PLA2G12A as a Novel Biomarker for Colorectal Cancer with Prognostic Relevance.

Metastasis is the leading cause of colorectal cancer (CRC)-related deaths. Therefore, the identification of accurate biomarkers predictive of metastasis is needed to better stratify high-risk patients to provide preferred management and reduce mortality. In this study, we identified 13 new genes that modified circulating tumor cell numbers using a genome-wide genetic screen in a whole animal CRC model. Candidate genes were subsequently evaluated at the gene expression level in both an internal human CRC cohort of 153 patients and an independent cohort from the TCGA including 592 patients. Interestingly, the expression of one candidate, PLA2G12A, significantly correlated with both the time to recurrence and overall survival in our CRC cohort, with its low expression being an indicator of a poor clinical outcome. By examining the TCGA cohort, we also found that low expression of PLA2G12A was significantly enriched in epithelial-mesenchymal transition signatures. Finally, the candidate functionality was validated in vitro using three different colon cancer cell lines, revealing that PLA2G12A deficiency increases cell proliferation, migration, and invasion. Overall, our study identifies PLA2G12A as a prognostic biomarker of early-stage CRC, providing evidence that its deficiency promotes tumor growth and dissemination.

Sensitive High-Throughput Assays for Tumour Burden Reveal the Response of a Drosophila melanogaster Model of Colorectal Cancer to Standard Chemotherapies.

Drosophila melanogaster (Drosophila) models of cancer are emerging as powerful tools to investigate the basic mechanisms underlying tumour progression and identify novel therapeutics. Rapid and inexpensive, it is possible to carry out genetic and drug screens at a far larger scale than in vertebrate organisms. Such whole-organism-based drug screens permits assessment of drug absorption and toxicity, reducing the possibility of false positives. Activating mutations in the Wnt and Ras signalling pathways are common in many epithelial cancers, and when driven in the adult Drosophila midgut, it induces aggressive intestinal tumour-like outgrowths that recapitulate many aspects of human colorectal cancer (CRC). Here we have taken a Drosophila CRC model in which tumourous cells are marked with both GFP and luciferase reporter genes, and developed novel high-throughput assays for quantifying tumour burden. Leveraging these assays, we find that the Drosophila CRC model responds rapidly to treatment with standard CRC-drugs, opening the door to future rapid genetic and drug screens.

Deciphering the genetic programme triggering timely and spatially-regulated chitin deposition.

Organ and tissue formation requires a finely tuned temporal and spatial regulation of differentiation programmes. This is necessary to balance sufficient plasticity to undergo morphogenesis with the acquisition of the mature traits needed for physiological activity. Here we addressed this issue by analysing the deposition of the chitinous extracellular matrix of Drosophila, an essential element of the cuticle (skin) and respiratory system (tracheae) in this insect. Chitin deposition requires the activity of the chitin synthase Krotzkopf verkehrt (Kkv). Our data demonstrate that this process equally requires the activity of two other genes, namely expansion (exp) and rebuf (reb). We found that Exp and Reb have interchangeable functions, and in their absence no chitin is produced, in spite of the presence of Kkv. Conversely, when Kkv and Exp/Reb are co-expressed in the ectoderm, they promote chitin deposition, even in tissues normally devoid of this polysaccharide. Therefore, our results indicate that both functions are not only required but also sufficient to trigger chitin accumulation. We show that this mechanism is highly regulated in time and space, ensuring chitin accumulation in the correct tissues and developmental stages. Accordingly, we observed that unregulated chitin deposition disturbs morphogenesis, thus highlighting the need for tight regulation of this process. In summary, here we identify the genetic programme that triggers the timely and spatially regulated deposition of chitin and thus provide new insights into the extracellular matrix maturation required for physiological activity.

Iro/IRX transcription factors negatively regulate Dpp/TGF-ß pathway activity during intestinal tumorigenesis.

Activating mutations in Wnt and EGFR/Ras signaling pathways are common in colorectal cancer (CRC). Remarkably, clonal co-activation of these pathways in the adult Drosophila midgut induces "tumor-like" overgrowths. Here, we show that, in these clones and in CRC cell lines, Dpp/TGF-ß acts as a tumor suppressor. Moreover, we discover that the Iroquois/IRX-family-protein Mirror downregulates the transcription of core components of the Dpp pathway, reducing its tumor suppressor activity. We also show that this genetic interaction is conserved in human CRC cells, where the Iro/IRX proteins IRX3 and IRX5 diminish the response to TGF-ß. IRX3 and IRX5 are upregulated in human adenomas, and their levels correlate inversely with the gene expression signature of response to TGF-ß. In addition, Irx5 expression confers a growth advantage in the presence of TGF-ß, but is selected against in its absence. Together, our results identify a set of Iro/IRX proteins as conserved negative regulators of Dpp/TGF-ß activity. We propose that during the characteristic adenoma-to-carcinoma transition of human CRC, the activity of IRX proteins could reduce the sensitivity to the cytostatic effect of TGF-ß, conferring a growth advantage to tumor cells prior to the acquisition of mutations in TGF-ß pathway components.

Multiple regulatory safeguards confine the expression of the GATA factor Serpent to the hemocyte primordium within the Drosophila mesoderm.

serpent (srp) encodes a GATA-factor that controls various aspects of embryogenesis in Drosophila, such as fatbody development, gut differentiation and hematopoiesis. During hematopoiesis, srp expression is required in the embryonic head mesoderm and the larval lymph gland, the two known hematopoietic tissues of Drosophila, to obtain mature hemocytes. srp expression in the hemocyte primordium is known to depend on snail and buttonhead, but the regulatory complexity that defines the primordium has not been addressed yet. Here, we find that srp is sufficient to transform trunk mesoderm into hemocytes. We identify two disjoint cis-regulatory modules that direct the early expression in the hemocyte primordium and the late expression in mature hemocytes and lymph gland, respectively. During embryonic hematopoiesis, a combination of snail, buttonhead, empty spiracles and even-skipped confines the mesodermal srp expression to the head region. This restriction to the head mesoderm is crucial as ectopic srp in mesodermal precursors interferes with the development of mesodermal derivates and promotes hemocytes and fatbody development. Thus, several genes work in a combined fashion to restrain early srp expression to the head mesoderm in order to prevent expansion of the hemocyte primordium.

Modelling Cancer Metastasis in Drosophila melanogaster.

Cancer metastasis, the process by which tumour cells spread throughout the body and form secondary tumours at distant sites, is the leading cause of cancer-related deaths. The metastatic cascade is a highly complex process encompassing initial dissemination from the primary tumour, travel through the blood stream or lymphatic system, and the colonisation of distant organs. However, the factors enabling cells to survive this stressful process and adapt to new microenvironments are not fully characterised. Drosophila have proven a powerful system in which to study this process, despite important caveats such as their open circulatory system and lack of adaptive immune system. Historically, larvae have been used to model cancer due to the presence of pools of proliferating cells in which tumours can be induced, and transplanting these larval tumours into adult hosts has enabled tumour growth to be monitored over longer periods. More recently, thanks largely to the discovery that there are stem cells in the adult midgut, adult models have been developed. We focus this review on the development of different Drosophila models of metastasis and how they have contributed to our understanding of important factors determining metastatic potential, including signalling pathways, the immune system and the microenvironment.

Methods to Generate and Assay for Distinct Stages of Cancer Metastasis in Adult Drosophila melanogaster.

Metastasis underlies the majority of cancer-related deaths. Until recently, research on this complex multi-step process has been hindered by a lack of genetically tractable experimental models amenable to high-throughput analyses. This was recently overcome with the development of a model of metastatic colorectal cancer (CRC) in adult flies, which relies on the activation of a partial-epithelial-to-mesenchymal transition (EMT) in intestinal tumors. In this model, tumor cells are labeled with both GFP and luciferase reporters, enabling high-throughput analyses. We report here the detailed protocol for generating the model, and assaying for primary tumor burden and distinct stages of metastasis, including the number of circulating tumor cells and secondary metastases.

Reading the Hedgehog morphogen gradient by measuring the ratio of bound to unbound Patched protein.

Morphogens are 'form-generating' substances that spread from localized sites of production and specify distinct cellular outcomes at different concentrations. A cell's perception of morphogen concentration is thought to be determined by the number of active receptors, with inactive receptors making little if any contribution. Patched (Ptc), the receptor for the morphogen Hedgehog (Hh), is active in the absence of ligand and blocks the expression of target genes by inhibiting Smoothened (Smo), an essential transducer of the Hh signal. Hh binding to Ptc abrogates the ability of Ptc to inhibit Smo, thereby unleashing Smo activity and inducing target gene expression. Here, we show that a cell's measure of ambient Hh concentration is not determined solely by the number of active (unliganded) Ptc molecules. Instead, we find that Hh-bound Ptc can titrate the inhibitory action of unbound Ptc. Furthermore, we demonstrate that this effect is sufficient to allow normal reading of the Hh gradient in the presence of a form of Ptc that cannot bind the ligand but retains its ability to inhibit Smo. These results support a model in which the ratio of bound to unbound Ptc molecules determines the cellular response to Hh.

Collective cell migration and metastases induced by an epithelial-to-mesenchymal transition in Drosophila intestinal tumors.

Metastasis underlies the majority of cancer-related deaths yet remains poorly understood due, in part, to the lack of models in vivo. Here we show that expression of the EMT master inducer Snail in primary adult Drosophila intestinal tumors leads to the dissemination of tumor cells and formation of macrometastases. Snail drives an EMT in tumor cells, which, although retaining some epithelial markers, subsequently break through the basal lamina of the midgut, undergo a collective migration and seed polyclonal metastases. While metastases re-epithelialize over time, we found that early metastases are remarkably mesenchymal, discarding the requirement for a mesenchymal-to-epithelial transition for early stages of metastatic growth. Our results demonstrate the formation of metastases in adult flies, and identify a key role for partial-EMTs in driving it. This model opens the door to investigate the basic mechanisms underlying metastasis, in a powerful in vivo system suited for rapid genetic and drug screens.

Social environment mediates cancer progression in Drosophila.

The influence of oncogenic phenomena on the ecology and evolution of animal species is becoming an important research topic. Similar to host-pathogen interactions, cancer negatively affects host fitness, which should lead to the selection of host control mechanisms, including behavioral traits that best minimize the proliferation of malignant cells. Social behavior is suggested to influence tumor progression. While the ecological benefits of sociality in gregarious species are widely acknowledged, only limited data are available on the role of the social environment on cancer progression. Here, we exposed adult Drosophila, with colorectal-like tumors, to different social environments. We show how subtle variations in social structure have dramatic effects on the progression of tumor growth. Finally, we reveal that flies can discriminate between individuals at different stages of tumor development and selectively choose their social environment accordingly. Our study demonstrates the reciprocal links between cancer and social interactions and how sociality may impact health and fitness in animals and its potential implications for disease ecology.

EXD2 governs germ stem cell homeostasis and lifespan by promoting mitoribosome integrity and translation.

Mitochondria are subcellular organelles that are critical for meeting the bioenergetic and biosynthetic needs of the cell. Mitochondrial function relies on genes and RNA species encoded both in the nucleus and mitochondria, and on their coordinated translation, import and respiratory complex assembly. Here, we characterize EXD2 (exonuclease 3'-5' domain-containing 2), a nuclear-encoded gene, and show that it is targeted to the mitochondria and prevents the aberrant association of messenger RNAs with the mitochondrial ribosome. Loss of EXD2 results in defective mitochondrial translation, impaired respiration, reduced ATP production, increased reactive oxygen species and widespread metabolic abnormalities. Depletion of the Drosophila melanogaster EXD2 orthologue (CG6744) causes developmental delays and premature female germline stem cell attrition, reduced fecundity and a dramatic extension of lifespan that is reversed with an antioxidant diet. Our results define a conserved role for EXD2 in mitochondrial translation that influences development and ageing.

Cancer brings forward oviposition in the fly Drosophila melanogaster.

Hosts often accelerate their reproductive effort in response to a parasitic infection, especially when their chances of future reproduction decrease with time from the onset of the infection. Because malignancies usually reduce survival, and hence potentially the fitness, it is expected that hosts with early cancer could have evolved to adjust their life-history traits to maximize their immediate reproductive effort. Despite the potential importance of these plastic responses, little attention has been devoted to explore how cancers influence animal reproduction. Here, we use an experimental setup, a colony of genetically modified flies Drosophila melanogaster which develop colorectal cancer in the anterior gut, to show the role of cancer in altering life-history traits. Specifically, we tested whether females adapt their reproductive strategy in response to harboring cancer. We found that flies with cancer reached the peak period of oviposition significantly earlier (i.e., 2 days) than healthy ones, while no difference in the length and extent of the fecundity peak was observed between the two groups of flies. Such compensatory responses to overcome the fitness-limiting effect of cancer could explain the persistence of inherited cancer-causing mutant alleles in the wild.

Evolutionary perspective of cancer: myth, metaphors, and reality.

The evolutionary perspective of cancer (which origins and dynamics result from evolutionary processes) has gained significant international recognition over the past decade and generated a wave of enthusiasm among researchers. In this context, several authors proposed that insights into evolutionary and adaptation dynamics of cancers can be gained by studying the evolutionary strategies of organisms. Although this reasoning is fundamentally correct, in our opinion, it contains a potential risk of excessive adaptationism, potentially leading to the suggestion of complex adaptations that are unlikely to evolve among cancerous cells. For example, the ability of recognizing related conspecifics and adjusting accordingly behaviors as in certain free-living species appears unlikely in cancer. Indeed, despite their rapid evolutionary rate, malignant cells are under selective pressures for their altered lifestyle for only few decades. In addition, even though cancer cells can theoretically display highly sophisticated adaptive responses, it would be crucial to determine the frequency of their occurrence in patients with cancer, before therapeutic applications can be considered. Scientists who try to explain oncogenesis will need in the future to critically evaluate the metaphorical comparison of selective processes affecting cancerous cells with those affecting organisms. This approach seems essential for the applications of evolutionary biology to understand the origin of cancers, with prophylactic and therapeutic applications.

Conserved mechanisms of tumorigenesis in the Drosophila adult midgut.

Whereas the series of genetic events leading to colorectal cancer (CRC) have been well established, the precise functions that these alterations play in tumor progression and how they disrupt intestinal homeostasis remain poorly characterized. Activation of the Wnt/Wg signaling pathway by a mutation in the gene APC is the most common trigger for CRC, inducing benign lesions that progress to carcinomas due to the accumulation of other genetic alterations. Among those, Ras mutations drive tumour progression in CRC, as well as in most epithelial cancers. As mammalian and Drosophila's intestines share many similarities, we decided to explore the alterations induced in the Drosophila midgut by the combined activation of the Wnt signaling pathway with gain of function of Ras signaling in the intestinal stem cells. Here we show that compound Apc-Ras clones, but not clones bearing the individual mutations, expand as aggressive intestinal tumor-like outgrowths. These lesions reproduce many of the human CRC hallmarks such as increased proliferation, blockade of cell differentiation and cell polarity and disrupted organ architecture. This process is followed by expression of tumoral markers present in human lesions. Finally, a metabolic behavioral assay shows that these flies suffer a progressive deterioration in intestinal homeostasis, providing a simple readout that could be used in screens for tumor modifiers or therapeutic compounds. Taken together, our results illustrate the conservation of the mechanisms of CRC tumorigenesis in Drosophila, providing an excellent model system to unravel the events that, upon mutation in Apc and Ras, lead to CRC initiation and progression.

Self-induced patched receptor down-regulation modulates cell sensitivity to the hedgehog morphogen gradient.

Morphogens form signaling gradients that control patterning processes during development. Responding cells must perceive and interpret the concentration-dependent information provided by the morphogen to generate precise patterns of gene expression and cell differentiation in developing tissues. Generally, the absolute number of activated, ligand-bound receptors determines cell perception of the morphogen. In contrast, cells interpret the morphogen Hedgehog (Hh) by measuring the ratio of bound to unbound molecules of its receptor Patched (Ptc). This ratio depends on both the Hh concentration and the absolute number of Ptc molecules. Here, I describe a posttranscriptional process that controls the absolute amount of Ptc present in a cell, which regulates gradient interpretation, wherein self-induced receptor down-regulation that is independent of ligand binding dictates the cell response to a morphogen gradient.

Intestinal stem cells in mammals and Drosophila.

In the last few years, our knowledge of intestinal stem cell biology has flourished. Here we review recent developments in this exciting field, paying special attention to the characterization of Drosophila and mammalian intestinal stem cells.