A Tumor-Specific Molecular Network Promotes Tumor Growth in Drosophila by Enforcing a Jun N-Terminal Kinase-Yorkie Feedforward Loop.

Cancer cells expand rapidly in response to altered intercellular and signaling interactions to achieve the hallmarks of cancer. Impaired cell polarity combined with activated oncogenes is known to promote several hallmarks of cancer, e.g., activating invasion by increased activity of Jun N-terminal kinase (JNK) and sustained proliferative signaling by increased activity of Hippo effector Yorkie (Yki). Thus, JNK, Yki, and their downstream transcription factors have emerged as synergistic drivers of tumor growth through pro-tumor signaling and intercellular interactions like cell competition. However, little is known about the signals that converge onto JNK and Yki in tumor cells and enable tumor cells to achieve the hallmarks of cancer. Here, using mosaic models of cooperative oncogenesis (RasV12,scrib-) in Drosophila, we show that RasV12,scrib- tumor cells grow through the activation of a previously unidentified network comprising Wingless (Wg), Dronc, JNK, and Yki. We show that RasV12,scrib- cells show increased Wg, Dronc, JNK, and Yki signaling, and all these signals are required for the growth of RasV12,scrib- tumors. We report that Wg and Dronc converge onto a JNK-Yki self-reinforcing positive feedback signal-amplification loop that promotes tumor growth. We found that the Wg-Dronc-Yki-JNK molecular network is specifically activated in polarity-impaired tumor cells and not in normal cells, in which apical-basal polarity remains intact. Our findings suggest that the identification of molecular networks may provide significant insights into the key biologically meaningful changes in signaling pathways and paradoxical signals that promote tumorigenesis.

A Tumour-Specific Molecular Network Promotes Tumour Growth in Drosophila by Enforcing a JNK-YKI Feedforward Loop.

Cancer cells expand rapidly in response to altered intercellular and signalling interactions to achieve hallmarks of cancer. Impaired cell polarity combined with activated oncogenes is known to promote several hallmarks of cancer e.g., activating invasion by increased activity of Jun N-terminal kinase (JNK), and sustained proliferative signalling by increased activity of Hippo effector Yorkie (Yki). Thus, JNK, Yki, and their downstream transcription factors have emerged as synergistic drivers of tumour growth through pro-tumour signalling and intercellular interactions like cell-competition. However, little is known about the signals that converge onto JNK and Yki in tumour cells that enable the tumour cells to achieve hallmarks of cancer. Here, using mosaic models of cooperative oncogenesis ( Ras V12 , scrib - ) in Drosophila , we show that Ras V12 , scrib - tumour cells grow by activation of a previously unidentified network comprising Wingless (Wg), Dronc, JNK and Yki. We show that Ras V12 , scrib - cells show increased Wg, Dronc, JNK, and Yki signalling, and all of these signals are required for the growth of Ras V12 , scrib - tumours. We report that Wg and Dronc converge onto a JNK-Yki self-reinforcing positive feedback signal-amplification loop that promotes tumour growth. We found that Wg-Dronc-Yki-JNK molecular network is specifically activated in polarity-impaired tumour cells and not in normal cells where apical basal polarity is intact. Our findings suggest that identification of molecular networks may provide significant insights about the key biologically meaningful changes in signalling pathways, and paradoxical signals that promote Tumourigenesis.

The matricellular protein Drosophila Cellular Communication Network Factor is required for synaptic transmission and female fertility.

Within the extracellular matrix, matricellular proteins are dynamically expressed nonstructural proteins that interact with cell surface receptors, growth factors, and proteases, as well as with structural matrix proteins. The cellular communication network factors family of matricellular proteins serve regulatory roles to regulate cell function and are defined by their conserved multimodular organization. Here, we characterize the expression and neuronal requirement for the Drosophila cellular communication network factor family member. Drosophila cellular communication network factor is expressed in the nervous system throughout development including in subsets of monoamine-expressing neurons. Drosophila cellular communication network factor-expressing abdominal ganglion neurons innervate the ovaries and uterus and the loss of Drosophila cellular communication network factor results in reduced female fertility. In addition, Drosophila cellular communication network factor accumulates at the synaptic cleft and is required for neurotransmission at the larval neuromuscular junction. Analyzing the function of the single Drosophila cellular communication network factor family member will enhance our potential to understand how the microenvironment impacts neurotransmitter release in distinct cellular contexts and in response to activity.

Regulation of Wnt distribution and function by Drosophila glypicans.

The extracellular distribution of secreted Wnt proteins is crucial for their ability to induce a response in target cells at short and long ranges to ensure proper development. Wnt proteins are evolutionarily conserved ligands that are lipid-modified, and their hydrophobic nature interferes with their solubility in the hydrophilic extracellular environment. This raises the question of how Wnt proteins spread extracellularly despite their lipid modifications, which are essential for both their secretion and function. Seminal studies on Drosophila Wingless (Wg), a prototypical Wnt, have discovered multiple mechanisms by which Wnt proteins spread. A central theme emerges from these studies: the Wnt lipid moiety is shielded from the aqueous environment, allowing the ligands to spread and remain viable for signaling. Wnt distribution in vivo is primarily facilitated by glypicans, which are cell-surface heparan sulfate proteoglycans, and recent studies have further provided mechanistic insight into how glypicans facilitate Wnt distribution. In this Review, we discuss the many diverse mechanisms of Wnt distribution, with a particular focus on glypican-mediated mechanisms.

Glypicans and cytonemes unite to distribute Wnt ligands.

Hu et al. (2021. J. Cell Biol.https://doi.org/10.1083/jcb.202009082) show that Glypican 4 participates in filopodia-mediated Wnt transport from endoderm to mesoderm in zebrafish embryos to facilitate intercellular communication between germ layers.

Extracellular spreading of Wingless is required for Drosophila oogenesis.

Recent studies have investigated whether the Wnt family of extracellular ligands can signal at long range, spreading from their source and acting as morphogens, or whether they signal only in a juxtacrine manner to neighboring cells. The original evidence for long-range Wnt signaling arose from studies of Wg, a Drosophila Wnt protein, which patterns the wing disc over several cell diameters from a central source of Wg ligand. However, the requirement of long-range Wg for patterning was called into question when it was reported that replacing the secreted protein Wg with a membrane-tethered version, NRT-Wg, results in flies with normally patterned wings. We and others previously reported that Wg spreads in the ovary about 50 µm or 5 cell diameters, from the cap cells to the follicle stem cells (FSCs) and that Wg stimulates FSC proliferation. We used the NRT-wg flies to analyze the consequence of tethering Wg to the cap cells. NRT-wg homozygous flies are sickly, but we found that hemizygous NRT-wg/null flies, carrying only one copy of tethered Wingless, were significantly healthier. Despite their overall improved health, these hemizygous flies displayed dramatic reductions in fertility and in FSC proliferation. Further, FSC proliferation was nearly undetectable when the wg locus was converted to NRT-wg only in adults, and the resulting germarium phenotype was consistent with a previously reported wg loss-of-function phenotype. We conclude that Wg protein spreads from its source cells in the germarium to promote FSC proliferation.

Dally-like protein sequesters multiple Wnt ligands in the Drosophila germarium.

Cells in multicellular organisms rely on secreted ligands for development and morphogenesis. Several mechanisms modulate the availability and distribution of secreted ligands, determining their ability to signal locally and at long range from their source. One of these mechanisms is Dally-like protein (Dlp), a cell-surface glypican that exhibits biphasic functions in Drosophila wing discs, promoting Wg signaling at long-range from Wg source cells and inhibiting Wg signaling near source cells. In the germarium at the tip of the ovary, Dlp promotes long-range distribution of Wg from cap cells to follicle stem cells. However, the germarium also expresses other Wnts - Wnt2, Wnt4, and Wnt6 - that function locally in escort cells to promote oogenesis. Whether and how local functions of these Wnts are regulated remains unknown. Here we show that the dlp overexpression phenotype is multifaceted and phenocopies multiple Wnt loss-of-function phenotypes. Each aspect of dlp overexpression phenotype is suppressed by co-expression of individual Wnts, and the suppression pattern exhibited by each Wnt suggests that Wnts have functional specificity in the germarium. Further, dlp knockdown phenocopies Wnt gain-of-function phenotypes. Together these data show that Dlp inhibits the functions of each Wnt. All four Wnts co-immunoprecipitate with Dlp in S2R+ â€‹cells, suggesting that in the germarium, Dlp sequesters Wnts to inhibit local paracrine Wnt signaling. Our results indicate that Dlp modulates the availability of multiple extracellular Wnts for local paracrine Wnt signaling in the germarium.

Phenotypic Plasticity of Invasive Edge Glioma Stem-like Cells in Response to Ionizing Radiation.

Unresectable glioblastoma (GBM) cells in the invading tumor edge can act as seeds for recurrence. The molecular and phenotypic properties of these cells remain elusive. Here, we report that the invading edge and tumor core have two distinct types of glioma stem-like cells (GSCs) that resemble proneural (PN) and mesenchymal (MES) subtypes, respectively. Upon exposure to ionizing radiation (IR), GSCs, initially enriched for a CD133+ PN signature, transition to a CD109+ MES subtype in a C/EBP-ß-dependent manner. Our gene expression analysis of paired cohorts of patients with primary and recurrent GBMs identified a CD133-to-CD109 shift in tumors with an MES recurrence. Patient-derived CD133-/CD109+ cells are highly enriched with clonogenic, tumor-initiating, and radiation-resistant properties, and silencing CD109 significantly inhibits these phenotypes. We also report a conserved regulation of YAP/TAZ pathways by CD109 that could be a therapeutic target in GBM.

Wnt Signaling in Stem Cell Maintenance and Differentiation in the Drosophila Germarium.

Wnt signaling is a conserved regulator of stem cell behaviors, and the Drosophila germarium has been an important model tissue for the study of stem cell maintenance, differentiation, and proliferation. Here we review Wnt signaling in the germarium, which houses two distinct types of ovarian stem cells: the anteriorly located germline stem cells (GSCs), which give rise to oocytes; and the mid-posteriorly located follicle stem cells (FSCs), which give rise to the somatic follicle cells that cover a developing oocyte. The maintenance and proliferation of GSCs and FSCs is regulated by the stem cell niches, whereas differentiation of the germline is regulated by the differentiation niche. Four distinct Wnt ligands are localized in the germarium, and we focus review on how these Wnt ligands and Wnt signaling affects maintenance and differentiation of both germline and follicle stem cells in their respective niches.

FOXD1-ALDH1A3 Signaling Is a Determinant for the Self-Renewal and Tumorigenicity of Mesenchymal Glioma Stem Cells.

Glioma stem-like cells (GSC) with tumor-initiating activity orchestrate the cellular hierarchy in glioblastoma and engender therapeutic resistance. Recent work has divided GSC into two subtypes with a mesenchymal (MES) GSC population as the more malignant subtype. In this study, we identify the FOXD1-ALDH1A3 signaling axis as a determinant of the MES GSC phenotype. The transcription factor FOXD1 is expressed predominantly in patient-derived cultures enriched with MES, but not with the proneural GSC subtype. shRNA-mediated attenuation of FOXD1 in MES GSC ablates their clonogenicity in vitro and in vivo Mechanistically, FOXD1 regulates the transcriptional activity of ALDH1A3, an established functional marker for MES GSC. Indeed, the functional roles of FOXD1 and ALDH1A3 are likely evolutionally conserved, insofar as RNAi-mediated attenuation of their orthologous genes in Drosophila blocks formation of brain tumors engineered in that species. In clinical specimens of high-grade glioma, the levels of expression of both FOXD1 and ALDH1A3 are inversely correlated with patient prognosis. Finally, a novel small-molecule inhibitor of ALDH we developed, termed GA11, displays potent in vivo efficacy when administered systemically in a murine GSC-derived xenograft model of glioblastoma. Collectively, our findings define a FOXD1-ALDH1A3 pathway in controling the clonogenic and tumorigenic potential of MES GSC in glioblastoma tumors. Cancer Res; 76(24); 7219-30. ©2016 AACR.

Loss of Cell Adhesion Increases Tumorigenic Potential of Polarity Deficient Scribble Mutant Cells.

Epithelial polarity genes are important for maintaining tissue architecture, and regulating growth. The Drosophila neoplastic tumor suppressor gene scribble (scrib) belongs to the basolateral polarity complex. Loss of scrib results in disruption of its growth regulatory functions, and downregulation or mislocalization of Scrib is correlated to tumor growth. Somatic scribble mutant cells (scrib-) surrounded by wild-type cells undergo apoptosis, which can be prevented by introduction of secondary mutations that provide a growth advantage. Using genetic tools in Drosophila, we analyzed the phenotypic effects of loss of scrib in different growth promoting backgrounds. We investigated if a central mechanism that regulates cell adhesion governs the growth and invasive potential of scrib mutant cells. Here we show that increased proliferation, and survival abilities of scrib- cells in different genetic backgrounds affect their differentiation, and intercellular adhesion. Further, loss of scrib is sufficient to cause reduced cell survival, activation of the JNK pathway and a mild reduction of cell adhesion. Our data show that for scrib cells to induce aggressive tumor growth characterized by loss of differentiation, cell adhesion, increased proliferation and invasion, cooperative interactions that derail signaling pathways play an essential role in the mechanisms leading to tumorigenesis. Thus, our study provides new insights on the effects of loss of scrib and the modification of these effects via cooperative interactions that enhance the overall tumorigenic potential of scrib deficient cells.

Drosophila C-terminal Src kinase regulates growth via the Hippo signaling pathway.

The Hippo signaling pathway is involved in regulating tissue size by inhibiting cell proliferation and promoting apoptosis. Aberrant Hippo pathway function is often detected in human cancers and correlates with poor prognosis. The Drosophila C-terminal Src kinase (d-Csk) is a genetic modifier of warts (wts), a tumor-suppressor gene in the Hippo pathway, and interacts with the Src oncogene. Reduction in d-Csk expression and the consequent activation of Src are frequently seen in several cancers including hepatocellular and colorectal tumors. Previous studies show that d-Csk regulates cell proliferation and tissue size during development. Given the similarity in the loss-of-function phenotypes of d-Csk and wts, we have investigated the interactions of d-Csk with the Hippo pathway. Here we present multiple lines of evidence suggesting that d-Csk regulates growth via the Hippo signaling pathway. We show that loss of dCsk caused increased Yki activity, and our genetic epistasis places dCsk downstream of Dachs. Furthermore, dCsk requires Yki for its growth regulatory functions, suggesting that dCsk is another upstream member of the network of genes that interact to regulate Wts and its effector Yki in the Hippo signaling pathway.

Intercellular cooperation and competition in brain cancers: lessons from Drosophila and human studies.

Glioblastoma (GBM) is a primary brain cancer with an extremely poor prognosis. GBM tumors contain heterogeneous cellular components, including a small subpopulation of tumor cells termed glioma stem cells (GSCs). GSCs are characterized as chemotherapy- and radiotherapy-resistant cells with prominent tumorigenic ability. Studies in Drosophila cancer models demonstrated that interclonal cooperation and signaling from apoptotic clones provokes aggressive growth of neighboring tumorigenic clones, via compensatory proliferation or apoptosis induced proliferation. Mechanistically, these aggressive tumors depend on activation of Jun-N-terminal kinase (upstream of c-JUN), and Drosophila Wnt (Wg) in the apoptotic clones. Consistent with these nonmammalian studies, data from several mammalian studies have shown that c-JUN and Wnt are hyperactivated in aggressive tumors (including GBM). However, it remains elusive whether compensatory proliferation is an evolutionarily conserved mechanism in cancers. In the present report, we summarize recent studies in Drosophila models and mammalian models (e.g., xenografts of human cancer cells into small animals) to elucidate the intercellular interactions between the apoptosis-prone cancer cells (e.g., non-GSCs) and the hyperproliferative cancer cells (e.g., GSCs). These evolving investigations will yield insights about molecular signaling interactions in the context of post-therapeutic phenotypic changes in human cancers. Furthermore, these studies are likely to revise our understanding of the genetic changes and post-therapeutic cell-cell interactions, which is a vital area of cancer biology with wide applications to many cancer types in humans.

Scribble acts in the Drosophila fat-hippo pathway to regulate warts activity.

Epithelial cells are the major cell-type for all organs in multicellular organisms. In order to achieve correct organ size, epithelial tissues need mechanisms that limit their proliferation, and protect tissues from damage caused by defective epithelial cells. Recently, the Hippo signaling pathway has emerged as a major mechanism that orchestrates epithelial development. Hippo signaling is required for cells to stop proliferation as in the absence of Hippo signaling tissues continue to proliferate and produce overgrown organs or tumors. Studies in Drosophila have led the way in providing a framework for how Hippo alters the pattern of gene transcription in target cells, leading to changes in cell proliferation, survival, and other behaviors. Scribble (Scrib) belongs to a class of neoplastic tumor suppressor genes that are required to establish apical-basal cell polarity. The disruption of apical-basal polarity leads to uncontrolled cell proliferation of epithelial cells. The interaction of apical basal polarity genes with the Hippo pathway has been an area of intense investigation. Loss of scrib has been known to affect Hippo pathway targets, however, its functions in the Hippo pathway still remain largely unknown. We investigated the interactions of Scrib with the Hippo pathway. We present data suggesting that Drosophila scrib acts downstream of the Fat (Ft) receptor, and requires Hippo signaling for its growth regulatory functions. We show that Ft requires Scrib to interact with Expanded (Ex) and Dachs (D), and for regulating Warts (Wts) levels and stability, thus placing Scrib in the Hippo pathway network.