Toll-mediated airway homeostasis is essential for fly survival upon injection of RasV12-GFP oncogenic cells.

Toll signaling is well known for its pivotal role in the host response against the invasion of external pathogens. Here, we investigate the potential involvement of Toll signaling in the intersection between the host and oncogenic cells. We show that loss of myeloid differentiation factor 88 (Myd88) leads to drastic fly death after the injection of RasV12-GFP oncogenic cells. Transcriptomic analyses show that challenging flies with oncogenic cells or bacteria leads to distinct inductions of Myd88-dependent genes. We note that downregulation of Myd88 in the tracheal system accounts for fly mortality, and ectopic tracheal complementation of Myd88 rescues the survival defect in Myd88 loss-of-function mutants following RasV12-GFP injection. Further, molecular and genetic evidence indicate that Toll signaling modulates fly resistance to RasV12-GFP cells through mediating airway function in a rolled-dependent manner. Collectively, our data indicate a critical role of Toll signaling in tracheal homeostasis and host survival after the injection of oncogenic cells.

Accumulation of annexin A2 and S100A10 prevents apoptosis of apically delaminated, transformed epithelial cells.

In various epithelial tissues, the epithelial monolayer acts as a barrier. To fulfill its function, the structural integrity of the epithelium is tightly controlled. When normal epithelial cells detach from the basal substratum and delaminate into the apical lumen, the apically extruded cells undergo apoptosis, which is termed anoikis. In contrast, transformed cells often become resistant to anoikis and able to survive and grow in the apical luminal space, leading to the formation of multilayered structures, which can be observed at the early stage of carcinogenesis. However, the underlying molecular mechanisms still remain elusive. In this study, we first demonstrate that S100A10 and ANXA2 (Annexin A2) accumulate in apically extruded, transformed cells in both various cell culture systems and murine epithelial tissues in vivo. ANXA2 acts upstream of S100A10 accumulation. Knockdown of ANXA2 promotes apoptosis of apically extruded RasV12-transformed cells and suppresses the formation of multilayered epithelia. In addition, the intracellular reactive oxygen species (ROS) are elevated in apically extruded RasV12 cells. Treatment with ROS scavenger Trolox reduces the occurrence of apoptosis of apically extruded ANXA2-knockdown RasV12 cells and restores the formation of multilayered epithelia. Furthermore, ROS-mediated p38MAPK activation is observed in apically delaminated RasV12 cells, and ANXA2 knockdown further enhances the p38MAPK activity. Moreover, the p38MAPK inhibitor promotes the formation of multilayered epithelia of ANXA2-knockdown RasV12 cells. These results indicate that accumulated ANXA2 diminishes the ROS-mediated p38MAPK activation in apically extruded transformed cells, thereby blocking the induction of apoptosis. Hence, ANXA2 can be a potential therapeutic target to prevent multilayered, precancerous lesions.

Calcium sparks enhance the tissue fluidity within epithelial layers and promote apical extrusion of transformed cells.

In vertebrates, newly emerging transformed cells are often apically extruded from epithelial layers through cell competition with surrounding normal epithelial cells. However, the underlying molecular mechanism remains elusive. Here, using phospho-SILAC screening, we show that phosphorylation of AHNAK2 is elevated in normal cells neighboring RasV12 cells soon after the induction of RasV12 expression, which is mediated by calcium-dependent protein kinase C. In addition, transient upsurges of intracellular calcium, which we call calcium sparks, frequently occur in normal cells neighboring RasV12 cells, which are mediated by mechanosensitive calcium channel TRPC1 upon membrane stretching. Calcium sparks then enhance cell movements of both normal and RasV12 cells through phosphorylation of AHNAK2 and promote apical extrusion. Moreover, comparable calcium sparks positively regulate apical extrusion of RasV12-transformed cells in zebrafish larvae as well. Hence, calcium sparks play a crucial role in the elimination of transformed cells at the early phase of cell competition.

Basal extrusion of single-oncogenic mutant cells induces dome-like structures with altered microenvironments.

At the initial stage of carcinogenesis, oncogenic transformation occurs in single cells within epithelial layers. However, the behavior and fate of the newly emerging transformed cells remain enigmatic. Here, using originally established mouse models, we investigate the fate of RasV12-transformed cells that appear in a mosaic manner within epithelial tissues. In the lung bronchial epithelium, most majority of RasV12-transformed cells are apically extruded, whereas noneliminated RasV12 cells are often basally delaminated leading to various noncell-autonomous changes in surrounding environments; macrophages and activated fibroblasts are accumulated, and normal epithelial cells overlying RasV12 cells overproliferate and form a convex multilayer, which is termed a 'dome-like structure'. In addition, basally extruded RasV12 cells acquire certain features of epithelial-mesenchymal transition (EMT). Furthermore, the expression of COX-2 is profoundly elevated in RasV12 cells in dome-like structures, and treatment with the COX inhibitor ibuprofen suppresses the recruitment of activated fibroblasts and moderately diminishes the formation of dome-like structures. Therefore, basal extrusion of single-oncogenic mutant cells can induce a tumor microenvironment and EMT and generate characteristic precancerous lesions, providing molecular insights into the earlier steps of cancer development.

Ha-RasV12-Induced Multilayer Cellular Aggregates Is Mediated by Rac1 Activation Rather Than YAP Activation.

We demonstrate that Ha-RasV12 overexpression induces the nuclear translocation of Hippo effector Yes-associated protein (YAP) in MDCK cells via the hippo-independent pathway at the confluent stage. Ha-RasV12 overexpression leads to the downregulation of Caveolin-1 (Cav1) and the disruption of junction integrity. It has been shown that the disruption of actin belt integrity causes YAP nuclear translocation in epithelial cells at high density. Therefore, we hypothesized that Ha-RasV12-decreased Cav1 leads to the disruption of cell junction integrity, which subsequently facilitates YAP nuclear retention. We revealed that Ha-RasV12 downregulated Cav1 through the ERK pathway. Furthermore, the distribution and expression of Cav1 mediated the cell junction integrity and YAP nuclear localization. This suggests that the downregulation of Cav1 induced by Ha-RasV12 disrupted the cell junction integrity and promoted YAP nuclear translocation. We further indicated the consequence of Ha-RasV12-induced YAP activation. Surprisingly, the activation of YAP is not required for Ha-RasV12-induced multilayer cellular aggregates. Instead, Ha-RasV12 triggered the ERK-Rac pathway to promote cellular aggregate formation. Moreover, the overexpression of constitutively active Rac is sufficient to trigger cellular aggregation in MDCK cells at the confluent stage. This highlights that Rac activity is essential for cellular aggregates.

Extracellular ATP facilitates cell extrusion from epithelial layers mediated by cell competition or apoptosis.

For the maintenance of epithelial homeostasis, various aberrant or dysfunctional cells are actively eliminated from epithelial layers. This cell extrusion process mainly falls into two modes: cell-competition-mediated extrusion and apoptotic extrusion. However, it is not clearly understood whether and how these processes are governed by common molecular mechanisms. In this study, we demonstrate that the reactive oxygen species (ROS) levels are elevated within a wide range of epithelial layers around extruding transformed or apoptotic cells. The downregulation of ROS suppresses the extrusion process. Furthermore, ATP is extracellularly secreted from extruding cells, which promotes the ROS level and cell extrusion. Moreover, the extracellular ATP and ROS pathways positively regulate the polarized movements of surrounding cells toward extruding cells in both cell-competition-mediated and apoptotic extrusion. Hence, extracellular ATP acts as an "extrude me" signal and plays a prevalent role in cell extrusion, thereby sustaining epithelial homeostasis and preventing pathological conditions or disorders.

Pilot RNAi Screen in Drosophila Neural Stem Cell Lineages to Identify Novel Tumor Suppressor Genes Involved in Asymmetric Cell Division.

A connection between compromised asymmetric cell division (ACD) and tumorigenesis was proven some years ago using Drosophila larval brain neural stem cells, called neuroblasts (NBs), as a model system. Since then, we have learned that compromised ACD does not always promote tumorigenesis, as ACD is an extremely well-regulated process in which redundancy substantially overcomes potential ACD failures. Considering this, we have performed a pilot RNAi screen in Drosophila larval brain NB lineages using RasV12 scribble (scrib) mutant clones as a sensitized genetic background, in which ACD is affected but does not cause tumoral growth. First, as a proof of concept, we have tested known ACD regulators in this sensitized background, such as lethal (2) giant larvae and warts. Although the downregulation of these ACD modulators in NB clones does not induce tumorigenesis, their downregulation along with RasV12 scrib does cause tumor-like overgrowth. Based on these results, we have randomly screened 79 RNAi lines detecting 15 potential novel ACD regulators/tumor suppressor genes. We conclude that RasV12 scrib is a good sensitized genetic background in which to identify tumor suppressor genes involved in NB ACD, whose function could otherwise be masked by the high redundancy of the ACD process.

Sequential oncogenic mutations influence cell competition.

At the initial stage of carcinogenesis, newly emerging transformed cells are often eliminated from epithelial layers via cell competition with the surrounding normal cells. For instance, when surrounded by normal cells, oncoprotein RasV12-transformed cells are extruded into the apical lumen of epithelia. During cancer development, multiple oncogenic mutations accumulate within epithelial tissues. However, it remains elusive whether and how cell competition is also involved in this process. In this study, using a mammalian cell culture model system, we have investigated what happens upon the consecutive mutations of Ras and tumor suppressor protein Scribble. When Ras mutation occurs under the Scribble-knockdown background, apical extrusion of Scribble/Ras double-mutant cells is strongly diminished. In addition, at the boundary with Scribble/Ras cells, Scribble-knockdown cells frequently undergo apoptosis and are actively engulfed by the neighboring Scribble/Ras cells. The comparable apoptosis and engulfment phenotypes are also observed in Drosophila epithelial tissues between Scribble/Ras double-mutant and Scribble single-mutant cells. Furthermore, mitochondrial membrane potential is enhanced in Scribble/Ras cells, causing the increased mitochondrial reactive oxygen species (ROS). Suppression of mitochondrial membrane potential or ROS production diminishes apoptosis and engulfment of the surrounding Scribble-knockdown cells, indicating that mitochondrial metabolism plays a key role in the competitive interaction between double- and single-mutant cells. Moreover, mTOR (mechanistic target of rapamycin kinase) acts downstream of these processes. These results imply that sequential oncogenic mutations can profoundly influence cell competition, a transition from loser to winner. Further studies would open new avenues for cell competition-based cancer treatment, thereby blocking clonal expansion of more malignant populations within tumors.

The CD44/COL17A1 pathway promotes the formation of multilayered, transformed epithelia.

At the early stage of cancer development, oncogenic mutations often cause multilayered epithelial structures. However, the underlying molecular mechanism still remains enigmatic. By performing a series of screenings targeting plasma membrane proteins, we have found that collagen XVII (COL17A1) and CD44 accumulate in RasV12-, Src-, or ErbB2-transformed epithelial cells. In addition, the expression of COL17A1 and CD44 is also regulated by cell density and upon apical cell extrusion. We further demonstrate that the expression of COL17A1 and CD44 is profoundly upregulated at the upper layers of multilayered, transformed epithelia in vitro and in vivo. The accumulated COL17A1 and CD44 suppress mitochondrial membrane potential and reactive oxygen species (ROS) production. The diminished intracellular ROS level then promotes resistance against ferroptosis-mediated cell death upon cell extrusion, thereby positively regulating the formation of multilayered structures. To further understand the functional role of COL17A1, we performed comprehensive metabolome analysis and compared intracellular metabolites between RasV12 and COL17A1-knockout RasV12 cells. The data imply that COL17A1 regulates the metabolic pathway from the GABA shunt to mitochondrial complex I through succinate, thereby suppressing the ROS production. Moreover, we demonstrate that CD44 regulates membrane accumulation of COL17A1 in multilayered structures. These results suggest that CD44 and COL17A1 are crucial regulators for the clonal expansion of transformed cells within multilayered epithelia, thus being potential targets for early diagnosis and preventive treatment for precancerous lesions.

A time course transcriptomic analysis of host and injected oncogenic cells reveals new aspects of Drosophila immune defenses.

Oncogenic RasV12 cells [A. Simcox et al., PLoS Genet 4, e1000142 (2008)] injected into adult males proliferated massively after a lag period of several days, and led to the demise of the flies after 2 to 3 wk. The injection induced an early massive transcriptomic response that, unexpectedly, included more than 100 genes encoding chemoreceptors of various families. The kinetics of induction and the identities of the induced genes differed markedly from the responses generated by injections of microbes. Subsequently, hundreds of genes were up-regulated, attesting to intense catabolic activities in the flies, active tracheogenesis, and cuticulogenesis, as well as stress and inflammation-type responses. At 11 d after the injections, GFP-positive oncogenic cells isolated from the host flies exhibited a markedly different transcriptomic profile from that of the host and distinct from that at the time of their injection, including in particular up-regulated expression of genes typical for cells engaged in the classical antimicrobial response of Drosophila.

Calcium Wave Promotes Cell Extrusion.

When oncogenic transformation or apoptosis occurs within epithelia, the harmful or dead cells are apically extruded from tissues to maintain epithelial homeostasis. However, the underlying molecular mechanism still remains elusive. In this study, we first show, using mammalian cultured epithelial cells and zebrafish embryos, that prior to apical extrusion of RasV12-transformed cells, calcium wave occurs from the transformed cell and propagates across the surrounding cells. The calcium wave then triggers and facilitates the process of extrusion. IP3 receptor, gap junction, and mechanosensitive calcium channel TRPC1 are involved in calcium wave. Calcium wave induces the polarized movement of the surrounding cells toward the extruding transformed cells. Furthermore, calcium wave facilitates apical extrusion, at least partly, by inducing actin rearrangement in the surrounding cells. Moreover, comparable calcium propagation also promotes apical extrusion of apoptotic cells. Thus, calcium wave is an evolutionarily conserved, general regulatory mechanism of cell extrusion.

Normal epithelial cells trigger EphA2-dependent RasV12 cell repulsion at the single cell level.

Epithelial cells expressing oncogenic Ras (RasV12) are detected by normal neighbors and are often extruded from tissues. We recently demonstrated that differential EphA2 signaling drives the segregation of mutant cells from normal monolayers via cell repulsion and increased RasV12 cell contractility. EphA2 signaling on RasV12 cells is triggered by ephrin-A ligands presented by normal cells. Here, we show that normal epithelial cells trigger the repulsion and enhanced contractility of Ras-transformed epithelial cells at the single cell level. We also reveal that ephrin-A ligands expressed on RasV12 cells are not required to drive RasV12 cell segregation following interaction with normal cells. Thus, normal-RasV12 cell-cell interaction triggers EphA2 forward signaling in RasV12 cells to drive repulsion and segregation of the transformed cells.

Mutant p53-Expressing Cells Undergo Necroptosis via Cell Competition with the Neighboring Normal Epithelial Cells.

p53 is a tumor suppressor protein, and its missense mutations are frequently found in human cancers. During the multi-step progression of cancer, p53 mutations generally accumulate at the mid or late stage, but not in the early stage, and the underlying mechanism is still unclear. In this study, using mammalian cell culture and mouse ex vivo systems, we demonstrate that when p53R273H- or p53R175H-expressing cells are surrounded by normal epithelial cells, mutant p53 cells undergo necroptosis and are basally extruded from the epithelial monolayer. When mutant p53 cells alone are present, cell death does not occur, indicating that necroptosis results from cell competition with the surrounding normal cells. Furthermore, when p53R273H mutation occurs within RasV12-transformed epithelia, cell death is strongly suppressed and most of the p53R273H-expressing cells remain intact. These results suggest that the order of oncogenic mutations in cancer development could be dictated by cell competition.

Obesity Suppresses Cell-Competition-Mediated Apical Elimination of RasV12-Transformed Cells from Epithelial Tissues.

Recent studies have revealed that newly emerging transformed cells are often eliminated from epithelial tissues via cell competition with the surrounding normal epithelial cells. This cancer preventive phenomenon is termed epithelial defense against cancer (EDAC). However, it remains largely unknown whether and how EDAC is diminished during carcinogenesis. In this study, using a cell competition mouse model, we show that high-fat diet (HFD) feeding substantially attenuates the frequency of apical elimination of RasV12-transformed cells from intestinal and pancreatic epithelia. This process involves both lipid metabolism and chronic inflammation. Furthermore, aspirin treatment significantly facilitates eradication of transformed cells from the epithelial tissues in HFD-fed mice. Thus, our work demonstrates that obesity can profoundly influence competitive interaction between normal and transformed cells, providing insights into cell competition and cancer preventive medicine.

Caveolin-1 down-regulation is required for Wnt5a-Frizzled 2 signalling in Ha-RasV12 -induced cell transformation.

Caveolin-1 (Cav1) is down-regulated during MK4 (MDCK cells harbouring inducible Ha-RasV12 gene) transformation by Ha-RasV12 . Cav1 overexpression abrogates the Ha-RasV12 -driven transformation of MK4 cells; however, the targeted down-regulation of Cav1 is not sufficient to mimic this transformation. Cav1-silenced cells, including MK4/shCav1 cells and MDCK/shCav1 cells, showed an increased cell area and discontinuous junction-related proteins staining. Cellular and mechanical transformations were completed when MDCK/shCav1 cells were treated with medium conditioned by MK4 cells treated with IPTG (MK4+I-CM) but not with medium conditioned by MK4 cells. Nanoparticle tracking analysis showed that Ha-RasV12 -inducing MK4 cells increased exosome-like microvesicles release compared with their normal counterparts. The cellular and mechanical transformation activities of MK4+I-CM were abolished after heat treatment and exosome depletion and were copied by exosomes derived from MK4+I-CM (MK4+I-EXs). Wnt5a, a downstream product of Ha-RasV12 , was markedly secreted by MK4+I-CM and MK4+I-EXs. Suppression of Wnt5a expression and secretion using the porcupine inhibitor C59 or Wnt5a siRNA inhibited the Ha-RasV12 - and MK4+I-CM-induced transformation of MK4 cells and MDCK/shCav1 cells, respectively. Cav1 down-regulation, either by Ha-RasV12 or targeted shRNA, increased frizzled-2 (Fzd2) protein levels without affecting its mRNA levels, suggesting a novel role of Cav1 in negatively regulating Fzd2 expression. Additionally, silencing Cav1 facilitated the internalization of MK4+I-EXs in MDCK cells. These data suggest that Cav1-dependent repression of Fzd2 and exosome uptake is potentially relevant to its antitransformation activity, which hinders the activation of Ha-RasV12 -Wnt5a-Stat3 pathway. Altogether, these results suggest that both decreasing Cav1 and increasing exosomal Wnt5a must be implemented during Ha-RasV12 -driven cell transformation.

Rab5-regulated endocytosis plays a crucial role in apical extrusion of transformed cells.

Newly emerging transformed cells are often eliminated from epithelial tissues. Recent studies have revealed that this cancer-preventive process involves the interaction with the surrounding normal epithelial cells; however, the molecular mechanisms underlying this phenomenon remain largely unknown. In this study, using mammalian cell culture and zebrafish embryo systems, we have elucidated the functional involvement of endocytosis in the elimination of RasV12-transformed cells. First, we show that Rab5, a crucial regulator of endocytosis, is accumulated in RasV12-transformed cells that are surrounded by normal epithelial cells, which is accompanied by up-regulation of clathrin-dependent endocytosis. Addition of chlorpromazine or coexpression of a dominant-negative mutant of Rab5 suppresses apical extrusion of RasV12 cells from the epithelium. We also show in zebrafish embryos that Rab5 plays an important role in the elimination of transformed cells from the enveloping layer epithelium. In addition, Rab5-mediated endocytosis of E-cadherin is enhanced at the boundary between normal and RasV12 cells. Rab5 functions upstream of epithelial protein lost in neoplasm (EPLIN), which plays a positive role in apical extrusion of RasV12 cells by regulating protein kinase A. Furthermore, we have revealed that epithelial defense against cancer (EDAC) from normal epithelial cells substantially impacts on Rab5 accumulation in the neighboring transformed cells. This report demonstrates that Rab5-mediated endocytosis is a crucial regulator for the competitive interaction between normal and transformed epithelial cells in mammals.

CARF activates beta-catenin/TCF signaling in the hepatocellular carcinoma.

Overactivation of Ras signaling is very common in the hepatocellular carcinoma (HCC) due to its constitutive active mutation, which makes it a big challenge to target Ras signaling. Therefore, identifying effectors downstream of Ras signaling would benefit the development of novel therapeutic strategies. In this study, it was found that the expression of CARF (collaborate of ARF) was induced by oncogenic RasV12. The expression of CARF was up-regulated in both HCC mouse model (Alb-Cre; P53f/f; Loxp-Stop-Loxp-RasG12D) and human HCC clinical samples. Overexpression of CARF promoted the growth and migration of HCC cells, while knocking down the expression of CARF inhibited the growth and migration of HCC cells. In the mechanism study, CARF was found to interact with beta-catenin, impaired the interaction between beta-catenin and ICAT, and activated beta-catenin/TCF signaling. Moreover, knocking down the expression of CARF inhibited the tumorigenesis in the HCC mouse model. Taken together, this study revealed the oncogenic functions of CARF in the tumorigenesis of HCC by activating beta-catenin/TCF signaling, and suggested CARF might be a therapeutic target in the treatment of HCC.

Opposing activities of the Ras and Hippo pathways converge on regulation of YAP protein turnover.

Cancer genomes accumulate numerous genetic and epigenetic modifications. Yet, human cellular transformation can be accomplished by a few genetically defined elements. These elements activate key pathways required to support replicative immortality and anchorage independent growth, a predictor of tumorigenesis in vivo. Here, we provide evidence that the Hippo tumor suppressor pathway is a key barrier to Ras-mediated cellular transformation. The Hippo pathway targets YAP1 for degradation via the ßTrCP-SCF ubiquitin ligase complex. In contrast, the Ras pathway acts oppositely, to promote YAP1 stability through downregulation of the ubiquitin ligase complex substrate recognition factors SOCS5/6. Depletion of SOCS5/6 or upregulation of YAP1 can bypass the requirement for oncogenic Ras in anchorage independent growth in vitro and tumor formation in vivo. Through the YAP1 target, Amphiregulin, Ras activates the endogenous EGFR pathway, which is required for transformation. Thus, the oncogenic activity of Ras(V12) depends on its ability to counteract Hippo pathway activity, creating a positive feedback loop, which depends on stabilization of YAP1.

Autophagy activity is inhibited in human fibroblast cells stably overexpressing H-RasV12 / 第二军医大学学报

Objective: To investigate the relationship between oncogenic H-RasV12 overexpression/activation and the autophagic activity by observing the effect of Ras overexpression on autophagic activity in human fibroblast cells. Methods: Human BJ fibroblast cells were transfected with H-RasV12 or control vector, and then the cellular responses to H-RasV12 overexpression were analyzed by observing the morphology, cell growth curve, senescence-associated β-Gal staining, Western blotting analysis, flow cytometry, and suppression of autophagy-related protein 5 (ATG5) by siRNA. Results: Compared with control group, BJ cells overexpressing H-RasV12 developed prominent premature senescence and inhibited autophagic activity, as manifested by significant accumulation of p62 and light chain 3 II (LC3 II). The autophagy inhibition by H-RasV12 remained stable during the study period; the apoptosis rate was increased in H-RasV12overexpressing BJ cells compared with that in the control cells. Suppression of ATG5 by siRNA led to more severe senescence in Ras-overexpressing BJ cells. Conclusion: Our results suggest that the autophagy activity is inhibited in human fibroblast cells stably overexpressing oncogenic H-RasV12, and the inhibition is in the later stage of autophagy, which may be related to H-RasV12-related tumorigenesis.