Notch signals modulate lgl mediated tumorigenesis by the activation of JNK signaling
Oncogenic potential of Notch signaling and its cooperation with other factors to affect proliferation are widely established. Notch exhibits a cooperative effect with loss of a cell polarity gene, scribble to induce neoplastic overgrowth. Oncogenic Ras also show cooperative effect with loss of cell polarity genes such as scribble (scrib), lethal giant larvae (lgl) and discs large to induce neoplastic overgrowth and invasion. Our study aims at assessing the cooperation of activated Notch with loss of function of lgl in tumor overgrowth, and the mode of JNK signaling activation in this context.
In the present study, we use Drosophila as an in vivo model to show the synergy between activated Notch (N act ) and loss of function of lgl (lgl-IR) in tumor progression. Coexpression of N act and lgl-IR results in massive tumor overgrowth and displays hallmarks of cancer, such as MMP1 upregulation and loss of epithelial integrity. We further show activation of JNK signaling and upregulation of its receptor, Grindelwald in N act /lgl-IR tumor. In contrast to previously described Notch act /scrib−/− tumor, our experiments in N act /lgl-IR tumor showed the presence of dying cells along with tumorous overgrowth.
KeywordsNotch lgl Drosophila Tumor overgrowth JNK signaling Cell death
lethal giant larvae
ventran nerve cord
Notch dominant negative
In the past decade, a keen interest has been shown to explore the oncogenic cooperation with loss of cell polarity in tumor progression and malignancy. Studies in Drosophila have revealed that the oncogenic form of Ras cooperates with loss of tumor suppressors, namely scrib, lgl and dlg to cause tumor cell invasion [1, 2]. The oncogenic form of Notch has also shown to cooperate with scrib−/− to induce neoplastic overgrowth . The loss-of-function mutation of Scribble complex genes (scrib, lgl and dlg) results in disruption of epithelial integrity followed by neoplastic tissue hyperproliferation [3, 4, 5]. However, the tumor formation caused by loss of scrib, lgl and dlg has been found to be restricted by the compensatory JNK mediated apoptosis [2, 6, 7, 8]. Among the Scrib complex genes, lgl was the first neoplastic tumor suppressor gene described in Drosophila . The phenotypes of lgl mutant tissues show close similarity with that of the human epithelial cancers [10, 11, 12]. Although it has been shown that Notch cooperates with scrib−/− to induce neoplastic growth, it is still unknown whether Notch works in the same way with loss-of-function of lgl also. Recently, Lgl has been shown to regulate Notch signaling via endocytosis . However, it gives no substantial evidence on coupling of lgl-Notch effect on tumorigenesis. In the present study, we checked the effect of a tumor suppressor gene mutation, lgl, in activated Notch background, and found that lgl downregulation synergizes with activated Notch to induce overgrowth and migratory behavior. Here, we show that N act /lgl-IR tissues display the hallmarks of tumor overgrowth. Moreover, our study revealed that the effect of N act /lgl-IR tumor is mediated by the activation of JNK signaling through the upregulation of its receptor, Grindelwald.
Detailed description of methods used in this study is provided in Additional file 1.
Oncogenic Notch synergizes with RNAi mediated downregulation of lgl to promote tissue overgrowth
In order to examine the cytoskeleton network and cell–cell adhesion, we marked the tissues with phalloidin and adherens junction marker proteins, Armadillo (Arm) and Cadherin (DE-Cad). The F-actin network marked by phalloidin revealed a defective actin cytoskeleton network in N act /lgl-IR tumor tissues compared to that of controls (Additional file 3: Figure S2). In the same way, the localization of DE-Cad and Arm were also deregulated in N act /lgl-IR tumorous eye discs (Additional file 4: Figure S3a–d, e–h). We, next, determined if neuronal differentiation was defective in N act /lgl-IR tumor using a neuronal marker, Elav that marks the differentiated neurons in eye disc and brain. Remarkably, coexpression of N act and lgl-IR led to severe loss of Elav positive cells in the eye disc and abnormal expression of Elav in the optic lobes indicating an impaired neuronal differentiation (Additional file 4: Figure S3i–l, m–p).
In parallel, we also used dominant-negative version of Notch to see the effect of depletion of Notch signaling on lgl-IR tumors. Previously, expression of mam DN in lgl− tissues partially rescued the lgl− mosaic adult eye phenotype . Our analysis also found that reduction of Notch signaling partially rescued the phenotypes of lgl loss-of-function flies (Additional file 5: Figure S4). Thus, our analysis support the notion that the lgl loss-of-function wing phenotype is dependent on elevated Notch signaling, consistent with the previous study .
Involvement of JNK pathway in N act /lgl-IR tumor
To check the mode of activation of JNK signaling, we examined the transcript level expression of ligand eiger (egr), and its receptor wengen (wgn), in N act /lgl-IR tumor. egr and wgn transcript levels were found to be depleted in case of N act /lgl-IR tumor as compared to that of the controls (Fig. 2j). Recently, another member in tumor necrosis factor receptor superfamily, Grindelwald (Grnd), found to be associated with loss of cell polarity and neoplastic growth . Interestingly, a significant upregulation of grnd transcripts in N act /lgl-IR tumor was found, when compared to that of the wild-type, only N act and only lgl-IR tissues (Fig. 2j). We went on to check the protein level expression of Egr in N act /lgl-IR tumors. Immunostaining with anti-Egr antibody  revealed that there is no change in the level of Egr protein expression in Nact/lgl-IR tumor (Fig. 2h) as compared to that of the wild-type, only N act and only lgl-IR tissues (Fig. 2e–g). As Egr is known to be also expressed by the tumor-associated hemocytes, leading to signaling activation , these immune cells may be in this case responsible for Grnd activation, but their poor adhesion to the tumor tissue may make them escape Immunofluorescence detection.
To further confirm the involvement of JNK signaling as a downstream event of N act /lgl-IR cooperation, we blocked JNK signaling in N act /lgl-IR tumor, and checked whether blocking JNK could affect the N act /lgl-IR tumor. The massive upregulation of MMP1 in N act /lgl-IR tumor (Additional file 6: Figure S5a) was drastically suppressed, when bsk-DN (a dominant negative allele of Drosophila JNK gene, basket) was expressed in the background (Additional file 6: Figure S5b). In addition, coexpression of bsk-DN with N act ; lgl-IR resulted in a reduced wing disc size as compared to N act /lgl-IR overexpressed wing disc (Additional file 6: Figure S5c). These results indicate that JNK signaling may be involved in the tumorous overgrowth of N act /lgl-IR tissues.
N act /lgl-IR tumor induces cell death
In the present study, we unveil a cooperation of Notch with RNAi-mediated downregulation of a polarity cum tumor suppressor gene, lgl to promote tumor overgrowth. Our data, presented here, illustrate that coexpression of N act and lgl-IR in Drosophila eye disc results in overgrowth, loss of positional clues and upregulation of MMP1 expression, which is less prevalent in only N act overexpression or only lgl-IR overexpression. Earlier the loss of polarity gene scribble found to cooperate with Notch signaling to promote neoplastic overgrowth . Another two independent studies of similar context show that oncogenic Ras cooperates with loss of cell polarity genes (lgl, scrib, dlg) to induce metastasis and secondary tumor formation at distant sites [7, 14]. Interestingly, we found that Notch synergizes with loss of lgl to promote tumorous overgrowth and elevated expression of MMP1, and inhibiting Notch signaling rescues the defects caused by loss of lgl. It indicates the potential function of Notch signaling during lgl mediated tumor development. Our data also show distorted epithelial integrity in N act /lgl-IR tumor that point towards epithelial to mesenchymal transition, where tightly joined epithelial cells with regularly spaced cell–cell junctions convert to mesenchymal cells which are of irregular shape without tight intracellular adhesion .
Further, we found upregulation of JNK signaling and its receptor Grindelwald in N act /lgl-IR tumor. Two previous studies have shown that Notch cooperates with two different proteins to induce proliferation and metastasis by the activation of JNK signaling in ligand-dependent and -independent manner [25, 26]. In case of N act /lgl-IR tumor, we show that the transcript levels of egr (ligand) and wgn (receptor) were not upregulated, whereas a significant upregulation of grnd transcripts in the N act /lgl-IR tumor was observed. Earlier the active form of Grnd has shown to activate JNK signaling in vivo . Thus, in case of N act /lgl-IR tumor, JNK signaling might get activated through Grindelwald. Previously, it has been shown that JNK signaling can initiate tumor initiation and growth in Eiger-independent manner also .
Another most important hallmark of almost all types of cancer is the ability to evade apoptosis that, in turn, helps tumor cell population to increase in number . In other similar tumor models such as Ras v12 /dlg−/−, dying cells of dlg−/− clones evade apoptosis in presence of oncogenic Ras, where JNK signaling switches its role from proapoptotic to progrowth . In contrast, Ras/scrib−/− and Ras/lgl−/−tumors were reported to show apoptosis [22, 28]. However, Notch/scrib−/− tumor did not show the presence of apoptosis . In our case, N act /lgl-IR tumor resulted in severe apoptosis along with strong overgrowth and MMP1 expression. These dying cells in N act /lgl-IR tumor might be the indication of cell competition as there is a strong proliferation and overgrowth. In case of N act /scrib−/− tumor, Notch is giving growth advantage to scrib−/− tissues by preventing cell death. However, in case of N act /lgl-IR wing discs, activation of Notch failed to restrict the cell death caused by loss of lgl; rather its activation induces further cell death. These differences indicate that although oncogenic cooperation with loss of cell polarity results in similar tumor cell migration but certain property like cell death occurs depending on the context.
The present study is not the first one to show the cooperation between Notch and loss of cell polarity genes. Activated Notch is known to cooperate with another cell polarity gene, scribble, to induce neoplastic overgrowth.
In the present study, experiments were performed using RNAi line of lgl, but not with the lgl loss-of-function mutants.
AM and MSP involved in conception and design of the study. MSP performed the experiments, analyzed the data and drafted the manuscript. DD involved in critically revising the original draft. AS and MM were involved in analysis and interpretation of the data. All authors read and approved the final manuscript.
The authors wish to thank Spyros Artavanis-Tsakonas, Estee Kurant, Konrad Basler and the Bloomington Stock Center for fly stocks. Some of the antibodies used in this work were obtained from DSHB. Confocal microscopy and Real-time PCR facility at DBT-BHU-ISLS are duly acknowledged.
The authors declare that they have no competing interests.
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This work was supported by funds from DBT, India (BT/PR14082/BRB/10/806/2010 and BT/PR14080/BRB/10/805/2010) and UGC-UPE, BHU to AM and MM. MSP was supported by the fellowship from JNMF while AS and DD were supported by CSIR, Government of India.
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