scribbled: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

Gene name - scribbled

Synonyms - scribble

Cytological map position - 97B8-9

Function - scaffold protein

Keywords - apical/basal polarity, epidermis

Symbol - scrib

FlyBase ID: FBgn0263289

Genetic map position -

Classification - Multiple PDZ with LLR regions

Cellular location - cytoplasmic



NCBI links: Entrez Gene | UniGene



Recent literature
Felix, M., Chayengia, M., Ghosh, R., Sharma, A. and Prasad, M. (2015). dPak3 regulates apical-basal polarity in migrating border cells during Drosophila oogenesis. Development [Epub ahead of print]. PubMed ID: 26395489
Summary:
During group cell migration collectively moving cells are physically attached to each other and retain some degree of apical-basal polarity during the migratory phase. Though much is known about direction sensing, it is far from clear how polarity is regulated in diverse instances of multicellular movement. This study reports the role of dPak3, a group I p21 activated serine-threonine protein kinase, in maintaining apical-basal polarity in migrating border cell cluster during Drosophila oogenesis. dPak3 is enriched in border cells and downregulation of its function impedes border cell movement. Time-lapse imaging suggests that dPak3 affects protrusive behavior of the border cell cluster specifically regulating the stability and directionality of the protrusions. This study shows that dPak3 functions downstream of the guidance receptor signaling to regulate the level and distribution of F-actin in the migrating border cells. Further evidence is provided that dPak3 genetically interacts with lateral polarity marker Scribble and regulates c-Jun N-terminal kinase (JNK) signaling in the moving border cells. Since dPak3 depletion results in mislocalization of several apical-basal polarity markers including Stardust, Crumbs and Coracle and overexpression of D-jun rescues the polarity of the dPak3-depleted cluster, it is proposed that dPak3 functions through the JNK signaling to modulate the apical-basal polarity of the migrating border cell cluster. Interestingly, loss of apical-basal polarity was also observed in Rac1 depleted border cell cluster suggesting that the guidance receptor signaling functions through Rac GTPase and dPak3 to regulate overall polarity of the cluster to mediate efficient collective movement of the border cells to the oocyte boundary.

Zhou, Y., Chang, R., Ji, W., Wang, N., Qi, M., Xu, Y., Guo, J. and Zhan, L. (2015). Loss of Scribble promotes Snail translation through translocation of HuR and enhances cancer drug resistance. J Biol Chem [Epub ahead of print]. PubMed ID: 26527679
Summary:
Drug resistance of cancer cells to various therapeutic agents and molecular targets is a major problem facing current cancer research. The tumor suppressor gene Scribble encodes a polarity protein that is conserved between Drosophila and mammals; loss of the locus disrupts cell polarity, inhibits apoptosis, and mediates cancer process. However, the role of Scribble in drug resistance remains unknown. This study shows that knockdown of Scribble enhances drug resistance by permitting accumulation of Snail, which functions as a transcription factor during the epithelial-mesenchymal transition. Then, loss of Scribble activates the mRNA binding protein HuR (ELAV in Drosophila) by facilitating translocation of HuR from the nucleus to the cytoplasm. Furthermore, HuR can recognize AU-rich elements (AREs) of the Snail-encoding mRNA, thereby regulating Snail translation. Moreover, Loss of Scribble induced HuR translocation mediates the accumulation of Snail via activation of the p38 MAPK pathway. Thus, this work clarifies the role of polarity protein Scribble, which is directly implicated in the regulation of developmental transcription factor Snail, and suggesting a mechanism for Scribble mediating cancer drug resistance.

Cervantes-Sandoval, I., Chakraborty, M., MacMullen, C. and Davis, R.L. (2016). Scribble scaffolds a signalosome for active forgetting. Neuron [Epub ahead of print]. PubMed ID: 27263975
Summary:
Forgetting, one part of the brain's memory management system, provides balance to the encoding and consolidation of new information by removing unused or unwanted memories or by suppressing their expression. Recent studies have identified the small G protein, Rac1, as a key player in the Drosophila mushroom bodies neurons (MBn) for active forgetting. It has also been shown that a few dopaminergic neurons (DAn) that innervate the MBn mediate forgetting. This study shows that Scribble, a scaffolding protein known primarily for its role as a cell polarity determinant, orchestrates the intracellular signaling for normal forgetting. Knocking down scribble expression in either MBn or DAn impairs normal memory loss. Scribble interacts physically and genetically with Rac1, Pak3, and Cofilin within MBn, nucleating a forgetting signalosome that is downstream of dopaminergic inputs that regulate forgetting. These results bind disparate molecular players in active forgetting into a single signaling pathway: Dopamine→ Dopamine Receptor→ Scribble→ Rac→ Cofilin.

Waghmare, I. and Kango-Singh, M. (2016). Loss of cell adhesion increases tumorigenic potential of polarity deficient Scribble mutant cells. PLoS One 11: e0158081. PubMed ID: 27327956
Summary:
Epithelial polarity genes are important for maintaining tissue architecture, and regulating growth. The Drosophila neoplastic tumor suppressor gene scribble (scrib) belongs to the basolateral polarity complex. Loss of scrib results in disruption of its growth regulatory functions, and downregulation or mislocalization of Scrib is correlated to tumor growth. Somatic scribble mutant cells (scrib-) surrounded by wild-type cells undergo apoptosis, which can be prevented by introduction of secondary mutations that provide a growth advantage. Using genetic tools in Drosophila, the phenotypic effects were analyzed of loss of scrib in different growth promoting backgrounds. This study investigated if a central mechanism that regulates cell adhesion governs the growth and invasive potential of scrib mutant cells. Increased proliferation, and survival abilities of scrib- cells in different genetic backgrounds affect their differentiation, and intercellular adhesion. Further, loss of scrib is sufficient to cause reduced cell survival, activation of the JNK pathway and a mild reduction of cell adhesion. These data show that for scrib cells to induce aggressive tumor growth characterized by loss of differentiation, cell adhesion, increased proliferation and invasion, cooperative interactions that derail signaling pathways play an essential role in the mechanisms leading to tumorigenesis. Thus, this study provides new insights on the effects of loss of scrib and the modification of these effects via cooperative interactions that enhance the overall tumorigenic potential of scrib deficient cells.
Schimizzi, G. V., Maher, M. T., Loza, A. J. and Longmore, G. D. (2016). Disruption of the Cdc42/Par6/aPKC or Dlg/Scrib/Lgl polarity complex promotes epithelial proliferation via overlapping mechanisms. PLoS One 11: e0159881. PubMed ID: 27454609
Summary:
The establishment and maintenance of apical-basal polarity is a defining characteristic and essential feature of functioning epithelia. Apical-basal polarity (ABP) proteins are also tumor suppressors that are targeted for disruption by oncogenic viruses and are commonly mutated in human carcinomas. Using the proliferating Drosophila wing disc epithelium, this study demonstrates that disruption of the junctional [Cdc42/Par6/Par3/Atypical PKC (aPKC)] complex vs. basolateral polarity complex [Scribble (Scrib)/Discs Large (Dlg)/Lethal Giant Larvae (Lgl)] complex results in increased epithelial proliferation via distinct downstream signaling pathways. Disruption of the basolateral polarity complex results in JNK-dependent proliferation, while disruption of the junctional complex primarily results in p38-dependent proliferation. Surprisingly, the Rho-Rok-Myosin contractility apparatus appears to play opposite roles in the regulation of the proliferative phenotype based on which polarity complex is disrupted. In contrast, non-autonomous Tumor Necrosis Factor (TNF; Eiger) signaling appears to suppress the proliferation that results from apical-basal polarity disruption, regardless of which complex is disrupted. Finally it was demonstrated that disruption of the junctional polarity complex activates JNK via the Rho-Rok-Myosin contractility apparatus independent of the cortical actin regulator, Moesin.
Vaughen, J. and Igaki, T. (2016). Slit-Robo repulsive signaling extrudes tumorigenic cells from epithelia. Dev Cell 39: 683-695. PubMed ID: 27997825
Summary:
Cells dynamically interact throughout animal development to coordinate growth and deter disease. For example, cell-cell competition weeds out aberrant cells to enforce homeostasis. In Drosophila, tumorigenic cells mutant for the cell polarity gene scribble (scrib) are actively eliminated from epithelia when surrounded by wild-type cells. While scrib cell elimination depends critically on JNK signaling, JNK-dependent cell death cannot sufficiently explain scrib cell extirpation. Thus, how JNK executed cell elimination remained elusive. This study shows that repulsive Slit-Robo2-Ena signaling exerts an extrusive force downstream of JNK to eliminate scrib cells from epithelia by disrupting E-cadherin. While loss of Slit-Robo2-Ena in scrib cells potentiates scrib tumor formation within the epithelium, Robo2-Ena hyperactivation surprisingly triggers luminal scrib tumor growth following excess extrusion. This extrusive signaling is amplified by a positive feedback loop between Slit-Robo2-Ena and JNK. These observations provide a potential causal mechanism for Slit-Robo dysregulation in numerous human cancers.

Rives-Quinto, N., Franco, M., de Torres-Jurado, A. and Carmena, A. (2017). canoe and scribble loss synergizes causing tumor-like overgrowth via Ras activation in neural stem cells and epithelia. Development [Epub ahead of print]. PubMed ID: 28619817
Summary:
Over the past decade an intriguing connection between asymmetric cell division, stem cells and tumorigenesis has emerged. Neuroblasts, the neural stem cells of the Drosophila central nervous system, divide asymmetrically and constitute an excellent paradigm for further investigating that connection. This study shows that the simultaneous loss of the asymmetric cell division regulators Canoe (Afadin in mammals) and Scribble in neuroblast clones leads to tumor-like overgrowth through both a severe disruption of the asymmetric cell division process and a canoe loss-mediated Ras-PI3K-Akt activation. Moreover, canoe loss also interacts synergistically with scribble to promote overgrowth in epithelial tissues, here just by activating the Ras-Raf-MAPK pathway. Finally scribble functionally related genes discs large and lethal (2) giant larvae were shown to contribute to repress the Ras-MAPK signaling cascade in epithelia. Hence, this work uncovers novel cooperative interactions between all these well-conserved tumor suppressors to ensure a tight regulation of the Ras signaling pathway.
Rui, M., Qian, J., Liu, L., Cai, Y., Lv, H., Han, J., Jia, Z. and Xie, W. (2017). The neuronal protein Neurexin directly interacts with the Scribble-Pix complex to stimulate F-actin assembly for synaptic vesicle clustering. J Biol Chem [Epub ahead of print]. PubMed ID: 28710284
Summary:
Synaptic vesicles (SVs) form distinct pools at synaptic terminals, and this well-regulated separation is necessary for normal neuro-transmission. However, how SV cluster in particular synaptic compartments to maintain normal neurotransmitter release remains a mystery. The presynaptic protein Neurexin (NRX) plays a significant role in synaptic architecture and function, and some evidences suggest that NRX is associated with neurological disorders, including autism spectrum disorders. However, the role of NRX in SV clustering is unclear. Using the neuromuscular junction at the 2-3 instar stages of Drosophila larvae as a model and biochemical, imaging, and electrophysiology techniques, this study demonstrate that Drosophila NRX (DNRX) plays critical roles in regulating synaptic terminal clustering and release of SVs. DNRX controls the terminal clustering and release of SVs by stimulating presynaptic F-actin. Furthermore, the results indicate that DNRX functions through the scaffold protein Scribble and the GEF protein Pixie to activate the small GTPase Rac1. A direct interaction was observed between the C-terminal PDZ-binding motif of DNRX and the PDZ domains of Scribble and that Scribble bridges DNRX to DPix, forming a DNRX/Scribble/DPix complex that activates Rac1 and subsequently stimulates presynaptic F-actin assembly and SV clustering. Taken together, this work provides important insights into the function of DNRX in regulating SV clustering, which could help inform further research into pathological neurexin-mediated mechanisms in neurological disorders such as autism.
BIOLOGICAL OVERVIEW

Loss of cell polarity and tissue architecture are characteristics of malignant cancers derived from epithelial tissues. Cells in epithelial sheets are characterized by columnar or cuboidal shape, strong cell-cell adhesion, and pronounced apicobasal polarity. However, tumors of epithelial origin lose these characteristics as they progress from benign growth to malignant carcinoma, and this loss is associated with poor clinical prognosis. Evidence is provided that a group of membrane-associated proteins act in concert to regulate both epithelial structure and cell proliferation. Scribbled (Scrib) is a cell junction-localized protein required for polarization of embryonic and imaginal disc and follicular epithelia. The tumor suppressor scrib was isolated in a screen for maternal effect mutations that disrupt aspects of epithelial morphogenesis such as cell adhesion, shape and polarity. Commonly referred to as 'scribble' both here and in the literature, the gene name is maintained as 'scribbled' by FlyBase, to avoid (or enhance) confusion. scrib encodes a multi-PDZ (PSD-95, Discs-large and ZO-1) and leucine-rich-repeat protein. The structure of the embryonic cuticle was used to reflect the organization of the underlying epithelial epidermis that secretes it. The wild-type cuticle forms a smooth, continuous sheet, but embryos that are maternally and zygotically mutant for scrib produce a corrugated cuticular surface that is riddled with holes, hence the name scribbled (Bilder, 2000a).

To place Scrib within the known pathway for Drosophila epithelial polarity determination, the effect of scrib mutations on Crumbs (Crb) was examined. Crb is an apically localized transmembrane protein that is necessary and sufficient to confer apical character on plasma membrane. In scrib embryos, Crb shows unrestricted localization in both apical and basolateral regions. Whether scrib mutants are identical to a gain-of-function crb phenotype was examined by comparing them with embryos in which GAL4-driven Crb is present throughout the cell membrane. Ectopic Crb that is produced in this manner is sufficient to phenocopy several aspects of scrib embryos, including mislocalization of apical proteins and the cuticle pattern. These data indicate that a major function of Scrib in epithelial polarity is to exclude Crb from the basolateral domain. Since ectopic Crb does not cause the epithelial morphology and multilayering defects seen in scrib embryos, Scrib may be required for the localization of additional epithelial determinants as well (Bilder, 2000a).

Analysis of the morphological and polarization phenotypes exhibited by scrib embryos shows that Scrib is a critical component of epithelial architecture in the Drosophila ectoderm, and suggests that its function is closely linked to that of Crb. Scrib is not required for the early localization of basal Discs lost (now redefined as Drosophila Patj) or apical Armadillo during blastoderm formation, and scrib mutants do not exhibit the defective cellularization or precipitous loss of cell adhesion seen when Discs lost or Armadillo, respectively, are depleted in the embryo. The increasingly severe cell shape, polarity and epithelial organization defects of scrib embryos are first manifested after gastrulation, coincident with the onset of defects in crb and stardust embryos. Loss of Crb results in loss of apical proteins from the plasma membrane and a failure to consolidate early adherens junction material into an apical band of zonula adherins (ZAs), while in scrib embryos early adherens junctions become misdistributed basolaterally. Together with the similarities between scrib loss-of-function and crb gain-of-function phenotypes, these data place Scrib and Crb in a pathway required for the progression from the initially differentiated blastoderm membrane domains into a fully polarized epithelium with mature junctions (Bilder, 2000a).

These results show that the junctional protein Scrib specifically restricts apical membrane determinants to the apical cell surface. This restriction allows the proper segregation of apical and basolateral domain components, and the appropriate placement of the adherens junction, resulting in full epithelial polarization. How does Scrib, a putative scaffolding protein whose localizaton bounds the apical domain, dictate the proper confinement of apical proteins? Two models suggest themselves. Scrib could assemble a diffusion barrier that physically separates apical and basolateral compartments, similar to the 'fence' function proposed for the vertebrate tight junction. To date, such a barrier has been shown to exist only for lipid diffusion in the outer leaflet of the plasma membrane. If scrib mutations disrupt such a mechanical barrier, then secondary retention systems must serve to maintain basolateral protein restriction from the apical cell surface. An alternative is that Scrib has a role in the polarized targeting of transport vesicles carrying apical proteins. The junctional complex, and in particular the tight junction, has been proposed to be a key sorting site for a subset of Golgi-derived vesicles. In this model, Scrib might interact with the 'exocyst', a secretory targeting apparatus localized to the tight junction and involved in polarized segregation of transmembrane proteins. PDZ domain proteins have been implicated at several different sites of the protein trafficking pathway, and occasional punctate intracellular staining of Scrib is reminiscent of vesicles. Distinction between these models will rely on the identification of binding partners for Scrib (Bilder, 2000a).

Two other tumor suppressors, lethal giant larvae (lgl) and discs-large (dlg), have the identical effects as scrib mutation on epithelial structure. Scrib and Dlg colocalize and overlap with Lgl in epithelia; activity of all three genes is required for cortical localization of Lgl and junctional localization of Scrib and Dlg. scrib, dlg, and lgl show strong genetic interactions. Thus, these three tumor suppressors act together in a common pathway to regulate cell polarity and growth control (Bilder, 2000b).

To test whether the requirement for Scrib is limited to the embryo, the role of scrib in follicle cells, a monolayered epithelium of somatic cells that encases the germ line in the adult female ovary, was examined. Scrib is localized to lateral follicle cell membranes, and clones of cells that lack scrib function become round and multilayered with polarity defects, similar to the phenotype of embryonic epithelia lacking scrib function. The epithelial defects of scrib mutant follicle cells are cell autonomous. These data indicate that scrib is required within cells from multiple tissues for proper epithelial structure (Bilder, 2000b).

Because follicle cell epithelia require scrib, lgl, and dlg, the functions of lgl and dlg were examined in the embryonic epidermis, where scrib acts to restrict apical proteins and adherens junctions to their appropriate positions within the cell membrane (Bilder, 2000a). Embryos lacking both maternal and zygotic contributions of lgl and dlg, (hereafter referred to as lgl and dlg embryos) were stained with antibodies to polarized proteins and cellular junction components. During mid-embryogenesis, lgl and dlg embryos show defects in apicobasal polarity, revealed by aberrant distribution of the apical protein Crumbs (Crb) and disruption of adherens junctions. These defects are similar to those of scrib embryos; the terminal phenotypes of scrib, lgl, and dlg embryos, as indicated by cuticle deposition, are also nearly identical. Thus, lgl and dlg, like scrib, act to properly localize apical proteins and adherens junctions to organize epithelial architecture in embryos (Bilder, 2000b).

The similarity of mutant phenotypes in different epithelia suggests that the three proteins are components of the fundamental machinery that creates the distinctive architecture of epithelial cells and tissues. To test this assertion, the scrib phenotype was compared to that of lgl and dlg in a third major epithelium, the larval imaginal disc. Discs isolated from late third instar larvae zygotically mutant for scrib are profoundly disorganized and also massively overgrown. scrib discs contain 4.7 times as many cells as wild-type (WT) discs and consist of spherical masses of tightly packed cells, as opposed to the folded monolayer epithelium seen in WT larvae. The apical polarization of actin evident in WT discs is absent in scrib discs. This loss of epithelial organization accompanied by overproliferation corresponds to the phenotype described for lgl and dlg zygotic mutant discs. Additional features of lgl and dlg larval phenotypes, such as overgrowth of brain tissue, are also present in scrib larvae. Together, these data indicate that scrib and the two previously characterized Drosophila malignant neoplastic tumor suppressors, lgl and dlg, share a role in growth control as well as epithelial polarity. Epistatic relations between scrib, lgl, and dlg were investigated by determining the localization of each protein in embryos mutant for the other two genes. These experiments have shown that dlg is required for the stable association of Scrib with the cell membrane and scrib is required for the cortical association of Lgl; all three genes act to localize Scrib and Dlg to the apical margin of the lateral membrane (ALM) of the embryonic epidermal epithelium (Bilder, 2000b).

These results provide strong evidence that Scrib, Dlg, and Lgl act in a common pathway to regulate cell architecture and cell proliferation control. Of the ~50 Drosophila genes in which mutation gives rise to overproliferation, only scrib shares with dlg and lgl the concomitant loss of tissue organization that groups the three together as malignant neoplastic tumor suppressors. Previous analyses have described a role for dlg and lgl in imaginal disc polarity; the demonstration in this work of genetic interactions with scrib and codependence for protein localization indicates a functional link between the three tumor suppressors. Furthermore, involvement of the tumor suppressors in embryonic epithelial polarity provides a well-studied context in which to understand their activities. These findings suggest that, in the WT gastrula, intrinsic, perhaps adhesion-based cues localize Dlg at the ALM; Dlg stabilizes Scrib at this position, and finally Scrib acts on the cortical cytoskeleton to bring Lgl to the membrane. The three proteins may then collaborate to maintain the proper distribution of polarized factors, including themselves (Bilder, 2000b).

The correlation between loss of membrane-associated Lgl in scrib and dlg mutants and defective cell polarity suggests models of action for this group of proteins. Whereas the PDZ domains of Scrib and Dlg are likely to bind to transmembrane proteins that organize the epithelial cell surface, the role of Lgl in polarity determination may derive from its function in targeted secretion of membrane proteins. Lgl homologs from humans and yeast can bind to plasma membrane t-SNARE proteins and promote the fusion of cargo-carrying vesicles with target membranes (Fujita, 1998; Lehman, 1999). In yeast undergoing polarized growth, the broadly distributed Lgl homologs function primarily at the bud tip, the site of the 'exocyst' complex required for vesicle trafficking and addition (TerBush, 1996). In vertebrate epithelia, exocyst components are found at the tight junction, a structure analogous to the septate junction where Dlg and Scrib localize. In Drosophila epithelia, recruitment of Lgl into the proximity of membrane t-SNAREs requires proper localization of Scrib and Dlg, thus potentially linking the transmembrane proteins that establish polarity to the protein-targeting system that preserves it (Bilder, 2000b and references therein).

In many epithelial-derived cancers, cytoarchitectural changes are hallmarks of oncogenic transformation. The disruption of epithelial architecture seen in scrib, dlg, and lgl animals could affect growth control by several mechanisms. Many growth factor receptors are polarized to a specific membrane domain, and mislocalization of such proteins may affect signaling pathways that maintain cells in a differentiated, nonproliferative state. Additionally, the aberrant cell-cell junctions formed in scrib, dlg, and lgl mutants could compromise contact inhibition. Finally, disruption of cell-cell contacts may release junction-localized signaling components, such as Arm or APC, that have been implicated in regulating cell proliferation; indeed, a human Dlg homolog has been shown to bind APC and associate with beta-catenin, the human homolog of Arm. Because the modes of action of Scrib, Dlg, and Lgl are likely to be conserved between vertebrates and invertebrates, investigation into a tumorigenic role for the multiple human homologs of these genes is warranted. Further analysis of the mechanisms by which Scrib, Dlg, and Lgl keep Drosophila cell growth in check will likely enhance an understanding of mammalian oncogenesis as well (Bilder, 2000b).

BTB-zinc finger oncogenes are required for Ras and Notch-driven tumorigenesis in Drosophila

During tumorigenesis, pathways that promote the epithelial-to-mesenchymal transition (EMT) can both facilitate metastasis and endow tumor cells with cancer stem cell properties. To gain a greater understanding of how these properties are interlinked in cancers, Drosophila epithelial tumor models were used, that are driven by orthologues of human oncogenes (activated alleles of Ras and Notch) in cooperation with the loss of the cell polarity regulator, scribbled (scrib). Within these tumors, both invasive, mesenchymal-like cell morphology and continual tumor overgrowth, are dependent upon Jun N-terminal kinase (JNK) activity. To identify JNK-dependent changes within the tumors a comparative microarray analysis was used to define a JNK gene signature common to both Ras and Notch-driven tumors. Amongst the JNK-dependent changes was a significant enrichment for BTB-Zinc Finger (ZF) domain genes, including chronologically inappropriate morphogenesis (chinmo). chinmo was upregulated by JNK within the tumors, and overexpression of chinmo with either RasV12 or Nintra was sufficient to promote JNK-independent epithelial tumor formation in the eye/antennal disc, and, in cooperation with RasV12, promote tumor formation in the adult midgut epithelium. Chinmo primes cells for oncogene-mediated transformation through blocking differentiation in the eye disc, and promoting an escargot-expressing stem or enteroblast cell state in the adult midgut. BTB-ZF genes are also required for Ras and Notch-driven overgrowth of scrib mutant tissue, since, although loss of chinmo alone did not significantly impede tumor development, when loss of chinmo was combined with loss of a functionally related BTB-ZF gene, abrupt, tumor overgrowth was significantly reduced. abrupt is not a JNK-induced gene, however, Abrupt is present in JNK-positive tumor cells, consistent with a JNK-associated oncogenic role. As some mammalian BTB-ZF proteins are also highly oncogenic, this work suggests that EMT-promoting signals in human cancers could similarly utilize networks of these proteins to promote cancer stem cell states (Doggett, 2015).

This report has defined the transcriptional changes induced by JNK signaling within both scrib>RasACT and scrib>NACT tumors by carrying out comparative microarray expression arrays. This analysis that JNK exerts a profound effect upon the transcriptional profile of both Ras and Notch-driven tumor types. The expression of nearly 1000 genes was altered by the expression of bskDN in either Ras or Notch-driven tumors, and less than half of these changes were shared between the two tumor types, indicating that JNK signaling elicits unique tumorigenic expression profiles depending upon the cooperating oncogenic signal. Nevertheless, of the 399 JNK-regulated probe sets shared between Ras and Notch-driven tumors, it is hypothesized that these had the potential to provide key insights into JNK's oncogenic activity, and to prioritize these targets, it was considered that the expression of the critical oncogenic regulators would not just be altered by bskDN, but would be normalized to close to wild type levels. This subset of the 399 probe set was identified by comparing the expression profile of each genotype back to control tissue, thereby producing a more focussed JNK signature of 103 genes. Notably, this included previously characterized targets of JNK in the tumors, such as Mmp1,cherand Pax, thereby providing validation of the approach. Also amongst these candidates were 4 BTB-ZF genes; two of which were upregulated by JNK in the tumors (chinmo and fru), and two downregulated (br and ttk) (Doggett, 2015).

Focussing upon chinmo, chinmo overexpression was shown to be sufficient to prime epithelial cells for cooperation with RasACT in both the eye antennal disc and in the adult midgut epithelium, and that chinmo is required for cooperative RasACTor NACT-driven tumor overgrowth, although its function was only exposed when its knockdown was combined with knockdown of a functionally similar BTB-ZF transcription factor, abrupt. This family of proteins is highly oncogenic in Drosophila, since previous work has shown that ab overexpression can cooperate with loss of scrib to promote neoplastic overgrowth, and in these studies, it was also shown that overexpression of a fru isoform normally expressed in the eye disc is capable of promoting cooperation with RasACT and NACT in the eye-antennal disc, in a similar manner to chinmo overexpression. Thus, whether fru also plays a role in driving Ras or Notch-driven tumorigenesis warrants further investigation. Indeed, a deeper understanding of the oncogenic activity of these genes is likely to be highly relevant to human tumors, since of the over 40 human BTB-ZF family members, many are implicated in both haematopoietic and epithelial cancers, functioning as either oncogenes (eg., Bcl6, BTB7) or tumor suppressors (eg., PLZF, HIC1). Furthermore, over-expression of BTB7, can also cooperate with activated Ras in transforming primary cells, and its loss makes MEFs refractory to transformation by various key oncogenes such as Myc, H-rasV12 and T-Ag, suggesting that cooperating mechanisms between BTB-ZF proteins and additional oncogenic stimuli might be conserved (Doggett, 2015).

JNK signaling in Drosophila tumors is known to promote tumor overgrowth through both the STAT and Hippo pathways. Deregulation of the STAT pathway was evident in the arrays through the upregulation of Upd ligands by JNK in both Ras and Notch-driven tumors. In contrast, although cher was identified in the arrays as being upregulated in both tumor types and previous studies have shown that cher is partly required for the deregulation of the Hippo pathway in scrib>RasACT tumors, more direct evidence for Hippo pathway deregulation amongst the JNK signature genes was lacking. In part, this could be due to JNK regulating the pathway through post-transcriptional mechanisms involving direct phosphorylation of pathway components. However, the failure to identify known Hippo pathway target genes, and proliferation response genes in general, may simply highlight limitations in the sensitivity of the array assay and the cut-offs used for determining significance, despite its obvious success in correctly identifying many known JNK targets (Doggett, 2015).

Whether tumor overgrowth through STAT and Yki activity is somehow associated with a stem cell or progenitor-like state remains uncertain. Although imaginal discs exhibit developmental plasticity and regeneration potential, and JNK signaling is required for both of these stem-like properties, there is no positive evidence for the existence of a population of asymmetrically dividing stem cells within imaginal discs. Instead, symmetrical divisions of progenitor cells may be the means by which imaginal discs can rapidly generate enough cells to form the differentiated structures of the adult fly. To date, progenitor cells have only been characterized in the eye disc neuroepithelium. These cells have a pseudostratified columnar epithelial morphology and express the MEIS family transcription factor, Hth, which is downregulated as cells initiate differentiation and begin expressing Dac and Eya. Interestingly, they also require Yki for their proliferation, and can be induced to overproliferate in response to increased STAT activity. However, analysis of cell fate markers indicated that tumor overgrowth was not likley to be solely due to the overproliferation of these undifferentiated progenitor cells. Although scrib>RasACT/NACT tumors, were characterized by the failure to transition to Dac/Eya expression in the eye disc, blocking JNK in scrib > RasACT/NACT tumors did not restore tumor cell differentiation, despite overgrowth being curtailed, and Hth expression was not maintained in the tumors in a JNK-dependent manner. Nevertheless, a JNK-induced gene such as chinmo is likely to be associated with promoting a progenitor-like state, since it is a potential STAT target gene required for adult eye development that is expressed in eye disc progenitor cells in response to increased Upd activity and its overexpression alone is sufficient to block Dac/Eya expression. Furthermore, chinmo is also required for cyst stem cell maintenance in the Drosophila testis, and the current work has shown that chinmo overexpression promotes increased numbers of esgGFP expressing stem cells or enteroblasts in the adult midgut. As the BTB-ZF protein Ab is also highly oncogenic and expressed in the eye disc progenitor cells, it is hypothesize that the JNK-induced expression ofchinmo in scrib>RasACT/NACT tumors could cooperate with Ab to maintain a progenitor-like cell state in the eye disc, and that this is required for scrib->RasACT/NACT tumor overgrowth. However, although Ab was expressed in chinmo-expressing, JNK positive tumor cells, Ab does not appear to be a JNK-induced gene. What JNK-independent mechanisms control ab expression will therefore require further analysis (Doggett, 2015).

Interestingly, previous studies have observed that ab overexpression in eye disc clones upregulates chinmo expression and although the effect of chinmo expression upon ab is yet to be described, the data at least suggest that the control of their expression is interlinked in a yet to be defined manner (Doggett, 2015).

Consistent with Chinmo being important for scrib->RasACT/NACTv tumor overgrowth, chinmo overexpression itself is also highly oncogenic. Over-expression of chinmo with RasACT or NACT drives tumorigenesis in the eye-antennal disc, and also resulted in enlarged brain lobes, presumably due to the generation of overexpressing clones within the neuroepithelium of the optic lobes. In the adult midgut, the overexpression of chinmo with RasACT in the stem cell and its immediate progeny, the enteroblast, promoted massive tumor overgrowth, resulting in esgGFP expressing cells completely filling the lumen of the gut, and eventual host lethality. The luminal filling of esgGFP cells is reminiscent of the effects of RasACT expression in larval adult midgut progenitor cells. Together with the data linking Chinmo function to stem or progenitor cells, these data reinforce the idea that epithelial tumorigenesis can be primed by signals, such as chinmo over-expression, that promote a stem or progenitor cell state (Doggett, 2015).

The function of some Drosophila BTB-ZF proteins including Chinmo and Ab, has also been linked to heterochronic roles involving the conserved let-7 miRNA pathway and hormone signals, to regulate the timing of differentiation. Indeed, Ab can directly bind to the steroid hormone receptor co-activator Taiman (Tai or AIB1/SRC3 in humans), to represses the transcriptional response to ecdysone signaling. Thus, the capacity of BTB-ZF proteins to influence the timing of developmental transitions, particularly if they impede developmental transitions within stem or progenitor cells, could help account for their potent oncogenic activity. Indeed, ecdysone-response genes were repressed by JNK in the tumorigenic state, consistent with the failure of the larvae to pupate and a delay in developmental timing. Whether repressing the ecdysone response cell autonomously might contribute to tumor overgrowth and/or invasion will be an interesting area of future investigation, given the complex role of hormone signaling in mammalian stem cell biology and cancers (Doggett, 2015).

Previous studies have suggested that JNK-dependent tumor cell invasion is developmentally similar to the JNK-induced EMT-like events occurring during imaginal disc eversion. Thus the capacity of JNK to also promote tumor overgrowth is reminiscent of how EMT inducers such as Twist (Twi) and Snail (Sna) are associated with the acquisition of cancer stem cell properties. In Drosophila, however, twi and snawere not induced by JNK in the tumors, although transcription factors involved in mesoderm specification, including the NF-kappaB homologue, dl (a member of the 103 JNK signature), and Mef2 (a member of the 399 JNK signature), were amongst the up-regulated JNK targets. Mesoderm specification is not necessarily associated with a mesenchymal-like cell morphology, however, dl is involved in the induction of EMT during embryonic development, and both dl and Mef2 act with Twi and Sna to coordinate mesoderm formation. Interestingly, recent studies have identified dl in an overexpression screen for genes capable of cooperating with scrib > in Drosophila tumorigenesis, and Mef2 has been identified as a cooperating oncogene in Drosophila, and possibly also in humans, where a correlation exists between the expression of Notch and Mef2 paralogues in human breast tumor samples. It is therefore possible that dl and Mef2 either act in combination with Twi or Sna, or independently of them but in a similar oncogenic capacity, to promote a mesodermal cell fate in scrib > RasACT/NACT tumors. The potential relevance of this to the mesenchymal cell morphology associated with tumor cell invasion, as well as the acquisition of progenitor states is worthy of further investigation (Doggett, 2015).

In mef2-driven tumors both overgrowth and invasion depend upon activation of JNK signaling, suggesting that Mef2 is not capable of promoting invasive capabilities independent of JNK. In contrast, chinmo+RasACT/NACT tumors appeared non-invasive and retained epithelial morphology despite the massive overgrowth, although closer examination of cell polarity markers will be required to confirm this. Furthermore, the overgrowth of chinmo+RasACT/NACT tumors was not dependent upon JNK signaling, suggesting that the maintenance of a progenitor-like state could be uncoupled from JNK-induced EMT-effectors associated with invasion. Whether clear divisions between mesenchymal behaviour and progenitor states in tumors can be clearly separated in this manner is not yet clear, however, overall, it is likely that multiple JNK-regulated genes will participate in both promoting tumor overgrowth as well as migration/invasion. Although this study used the 103 JNK signature as a means to focus upon potential key candidates, an analysis of the 399 JNK-regulated probe sets common to both Ras and Notch-driven tumours has the potential to provide deeper insights into the multiple effectors of JNK signaling during tumorigenesis. Whilst the individual role of these genes can be probed with knockdowns, the complexity of the response, potentially with multiple redundancies and cross-talk, will ultimately need a network level of understanding to more fully expose key nodes participating in overgrowth and invasion. This approach has considerable potential to further expose core principles and mechanisms that drive human tumorigenesis, since it is clear that many fundamental commonalities underlie the development of tumors in Drosophila and mammals (Doggett, 2015).


PROTEIN STRUCTURE

Amino Acids - 1731

Structural Domains

Sequence analysis of Scribbled reveals the presence of two known protein-protein interaction motifs. At the amino terminus of Scrib is a set of 16 leucine-rich repeats (LRRs). This region is very similar to the Ras-binding LRRs of the proteins Sur-8 from Caenorhabditis elegans and adenylate cyclase from yeast. Distributed throughout the remainder of the protein are four PDZ domains. The PDZ domains of Scrib are highly similar to those of PDZ3 from PSD-95, a human homolog of fly Discs-large. Comparison of Scrib residues with those of PSD-95 indicates that all four Scrib PDZ domains are Type 1A; these domains predicted to bind to the consensus S/TXV at the carboxy terminus of proteins. Scrib has at least one human homolog, encoded by the KIAA0147 cDNA, which is called here hscrib1. No obvious signal sequences or transmembrane domains are predicted in either Scrib or hScrib1 by standard algorithms, suggesting that Scrib is a cytosolic protein. Densin, a protein with 16 LRRs and one PDZ domain, has been suggested to have transmembrane topology. The possibility that Scrib is an atypical transmembrane protein cannot be excluded (Bilder, 2000a).


scribbled:
Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

date revised: 20 August 2000

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