EGF receptor : Evolutionary Homologs | Regulation | Protein Interactions | Developmental Biology | Effects of Mutation | References

Gene name - EGF receptor

Synonyms - DER, Ellipse, torpedo

Cytological map position - 57F1

Function - transmembrane signaling

Keywords - Epidermal growth factor pathway

Symbol - Egfr

FlyBase ID:FBgn0003731

Genetic map position - 2-[97]

Classification - receptor tyrosine kinase

Cellular location - surface TM protein

NCBI link: Entrez Gene
Egfr orthologs: Biolitmine

Recent literature
Seong, K.H., Tsuda, M., Tsuda-Sakurai, K. and Aigaki, T. (2015). The plant homeodomain finger protein MESR4 is essential for embryonic development in Drosophila. Genesis [Epub ahead of print]. PubMed ID: 26467775
Misexpression Suppressor of Ras 4 (MESR4), a PHD finger protein with nine zinc-finger motifs has been implicated in various biological processes including the regulation of fat storage and innate immunity in Drosophila. However, the role of MESR4 in the context of development remains unclear. This study shows that MESR4 is a nuclear protein essential for embryonic development. Immunostaining of polytene chromosomes using anti-MESR4 antibody revealed that MESR4 binds to numerous bands along the chromosome arms. The most intense signal was detected at the 39E-F region, which is known to contain the histone gene cluster. P-element insertions in the MESR4 locus are homozygous lethal during embryogenesis with defects in ventral ectoderm formation and head encapsulation. In the mutant embryos, expression of Fasciclin 3 (Fas3), an EGFR signal target gene is greatly reduced, and the level of EGFR signal-dependent double phosphorylated ERK (dp-ERK) remains low. However, in the context of wing vein formation, genetic interaction experiments suggest that MESR4 is involved in the EGFR signaling as a negative regulator. These results suggest that MESR4 is a novel chromatin-binding protein required for proper expression of genes including those regulated by the EGFR signaling pathway during development.

Jin, Y., Ha, N., Fores, M., Xiang, J., Glasser, C., Maldera, J., Jimenez, G. and Edgar, B. A. (2015). EGFR/Ras signaling controls Drosophila intestinal stem cell proliferation via Capicua-regulated genes. PLoS Genet 11: e1005634. PubMed ID: 26683696
Epithelial renewal in the Drosophila intestine is orchestrated by Intestinal Stem Cells (ISCs). Following damage or stress the intestinal epithelium produces ligands that activate the epidermal growth factor receptor (EGFR) in ISCs. This promotes their growth and division and, thereby, epithelial regeneration. This study demonstrates that the HMG-box transcriptional repressor, Capicua (Cic), mediates these functions of EGFR signaling. Depleting Cic in ISCs activated them for division, whereas overexpressed Cic inhibited ISC proliferation and midgut regeneration. Epistasis tests showed that Cic acted as an essential downstream effector of EGFR/Ras signaling, and immunofluorescence showed that Cic's nuclear localization was regulated by EGFR signaling. ISC-specific mRNA expression profiling and DNA binding mapping using DamID indicated that Cic represses cell proliferation via direct targets including string (Cdc25), Cyclin E, and the ETS domain transcription factors Ets21C and Pointed (pnt). pnt was required for ISC over-proliferation following Cic depletion, and ectopic pnt restored ISC proliferation even in the presence of overexpressed dominant-active Cic. These studies identify Cic, Pnt, and Ets21C as critical downstream effectors of EGFR signaling in Drosophila ISCs.

Eichenlaub, T., Cohen, S.M. and Herranz, H. (2016). Cell competition drives the formation of metastatic tumors in a Drosophila model of epithelial tumor formation. Curr Biol [Epub ahead of print]. PubMed ID: 26853367
Cell competition is a homeostatic process in which proliferating cells compete for survival. Elimination of otherwise normal healthy cells through competition is important during development and has recently been shown to contribute to maintaining tissue health during organismal aging. The mechanisms that allow for ongoing cell competition during adult life could, in principle, contribute to tumorigenesis. However, direct evidence supporting this hypothesis has been lacking. This study provides evidence that cell competition drives tumor formation in a Drosophila model of epithelial cancer. Cells expressing EGFR together with the conserved microRNA miR-8 acquire the properties of supercompetitors. Neoplastic transformation and metastasis depend on the ability of these cells to induce apoptosis and engulf nearby cells. miR-8 expression causes genome instability by downregulating expression of the Septin family protein Peanut. Cytokinesis failure due to downregulation of Peanut is required for tumorigenesis. The study provides evidence that the cellular mechanisms that drive cell competition during normal tissue growth can be co-opted to drive tumor formation and metastasis. Analogous mechanisms for cytokinesis failure may lead to polyploid intermediates in tumorigenesis in mammalian cancer models.

Chen, L.P., Wang, P., Sun, Y.J. and Wu, Y.J. (2016). Direct interaction of avermectin with epidermal growth factor receptor mediates the penetration resistance in Drosophila larvae. Open Biol 6. PubMed ID: 27249340
With the widespread use of avermectins (AVMs) for managing parasitic and agricultural pests, the resistance of worms and insects to AVMs has emerged as a serious threat to human health and agriculture worldwide. The reduced penetration of AVMs is one of the main reasons for the development of the resistance to the chemicals. However, the detailed molecular mechanisms remain elusive. This study used the larvae of Drosophila melanogaster as a model system to explore the molecular mechanisms underlying the development of penetration resistance to AVMs. It was shown that the chitin layer is thickened and the efflux transporter P-glycoprotein (P-gp) is overexpressed in the AVM-resistant larvae epidermis. The activation of the transcription factor Relish by the over-activated Epidermal growth factor receptor (EGFR)/AKT/ERK pathway was found to induce the overexpression of the chitin synthases DmeCHS1/2 and P-gp in the resistant larvae. Interestingly, AVM was found to directly interact with EGFR and lead to the activation of the EGFR/AKT/ERK pathway, which activates the transcription factor Relish and induces the overexpression of DmeCHS1/2 and P-gp. These findings provide new insights into the molecular mechanisms underlying the development of penetration resistance to drugs. 

Fregoso Lomas, M., De Vito, S., Boisclair Lachance, J.F., Houde, J. and Nilson, L.A. (2016). Determination of EGFR signaling output by opposing gradients of BMP and JAK/STAT activity. Curr Biol [Epub ahead of print]. PubMed ID: 27593379
A relatively small number of signaling pathways drive a wide range of developmental decisions, but how this versatility in signaling outcome is generated is not clear. In the Drosophila follicular epithelium, localized epidermal growth factor receptor (EGFR) activation induces distinct cell fates depending on its location. Posterior follicle cells respond to EGFR activity by expressing the T-box transcription factors Midline and H15, while anterior cells respond by expressing the homeodomain transcription factor Mirror. This study shows that the choice between these alternative outputs of EGFR signaling is regulated by antiparallel gradients of JAK/STAT and BMP pathway activity and that mutual repression between Midline/H15 and Mirror generates a bistable switch that toggles between alternative EGFR signaling outcomes. JAK/STAT and BMP pathway input is integrated through their joint and opposing regulation of both sides of this switch. By converting this positional information into a binary decision between EGFR signaling outcomes, this regulatory network ultimately allows the same ligand-receptor pair to establish both the anterior-posterior (AP) and dorsal-ventral (DV) axes of the issue.

Jussen, D., von Hilchen, J. and Urbach, R. (2016). Genetic regulation and function of epidermal growth factor receptor signalling in patterning of the embryonic Drosophila brain. Open Biol 6(12). PubMed ID: 27974623
The specification of distinct neural cell types in central nervous system development crucially depends on positional cues conferred to neural stem cells in the neuroectoderm. This study investigated the regulation and function of the epidermal growth factor receptor (EGFR) signalling pathway in early development of the Drosophila brain. Localized EGFR signalling in the brain neuroectoderm was found to rely on a neuromere-specific deployment of activating (Spitz, Vein) and inhibiting (Argos) ligands. Activated EGFR controls the spatially restricted expression of all dorsoventral (DV) patterning genes in a gene- and neuromere-specific manner. Further, a novel role of DV genes-ventral nervous system defective (vnd), intermediate neuroblast defective (ind), Nkx6-in regulating the expression of vein and argos, which feed back on EGFR, indicating that EGFR signalling stands not strictly atop the DV patterning genes. Within this network of genetic interactions, Vnd acts as a positive EGFR feedback regulator. Further, it was shown that EGFR signalling becomes dependent on single-minded-expressing midline cells in the posterior brain (tritocerebrum), but remains midline-independent in the anterior brain (deuto- and protocerebrum). Finally, it was demonstrated that activated EGFR controls the proper formation of brain neuroblasts by regulating the number, survival and proneural gene expression of neuroectodermal progenitor cells. These data demonstrate that EGFR signalling is crucially important for patterning and early neurogenesis of the brain.
Chabu, C., Li, D. M. and Xu, T. (2017). EGFR/ARF6 regulation of Hh signalling stimulates oncogenic Ras tumour overgrowth. Nat Commun 8: 14688. PubMed ID: 28281543
Multiple signalling events interact in cancer cells. Oncogenic Ras cooperates with Egfr, which cannot be explained by the canonical signalling paradigm. In turn, Egfr cooperates with Hedgehog signalling. How oncogenic Ras elicits and integrates Egfr and Hedgehog signals to drive overgrowth remains unclear. Using a Drosophila tumour model, this study shows that Egfr cooperates with oncogenic Ras via Arf6, which functions as a novel regulator of Hh signalling. Oncogenic Ras induces the expression of Egfr ligands. Egfr then signals through Arf6, which regulates Hh transport to promote Hh signalling. Blocking any step of this signalling cascade inhibits Hh signalling and correspondingly suppresses the growth of both, fly and human cancer cells harbouring oncogenic Ras mutations. These findings highlight a non-canonical Egfr signalling mechanism, centered on Arf6 as a novel regulator of Hh signalling. This explains both, the puzzling requirement of Egfr in oncogenic Ras-mediated overgrowth and the cooperation between Egfr and Hedgehog.
Xiang, J., Bandura, J., Zhang, P., Jin, Y., Reuter, H. and Edgar, B. A. (2017). EGFR-dependent TOR-independent endocycles support Drosophila gut epithelial regeneration. Nat Commun 8: 15125. PubMed ID: 28485389
Following gut epithelial damage, epidermal growth factor receptor/mitogen-activated protein kinase (EGFR/MAPK) signalling triggers Drosophila intestinal stem cells to produce enteroblasts (EBs) and enterocytes (ECs) that regenerate the gut. As EBs differentiate into ECs, they become postmitotic, but undergo extensive growth and DNA endoreplication. This study reports that EGFR/RAS/MAPK signalling is required and sufficient to drive damage-induced EB/EC growth. Endoreplication occurs exclusively in EBs and newborn ECs that inherit EGFR and active MAPK from fast-dividing progenitors. Mature ECs lack EGF receptors and are refractory to growth signalling. Genetic tests indicated that stress-dependent EGFR/MAPK promotes gut regeneration via a novel mechanism that operates independently of Insulin/Pi3K/TOR signalling, which is nevertheless required in nonstressed conditions. The E2f1 transcription factor is required for and sufficient to drive EC endoreplication, and Ras/Raf signalling upregulates E2f1 levels posttranscriptionally. This study illustrates how distinct signalling mechanisms direct stress-dependent versus homeostatic regeneration, and highlight the importance of postmitotic cell growth in gut epithelial repair.
Kim, S., Nahm, M., Kim, N., Kwon, Y., Kim, J., Choi, S., Choi, E. Y., Shim, J., Lee, C. and Lee, S. (2017). Graf regulates hematopoiesis through GEEC endocytosis of EGFR. Development 144(22): 4159-4172. PubMed ID: 28993397
GTPase regulator associated with focal adhesion kinase 1 (GRAF1) is an essential component of the GPI-enriched endocytic compartment (GEEC) endocytosis pathway. Mutations in the human GRAF1 gene are associated with acute myeloid leukemia, but its normal role in myeloid cell development remains unclear. This study shows that Graf, the Drosophila ortholog of GRAF1, is expressed and specifically localizes to GEEC endocytic membranes in macrophage-like plasmatocytes. Loss of Graf impairs GEEC endocytosis, enhances EGFR signaling and induces a plasmatocyte overproliferation phenotype that requires the EGFR signaling cascade. Mechanistically, Graf-dependent GEEC endocytosis serves as a major route for EGFR internalization at high, but not low, doses of the predominant Drosophila EGFR ligand Spitz (Spi), and is indispensable for efficient EGFR degradation and signal attenuation. Finally, Graf interacts directly with EGFR in a receptor ubiquitylation-dependent manner, suggesting a mechanism by which Graf promotes GEEC endocytosis of EGFR at high Spi. Based on these findings, a model is proposed in which Graf functions to downregulate EGFR signaling by facilitating Spi-induced receptor internalization through GEEC endocytosis, thereby restraining plasmatocyte proliferation.
Crespo-Yanez, X., Aguilar-Gurrieri, C., Jacomin, A. C., Journet, A., Mortier, M., Taillebourg, E., Soleilhac, E., Weissenhorn, W. and Fauvarque, M. O. (2018). CHMP1B is a target of USP8/UBPY regulated by ubiquitin during endocytosis. PLoS Genet 14(6): e1007456. PubMed ID: 29933386
Integration and down-regulation of cell growth and differentiation signals rely on plasma membrane receptors endocytosis and sorting towards either recycling vesicles or degradative lysosomes via multivesicular bodies (MVB). In this process, the endosomal sorting complex-III required for transport (ESCRT-III) controls membrane deformation and scission triggering intraluminal vesicle (ILV) formation at early endosomes. This study shows that the ESCRT-III member CHMP1B can be ubiquitinated within a flexible loop known to undergo conformational changes during polymerization. This study demonstrates further that CHMP1B is deubiquitinated by the ubiquitin specific protease USP8 (syn. UBPY) and found fully devoid of ubiquitin in a ~500 kDa large complex that also contains its known ESCRT-III partner IST1. Moreover, EGF stimulation induces the rapid and transient accumulation of ubiquitinated forms of CHMP1B on cell membranes. Accordingly, CHMP1B ubiquitination is necessary for CHMP1B function in both EGF receptor trafficking in human cells and wing development in Drosophila. From these observations, it is proposed that CHMP1B is dynamically regulated by ubiquitination in response to EGF and that USP8 triggers CHMP1B deubiquitination possibly favoring its subsequent assembly into a membrane-associated ESCRT-III polymer.
Courgeon, M., He, D., Liu, H. H., Legent, K. and Treisman, J. E. (2018). The Drosophila Epidermal Growth Factor Receptor does not act in the nucleus. J Cell Sci. PubMed ID: 30158176
Mammalian members of the ErbB family, including the Epidermal Growth Factor Receptor (EGFR), can regulate transcription, DNA replication and repair through nuclear entry of either the full-length proteins or their cleaved cytoplasmic domains. In cancer cells, these nuclear functions contribute to tumor progression and drug resistance. This study examined whether the single Drosophila EGFR can also localize to the nucleus. A chimeric EGFR protein fused at its cytoplasmic C-terminus to DNA-binding and transcriptional activation domains strongly activated transcriptional reporters when overexpressed in cultured cells or in vivo. However, this activity was independent of cleavage and endocytosis. Without an exogenous activation domain, EGFR fused to a DNA-binding domain did not activate or repress transcription. Addition of the same DNA-binding and transcriptional activation domains to the endogenous Egfr locus by genome editing produced no detectable reporter expression in wild type or oncogenic contexts. These results show that when expressed at physiological levels, the cytoplasmic domain of the Drosophila EGFR does not have access to the nucleus. Nuclear EGFR functions are likely to have evolved after vertebrates and invertebrates diverged.
Meschi, E., Leopold, P. and Delanoue, R. (2019). An EGF-responsive neural circuit couples insulin secretion with nutrition in Drosophila. Dev Cell 48(1): 76-86.e75. PubMed ID: 30555002
Developing organisms use fine-tuning mechanisms to adjust body growth to ever-changing nutritional conditions. In Drosophila, the secretory activity of insulin-producing cells (IPCs) is central to couple systemic growth with amino acids availability. This study identified a subpopulation of inhibitory neurons contacting the IPCs (IPC-connecting neurons or ICNs) that play a key role in this coupling. ICNs respond to growth-blocking peptides (GBPs), a family of fat-body-derived signals produced upon availability of dietary amino acids. GBPs are atypical ligands for the fly EGF receptor (EGFR). Upon activation of EGFR by adipose GBPs, ICN-mediated inhibition of IPC function is relieved, allowing insulin secretion. Tnis study reveals an unexpected role for EGF-like metabolic hormones and EGFR signaling as critical modulators of neural activity, coupling insulin secretion to the nutritional status.
Jorg, D. J., Caygill, E. E., Hakes, A. E., Contreras, E. G., Brand, A. H. and Simons, B. D. (2019). The proneural wave in the Drosophila optic lobe is driven by an excitable reaction-diffusion mechanism. Elife 8. PubMed ID: 30794154
In living organisms, self-organised waves of signalling activity propagate spatiotemporal information within tissues. During the development of the largest component of the visual processing centre of the Drosophila brain, a travelling wave of proneural gene expression initiates neurogenesis in the larval optic lobe primordium and drives the sequential transition of neuroepithelial cells into neuroblasts. This study proposes that this 'proneural wave' is driven by an excitable reaction-diffusion system involving epidermal growth factor receptor (EGFR) signalling interacting with the proneural gene l'sc. Within this framework, a propagating transition zone emerges from molecular feedback and diffusion. Ectopic activation of EGFR signalling in clones within the neuroepithelium demonstrates that a transition wave can be excited anywhere in the tissue by inducing signalling activity, consistent with a key prediction of the model. This model illuminates the physical and molecular underpinnings of proneural wave progression and suggests a generic mechanism for regulating the sequential differentiation of tissues.
Mao, Y., Tu, R., Huang, Y., Mao, D., Yang, Z., Lau, P. K., Wang, J., Ni, J., Guo, Y. and Xie, T. (2019). The exocyst functions in niche cells to promote germline stem cell differentiation by directly controlling EGFR membrane trafficking. Development. PubMed ID: 31142545
The niche controls stem cell self-renewal and differentiation in animal tissues. Although the exocyst is known to be important for protein membrane trafficking and secretion, its role in stem cells and niches has never been reported. This study shows that the exocyst functions in the niche to promote germline stem cell (GSC) progeny differentiation in the Drosophila ovary by directly regulating EGFR membrane trafficking and signaling. Inactivating exocyst components in inner germarial sheath cells, which form the differentiation niche, causes a severe GSC differentiation defect. The exocyst is required for maintaining niche cells and preventing BMP signaling in GSC progeny by promoting EGFR membrane targeting and signaling through direct association with EGFR. Finally, it is also required for EGFR membrane targeting, recycling and signaling in human cells. Therefore, this study has revealed a novel function of the exocyst in niche cells to promote stem cell progeny differentiation by directly controlling EGFR membrane trafficking and signaling in vivo and has also provided important insight into how the niche controls stem cell progeny differentiation at the molecular level.
Senos Demarco, R., Uyemura, B. S. and Jones, D. L. (2020). EGFR Signaling Stimulates Autophagy to Regulate Stem Cell Maintenance and Lipid Homeostasis in the Drosophila Testis. Cell Rep 30(4): 1101-1116.e1105. PubMed ID: 31995752
Although typically upregulated upon cellular stress, autophagy can also be utilized under homeostatic conditions as a quality control mechanism or in response to developmental cues. This study reports that autophagy is required for the maintenance of somatic cyst stem cells (CySCs) in the Drosophila testis. Disruption of autophagy in CySCs and early cyst cells (CCs) by the depletion of autophagy-related (Atg) genes reduced early CC numbers and affected CC function, resembling decreased epidermal growth factor receptor (EGFR) signaling. Indeed, the data indicate that EGFR acts to stimulate autophagy to preserve early CC function, whereas target of rapamycin (TOR) negatively regulates autophagy in the differentiating CCs. Finally, this study shows that the EGFR-mediated stimulation of autophagy regulates lipid levels in CySCs and CCs. These results demonstrate a key role for autophagy in regulating somatic stem cell behavior and tissue homeostasis by integrating cues from both the EGFR and TOR signaling pathways to control lipid metabolism.
Revaitis, N. T., Niepielko, M. G., Marmion, R. A., Klein, E. A., Piccoli, B. and Yakoby, N. (2020). Quantitative analyses of EGFR localization and trafficking dynamics in the follicular epithelium. Development. PubMed ID: 32680934
To bridge the gap between qualitative and quantitative analyses of the epidermal growth factor receptor (EGFR) in tissues, this study generated a sfGFP-tagged EGF receptor (EGFR-sfGFP) in Drosophila. The homozygous fly appears wild type with EGFR expression and activation patterns that are consistent with previous reports in the ovary, early embryo, and imaginal discs. Using ELISA, an average of 1100, 6200, and 2500 receptors was quantified per follicle cell (FC) at stages 8/9, 10, and ≥11 of oogenesis, respectively. Interestingly, the spatial localization of the EGFR to the apical side of the FCs at early stages depended on the TGF-alpha-like ligand Gurken. At later stages, EGFR localized to basolateral positions of the FCs. Finally, the endosomal localization of EGFR was followed in the FCs. The EGFR co-localized with the late endosome, but no significant co-localization of the receptor was found with the early endosome. The EGFR-sfGFP fly is an exciting new resource to study cellular localization and regulation of EGFR in tissues.
Spierer, A. N., Mossman, J. A., Smith, S. P., Crawford, L., Ramachandran, S. and Rand, D. M. (2021). Natural variation in the regulation of neurodevelopmental genes modifies flight performance in Drosophila. PLoS Genet 17(3): e1008887. PubMed ID: 33735180
The winged insects of the order Diptera are colloquially named for their most recognizable phenotype: flight. These insects rely on flight for a number of important life history traits, such as dispersal, foraging, and courtship. Despite the importance of flight, relatively little is known about the genetic architecture of flight performance. Accordingly, this study sought to uncover the genetic modifiers of flight using a measure of flies' reaction and response to an abrupt drop in a vertical flight column. A genome wide association study (GWAS) was conducted using 197 of the Drosophila Genetic Reference Panel (DGRP) lines, and a combination was identified of additive and marginal variants, epistatic interactions, whole genes, and enrichment across interaction networks. Egfr, a highly pleiotropic developmental gene, was among the most significant additive variants identified. 13 of the additive candidate genes (Adgf-A/Adgf-A2/CG32181, bru1, CadN, flapper (CG11073), CG15236, flippy (CG9766), CREG, Dscam4, form3, fry, Lasp/CG9692, Pde6, Snoo) were functionally validated, and a novel approach was introduced to whole gene significance screens: PEGASUS_flies. Additionally, ppk23, an Acid Sensing Ion Channel (ASIC) homolog, was identified as an important hub for epistatic interactions. A model is proposed that suggests genetic modifiers of wing and muscle morphology, nervous system development and function, BMP signaling, sexually dimorphic neural wiring, and gene regulation are all important for the observed differences flight performance in a natural population. Additionally, these results represent a snapshot of the genetic modifiers affecting drop-response flight performance in Drosophila, with implications for other insects.
Maravat, M., Bertrand, M., Landon, C., Fayon, F., Morisset-Lopez, S., Sarou-Kanian, V. and Decoville, M. (2021). Complementary Nuclear Magnetic Resonance-Based Metabolomics Approaches for Glioma Biomarker Identification in a Drosophila melanogaster Model. J Proteome Res. PubMed ID: 34286978
Human malignant gliomas are the most common type of primary brain tumor. Composed of glial cells and their precursors, they are aggressive and highly invasive, leading to a poor prognosis. Due to the difficulty of surgically removing tumors and their resistance to treatments, novel therapeutic approaches are needed to improve patient life expectancy and comfort. Glioma has been induced in Drosophila by co-activating the epidermal growth factor receptor and the phosphatidyl-inositol-3 kinase signaling pathways. Complementary nuclear magnetic resonance (NMR) techniques were used to obtain metabolic profiles in the third instar larvae brains. Fresh organs were directly studied by (1)H high resolution-magic angle spinning (HR-MAS) NMR, and brain extracts were analyzed by solution-state (1)H-NMR. Statistical analyses revealed differential metabolic signatures, impacted metabolic pathways, and glioma biomarkers. Each method was efficient to determine biomarkers. The highlighted metabolites including glucose, myo-inositol, sarcosine, glycine, alanine, and pyruvate for solution-state NMR and proline, myo-inositol, acetate, and glucose for HR-MAS show very good performances in discriminating samples according to their nature with data mining based on receiver operating characteristic curves. Combining results allows for a more complete view of induced disturbances and opens the possibility of deciphering the biochemical mechanisms of these tumors. The identified biomarkers provide a means to rebalance specific pathways through targeted metabolic therapy and to study the effects of pharmacological treatments using Drosophila as a model organism.
Beatty, J. S., Molnar, C., Luque, C. M., de Celis, J. F. and Martin-Bermudo, M. D. (2021). EGFRAP encodes a new negative regulator of the EGFR acting in both normal and oncogenic EGFR/Ras-driven tissue morphogenesis. PLoS Genet 17(8): e1009738. PubMed ID: 34411095
Activation of Ras signaling occurs in ~30% of human cancers. However, activated Ras alone is insufficient to produce malignancy. Thus, it is imperative to identify those genes cooperating with activated Ras in driving tumoral growth. This work identified a novel EGFR inhibitor, which was named EGFRAP, for EGFR adaptor protein. Elimination of EGFRAP potentiates activated Ras-induced overgrowth in the Drosophila wing imaginal disc. EGFRAP interacts physically with the phosphorylated form of EGFR via its SH2 domain. EGFRAP is expressed at high levels in regions of maximal EGFR/Ras pathway activity, such as at the presumptive wing margin. In addition, EGFRAP expression is up-regulated in conditions of oncogenic EGFR/Ras activation. Normal and oncogenic EGFR/Ras-mediated upregulation of EGRAP levels depend on the Notch pathway. Elimination of EGFRAP does not affect overall organogenesis or viability. However, simultaneous downregulation of EGFRAP and its ortholog PVRAP results in defects associated with increased EGFR function. Based on these results, it is proposed that EGFRAP is a new negative regulator of the EGFR/Ras pathway, which, while being required redundantly for normal morphogenesis, behaves as an important modulator of EGFR/Ras-driven tissue hyperplasia. It is suggested that the ability of EGFRAP to functionally inhibit the EGFR pathway in oncogenic cells results from the activation of a feedback loop leading to increase EGFRAP expression. This could act as a surveillance mechanism to prevent excessive EGFR activity and uncontrolled cell growth.
Resnik-Docampo, M., Cunningham, K. M., Ruvalcaba, S. M., Choi, C., Sauer, V. and Jones, D. L. (2021). Neuroglian regulates Drosophila intestinal stem cell proliferation through enhanced signaling via the epidermal growth factor receptor. Stem Cell Reports. PubMed ID: 33961791
The Drosophila intestine is an excellent system for elucidating mechanisms regulating stem cell behavior. This study shows that the septate junction (SJ) protein Neuroglian (Nrg) is expressed in intestinal stem cells (ISCs) and enteroblasts (EBs) within the fly intestine. SJs are not present between ISCs and EBs, suggesting Nrg plays a different role in this tissue. This study reveals that Nrg is required for ISC proliferation in young flies, and depletion of Nrg from ISCs and EBs suppresses increased ISC proliferation in aged flies. Conversely, overexpression of Nrg in ISC and EBs promotes ISC proliferation, leading to an increase in cells expressing ISC/EB markers; in addition, an increase was observed in epidermal growth factor receptor (Egfr) activation. Genetic epistasis experiments reveal that Nrg acts upstream of Egfr to regulate ISC proliferation. As Nrg function is highly conserved in mammalian systems, this work characterizing the role of Nrg in the intestine has implications for the treatment of intestinal disorders that arise due to altered ISC behavior.
Proske, A., Bossen, J., von Frieling, J. and Roeder, T. (2021). Low-protein diet applied as part of combination therapy or stand-alone normalizes lifespan and tumor proliferation in a model of intestinal cancer. Aging (Albany NY) 13:. PubMed ID: 34766923
Tumors of the intestinal tract are among the most common tumor diseases in humans, but, like many other tumor entities, show an unsatisfactory prognosis with a need for effective therapies. To test whether nutritional interventions and a combination with a targeted therapy can effectively cure these cancers, the fruit fly Drosophila was used as a model. In this system, tumors were introduced by EGFR overexpression in intestinal stem cells. Limiting the amount of protein in the diet restored life span to that of control animals. In combination with a specific EGFR inhibitor, all major tumor-associated phenotypes could be rescued. This form of treatment was also successful in a real treatment scenario, which means when they started after the full tumor phenotype was expressed. In conclusion, reduced protein administration can be a very promising form of adjuvant cancer therapy.
Greenspan, L. J., de Cuevas, M., Le, K. H., Viveiros, J. M. and Matunis, E. L. (2022). Activation of the EGFR/MAPK pathway drives transdifferentiation of quiescent niche cells to stem cells in the Drosophila testis niche. Elife 11. PubMed ID: 35468055
Adult stem cells are maintained in niches, specialized microenvironments that regulate their self-renewal and differentiation. In the adult Drosophila testis stem cell niche, somatic hub cells produce signals that regulate adjacent germline stem cells (GSCs) and somatic cyst stem cells (CySCs). Hub cells are normally quiescent, but after complete genetic ablation of CySCs, they can proliferate and transdifferentiate into new CySCs. This study found that Epidermal growth factor receptor (EGFR) signaling is upregulated in hub cells after CySC ablation and that the ability of testes to recover from ablation is inhibited by reduced EGFR signaling. In addition, activation of the EGFR pathway in hub cells is sufficient to induce their proliferation and transdifferentiation into CySCs. It is proposed that EGFR signaling, which is normally required in adult cyst cells, is actively inhibited in adult hub cells to maintain their fate but is repurposed to drive stem cell regeneration after CySC ablation.
Velasquez, E., Gomez-Sanchez, J. A., Donier, E., Grijota-Martinez, C., Cabedo, H. and Garcia-Alonso, L. (2022). Fasciclin 2 engages EGFR in an auto-stimulatory loop to promote imaginal disc cell proliferation in Drosophila. PLoS Genet 18(6): e1010224. PubMed ID: 35666718
How cell to cell interactions control local tissue growth to attain a species-specific organ size is a central question in developmental biology. The Drosophila Neural Cell Adhesion Molecule, Fasciclin 2, is expressed during the development of neural and epithelial organs. Fasciclin 2 is a homophilic-interaction protein that shows moderate levels of expression in the proliferating epithelia and high levels in the differentiating non-proliferative cells of imaginal discs. Genetic interactions and mosaic analyses reveal a cell autonomous requirement of Fasciclin 2 to promote cell proliferation in imaginal discs. This function is mediated by the EGFR, and indirectly involves the JNK and Hippo signaling pathways. it was further shown that Fasciclin 2 physically interacts with EGFR and that, in turn, EGFR activity promotes the cell autonomous expression of Fasciclin 2 during imaginal disc growth. It is proposed that this auto-stimulatory loop between EGFR and Fasciclin 2 is at the core of a cell to cell interaction mechanism that controls the amount of intercalary growth in imaginal discs.
Dutta, S. B., Linneweber, G. A., Andriatsilavo, M., Hiesinger, P. R. and Hassan, B. A. (2023). TEGFR-dependent suppression of synaptic autophagy is required for neuronal circuit development. Curr Biol. PubMed ID: 36640763
The development of neuronal connectivity requires stabilization of dynamic axonal branches at sites of synapse formation. Models that explain how axonal branching is coupled to synaptogenesis postulate molecular regulators acting in a spatiotemporally restricted fashion to ensure branching toward future synaptic partners while also stabilizing the emerging synaptic contacts between such partners. This question was investigated using neuronal circuit development in the Drosophila brain as a model system. Epidermal growth factor receptor (EGFR) activity was shown to be required in presynaptic axonal branches during two distinct temporal intervals to regulate circuit wiring in the developing Drosophila visual system. EGFR is required early to regulate primary axonal branching. EGFR activity is then independently required at a later stage to prevent degradation of the synaptic active zone protein Bruchpilot (Brp). Inactivation of EGFR results in a local increase of autophagy in presynaptic branches and the translocation of active zone proteins into autophagic vesicles. The protection of synaptic material during this later interval of wiring ensures the stabilization of terminal branches, circuit connectivity, and appropriate visual behavior. Phenotypes of EGFR inactivation can be rescued by increasing Brp levels or downregulating autophagy. In summary, a temporally restricted molecular mechanism required for coupling axonal branching and synaptic stabilization was demonstrated that contributes to the emergence of neuronal wiring specificity.
Zhou, S., Li, P., Liu, J., Liao, J., Li, H., Chen, L., Li, Z., Guo, Q., Belguise, K., Yi, B. and Wang, X. (2022). Two Rac1 pools integrate the direction and coordination of collective cell migration. Nat Commun 13(1): 6014. PubMed ID: 36224221
Integration of collective cell direction and coordination is believed to ensure collective guidance for efficient movement. Previous studies demonstrated that chemokine receptors PVR and EGFR govern a gradient of Rac1 activity essential for collective guidance of Drosophila border cells, whose mechanistic insight is unknown. By monitoring and manipulating subcellular Rac1 activity, this study reveal two switchable Rac1 pools at border cell protrusions and supracellular cables, two important structures responsible for direction and coordination. Rac1 and Rho1 form a positive feedback loop that guides mechanical coupling at cables to achieve migration coordination. Rac1 cooperates with Cdc42 to control protrusion growth for migration direction, as well as to regulate the protrusion-cable exchange, linking direction and coordination. PVR and EGFR guide correct Rac1 activity distribution at protrusions and cables. Therefore, these studies emphasize the existence of a balance between two Rac1 pools, rather than a Rac1 activity gradient, as an integrator for the direction and coordination of collective cell migration.
Taniguchi, K. and Igaki, T. (2023). Sas-Ptp10D shapes germ-line stem cell niche by facilitating JNK-mediated apoptosis. PLoS Genet 19(3): e1010684. PubMed ID: 36972315
The function of the stem cell system is supported by a stereotypical shape of the niche structure. In Drosophila ovarian germarium, somatic cap cells form a dish-like niche structure that allows only two or three germ-line stem cells (GSCs) reside in the niche. Despite extensive studies on the mechanism of stem cell maintenance, the mechanisms of how the dish-like niche structure is shaped and how this structure contributes to the stem cell system have been elusive. This study shows that a transmembrane protein Stranded at second (Sas) and its receptor Protein tyrosine phosphatase 10D (Ptp10D), effectors of axon guidance and cell competition via Epidermal growth factor receptor (Egfr) inhibition, shape the dish-like niche structure by facilitating c-Jun N-terminal kinase (JNK)-mediated apoptosis. Loss of Sas or Ptp10D in gonadal apical cells, but not in GSCs or cap cells, during the pre-pupal stage results in abnormal shaping of the niche structure in the adult, which allows excessive, four to six GSCs reside in the niche. Mechanistically, loss of Sas-Ptp10D elevates Egfr signaling in the gonadal apical cells, thereby suppressing their naturally-occurring JNK-mediated apoptosis that is essential for the shaping of the dish-like niche structure by neighboring cap cells. Notably, the abnormal niche shape and resulting excessive GSCs lead to diminished egg production. These data propose a concept that the stereotypical shaping of the niche structure optimizes the stem cell system, thereby maximizing the reproductive capacity.
Yoshida, K. and Hayashi, S. (2023). Epidermal growth factor receptor signaling protects epithelia from morphogenetic instability and tissue damage in Drosophila. Development 150(5). PubMed ID: 36897356
Dying cells in the epithelia communicate with neighboring cells to initiate coordinated cell removal to maintain epithelial integrity. Naturally occurring apoptotic cells are mostly extruded basally and engulfed by macrophages. This study has investigated the role of Epidermal growth factor (EGF) receptor (EGFR) signaling in the maintenance of epithelial homeostasis. In Drosophila embryos, epithelial tissues undergoing groove formation preferentially enhanced extracellular signal-regulated kinase (ERK) signaling. In EGFR mutant embryos at stage 11, sporadic apical cell extrusion in the head initiates a cascade of apical extrusions of apoptotic and non-apoptotic cells that sweeps the entire ventral body wall. This study shows that this process is apoptosis dependent, and clustered apoptosis, groove formation, and wounding sensitize EGFR mutant epithelia to initiate massive tissue disintegration. It was further shown that tissue detachment from the vitelline membrane, which frequently occurs during morphogenetic processes, is a key trigger for the EGFR mutant phenotype. These findings indicate that, in addition to cell survival, EGFR plays a role in maintaining epithelial integrity, which is essential for protecting tissues from transient instability caused by morphogenetic movement and damage.
Yin, H., Wang, Z., Wang, D., Nuer, M., Han, M., Ren, P., Ma, S., Lin, C., Chen, J., Xian, H., Ai, D., Li, X., Ma, S., Lin, Z. and Pan, Y. (2023). TIMELESS promotes the proliferation and migration of lung adenocarcinoma cells by activating EGFR through AMPK and SPHK1 regulation. Eur J Pharmacol 955: 175883. PubMed ID: 37433364
Lung adenocarcinoma (LUAD) has high morbidity and is prone to recurrence. TIMELESS (TIM), which regulates circadian rhythms in Drosophila, is highly expressed in various tumors. Tumor samples from patients with LUAD patient data from public databases were used to confirm the relationship of TIM expression with lung cancer. LUAD cell lines were used and siRNA of TIM was adopted to knock down TIM expression in LUAD cells, and further cell proliferation, migration and colony formation were analyzed. By using Western blot and qPCR, the influence was detected of TIM on epidermal growth factor receptor (EGFR), sphingosine kinase 1 (SPHK1) and AMP-activated protein kinase (AMPK). With proteomics analysis, this study comprehensively inspected the different changed proteins influenced by TIM, and global bioinformatic analysis was performed. TIM expression was found to be elevated in LUAD and that this high expression was positively correlated with more advanced tumor pathological stages and shorter overall and disease-free survival. TIM knockdown inhibited EGFR activation and also AKT/mTOR phosphorylation. This study also clarified that TIM regulated the activation of SPHK1 in LUAD cells. And with SPHK1 siRNA to knock down the expression level of SPHK1, it was found that EGFR activation were inhibited greatly too. Quantitative proteomics techniques combined with bioinformatics analysis clarified the global molecular mechanisms regulated by TIM in LUAD. The results of proteomics suggested that mitochondrial translation elongation and termination were altered, which were closely related to the process of mitochondrial oxidative phosphorylation. It was further confirmed that TIM knockdown reduced ATP content and promoted AMPK activation in LUAD cells. This study revealed that siTIM could inhibit EGFR activation through activating AMPK and inhibiting SPHK1 expression, as well as influencing mitochondrial function and altering the ATP level; TIM's high expression in LUAD is an important factor and a potential key target in LUAD.

The Drosophila EGF receptor homolog, commonly called Torpedo or DER, has a complex biology. It is found in multiple sites and at varying times during development; in signaling processes that determine egg polarity, during early development to determine the identity of cells in the ventral ectoderm, during neurogenesis, in the development of the Malpighian tubules, and during larval stages in the development of the eye and wing.

Egfr interacts with three different ligands: Gurken, Spitz (the principle ligand), and Argos. Two accessory proteins modulate Egfr signaling: Rhomboid and Star. The signaling molecules downstream of Egfr include Shc ( the Drosophila homolog of a mammalian oncogene), DRK (an adaptor protein that docks onto Shc bound to Egfr and a homolog of mammalian GRB2), a guanine nucleotide exchange factor (SOS) activated by DRK, and downstream targets like Ras, Raf and Rolled (Map kinase)(Lai, 1995).

All these downstream signaling molecules are members of the RAS-RAF-MAPK pathway that amplifies and transmits receptor signals to various parts of the cell. Signals to the cytoskeleton result in changes in cell shape; signals to the nucleus result in gene activation.

The signaling pathway used by Egfr is also used by other receptor tyrosine kinases, including the Torso receptor and Sevenless, another Drosophila EFG receptor homolog. How does a single pathway used by a number of different receptors permit the wide variety of specific cell differentiation effects that are receptor-dependent? This question is currently being investigated in a number of developmental pathways, but as yet, no answer is available. The immune system, that most intensely studied cell interaction system, is not driven by a single ligand receptor interaction, but by numerous signaling events involving a number of receptors and a number of ligands. There is no reason to believe that Drosophila differentiation will be any less complex. Thus signals through the Egfr will be one of a number of signals integrated into differentiation decisions.

The complexity of receptor tyrosine kinase signaling is revealed by studies of eye and wing differentiation. Signaling in the eye involves three ligands and two receptors as well as Rhomboid and Star, the two accessory proteins of Egfr signaling. The Boss-Sevenless receptor-ligand pair is involved, as well as the Spitz-Egfr receptor-ligand pair. An additional ligand, Argos, functions either directly or indirectly to inhibit the Egfr signaling (Freeman, 1994).

Rhomboid's particiption in Egfr signaling in the wing currently presents a fascinating mystery. Rhomboid is expressed in a geometric pattern that establishes the future sites of wing vein differentiation. Rhomboid enhances Egfr signaling at the sites of future veins, but exactly how this enhancement occurs is currently unknown. It appears that Rhomboid sets the stage for Egfr signaling and the resultant induction of wing veins (Sturtevant, 1995).

A more detailed review of Gurken's role in signaling dorsoventral and anterior-posterior polarity in the developing oocyte will be found at the gurken site. Maternally transcribed Egfr is used in this signaling process. Egfr has two promoters and two first exons. The different developmental roles for each of the two transcripts have not yet been documented (Clifford, 1994).

Egfr and the determination of wing and leg cell fates

Wing and leg precursors of Drosophila are recruited from a common pool of ectodermal cells expressing the homeobox gene Dll. Induction by Dpp promotes this cell fate decision toward the wing and proximal leg. The receptor tyrosine kinase Egfr antagonizes the wing-promoting function of Dpp and allows recruitment of leg precursor cells from uncommitted ectodermal cells. By monitoring the spatial distribution of cells responding to Dpp and Egfr, it has been shown that nuclear transduction of the two signals peaks at different positions along the dorsoventral axis when the fates of wing and leg discs are specified and that the balance of the two signals assessed within the nucleus determines the number of cells recruited to the wing. Differential activation of the two signals and the cross talk between them critically affect this cell fate choice (Kubota, 2000).

In a screen for genes expressed in the embryonic limb primordia, rhomboid was found to be transiently expressed in the central part of Dll-expressing limb primordia in stage 11 embryos. rho transcription is the rate-limiting step of the activation of an EGFR ligand Spitz. As expected from the role of rho as a stimulator of Egfr, a transient expression of an activated, phosphorylated form of MAPK (dpMAPK) is detected in the nucleus of limb primordial cells surrounding the rho-expressing cells. The dpMAPK expression starts after the initiation of Dll transcription and diminishes before the separation of the wing and leg disc primordium. The dpMAPK expression is undetectable in null mutants of rho or Egfr. The peak of dpMAPK expression is located ventrally to the cells expressing dpp. The results suggest that rho-mediated stimulation of Egfr and MAPK occurs at the time of cell fate specification of wing and leg discs (Kubota, 2000).

The spatial distribution of cells responding to Dpp and its relationship to Egfr signals was studied. To this end, an antibody specific to phosphorylated C-terminal sequence of Mad was produced. The phosphorylated sequence corresponds to the site at which the type I BMP receptor phosphorylates SMad1. The antibody detects an antigen distributed in a pattern similar to, but broader than, that of DPP mRNA. This immunoreactivity is dependent on Dpp signaling, as it is absent in stage 11 mutants of thick veins encoding type I Dpp receptor and in dpp mutants. This indicates that other extant TGFbeta-related signaling molecules present in Drosophila embryos do not substitute for Dpp to induce this immunoreactivity. Conversely, ectopic expression of Dpp results in high accumulation of this immunoreactivity. These results suggest that the antibody detects a Dpp-specific signaling event, most likely the phosphorylation and nuclear transport of Mad. Hereafter, the immunoreactivity detected by this antibody is called pSSVS (Kubota, 2000).

pSSVS is found mainly localized in the nucleus and distributed in regions a few cells wider in diameter than those of dpp-expressing cells. These properties are consistent with the previous findings that Mad transduces the Dpp signal to the nucleus. Double labeling of pSSVS and DLL mRNA shows that pSSVS expression is higher in the dorsal region of Dll-expressing cells. Combined with the double-labeling results of dpMAPK and Dll or dpp, it is concluded that cells responding to Dpp and Egfr overlap, but the peak of the responses are shifted. Such differential distribution of the two signals results in an arrangement of cells responding to a different strength of Dpp and Egfr along the dorsoventral axis (Kubota, 2000).

To study the role of Egfr at the stage of wing and leg cell fate determination, specific marker gene expression was examined in Egfr signaling mutants. DLL mRNA is expressed in the entire limb primordium at stage 11 and becomes restricted to distal leg cells at stage 15. Esg protein expression was used to detect both wing and proximal leg cells. In rho mutants, the size of limb primordia at stage 11 is the same as the control, but the later development of leg discs is abnormal. The number of leg disc cells expressing Dll and/or Esg at stage 15 is reduced, and these cells no longer show the circular arrangement typical of leg disc precursors. Amorphic mutation of Egfr cause a ventral expansion of limb primordia as a result of a loss of the early function of Egfr, but the expression of leg markers is severely reduced at stage 15. A similar phenotype is observed in mutants lacking both maternal and paternal copies of Dsor1, which encodes a MAP kinase kinase. In all cases described above, Esg-expressing cells at the dorsal part of leg discs are most frequently lost, suggesting that the development of dorsoproximal leg cells is most sensitive to the loss of Egfr activity. In contrast, wing and leg disc development is normal in vein mutants, suggesting the putative ligand of Egfr encoded by this gene is dispensable. These results suggest that MAPK activation induced by Rho and Egfr is essential for normal leg development (Kubota, 2000).

The temporal requirement for Egfr was studied by the temperature-sensitive allele Egfrf1. When the temperature is increased to the restrictive temperature at 5 hours after egg laying (AEL) prior to the induction of the limb primordium, the expression of Dll is expanded to the ventral midline, as was also observed with the strong Egfr mutants. When the temperature is increased at 6 hour AEL, the initial Dll expression is not altered, but the leg disc development is severely affected. Only mild defect is found in leg discs when the temperature is increased at 7 hours AEL, suggesting that Egfr must function between 6 and 7 hours AEL to correctly specify the leg cell fate. This is the time when the transient activation of MAPK is observed. Furthermore, whether Egfr is required autonomously in limb primordial cells was examined by expressing a dominant-negative form of Egfr using Dll-Gal4. Expression of this driver starts in the limb primordium at stage 11 and persists in a subset of wing discs and in entire leg discs at stage 15 because of the persistence of Gal4 activity. Imaginal disc-specific inhibition of Egfr interfers with leg disc development, while leaving the wing disc intact. These results demonstrated that a transient activation of Egfr in stage 11 limb primordia is essential for the leg disc development (Kubota, 2000).

In contrast to the severe defects in leg discs, none of the mutations in Egfr signaling interfer with wing disc formation. In these mutants, wing primordia consistently express Esg and another wing disc marker Vestigial, and invaginate to form discs. However, an increase in the number of wing disc cells has been noted in Egfr signaling mutants. This effect was analyzed in rho mutants; unlike Egfr mutants, in rho mutants the number of limb primordial cells at stage 11 is the same as the control. The number of Esg-expressing wing disc cells in rho mutants is increased compared to the control, while the number of the proximal leg disc cells is severely reduced. It is concluded that Egfr signaling is required to limit wing disc cell differentiation in limb primordial cells that are not yet fully committed. It is inferred that a subset of prospective leg cells that do not receive a sufficient amount of Egfr signaling fail to differentiate as proximal leg and instead adopt a wing fate (Kubota, 2000).

The increase in the number of wing disc cells in rho mutants resembles the overexpression phenotype of Dpp and raises a possibility that Egfr might prevent wing disc development by negatively regulating Dpp signaling. Such a cross talk could occur at several levels including the following: (1) regulation of dpp transcription, (2) signal transduction from Dpp receptors to the nucleus, and (3) transcriptional regulation of downstream target genes. The analyses excluded the first two possibilities for two reasons. (1) The expression pattern of DPP mRNA is unaffected by the mutation of rho. A previous report showing an expansion of dpp expression in Egfr mutants probably reflects the global patterning role of Egfr in the earlier stage. (2) pSSVS expression around limb primordia does not change in rho mutants. Conversely, the expression pattern of dpMAPK is not changed by a null mutation of tkv. These results suggest that the differential distribution of cells responding to Dpp and Egfr is set up independently of each other's activity (Kubota, 2000).

Dpp and Egfr were found to antagonize each other after signal transduction into the nucleus. Hyperactivation of Egfr by an ectopic expression of an Egfr ligand Spitz causes a great accumulation of dpMAPK. As expected from the negative effect of Egfr on the wing development, this treatment completely eliminates wing disc formation and, in addition, causes a malformation of the leg disc. Since it was found that cells migrating out of the leg primordium express dpMAPK, it is unlikely that the failure in wing disc formation is due to the prevention of cell migration or to cell death. It has been suggested that hyperactivation of Egfr prevents limb primordial cells from adopting the wing cell fate. It is likely that those cells adopt the epidermal fate instead. Overexpression of Dpp causes an accumulation of pSSVS and an increase in the number of wing disc cells. Coexpression of Dpp with Spi partially restores the development of both wing and leg discs, suggesting that wing disc development overcomes the negative effect of Egfr if provided with a sufficient amount of Dpp. The restored wing primordia migrate with high levels of pSSVS and dpMAPK, further supporting the notion that Dpp and Egfr signals are transduced independently of one another (Kubota, 2000).

dad is an immediate transcriptional target gene of Dpp, the expression of which closely parallels that of pSSVS expression in embryos and is inducible by Dpp. dad expression is not affected in Egfr or rho mutants. Furthermore, elevated dad expression induced by Dpp is not affected by sSpi, suggesting that at least one of the immediate transcriptional responses to Dpp is unaffected by elevated Egfr signaling (Kubota, 2000).

The antagonism between Dpp and Egfr during wing disc development raises a question: what is the default state of the wing and leg primordia in the absence of the two signals? Double mutant phenotypes of Dpp and Egfr signaling were examined. tkv mutants lack wing discs and their leg discs are malformed. This phenotype reflects a disc cell autonomous requirement for Dpp signaling, because the phenotype is reproduced by the disc-specific inhibition of Dpp signaling by dad, which inhibits Mad. The phenotype of either tkv;rho or tkv;Egfr double mutants is a simple addition of each mutation, in which wing discs are lost completely and leg discs are severely reduced. Since Dll-expressing limb primordial cells are present in tkv;Egfr double mutants in stage 11, it has been concluded that these cells fail to differentiate as wing discs and their ability to differentiate as leg discs is also compromised. A few Esg-positive cells remain at the position of the leg, and it is speculated that this reflects the presence of a second leg-inducing signal. These results suggest that Dpp is absolutely required for wing disc development irrespective of the activity of Egfr (Kubota, 2000).

Egfr affects the choice of wing vs. leg developmental options differently; it promotes leg development while it inhibits wing development. These two activities of Egfr are the earliest of known events of leg specification, and occur prior to the establishment of proximodistal axis in the leg. In the absence of late functions of Dpp and Egfr, limb primordia are specified but fail to differentiate into wing disc and most of leg disc. Thus it is proposed that early limb primordium at stage 11 consists of cells not yet fully committed to either wing or leg disc fate, and the cells are exposed to different amounts of Dpp and Egfr signaling according to their dorsoventral location. Dpp recruits the cells to the wing disc fate. Egfr antagonizes the cellular response to the wing-inducing function of Dpp and allows the development of wing discs only in the dorsal region. Thus the dorsoventral difference in Dpp and Egfr signaling in the limb primordium provides key information to the separation and differentiation of the wing and leg discs. In contrast to the opposing roles of Dpp and Egfr on wing disc development, leg discs requires both signals. The effect of the loss of Egfr activity on leg disc development is not compensated for by a simultaneous loss of Dpp signaling, indicating that Egfr has an additional activity to promote leg development separately from its role to antagonize Dpp. Because dorsal and ventral limb primordial cells respond to Egfr differently, it is speculated that at least one additional dorsoventral factor influences leg disc formation at stage 11. This idea is consistent with the fact that residual proximal leg cells can still be induced in the almost complete absence of Egfr and Dpp activity. One candidate for the factor is Wg, which is expressed in the limb primordium (Kubota, 2000).

The nuclear transduction of the Dpp signal, as visualized by the distribution of pSSVS and expression of dad, is unaffected by Egfr. The results suggest that the antagonistic effect of Egfr on Dpp signaling occurs after transduction into the nucleus. Therefore, the mechanism of SMad inhibition by direct phosphorylation by MAP kinase does not play a major role in this case (Kubota, 2000).

The finding that Egfr is activated in the limb primordium and prevents wing disc formation suggests that Egfr is a key factor in the diversification of the wing and leg fate. It is proposed that the differential activation of Dpp and Egfr, and the dorsal cell migration brings a subset of limb primordial cells out of the range of Egfr signaling, and thereby allows Dpp to induce wing development. It follows that dorsally migrating cells acquire the wing cell identity only after the separation from leg-promoting signals. Consistent with this idea, expression of wing-specific markers Vg and Sna, start only after the separation of the two primordia. Mechanisms that promote the dorsal cell migration remain to be identified. Given that the basic genetic components for the induction of the wing and leg have been identified in the model organism Drosophila, it can now be asked how the genetic mechanism of wing and leg specification has evolved by comparing the expression and function of these genes in limb primordial cells of primitive insects (Kubota, 2000).

Warburg effect metabolism drives neoplasia in a Drosophila genetic model of epithelial cancer

Cancers develop in a complex mutational landscape. Genetic models of tumor formation have been used to explore how combinations of mutations cooperate to promote tumor formation in vivo. This study identified lactate dehydrogenase (LDH), a key enzyme in Warburg effect metabolism, as a cooperating factor that is both necessary and sufficient for epidermal growth factor receptor (EGFR)-driven epithelial neoplasia and metastasis in a Drosophila model. LDH is upregulated during the transition from hyperplasia to neoplasia, and neoplasia is prevented by LDH depletion. Elevated LDH is sufficient to drive this transition. Notably, genetic alterations that increase glucose flux, or a high-sugar diet, are also sufficient to promote EGFR-driven neoplasia, and this depends on LDH activity. This study provides evidence that increased LDHA expression promotes a transformed phenotype in a human primary breast cell culture model. Furthermore, analysis of publically available cancer data showed evidence of synergy between elevated EGFR and LDHA activity linked to poor clinical outcome in a number of human cancers. Altered metabolism has generally been assumed to be an enabling feature that accelerates cancer cell proliferation. These findings provide evidence that sugar metabolism may have a more profound role in driving neoplasia than previously appreciated (Eichenlaub, 2018).

Cancers develop in a complex mutational landscape. Individual tumors carry hundreds, even thousands, of mutations. Specific tumor types have identifiable signatures, consisting of a small number of relatively common 'driver' mutations. The mutational spectrum can vary in different regions of any given tumor, indicating clonal heterogeneity. This heterogeneity poses a challenge to identify which among the many mutational changes contribute to disease (Eichenlaub, 2018).

Genetic models of tumor formation have been used to explore how combinations of mutations can cooperate to promote neoplasia. Excess epidermal growth factor (EGF) receptor activity is causally linked to many epithelial cancers, including breast cancer. In Drosophila tumor models, EGF receptor (EGFR) overexpression drives hyperplastic growth, but the tissue does not normally progress to neoplasia. When combined with additional genetic alterations, the hyperplastic imaginal disc tissues can undergo neoplastic transformation and metastasis. Interestingly, specific genetic combinations produce tumors with different phenotypic characteristics, suggesting that these models may provide the means to explore specific cancer phenotypes (Eichenlaub, 2018).

A growing body of evidence has suggested an association between altered sugar metabolism and cancer risk. In cancer cells, glucose metabolism shifts away from using pyruvate to feed oxidative phosphorylation toward use of lactate in aerobic glycolysis (the Warburg effect). The lactate dehydrogenase enzyme plays a key role in the shift to Warburg metabolism. Altered metabolism is thought to enhance the growth potential of cancer cells by diverting glucose to produce building blocks for increased biomass in the form of amino acids, at the expense of efficiency in ATP production via the tricarboxylic acid (TCA) cycle. Depletion of lactate dehydrogenase (LDH) can reduce tumorigenesis in EGFR (Neu)-dependent breast cancer as well as c-Myc-mediated transformation, indicating an important role for this metabolic shift. LDH was found to be upregulated in a Drosophila tumor model driven by overexpressing the activated vascular endothelial growth factor (VEGF) or platelet-derived growth factor (PDGF) receptor, Pvr, but its contribution to tumor formation was not assessed. This report has identified LDH as a cooperating factor that is both necessary and sufficient for EGFR-driven epithelial neoplasia in vivo. Genetic alterations that increase glucose flux, or a high-sugar diet, were sufficient to promote EGFR-driven neoplasia, and this depends on LDH. These findings provide evidence that Warburg effect metabolism may have a more fundamental role in driving neoplasia than previously appreciated (Eichenlaub, 2018).

This study shows that LDH overexpression is sufficient to drive neoplasia in combination with EGFR expression. Overexpression of LDHA in a human primary breast cancer cell model promoted a more transformed cellular phenotype. The possible significance of synergy between high LDH in a background of high EGFR activity in human cancer is supported by analysis of the cancer genome atlas (TCGA) datasets: evidence that patients with higher LDHA activity and higher EGFR activity show earlier disease progression in breast cancer, sarcoma, and lower grade gliomas. These effects were only seen when the two factors occurred together, suggesting synergy between EGFR activity and the metabolic shift toward aerobic glycolysis (Eichenlaub, 2018).

Another study has reported that increases in LDHA were able to promote EMT and invasiveness in renal clear cell carcinoma (ccRCC) and that blocking LDH activity could suppress these phenotypes as well as metastasis of ccRCC in xenografts. Although no evidence was found for an effect of LDHA alone or of LDHA/EGFR synergy in the TCGA ccRCC data, these findings merit further attention (Eichenlaub, 2018).

The observations reported in this study provide evidence that increased sugar flux, whether dietary or due to increased absorption, can promote neoplastic transformation of EGFR-expressing epithelial tissue. The underlying metabolic changes appear to elicit these effects via the lactate shunt, because the effects of high sugar were abrogated by lowering the level of LDH expression in the tissue. A number of recent studies have begun to link elevated sugar flux to the metastatic phenotype. Together with the current findings, these studies may provide a molecular framework to better understand the links between diet, obesity, and cancer and may help to select patient populations who might benefit from future therapeutic agents targeting lactate dehydrogenase activity (Eichenlaub, 2018).

Notch mediates inter-tissue communication to promote tumorigenesis

Disease progression in many tumor types involves the interaction of genetically abnormal cancer cells with normal stromal cells. Neoplastic transformation in a Drosophila genetic model of Epidermal growth factor receptor (EGFR)-driven tumorigenesis similarly relies on the interaction between epithelial and mesenchymal cells, providing a simple system to investigate mechanisms used for the cross-talk. Using the Drosophila model, this study shows that the transformed epithelium hijacks the mesenchymal cells through Notch signaling, which prevents their differentiation and promotes proliferation. A key downstream target in the mesenchyme is Zfh1/ZEB. When Notch or zfh1 are depleted in the mesenchymal cells, tumor growth is compromised. The ligand Delta is highly upregulated in the epithelial cells where it is found on long cellular processes. By using a live transcription assay in cultured cells and by depleting actin-rich processes in the tumor epithelium, this study provides evidence that signaling can be mediated by cytonemes from Delta-expressing cells. It is thus proposed that high Notch activity in the unmodified mesenchymal cells is driven by ligands produced by the cancerous epithelial. This long-range Notch signaling integrates the two tissues to promote tumorigenesis, by co-opting a normal regulatory mechanism that prevents the mesenchymal cells from differentiating (Boukhatmi, 2020).

Normal tissue mesenchymal cells are thought to have important roles in promoting the growth and metastasis of many tumors. To do so, they must be educated by the aberrant cancerous cells to acquire the properties needed to sustain tumorigenesis. Using a Drosophila model of EGFR/Ras-driven tumorigenesis, this study demonstrates that Notch activity in the unmodified mesenchymal cells is essential for tumor growth. Downregulating Notch specifically in mesenchymal cells reduced their proliferation rates, promoted their differentiation, and significantly compromised the size of tumors that developed. Strikingly, the activation of Notch in these supporting cells appears to rely on direct communication from the cancerous epithelial cells, illustrating that this pathway can operate in long-range signaling between tissue layers (Boukhatmi, 2020).

The conclusion that Notch receptors in the mesenchymal cells are activated from ligands presented by nearby epithelial cells is unexpected because most examples of Notch signaling occur between cells within an epithelial cell layer. The fact that the ligands are transmembrane proteins means that direct cell-cell contacts are required to elicit signaling and that signaling usually occurs between neighboring cells. More recently, examples have emerged where signaling occurs across longer distances that appear to involve contacts mediated by cell protusions, such as filopodia or cytonemes. Evidence indicates that a similar mechanism operates in the tumors. Delta is produced in the epithelial cells and can be detected in fine processes that extend through the nearby mesenchymal cells, which is consistent with a recent report describing cytonemes in these EGFR-psqRNAi tumors. In a heterologous system, it was found there was robust activation of a Notch target gene rapidly after ligand-expressing cells made contact through cell processes. Likewise, ectopic patches of Delta in the disc epithelium led to the expression of the Notch-regulated m6-GFP in the underlying mesenchyme. Thus, it is proposed that the widespread upregulation of Delta in the epithelial compartment of the tumorous wing discs, in turn, activates the Notch pathway in the neighboring mesenchymal cells by long cellular processes. As a consequence, the mesenchymal cells become coordinated with the cancer epithelial cells and are maintained in an undifferentiated state (Boukhatmi, 2020).

Although the data demonstrate that Delta-Notch-mediated inter-tissue signaling is important for sustaining tumor growth, it is evident that other signals are also required. First, it was previously shown that Dpp from the cancerous epithelium is essential for these tumors to grow. Because the Dpp pathway was still activated in the mesenchyme when Notch was depleted, it is proposed that Dpp and Notch operate in parallel. This may explain why apicobasal polarity was not fully restored when Notch activity was impaired and highlights the likelihood that several different pathways are coopted to drive tumorigenesis. Second, the fact that tumorigenesis is rescued by perturbing Notch or Dpp signaling in the mesenchyme argues that there must be a reciprocal signal to the epithelium. Notably, the relative growth of the two populations appears highly co-ordinated in the tumors, unlike the wild type where the epithelial growth predominates. A plausible model is that combined inputs from Notch and Dpp are required to produce reciprocal signal(s), and it will be interesting to discover whether the reciprocal signaling also operates through cytonemes, given that the mesenchymal cells emit processes (Boukhatmi, 2020).

One of the key effectors of Notch activity in the tumor mesenchyme is Zfh1/ZEB, which is important for maintaining the muscle progenitors in normal conditions. In a similar manner, its expression is kept high in the tumor mesenchyme, due to Notch activity, where it helps prevent their differentiation. Downregulating zfh1 in mesenchymal cells induces their premature differentiation and prevents tumor growth. The role of Zfh1/ ZEB in promoting progenitors and stem cell proliferation appears to be widespread. Furthermore, ZEB1 is upregulated in many cancers, where it can cause the expansion of cancer stem cells and frequently drives epithelial-to-mesechymal transition to promote metastasis. Whether its activation in these conditions also involves Notch activation and inter-tissue signaling remains to be determined (Boukhatmi, 2020).

Egfr signaling is a major regulator of ecdysone biosynthesis in the Drosophila prothoracic gland

Understanding the mechanisms that determine final body size of animals is a central question in biology. In animals with determinate growth, such as mammals or insects, the size at which the immature organism transforms into the adult defines the final body size, as adult individuals do not grow. In Drosophila, the growth period ends when the immature larva undergoes the metamorphic transition to develop the mature adult. This metamorphic transition is triggered by a sharp increase of the steroid ecdysone, synthetized in the prothoracic gland (PG), that occurs at the end of the third instar larvae (L3). It is widely accepted that ecdysone biosynthesis in Drosophila is mainly induced by the activation of tyrosine kinase (RTK) Torso by the prothoracicotropic hormone (Ptth) produced into two pairs of neurosecretory cells that project their axons onto the PG. However, the fact that neither Ptth nor torso-null mutant animals arrest larval development but only present a delay in the larva-pupa transition mandates for a reconsideration of the conventional model. This study shows that Egfr signaling, rather than Ptth/torso, is the major contributor of ecdysone biosynthesis in Drosophila. Egfr signaling was found to be activated in the PG in an autocrine mode by the EGF ligands spitz and vein, which in turn are regulated by the levels of ecdysone. This regulatory positive feedback loop ensures the production of ecdysone to trigger metamorphosis by a progressive Egfr-dependent activation of MAPK/ERK pathway, thus determining the animal final body size (Cruz, 2020).

In contrast to the developmental delay phenotype observed in larvae with reduced Ptth or torso, this study foudn that specific depletion of Drosophila homolog transducers ras(ras85D), Raf oncogene (Raf), and ERK, the core components of the MAPK/ERK pathway, in the prothoracic gland (PG) using the phmGal4 driver (phm>) induced developmental arrest at L3. This result suggests that additional RTKs might play important roles in ecdysone production. To study this possibility, all known Drosophila RTKs in the PG were knocked down and found that only depletion of Egfr phenocopied L3 arrested development observed in phm > ras85DRNAi larvae. Likewise, overexpression in the PG of a dominant-negative form of Egfr (EgfrDN) or depletion of the transcription factor pointed (pnt), the principal nuclear mediator of the Egfr signaling pathway, also resulted in arrested L3 larvae. The same results were obtained upon inactivation of Egfr or different components of the MAPK/ERK pathway using an alternative PG specific driver, amnc651Gal4. Consistent with the observed phenotypes, overexpression of a constitutively activated form of either Egfr (Egfract) or Pnt (PntP2VP16) in the PG induced premature pupariation and reduced pupal size. These results are in agreement with a previous report showing that overexpression of a constitutively activated form of Ras (RasV12) in the PG produced the same phenotype. Furthermore, overexpression of RasV12 in Egfr-depleted larvae rescued the developmental arrest phenotype and forced premature pupation. These results strongly suggest that Egfr signaling in the PG is required for the synthesis of the ecdysone pulse that triggers metamorphosis. Confirming this hypothesis, ecdysone titers in larvae depleted of either Egfr or pnt in the PG were dramatically reduced. Accordingly, Hr3 and Hr4 expression, two direct target genes of the hormone that have been used as readouts for ecdysone levels, was completely abolished in phm > EgfrRNAi and phm > pntRNAi L3 larvae compared to control animals. Moreover, addition of the active form of ecdysone, 20-hydroxyecdysone (20E), to the food rescued the developmental arrest phenotype induced by inactivation of Egfr signaling in the PG. Altogether, these results indicate that Egfr signaling in the PG endocrine cells is required for the production of the ecdysone pulse that triggers pupariation and fixes adult body size (Cruz, 2020).

Since Egfr signaling is involved in cell proliferation and survival, this study analyzed whether the above-described phenotype was due to compromised viability of PG cells. Although reduced activation of Egfr signaling diminished cell size, PG cell number and viability were not affected. Interestingly, ecdysone synthesis has been recently shown to correlate with endocycle progression and therefore cell size of PG cells. PG cells undergo three rounds of endoreplication during larval development resulting in chromatin values (C values) of 32-64 C by late L3. Remarkably, a clear reduction was observed in the C value of PG cells of phm > EgfrRNAi larvae at 120 h AEL, with most cells at 8-16 C, indicating that Egfr activation is also required to promote polyploidy in the PG cells (Cruz, 2020).

This result raised the possibility that Egfr signaling regulates ecdysone production by determining the size of the PG. To analyze this hypothesis, the effect of Egfr signaling in ecdysone production was examined. Steroidogenesis in the PG cells depends on the timely expression of ecdysone biosynthesis enzyme-encoding genes that mediate the conversion of cholesterol to ecdysone. To analyze whether Egfr signaling controls ecdysone synthesis by regulating the expression of these genes, qRT-PCR was performed in early (72 h after egg laying [AEL]), mid (96 h AEL), and late (120 h AEL) phm > EgfrRNAi and phm > pntRNAi L3 larvae. Whereas expression of the six ecdysone biosynthetic genes increased gradually from mid to late L3 in control larvae, correlating with the production of the high-level ecdysone pulse that triggers metamorphosis, inactivation of the Egfr pathway in the PG resulted in a dramatic reduction in the expression levels of neverland (nvd), spook (spo), shroud (sro), and phantom (phm) in late L3 larvae. In contrast, the expression of disembodied (dib) and shadow (sad) was not significantly reduced in Egfr-depleted larvae, which suggests that compromising Egfr signaling in the PG does not result in a general reduction in the transcriptional activity by its minor C value, as previously shown, but rather by a specific transcriptional effect. Further confirming this point, the overexpression of CycE in Egfr-depleted PGs was unable to restore normal expression of ecdysteroid biosynthetic genes nor induced proper pupariation of these animals, indicating that Egfr signaling is required for proper expression of ecdysone enzyme-encoding genes independently of promoting polyploidy of PG cells (Cruz, 2020).

As the levels of circulating ecdysone are influenced by the rates of hormone production and release, whether Egfr signaling also regulates ecdysone secretion was studied. Recently, it has been shown that ecdysone secretion from the PG cells is mediated by a vesicular regulated transport mechanism. After its synthesis, ecdysone is loaded through an ATP-binding cassette (ABC) transporter, Atet, into Syt1-positive secretory vesicles that fuse to the cytoplasmic membrane for release of the hormone in a calcium-dependent signaling. To analyze the role of Egfr signaling in this process, secretory vesicles were visualized in PG cells of phm > EgfrRNAi and phm > pntRNAi L3 larvae by expressing eGFP-tagged Syt1 (Syt-GFP) in these glands. Whereas Syt-GFP vesicles accumulate at the plasma membrane with a small number of vesicles in the cytoplasm in wild-type L3 larval PGs, a dramatic accumulation of Syt-GFP vesicles in the cytoplasm was observed in PGs with reduced Egfr signaling. Similar results were obtained when the subcellular localization of the ecdysone transporter Atet-GFP was analyzed. Consistently, overexpression of rasV12 in PGs of phm > EgfrRNAi larvae restored the subcellular localization of both Syt and Atet-GFP. Furthermore, mRNA levels of several genes involved in vesicle-mediated release of ecdysone, including Syt and Atet, were dramatically downregulated in the PG of phm > EgfrRNAi and phm > pntRNAi larvae. Therefore, the results show that Egfr signaling is also required for the vesicle-mediated release of ecdysone from PG cells. Interestingly, direct effects of Egfr signaling on the endocytic machinery have been already described in Drosophila tracheal cells as well as in human cells (Cruz, 2020).

The next question was to determine which of the EGF ligands were responsible for the Egfr pathway activation in the PG. In Drosophila, Gurken (Gur), Spitz (Spi), Keren (Krn), and Vein (Vn) serve as ligands for Egfr. Expression analysis of the four ligands revealed that only vn and spi were expressed in the PGs. Consistently, the intramembrane protease rhomboid (rho), which is necessary for the proteolytic activation of Spi, was also expressed in the PG cells. A temporal expression pattern of staged L3 PGs revealed that rho expression progressively increased during the last larval stage, while the expression of spi and vn increased sequentially, with vn upregulated at mid L3 and spi at late L3. Consistent with the expression of the ligands, mRNA levels of Egfr also showed a clear upregulation by late L3. Likewise, a specific expression of PntP2 isoform was also observed in the PG of late L3 larvae. Altogether, these results suggest that Vn and Spi might activate Egfr signaling in an autocrine manner to induce ecdysone production (Cruz, 2020).

To determine the functional relevance of each ligand, vn, spi, or both simultaneously were knocked down in the PG. As in the case of phm > EgfrRNAi, depletion of spi, vn, or both ligands at the same time caused developmental arrest in L3, although Spi appeared to have a minor effect as around 40% of phm > spiRNAi larvae underwent delayed pupariation. The attenuated effect of spi-depleted animals was probably due to a weaker effect of the spiRNAi lines as depletion of the Spi-processing protease rho in the PG resulted in all phm > rhoRNAi animals arresting development at L3. Importantly, ecdysteroid levels in mid and late L3 were significantly reduced in animals depleted of either vn or spi. Consistent with their role in controlling ecdysone production, overexpression of either Vn or an active-cleaved form of Spi in the PG induced precocious pupariation and smaller pupae. Altogether, these findings show that spi and vn act in an autocrine manner as Egfr ligands in the PG to induce ecdysone biosynthesis during the last larval stage. In fact, the correlation between vn and spi expression with the occurrence of increasing levels of ecdysteroids points to a possible positive-feedback loop regulation with 20E inducing vn and spi expression. Consistent with this possibility, vn and spi mRNA levels were reduced in PGs of ecdysteroid deficient larvae that were generated by depleting spo (phm > spoRNAi) or by overexpressing a dominant-negative form of the ecdysone receptor (phm > EcRDN). Moreover, staged PGs were cultured for 6 h ex vivo in presence or absence of 20E, and vn and spi mRNA levels were found to be significantly upregulated in the presence of the hormone. Altogether, these observations demonstrate that ecdysone exerts a positive-feedback effect on PG cells amplifying its own synthesis by inducing the expression of vn and spi. This result is consistent with a previous proposed model of ecdysone regulation in an autonomous mechanism by a positive feedback and biogenic amines. Thus, a model is proposed in which increasing levels of ecdysone promote the expression of vn and spi in the PG cells, which, in turn, increases Egfr signaling in this gland in an autocrine manner to further promote the production of ecdysone. Interestingly, it has been already shown that expression of Spi and Vn in midgut cells of Drosophila depends on ecdysone activity during metamorphosis. In addition, in vertebrates, other hormones have been postulated to control Egfr activity, such as Thyrotropin-releasing hormone, which induced the phosphorylation and activation of the Egf receptor, leading to specific transcriptional events in GH3 pituitary cells. Likewise, the Growth Hormone modulates Egfr trafficking and signaling by activating ERKs (Cruz, 2020).

Thus far, the results above show that MAPK/ERK pathway is a central regulatory element in the control of ecdysone biosynthesis in the PG, with Egfr signaling chiefly contributing to its activity. However, since Ptth/torso signaling operates through the same MAPK/ERK pathway the relative contribution of this signaling pathway in the overall activity of the PG was investigated. The fact that inactivation of Egfr signaling in the PG did not affect the mRNA expression levels of either Ptth or torso points to a minor contribution of Ptth/torso signaling in the overall MAPK/ERK activity. To analyze this possibility, the levels were compared of dpERK, a readout of MAPK/ERK activity, in PGs of phm > EgfrRNAi and phm > torsoRNAi larvae. A dramatic reduction of dpERK levels was observed in PGs of phm > EgfrRNAi larvae. Importantly, dpERK levels were also reduced in phm > torsoRNAi PGs, although to a significant lesser extent when compared to phm > EgfrRNAi larvae. Similar results were observed when nuclear accumulation of dpERK was analyzed in both larvae. Consistently, the level of activity of the MAPK/ERK pathway in phm > pntRNAi and phm > torsoRNAi larvae correlated very well with expression of the biosynthetic enzyme phm and the ecdysone-responsive genes Hr3, Hr4, and Broad-Complex (BrC), although the levels of ecdysone were significantly reduced in both cases. The different level of activation of dpERK by Egfr and Ptth/torso signaling was also consistent with the respective accumulation of Syt-GFP and Atet-GFP vesicles at the cytoplasm and the reduction of the C value of PG cells. Finally, it is important to note that the level of activity of the MAPK/ERK pathway correlated with the respective phenotypes upon inactivation of each pathway, with phm > EgfrRNAi larvae arresting development at L3 and phm > torsoRNAi larvae presenting only a delay in the pupariation time. In line with this, whereas over-activation of Egfr pathway in the PG of phm > torsoRNAi larvae induced a significant advancement in pupariation, the expression of a constitutively activated form of Torso (torsoD4021 mutants) in PGs with depleted Egfr (EgfrRNAi; torso D4021) was not able to induce precocious pupariation (Cruz, 2020).

Overall, these results show that the Egfr signaling pathway plays the main role in the biosynthesis of ecdysone by activating the MAPK/ERK pathway in the PG during mid-late L3, whereas Ptth/torso signaling acts synergistically only to increase the MAPK/ERK pathway activity thus accelerating developmental timing. In this regard, it is possible that the different strength of MAPK/ERK activation by the two signaling pathways might underline this distinct requirement of each pathway. Furthermore, temporal expression of the Egfr and Torso ligands may also contribute to the difference strengths of MAPK/ERK activation, as EGF ligands vn and spi are highly expressed during L3, whereas Ptth is only upregulated at a specific developmental time, the wandering stage. Taken together, these data suggest a model in which the increasing circulating levels of ecdysone during the last larval stage are induced by a progressive Egfr dependent activation of MAPK/ERK in the PG, whereas Ptth/torso signaling further regulates ecdysone production by integrating different environmental signals such as nutritional status, crowding conditions, and light. It is important to note that, in addition to the Egfr and Ptth/torso pathways, ecdysone biosynthesis is also regulated by the insulin/insulin-like growth factor signaling (IIS)/target of Rapamycin (TOR) signaling pathway. However, in contrast to the major role of Egfr controlling ecdysteroid levels during mid-late L3, including the strong ecdysteroid pulse that triggers pupariation, the main effect of IIS/TOR pathway is to control the production of the small ecdysteroid peak that is associated to the nutrition-dependent critical weight checkpoint that occurs at the very early L3. Thus, decreasing the IIS/TOR activity in the PG delays the critical weight checkpoint, slowing development and delaying pupariation, while increasing IIS/TOR activity in the gland induces precocious critical weight and accelerates the onset of metamorphosis. Nevertheless, it is conceivable that the increasing levels of ecdysone at the critical weight checkpoint might initiate the expression of the Egf ligands, that in turn activates the ecdysone production during mid-late L3 (Cruz, 2020).

Finally, since no role of Ptth/torso signaling has been characterized in hemimetabolous insects, it is postulated that Egfr signaling might be the ancestral ecdysone biosynthesis regulator, whereas Ptth/torso signaling has probably been co-opted in holometabolous insects during evolution to fine-tune the timing of pupariation in response to changing environmental cues. Consistent with this view, depletion of Gb-Egfr in the hemimetabolous insect Gryllus bimaculatus, where no Ptth/torso has been described, results in arrested development by the last nymphal instar. Therefore, this double regulation in holometabolous insects might provide developmental timing plasticity contributing to an appropriated adaptation to a time-limited food supply (Cruz, 2020).

Buffered EGFR signaling regulated by spitz-to-argos expression ratio is a critical factor for patterning the Drosophila eye

Developmental pathways have evolved mechanisms to monitor positional information in order to generate reproducible organismal patterns. These pathways are robust and insensitive to small changes in individual processes involved. Spatial differentiation, where a population of cells undergo deterministic molecular differentiation, brings about spatial patterns. Redundancy of mechanism and negative feedback are two ways in which reliability in pattern formation is brought about. Lateral inhibition by diffusible molecules is another mechanism that can be used to generate patterns. For systems which do not depend on developmental history, environmental makeup determines their molecular differentiation contributing towards generating a pattern (Pasnuri, 2023).

This paper proposes a mechanism where relative expression levels of principal EGFR ligand, Spitz and negative feedback regulator Argos determines the extent of EGFR activation which is crucial for the periodic ommatidial pattern. The data suggests that it is not the absolute gene expression but the balance between gene networks on the whole which may contribute towards pattern formation. GMR-Gal4 is expressed in all cells posterior to the morphogenetic furrow and any expression cassette under the UAS is expressed strongly. Whereas, Elav-Gal4 is expressed only in neuronal cells and the expression is strongest towards the posterior end of the eye disc. While Elav-Gal4 expression occurs in differentiated neurons starting with R8 in the eye disc (for which EGFR signaling is not needed), but beyond R8 specification, Spitz and Argos levels are important for subsequent PR cell differentiation and also in the pupal stages. Although an equally drastic reduction was observed in argos expression when UAS-spitz dsRNA driven by GMR-Gal4 and Elav-Gal4, the eye discs show reduced dpERK staining in both cases. The reduction was lower when Elav-Gal4 driver was used, corresponding to the absence of phenotype in the adult eye. The eye discs expressing EGFRCA construct under GMR-Gal4 Gal80ts with spitz-to-argos ratio near 1, showed a discontinuous S-phase band after the morphogenetic furrow indicating a lower population of cells entering the second mitotic wave. Fewer cells entering the second mitotic wave leaves the tissue field with fewer uncommitted cells to make cell fate decisions. This can affect pattern formation to a great extent. This could also explain fewer number of bristle cells in the rough adult eyes as bristles cell fate is assigned from cells arising from the second mitotic wave. It should also be noted that the rough eye phenotype for EGFRDN is rather different from EGFRCA and shows a profusion of bristles. This mechanism of relative expression determining phenotype supports older work on the importance of spitz-to-argos dose as a critical determinant of eye patterning. Indeed mRNA levels may not be always predictive of protein levels, and both translational regulation and post-translational modifications may well affect biological function. However, under conditions of stress or where specific transcriptional programmes bring about developmental outcomes, transcript levels may be thought to be well-correlated to protein levels. In addition, experiments using smFISH allows clear identification of the cells that are expressing specific genes, where the diffusible protein end-products may not provide as conclusive answers. We did attempt performing antibody staining for Spitz and Argos but such relative measures cannot be used to comment on expression stoichiometry. This study shows a clear differential expression of spitz and argos mRNA in the early eye field contributing to photoreceptor fate determination and also addresses the sensitivity of the system to the heterogeneity in the expression levels of gene networks and makes developmental programs robust. It has to be noted of course that signaling via EGFR is not the only pathway determining the ommatidial pattern in the eye. For example, Notch is known to play an important role in the initiation of neural development and also in ommatidial rotation. Buffered regulation of genes in different developmental pathways that crosstalk can decrease sensitivity to variations in a gene network and can help explain other reproducible and stereotypical patterns generated throughout the development (Pasnuri, 2023).

Sequential Ras/MAPK and PI3K/AKT/mTOR pathways recruitment drives basal extrusion in the prostate-like gland of Drosophila

One of the most important but less understood step of epithelial tumourigenesis occurs when cells acquire the ability to leave their epithelial compartment. This phenomenon, described as basal epithelial cell extrusion (basal extrusion), represents the first step of tumour invasion. However, due to lack of adequate in vivo model, implication of emblematic signalling pathways such as Ras/Mitogen-Activated Protein Kinase (MAPK) and phosphoinositide 3 kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) signalling pathways, is scarcely described in this phenomenon. This paper reports a unique model of basal extrusion in the Drosophila accessory gland. There, it was demonstrated that both Ras/MAPK and PI3K/AKT/mTOR pathways are necessary for basal extrusion. Furthermore, as in prostate cancer, this study shows that these pathways are co-activated. This occurs through set up of Epidermal Growth Factor Receptor (EGFR) and Insulin Receptor (InR) dependent autocrine loops, a phenomenon that, considering human data, could be relevant for prostate cancer (Rambur, 2020).

Worldwide, a large majority of cancers originates from epithelial tissues such as lung, breast and prostate1. Despite reinforced prevention, most of the tumours are detected at late stages, and patient care is centred on invasive adenocarcinomas, resistant forms of these carcinomas and metastatic carcinomas. As late stages of cancer progression have been under intense scrutiny in the last decades, the molecular mechanisms associated to such progression are largely described, showing for example the major role of receptor tyrosine kinase (RTK)-dependent signalling pathways in these mechanisms. Typically, for prostate adenocarcinoma, the second most common cancer in men, both PI3K/AKT/mTOR pathway and Ras/MAPK pathways are associated with tumour progression. In the prostate adenocarcinoma, Ras/MAPK and PI3K/AKT/mTOR pathways display activating genetic alterations in more than 40% of primary tumours and in virtually all metastatic prostate tumours, and phosphoproteomic studies confirmed a strong correlation in the activation of these two pathways. Furthermore, pre-clinical mouse models reproducing alteration of either one or the other pathway in the prostate epithelium display tumourigenesis that mimics histopathological features of the human adenocarcinoma. Moreover, advanced tumour progression is obtained when combining alterations in both pathways. These different data emphasise that in one hand, Ras/MAPK or PI3K/AKT/mTOR pathways can initiate prostate tumour development, and in the other hand that these pathways are implicated in late phases of tumour progression. However, they explain neither their respective or combined role in actual adenocarcinoma formation nor the molecular mechanisms that could couple these two pathways to promote this phenomenon in vivo (Rambur, 2020).

Adenocarcinoma formation occurs when pre-invasive epithelial cells acquire the ability to leave their epithelial compartment. This implicates that these cells are able to extrude from the normal epithelium and to cross the basement membrane which is the limit of the epithelial compartment. These phenomena can be described as basal extrusion and are resulting in early invasion, as opposed to late invasion associated to the metastatic process. Due to the difficulty to precisely visualise basal extrusion in animals, mechanistic associated to this phenomenon has been essentially described in cellular models or in developing tissues such as Drosophila imaginal disc of zebrafish embryo, even though the role of P120 catenin in basal extrusion has been shown in a mouse model of pancreatic neoplasia. The role of Ras/MAPK pathway in basal extrusion has only been described through the use of RasV12 as an oncogenic hit, and role of PI3K/AKT/mTOR pathway has never been assessed (Rambur, 2020).

To determine the role of Ras/MAPK and PI3K/AKT/mTOR pathways in basal extrusion and understand the underlying mechanisms that may coordinate their hyperactivation in prostate cancer, this study has developed a new model of in vivo early invasive adenocarcinoma in the Drosophila prostate-like accessory gland. Drosophila is a powerful genetic model where more than 70% of genes implicated in human diseases display orthologs and where Ras/MAPK and PI3K/AKT/mTOR signalling pathways are well conserved. Drosophila has already proven its pertinence as cancer model for brain, lung, and colon. The Drosophila accessory gland is a functional equivalent for the prostate, playing a role in fertility by secreting seminal fluid. Secretions come from a monolayer of epithelial cells that are well differentiated and quiescent at the adult age, and there is no evidence of stem cells in this tissue. Considering that a majority of prostate adenocarcinoma is thought to originate from luminal cells, epithelial cells from the accessory gland represent a valuable model to study the mechanisms of epithelial prostate tumourigenesis (Rambur, 2020).

This study describes this unique model of basal extrusion and tumour formation in the accessory gland that recapitulates most aspects of cancer development. Both Ras/MAPK and PI3K/Akt/TOR pathways are overactivated in the produced tumours, and these pathways cooperate to induce basal extrusion and subsequent tumour formation. Furthermore, the mechanism is described that allows the coactivation of these pathways, which relies on the sequential recruitment of a double autocrine feedback loop dependent on Epidermal (EGF/Spitz) and Insulin-like (IGF/Ilp6) Growth Factors and their respective receptors. Finally, using publicly available data of prostate cancer samples and migration assay in human pre-tumoural prostate epithelial cell line, the possible role of these findings in the actual human pathology is assessed (Rambur, 2020).

To faithfully reproduce what is thought to happen in the earliest stages of tumour formation in patients, a single genetic alteration was produced in few clones of randomly selected and mostly differentiated cells. Furthermore, accessory gland epithelium, shown to be adjacent to a basement membrane, is surrounded by a stromal-like sheet of muscle fibres, and oncogene-induced epithelial cells are able to cross both layers to form external tumours. This recapitulates the phenomenon of basal epithelial cell extrusion, which is thought to be central to cell invasion. Basal extrusion has been described in cell culture, in Drosophila imaginal discs, in zebrafish embryos and in mouse. However, implication of Ras/MAPK and PI3K/AKT/mTOR pathways has never been assessed in this phenomenon, despite the fact that these pathways are among the most deregulated in cancers, and especially in epithelial cancers such as prostate adenocarcinoma. This study shows in a new model of accessory gland tumourigenesis that both pathways are implicated in basal extrusion, indicating that this step demands a particular state of activation for the cell that undergoes this basal extrusion. Furthermore, this finding correlates with the fact that the two considered pathways are already frequently co-deregulated in primary tumours. From these experiments, where oncogene expression is restricted to few cells and intra-tumoural inhibition of the pathways decreases invasion, it is infered that the mechanisms of basal cell extrusion are cell autonomous, as previously shown in cell lines. Indeed, this study shows that this cell-autonomous mechanism relies on the production of two growth factors, and subsequent activation of two autocrine loops. Role of autocrine loops has been hypothetized in late tumourigenesis, as higher levels of growth factors have been found in tumoural tissues, and has been studied in cell models where inhibition of these loops decreases tumourigenic features such as migration or proliferation capacity as their activation have been linked to transformation of various epithelial cells. However, a role of autocrine loops has never been demonstrated for basal extrusion in vivo. If these loops seem implicated in tumour late progression, so could they be more important for early human tumour development. In fact, many strategies have been attempted to treat cancer patients especially by blocking EGF/EGFR autocrine loop. However, for advanced prostate cancer, these strategies have shown poor results, as well for monotherapies as for combined treatments with classical anti-prostate cancer agents. It could be logical if autocrine loops are less implicated in late stages of cancer but more in the capacity for tumour cells to leave the epithelial compartment. In later stages, higher rates of activating mutations in the Ras/MAPK and PI3K/AKT/mTOR pathways could suppress the need for RTK-driven activation. In contrast, in early tumourigenesis, as fewer genetic alterations are present, activation of signalling pathways must rely on different mechanisms. As is shown in the accessory gland, this recruitment could be efficiently done in tumour cells by autocrine production of growth factors, autocrine activation of their RTK and subsequent activation of the pathways necessary for the tumour development. In a human cohort of prostate cancer samples, it was found that EGF is more expressed in primary tumours than either in normal tissue or in metastases. This could correlate with an early requirement for such growth factor in the formation of adenocarcinoma. Contrary to the observations in Drosophila, no early overexpression of IGFs can be detected in human samples. However, in human, EGFR is able to recruit both Ras/MAPK and PI3K/AKT/mTOR pathway, and EGF overexpression could drive their activation and act in the same way as EGF/Spitz and IGF/Ilp6 in Drosophila (Rambur, 2020).

To study early phases of tumourigenesis remains difficult in vivo, especially for epithelial cells that can develop into benign tumours still in the epithelial compartment such as benign prostatic intraepithelial neoplasia, or into adenocarcinoma that are characterised by an expansion out of the epithelial compartment. The model developed in the Drosophila accessory gland represents a unique in vivo model to explore basal extrusion and early invasion. Two major pathways of cancer progression are implicated in this basal extrusion, and these two pathways are co-recruited by autocrine loops. Further investigation will be necessary to test whether other pathways implicated in late tumourigenesis are important in this phenomenon. Furthermore, it will also be important to determine which genes are activated or inhibited by these pathways and which mechanisms are recruited to promote the actual extrusion (Rambur, 2020).

Egfr and midline glia maturation

A good example of the function of Egfr in regulating cell development is found by examining the role of Egfr in midline glia maturation. The midline glial cells are required for correct formation of the axonal pattern in the embryonic ventral nerve cord. Initially, six midline cells form an equivalence group with the capacity to develop as glial cells. By the end of embryonic development three to four cells are singled out as midline glial cells. Midline glia development occurs in two steps, both of which depend on activation of the Egfreceptor and subsequent Ras1/Raf-mediated signal transduction (See Drosophila Ras1). In the first step six midline cells in each segment, originating from the anterior-most three of a total of eight midline progenitor cells, are determined as the midline glia equivalence group. The process of generation of the midline glia equivalence group involves Notch function and segmentation genes. It might also depend on the function of single minded, the master regulatory gene of midline development. The single minded transcript accumulates in the midline glia and, depending on the context, can act either as a transcriptional activator or repressor. By the end of embryogenesis the final number of midline glial cells is about 3 to 4. Thus, the final number of cells has to be selected from the initially defined equivalence group in a second step (Scholz, 1997).

Egfr mutants show a reduced number of midline glial cells and argos mutants, which possibly exhibit an increased activation of Egfr in the midline, show an increased number of midline glial cells. Furthermore, expression of activated ras1 (or activated raf) in the midline results in the appearance of extra midline cells. This model suggests that activation of ras1 signaling in the entire midline glial equivalence group promotes survival of all cells in this cluster. Thus, in wild-type flies, about 2-3 cells in each group down-regulate Egfr signaling and are destined for cell death. Both Rhomboid and Argos control activation of the Egfr during midline glia development. It is thought that a graded activation of Egfr is brought about by the activity of Rhomboid, which is thought to promote EGF receptor signaling, possibly by cell autonomous activation of the Egfr ligand Spitz. Ectopic rhomboid leads to extra midline glial cells. Egfr activates PointedP2 through phosphorylation; Pointed in turn activates the transcription of argos. Argos negatively regulates Egfr signaling non-cell autonomously and competes with Spitz function. pointed mutants form extra glial cells. Yan antagonizes PointedP2A in midline glial cells, just as it does in the developing photoreceptor cells (Scholz, 1997).

Egfr and head midline development

Egfr signaling is required in a narrow medial domain of the head ectoderm (here called head midline) that includes the anlagen of the medial brain (including the dorsomedial and ventral medial domain of the brain, termed DMD and VMD respectively), the visual system (optic lobe, larval eye) and the stomatogastric nervous system (SNS). These head midline cells differ profoundly from their lateral neighbors in the way they develop. Three differences are noteworthy: (1) Like their counterparts in the mesectoderm, the head midline cells do not give rise to typical neuroblasts by delamination, but stay integrated in the surface ectoderm for an extended period of time. (2) The proneural gene l’sc, which transiently (for approximately 30 minutes) comes on in all parts of the procephalic neurectoderm while neuroblasts delaminate, is expressed continuously in the head midline cells for several hours. (3) Head midline cells, similar to ventral midline cells of the trunk, require the Egfr pathway. In embryos carrying loss-of-function mutations in Egfr, spi, rho, S and pnt, most of the optic lobe, larval eye, SNS and dorsomedial brain are absent. This phenotype arises by a failure of many neurectodermal cells to segregate (i.e., invaginate) from the ectoderm; in addition, around the time when segregation should take place, there is an increased amount of apoptotic cell death, accompanied by reaper expression, which removes many head midline cells. In embryos where Egfr signaling is activated ectopically by inducing rho, or by argos (aos) or yan loss-of-function, head midline structures are variably enlarged. A typical phenotype resulting from the overactivity of Egfr signaling is a cyclops like malformation of the visual system, in which the primordia of the visual system stay fused in the dorsal midline. The early expression of cell fate markers, such as sine oculis in Spitz-group mutants, is unaltered (Dumstrei, 1998).

About the time of gastrulation, Egfr signaling is activated in both mesectoderm and the head midline. The ventromedial domain of Egfr activation, as monitored by rho, aos and phosphorylated ERK expression, continues from the ventral mesectodermal domain into the head ectoderm. The ventromedial stripe of aos expression bifurcates at the level of the stomodeal plate and continues dorsally. Approaching the dorsal midline, it turns posteriorly and continues all the way to the posterior boundary of the head neurectoderm. There is an anterior-median patch of aos expression corresponding to the anlage of the stomatogastric nervous system. At later stages (stage 10-12) expression of aos in head midline structures becomes more complex; however, a high level of expression persists in large parts of the optic lobe, SNS, VMD and DMD. The domain of rho expression and ERK phosphorylation matches closely the aos expression domain. pnt and yan, two transcription factors executing the signal passed to the nucleus by the Egfr signaling cascade, are also expressed in structures of the head midline The Egfr pathway is required in the head midline for proper cytodifferentiation and epithelial maintenance. Both Egfr and its ligand Spitz are ubiquitously expressed in the embryo (Dumstrei, 1998).

The Egfr loss-of-function phenotype in the head midline and the mesectoderm is quite compatible with the postulated function of Egfr signaling in the Drosophila compound eye and in various vertebrate systems where Egfr signaling also promotes differentiation and prevents cell death. In the head midline, no evidence for a transformation of cell fate is observed similar to the that takes place in the ventral neurectoderm of the Drosophila embryo. A transformation of fate would imply that the structures missing (e.g., optic lobe, dorsomedial brain) are replaced by other structures, which then would have to expand in size in order to occupy the space normally allotted to them, as well as the additional space normally taken up by the midline structures. However, there does not appear to be an expansion of the lateral neurectoderm, such as takes place in ventral neurectoderm: for example, the number and pattern of neuroblasts delaminating from the lateral head neurectoderm in Egfr or Spi mutant embryos is normal. Thus, it appears that the loss of head midline structures observed in Egfr-signaling-deficient embryos is effected mainly by cell death (Dumstrei, 1998).

The gain of function of EGFR signaling causes a complex phenotype. In yan loss-of-function mutants, a hyperplasia of both dorsomedial brain and stomatogastric nervous system is observed. In case of the latter, cells of the DMD clearly overproliferate; ultimately, these cells express neural markers and become incorporated into the brain. The optic lobe phenotype is more difficult to interpret. Thus, cells located medial to the optic lobes express fasII, resulting in a single optic lobe ('cyclops' phenotype) overgrown by the enlarged brain. One might interpret this phenotype as a cell fate transformation: dorsomedial cells, which would normally not express fasII (and would not become part of the optic lobe) turn on this gene if the output of EGFR signaling is increased. However, to clearly interpret this phenotype, one would have to know much better what normally happens to the dorsomedial cells located between the optic lobes. At the blastoderm stage, the anlagen of the optic lobe map to a dorsomedial position. Shortly thereafter, dorsomedial cells move laterally, generating a thin membrane of amnioserosa like cells in between them. A great deal of cell death takes place in this region, prompting one to speculate that cell death is important for the lateral shift of the optic lobe primordia. Supporting this idea it has been found that in embryos in which no cell death occurs due to a deficiency that removes the reaper complex of genes, the optic lobe primordia are frequently extended toward the midline, similar to what can be observed in yan or aos loss-of-function. It is therefore possible that increased activity of Egfr signaling, rather than inducing another fate in the dorsomedial cells, rescues dorsomedial cells from cell death; the expression of FasII might reflect the fate that these cells would normally show if they were permitted to live. Clearly, more experimental studies are required to grasp the effect of Egfr signaling in this region (Dumstrei, 1998).

In head midline structures, in particular the optic lobe and SNS, there may be a late phase of EGFR signaling (as assayed by the expression of aos and activated ERK) whose significance is not yet known. EGFR signaling could be involved in modifying the inhibitory feed-back loop between neurogenic and proneural genes that exists in other neurectoderm cells. In the head midline neurectoderm, regulation of proneural and neurogenic genes has to be different. Thus, instead of a short burst of proneural gene expression in proneural clusters that is resolved into expression in individual neuroblasts, proneural genes are expressed for a long period of time; at the same time, the expression is never restricted to single neuroblasts. Since genes of the E(spl) complex are expressed in the same cells that express l’sc, the inhibitory loop between E(spl)-C and proneural genes must be interrupted at some level. It is possible that Egfr signaling is causing the interruption of this inhibitory loop. Based on genetic studies of Notch and Egfr signaling in the compound eye, it has been speculated that one of the consequences of Egfr activation (which ultimately is required for all ommatidial cell types to differentiate) is to inhibit N signaling, since constitutively active N inhibits ommatidial cell differentiation by preventing response to differentiative signals. However, the same effect could be achieved if Egfr signaling, similar to what is proposed here for the midline neurectoderm, interrupts the inhibition of proneural genes by E(spl). Although this would not prevent N signaling, it would cancel the effect of N signaling on downregulating proneural genes and thereby keep cells in a state of competency to respond to signals (Dumstrei, 1998).

Coordinated sequential action of EGFR and Notch signaling pathways regulates proneural wave progression in the Drosophila optic lobe.

During neurogenesis in the medulla of the Drosophila optic lobe, neuroepithelial cells are programmed to differentiate into neuroblasts at the medial edge of the developing optic lobe. The wave of differentiation progresses synchronously in a row of cells from medial to the lateral regions of the optic lobe, sweeping across the entire neuroepithelial sheet; it is preceded by the transient expression of the proneural gene lethal of scute [l(1)sc] and is thus called the proneural wave. This study found that the epidermal growth factor receptor (EGFR) signaling pathway promotes proneural wave progression. EGFR signaling is activated in neuroepithelial cells and induces l(1)sc expression. EGFR activation is regulated by transient expression of Rhomboid (Rho), which is required for the maturation of the EGF ligand Spitz. Rho expression is also regulated by the EGFR signal. The transient and spatially restricted expression of Rho generates sequential activation of EGFR signaling and assures the directional progression of the differentiation wave. This study also provides new insights into the role of Notch signaling. Expression of the Notch ligand Delta is induced by EGFR, and Notch signaling prolongs the proneural state. Notch signaling activity is downregulated by its own feedback mechanism that permits cells at proneural states to subsequently develop into neuroblasts. Thus, coordinated sequential action of the EGFR and Notch signaling pathways causes the proneural wave to progress and induce neuroblast formation in a precisely ordered manner (Yasugi, 2010).

Loss of EGFR function in progenitor cells caused failure of L(1)sc expression and differentiation into neuroblasts (see A model of progression of the proneural wave). In addition, elevated EGFR signaling resulted in faster proneural wave progression and induced earlier neuroblast differentiation. The activation of the EGFR signal is regulated by a transient expression of Rho, which cleaves membrane-associated Spi to generate secreted active Spi. This study also demonstrated that Rho expression itself depends on EGFR function, and thus the sequential induction of the EGFR signal progresses the proneural wave. Clones of cells mutant for pnt were not recovered unless Minute was employed, suggesting that the EGFR pathway is required for the proliferation of neuroepithelial cells. However, the progression of the proneural wave is not regulated by the proliferation rate per se (Yasugi, 2010).

The function of the Notch signaling pathway in neurogenesis is known as the lateral inhibition. A revision of this notion has recently been proposed for mouse neurogenesis, in which levels of the Notch signal oscillate in neural progenitor cells during early stages of embryogenesis, and thus no cell maintains a constant level of the signal. The oscillation depends mainly on a short lifetime and negative-feedback regulation of the Notch effecter protein Hes1, a homolog of Drosophila E(spl). This prevents precocious neuronal fate determination. The biggest difficulty in analysis of Notch signaling is the random distribution of different stages of cells in the developing ventricular zone, which is thus called a salt-and-pepper pattern. In medulla neurogenesis, however, cell differentiation is well organized spatiotemporally and the developmental process of medulla neurons can be viewed as a medial-lateral array of progressively aged cells across the optic lobe. Such features allowed the functions of Notch to be precisely analyzed. Cells are classified into four types according to their developmental stages: neuroepithelial cells expressing PatJ, neuronal progenitor I expressing a low level of Dpn, neuronal progenitor II expressing L(1)sc and neuroblasts expressing high levels of Dpn. The Notch signal is activated in neuronal progenitor I and II. The EGFR signal turns on in the neuronal progenitor II stage and progresses the stage by activating L(1)sc expression. Cells become neuroblasts when the Notch and EGFR signals are shut off. Cells stay as neuronal progenitor I when Notch signal alone is activated, whereas cells stay as neuronal progenitor II when the Notch signal is activated in conjunction with the EGFR signal. Although the Notch signal is once activated, it must be turned off to let cells differentiate into neuroblasts. In neuronal progenitor II, E(spl)-C expression is induced by Notch signaling, and the increased E(spl)-C next downregulates Dl expression and subsequent activation of the Notch signal (Yasugi, 2010).

What does Notch do in medulla neurogenesis? It is infered that the Notch signal sustains cell fates, whereas the EGFR signal progresses the transitions of cell fate. This was well documented when a constitutively active form of each signal component was induced. EGFR, or its downstream Ras, induces expression of L(1)sc but does not fix its state, even though the constitutively active form is employed. At the same time, a constitutively active Notch sustains cell fates in a cell-autonomous manner. Constitutively active N receptors, by contrast, autonomously determine cell fates depending on the context: cells become neuronal progenitor I in the absence of EGFR and neuronal progenitor II in the presence of EGFR. The precocious neurogenesis caused by the impairment of Notch signaling suggests that Notch keeps cells in the progenitor state for a certain length of time in order to allow neuroepithelial cells to grow into a sufficient population. In the prospective spinal cord of chick embryo (Hammerle, 2007), the development from neural stem cells to neurons progresses rostrocaudally, during which the transition from proliferating progenitors to neurogenic progenitors is regulated by Notch signaling (Yasugi, 2010).

Although Notch plays a pivotal role in determining cell fate between neural and non-neural cells, the function may be context dependent and can be classified into three categories. (1) Classical lateral inhibition is seen in CNS formation in embryogenesis and SOP formation in Drosophila. Cells that once expressed a higher level of the Notch ligand maintain their cell states and become neuroblasts. (2) Oscillatory activations are found in early development of the mouse brain (Shimojo, 2008). Progenitor cells are not destined to either cell types. (3) An association with the proneural wave found in Drosophila medulla neurogenesis as is described in this study. The Notch signal is transiently activated only once and then shuts off in a synchronized manner. The notable difference in the outcome is the ratio of neural to non-neural cells; a small number of cells from the entire population become neuroblasts or neural stem cells in the former cases (1 and 2), whereas most of the cells become neuroblasts in the latter case (3). The differences between (1) and (2) can be ascribed at least in part to the duration of development. Hes1 expression has been shown to oscillate within a period of 2 hours in the mouse, whereas in Drosophila embryogenesis, selection of neuroblasts from neuroectodermal cells takes place within a few hours. Thus, even if Drosophila E(spl) has a half-life time equivalent to Hes1, the selection process during embryogenesis probably finishes within a cycle of the oscillation. The process of medulla neuroblast formation continues for more than 1 day, but Notch signaling is activated for a much shorter period in any given cell. This raises the possibility that E(spl)/Hes1 may have a similarly short half-life but outcome would depend on the developmental context (Yasugi, 2010).

The functions of EGFR and Notch described in this study resemble their roles in SOP formation of adult chordotonal organ development; the EGFR signal provides an inductive cue, whereas the Notch signal prevents premature SOP formation. In addition, restricted expression of rho and activation of the EGFR signal assure reiterative SOP commitment. Several neuroblasts are also sequentially differentiated from epidermal cells in adult chordotonal organs (Yasugi, 2010).

Unpaired, a ligand of the JAK/STAT pathway is expressed in lateral neuroepithelial cells and shapes an activity gradient that is higher in lateral and lower in the medial neuroepithelium. The JAK/STAT signal acts as a negative regulator of the progression of the proneural wave (Yasugi, 2008). This report has shown that activation of both EGFR and Notch signaling pathways depends on the activity of the JAK/STAT signal. The JAK/STAT signal probably acts upstream of EGFR and Notch signals in a non-autonomous fashion. These three signals coordinate and precisely regulate the formation of neuroblasts (Yasugi, 2010).

Protein tyrosine phosphatase PTPN3 inhibits lung cancer cell proliferation and migration by promoting EGFR endocytic degradation

Epidermal growth factor receptor (EGFR) regulates multiple signaling cascades essential for cell proliferation, growth and differentiation. Using a genetic approach, this study found that Drosophila FERM and PDZ domain-containing protein tyrosine phosphatase, dPtpmeg, negatively regulates border cell migration and inhibits the EGFR/Ras/mitogen-activated protein kinase signaling pathway during wing morphogenesis. EGFR pathway substrate 15 (Eps15) was further identified as a target of dPtpmeg and its human homolog PTPN3. Eps15 is a scaffolding adaptor protein known to be involved in EGFR endocytosis and trafficking. Interestingly, PTPN3-mediated tyrosine dephosphorylation of Eps15 promotes EGFR for lipid raft-mediated endocytosis and lysosomal degradation. PTPN3 and the Eps15 tyrosine phosphorylation-deficient mutant suppress non-small-cell lung cancer cell growth and migration in vitro and reduce lung tumor xenograft growth in vivo. Moreover, depletion of PTPN3 impairs the degradation of EGFR and enhances proliferation and tumorigenicity of lung cancer cells. Taken together, these results indicate that PTPN3 may act as a tumor suppressor in lung cancer through its modulation of EGFR signaling (Li, 2014).

Reversible tyrosine protein phosphorylation by protein tyrosine kinases and protein tyrosine phosphatases (PTPs) acts as a molecular switch that regulates a variety of biological processes. The receptor tyrosine kinase epidermal growth factor receptor (EGFR), the best characterized member of the ErbB family receptors, acts as a critical regulator of numerous cellular processes, including growth, proliferation and differentiation. Upon activation by its growth factor ligands, EGFR undergoes dimerization and activation, leading to tyrosine phosphorylation of the intracellular region of the receptor as well as many cytoplasmic substrates. The activated EGFR is then internalized by clathrin-mediated endocytosis and sorted into the endosomal compartments, through which it is either recycled back to the plasma membrane or transported to the lysosome for degradation. Because overexpression or constitutive activation of EGFR has been implicated in the pathogenesis and progression of a variety of human malignancies, it is therefore crucial to understand how EGFR signaling is regulated. Several PTPs have been implicated in the regulation of EGFR signaling. Among them, PTPrk, DEP-1 (PTPRJ), PTP1B (PTPN1), SHP-1 (PTPN6), TCPTP (PTPN2), PTPN9 and PTPN12 have been shown to downregulate EGFR signaling by dephosphorylating EGFR. The receptor-type PTP DEP-1 dephosphorylates EGFR on the cell surface and inhibits its internalization. On the other hand, the endoplasmic reticulum-localized PTP1B has been reported to regulate EGFR signaling from endosomes. PTP1B promotes the sequestration of EGFR onto internal vesicles of multivesicular bodies. The ESCRT (endosomal sorting complex required for transport) complexes are known to play an important role in sorting EGFR to multivesicular bodies. Recently, it has been shown that the ESCRT accessory protein HD-PTP/PTPN23 coordinates with the ubiquitin-specific peptidase UBPY to drive EGFR sorting to the multivesicular bodies. A better understanding of the role of PTPs in regulating EGFR signaling will help to provide insights into the molecular mechanisms behind EGFR-mediated tumorigenesis (Li, 2014).

PTPN3 (PTPH1) and the closely-related PTPN4 (PTPMEG) are non-transmembrane PTPs that contain an N-terminal FERM (Band 4.1, Ezrin, Radixin, Moesin homology) domain followed by a single PDZ (PSD95, Dlg, ZO1) domain and the C-terminal PTP domain. They have been implicated in the regulation of cell growth and proliferation. However, their role in receptor protein tyrosine kinase signaling is not clear. The dPtpmeg is the Drosophila homolog of mammalian PTPN3 and PTPN4. Phenotypic analyses have revealed that dptpmeg mutants exhibit aberrant mushroom body axon projection patterns in the brain. Besides its role in regulating neuronal wiring, the molecular function of dPtpmeg has remained largely unknown. This study has identified EGFR pathway substrate 15 (Eps15) as a substrate of dPtpmeg and PTPN3. Eps15 is known to be an endocytic adaptor involved in the regulation of EGFR trafficking. PTPN3 dephosphorylated Eps15 and promoted EGFR for lipid raft-mediated endocytosis and lysosomal degradation. The ectopic expression of PTPN3 or Eps15-Y850F mutant in the non-small-cell lung cancer (NSCLC) cells inhibited cell proliferation, migration and tumor growth. These findings uncover a novel role for PTPN3 in the regulation of EGFR endocytic trafficking, degradation and signaling (Li, 2014).

The EGFR belongs to the ErbB family of protein tyrosine kinases and is a major regulator for both normal development and cancer progression. PTPs, which include receptor-like PTPs and nonreceptor PTPs, are a group of tightly regulated enzymes thought to regulate tyrosine phosphorylation by antagonizing the action protein tyrosine kinases. This study provides the first evidence that PTPN3 inhibits the EGFR signaling by targeting the receptor for lysosomal degradation. In Drosophila, dPtpmeg antagonizes receptor tyrosine kinase activity and plays a role in controlling border cell migration during oogenesis. Moreover, dPtpmeg negatively regulates the EGFR/Ras/MAPK pathway during wing morphogenesis. Substrate-trapping and biochemical analysis further identified Eps15 as a substrate for dPTPmeg and PTPN3. The results demonstrate that PTPN3 dephosphorylates Eps15 and promotes EGFR for degradation in lung cancer cells (Li, 2014).

Eps15 is a multidomain adaptor protein that plays an important role in regulating endocytic trafficking. In mammalian cells, Eps15 can be phosphorylated by EGFR at tyrosine residue 850 upon EGF stimulation. It has been shown that ectopic expression of Eps15-Y850F mutant impairs the internalization of EGFR. On the contrary, using immunofluorescence and flow cytometry, this study shows that ectopic expression of PTPN3 and Eps15-Y850F does not affect the internalization of EGFR. Since a dramatic reduction of EGFR levels was found in cells expressing PTPN3 and Eps15-Y850F, one explanation for the results being contradictory to previous findings might be because of the difference in detection sensitivity. In addition to its role in endocytic trafficking, Eps15 was reported to localize at the trans-Golgi network and regulate vesicle trafficking during the secretory process. However, immunofluorescence analysis of the intracellular distribution of endogenous PTPN3 or exogenously expressed HA-tagged PTPN3 indicated no significant localization at the trans-Golgi network, and this might suggest that PTPN3 is not involved in Eps15-mediated protein sorting and vesicle trafficking at the trans-Golgi network (Li, 2014).

The ligand-activated EGFR and the transforming growth factor-β receptor have been reported to be endocytosed through a clathrin-dependent as well as a clathrin-independent pathway. Segregation of these cell surface receptors through distinct endocytic pathways is known to regulate downstream signal duration and receptor trafficking, although it is unclear how it does this. Several lines of evidence indicate that PTPN3 and Eps15-Y850F accelerate downregulation of EGFR via a clathrin-independent but lipid raft-dependent pathway. First, EGF-488 trafficking assay revealed that EGF-488 was largely colocalized with lipid raft-associated protein caveolin-1 but not with clathrin in cells expressing PTPN3 or Eps15-Y850F. Second, analysis of EGFR profile by sucrose gradient fractionation showed that EGFR was concentrated in clathrin-enriched non-lipid fractions in control cells. However, overexpression of PTPN3 and Eps15-Y850F led to a redistribution of EGFR to caveolin-1-enriched lipid raft fractions. Third, disruption of lipid rafts with MβCD and filipin suppressed PTPN3- or Eps15-Y850F-induced EGFR degradation. How does PTPN3-mediated tyrosine dephosphorylation of Eps15 regulate EGFR for lipid raft-dependent endocytosis and lysosomal degradation? Accumulating evidence has shown that EGFR ubiquitination is not essential for its internalization, but appears to play an important role in endosomal sorting and lysosomal targeting of the receptor. One possibility is that tyrosine dephosphorylation of Eps15 by PTPN3 may affect the ubiquitination status of EGFR, accelerating EGFR for lysosomal degradation. Recently, an endosomally localized Eps15 isoform (Eps15b) has been identified that interacts with the Hrs (hepatocyte growth factor-regulated tyrosine kinase substrate) complex to mediate EGFR degradation. This study found that like Eps15, Eps15b is also a substrate of PTPN3, indicating a dual role for PTPN3 in the regulation of endocytic trafficking and endosomal sorting of EGFR (Li, 2014).

Accumulating evidence has indicated that PTPs can function as tumor suppressors or oncogenes depending on the substrate involved and the cellular context. It has been reported that PTPN3 expresses in gastric cancer cells and may play a role in gastric cancer progression and differentiation. PTPN3 has been found to coordinate with p38γ MAPK to promote Ras oncogenesis in colon cancer, and it has been found to stimulate breast cancer growth by inducing and stabilizing the protein expression of vitamin D receptor. Interestingly, recent studies have also indicated that PTPN3 plays a role in tumor suppression. Mutational analysis of the tyrosine phosphatome found PTPN3 along with five other PTPs (PTPRF, PTPRG, PTPRT, PTPN13 and PTPN14) are mutated in colorectal cancer. Moreover, the transcriptome of two NSCLC cell lines were analyzed, and one allele of PTPN3 was found to be mutated in the NSCLC cell line H2228. It was further shown that ectopic expression of PTPN3 inhibits the growth of NSCLC cells, although the molecular mechanisms underlying the growth inhibition remain unknown. The current data support a model in which PTPN3-mediated tyrosine dephosphorylation of Eps15 leads to EGFR degradation and tumor suppression in NSCLC cells. This study demonstrated that PTPN3 and Eps15-Y850F overexpression reduced EGFR protein levels and impeded the proliferation and migration of NSCLC cells. Moreover, PTPN3 and Eps15-Y850F significantly suppressed NSCLC tumor growth in a subcutaneous xenograft model. Conversely, depletion of PTPN3 enhanced EGFR stabilization and promoted NSCLC tumorigenicity both in vitro and in vivo. The findings are consistent with the idea that PTPN3 acts as a tumor suppressor in NSCLC (Li, 2014).

In conclusion, this study has identified Eps15 as an evolutionarily conserved dPtpmeg/PTPN3 substrate that regulates EGFR signaling. The finding that PTPN3-mediated tyrosine dephosphorylation of Eps15 modulates EGFR-dependent cancer progression may help contribute to the development of a targeting intervention in NSCLC (Li, 2014).


The structure of the cDNAs indicates the use of alternative 5' exons. Thus the gene encodes two proteins differing at their N-terminus. The splicing alternatives show similar distribution during development. Most of the coding region of Egfr is located within four exons separated by three small introns (Schejter, 1986).

The alternative 3' exons, termed Type 1 and Type 2 exons, are separated from the downstream common sequences by 45 kb. Each alternative exon codes for a signal sequence. The Type 1 exon codes for 101 amino acids while the Type 2 exon codes for 52 amino acids (Clifford, 1994).

Genomic DNA length - 60 kb


Amino Acids - 1458 and 1409

Structural Domains

cDNA clones of Egfr were isolated and sequenced. The deduced amino acid sequence shows a similar degree of homology to the human epidermal growth factor receptor and to the rat and human neu proteins; the most striking difference is the addition of a third cysteine-rich extracellular domain in Egfr. In the extracellular region the homology between Egfr and HER (human EGF receptor) is 37% (Schejter, 1986 and Price, 1989). The intracellular region contains one kinase domain (Clifford, 1994).

EGF receptor: Evolutionary Homologs | Regulation | Protein Interactions | Developmental Biology | Effects of Mutation | References
date revised: 25 August 2023 

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