Ras oncogene at 85D
Complications from metastasis are responsible for the majority of cancer-related deaths. Despite the outsized medical impact of metastasis, remarkably little is known about one of the key early steps of metastasis: departure of a tumor cell from its originating tissue. It is well documented that cellular delamination in the basal direction can induce invasive behaviors, but it remains unknown if apical cell delamination can induce migration and invasion in a cancer context. To explore this feature of cancer progression, a genetic screen was performed in Drosophila, and mutations in the protein M6 were found to synergize with oncogenic Ras to drive invasion following apical delamination without crossing a basement membrane. Mechanistically, it was observed that M6-deficient Ras(V12) clones delaminate as a result of alterations in a Canoe-RhoA-myosin II axis that is necessary for both the delamination and invasion phenotypes. To uncover the cellular roles of M6, this study showed that it localizes to tricellular junctions in epithelial tissues where it is necessary for the structural integrity of multicellular contacts. This work provides evidence that apical delamination can precede invasion and highlights the important role that tricellular junction integrity can play in this process (Dunn, 2018).
Cells are known to delaminate from their tissues in both the apical and basal directions during development and in disease conditions. Importantly, cell delamination plays a vital role in cancer progression as it is one way that a cancer cell can escape its originating tissue before spreading to more distant sites. During tumor progression, different models have revealed that cell delamination in the apical direction can lead either to the elimination of the delaminated cells or to the overgrowth of those cells. However, invasive behaviors have not been observed to follow apical delamination but instead have been shown to occur only through basal delamination (Dunn, 2018).
During basal delamination-induced invasion, basement membrane degradation and cell invasion into the underlying tissue can be observed in fixed tissues. On the other hand, if cancer cells leave the tissue by migrating and invading following apical delamination, the invasion would not leave such a histologically visible trail as this invasion could occur without crossing the basement membrane but instead through migration along connected tissues. As such, alternate methods in a suitable system would be needed to recognize if apical delamination is able to induce invasion. Thus, although previous work has documented direct basal delamination and invasion during metastasis in animal models and human patients, it does not preclude the possibility that invasion can be initiated by apical delamination as well. Drosophila cancer models are well suited to address the role of apical delamination in inducing invasive behaviors due to their simple tissue architecture that allows for the easy identification of an apical delamination event, as well as established techniques to image intact living tissues over time to follow the fates of apically delaminated cells. This study documents that cell migration and invasion can be induced via apical delamination through the characterization of a tumor suppressor, M6, in Drosophila (Dunn, 2018).
While bicellular junctions have been well studied for their roles in tissue integrity and signaling, the importance of TCJs has been gradually coming to light in recent years as they have been shown to be key players in ionic barrier formation and maintenance, pathogen spread, and orientation of cell division. This study demonstrates that inactivation of a TCJ protein, M6, disrupts the structural integrity of multicellular contacts and induces apical delamination and invasion of otherwise benign RasV12 tumors in a manner dependent upon a Cno-RhoA-MyoII axis. This study thus provides a causative role for TCJ mutations in driving delamination and invasion in vivo, highlighting the importance of these junctions in tissue integrity and cancer biology (Dunn, 2018).
This study demonstrates a functional link between tricellular junctions and RhoA, which is a known cytoskeletal regulator. This finding adds to recent work that has begun suggesting that TCJs act as centers for cytoskeletal organization. It will be interesting to further learn the mechanisms and consequences of functionally linking RhoA and cytoskeletal components to TCJs. Additionally, RhoA is known to affect a variety of proteins and cellular processes in addition to Sqh. As such, it is highly possible that RhoA is inducing apical delamination and invasion through multiple routes in addition to its effects on sqh. Also, since Cno localizes at the adherens junctions, which are apical to M6, it is plausible that M6 only indirectly affects Cno, and thus RhoA, through alterations in epithelial integrity rather than through direct means (Dunn, 2018).
Finally, invasion of cancer cells into surrounding tissues was previously thought to occur only through direct basal delamination and subsequent invasion. This work shows that apical delamination can also precede migration and invasion to distant tissues. Furthermore, since no basement membrane degradation was observed in invasive RasV12; M6-/- clones, the invasion most likely occurs along connected tissues rather than through an apical delamination to the basal penetration route, but further experiments are needed to confirm this hypothesis. Although mammalian anatomy differs markedly from that of the simple architecture of the Drosophila imaginal discs, it will be interesting to learn if apical delamination, such as is observed in early stage human breast cancer, can also precede invasion in mammalian models. Further investigation into this paradigm of apical delamination-induced invasion could aid in understanding of the mechanisms underlying cancer progression and metastasis (Dunn, 2018).
The use of time series profiling to identify groups of functionally
related genes (synexpression groups) is a powerful approach for the
discovery of gene function. This study applies this strategy during RasV12
immortalization of Drosophila embryonic cells, a phenomenon not
well characterized. Using high-resolution transcriptional time-series
datasets, a gene network based on temporal expression profile similarities
was generated. This analysis revealed that common immortalized cells are
related to adult muscle precursors
(AMPs), a stem cell-like population contributing to adult muscles and
sharing properties with vertebrate satellite cells. Remarkably, the
immortalized cells retain the capacity for myogenic differentiation when
treated with the steroid hormone ecdysone (Dequéant, 2015).
How cancer cells sense and promote growth in the nutrient favorable conditions remain incompletely understood. Epidemiological studies have indicated that obesity is a risk factor for various types of cancers. Feeding Drosophila a high dietary sugar not only directs metabolic defects including obesity and organismal insulin resistance, but also transform Ras/Src-activated cells into aggressive tumors. This study demonstrates that Ras/Src-activated cells are sensitive to perturbations in the Hippo signaling pathway. Evidence is provided that nutritional cues activate Salt-inducible kinase, leading to Hippo pathway downregulation in Ras/Src-activated cells. The result is Yorkie-dependent increase in Wingless signaling, a key mediator that promotes diet-enhanced Ras/Src-tumorigenesis in an otherwise insulin-resistant environment. Through this mechanism, Ras/Src-activated cells are positioned to efficiently respond to nutritional signals and ensure tumor growth upon nutrient rich condition including obesity (Hirabayashi, 2015).
Germline genes often become re-expressed in soma-derived human cancers as 'cancer/testis antigens' (CTAs), and piRNA (PIWI-interacting RNA) pathway proteins are found among CTAs. However, whether and how the piRNA pathway contributes to oncogenesis in human neoplasms remain poorly understood. This study found that oncogenic Ras combined with loss of the Hippo tumor suppressor pathway reactivates a primary piRNA pathway in Drosophila somatic cells coincident with oncogenic transformation. In these cells, Piwi becomes loaded with piRNAs derived from annotated generative loci, which are normally restricted to either the germline or the somatic follicle cells. Negating the pathway leads to increases in the expression of a wide variety of transposons and also altered expression of some protein-coding genes. This correlates with a reduction in the proliferation of the transformed cells in culture, suggesting that, at least in this context, the piRNA pathway may play a functional role in cancer (Fagegaltier, 2015).
RAS genes are frequently mutated in cancers, yet an effective treatment has not been developed, partly because of an incomplete understanding of signaling within Ras-related tumors. To address this, a genetic screen was performed in Drosophila, aiming to find mutations that cooperate with oncogenic Ras (RasV12) to induce tumor overgrowth and invasion. fiery mountain (fmt; CG10289), a regulatory subunit of the protein phosphatase 6 (PP6) complex, was identified as a tumor suppressor that synergizes with RasV12 to drive c-Jun N-terminal kinase (JNK)-dependent tumor growth and invasiveness. Fmt was shown to negatively regulate JNK upstream of dTAK1. It was further demonstrated that disruption of PpV, the catalytic subunit of PP6, mimics fmt loss-of-function-induced tumorigenesis. Finally, Fmt synergizes with PpV to inhibit JNK-dependent tumor progression. These data here further highlight the power of Drosophila as a model system to unravel molecular mechanisms that may be relevant to human cancer biology (Ma, 2017).
Shu, Z., Huang, Y. C., Palmer, W. H., Tamori, Y., Xie, G., Wang, H., Liu, N. and Deng, W. M. (2017). Systematic analysis reveals tumor-enhancing and -suppressing microRNAs in Drosophila epithelial tumors. Oncotarget 8(65): 108825-108839. PubMed ID: 29312571
Despite their emergence as an important class of noncoding RNAs involved in cancer cell transformation, invasion, and migration, the precise role of microRNAs (miRNAs) in tumorigenesis remains elusive. To gain insights into how miRNAs contribute to primary tumor formation, an RNA sequencing (RNA-Seq) analysis was conducted of Drosophila wing disc epithelial tumors induced by knockdown of a neoplastic tumor-suppressor gene (nTSG) lethal giant larvae (lgl), combined with overexpression of an active form of oncogene Ras (Ras(V12)), and 51 mature miRNAs were identified that changed significantly in tumorous discs. Followed by in vivo tumor enhancer and suppressor screens in sensitized genetic backgrounds, ten tumor-enhancing (TE) miRNAs and eleven tumor-suppressing (TS) miRNAs were identified that contributed to the nTSG defect-induced tumorigenesis. Among these, four TE and three TS miRNAs have human homologs. From this study, 29 miRNAs were identified that individually had no obvious role in enhancing or alleviating tumorigenesis despite their changed expression levels in nTSG tumors. This systematic analysis, which includes both RNA-Seq and in vivo functional studies, helps to categorize miRNAs into different groups based on their expression profile and functional relevance in epithelial tumorigenesis, whereas the evolutionarily conserved TE and TS miRNAs provide potential therapeutic targets for epithelial tumor treatment (Shu, 2017).
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 (Chabu, 2017).
Cancer cells demand excessive nutrients to support their proliferation but how cancer cells sense and promote growth in the nutrient favorable conditions remain incompletely understood. Epidemiological studies have indicated that obesity is a risk factor for various types of cancers. Feeding Drosophila a high dietary sugar was previously demonstrated to not only direct metabolic defects including obesity and organismal insulin resistance, but also transform Ras/Src-activated cells into aggressive tumors. This study demonstrates that Ras/Src-activated cells are sensitive to perturbations in the Hippo signaling pathway. Evidence that nutritional cues activate Salt-inducible kinase, leading to Hippo pathway downregulation in Ras/Src-activated cells. The result is Yorkie-dependent increase in Wingless signaling, a key mediator that promotes diet-enhanced Ras/Src-tumorigenesis in an otherwise insulin-resistant environment. Through this mechanism, Ras/Src-activated cells are positioned to efficiently respond to nutritional signals and ensure tumor growth upon nutrient rich condition including obesity (Hirabayashi, 2013).
The prevalence of obesity is increasing globally. Obesity impacts whole-body homeostasis and is a risk factor for severe health complications including type 2 diabetes and cardiovascular disease. Accumulating epidemiological evidence indicates that obesity also leads to elevated risk of developing several types of cancers. However, the mechanisms that link obesity and cancer remain incompletely understood. Using Drosophila, a whole-animal model system has been developed to study the link between diet-induced obesity and cancer: this model has provided a potential explanation for how obese and insulin resistant animals are at increased risk for tumor progression (Hirabayashi, 2013).
Drosophila fed a diet containing high levels of sucrose (high dietary sucrose or ‘HDS') developed sugar-dependent metabolic defects including accumulation of fat (obesity), organismal insulin resistance, hyperglycemia, hyperinsulinemia, heart defects and liver (fat body) dysfunctions. Inducing activation of oncogenic Ras and Src together in the Drosophila eye epithelia led to development of small benign tumors within the eye epithelia. Feeding animals HDS transformed Ras/Src-activated cells from benign tumor growths to aggressive tumor overgrowth with tumors spread into other regions of the body (Hirabayashi, 2013). While most tissues of animals fed HDS displayed insulin resistance, Ras/Src-activated tumors retained insulin pathway sensitivity and exhibited an increased ability to import glucose. This is reflected by increased expression of the Insulin Receptor (InR), which was activated through an increase in canonical Wingless (Wg)/dWnt signaling that resulted in evasion of diet-mediated insulin resistance in Ras/Src-activated cells. Conversely, expressing a constitutively active isoform of the Insulin Receptor in Ras/Src-activated cells (InR/Ras/Src) was sufficient to elevate Wg signaling, promoting tumor overgrowth in animals fed a control diet. These results revealed a circuit with a feed-forward mechanism that directs elevated Wg signaling and InR expression specifically in Ras/Src-activated cells. Through this circuit, mitogenic effects of insulin are not only preserved but are enhanced in Ras/Src-activated cells in the presence of organismal insulin resistance (Hirabayashi, 2013).
These studies provide an outline for a new mechanism by which tumors evade insulin resistance, but several questions remain: (1) how Ras/Src-activated cells sense the organism's increased insulin levels, (2) how nutrient availability is converted into growth signals, and (3) the trigger for increased Wg protein levels, a key mediator that promotes evasion of insulin resistance and enhanced Ras/Src-tumorigenesis consequent to HDS. This study identifies the Hippo pathway effector Yorkie (Yki) as a primary source of increased Wg expression in diet-enhanced Ras/Src-tumors. Ras/Src-activated cells are sensitized to Hippo signaling, and even a mild perturbation in upstream Hippo pathway is sufficient to dominantly promote Ras/Src-tumor growth. Functional evidence is provided that increased insulin signaling promotes Salt-inducible kinases (SIKs) activity in Ras/Src-activated cells, revealing a SIKs-Yki-Wg axis as a key mediator of diet-enhanced Ras/Src-tumorigenesis. Through this pathway, Hippo-sensitized Ras/Src-activated cells are positioned to efficiently respond to insulin signals and promote tumor overgrowth. These mechanisms act as a feed-forward cassette that promotes tumor progression in dietary rich conditions, evading an otherwise insulin resistant state (Hirabayashi, 2013).
Previously work has demonstrated that Ras/Src-activated cells preserve mitogenic effects of insulin under the systemic insulin resistance induced by HDS-feeding of Drosophila (Hirabayashi, 2013). Evasion of insulin resistance in Ras/Src-activated cells is a consequence of a Wg-dependent increase in InR gene expression (Hirabayashi, 2013). This study identified the Hippo pathway effector Yki as a primary source of the Wnt ortholog Wg in diet-enhanced Ras/Src-tumors. Mechanistically, functional evidence is provided that activation of SIKs promotes Yki-dependent Wg-activation and reveal a SIK-Yki-Wg-InR axis as a key feed-forward signaling pathway that underlies evasion of insulin resistance and promotion of tumor growth in diet-enhanced Ras/Src-tumors (Hirabayashi, 2013).
In animals fed a control diet, at most a mild increase was observed in Yki reporter activity within ras1G12V;csk-/- cells. A previous report indicates that activation of oncogenic Ras (ras1G12V) led to slight activation of Yki in eye tissue. Activation of Src through over-expression of the Drosophila Src ortholog Src64B has been shown to induce autonomous and non-autonomous activation of Yki. In contrast, inducing activation of Src through loss of csk (csk-/-) failed to elevate diap1 expression. The results indicate that activation of Yki is an emergent property of activating Ras plus Src (ras1G12V;csk-/-). However, this level of Yki-activation was not sufficient to promote stable tumor growth of Ras/Src-activated cells in the context of a control diet: Ras/Src-activated cells were progressively eliminated from the eye tissue (Hirabayashi, 2013). It was, however, sufficient to sensitize Ras/Src-activated cells to upstream Hippo pathway signals: loss of a genetic copy of ex-which was not sufficient to promote growth by itself-dominantly promoted tumor growth of Ras/Src-activated cells even in animals fed a control diet. These data provide compelling evidence that Ras/Src-transformed cells are sensitive to upstream Hippo signals (Hirabayashi, 2013).
SIK was recently demonstrated to phosphorylate Sav at Serine-413, resulting in dissociation of the Hippo complex and activation of Yki (Wehr, 2013). SIKs are required for diet-enhanced Ras/Src-tumor growth in HDS. Conversely, expression of a constitutively activated isoform of SIK was sufficient to promote Ras/Src-tumor overgrowth even in a control diet. Mammalian SIKs are regulated by glucose and by insulin signaling. However, a recent report indicated that glucagon but not insulin regulates SIK2 activity in the liver. The current data demonstrate that increased insulin signaling is sufficient to promote SIK activity through Akt in Ras/Src-activated cells. It is concluded that SIKs couple nutrient (insulin) availability to Yki-mediated evasion of insulin resistance and tumor growth, ensuring Ras/Src-tumor growth under nutrient favorable conditions (Hirabayashi, 2013).
The results place SIKs as key sensors of nutrient and energy availability in Ras/Src-tumors through increased insulin signaling and, hence, increased glucose availability. SIK activity promotes Ras/Src-activated cells to efficiently respond to upstream Hippo signals, ensuring tumor overgrowth in organisms that are otherwise insulin resistant. One interesting question is whether this mechanism is relevant beyond the context of an obesity-cancer connection: both Ras and Src have pleiotropic effects on developmental processes including survival, proliferation, morphogenesis, differentiation, and invasion, and these mechanisms may facilitate these processes under nutrient favorable conditions. From a treatment perspective the current data highlight SIKs as potential therapeutic targets. Limiting SIK activity through compounds such as HG-9-91-01 may break the connection between oncogenes and diet, targeting key aspects of tumor progression that are enhanced in obese individuals (Hirabayashi, 2013).
Cancer is a multistep process involving cooperation between oncogenic or tumor suppressor mutations and interactions between the tumor and surrounding normal tissue. This study is the first description of cooperative tumorigenesis in Drosophila, and uses a system that mimics the development of tumors in mammals. The MARCM system was used to generate mutant clones of the apical-basal cell polarity tumor suppressor gene, scribbled, in the context of normal tissue. scribbled mutant clones in the eye disc exhibit ectopic expression of cyclin E and ectopic cell cycles, but do not overgrow due to increased cell death mediated by the JNK pathway and the surrounding wild-type tissue. In contrast, when oncogenic Ras or Notch is expressed within the scribbled mutant clones, cell death is prevented and neoplastic tumors develop. This demonstrates that, in Drosophila, activated alleles of Ras and Notch can act as cooperating oncogenes in the development of epithelial tumors, and highlights the importance of epithelial polarity regulators in restraining oncogenes and preventing tumor formation (Brumby, 2003).
A clonal approach, more closely resembling the clonal nature of mammalian cancer, was used to analyze the effects of removing Scrib function on tumor formation. This analysis indicates that Drosophila scrib- tumors: (1) lose tissue architecture, including apical-basal cell polarity; (2) fail to differentiate properly; (3) exert non-cell-autonomous effects upon the surrounding wild-type tissue; (4) upregulate cyclin E and undergo excessive cell proliferation; (5) are restrained from overgrowing by the surrounding wild-type tissue via a JNK-dependent apoptotic response, and (6) show strong cooperation with oncogenic alleles of Ras and Notch to produce large amorphous tumors. These conclusions are summarized in a model for tumor development in Drosophila. It is suggested that the role of epithelial cell polarity regulators in restraining oncogenes is likely to be of general significance in mammalian tumorigenesis (Brumby, 2003).
The model suggests that the wild-type larval eye disc is a monolayered columnar epithelium, in which cell proliferation is tightly regulated. Cell architecture is maintained by the formation of adherens junctions, the apical localization of Scribbled, and adhesion to the basement membrane. Mutation of scrib results in loss of apical-basal polarity, leading to multilayering and rounding up of cells. scrib- tissue also shows impaired differentiation, and ectopic cyclin E expression (by an unknown mechanism) leads to ectopic cell proliferation. Unrestrained overgrowth and tumor formation of scrib- cells is held in check by compensatory JNK-mediated apoptosis, dependent upon the presence of surrounding wild-type cells. Secondary mutations are required to avoid this apoptotic fate. If JNK activity is blocked within scrib- cells, by expressing a dominant-negative form of JNK, apoptosis is prevented, resulting in tissue overgrowth and lethality. Even more aggressive overgrowth results from the addition of activating oncogenic alleles of Ras or Notch. In addition to promoting cell survival, these oncogenes must also promote tumor cell proliferation; however, it is proposed that other downstream effectors of these oncogenes are likely also to be important, since it was not possible to mimic the cooperative overgrowth effects of RasACT or NACT on scrib- tissue by simply blocking apoptosis and enhancing cell proliferation (Brumby, 2003).
In Drosophila, activated Ras exerts its oncogenic effects through Raf and the MAPK pathway. Downstream targets of MAPK in the eye disc promote differentiation, cell survival and cell proliferation. This work also demonstrates that Ras can increase cyclin E protein levels in the eye disc. In combination with scrib-, the differentiation output of RasACT signaling appears to be attenuated, and the proliferative and anti-apoptotic responses prevail (Brumby, 2003).
Activated Notch also cooperates with scrib-, resulting in neoplastic overgrowth, and although no anti-apoptotic role for Notch signaling in the eye has been described previously, NACT exerts hyperproliferative effects in flies, and Notch signaling is required for proliferation of eye disc cells. Although it is not known if NACT induces the same critical downstream targets as RasACT to cause overgrowth of scrib- tissue, removing ras function in scrib- cells overexpressing NACT rescues the overgrowth phenotype, suggesting that the effects of NACT are at least partially dependent on Ras (Brumby, 2003).
Initially it seemed likely that the cooperative effects of RasACT or NACT on scrib- tissue could be explained by the ability of these oncogenes to promote cell proliferation while blocking apoptosis. However, the expression of neither cyclin E nor E2F1/DP, in combination with the apoptosis inhibitor p35 (or with the inhibitor of JNK pathway activity, BskDN), was capable of phenocopying the effect of RasACT or NACT in scrib- clones. It is therefore suggested that other downstream effectors, apart from anti-apoptotic and cell cycle regulators, must be important in mediating the oncogenic effects of RasACT or NACT. In fact, in Drosophila, Ras has also been shown to be a potent inducer of cellular growth, while cyclin E and E2F1 mainly promote cell cycle progression. Whether NACT also promotes cell growth in Drosophila has not been examined in detail. If growth promotion targets downstream of RasACT or NACT are critical in promoting the overgrowth of scrib- tumors, these are likely to be independent of the PI3 kinase pathway since ectopic PI3 kinase signaling in scrib- clones does not induce synergistic overgrowth, and RafACT is able to induce overgrowth as equally extensive as RasACT (Brumby, 2003).
Finally, it is noted that in mammalian systems, evidence exists for a role for Ras signaling in modulating cell junction complexes and enhancing epithelial to mesenchymal transitions, and in Drosophila also, constitutive RasACT signaling in clones alters cell affinities and changes the levels of E-cadherin and ß-catenin. Whether RasACT or NACT signaling destabilizes adherens junctions in Drosophila and this potentiates scrib- neoplastic overgrowth or whether alterations in the structure of the adherens junction resulting from the absence of Scrib alters a cells response to constitutive activation of these oncogenes are important future questions (Brumby, 2003).
This study has described a novel multi-hit model of tumorigenesis in Drosophila. Furthermore, although it has been suspected that disruptions to cell polarity could potentiate tumor progression and metastasis, this work demonstrates for the first time how the oncogenic effects of activated Ras and Notch are unleashed in the absence of epithelial polarity regulators. It is predicted that in mammals also, defects in apical-basal polarity could cooperate with oncogenes during neoplastic development. This approach in Drosophila can now be used to screen for novel oncogenes that, when specifically overexpressed in scrib- clones, are capable of inducing cooperative tumorigenesis, and can also be extended to identify cooperative interactions between other tumor suppressors and oncogenes within a whole animal context (Brumby, 2003).
Human tumours have a large degree of cellular and genetic heterogeneity. Complex cell interactions in the tumour and its microenvironment are thought to have an important role in tumorigenesis and cancer progression. Furthermore, cooperation between oncogenic genetic lesions is required for tumour development; however, it is not known how cell interactions contribute to oncogenic cooperation. The genetic techniques available in the fruitfly Drosophila melanogaster allow analysis of the behaviour of cells with distinct mutations, making this the ideal model organism with which to study cell interactions and oncogenic cooperation. In Drosophila eye-antennal discs, cooperation between the oncogenic protein RasV12 and loss-of-function mutations in the conserved tumour suppressor scribbled (scrib) gives rise to metastatic tumours that display many characteristics observed in human cancers. This study shows that clones of cells bearing different mutations can cooperate to promote tumour growth and invasion in Drosophila. The RasV12 and scrib- mutations can also cause tumours when they affect different adjacent epithelial cells. This interaction between RasV12 and scrib- clones involves JNK signalling propagation and JNK-induced upregulation of JAK/STAT-activating cytokines, a compensatory growth mechanism for tissue homeostasis. The development of RasV12 tumours can also be triggered by tissue damage, a stress condition that activates JNK signalling. Given the conservation of the pathways examined in this study, similar cooperative mechanisms could have a role in the development of human cancers (Wu, 2010).
Clones of mutant cells marked with green fluorescent protein (GFP) can be generated in the eye-antennal imaginal discs of Drosophila larvae by mitotic recombination. Clones expressing RasV12, an oncogenic form of the Drosophila Ras85D protein, moderately overgrow. Clones mutant for scrib lose apico-basal polarity and die. In contrast, scrib clones simultaneously expressing RasV12 grow into large metastatic tumours. To understand better the cooperation between these two mutations, animals were produced in which cell division after a mitotic recombination event creates two daughter cells: one expressing RasV12 and the other mutant for scrib. Discs containing adjacent RasV12 (GFP-positive) and scrib- clones developed into large tumours, capable of invading the ventral nerve cord. This shows that RasV12 and scrib also cooperate for tumour induction when they occur in different cells. These tumours are referred to as RasV12//scrib- tumours, to denote interclonal oncogenic cooperation and distinguish them from RasV12scrib- tumours, in which cooperation occurs in the same cells intraclonally (Wu, 2010).
This study has used Drosophila to investigate how oncogenic cooperation between different cells can promote tumour growth and invasion. These experiments, addressed to understanding interclonal cooperation in RasV12//scrib- tumours, uncovered a two-tier mechanism by which scrib- cells promote neoplastic development of RasV12 cells: (1) propagation of stress-induced JNK activity from scrib- cells to RasV12 cells; and (2) expression of the JAK/STAT-activating Unpaired cytokines downstream of JNK. These findings, therefore, highlight the importance of cell interactions in oncogenic cooperation and tumour development. It was also shown that stress-induced JNK signalling and epigenetic factors such as tissue damage can contribute to tumour development in flies. Notably, tissue damage caused by conditions such as chronic inflammation has been linked to tumorigenesis in humans. Furthermore, expression of the Unpaired cytokines promotes tumour growth as well as an antitumoural immune response, which parallels the situation in mice and humans. Future research into phenomena such as compensatory growth and interclonal cooperation in Drosophila will provide valuable insights into the biology of cancer (Wu, 2010).
This study defines TF network that triggers an abnormal gene expression program promoting malignancy of clonal tumors, generated in Drosophila imaginal disc epithelium by gain of oncogenic Ras (RasV12) and loss of the tumor suppressor Scribble (scrib1). Malignant transformation of the rasV12scrib1 tumors requires TFs of distinct families, namely the bZIP protein Fos, the ETS-domain factor Ets21c and the nuclear receptor Ftz-F1, all acting downstream of Jun-N-terminal kinase (JNK). Depleting any of the three TFs improves viability of tumor-bearing larvae, and this positive effect can be enhanced further by their combined removal. Although both Fos and Ftz-F1 synergistically contribute to rasV12scrib1 tumor invasiveness, only Fos is required for JNK-induced differentiation defects and Matrix metalloprotease (MMP1) upregulation. In contrast, the Fos-dimerizing partner Jun is dispensable for JNK to exert its effects in rasV12scrib1 tumors. Interestingly, Ets21c and Ftz-F1 are transcriptionally induced in these tumors in a JNK- and Fos-dependent manner, thereby demonstrating a hierarchy within the tripartite TF network, with Fos acting as the most upstream JNK effector. Of the three TFs, only Ets21c can efficiently substitute for loss of polarity and cooperate with Ras(V12) in inducing malignant clones that, like rasV12scrib1 tumors, invade other tissues and overexpress MMP1 and the Drosophila insulin-like peptide 8 (Dilp8). While rasV12ets21c tumors require JNK for invasiveness, the JNK activity is dispensable for their growth. In conclusion, this study delineates both unique and overlapping functions of distinct TFs that cooperatively promote aberrant expression of target genes, leading to malignant tumor phenotypes.
(Kulshammer, 2015).
Genome-wide transcriptome profiling in the Drosophila epithelial tumor model has generated a comprehensive view of gene expression changes induced by defined oncogenic lesions that cause tumors of an increasing degree of malignancy. These data allowed discovery of how a network of collaborating transcription factors confers malignancy to RasV12scrib1 tumors (Kulshammer, 2015).
This study revealed that the response of transformed RasV12scrib1 epithelial cells is more complex in comparison to those with activated RasV12 alone with respect to both the scope and the magnitude of expression of deregulated genes.
Aberrant expression of more than half of the genes in RasV12scrib1 tumors requires JNK activity, highlighting the significance of JNK signaling in malignancy. Importantly, the tumor-associated, JNK-dependent transcripts cluster with biological functions and processes that tightly match the phenotypes of previously described tumor stages. Furthermore, the RasV12scrib1 transcriptome showed significant overlap (27% upregulated and 15% downregulated genes) with microarray data derived from mosaic EAD in which tumors were induced by overexpressing the BTB-zinc finger TF Abrupt (Ab) in scrib1 mutant clones as well as with a transcriptome of scrib1 mutant wing discs. It is proposed that 429 misregulated transcripts (e.g. cher, dilp8, ets21c, ftz-f1, mmp1, upd), shared among all the three data sets irrespective of epithelial type (EAD versus wing disc) or cooperating lesion (RasV12 or Ab), represent a 'polarity response transcriptional signature' that characterizes the response of epithelia to tumorigenic polarity loss. Genome-wide profiling and comparative transcriptome analyses thus provide a foundation to identify novel candidates that drive and/or contribute to tumor development and malignancy while unraveling their connection to loss of polarity and JNK signaling (Kulshammer, 2015).
In agreement with a notion of combinatorial control of gene expression by an interplay among multiple TFs, this study identified overrepresentation of cis-acting DNA elements for STAT, GATA, bHLH, ETS, BTB, bZIP factors and NRs in genes deregulated in RasV12scrib1 mosaic EAD, implying that transcriptome anomalies result from a cross-talk among TFs of different families. Many of the aberrantly expressed genes contained binding motifs for AP-1, Ets21c and Ftz-F1, indicating that these three TFs may regulate a common set of targets and thus cooperatively promote tumorigenesis. This is consistent with the occurrence of composite AP-1-NRRE (nuclear receptor response elements), ETS-NRRE and ETS-AP-1 DNA elements in the regulatory regions of numerous human cancer-related genes, such as genes for cytokines, MMPs (e.g., stromelysin, collagenase) and MMP inhibitors (e.g., TIMP) (Kulshammer, 2015).
Interestingly, Drosophila ets21c and ftz-f1 gene loci themselves contain AP-1 motifs and qualify as polarity response transcriptional signature transcripts. Indeed, this study has detected JNK- and Fos-dependent upregulation of ets21c and ftz-f1 mRNAs in RasV12scrib1 tumors. While JNK-mediated control of ftz-f1 transcription has not been reported previously, upregulation of ets21c in the current tumor model is consistent with JNK requirement for infection-induced expression of ets21c mRNA in Drosophila S2 cells and in vivo. Based on these data, it is proposed that Ftz-F1 and Ets21c are JNK-Fos-inducible TFs that together with AP-1 underlie combinatorial transcriptional regulation and orchestrate responses to cooperating oncogenes. Such an interplay between AP-1 and Ets21c is further supported by a recent discovery of physical interactions between Drosophila Ets21c and the AP-1 components Jun and Fos (Rhee, 2014). Whether regulatory interactions among AP-1, Ets21c and Ftz-F1 require their direct physical contact and/or the presence of composite DNA binding motifs of a particular arrangement to control the tumor-specific transcriptional program remains to be determined (Kulshammer, 2015).
Importantly, some of the corresponding DNA elements, namely AP-1 and STAT binding sites, have recently been found to be enriched in regions of chromatin that become increasingly accessible in RasV12scrib1 mosaic EAD relative to control. This demonstrates that comparative transcriptomics and open chromatin profiling using ATAC-seq and FAIRE-seq are suitable complementary approaches for mining the key regulatory TFs responsible for controlling complex in vivo processes, such as tumorigenesis (Kulshammer, 2015).
The prototypical form of AP-1 is a dimer comprising Jun and Fos proteins. In mammals, the Jun proteins occur as homo- or heterodimers, whereas the Fos proteins must interact with Jun in order to bind the AP-1 sites. In contrast to its mammalian orthologs, the Drosophila Fos protein has been shown to form a homodimer capable of binding to and activating transcription from an AP-1 element, at least in vitro (Kulshammer, 2015).
The role of individual AP-1 proteins in neoplastic transformation and their involvement in pathogenesis of human tumors remain somewhat elusive. While c-Jun, c-Fos and FosB efficiently transform mammalian cells in vitro, only c-Fos overexpression causes osteosarcoma formation, whereas c-Jun is required for development of chemically induced skin and liver tumors in mice. In contrast, JunB acts as a context-dependent tumor suppressor. Thus, cellular and genetic context as well as AP-1 dimer composition play essential roles in dictating the final outcome of AP-1 activity in tumors (Kulshammer, 2015).
This study shows that, similar to blocking JNK with its dominant-negative form, Bsk, removal of Fos inhibits ets21c and ftz-f1 upregulation, suppresses invasiveness, improves epithelial organization and differentiation within RasV12scrib1 tumors and allows larvae to pupate. Strikingly, depletion of Jun had no such tumor-suppressing effects. It is therefore concluded that in the malignant RasV12scrib1 tumors, Fos acts independently of Jun, either as a homodimer or in complex with another, yet unknown partner. A Jun-independent role for Fos is further supported by additional genetic evidence. Fos, but not Jun, is involved in patterning of the Drosophila endoderm and is required for expression of specific targets, e.g., misshapen (msn) and dopa decarboxylase (ddc), during wound healing. Future studies should establish whether the JNK-responsive genes containing AP-1 motifs, identified in this study, are indeed regulated by Fos without its 'canonical' partner (Kulshammer, 2015).
The current data identify Fos as a key mediator of JNK-induced MMP1 expression and differentiation defects in RasV12scrib1 tumors. Only Fos inhibition caused clear suppression of MMP1 levels and restoration of neurogenesis within clonal EAD tissue, thus mimicking effects of JNK inhibition. Improved differentiation and reduced invasiveness are, however, not sufficient for survival of animals to adulthood, because interfering with Fos function in RasV12scrib1 clones always resulted in pupal lethality (Kulshammer, 2015).
The systems approach of this paper, followed by genetic experiments, identified Ets21c and Ftz-F1 as being essential for RasV12scrib1-driven tumorigenesis. It was further shown that mutual cooperation of both of these TFs with Fos is required to unleash the full malignancy of RasV12scrib1 tumors (Kulshammer, 2015).
TFs of the ETS-domain family are key regulators of development and homeostasis in all metazoans, whereas their aberrant activity has been linked with cancer. ets21c encodes the single ortholog of human Friend leukemia insertion1 (FLI1) and ETS-related gene (ERG) that are commonly overexpressed or translocated in various tumor types. While FLI1 is considered pivotal to development of Ewing's sarcoma, ERG has been linked to leukemia and prostate cancer. As for Ftz-F1 orthologs, the human liver receptor homolog-1 (LRH-1) has been associated with colonic, gastric, breast and pancreatic cancer, whereas steroidogenic factor 1 (SF-1) has been implicated in prostate and testicular cancers and in adrenocortical carcinoma. However, the molecular mechanisms underlying oncogenic activities of either the ERG/FLI1 or the SF-1/LRH-1 proteins are not well understood (Kulshammer, 2015).
This study shows that removal of Ftz-F1 markedly suppressed invasiveness of RasV12scrib1 tumors, restoring the ability of tumor-bearing larvae to pupate. Additionally, and in contrast to Fos, Ftz-F1 inhibition also partly reduced tumor growth in the third-instar EAD and allowed emergence of adults with enlarged, rough eyes composed predominantly of non-clonal tissue. The reduced clonal growth coincided with downregulation of the well-established Yki target, expanded, implicating Ftz-F1 as a potential novel growth regulator acting on the Hpo/Yki pathway. It is further speculated that reduced viability of RasV12scrib1ftz-f1RNAi clones and induction of non-autonomous compensatory proliferation by apoptotic cells during the pupal stage could explain the enlargement of the adult eyes. The precise mechanism underlying compromised growth and invasiveness of RasV12scrib1ftz-f1RNAi tumors and improved survival of the host remains to be identified (Kulshammer, 2015).
In contrast, effects of Ets21cLONG knockdown in RasV12scrib1 tumors appeared moderate relative to the clear improvement conferred by either Fos or Ftz-F1 elimination. ets21cLONG RNAi neither reduced tumor mass nor suppressed invasiveness, and pupation was rescued only partly. However, unlike ftz-f1RNAi, ets21cLONG RNAi significantly reduced expression of dilp8 mRNA. Based on abundance of Ets21c binding motifs in the regulatory regions of tumor-associated genes and the normalized expression of >20% of those genes upon removal of Ets21c, it is further suggested that Ets21c acts in RasV12scrib1 tumors to fine-tune the tumor gene-expression signature (Kulshammer, 2015).
Dilp8 is known to be secreted by damaged, wounded or tumor-like tissues to delay the larval-to-pupal transition. This study has corroborated the role of JNK in stimulating dilp8 expression in RasV12scrib1 tumor tissue, and has further implicated Ets21c and Fos as novel regulators of dilp8 downstream of JNK. However, the data also show that elevated dilp8 transcription per se is not sufficient to delay metamorphosis. Unlike the permanent larvae bearing RasV12scrib1 tumors, those with RasV12scrib1ftz-f1RNAi tumors pupated despite the excessive dilp8 mRNA. Likewise, pupation was not blocked by high dilp8 levels in larvae bearing EAD clones overexpressing Abrupt. As Dilp8 secretion appears critical for its function, it is proposed that loss of Ftz-F1 might interfere with Dilp8 translation, post-translational processing or secretion (Kulshammer, 2015).
Consistent with the individual TFs having unique as well as overlapping functions in specifying properties of RasV12scrib1 tumors, knocking down pairwise combinations of the TFs had synergistic effects on tumor suppression compared with removal of single TF. This evidence supports the view that malignancy is driven by a network of cooperating TFs, and elimination of several tumor hallmarks dictated by this network is key to animal survival. An interplay between AP-1, ETS-domain TFs and NRs is vital for development. For example, the ETS-factor Pointed has been shown to cooperate with Jun to promote R7 photoreceptor formation in the Drosophila adult eye. In mosquitoes, synergistic activity of another ETS-factor, E74B, with the ecdysone receptor (EcR/USP) promotes vitellogenesis. It is thus proposed that tumors become malignant by hijacking the developmental mechanism of combinatorial control of gene activity by distinct TFs (Kulshammer, 2015).
Despite the minor impact of ets21cLONG knockdown on suppressing RasV12scrib1 tumors, Ets21cLONG is the only one of the tested TFs that was capable of substituting for loss of scrib in inducing malignant clonal overgrowth when overexpressed with oncogenic RasV12 in EAD. While invasiveness of such RasV12ets21cLONG tumors required JNK activity, JNK signaling appeared dispensable for tumor growth. Importantly, the overgrowth of RasV12ets21cLONG tumors was primarily independent of a prolonged larval stage, because dramatic tumor mass expansion was detected already on day 6 AEL. How cooperativity between Ets21cLONG and RasV12 ensures sufficient JNK activity and the nature of the downstream effectors driving tumor overgrowth remain to be determined. In contrast, co-expression of either Ftz-F1 or Fos with RasV12 resulted in a non-invasive, RasV12-like hyperplastic phenotype (Kulshammer, 2015).
Why does Ets21cLONG exert its oncogenic potential while Fos and Ftz-F1 do not? Simple overexpression of a TF may not be sufficient, because many TFs require activation by a post-translational modification (e.g., phosphorylation), interaction with a partner protein and/or binding of a specific ligand. Full activation of Fos in response to a range of stimuli is achieved through hyperphosphorylation by mitogen-activated protein kinases (MAPKs), including ERK and JNK. Indeed, overexpression of a FosN-Ala mutated form that cannot be phosphorylated by JNK was sufficient to phenocopy fos deficiency, indicating that Fos must be phosphorylated by JNK in order to exert its oncogenic function. Consistent with the current data, overexpression of FosN-Ala partly restored polarity of lgl mutant EAD cells. It is therefore conclude that the tumorigenic effect of Fos requires a certain level of JNK activation, which is lacking in EAD co-expressing Fos with RasV12. Nevertheless, the absence of an unknown Fos-interacting partner cannot be excluded (Kulshammer, 2015).
Interestingly, MAPK-mediated phosphorylation also greatly enhances the ability of SF-1 and ETS proteins to activate transcription. Two potential MAPK sites can be identified in the hinge region of Ftz-F1, although their functional significance is unknown. Whether Ets21c or Ftz-F1 requires phosphorylation and how this would impact their activity in the tumor context remains to be determined. Genetic experiments demonstrate that at least the overgrowth of RasV12ets21cLONG tumors does not require Ets21c phosphorylation by JNK (Kulshammer, 2015).
In addition, previous crystallography studies revealed the presence of phosphoinositides in the ligand binding pocket of LHR-1 and SF-1 and showed their requirement for the NR transcriptional activity. Although developmental functions of Drosophila Ftz-F1 seem to be ligand independent, it is still possible that Ftz-F1 activity in the tumor context is regulated by a specific ligand. An effect of Ftz-F1 SUMOylation cannot be ruled out (Kulshammer, 2015).
In summary, this work demonstrates that malignant transformation mediated by RasV12 and scrib loss depends on MAPK signaling and at least three TFs of different families, Fos, Ftz-F1 and Ets21c. While their coordinated action ensures precise transcriptional control during development, their aberrant transcriptional (Ets21c, Ftz-F1) and/or post-translational (Fos, Ftz-F1, Ets21c) regulation downstream of the cooperating oncogenes contributes to a full transformation state. The data implicate Fos as a primary nuclear effector of ectopic JNK activity downstream of disturbed polarity that controls ets21c and ftz-f1 expression. Through combinatorial interactions on overlapping sets of target genes and acting on unique promoters, Fos, Ftz-F1 and Ets21c dictate aberrant behavior of RasV12scrib1 tumors. Although originally described in Drosophila, detrimental effects of cooperation between loss of Scrib and oncogenic Ras has recently been demonstrated in mammalian tumor models of prostate and lung cancer. This study and further functional characterization of complex TF interactions in the accessible Drosophila model are therefore apt to provide important insight into processes that govern cancer development and progression in mammals (Kulshammer, 2015).
An emerging interest in oncology is to tailor treatment to particular cancer genotypes, i.e. oncogenic mutations present in the tumor, and not the tissue of cancer incidence. Integral to such a practice is the idea that the same oncogenic mutation(s) produces similar outcomes in different tissues. To test this idea experimentally, tumors were studied driven by a combination of RasV12 and scrib1 mutations in Drosophila larvae. Tumors induced in tissues of neural ectodermal and mesodermal origins were found to behave similarly in every manner examined: cell cycle checkpoints, apoptosis, cellular morphology, increased aneuploidy and response to Taxol. It is concluded that oncogenic effects override tissue-specific differences, at least for the mutations, tissues, and phenotypes in this study (Stickel, 2014).
Oncogenic stress provokes tumor suppression by p53 but the extent to which this regulatory axis is conserved remains unknown. Using a biosensor to visualize p53 action, this study found that Drosophila p53 is selectively active in gonadal stem cells after exposure to stressors that destabilize the genome. Similar p53 activity occurred in hyperplastic growths that were triggered either by the Ras(V12) oncoprotein or by failed differentiation programs. In a model of transient sterility, p53 was required for the recovery of fertility after stress, and entry into the cell cycle was delayed in p53(-) stem cells. Together, these observations establish that the stem cell compartment of the Drosophila germline is selectively licensed for stress-induced activation of the p53 regulatory network. Furthermore, the findings uncover ancestral links between p53 and aberrant proliferation that are independent of DNA breaks and predate evolution of the ARF/Mdm2 axis (Wylie, 2014).
Tumorigenesis is a complex process, which requires alterations in several tumor suppressor or oncogenes. This study used a Drosophila tumor model to identify genes, which are specifically required for tumor growth. Reduction of phosphoinositide 3-kinase (PI3K) activity was found to result in very small tumors while only slightly affecting growth of wild-type tissue. The observed inhibition on tumor growth occurred at the level of cell-cycle progression. It is concluded that tumor cells become dependent on PI3K function and that reduction of PI3K activity synthetically interferes with tumor growth. The results of this study broaden insights into the intricate mechanisms underling tumorigenesis and illustrate the power of Drosophila genetics in revealing weak points of tumor progression (Willecke, 2011).
This study employed a genetic approach to identify genes required for the neoplastic growth phenotype of RasV12, DlgRNAi tumors. It was found that RasV12, DlgRNAi tumors are highly sensitive to reductions of the PI3K pathway and that changes in PI3K activity block cell-cycle progression (Willecke, 2011).
In agreement with previous reports, this study found that RasV12 induces the PI3K pathway in Drosophila. Yet curiously, RasV12, DlgRNAi tumors exhibit low levels of PI3K signaling. A possible reason for the paradoxical result may be derived from the cell polarity defects of RasV12, DlgRNAi tumors, which are not induced when RasV12 is expressed in otherwise wild-type cells or in combination with Upd. This interpretation implies that the activation of PI3K through RasV12 depends on proper cell polarity. In support of this explanation, it has been reported that RasV12 activates the PIP3 reporter only at the apical side of cells. Additionally, studies have shown that PTEN directly binds to the polarity gene Bazooka and that the activity of the PI3K pathway is polarized in Drosophila oocyte cells. The activation of the PI3K pathway might, therefore, require the preservation of cell polarity, which could explain why RasV12 does not induce the PI3K pathway if Dlg is lost (Willecke, 2011).
Why are RasV12, DlgRNAi tumors sensitive to changes in PI3K signaling? RasV12, DlgRNAi tumor cells receive mitogenic signals from the JAK-STAT and MAPK pathways, which promote extensive tumor growth. RasV12, DlgRNAi tumors require a higher metabolic rate compared with wild-type cells, without gaining extra activation of PI3K through RasV12. As a result, PI3K signaling might be absolutely limiting for the growth of RasV12, DlgRNAi tumors. Expression of PI3KRNAi then causes PI3K activity to drop below the threshold for such cells, triggering a block in cell-cycle progression. Tumors which express RasV12 together with Upd are not sensitive to changes in PI3K levels even though they overgrow as much as RasV12, DlgRNAi tumors. An pAkt western blot shows, however, that these tumors have high levels of PI3K signaling. Expression of PI3KRNAi in this background might not cause PI3K activity to drop below a threshold for cell-cycle progression (Willecke, 2011).
Compounds that block PI3K pathway activity are known to be potent inhibitors of mammalian tumor growth and inhibitors that target PI3K, and other members of the pathway are currently being tested in clinical trials. Preclinical and clinical trials focus mainly on tumors that carry mutations in PI3K pathway components or that display abnormal levels of the biomarkers pAKT and PS6k1. The current results are, however, an example for a case where tumors with no genetic alterations in PI3K signaling components are also highly susceptible to reduction of PI3K levels. Understanding the molecular networks that create such PI3K dependency is a central topic in cancer research as it is highly relevant to identify PI3K-dependent tumors to predict the potential effectiveness of PI3K inhibitors. Genetic studies in Drosophila may therefore complement mammalian studies to more precisely determine which tumor-initiating pathways create PI3K sensitivity (Willecke, 2011).
In conclusion, this study has uncovered a synthetic interaction between the Drosophila PI3K signaling and RasV12, DlgRNAi tumor-initiating pathways. The results provide insights into the complex mechanisms underlying tumorigenesis and illustrate the power of Drosophila genetics in revealing the vulnerabilities of tumors. Identification of additional synthetic interactions through genetic screening in Drosophila may serve as a valuable resource for identifying potential drug targets in cancer therapy (Willecke, 2011).
Loss of the cell polarity gene could cooperate with oncogenic Ras to drive tumor growth and invasion, which critically depends on the c-Jun N-terminal Kinase (JNK) signaling pathway in Drosophila. By performing a genetic screen, this study identified Src42A, the ortholog of mammalian Src, as a key modulator of both RasV12/lgl -/-triggered tumor invasion and loss of cell polarity gene-induced cell migration. The genetic study further demonstrated that the Bendless (Ben)/dUev1a ubiquitin E2 complex is an essential regulator of Src42A-induced, JNK-mediated cell migration. Furthermore, this study showed that ectopic Ben/dUev1a expression induced invasive cell migration along with increased MMP1 production in wing disc epithelia. Moreover, Ben/dUev1a could cooperate with RasV12 to promote tumor overgrowth and invasion. In addition, it was found that the Ben/dUev1a complex is required for ectopic Src42A-triggered cell death and endogenous Src42A-dependent thorax closure. These data not only provide a mechanistic insight into the role of Src in development and disease but also propose a potential oncogenic function for Ubc13 and Uev1a, the mammalian homologs of Ben and dUev1a (Ma, 2013).
During tumorigenesis, pathways that promote the epithelial-to-mesenchymal transition (EMT) can both facilitate metastasis and endow tumor cells with cancer stem cell properties. To gain a greater understanding of how these properties are interlinked in cancers, Drosophila epithelial tumor models were used, that are driven by orthologues of human oncogenes (activated alleles of Ras and Notch) in cooperation with the loss of the cell polarity regulator, scribbled (scrib). Within these tumors, both invasive, mesenchymal-like cell morphology and continual tumor overgrowth, are dependent upon Jun N-terminal kinase (JNK) activity. To identify JNK-dependent changes within the tumors a comparative microarray analysis was used to define a JNK gene signature common to both Ras and Notch-driven tumors. Amongst the JNK-dependent changes was a significant enrichment for BTB-Zinc Finger (ZF) domain genes, including chronologically inappropriate morphogenesis (chinmo). chinmo was upregulated by JNK within the tumors, and overexpression of chinmo with either RasV12 or Nintra was sufficient to promote JNK-independent epithelial tumor formation in the eye/antennal disc, and, in cooperation with RasV12, promote tumor formation in the adult midgut epithelium. Chinmo primes cells for oncogene-mediated transformation through blocking differentiation in the eye disc, and promoting an escargot-expressing stem or enteroblast cell state in the adult midgut. BTB-ZF genes are also required for Ras and Notch-driven overgrowth of scrib mutant tissue, since, although loss of chinmo alone did not significantly impede tumor development, when loss of chinmo was combined with loss of a functionally related BTB-ZF gene, abrupt, tumor overgrowth was significantly reduced. abrupt is not a JNK-induced gene, however, Abrupt is present in JNK-positive tumor cells, consistent with a JNK-associated oncogenic role. As some mammalian BTB-ZF proteins are also highly oncogenic, this work suggests that EMT-promoting signals in human cancers could similarly utilize networks of these proteins to promote cancer stem cell states (Doggett, 2015).
This report has defined the transcriptional changes induced by JNK signaling within both scrib>RasACT and scrib>NACT tumors by carrying out comparative microarray expression arrays. This analysis that JNK exerts a profound effect upon the transcriptional profile of both Ras and Notch-driven tumor types. The expression of nearly 1000 genes was altered by the expression of bskDN in either Ras or Notch-driven tumors, and less than half of these changes were shared between the two tumor types, indicating that JNK signaling elicits unique tumorigenic expression profiles depending upon the cooperating oncogenic signal. Nevertheless, of the 399 JNK-regulated probe sets shared between Ras and Notch-driven tumors, it is hypothesized that these had the potential to provide key insights into JNK's oncogenic activity, and to prioritize these targets, it was considered that the expression of the critical oncogenic regulators would not just be altered by bskDN, but would be normalized to close to wild type levels. This subset of the 399 probe set was identified by comparing the expression profile of each genotype back to control tissue, thereby producing a more focussed JNK signature of 103 genes. Notably, this included previously characterized targets of JNK in the tumors, such as Mmp1,cherand Pax, thereby providing validation of the approach. Also amongst these candidates were 4 BTB-ZF genes; two of which were upregulated by JNK in the tumors (chinmo and fru), and two downregulated (br and ttk) (Doggett, 2015).
Focussing upon chinmo, chinmo overexpression was shown to be sufficient to prime epithelial cells for cooperation with RasACT in both the eye antennal disc and in the adult midgut epithelium, and that chinmo is required for cooperative RasACTor NACT-driven tumor overgrowth, although its function was only exposed when its knockdown was combined with knockdown of a functionally similar BTB-ZF transcription factor, abrupt. This family of proteins is highly oncogenic in Drosophila, since previous work has shown that ab overexpression can cooperate with loss of scrib to promote neoplastic overgrowth, and in these studies, it was also shown that overexpression of a fru isoform normally expressed in the eye disc is capable of promoting cooperation with RasACT and NACT in the eye-antennal disc, in a similar manner to chinmo overexpression. Thus, whether fru also plays a role in driving Ras or Notch-driven tumorigenesis warrants further investigation. Indeed, a deeper understanding of the oncogenic activity of these genes is likely to be highly relevant to human tumors, since of the over 40 human BTB-ZF family members, many are implicated in both haematopoietic and epithelial cancers, functioning as either oncogenes (eg., Bcl6, BTB7) or tumor suppressors (eg., PLZF, HIC1). Furthermore, over-expression of BTB7, can also cooperate with activated Ras in transforming primary cells, and its loss makes MEFs refractory to transformation by various key oncogenes such as Myc, H-rasV12 and T-Ag, suggesting that cooperating mechanisms between BTB-ZF proteins and additional oncogenic stimuli might be conserved (Doggett, 2015).
JNK signaling in Drosophila tumors is known to promote tumor overgrowth through both the STAT and Hippo pathways. Deregulation of the STAT pathway was evident in the arrays through the upregulation of Upd ligands by JNK in both Ras and Notch-driven tumors. In contrast, although cher was identified in the arrays as being upregulated in both tumor types and previous studies have shown that cher is partly required for the deregulation of the Hippo pathway in scrib>RasACT tumors, more direct evidence for Hippo pathway deregulation amongst the JNK signature genes was lacking. In part, this could be due to JNK regulating the pathway through post-transcriptional mechanisms involving direct phosphorylation of pathway components. However, the failure to identify known Hippo pathway target genes, and proliferation response genes in general, may simply highlight limitations in the sensitivity of the array assay and the cut-offs used for determining significance, despite its obvious success in correctly identifying many known JNK targets (Doggett, 2015).
Whether tumor overgrowth through STAT and Yki activity is somehow associated with a stem cell or progenitor-like state remains uncertain. Although imaginal discs exhibit developmental plasticity and regeneration potential, and JNK signaling is required for both of these stem-like properties, there is no positive evidence for the existence of a population of asymmetrically dividing stem cells within imaginal discs. Instead, symmetrical divisions of progenitor cells may be the means by which imaginal discs can rapidly generate enough cells to form the differentiated structures of the adult fly. To date, progenitor cells have only been characterized in the eye disc neuroepithelium. These cells have a pseudostratified columnar epithelial morphology and express the MEIS family transcription factor, Hth, which is downregulated as cells initiate differentiation and begin expressing Dac and Eya. Interestingly, they also require Yki for their proliferation, and can be induced to overproliferate in response to increased STAT activity. However, analysis of cell fate markers indicated that tumor overgrowth was not likley to be solely due to the overproliferation of these undifferentiated progenitor cells. Although scrib>RasACT/NACT tumors, were characterized by the failure to transition to Dac/Eya expression in the eye disc, blocking JNK in scrib > RasACT/NACT tumors did not restore tumor cell differentiation, despite overgrowth being curtailed, and Hth expression was not maintained in the tumors in a JNK-dependent manner. Nevertheless, a JNK-induced gene such as chinmo is likely to be associated with promoting a progenitor-like state, since it is a potential STAT target gene required for adult eye development that is expressed in eye disc progenitor cells in response to increased Upd activity and its overexpression alone is sufficient to block Dac/Eya expression. Furthermore, chinmo is also required for cyst stem cell maintenance in the Drosophila testis, and the current work has shown that chinmo overexpression promotes increased numbers of esgGFP expressing stem cells or enteroblasts in the adult midgut. As the BTB-ZF protein Ab is also highly oncogenic and expressed in the eye disc progenitor cells, it is hypothesize that the JNK-induced expression ofchinmo in scrib>RasACT/NACT tumors could cooperate with Ab to maintain a progenitor-like cell state in the eye disc, and that this is required for scrib->RasACT/NACT tumor overgrowth. However, although Ab was expressed in chinmo-expressing, JNK positive tumor cells, Ab does not appear to be a JNK-induced gene. What JNK-independent mechanisms control ab expression will therefore require further analysis (Doggett, 2015).
Interestingly, previous studies have observed that ab overexpression in eye disc clones upregulates chinmo expression and although the effect of chinmo expression upon ab is yet to be described, the data at least suggest that the control of their expression is interlinked in a yet to be defined manner (Doggett, 2015).
Consistent with Chinmo being important for scrib->RasACT/NACTv tumor overgrowth, chinmo overexpression itself is also highly oncogenic. Over-expression of chinmo with RasACT or NACT drives tumorigenesis in the eye-antennal disc, and also resulted in enlarged brain lobes, presumably due to the generation of overexpressing clones within the neuroepithelium of the optic lobes. In the adult midgut, the overexpression of chinmo with RasACT in the stem cell and its immediate progeny, the enteroblast, promoted massive tumor overgrowth, resulting in esgGFP expressing cells completely filling the lumen of the gut, and eventual host lethality. The luminal filling of esgGFP cells is reminiscent of the effects of RasACT expression in larval adult midgut progenitor cells. Together with the data linking Chinmo function to stem or progenitor cells, these data reinforce the idea that epithelial tumorigenesis can be primed by signals, such as chinmo over-expression, that promote a stem or progenitor cell state (Doggett, 2015).
The function of some Drosophila BTB-ZF proteins including Chinmo and Ab, has also been linked to heterochronic roles involving the conserved let-7 miRNA pathway and hormone signals, to regulate the timing of differentiation. Indeed, Ab can directly bind to the steroid hormone receptor co-activator Taiman (Tai or AIB1/SRC3 in humans), to represses the transcriptional response to ecdysone signaling. Thus, the capacity of BTB-ZF proteins to influence the timing of developmental transitions, particularly if they impede developmental transitions within stem or progenitor cells, could help account for their potent oncogenic activity. Indeed, ecdysone-response genes were repressed by JNK in the tumorigenic state, consistent with the failure of the larvae to pupate and a delay in developmental timing. Whether repressing the ecdysone response cell autonomously might contribute to tumor overgrowth and/or invasion will be an interesting area of future investigation, given the complex role of hormone signaling in mammalian stem cell biology and cancers (Doggett, 2015).
Previous studies have suggested that JNK-dependent tumor cell invasion is developmentally similar to the JNK-induced EMT-like events occurring during imaginal disc eversion. Thus the capacity of JNK to also promote tumor overgrowth is reminiscent of how EMT inducers such as Twist (Twi) and Snail (Sna) are associated with the acquisition of cancer stem cell properties. In Drosophila, however, twi and snawere not induced by JNK in the tumors, although transcription factors involved in mesoderm specification, including the NF-kappaB homologue, dl (a member of the 103 JNK signature), and Mef2 (a member of the 399 JNK signature), were amongst the up-regulated JNK targets. Mesoderm specification is not necessarily associated with a mesenchymal-like cell morphology, however,
dl is involved in the induction of EMT during embryonic development, and both dl and Mef2 act with Twi and Sna to coordinate mesoderm formation. Interestingly, recent studies have identified dl in an overexpression screen for genes capable of cooperating with scrib > in Drosophila tumorigenesis, and Mef2 has been identified as a cooperating oncogene in Drosophila, and possibly also in humans, where a correlation exists between the expression of Notch and Mef2 paralogues in human breast tumor samples. It is therefore possible that dl and Mef2 either act in combination with Twi or Sna, or independently of them but in a similar oncogenic capacity, to promote a mesodermal cell fate in scrib > RasACT/NACT tumors. The potential relevance of this to the mesenchymal cell morphology associated with tumor cell invasion, as well as the acquisition of progenitor states is worthy of further investigation (Doggett, 2015).
In mef2-driven tumors both overgrowth and invasion depend upon activation of JNK signaling, suggesting that Mef2 is not capable of promoting invasive capabilities independent of JNK. In contrast, chinmo+RasACT/NACT tumors appeared non-invasive and retained epithelial morphology despite the massive overgrowth, although closer examination of cell polarity markers will be required to confirm this. Furthermore, the overgrowth of chinmo+RasACT/NACT tumors was not dependent upon JNK signaling, suggesting that the maintenance of a progenitor-like state could be uncoupled from JNK-induced EMT-effectors associated with invasion. Whether clear divisions between mesenchymal behaviour and progenitor states in tumors can be clearly separated in this manner is not yet clear, however, overall, it is likely that multiple JNK-regulated genes will participate in both promoting tumor overgrowth as well as migration/invasion. Although this study used the 103 JNK signature as a means to focus upon potential key candidates, an analysis of the 399 JNK-regulated probe sets common to both Ras and Notch-driven tumours has the potential to provide deeper insights into the multiple effectors of JNK signaling during tumorigenesis. Whilst the individual role of these genes can be probed with knockdowns, the complexity of the response, potentially with multiple redundancies and cross-talk, will ultimately need a network level of understanding to more fully expose key nodes participating in overgrowth and invasion. This approach has considerable potential to further expose core principles and mechanisms that drive human tumorigenesis, since it is clear that many fundamental commonalities underlie the development of tumors in Drosophila and mammals (Doggett, 2015).
Although developmental signalling pathways control tumourigenic growth, the cellular mechanisms that abnormally proliferating cells rely on are still largely unknown. Drosophila melanogaster is a genetically tractable model that is used to study how specific genetic changes confer advantageous tumourigenic traits. Despite recent efforts, the role of deubiquitylating enzymes in cancer is particularly understudied. This study performed a Drosophila in vivo RNAi screen to identify deubiquitylating enzymes that modulate Ras(V12)-induced hyperplastic growth. The spliceosome core component Prp8 was identified as a crucial regulator of Ras-, EGFR-, Notch- or RET-driven hyperplasia. Loss of prp8 function alone decreased cell proliferation, increased cell death, and affected cell differentiation and polarity. In hyperplasia, Prp8 supported tissue overgrowth independently of caspase-dependent cell death. The depletion of prp8 efficiently blocked Ras-, EGFR- and Notch-driven tumours but, in contrast, enhanced tumours that were driven by oncogenic RET, suggesting a context-specific role in hyperplasia. These data show, for the first time, that Prp8 regulates hyperplasia, and extend recent observations on the potential role of the spliceosome in cancer. These findings suggest that targeting Prp8 could be beneficial in specific tumour types (Fernandez-Espartero, 2018).
Several oncogenes induce untimely entry into S phase and alter replication timing and progression, thereby generating replicative stress, a well-known source of genomic instability and a hallmark of cancer. Using an epithelial model in Drosophila, this study shows that the RAS oncogene, which triggers G1/S transition, induces DNA damage and, at the same time, silences the DNA damage response pathway. RAS compromises ATR-mediated phosphorylation of the histone variant H2Av and ATR-mediated cell-cycle arrest in G2 and blocks, through ERK, Dp53-dependent induction of cell death. ERK is also activated in normal tissues by an exogenous source of damage, and this activation is necessary to dampen the pro-apoptotic role of Dp53. This study exploits the pro-survival role of ERK activation upon endogenous and exogenous sources of DNA damage to present evidence that its genetic or chemical inhibition can be used as a therapeutic opportunity to selectively eliminate RAS-malignant tissues (Murcia, 2019).
Eukaryotic elongation factor 2 (eEF2) undergoes a unique post-translational modification called diphthamidation. Although eEF2 diphthamidation is highly conserved, its pathophysiological function is still largely unknown. To elucidate the function of diphthamidation in tumor, this study examined the involvement of diphthamidation pathway enzyme Dph5 in tumor progression in Drosophila adult gut. Expression of oncogenic Ras(V12) in gut intestinal stem cells (ISCs) and enteroblasts (EBs) causes hypertrophy and disruption of gut epithelia, and shortened lifespan. Knockdown of Dph5 ameliorated these pathogenic phenotypes. Dph5 is required for gross translation activation and high dMyc protein level in Ras(V12) tumor-like hyperplasia. Transcriptome analysis revealed that Dph5 is involved in the regulation of ribosome biogenesis genes. These results suggest that diphthamidation is required for translation activation partly through the regulation of ribosome biogenesis in Ras-induced tumor-like hyperplasia model in Drosophila gut (Tsuda-Sakurai, 2019).
Ras85D:
Biological Overview
| Evolutionary Homologs
| Regulation
| Protein Interactions
| Effects of Mutation
| Ras as Oncogene
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