levels of DIAP1 (A), however, levels of DIAP1 are increased in scrib1 clones expressing bskDN (B). (C-F) ex-lacZ is expressed throughout the eye/antennal disc and is higher anterior to the MF in the eye disc; and fj-lacZ is expressed in a gradient in the eye disc with highest levels in the anterior centre, decreasing in levels along the dorsal, ventral and posterior axes. The expression of bskDN does not alter ex-lacZ (C) or fj-lacZ (E) expression, however, scrib1 clones expressing bskDN show elevated expression levels of ex-lacZ (D; note particularly clones of tissue posterior to the MF) and fj-lacZ (F).
Figure 2
Figure 2
Sd and Yki are required for the ectopic cell proliferation in scrib mutant eye disc clones. Mutant eye/antennal disc clones were generated with ey-FLP and are positively marked by GFP expression (green, or magenta in the merges). Grayscale is BrdU. A white bar indicates the location of the MF. (A-D) RNAi-mediated knockdown of sd in clones does not alter the normal pattern of BrdU incorporation in the eye/antennal disc (A), whilst scrib1 clones expressing bskDN are large and ectopically proliferate posterior to the MF (B). Knockdown of sd in scrib1 clones expressing bskDN reduces the ectopic cell proliferation in the mutant clones posterior to the MF (C), and a similar rescue is shown by knockdown of yki (D).
Figure 3
Figure 3
Sd is not required for the cell morphology defects in scrib mutant clones. Cross sections of larval eye discs. Mutant clones (A-C) were generated with ey-FLP and are positively marked by GFP expression (green, or magenta and yellow in the merges). Grayscale is Elav and red is F-actin. A white bar indicates the location of the MF. (A-C) Control eye disc clones, generated using a wild type chromosome with a Flippase recognition target (FRT), show the columnar epithelial structure of the eye disc, with the apically localized nuclei of the developing photoreceptor cells marked by Elav (A). In contrast, scrib1 clones expressing bskDN show a disorganized epithelial structure resulting in Elav positive photoreceptor cell nuclei being mislocalized basally within the epithelium (B). Knockdown of sd in scrib1 clones expressing bskDN does not rescue the mutant cell morphology defects (C).
Figure 4
Figure 4
scrib mutant wing disc tissue overgrows through impaired Hippo signaling. Confocal sections through 3rd instar larval wing discs (A-H), posterior to the left. en-GAL4 driven expression of transgenes in the posterior half of the discs (A-D) is marked by GFP expression (green, or magenta in the merges). Grayscale is β-GAL (A-D) or F-actin (E-H). (A-D) en-GAL4 driven expression of scribRNAi in the posterior half of the wing disc increases ex-lacZ (A), fj-lacZ (B) and msn-lacZ (C) expression. Coexpression of bskDN with scribRNAi does not rescue the increased expression of fj-lacZ (D). (E-H) scrib1/scrib3 transheterozygous mutant wing discs over grow and become increasingly disorganized (E). Halving the gene dosage of yki dramatically reduces the size of homozygous scrib mutant wing discs (F). In contrast, neither halving the gene dosage of Ras85D (G) or bsk (H) reduces homozygous scrib mutant wing disc overgrowth.
Figure 5
Figure 5
Impaired Hippo pathway signaling promotes scrib1 + Rafgof neoplastic tumor overgrowth. Confocal sections through larval eye/antennal discs at day 5 (A-D) and eye/antennal discs still attached to the brain lobes (bl) at day 8 after egg laying (E-H). Mutant clones were generated with ey-FLP and are positively marked by GFP expression (green, or, in the merges, magenta in A-D and yellow in E-H). Grayscale is β-GAL (A-D), F-actin (A-D) or Elav (E-H). Red is F-actin (E-H). (A, B) Expression of Rafgof does not alter the normal pattern of ex-lacZ (A) or fj-lacZ (B) expression in the eye disc. (C-D) scrib1 + Rafgof tumors ectopically express ex-lacZ (C; arrows) and fj-lacZ (D; arrows) within the eye disc. (E-H) scrib1 + Rafgof tumors overgrow and fuse with the brain lobes throughout an extended larval stage of development (E). Knockdown of sd (F), halving the gene dosage of yki (G) or expression of ykiRNAi (H) reduces scrib1 + Rafgof tumor overgrowth.
Figure 6
Figure 6
aPKC signaling is required for the impaired Hippo pathway signaling in scrib mutants. Larval eye/antennal discs with ey-FLP induced mutant clones marked by GFP expression (green, or magenta in the merges). Grayscale is β-GAL. A white bar indicates the location of the MF. (A-D) Coexpression of aPKCCAAXDN and bskDN in clones does not alter ex-lacZ (A) or fj-lacZ (C) expression, but when aPKCCAAXDN and bskDN are coexpressed in scrib1 clones, the normal pattern of ex-lacZ (B) and fj-lacZ (D) expression is restored (compared with scrib1 clones expressing bskDN alone in Figure 1).
Figure 7
Figure 7
Ectopic aPKC signaling impairs Hippo pathway signaling even when JNK signaling is blocked. Larval eye/antennal discs with ey-FLP induced mutant clones marked by GFP expression (green, or magenta in the merges). Grayscale is β-GAL and F-actin. A white bar indicates the location of the MF. Apical (A, C) and basal (B, D) confocal sections are shown. (A-D) Coexpression of aPKCΔN with bskDN in clones results in large masses of tissue extruded basally from the epithelium which ectopically express ex-lacZ (A, B; arrows) and fj-lacZ (C, D; arrows).
Figure 8
Figure 8
wts mutant overgrowth is independent of aPKC signaling. Larval eye discs (A-D) with ey-FLP induced mutant clones (green, or magenta in the merges) and dorsal views of adult mosaic flies (E, F). Grayscale is CycE (A, C) and DIAP1 (B, D). A white bar indicates the location of the MF. (A-F) Expression of wtsRNAi in eye disc clones results in upregulation of CycE (A) and DIAP1 (B) posterior to the MF (example clones highlighted with arrows), resulting in adult flies eclosing with overgrown eyes (E). Coexpression of aPKCCAAXDN with wtsRNAi does not prevent upregulation of CycE (C) and DIAP1 (D) in mutant clones (example clones highlighted with arrows), and adult flies still eclose with overgrown eyes (F).
Figure 9
Figure 9
Loss of scrib impairs Hippo pathway signaling in parallel to expanded and fat. Larval eye/antennal disc clones generated with ey-FLP and marked by GFP expression (A-H), and wing disc clones generated by hs-FLP and marked by the absence of GFP (I, J). Green is GFP (or magenta in the merges). Grayscale is F-actin (A-F), CycE (G, H) and β-GAL (I, J). A white bar indicates the location of the MF in the eye disc (A-H), and a dashed white line marks the location of the Z-stacks shown below the wing discs (I, J). (A-C) scrib1 clones (B) are reduced in size compared to control clones (A), but expressing bskDN in the scrib1 clones dramatically increases clone size (C). (D-F) Overexpression of ex in otherwise wild type clones reduces clonal tissue size (D), and when ex is expressed in scrib1 (E), or scrib1 clones expressing bskDN (F), clonal tissue size is also greatly reduced. (G, H) Overexpression of ex in scrib1 clones expressing bskDN does not prevent ectopic expression of CycE in basally located mutant clones posterior to the MF (H, highlighted with arrow), as compared to control clones (G). (I, J) scrib1 clones generated in the wing disc (arrows) in either a transheterozygous d1/dGC13mutant background (I) or homozygous ftfd exe1 (J) mutant background show increased ex-lacZ compared to the surrounding tissue.
Figure 10
Figure 10
Model for how the loss of scrib affects Hippo pathway signaling. In an epithelial cell, the core Hippo pathway components Hpo (H), Wts (W), Sav (S) and Mats (M) inhibit the activity of Yki, which acts to regulate transcription through its DNA binding partner, Sd. Extracellular regulation of the pathway occurs through the two transmembrane proteins, Crb acting through Ex, and Ft, acting through both Ex and D. Ex can also directly bind to and inhibit Yki. Loss of scrib in the eye disc is associated with aPKC-dependent inhibition of the Hippo pathway. Although JNK signaling is activated by either the loss of scrib, or ectopic aPKC activity, JNK is not required for Hippo pathway inhibition. In the wing disc, loss of scrib is also associated with JNK activation, and although JNK signaling has been shown to be sufficient to downregulate the Hippo pathway in the wing disc [37], Hippo pathway signaling remains impaired in scrib mutants even when JNK is blocked. Epistasis experiments suggest that pathway inhibition in scrib mutants occurs, at least in part, downstream or in parallel to Ex and Ft, and could impact upon either the core components (H/S/M/W) or upon Yki activity itself.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Brumby AM, Richardson HE. Using Drosophila melanogaster to map human cancer pathways. Nat Rev Cancer. 2005;5:626–39. doi: 10.1038/nrc1671. - DOI - PubMed
    1. Hariharan IK, Bilder D. Regulation of imaginal disc growth by tumor-suppressor genes in Drosophila. Annu Rev Genet. 2006;40:335–61. doi: 10.1146/annurev.genet.39.073003.100738. - DOI - PubMed
    1. Harvey K, Tapon N. The Salvador-Warts-Hippo pathway - an emerging tumor-suppressor network. Nat Rev Cancer. 2007;7:182–91. doi: 10.1038/nrc2070. - DOI - PubMed
    1. Wu S, Liu Y, Zheng Y, Dong J, Pan D. The TEAD/TEF family protein Scalloped mediates transcriptional output of the Hippo growth-regulatory pathway. Dev Cell. 2008;14:388–98. doi: 10.1016/j.devcel.2008.01.007. - DOI - PubMed
    1. Zhang L, Ren F, Zhang Q, Chen Y, Wang B, Jiang J. The TEAD/TEF family of transcription factor Scalloped mediates Hippo signaling in organ size control. Dev Cell. 2008;14:377–87. doi: 10.1016/j.devcel.2008.01.006. - DOI - PMC - PubMed

Publication types

MeSH terms