Article

Concomitant Notch activation and p53 deletion trigger epithelial-to-mesenchymal transition and metastasis in mouse gut

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Epithelial-to-mesenchymal transition-like (EMT-like) is a critical process allowing initiation of metastases during tumour progression. Here, to investigate its role in intestinal cancer, we combine computational network-based and experimental approaches to create a mouse model with high metastatic potential. Construction and analysis of this network map depicting molecular mechanisms of EMT regulation based on the literature suggests that Notch activation and p53 deletion have a synergistic effect in activating EMT-like processes. To confirm this prediction, we generate transgenic mice by conditionally activating the Notch1 receptor and deleting p53 in the digestive epithelium (NICD/p53(-/-)). These mice develop metastatic tumours with high penetrance. Using GFP lineage tracing, we identify single malignant cells with mesenchymal features in primary and metastatic tumours in vivo. The development of such a model that recapitulates the cellular features observed in invasive human colorectal tumours is appealing for innovative drug discovery.
Characterization of NICD/​p53−/− primary tumours and metastases. (a) Kaplan–Meier analysis of NICD/​p53−/− mice and their single transgenic littermates NICD (pink) and ​p53−/− (green) over a period of 2 years. The triple transgenic mice (blue) are all dead by 15 months with gut adenocarcinoma, whereas control littermates (pink and green) are dying after 15 months. (b) Tumour intake in the intestinal tract of NICD/​p53−/− cohort mice and their relative control littermates at different time points after ​tamoxifen induction. NICD/​p53−/− mice (blue) develop intestinal adenocarcinoma faster and with a higher penetrance than control littermates (pink and green). (c) Macroscopic view of a primary invasive adenocarcinoma located in the jejunum of a NICD/​p53−/− mouse. Scale bar, 1 cm. (d) haematoxylin and eosin staining (H&E) staining on paraffin-embedded primary tumour from NICD/​p53−/− mice, representing main features of the tumour. Cancer cells are invading all layers of normal tissue to reach the serosa. We suspect that elongated, single cancer cells are present in desmoplastic areas. Scale bar, 40 μm. (e) H&E and (f) nuclear GFP stainings showing primary tumour, lymph node and liver metastases, and peritoneal carcinomatosis in NICD/​p53−/− compound mice. Notice the nonspecific staining due to autofluorescence in the surrounding tumour tissue of lymph node (LN) or liver metastasis and peritoneum carcinomatosis. Scale bars, 40 μm. (g) Immunohistochemical staining of ​β-catenin showing its nuclear delocalization in cancer cells and its overexpression at the tumour front. Scale bars, 40 μm.
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Characterization of an EMT-like phenotype in NICD/​p53−/− primary tumours. (a) Staining of the mesenchymal marker ​N-cadherin and the EMT transcription factors, ​SNAIL, ​SLUG and ​TWIST in NICD/​p53−/− primary tumours. Left panel: ​N-cadherin is overexpressed in tumour cells invading the desmoplastic area compared with those from the tumour bulk. Scale bars, 40 μm. Middle and right panels: a nuclear overexpression of EMT transcription factors is observed in cancer cells from the desmoplastic area compared with differentiated tubular structures. Scale bars, 100 μm. (b,c) Expression of the EMT-inducer ​ZEB1 in primary tumours of NICD/​p53−/−. (b) Left panel: triple immunofluorescence on NICD/​p53−/− primary tumours using 4,6-diamidino-2-phenylindole (​DAPI; grey) and GFP staining (green), ​ECAD (blue) and ​ZEB1 (red). Scale bars, 40 μm. Right panel: magnification of different tumour cell features coming from the same slide showing that cells undergoing EMT-like processes express the EMT-inducer ​ZEB1. *​Zeb1 negative epithelial tumour cells; ** and *** ​Zeb1 and GFP-positive, ​ECAD-negative cells, independently of their morphology. Scale bars, 4 μm. The white arrow labels individual ​ZEB1-negative cells with a non-epithelial origin. (c) The left bar graph shows the frequency of ​vimentin, ​SMA or ​Zeb1-positive cells among GFP-positive cells. Cells were separated in two groups presenting either an epithelial phenotype described by ​ECAD-positive cells (E+) or cytokeratin-positive cells (CK+), or an EMT-like phenotype characterized by ​ECAD negative (E−) or CK negative (CK−). The associated tables of contingency (Supplementary Table 4) depict a strong negative correlation (***) between ​ZEB1 and ​ECAD expressions (P<0.001), ​vimentin and ​ECAD expressions (χ2 test, P<0.001) or ​SMA and CK expressions (χ2 test, P<0.001). The right bar graph shows the distribution of ​Zeb1 and ​SMA among GFP positive cells. ​ZEB1 is expressed in ​SMA-negative cells in 27% of GFP-positive cells and in ​SMA-positive cells for 22.4% GFP-positive cells. Only 3% of GFP-positive are ​ZEB1-negative cells and ​SMA positive, suggesting that a small fraction of cells have fully completed the EMT process. (d) Triple immunofluorescence on NICD/​p53−/− metastatic tissues using ​DAPI (grey), GFP staining (green), ​ECAD (blue) and ​ZEB1 (red; scale bars, 20 μm) with magnifications of one area showing the presence of GFP-intestinal cancer cells with mesenchymal features in the secondary organs (scale bars, 4 μm).
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... In this model, tumors spontaneously arise in the intestine and metastasize to the lymph nodes, peritoneum and liver (Chanrion et al., 2014), reflecting the heterogeneity and growth timescales of naturally occurring tumors. Due to the inaccessibility of deep regions of intestinal tumors for intravital imaging, we imaged tumor explant slices using two-photon excitation microscopy (Staneva et al., 2018). ...
... To study the growth and migratory behaviors of cancer cells in spontaneously developing tumors, we used the NICD/p53 −/− tumor model (Chanrion et al., 2014). In this mouse model, the formation of invasive carcinoma is driven by the expression of a conditionally activated receptor Notch1 (NICD) and the deletion of the tumor suppressor p53 (also known as TP53) in the intestinal epithelium following tamoxifen injection. ...
... Present at the invasive front are E-cadherin-positive cancer cell clusters (Fig. 1B,upper left panel), as well as single cells. Single cells are positive for smooth muscle actin (α-SMA, also known as ACTA2) and ZEB-1 (Fig. 1B, upper middle and right panels); previous work with this model has shown that a subset of ZEB-1-positive cells at the invasive front are of epithelial origin and have undergone an epithelial-to-mesenchymal transition (EMT) (Chanrion et al., 2014). Deeper in the interior of the tumor is the tumor core, which is composed of densely packed epithelial tumor cells (Fig. 1A,inset 2;Fig. ...
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In the early stages of metastasis, cancer cells exit the primary tumor and enter the vasculature. Although most studies have focused on the tumor invasive front, cancer cells from the tumor core can also potentially metastasize. To address cell motility in the tumor core, we imaged tumor explants from spontaneously forming tumors in mice in real time using long-term two-photon microscopy. Cancer cells in the tumor core are remarkably dynamic and exhibit correlated migration patterns, giving rise to local ‘currents’ and large-scale tissue dynamics. Although cells exhibit stop-and-start migration with intermittent pauses, pausing does not appear to be required during division. Use of pharmacological inhibitors indicates that migration patterns in tumors are actively driven by the actin cytoskeleton. Under these conditions, we also observed a relationship between migration speed and correlation length, suggesting that cells in tumors are near a jamming transition. Our study provides new insight into the dynamics of cancer cells in the tumor core, opening new avenues of research in understanding the migratory properties of cancer cells and later metastasis. This article has an associated First Person interview with the first author of the paper.
... As input a mapping between each initial state and 44 the corresponding steady states is given. In an asynchronous update, as we consider here, the state of only one 45 species can be changed per step. This means that the Hamming distance of all states that are reachable in the 46 next step is equal to one [43]. ...
... The algorithm automatically simplifies these 53 results using symbolic manipulation. The algorithm proposed is described in the following sections for two examples: 54 an Enzyme-Substrate kinetics model and an established Epithelial to Mesenchymal Transitions (EMT) epithelial 55 mouse cancer cell metastasis [44,45]. ...
... Our implementation enables the user to start with either the forward-path network, or the full backward path 151 including network from which transitions can be added or removed as seen fit. Note, however, that not all removals 152 are valid to keep the network dynamics: adding and/or removing random transitions could result in the following 153 problems: To demonstrate our mechanism inference approach in a real-world system, we infer the Boolean logic mechanism for the EMT transition observed in [44,45]. The ruleset for the reference EMT model is: ...
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Modern analytical techniques enable researchers to collect data about cellular states, before and after perturbations. These states can be characterized using analytical techniques, but the inference of regulatory interactions that explain and predict changes in these states remains a challenge. Here we present a generalizable unsupervised approach to generate parameter-free, logic-based mechanistic hypotheses of cellular processes, described by multiple discrete states. Our algorithm employs a Hamming-distance based approach to formulate, test, and identify, the best mechanism that links two states. Our approach comprises two steps. First, a model with no prior knowledge except for the mapping between initial and attractor states is built. Second, we employ biological constraints to improve model fidelity. Our algorithm automatically recovers the relevant dynamics for the explored models and recapitulates all aspects of the original models biochemical species concentration dynamics. We then conclude by placing our results in the context of ongoing work in the field and discuss how our approach could be used to infer mechanisms of signaling, gene-regulatory, and any other input-output processes describable by logic-based mechanisms.
... proteins, DNA, RNA, etc.) than text-based-only database [16]. The map represents biological mechanisms and through analysis of the of map, either as a static network (topology) or dynamic one (simulations) structural details and regulations can be revealed behind certain biological phenomena [17][18][19][20][21]. ...
... The reduced network allows postulating a putative mechanism without the need to display the full details. Such strategy has been applied successfully before for proposing mechanisms behind observed biological phenomena [18]. ...
... The sub-network obtained after path analysis of the initial network was manually converted into an activity-flow diagram in order to understand the influence of iodide on the other nodes [18]. A protein catalysing a reaction will have an activating influence on the formed product; an enzyme that inhibits a reaction will have a negative influence on the formed product. ...
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Our group showed that repetitive dose of potassium iodide (KI)for eight days offers an efficient protection for exposure to repeated radioactive emissions without adverse effects on adult rats. However, differential expression of genes implicated in Wolff-Chaikoff effect was observed. To understand the Wolff-Chaikoff regulation and its molecular constituents during repetitive administration of KI, a biochemical reaction network was constructed as a “geographical” map of the thyrocyte depicting iodide and thyroid hormone synthesis. Path analysis of the network has been performed to investigate the presence of a regulatory circuit of the node iodide to the node “nis transcription”. NIS is responsible for the uptake of KI and plays an important role in the Wolff-Chaikoff effect. The map is a source for the most updated information about iodide and thyroid hormone metabolism. Based on this map, we propose a hypothesis that shows a putative mechanism behind NIS regulation and KI uptake.
... TWIST1 can enhance tumor initiation, conferring resistance to senescence and apoptosis, promoted by p53 [108]. In addition, the combination of Notch pathway activation with the loss of p53 function triggered the expression of EMT-TFs, including Twist1, in mouse gut, hence promoting an invasive and diffusive behavior [109]. ...
... A similar approach, was used in a mouse model for intestinal tumors with conditional activation of Notch receptor (labelled with green fluorescent protein (GFP)) and p53 inactivation [109], mimicking aggressive CRC. The expression of several EMT-TFs were detected in invasive and desmoplastic regions, while Zeb1 expression was detected in invasive GFP+ cells undergoing EMT [47]. ...
... The expression of several EMT-TFs were detected in invasive and desmoplastic regions, while Zeb1 expression was detected in invasive GFP+ cells undergoing EMT [47]. In vivo and ex vivo analysis allowed the identification of GFP+ EMT-like cells at metastatic sites supporting the validity of this genetic model to study epithelial plasticity, as well as the association of Notch activation (in the context of p53 downregulation) with metastatic CRC [109]. ...
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Resembling the development of cancer by multistep carcinogenesis, the evolution towards metastasis involves several passages, from local invasion and intravasation, encompassing surviving anoikis into the circulation, landing at distant sites and therein establishing colonization, possibly followed by the outgrowth of macroscopic lesions. Within this cascade, epithelial to mesenchymal transition (EMT) works as a pleiotropic program enabling cancer cells to overcome local, systemic, and distant barriers against diffusion by replacing traits and functions of the epithelial signature with mesenchymal-like ones. Along the transition, a full-blown mesenchymal phenotype may not be accomplished. Rather, the plasticity of the program and its dependency on heterotopic signals implies a pendulum with oscillations towards its reversal, that is mesenchymal to epithelial transition. Cells in intermixed EÛM states can also display stemness, enabling their replication together with the epithelial reversion next to successful distant colonization. If we aim to include the EMT among the hallmarks of cancer that could modify clinical practice, the gap between the results pursued in basic research by animal models and those achieved in translational research by surrogate biomarkers needs to be filled. We review the knowledge on EMT, derived from models and mechanistic studies as well as from translational studies, with an emphasis on gastrointestinal cancers (GI).
... In fact, the combination of Apc mutation with a dominant-negative P53 mutation leads to increased invasiveness of intestinal tumors with signs of epithelial to mesenchymal transition [139,140] . Also, loss of P53 in a constitutively active Notch signaling background leads to intestinal tumor formation and metastasis [141] , whereas Notch signaling does not cooperate with the Wnt-pathway [142] . Nevertheless, Apc deficiency seems to represent a key prerequisite of cancer progression, since Apc restoration leads to spontaneous tumor regression of Kras-mutated, P53-deficient adenocarcinomas [143] . ...
... In summary, GEMM have contributed enormously to the understanding of the molecular processes of CRC initiation, progression and crosstalk of common cancerassociated pathways. Besides the models for spontaneous CRC and common cancer syndromes, like Familiar Adenomatius Polyposis and LS, several models recapitulate metastatic disease, either as "classic" GEMM [141,148,166,198] or upon viral Cre delivery [129,130] . Yet, these models have several limitations. ...
... Lower tumor burden in C57/BL6 × 129/Sv Smad3 -/hybrids. Note, that Smad3 mutations occur only in 2% of CRC (Fleming et al [162] , 2013) 55 [179] Smad4 f/f ;Catnb lox(ex3)/+ ;Lgr5-Cre ERT2 -IRES-GFP Mosaic Cre-expression leads to adenoma formation 56 [178] Catnb +/lox(ex3) : Krt1-19 +/cre Constitutional Cre-mediated excision of β-catenin phosphorylation site leads to a plethora of small intestine adenomas Catnb +/lox(ex3) : Tg·Fabpl cre 57 [177] Villin-creER T2 /Catnb loxEx3/WT Expression of GSK3β-resistant β-catenin leads to substitution of enterocytes by highly proliferative crypt stem cells (rapid death) 58 [142] Nicd/Apc +/1638N NOTCH-signaling does not influence adenoma formation 59 [141] Nicd/P53 −/− Villin-CreER T2 tamoxifen-dependent P53 deletion in constitutively active NOTCH-signaling background leads to intestinal tumor formation and metastasis 60 [158] Car1 CreER/+ ; Apc fl/fl ; Kras LSL-G12D/+ ; P53 KO ; Smad4 fl/fl Rapid tumor formation in cecum and proximal colon, but high mortality in triple and quadruple mutants 61 [192] Msh2 Δ7N [185,186] Msh2 -/-Death due to lymphoma 66 [194] Apc 1638N/+ Exo1 +/− Fen1 +/− Increased tumor multiplicity and incidence, higher progression to malignancy, high incidence of hematopoietic cancers 67 [193] Fen1 null /Apc 1638N Increased malignancy of intestinal tumors compared to Apc 1638N mice through MSI 68 [184] Mlh1 -/-/Apc 1638N Increased tumor incidence and multiplicity, 30% adenocarcinomas, reduced lifespan of 3.3 mo. High amount of extraintestinal tumors 69 [187] Msh6 -/+ Reduced life span in hetero-and homozygotes due to lymphomas and gastrointestinal tumors. ...
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Colorectal cancer (CRC) is the third most common diagnosed malignancy among both sexes in the United States as well as in the European Union. While the incidence and mortality rates in western, high developed countries are declining, reflecting the success of screening programs and improved treatment regimen, a rise of the overall global CRC burden can be observed due to lifestyle changes paralleling an increasing human development index. Despite a growing insight into the biology of CRC and many therapeutic improvements in the recent decades, preclinical in vivo models are still indispensable for the development of new treatment approaches. Since the development of carcinogen-induced rodent models for CRC more than 80 years ago, a plethora of animal models has been established to study colon cancer biology. Despite tenuous invasiveness and metastatic behavior, these models are useful for chemoprevention studies and to evaluate colitis-related carcinogenesis. Genetically engineered mouse models (GEMM) mirror the pathogenesis of sporadic as well as inherited CRC depending on the specific molecular pathways activated or inhibited. Although the vast majority of CRC GEMM lack invasiveness, metastasis and tumor heterogeneity, they still have proven useful for examination of the tumor microenvironment as well as systemic immune responses; thus, supporting development of new therapeutic avenues. Induction of metastatic disease by orthotopic injection of CRC cell lines is possible, but the so generated models lack genetic diversity and the number of suited cell lines is very limited. Patient-derived xenografts, in contrast, maintain the pathological and molecular characteristics of the individual patient's CRC after subcutaneous implantation into immunodeficient mice and are therefore most reliable for preclinical drug development - even in comparison to GEMM or cell line-based analyses. However, subcutaneous patient-derived xenograft models are less suitable for studying most aspects of the tumor microenvironment and anti-tumoral immune responses. The authors review the distinct mouse models of CRC with an emphasis on their clinical relevance and shed light on the latest developments in the field of preclinical CRC models.
... The authors termed these three stages of temporal remodeling of the collagen microstructure the tumor-associated collagen signatures (TACS-1, -2, and -3) and intended to define visual hallmarks that characterize critical stages of tumor development. Tumor cell behavior is affected by the collagen architecture at every stage, including increased invasiveness associated with radially aligned collagen 8,10 . Although these thick bundled collagen strands were commonly associated with tumor progression in vivo, in vitro models often overlook these distinct architectural features. ...
... The patterned microscale collagen bundles resembled the alignment of thick collagen bundles observed in in vivo studies 10 (Figure 4d) because the spacing between the aligned collagen bundles was on the order of a few tens of micrometers, which is approximately the size of a single cell ( Figure 4e). With this assay we showed that the 3D aligned collagen bundles enabled cancer cell contact guidance. ...
... By varying temperature and ion content in the collagen precursor, we found high temperature and low ionic strength played key roles in the instant collagen bundle formation. As widely shown in in vivo studies, the tumor microenvironment often contains abundant microscale collagen fiber bundles that could directly guide cancer cell invasion as single cells or in a collective way 10 . This biological process may not be fully recapitulated by traditional collagen fiber gels due to the lack of the proper collagen architecture and dimension. ...
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Stromal collagen surrounding a solid tumor tends to present as dense, thick bundles. The collagen bundles are remodeled during tumor progression: first tangential to the tumor boundary (indicating growth) and later perpendicular to the tumor boundary (indicating likely metastasis). Current reconstituted-collagen in vitro tumor models are unable to recapitulate the in vivo structural features of collagen bundling and alignment. Here, we present a rapid yet simple procedure to fabricate collagen bundles with an average thickness of 9 μm, compared to the reticular dense collagen nanofiber (~900 nm-diameter, on average) prepared using common protocols. The versatility of the collagen bundles was demonstrated with their incorporation into two in vitro models where the thickness and alignment of the collagen bundles resembled the various in vivo arrangements. First, collagen bundles aligned by a microfluidic device elicited cancer cell contact guidance and enhanced their directional migration. Second, the presence of the collagen bundles in a bio-inert agarose hydrogel was shown to provide a highway for cancer cell invasion. The unique structural features of the collagen bundles advance the physiological relevance of in vitro collagen-based tumor models for accurately capturing cancer cell-stroma interactions.
... Activation of Notch signaling and loss of p53 in the intestinal epithelium (in NICD EYFPflox/flox /p53 flox/flox ; VillinCre ERT2 mice, here called NICD/p53 −/− mice) by inducible Cre-mediated recombination causes the production of the intracellular domain of the Notch receptor (NICD) and ablation of p53 (25). Mice can develop invasive intestinal tumors with metastases, thus recapitulating human tumors to a great extent (25). ...
... Activation of Notch signaling and loss of p53 in the intestinal epithelium (in NICD EYFPflox/flox /p53 flox/flox ; VillinCre ERT2 mice, here called NICD/p53 −/− mice) by inducible Cre-mediated recombination causes the production of the intracellular domain of the Notch receptor (NICD) and ablation of p53 (25). Mice can develop invasive intestinal tumors with metastases, thus recapitulating human tumors to a great extent (25). However, the tumors develop only after months and exhibit accumulation of mutations. ...
... Organoid Culture. Organoids were generated from small intestine of Villin-Cre ERT2 ; NICD flox/flox ; p53 flox/flox mice (25), cultured in small intestinal organoid media, and treated with the indicated compounds. Mutagenesis was induced in culture 4-OHT (details in SI Appendix, Supplementary Methods). ...
Article
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Specified intestinal epithelial cells reprogram and contribute to the regeneration and renewal of the epithelium upon injury. Mutations that deregulate such renewal processes may contribute to tumorigenesis. Using intestinal organoids, we show that concomitant activation of Notch signaling and ablation of p53 induce a highly proliferative and regenerative cell state, which is associated with increased levels of Yap and the histone methyltransferase Mll1. The induced signaling system orchestrates high proliferation, self-renewal, and niche-factor-independent growth, and elevates the trimethylation of histone 3 at lysine 4 (H3K4me3). We demonstrate that Yap and Mll1 are also elevated in patient-derived colorectal cancer (CRC) organoids and control growth and viability. Our data suggest that Notch activation and p53 ablation induce a signaling circuitry involving Yap and the epigenetic regulator Mll1, which locks cells in a proliferative and regenerative state that renders them susceptible for tumorigenesis.
... Tissue architecture, such as that provided by collagen and other fibrous proteins, profoundly influences cell behavior and function-in the human brain [8] , prostate [9] , and breast [10][11][12] as well as in mouse models of various different tumor types [13][14][15][16][17][18] . The upregulated collagen in the tumor environment is also remodeled into what is described as being bundles of linearized and aligned collagen fibers [12 , 19] with, for example, widths of bundled collagen fibers in brain tissue sections of glioblastoma multiform measuring over 2 μm [8] . ...
... By varying temperature and ion content in the collagen precursor, we found high temperature and low ionic strength played key roles in the instant collagen bundle formation. As widely shown in in vivo studies, the tumor microenvironment often contains abundant microscale collagen fiber bundles that could directly guide cancer cell invasion as single cells or in a collective way [15] . This biological process may not be fully recapitulated by traditional collagen fiber gels due to the lack of the proper collagen architecture. ...
... Although the presence of collagen nanofibers significantly enhanced the cell proliferation and elicited an invasive tendency of the mechanically constrained cells, cell invasion was hindered by the agarose mesh. On the other hand, in the collagen bundle-containing agarose, cancer cells formed multicellular cohorts along the collagen, which resembled collective invasion as seen in vivo [15] . In comparison with MDA-MB-231 cells, human colon cancer cells HCT-116, with strong cell-cell adhesion, did not appear to have as pronounced of an invasive tendency when surrounded by collagen nanofibers. ...
Article
Stromal collagen is upregulated surrounding a solid tumor and presents as dense, thick, linearized, and aligned bundles. The collagen bundles are continually remodeled during tumor progression, and their orientation with respect to the tumor boundary has been correlated with invasive state. Currently, reconstituted-collagen gels are the standard in vitro tumor cell-extracellular matrix interaction model. The reticular, dense, and isotropic nanofiber (∼900 nm-diameter, on average) gels do not, however, recapitulate the in vivo structural features of collagen bundling and alignment. Here, we present a rapid and simple method to fabricate bundles of collagen type I, whose average thickness may be varied between about 4 μm and 9 μm dependent upon diluent temperature and ionic strength. The durability and versatility of the collagen bundles was demonstrated with their incorporation into two in vitro models where the thickness and alignment of the collagen bundles resembled various in vivo arrangements. First, collagen bundles aligned by a microfluidic device elicited cancer cell contact guidance and enhanced their directional migration. Second, the presence of the collagen bundles in a bio-inert agarose hydrogel was shown to provide a route for cancer cell outgrowth. The unique structural features of the collagen bundles advance the physiological relevance of in vitro collagen-based tumor models for accurately capturing tumor cell-extracellular matrix interactions.
... The algorithm automatically simplifies these results using symbolic manipulation. The algorithm proposed is described in the following sections for two examples: an Enzyme-Substrate kinetics model and an established Epithelial to Mesenchymal Transitions (EMT) epithelial mouse cancer cell metastasis [55,56]. ...
... To demonstrate our mechanism inference approach in a real-world system, we infer the Boolean logic mechanism for the EMT transition observed in [55,56]. The ruleset for the reference EMT model is: ...
Article
Full-text available
Modern analytical techniques enable researchers to collect data about cellular states, before and after perturbations. These states can be characterized using analytical techniques, but the inference of regulatory interactions that explain and predict changes in these states remains a challenge. Here we present a generalizable, unsupervised approach to generate parameter-free, logic-based models of cellular processes, described by multiple discrete states. Our algorithm employs a Hamming-distance based approach to formulate, test, and identify optimized logic rules that link two states. Our approach comprises two steps. First, a model with no prior knowledge except for the mapping between initial and attractor states is built. We then employ biological constraints to improve model fidelity. Our algorithm automatically recovers the relevant dynamics for the explored models and recapitulates key aspects of the biochemical species concentration dynamics in the original model. We present the advantages and limitations of our work and discuss how our approach could be used to infer logic-based mechanisms of signaling, gene-regulatory, or other input-output processes describable by the Boolean formalism.
... (Cell death, Proliferation, Metastasis). (Adapted from [53]) . ...
... These mice develop digestive tumours with dissemination of EMTlike epithelial malignant cells to the lymph nodes, liver and peritoneum and generation of distant metastases ( Figure 10B). Exploration of early EMT program inducers in in invasive human colon cancer samples confirmed that EMT markers are associated with modulation of Notch and p53 gene expression in similar manner as in the mice model ( Figure 10C), supporting a synergy between these genes to permit EMT induction [53]. ...
Preprint
Initiation and progression of cancer involve multiple molecular mechanisms. The knowledge on these mechanisms is expanding and should be converted into guidelines for tackling the disease. We discuss here formalization of biological knowledge into a comprehensive resource Atlas of Cancer Signalling Network (ACSN) and Google Maps-based tool NaviCell that supports map navigation. The application of maps for omics data visualisation in the context of signalling maps is possible using NaviCell Web Service module and NaviCom tool for generation of network-based molecular portraits of cancer using multi-level omics data. We review how these resources and tools are applied for cancer pre-clinical studies among others for rationalizing synergistic effect of drugs and designing complex disease stage-specific druggable interventions following structural analysis of the maps together with omics data. Modules and maps of ACSN as signatures of biological functions, can help in cancer data analysis and interpretation. In addition, they can also be used to find association between perturbations in particular molecular mechanisms to the risk of a specific cancer type development. These approaches and beyond help to study interplay between molecular mechanisms of cancer, deciphering how gene interactions govern hallmarks of cancer in specific context. We discuss a perspective to develop a flexible methodology and a pipeline to enable systematic omics data analysis in the context of signalling network maps, for stratifying patients and suggesting interventions points and drug repositioning in cancer and other human diseases.
... However, also in some carcinomas, or tumors of epithelial origin, such as gastrointestinal (stomach, oesophagus) and colorectal cancers, GOF or oncogenic Notch mutations appear predominant. Activating mutant Notch receptors may further collaborate with frequent p53 mutations [32], thus promoting EMT and enhancing invasive or aggressive phenotypes in various cancer types such as colorectal carcinomas and pancreatic cancer. This will be taken up further below. ...
... GOF Notch mutations have been described as promoting EMT and invasive phenotypes in NSCLCs (non-small cell lung cancers) [33], as well as promoting a drug-resistant phenotype, e.g., against gefitinib in lung cancer [34]. Cooperation with recurrent mutations that occur early in tumor progression, maybe most prominently the p53 tumor suppressor, may further promote Notch-specific effects on EMT and thus enhance aggressive properties [32], as well as promote drug resistance, as demonstrated for cetuximab in HNSCC [35]. These findings in advanced lung cancers are in line with recent discoveries showing that EMT may be dispensable for metastasis, but promotes cell survival and drug resistance [36], possibly with the help of Notch. ...
Article
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The Notch signaling pathway is a critical player in embryogenesis but also plays various roles in tumorigenesis, with both tumor suppressor and oncogenic activities. Mutations, deletions, amplifications, or over-expression of Notch receptors, ligands, and a growing list of downstream Notch-activated genes have by now been described for most human cancer types. Yet, it often remains unclear what may be the functional impact of these changes for tumor biology, initiation, and progression, for cancer therapy, and for personalized medicine. Emerging data indicate that Notch signaling can also contribute to increased aggressive properties such as invasion, tumor heterogeneity, angiogenesis, or tumor cell dormancy within solid cancer tissues; especially in epithelial cancers, which are in the center of this review. Notch further supports the “stemness” of cancer cells and helps define the stem cell niche for their long-term survival, by integrating the interaction between cancer cells and the cells of the tumor microenvironment (TME). The complexity of Notch crosstalk with other signaling pathways and its roles in cell fate and trans-differentiation processes such as epithelial-to-mesenchymal transition (EMT) point to this pathway as a decisive player that may tip the balance between tumor suppression and promotion, differentiation and invasion. Here we not only review the literature, but also explore genomic databases with a specific focus on Notch signatures, and how they relate to different stages in tumor development. Altered Notch signaling hereby plays a key role for tumor cell survival and coping with a broad spectrum of vital issues, contributing to failed therapies, poor patient outcome, and loss of lives.
... The NOTCH signaling mechanism involves a variety of cellular events such as cell differentiation, development, and cancer progression via interaction with other signaling pathways. Activation of the NOTCH signal strongly induces the expression of several proteins involved in EMT and resistance to apoptosis by inhibiting p53 during tumorigenesis [205][206][207][208][209]. The NOTCH signaling is initiated by the binding of Notch to Deltex, which is located on the membrane of neighbouring, followed by subsequent cleavage via metalloproteinases (ADAM17 or ADAM10) and presenilin-dependent gamma secretase complex (S1, S2, S3 and S4), resulting in synthesis of NOTCH intracellular domain (NICD) in cytosol [206]. ...
... Activation of the NOTCH signal strongly induces the expression of several proteins involved in EMT and resistance to apoptosis by inhibiting p53 during tumorigenesis [205][206][207][208][209]. The NOTCH signaling is initiated by the binding of Notch to Deltex, which is located on the membrane of neighbouring, followed by subsequent cleavage via metalloproteinases (ADAM17 or ADAM10) and presenilin-dependent gamma secretase complex (S1, S2, S3 and S4), resulting in synthesis of NOTCH intracellular domain (NICD) in cytosol [206]. The nuclear translocation of NICD leads to transcriptional activation of target genes including p27 cip1/waf1 , SNAI1, VEGF, AKT, mTOR, NF-κB, c-Myc and cyclins [205][206][207][208][209]. The NOTCH signal interacts with multiple oncogenic proteins such as platelet-derived growth factor (PDGF), SNAI1, hedgehog, WNT, AKT, mTOR, RAS, NF-κB, and sonic hedgehog (Shh). ...
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Full-text available
Autophagy is a highly conserved metabolic process involved in the degradation of intracellular components including proteins and organelles. Consequently, it plays a critical role in recycling metabolic energy for the maintenance of cellular homeostasis in response to various stressors. In cancer, autophagy either suppresses or promotes cancer progression depending on the stage and cancer type. Epithelial-mesenchymal transition (EMT) and cancer metastasis are directly mediated by oncogenic signal proteins including SNAI1, SLUG, ZEB1/2, and NOTCH1, which are functionally correlated with autophagy. In this report, we discuss the crosstalk between oncogenic signaling pathways and autophagy followed by possible strategies for cancer treatment via regulation of autophagy. Although autophagy affects EMT and cancer metastasis, the overall signaling pathways connecting cancer progression and autophagy are still illusive. In general, autophagy plays a critical role in cancer cell survival by providing a minimum level of energy via self-digestion. Thus, cancer cells face nutrient limitations and challenges under stress during EMT and metastasis. Conversely, autophagy acts as a potential cancer suppressor by degrading oncogenic proteins, which are essential for cancer progression, and by removing damaged components such as mitochondria to enhance genomic stability. Therefore, autophagy activators or inhibitors represent possible cancer therapeutics. We further discuss the regulation of autophagy-dependent degradation of oncogenic proteins and its functional correlation with oncogenic signaling pathways, with potential applications in cancer therapy.
... However, our laboratory showed in the same study that Hes1, the main intestinal Notch target, was indeed expressed at high levels in mouse and human adenomas but not in carcinomas, suggesting a role for Notch in tumour initiation but not necessarily in tumour progression. On the other hand, it has been shown that Notch activation in p53-/-mice led to tumour metastasis, through Epithelial-Mesenchymal-Transition (EMT) of the epithelial cancer cell (Chanrion et al., 2014). The current model suggests a dual role for Notch signalling: first in promoting tumorigenesis by synergizing with Wnt and later in inducing cell invasion and metastasis, in tumours that also lost p53. ...
... Indeed, conditioned media prepared from different tumouroid cultures displayed a penetrance of transformation ranging from 5% to 30%. I have examined, in experiments that are not presented in this thesis, the conditioned media derived from Apc flox/flox (n=2) or bCatenin ACT (n=1) dysplastic organoid cultures and observed that neither of them was able to induce transformation, contrary to tumouroids derived from N1IC;p53 -/mice (Chanrion et al., 2014), which were highly transforming (n=2). In view of the observed toxic effect of THBS1 neutralisation on tumour organoids, these preliminary results must be revisited. ...
Thesis
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Epithelial homeostasis and tumorigenesis are two intertwined concepts. Indeed, the formation of a tumour and its progression to aggressive stages are the consequence of a loss of control of the spatial and mechanical interactions of epithelial cells with their environment. Such perturbed tissue homeostasis can have two origins: an intrinsic one, often due to genetic mutations, causing mutant cells to lose the ability to correctly interpret environment signals, and an extrinsic or non-cell autonomous cause, as the environment surrounding mutant cells can no longer provides coherent information to correctly orchestrate tissue homeostasis.Previous results in the lab indicated that some tumour cells transcriptionally resemble normal stem cells. I was intrigued by this observation and decided to study the molecular basis of intratumoral heterogeneity, keeping in mind that normal-like stem cells could be present within tumours. My PhD was focused on examining interactions between normal and tumour epithelial cells, using the stroma-free model of organotypic cultures. During these studies, I discovered and characterised a hitherto unknown mechanism of cellular communication between tumour epithelial cells and genetically wild type epithelial cells in the context of colorectal cancer.Taking advantage of the intestinal organoid model system, allowing in vitro study of epithelial cells organising in a defined micro-environmental context free of stromal cells, I identified a rapid "transforming" effect of tumour cells on genetically wild type intestinal stem cells. I then demonstrated that this fast and reversible transformation was mediated by a secreted protein, and evaluated by SILAC mass spectrometry which proteins were specifically secreted by tumour but not normal organoids. This high-throughput quantitative analysis allowed us to identify a factor that was necessary for the observed transformation: thrombospondin-1 (Thbs1). Indeed, inhibition of Thbs1 by neutralizing antibodies or by genetic knock-out was sufficient to abolish the transforming capacity of tumour organoids. Transformation of wild type organoids by tumour organoids is manifested by a morphological change resulting in loss of cell polarisation and formation of hollow cysts, but also by a loss of compartmentalisation of proliferative cells, normally restricted to the crypt regions of organoids. In order to understand how Thbs1 induced such a change, I then analysed the transcriptome of transformed organoids by RNA sequencing and showed a specific activation of the Hippo signalling pathway in response to tumour-derived conditioned medium. This study shows how tumour cells can induce genetically wild type cells to switch to a tumour-like behaviour, using signal such as Hippo pathway activation normally employed during regeneration, making non-mutant cells dangerously adapted to survive and proliferate in a tumoral context.This work provides original mechanistic insights into the processes of early tumour remodelling and epithelial cell communication independently of stromal cells. The molecular mechanisms we have unveiled support the hypothesis that wild type stem cells can co-exist with mutant cells in tumours and contribute to tumour growth and clonal expansion, thanks to paracrine factors (like Thbs1) secreted by surrounding tumour cells, which allow them to thrive in the tumour environment
... imLECs and Colon Cancer Organoid Cocultures in the 3-Lane OrganoPlate. Colon cancer organoids were created from dissociated tumor tissue, as previously described by Sato et al. 51 Tumor tissues from a spontaneously metastasizing murine colorectal cancer model by Chanrion et al. 52 were used. The Chanrion group created a transgenic murine model in which Notch1 receptor is conditionally activated and p53 is deleted in the epithelium of the digestive tract. ...
... After gelation, imLECs were seeded into the perfusion channel. (B) Cocultures with imLECs and two distinct colon cancer organoid lines, generated from a spontaneously metastasizing murine colorectal cancer model, 52 shown from day 2−9. The organoids show sustained growth in the model as well as concomitant maintenance of the main imLEC vessel. ...
... Organoids were derived from spontaneously formed tumours in a transgenic mouse model with conditional activation of the Notch1 receptor and deletion of p53 in the digestive tract. 27 Exome sequencing revealed that all tumours harbour mutations in either the Ctnnb1 or Apc genes, demonstrating classical Wnt pathway activation. 27 CRC organoids were transduced with a lentiviral vector expressing luciferase and dTomato (pUltra-Chilli-Luc, Addgene #48688) and were FACS-purified. ...
... 27 Exome sequencing revealed that all tumours harbour mutations in either the Ctnnb1 or Apc genes, demonstrating classical Wnt pathway activation. 27 CRC organoids were transduced with a lentiviral vector expressing luciferase and dTomato (pUltra-Chilli-Luc, Addgene #48688) and were FACS-purified. ...
Article
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Background Physical activity is associated with a lower risk of colorectal cancer (CRC) and CRC-specific mortality. However, evidence for a causal relationship between physical activity and disease progression is lacking. Here, we have used CRC organoids to create a novel mouse model for spontaneous metastasis formation to multiple organs. We have used this model to assess the influence of voluntary exercise on disease progression. Methods Collagen-embedded murine colorectal tumour organoids were transplanted into the livers of immunocompetent C57Bl/6 mice using microsurgery. Voluntary exercise in tumour-bearing mice was modelled by offering running wheels continuously (n = 12) or 3 h/day (n = 12) versus no wheel access (n = 12). Running wheel revolutions were cumulatively measured every 30 min and physical activity was continuously monitored by infrared cameras. Food intake was monitored throughout the experiment and body composition was assessed with echoMRI. Animals were sacrificed 14 weeks after tumour initiation. Tumour load was quantified by EpCAM immunohistochemistry staining. Systemic inflammation parameters were assessed in blood plasma by a multiplex immunoassay. Results Tumour growth was initiated by implantation of CRC organoids into the livers of immunocompetent mice. The resulting tumours spontaneously formed distant metastases to non-implanted liver lobes and to the lungs. Mice with access to the running wheels for 3 h/day ran relatively short distances (2.3 ± 0.3 km/night; 221 ± 29 km total distance) with relatively high intensity (wheel revolutions/h). Mice with continuous access to the running wheels ran significantly longer distances (6.6 ± 3.0 km/night; 600 ± 290 km total distance) with a significantly lower intensity. Both exercise groups showed increased lean body mass, and decreased fat mass and body weight compared with tumour-bearing control mice. Food intake was unaffected by exercise or tumour growth. Primary tumour growth was not significantly affected by exercise. However, mice with continuous wheel access (long distance-lower intensity group) displayed increased lung metastasis and decreased liver metastasis formation, when compared with the sedentary control group. Short distance-higher intensity exercise did not affect metastasis formation. Analysis of blood cytokine levels revealed that mice with continuous wheel access displayed signs of systemic inflammation. Conclusions These results suggest that exercise has the potential to influence the patterns and extent of metastasis in CRC, and that the degree and intensity of exercise are likely to be important variables. Confirmation of these results in additional preclinical models with or without systemic treatment is warranted.
... The heterogeneity of primary tumors and their microenvironments has made it difficult to determine if precise signals dictate the mode of migration; some studies suggest that a combination of microenvironment, genetics, and signaling cues influence the migration mode selected by a primary tumor cell (12,36). Curiously, the structure of the tumor microenvironment and ECM components seems to promote collective migration in spontaneous murine colorectal tumors (37). In invasive regions of tumors with thick and straight collagen bundles, collectively migrating sheets and strings of connected tumor cells are present. ...
... In invasive regions of tumors with thick and straight collagen bundles, collectively migrating sheets and strings of connected tumor cells are present. However, in invasive regions of tumors with poorly organized, short and curly collagen fibers, isolated single cancer cells are present (37). This is particularly interesting in light of findings that bundling of collagen fibers provides migration tracks for breast carcinoma cells, and bundled collagen correlates with poor patient survival (38). ...
Article
Our understanding of the cellular mechanisms governing carcinoma invasiveness and metastasis has evolved dramatically over the last several years. The previous emphasis on the epithelial–mesenchymal transition as a driver of the migratory properties of single cells has expanded with the observation that carcinoma cells often invade and migrate collectively as adherent groups. Moreover, recent analyses suggest that circulating tumor cells within the vasculature often exist as multicellular clusters and that clusters more efficiently seed metastatic lesions than single circulating tumor cells. While these observations point to a key role for collective cell migration in carcinoma metastasis, the molecular mechanisms driving collective tumor cell migration remain to be discerned. Wnt/PCP (planar cell polarity) signaling, one of the noncanonical Wnt signaling pathways, mediates collective migratory events such as convergent extension during developmental processes. Wnt/PCP signaling components are frequently dysregulated in solid tumors, and aberrant pathway activation contributes to tumor cell migratory properties. Here we summarize key studies that address the mechanisms by which Wnt/PCP signaling mediate collective cell migration in developmental and tumor contexts. We emphasize Wnt/PCP component localization within migrating cells and discuss how component asymmetry may govern the spatiotemporal control of downstream cytoskeletal effectors to promote collective cell motility.
... This finding can be also related to some previous studies on the ordered mutations between oncogenes and tumor suppressor genes. For example, the double mutation in the order of TP53 and NOTCH, which are representative tumorsuppressor and oncogenes, respectively, was frequently observed in early stage of esophageal carcinoma patients [53], whereas the reverse-ordered mutation is likely to lead to a metastasis progression in mouse experiments [63,64]. It was also shown that alteration of RAS, which is another oncogene, before loss of P53 formed a malignant tumor with metastatic behavior, but the reverseordered mutation resulted in benign tumors [2,65]. ...
Article
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Background: Many previous clinical studies have found that accumulated sequential mutations are statistically related to tumorigenesis. However, they are limited in fully elucidating the significance of the ordered-mutation because they did not focus on the network dynamics. Therefore, there is a pressing need to investigate the dynamics characteristics induced by ordered-mutations. Methods: To quantify the ordered-mutation-inducing dynamics, we defined the mutation-sensitivity and the order-specificity that represent if the network is sensitive against a double knockout mutation and if mutation-sensitivity is specific to the mutation order, respectively, using a Boolean network model. Results: Through intensive investigations, we found that a signaling network is more sensitive when a double-mutation occurs in the direction order inducing a longer path and a smaller number of paths than in the reverse order. In addition, feedback loops involving a gene pair decreased both the mutation-sensitivity and the order-specificity. Next, we investigated relationships of functionally important genes with ordered-mutation-inducing dynamics. The network is more sensitive to mutations subject to drug-targets, whereas it is less specific to the mutation order. Both the sensitivity and specificity are increased when different-drug-targeted genes are mutated. Further, we found that tumor suppressors can efficiently suppress the amplification of oncogenes when the former are mutated earlier than the latter. Conclusion: Taken together, our results help to understand the importance of the order of mutations with respect to the dynamical effects in complex biological systems.
... Meng et al. have shown that Hes1 and Notch1 are upregulated in colon cancer, similar to other genes involved in chemoresistance (such as BCL2, BIRC5/Survivin and cyclinD1) and that they are involved in the malignant transformation of normal colonic mucosa [181]. These mutations contribute to an increased CSCs self-renewal and metastasis formation, while a combination of Notch1 activation and p53 deletion caused metastatic disease in colon cancer [185,186]. This would explain why the inhibition of Notch by γ-secretase inhibitors hampers the tumor growth of colorectal cancer cells [176]. ...
Article
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The Notch signaling pathway regulates cell proliferation, cytodifferentiation and cell fate decisions in both embryonic and adult life. Several aspects of stem cell maintenance are dependent from the functionality and fine tuning of the Notch pathway. In cancer, Notch is specifically involved in preserving self-renewal and amplification of cancer stem cells, supporting the formation, spread and recurrence of the tumor. As the function of Notch signaling is context dependent, we here provide an overview of its activity in a variety of tumors, focusing mostly on its role in the maintenance of the undifferentiated subset of cancer cells. Finally, we analyze the potential of molecules of the Notch pathway as diagnostic and therapeutic tools against the various cancers.
... This gene is considered a hematopoietic proto-oncogene in T-cell acute lymphoblastic leukemia, while it has a tumor-suppressor role in solid tumors-such as basal cell carcinoma of the skin, hepatocellular carcinoma, and in some forms of leukemia 6 . In addition, it has been shown that concomitant Notch activation and p53 deletion trigger epithelial-tomesenchymal transition and metastasis 7 . ...
Article
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Cancer driver gene alterations influence cancer development, occurring in oncogenes, tumor suppressors, and dual role genes. Discovering dual role cancer genes is difficult because of their elusive context-dependent behavior. We define oncogenic mediators as genes controlling biological processes. With them, we classify cancer driver genes, unveiling their roles in cancer mechanisms. To this end, we present Moonlight, a tool that incorporates multiple -omics data to identify critical cancer driver genes. With Moonlight, we analyze 8000+ tumor samples from 18 cancer types, discovering 3310 oncogenic mediators, 151 having dual roles. By incorporating additional data (amplification, mutation, DNA methylation, chromatin accessibility), we reveal 1000+ cancer driver genes, corroborating known molecular mechanisms. Additionally, we confirm critical cancer driver genes by analysing cell-line datasets. We discover inactivation of tumor suppressors in intron regions and that tissue type and subtype indicate dual role status. These findings help explain tumor heterogeneity and could guide therapeutic decisions. Identification of cancer driver genes, especially those that can act as tumour suppressors or oncogenes depending on context, remains a challenge. Here, the authors introduce Moonlight, a tool that integrates multi-omic data to address this challenge and identify numerous dual-role cancer genes.
... As other studies show, similar resources are used for omics data visualization in the context maps that can provide networkbased molecular portraits of studied cases. Comprehensive maps are rich in molecular details carefully compiled together, therefore structural analysis of the maps can explain particular phenotypes, redundancies, and robustness 49,50 . Such analysis together with omics data can guide to design of complex druggable interventions 51 . ...
Article
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The lack of integrated resources depicting the complexity of the innate immune response in cancer represents a bottleneck for high-throughput data interpretation. To address this challenge, we perform a systematic manual literature mining of molecular mechanisms governing the innate immune response in cancer and represent it as a signalling network map. The cell-type specific signalling maps of macrophages, dendritic cells, myeloid-derived suppressor cells and natural killers are constructed and integrated into a comprehensive meta map of the innate immune response in cancer. The meta-map contains 1466 chemical species as nodes connected by 1084 biochemical reactions, and it is supported by information from 820 articles. The resource helps to interpret single cell RNA-Seq data from macrophages and natural killer cells in metastatic melanoma that reveal different anti- or pro-tumor sub-populations within each cell type. Here, we report a new open source analytic platform that supports data visualisation and interpretation of tumour microenvironment activity in cancer.
... Besides, Fas signaling promotes motility and metastasis in GC in an EMT-dependent manner [70]. GC EMT can be stimulated by Notch activation and p53 deletion [71]. Other mechanisms include GSK3β inhibition, EphA2 overexpression, Wnt/β-catenin signaling activation, aquaporin 3 (AQP3) upregulation, overexpression of epidermal growth factor-like domain-containing protein 7 (EGFL7) and CEACAM6 [72][73][74][75][76]. ...
Article
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Infection with Helicobacter pylori, a Gram-negative, microaerophilic pathogen often results in gastric cancer in a subset of affected individuals. This explains why H. pylori is the only bacterium classified as a class I carcinogen by the World Health Organization. Several studies have pinpointed mechanisms by which H. pylori alters signaling pathways in the host cell to cause diseases. In this article, the authors have reviewed 234 studies conducted over a span of 18 years (2002–2020). The studies investigated the various mechanisms associated with gastric cancer induction. For the past 1.5 years, researchers have discovered new mechanisms contributing to gastric cancer linked to H. pylori etiology. Alongside alteration of the host signaling pathways using oncogenic CagA pathways, H. pylori induce DNA damage in the host and alter the methylation of DNA as a means of perturbing downstream signaling. Also, with H. pylori, several pathways in the host cell are activated, resulting in epithelial-to-mesenchymal transition (EMT), together with the induction of cell proliferation and survival. Studies have shown that H. pylori enhances gastric carcinogenesis via a multifactorial approach. What is intriguing is that most of the targeted mechanisms and pathways appear common with various forms of cancer.
... Another elegant system designed to track endogenous E-cadherin in MMTV-PyMT breast cancer mouse model combined with high-resolution intravital imaging allowed the identification of a subpopulation of cells undergoing EMT with invasive and metastatic properties, exposing as well high the intrinsic plasticity of EMT cells at metastatic sites [54], similar to results obtained using a different breast cancer model [55]. Lineage tracing in a Notch-p53-based colorectal cancer (CRC) mouse model also provided evidence for invasive cells exhibiting a gradient of epithelial and mesenchymal phenotypes [56]. In addition, several mouse models based in the genetic manipulation of EMT-TFs have reported the implication of Snail or Twist1 in EMT induction in PyMT-breast cancer or skin squamous cell carcinomas (SCC), respectively [48,57]. ...
Article
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Therapy resistance is responsible for tumour recurrence and represents one of the major challenges in present oncology. Significant advances have been made in the understanding of the mechanisms underlying resistance to conventional and targeted therapies improving the clinical management of relapsed patients. Unfortunately, in too many cases, resistance reappears leading to a fatal outcome. The recent introduction of immunotherapy regimes has provided an unprecedented success in the treatment of specific cancer types; however, a good percentage of patients do not respond to immune-based treatments or ultimately become resistant. Cellular plasticity, cancer cell stemness and tumour heterogeneity have emerged as important determinants of treatment resistance. Epithelial-to-mesenchymal transition (EMT) is associated with resistance in many different cellular and preclinical models, although little evidence derives directly from clinical samples. The recognition of the presence in tumours of intermediate hybrid epithelial/mesenchymal states as the most likely manifestation of epithelial plasticity and their potential link to stemness and tumour heterogeneity, provide new clues to understanding resistance and could be exploited in the search for anti-resistance strategies. Here, recent evidence linking EMT/epithelial plasticity to resistance against conventional, targeted and immune therapy are summarized. In addition, future perspectives for related clinical approaches are also discussed.
... Atg7 −/− mice develop no spontaneous intestinal tumors for up to 12 mo after tamoxifen injection. As previously reported (13,14), we found that p53 knockout in intestinal epithelial cells was not sufficient to initiate intestinal neoplasia during the first 12 mo after tamoxifen injection, with only a few tumors detected at this time point. By contrast, Atg7 −/− p53 −/− mice harbored many adenomas 12 mo after tamoxifen injection (Fig. 7A). ...
Article
The intestinal epithelium acts as a barrier between the organism and its microenvironment, including the gut microbiota. It is the most rapidly regenerating tissue in the human body thanks to a pool of intestinal stem cells (ISCs) expressing Lgr5 . The intestinal epithelium has to cope with continuous stress linked to its digestive and barrier functions. Epithelial repair is crucial to maintain its integrity, and Lgr5-positive intestinal stem cell (Lgr5 ⁺ ISC) resilience following cytotoxic stresses is central to this repair stage. We show here that autophagy, a pathway allowing the lysosomal degradation of intracellular components, plays a crucial role in the maintenance and genetic integrity of Lgr5 ⁺ ISC under physiological and stress conditions. Using conditional mice models lacking the autophagy gene Atg7 specifically in all intestinal epithelial cells or in Lgr5 ⁺ ISC, we show that loss of Atg7 induces the p53-mediated apoptosis of Lgr5 ⁺ ISC. Mechanistically, this is due to increasing oxidative stress, alterations to interactions with the microbiota, and defective DNA repair. Following irradiation, we show that Lgr5 ⁺ ISC repair DNA damage more efficiently than their progenitors and that this protection is Atg7 dependent. Accordingly, we found that the stimulation of autophagy on fasting protects Lgr5 ⁺ ISC against DNA damage and cell death mediated by oxaliplatin and doxorubicin treatments. Finally, p53 deletion prevents the death of Atg7 -deficient Lgr5 ⁺ ISC but promotes genetic instability and tumor formation. Altogether, our findings provide insights into the mechanisms underlying maintenance and integrity of ISC and highlight the key functions of Atg7 and p53.
... 12 Notably, the loss of p53 function has been associated with EMT, invasion, and metastasis in colorectal cancer. [13][14][15] Although reciprocal cross-talk between numerous intracellular signaling pathways is known to regulate and maintain EMT, it now is emerging that extracellular factors provided by the tumor microenvironment also can influence the tumor cell state and invasive potential. Indeed, it has been shown that cells undergoing EMT enhance the secretion of various factors, especially growth factors (transforming growth factor b [TGFb], hepatocyte growth factor [HGF]) 16 and proinflammatory cytokines, such as interleukins 6 and 8, which induce EMT. ...
Article
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Background & Aims Intratumor heterogeneity is a common feature of colorectal cancer (CRC). Here, we analyzed whether mesenchymal-like CRC cells promote the progression of epithelial-like CRC cells via paracrine mechanisms. Methods Six CRC cell lines that show an epithelial phenotype were treated with conditioned media (CM) from CRC cell lines that show a mesenchymal phenotype, and effects on epithelial-mesenchymal transition (EMT), migration, invasion, and chemoresistance were determined. Secreted factors potentially mediating these effects were identified by using cytokine arrays. Associations of these factors with tumor progression and patient survival were determined. Results CM obtained from mesenchymal-like CRC cells induced EMT associated with increased migration, invasion, and chemoresistance in epithelial-like CRC cell lines. Notably, activation of p53 in mesenchymal-like CRC cells prevented these effects of CM. Increased concentrations of several cytokines were identified in CM from mesenchymal-like CRC cell lines and a subset of these cytokines showed repression by p53. The down-regulation of nidogen-1 (NID1) was particularly significant and was owing to p53-mediated induction of microRNA-192 and microRNA-215, which directly target the NID1 messenger RNA. NID1 was found to be required and sufficient for inducing EMT, invasion, and migration in epithelial-like CRC cells. In primary CRCs, increased NID1 expression was associated with p53 mutation and microRNA-192/215 down-regulation. Importantly, increased NID1 expression in CRCs correlated with enhanced tumor progression and poor patient survival. Conclusions Taken together, our results show that CRC cells promote tumor progression via secreting NID1, which induces EMT in neighboring tumor cells. Importantly, the interference of p53 with this paracrine signaling between tumor cells may critically contribute to tumor suppression.
... This gene is considered a hematopoietic proto-oncogene in T-cell acute lymphoblastic leukemia, while it has a tumor-suppressor role in solid tumors-such as basal cell carcinoma of the skin, hepatocellular carcinoma, and in some forms of leukemia 6 . In addition, it has been shown that concomitant Notch activation and p53 deletion trigger epithelial-tomesenchymal transition and metastasis 7 . ...
... Inducing metastasis in these murine models has been challenging, with a few models able to reliably drive distant metastasis patterns (Burtin et al., 2020). Among these, spatial and temporal control over the expression of Apc, Kras, and Tp53 has been used to drive primary colon tumor formation and distant metastasis, whether using 4-OH-tamoxifenethanol enemas in iKAP mice (Apc Lox/Lox ; p53 Lox/Lox ; Tet-O-LSL-Kras G12D ; VillinCre ERT2 ) or surgical adenoviral Cre administration in Apc CKO/CKO LSL-G12D; Kras tm4tyj/+ mice (Chanrion et al., 2014;Haigis et al., 2008;Hung et al., 2010). However, despite their usefulness in studying carcinogenesis, GEMMs tend to be costly, slow, and not necessarily representative of the diverse genetic drivers of clinical metastasis (Francia et al., 2011). ...
Article
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Colon cancer remains the third most common cause of cancer in the US, and the third most common cause of cancer death. Worldwide, colon cancer is the second most common cause of cancer and cancer deaths. At least 25% of patients still present with metastatic disease, and at least 25-30% will develop metastatic colon cancer in the course of their disease. While chemotherapy and surgery remain the mainstay of treatment, understanding the fundamental cellular niche and mechanical properties that result in metastases would facilitate both prevention and cure. Advances in biomaterials, novel 3D primary human cells, modelling using microfluidics and the ability to alter the physical environment, now offers a unique opportunity to develop and test impactful treatment.
... In the intestine, NOTCH maintains intestinal stem cell function and, in concert with other signaling pathways, regulates cell fate decisions (Fre et al., 2005;Milano et al., 2004;Stanger et al., 2005;van Es et al., 2005). Although NOTCH signaling can confer both tumorsuppressive and -promoting properties depending on cancer type (Siebel and Lendahl, 2017), in CRC, NOTCH appears to be protumorigenic (Chanrion et al., 2014;Chu et al., 2010;Fre et al., 2009;Sonoshita et al., 2011;Ueo et al., 2012). Activating NOTCH mutations are very rare in CRC, yet overexpression of NOTCH receptors and ligands has been described (Noah and Shroyer, 2013). ...
Article
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Recently, a transcriptome-based consensus molecular subtype (CMS) classification of colorectal cancer (CRC) has been established, which may ultimately help to individualize CRC therapy. However, the lack of animal models that faithfully recapitulate the different molecular subtypes impedes adequate preclinical testing of stratified therapeutic concepts. Here, we demonstrate that constitutive AKT activation in intestinal epithelial cells markedly enhances tumor invasion and metastasis in Trp53ΔIEC mice (Trp53ΔIECAktE17K) upon challenge with the carcinogen azoxymethane. Gene-expression profiling indicates that Trp53ΔIECAktE17K tumors resemble the human mesenchymal colorectal cancer subtype (CMS4), which is characterized by the poorest survival rate among the four CMSs. Trp53ΔIECAktE17K tumor cells are characterized by Notch3 up-regulation, and treatment of Trp53ΔIECAktE17K mice with a NOTCH3-inhibiting antibody reduces invasion and metastasis. In CRC patients, NOTCH3 expression correlates positively with tumor grading and the presence of lymph node as well as distant metastases and is specifically up-regulated in CMS4 tumors. Therefore, we suggest NOTCH3 as a putative target for advanced CMS4 CRC patients.
... In the case of gut cancer, simultaneous Notch overexpression and p53 deletion in a TGFβ-induced EMT network drives settling of cells exhibiting invasion or EMT or other phenotypic attractors into a metastatic one [125]. This suggests that the synergistic effect could have favored migration, as has been corroborated by experiments in mouse gut [126]. ...
Article
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Cancer is a multifactorial disease. Aberrant functioning of the underlying complex signaling network that orchestrates cellular response to external or internal cues governs incidence, progression, and recurrence of cancer. Detailed understanding of cancer's etiology can offer useful insights into arriving at novel therapeutic and disease management strategies. Such an understanding for most cancers is currently limited due to unavailability of a predictive large‐scale, integrated signaling model accounting for all tumor orchestrating factors. We suggest that the potential of Boolean dynamic (BD) modeling approaches, though qualitative, can be harnessed for developing holistic models capturing multi‐scale, multi‐cellular signaling processes involved in cancer incidence and progression. We believe that constraining such an integrated BD model with variety of omics data at different scales from laboratory and clinical settings could offer deeper insights into causal mechanisms governing the disease leading to better prognosis. We review the recent literature employing different BD modeling strategies to model variety of cancer signaling programs leading to identification of cancer‐specific prognostic markers such as SMAD proteins, which may also serve as early predictors of tumor cells hijacking the epithelial‐mesenchymal plasticity program. In silico simulations of BD models of different cancer signaling networks combined with attractor landscape analysis and validated with experimental data predicted the nature of short‐ and long‐term response of standard targeted therapeutic agents such as Nutlin‐3, a small molecule inhibitor for p53‐MDM2 interaction. BD simulations also offered a mechanistic view of emerging resistance to drugs such as Trastuzumab for HER+ breast cancer, analysis of which suggested new combination therapies to circumvent them. We believe future improvements in BD modeling techniques, and tools can lead to development of a comprehensive platform that can drive holistic approaches toward better decision‐making in the clinical settings, and thereby help identify novel therapeutic strategies for improved cancer treatment at personalised levels.
... Comparison of TGFBR OFF simulations showed differences between the original and our model in two cases: NOTCH or MEK KI led to EMT in the original but not in the compartmentalized model. In the case of NOTCH activation, our model better simulated the experimental results showing that Neurogenic locus notch homolog protein 1 (NOTCH) activation alone without the induction of TGFB was not sufficient to induce EMT 36,37 ( Supplementary Figs. 3 and 4). This result stemmed from the fact that in our model the activation of Zinc finger protein SNAI1 (SNAI1) is captured more complexly, and NOTCH alone could not activate this key transcription factor of EMT. ...
Article
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Regulation of translocating proteins is crucial in defining cellular behaviour. Epithelial-mesenchymal transition (EMT) is important in cellular processes, such as cancer progression. Several orchestrators of EMT, such as key transcription factors, are known to translocate. We show that translocating proteins become enriched in EMT-signalling. To simulate the compartment-specific functions of translocating proteins we created a compartmentalized Boolean network model. This model successfully reproduced known biological traits of EMT and as a novel feature it also captured organelle-specific functions of proteins. Our results predicted that glycogen synthase kinase-3 beta (GSK3B) compartment-specifically alters the fate of EMT, amongst others the activation of nuclear GSK3B halts transforming growth factor beta-1 (TGFB) induced EMT. Moreover, our results recapitulated that the nuclear activation of glioma associated oncogene transcription factors (GLI) is needed to achieve a complete EMT. Compartmentalized network models will be useful to uncover novel control mechanisms of biological processes. Our algorithmic procedures can be automatically rerun on the https://translocaboole.linkgroup.hu website, which provides a framework for similar future studies.
... In particular, the expression of Ki67 (MKI67), which has an integral role in cell cycle progression, was not detected, suggesting that CTCs are a cell population in which growth has been arrested. In general, mesenchymal cancer cells have lower proliferative potential than epithelial cancer cells (Fig. 3C) 45 . This low proliferative potential of CTCs may be due to EMT. ...
Article
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Transcriptome analysis of circulating tumor cells (CTCs), which migrate into blood vessels from primary tumor tissues, at the single-cell level offers critical insights into the biology of metastasis and contributes to drug discovery. However, transcriptome analysis of single CTCs has only been reported for a limited number of cancer types, such as multiple myeloma, breast, hepatocellular, and prostate cancer. Herein, we report the transcriptome analysis of gastric cancer single-CTCs. We utilized an antigen-independent strategy for CTC isolation from metastatic gastric cancer patients involving a size-dependent recovery of CTCs and a single cell isolation technique. The transcriptomic profile of single-CTCs revealed that a majority of gastric CTCs had undergone epithelial-mesenchymal transition (EMT), and indicated the contribution of platelet adhesion toward EMT progression and acquisition of chemoresistance. Taken together, this study serves to employ CTC characterization to elucidate the mechanisms of chemoresistance and metastasis in gastric cancer.
... CSCs dependent on β-catenin hyperactivation Wend et al, 2013). In addition, we showed that knockout of the epigenetic regulator Mll1 in the salivary gland cancer model (Zhu et al, 2019) and in a Wnt-high model of colorectal cancer (Grinat et al, 2020) as well as deletion of Yap in the breast cancer model (Quinn et al, 2021) (Chanrion et al, 2014). ...
Thesis
Organoide aus adulten Mäusen sind vielversprechende Modelle für die Nierenforschung. Ihre Charakterisierung wurde jedoch nicht auf ein zufriedenstellendes Niveau gebracht. Hier habe ich ein langfristiges 3D-Maus-Organoid (Tubuloid)-Modell etabliert und charakterisiert, das die Erneuerung und die Reparatur sowie die Architektur und die Funktionalität der adulten tubulären Epithelien rekapituliert. In der Zukunft wird das Modell detaillierte Untersuchungen der Trajektorien selbsterneuernder Zellen sowohl zur teilweisen Wiederherstellung der Niere als auch zur malignen Transformation der Niere ermöglichen. Das klarzellige Nierenzellkarzinom (ccRCC) ist der häufigste und aggressivste Nierenkrebs. Die Inaktivierung des Tumorsuppressorgens Von Hippel-Lindau (VHL) ist der Haupttreiber des ccRCCs. Zuvor hatten wir die Hochregulation der Wnt- und Notch-Signalübertragung in den CXCR4+MET+CD44+-Krebsstammzellen (CSC) aus primären humanen ccRCC-Tumoren identifiziert. Das Blockieren von Wnt und Notch in von Patienten stammenden Xenotransplantaten, Organoiden und nicht-anhaftenden Sphären unter Verwendung von niedermolekularen Inhibitoren beeinträchtigte die Selbsterneuerung der CSC und das Tumorwachstum. Um CSC-gesteuertes humanes ccRCC in genetischen Mausmodellen nachzuahmen, begann ich mit der Erzeugung von zwei Doppelmausmutanten; β-Catenin-GOF; Notch-GOF und Vhl-LOF; β-Catenin-GOF. Sowohl die β-Catenin-GOF; Notch-GOF Mausmutante als auch die Vhl-LOF; β-Catenin-GOF Mausmutante entwickelten innerhalb einiger Monate schwere Krankheitssymptome. Überraschenderweise beobachtete ich weder Tumore oder Tumorvorläuferläsionen noch höhere Zellproliferationsraten in den mutierten Nieren. Weitere Analysen ergaben, dass die Mausmutanten Merkmale chronischer Nierenerkrankung (CKD) aufwiesen.
... not cause any noticeable phenotype44,45 (Fig. 6cand SupplementaryFig. 6d). ...
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The R2TP chaperone cooperates with HSP90 to integrate newly synthesized proteins into multi-subunit complexes, yet its role in tissue homeostasis is unknown. Here, we generated conditional, inducible knock-out mice for Rpap3 to inactivate this core component of R2TP in the intestinal epithelium. In adult mice, Rpap3 invalidation caused destruction of the small intestinal epithelium and death within 10 days. Levels of R2TP substrates decreased, with strong effects on mTOR, ATM and ATR. Proliferative stem cells and progenitors deficient for Rpap3 failed to import RNA polymerase II into the nucleus and they induced p53, cell cycle arrest and apoptosis. Post-mitotic, differentiated cells did not display these alterations, suggesting that R2TP clients are preferentially built in actively proliferating cells. In addition, high RPAP3 levels in colorectal tumors from patients correlate with bad prognosis. Here, we show that, in the intestine, the R2TP chaperone plays essential roles in normal and tumoral proliferation. RPAP3 is a subunit of the R2TP complex, a co-chaperone of HSP90, with substrate proteins involved in transcription, ribosome biogenesis, DNA repair and cell growth. Here the authors report that deletion of Rpap3 abrogates cell proliferation and homeostasis in mouse intestine, partly through destabilization of PI3K-like kinases, while elevated RPAP3 levels in colorectal tumors are associated with poor prognosis.
... Alterations in the Notch signaling pathway (mutations in ligands and/or receptors, or the overexpression or impaired translocation of these proteins) have been described in several malignancies, including lymphoid leukemia, melanoma, glioblastoma, and cancers of the breast, ovary, lung, pancreas, colon, head and neck, cervix, and kidney (Radtke and Raj, 2003;Koch and Radtke, 2007;Puente et al., 2011). The Notch activation pathway might also promote an epithelial-to-mesenchymal cell transition, cancer progression, and metastasis (Chanrion et al., 2014). Gamma secretase inhibitors are the mainstay of Notch-related drug therapy (Krop et al., 2012;Lee et al., 2015;Messersmith et al., 2015;Massard et al., 2016). ...
Article
In humans, inhibition of Notch oncogenic signaling leads to tumor regression. Preclinical studies indicate that Notch signaling contributes to the maintenance of intestinal homeostasis. Here, we sought to describe the intestinal effects of a first-in-human Notch inhibitor in an indication of refractory cancer. Between 2014 and 2017, adult patients treated for refractory cancer with the novel Notch inhibitor LY3039478 and who had grade ≥ 2 diarrhea were referred to the gastroenterology department of a tertiary hospital in the Paris region of France. Eleven patients (median (range) age: 72 (29-83)) were included in the study. All patients had advanced cancer: adenoid cystic carcinoma (n=3, 27 %), sarcoma (n=3, 27 %), and other types (n=5, 46 %). In all cases, digestive tract endoscopy revealed abundant mucus in the intestinal lumen, and digestive tract biopsies showed an abnormally low proportion of enterocytes and marked elevation of the proportion of pseudostratified goblet cells. Microscopic inflammation was seen in colon biopsies from 2 of the 11 patients (18 %). The clinical, endoscopic and histological abnormalities were dependent on the dose of Notch inhibitor. All patients resolved their digestive signs or symptoms after discontinuing the dose and the median (range) time interval between discontinuation of the Notch inhibitor and resolution of all the gastrointestinal signs and symptoms was 7 days (4-24). Likewise, the median time interval between discontinuation and resolution of the histological abnormalities was 7 days (1-10). Blocking Notch signaling induces secretory cell metaplasia of the intestinal epithelium, which in turn leads to transient diarrhea. Our results confirm the role of Notch signaling in intestinal homeostasis in humans.
... In patients, TP53 is correlated with advanced cancer stages. In mice with a constitutively active Notch background, its combination with Apc loss increases invasiveness136 . ...
Thesis
The metastatic dissemination of cancer remains a major issue in patients’ treatment. Cell biology approaches, such as organoids, have the potential to bridge histological and molecular analyses to decipher the onco-morphogenetic programs that fuel cancer dissemination. The systematic prospective analysis of peritoneal effusions from patients with colorectal carcinoma (CRC) identified new tumoral intermediates called Tumour Spheres with Inverted polarity (TSIPs). TSIPs are cell clusters mediating the metastatic spread of CRC evolving from the serrated pathway, but their underlying biology is unknown. While all TSIPs harbor an inverted apico-basolateral polarity in fluids, here, we report two distinct topologies in tissues and organotypic culture: TSIPs either conserve the inverted “apical-out” polarity or switch to a conventional “apical-in” polarity. We identified TGFβ and focal adhesion signaling as the main drivers of polarity orientation. Moreover, the automated assessment of these phenotypes and the calculation of a polarity score proved that the apical-out histology is associated with poor patient survival. This study identifies new tumour cell behaviors and their associated oncogenic pathways that could be exploited to stratify mucinous CRC patients with high risk of metastatic spread.
... Detailed descriptions of disease mechanisms on the level of molecular processes have recently become available [1,2] , with many examples of practical applications in the field of cancer research [3][4][5][6][7] . These disease maps are needed for integrating scattered knowledge and for advanced data interpretation and hypothesis generation [1,2] . ...
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Detailed maps of the molecular basis of the disease are powerful tools for interpreting data and building predictive models. Modularity and composability are considered necessary network features for large-scale collaborative efforts to build comprehensive molecular descriptions of disease mechanisms. An effective way to create and manage large systems is to compose multiple subsystems. Composable network components could effectively harness the contributions of many individuals and enable teams to seamlessly assemble many individual components into comprehensive maps. We examine manually-built versions of the RAS-RAF-MEK-ERK cascade from the Atlas of Cancer Signalling Network, PANTHER and Reactome databases and review them in terms of their reusability and composability for assembling new disease models. We identify design principles for managing complex systems that could make it easier for investigators to share and reuse network components. We demonstrate the main challenges including incompatible levels of detail and ambiguous representation of complexes and highlight the need to address these challenges.
... Detailed descriptions of disease mechanisms on the level of molecular processes have recently become available [1,2], with many examples of practical applications in the field of cancer research [3][4][5][6][7]. These disease maps are needed for integrating scattered knowledge and for advanced data interpretation and hypothesis generation [1,2]. ...
Article
Full-text available
Detailed maps of the molecular basis of the disease are powerful tools for interpreting data and building predictive models. Modularity and composability are considered necessary network features for large-scale collaborative efforts to build comprehensive molecular descriptions of disease mechanisms. An effective way to create and manage large systems is to compose multiple subsystems. Composable network components could effectively harness the contributions of many individuals and enable teams to seamlessly assemble many individual components into comprehensive maps. We examine manually built versions of the RAS–RAF–MEK–ERK cascade from the Atlas of Cancer Signalling Network, PANTHER and Reactome databases and review them in terms of their reusability and composability for assembling new disease models. We identify design principles for managing complex systems that could make it easier for investigators to share and reuse network components. We demonstrate the main challenges including incompatible levels of detail and ambiguous representation of complexes and highlight the need to address these challenges.
... A study by Staneva et al. (2019) explored the migration pattern of cancer cells in the tumor core of Notch1 intracellular domain (NICD)/p53 À/À tumor model, reported that the invasive front of the tumor is comprised not only epithelial E-cadherin-positive cancer cell cluster, but also smooth muscle actin (a-SMA)-and ZEB-1-positive single cells. These ZEB-1 positive single cells were found to be of epithelial origin that have undergone an EMT (Chanrion et al. 2014), which actively migrate from the tumor core to the invasive front via actin cytoskeleton that is densely distributed in the tumor core, independent of division rate (Staneva et al. 2019). Interestingly, post-hypoxic tumor cells have the ability to acquire ROS-resistant phenotype which provides survival benefits as they enter the bloodstream in overt metastasis (Godet et al. 2019). ...
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Hypoxia is a common feature of the tumor microenvironment (TME) of nearly all solid tumors, leading to therapeutic failure. The changes in stiffness of the extracellular matrix (ECM), pH gradients, and chemical balance that contribute to multiple cancer hallmarks are closely regulated by intratumoral oxygen tension via its primary mediators, hypoxia-inducible factors (HIFs). HIFs, especially HIF-1α, influence these changes in the TME by regulating vital cancer-associated signaling pathways and cellular processes including MAPK/ERK, NF-κB, STAT3, PI3K/Akt, Wnt, p53, and glycolysis. Interestingly, research has revealed the involvement of epigenetic regulation by hypoxia-regulated microRNAs (HRMs) of downstream target genes involved in these signaling. Through literature search and analysis, we identified 48 HRMs that have a functional role in the regulation of 5 key cellular processes: proliferation, metabolism, survival, invasion and migration, and immunoregulation in various cancers in hypoxic condition. Among these HRMs, 17 were identified to be directly associated with HIFs which include miR-135b, miR-145, miR-155, miR-181a, miR-182, miR-210, miR-224, miR-301a, and miR-675-5p as oncomiRNAs, and miR-100-5p, miR-138, miR-138-5p, miR-153, miR-22, miR-338-3p, miR-519d-3p, and miR-548an as tumor suppressor miRNAs. These HRMs serve as a potential lead in the development of miRNA-based targeted therapy for advanced solid tumors. Future development of combined HIF-targeted and miRNA-targeted therapy is possible, which requires comprehensive profiling of HIFs-HRMs regulatory network, and improved formula of the delivery vehicles to enhance the therapeutic kinetics of the targeted cancer therapy (TCT) moving forward.
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Epithelial to mesenchymal transition (EMT) is a complex biological process that plays a key role in cancer progression and metastasis formation. Its activation results in epithelial cells losing adhesion and polarity and becoming capable of migrating from their site of origin. At this step the disease is generally considered incurable. As EMT execution involves several individual molecular components, connected by nontrivial relations, in vitro techniques are often inadequate to capture its complexity. Computational models can be used to complement experiments and provide additional knowledge difficult to build up in a wetlab. Indeed in silico analysis gives the user total control on the system, allowing to identify the contribution of each independent element. In the following, two kinds of approaches to the computational study of EMT will be presented. The first relies on signal transduction networks description and details how changes in gene expression could influence this process, both focusing on specific aspects of the EMT and providing a general frame for this phenomenon easily comparable with experimental data. The second integrates single cell and population level descriptions in a multiscale model that can be considered a more accurate representation of the EMT. The advantages and disadvantages of each approach will be highlighted, together with the importance of coupling computational and experimental results. Finally, the main challenges that need to be addressed to improve our knowledge of the role of EMT in the neoplastic disease and the scientific and translational value of computational models in this respect will be presented. This article is categorized under: • Analytical and Computational Methods > Computational Methods
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Epithelial tissues respond to a wide variety of environmental and genotoxic stresses. As an adaptive mechanism, cells can deviate from their natural paths to acquire new identities, both within and across lineages. Under extreme conditions, epithelial tissues can utilize "shape-shifting" mechanisms whereby they alter their form and function at a tissue-wide scale. Mounting evidence suggests that in order to acquire these alternate tissue identities, cells follow a core set of "tissue logic" principles based on developmental paradigms. Here, we review the terminology and the concepts that have been put forward to describe cell plasticity. We also provide insights into various cell intrinsic and extrinsic factors, including genetic mutations, inflammation, microbiota, and therapeutic agents that contribute to cell plasticity. Additionally, we discuss recent studies that have sought to decode the "syntax" of plasticity-i.e., the cellular and molecular principles through which cells acquire new identities in both homeostatic and malignant epithelial tissues-and how these processes can be manipulated for developing novel cancer therapeutics.
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Epithelial‐to‐mesenchymal transition (EMT), a process through which epithelial tumor cells acquire mesenchymal phenotypic properties, contributes to both metastatic dissemination and therapy resistance in cancer. Accumulating evidence indicates that non‐epithelial tumors, including melanoma, can also gain mesenchymal‐like properties that increase their metastatic propensity and decrease their sensitivity to therapy. In this review, we discuss recent findings, illustrating the striking similarities ‐but also knowledge gaps‐ between the biology of mesenchymal‐like state(s) in melanoma and mesenchymal state(s) from epithelial cancers. Based on this comparative analysis, we suggest hypothesis‐driven experimental approaches to further deepen our understanding of the EMT‐like process in melanoma and how such investigations may pave the way towards the identification of clinically‐relevant biomarkers for prognosis and new therapeutic strategies.
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Cancer is a complex and heterogeneous disease, and cancer cells dynamically interact with the mechanical microenvironment such as hydrostatic pressure, fluid shear, and interstitial flow. These factors play an essential role in cell fate and circulating tumor cell heterogeneity, and can influence the cellular phenotype. In this study, a peristaltic continuous flow reactor is designed and applied to HCT‐116 colorectal carcinoma cells to mimic the fluid dynamics of circulation. With this intervention, a CD44/CD24‐cell subpopulation emerges, and 100 genes are significantly regulated. The expression of cells at 4 h in the flow reactor is very similar to TGF‐ß treatment, which is an inducer of epithelial–mesenchymal transition. ATF3 and SERPINE1 are significantly upregulated in these groups, suggesting that the mesenchymal transition is induced through this signaling pathway. This flow reactor model is satisfactory on its own to reprogram colorectal cancer cells toward a more mesenchymal niche mimicking circulation of the blood. This work represents a broadly applicable method to model and predict how colorectal cancer cells respond to hemodynamic continuous flow conditions, which can enable adjusting biological and physical factors under well‐controlled parameters. This flow reactor model can reprogram colorectal cancer cells toward a more mesenchymal niche in circulation, revealing itself as a promising method to mimic fluid dynamics of circulation.
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Cancer cell identity and plasticity are required in transition states, such as epithelial–mesenchymal transition (EMT) and mesenchymal–epithelial transition (MET), in primary tumor initiation, progression, and metastasis. The functional roles of EMT, MET, and the partial state (referred to as pEMT) may vary based on the type of tumor, the state of dissemination, and the degree of metastatic colonization. Herein, we review EMT, MET, pEMT, and plasticity in the context of tumor metastasis.
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The metastatic process of colorectal cancer (CRC) is not fully understood and effective therapies are lacking. We show that activation of NOTCH1 signaling in the murine intestinal epithelium leads to highly penetrant metastasis (100% metastasis; with >80% liver metastases) in KrasG12D-driven serrated cancer. Transcriptional profiling reveals that epithelial NOTCH1 signaling creates a tumor microenvironment (TME) reminiscent of poorly prognostic human CRC subtypes (CMS4 and CRIS-B), and drives metastasis through transforming growth factor (TGF) β-dependent neutrophil recruitment. Importantly, inhibition of this recruitment with clinically relevant therapeutic agents blocks metastasis. We propose that NOTCH1 signaling is key to CRC progression and should be exploited clinically.
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The epithelial-mesenchymal transition (EMT) is a process of cell transformation under certain physiological and pathological states in which epithelial cells are transformed into mesenchymal cells with fibroblast-like properties, which confers upon them the increased invasion and migration capabilities of cancer cells. Previous studies have demonstrated that SRY-related high-mobility-group box 4 (Sox4) protein coordinates EMT-related pathways and EMT-related transcription factors, thereby regulating the EMT process. The focus of this review is to evaluate recent advances regarding the role of Sox4 protein in the cancer EMT. First, we provide an overview of the general background of Sox4 (structure and function) and the EMT in cancer. Next, we introduce the interactions between Sox4 protein and various factors during cancer EMT. Finally, we suggest directions for future investigations. In general, the information compiled in this paper should serve as a comprehensive repository of information on the subject matter and contribute to the design of other research and future efforts to develop therapeutic strategies that target the Sox4 protein.
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Cancer initiation and progression are associated with multiple molecular mechanisms. The knowledge of these mechanisms is expanding and should be converted into guidelines for tackling the disease. Here, we discuss the formalization of biological knowledge into a comprehensive resource: the Atlas of Cancer Signalling Network (ACSN) and the Google Maps-based tool NaviCell, which supports map navigation. The application of ACSN for omics data visualization, in the context of signalling maps, is possible via the NaviCell Web Service module and through the NaviCom tool. It allows generation of network-based molecular portraits of cancer using multilevel omics data. We review how these resources and tools are applied for cancer preclinical studies. Structural analysis of the maps together with omics data helps to rationalize the synergistic effects of drugs and allows design of complex disease stage-specific druggable interventions. The use of ACSN modules and maps as signatures of biological functions can help in cancer data analysis and interpretation. In addition, they empowered finding of associations between perturbations in particular molecular mechanisms and the risk to develop a specific type of cancer. These approaches are helpful, among others, to study the interplay between molecular mechanisms of cancer. It opens an opportunity to decipher how gene interactions govern the hallmarks of cancer in specific contexts. We discuss a perspective to develop a flexible methodology and a pipeline to enable systematic omics data analysis in the context of signalling network maps, for stratifying patients and suggesting interventions points and drug repositioning in cancer and other diseases.
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Notch plays a protumorigenic role in many cancers including prostate cancer (PCa). Global notch inhibition of multiple Notch family members using γ‐secretase inhibitors has shown efficacy in suppressing PCa growth in murine models. However, global Notch inhibition is associated with marked toxicity due to the widespread function of many different Notch family members in normal cell physiology. Accordingly, in the current study, we explored if specific inhibition of Notch1 would effectively inhibit PCa growth in a murine model. The androgen‐dependent VCaP and androgen‐independent DU145 cell lines were injected subcutaneously into mice. The mice were treated with either control antibody 1B7.11, anti‐Notch1 antibody (OMP‐A2G1), docetaxel or the combination of OMP‐A2G1 and docetaxel. Tumor growth was measured using calipers. At the end of the study, tumors were assessed for proliferative response, apoptotic response, Notch target gene expression, and DNA damage response (DDR) expression. OMP‐A2G1 alone inhibited tumor growth of both PCa cell lines to a greater extent than docetaxel alone. There was no additive or synergistic effect of OMP‐A2G1 and docetaxel. The primary toxicity was weight loss that was controlled with dietary supplementation. Proliferation and apoptosis were affected differentially in the two cell lines. OMP‐A2G1 increased expression of the DDR gene GADD45α in VCaP cells but downregulated GADD45α in Du145 cells. Taken together, these data show that Notch1 inhibition decreases PCa xenograft growth but does so through different mechanisms in the androgen‐dependent VCaP cell line vs the androgen‐independent DU145 cell line. These results provide a rationale for further exploration of targeted Notch inhibition for therapy of PCa.
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Generation and usage of high-quality molecular signalling network maps can be augmented by standardising notations, establishing curation workflows and application of computational biology methods to exploit the knowledge contained in the maps. In this manuscript, we summarize the major aims and challenges of assembling information in the form of comprehensive maps of molecular interactions. Mainly, we share our experience gained while creating the Atlas of Cancer Signalling Network. In the step-by-step procedure, we describe the map construction process and suggest solutions for map complexity management by introducing a hierarchical modular map structure. In addition, we describe the NaviCell platform, a computational technology using Google Maps API to explore comprehensive molecular maps similar to geographical maps, and explain the advantages of semantic zooming principles for map navigation. We also provide the outline to prepare signalling network maps for navigation using the NaviCell platform. Finally, several examples of cancer high-throughput data analysis and visualization in the context of comprehensive signalling maps are presented.
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Neuropilin-2 (Nrp2), an important regulator of lymphangiogenesis and lymphatic metastasis, has been associated with progression in colorectal cancer (CRC). However, the tumor cell-intrinsic role of Nrp2 in cancer progression is incompletely understood. To address this question, we employed CRISPR-Cas9 technology to generate Nrp2-knockout organoids derived from murine CRC tumors with a mesenchymal phenotype. Transcriptome profiling and tumor tissue analysis showed that Nrp2 loss resulted in mesenchymal-to-epithelial transition (MET), which was accompanied with restored polarity and tight junction stabilization. Signaling pathway analysis revealed that Nrp2-knockout organoids acquire de novo dependency on insulin receptor (IR) signaling and autophagy as alternative survival mechanisms. Combined inhibition of IR signaling and autophagy prevented the stabilization of cell-cell junctions, reduced metabolic activity, and caused profound cell death in Nrp2-knockout organoids. Collectively, the data demonstrate a key role for Nrp2 in maintaining the aggressive phenotype and survival of tumor-derived CRC organoids. The identified connection between Nrp2, insulin receptor signaling and autophagy may guide the development of novel combination-treatment strategies for aggressive CRC.
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The NOTCH gene was identified approximately 110 years ago. Classical studies have revealed that NOTCH signaling is an evolutionarily conserved pathway. NOTCH receptors undergo three cleavages and translocate into the nucleus to regulate the transcription of target genes. NOTCH signaling deeply participates in the development and homeostasis of multiple tissues and organs, the aberration of which results in cancerous and noncancerous diseases. However, recent studies indicate that the outcomes of NOTCH signaling are changeable and highly dependent on context. In terms of cancers, NOTCH signaling can both promote and inhibit tumor development in various types of cancer. The overall performance of NOTCH-targeted therapies in clinical trials has failed to meet expectations. Additionally, NOTCH mutation has been proposed as a predictive biomarker for immune checkpoint blockade therapy in many cancers. Collectively, the NOTCH pathway needs to be integrally assessed with new perspectives to inspire discoveries and applications. In this review, we focus on both classical and the latest findings related to NOTCH signaling to illustrate the history, architecture, regulatory mechanisms, contributions to physiological development, related diseases, and therapeutic applications of the NOTCH pathway. The contributions of NOTCH signaling to the tumor immune microenvironment and cancer immunotherapy are also highlighted. We hope this review will help not only beginners but also experts to systematically and thoroughly understand the NOTCH signaling pathway.
Article
Background Adenoid cystic carcinoma (ACC) is a rare salivary cancer. The highest rates of disease recurrence are in patients with NOTCH pathway activation, reported in up to 20%. Novel drugs targeting NOTCH signaling are under investigation in the recurrent/metastatic (R/M) setting. To understand their clinical utility, there is an urgent need to better characterize the disease course and outcomes following current standard of care treatment. Methods 120 patients with R/M ACC underwent clinical review at a single UK Cancer Centre. Patients were retrospectively assessed for tumor NOTCH pathway activation using next generation sequencing (NGS) targeting NOTCH1/2/3 genes and/or NOTCH1 intra-cellular domain (NICD1) immunohistochemistry. Demographic and treatment data were extracted from the clinical notes. Kaplan-Meier survival analysis was performed using log rank test. Results NOTCH pathway activation was identified in 13/120 patients (11 %). In 12/101 patients analyzed by NGS, NOTCH1/3 activating somatic mutations were identified, and a further patient was identified with NICD1 diffuse nuclear staining in whom NGS testing was not possible. Patients with NOTCH pathway activation had shorter median RFS (1.1 vs 3.4 years, p = 0.2032) and significantly reduced median OS from diagnosis (4.0 vs 16.3 years, p < 0.0001). There was significantly reduced median OS from time of disease recurrence/metastasis (1.9 vs 9.6 years, p < 0.0001). Conclusion This study clearly demonstrates a reduction in OS from time of first confirmed disease recurrence/metastasis for patients with NOTCH pathway activated ACC. This provides support for developing new drugs for this sub-group of patients, for whom clinical outcomes are significantly worse and effective treatments are lacking.
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To characterize somatic alterations in colorectal carcinoma, we conducted a genome-scale analysis of 276 samples, analysing exome sequence, DNA copy number, promoter methylation and messenger RNA and microRNA expression. A subset of these samples (97) underwent low-depth-of-coverage whole-genome sequencing. In total, 16% of colorectal carcinomas were found to be hypermutated: three-quarters of these had the expected high microsatellite instability, usually with hypermethylation and MLH1 silencing, and one-quarter had somatic mismatch-repair gene and polymerase μ (POLE) mutations. Excluding the hypermutated cancers, colon and rectum cancers were found to have considerably similar patterns of genomic alteration. Twenty-four genes were significantly mutated, and in addition to the expected APC, TP53, SMAD4, PIK3CA and KRAS mutations, we found frequent mutations in ARID1A, SOX9 and FAM123B. Recurrent copy-number alterations include potentially drug-targetable amplifications of ERBB2 and newly discovered amplification of IGF2. Recurrent chromosomal translocations include the fusion of NAV2 and WNT pathway member TCF7L1. Integrative analyses suggest new markers for aggressive colorectal carcinoma and an important role for MYC-directed transcriptional activation and repression.
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We show that BRAF(V600E) initiates an alternative pathway to colorectal cancer (CRC), which progresses through a hyperplasia/adenoma/carcinoma sequence. This pathway underlies significant subsets of CRCs with distinctive pathomorphologic/genetic/epidemiologic/clinical characteristics. Genetic and functional analyses in mice revealed a series of stage-specific molecular alterations driving different phases of tumor evolution and uncovered mechanisms underlying this stage specificity. We further demonstrate dose-dependent effects of oncogenic signaling, with physiologic Braf(V600E) expression being sufficient for hyperplasia induction, but later stage intensified Mapk-signaling driving both tumor progression and activation of intrinsic tumor suppression. Such phenomena explain, for example, the inability of p53 to restrain tumor initiation as well as its importance in invasiveness control, and the late stage specificity of its somatic mutation. Finally, systematic drug screening revealed sensitivity of this CRC subtype to targeted therapeutics, including Mek or combinatorial PI3K/Braf inhibition.
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We have examined the effects of inactivation of the p53 tumor suppressor gene on the incidence of apoptotic cell death in two stages of the adenoma-to-carcinoma progression in the intestine: in early adenomas where p53 mutations are rare and in highly dysplastic adenomas where loss of p53 occurs frequently. Homozygosity for an inactivating germ-line mutation of p53 had no effect on the incidence or the rate of progression of ApcMin/+-induced adenomas in mice and also did not affect the frequency of apoptosis in the cells of these adenomas. To examine the effect of p53 loss on apoptosis in late-stage adenomas, we compared the incidence of apoptotic cell death before and after the appearance of highly dysplastic cells in human colonic adenomas. The appearance of highly dysplastic cells, which usually coincides during colon tumor progression with loss of heterozygosity at the p53 locus, did not correlate with a reduction in the incidence of apoptosis. These studies suggest that p53 is only one of the genes that determine the incidence of apoptotic in colon carcinomas and that wild-type p53 retards the progression of many benign colonic adenoma to malignant carcinomas by mechanism(s) other than the promotion of apoptosis.
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The interaction between mutations in the tumor-suppressor genes Apc and p53 was studied in congenic mouse strains to minimize the influence of polymorphic modifiers. The multiplicity and invasiveness of intestinal adenomas of ApcMin/+ (Min) mice was enhanced by deficiency for p53. In addition, the occurrence of desmoid fibromas was strongly enhanced by p53 deficiency. The genetic modifier Mom1 and the pharmacological agents piroxicam and difluoromethylornithine each reduced intestinal adenoma multiplicity in the absence of p53 function. Mom1 showed no influence on the development of desmoid fibromas, whereas the combination of piroxicam and difluoromethylornithine exerted a moderate effect. The ensemble of tumor suppressors and modifiers of a neoplastic process can be usefully analyzed in respect to tissue specificity and synergy.
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Epithelial to mesenchymal transition (EMT) and pulmonary fibrogenesis require epithelial integrin α3β1-mediated cross-talk between TGFβ1 and Wnt signaling pathways. One hallmark of this cross-talk is pY654-β-catenin accumulation, but whether pY654-β-catenin is a biomarker of fibrogenesis or functionally important is unknown. To clarify further the role of β-catenin in fibrosis, we explored pY654-β-catenin generation and function. α3β1 was required for TGFβ1-mediated activation of Src family kinases, and Src inhibition blocked both pY654 and EMT in primary alveolar epithelial cells (AECs). TGFβ1 stimulated β-catenin/Lef1-dependent promoter activity comparably in immortalized AECs stably expressing WT β-catenin as well as Y654E or Y654F β-catenin point mutants. But EMT was abrogated in the Tyr to Phe mutant. pY654-β-catenin was sensitive to the axin β-catenin turnover pathway as inhibition of tankyrase 1 led to high AEC axin levels, loss of pY654-β-catenin, and inhibition of EMT ex vivo. Mice given a tankyrase inhibitor (50 mg/kg orally) daily for 7 days beginning 10 days after intratracheal bleomycin had improved survival over controls. Treated mice developed raised axin levels in the lung that abrogated pY654-β-catenin and attenuated lung Snail1, Twist1, α-smooth muscle actin, and type I collagen accumulation. Total β-catenin levels were unaltered. These findings identify Src kinase(s) as a mediator of TGFβ1-induced pY654-β-catenin, provide evidence that pY654-β-catenin levels are a critical determinant of EMT and fibrogenesis, and suggest regulation of axin levels as a novel therapeutic approach to fibrotic disorders.
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The epithelial-mesenchymal transition (EMT) is an embryonic transdifferentiation process consisting of conversion of polarized epithelial cells to motile mesenchymal ones. EMT-inducing transcription factors are aberrantly expressed in multiple tumor types and are known to favor the metastatic dissemination process. Supporting oncogenic activity within primary lesions, the TWIST and ZEB proteins can prevent cells from undergoing oncogene-induced senescence and apoptosis by abolishing both p53- and RB-dependent pathways. Here we show that they also downregulate PP2A phosphatase activity and efficiently cooperate with an oncogenic version of H-RAS in malignant transformation of human mammary epithelial cells. Thus, by down-regulating crucial tumor suppressor functions, EMT inducers make cells particularly prone to malignant conversion. Importantly, by analyzing transformed cells generated in vitro and by characterizing novel transgenic mouse models, we further demonstrate that cooperation between an EMT inducer and an active form of RAS is sufficient to trigger transformation of mammary epithelial cells into malignant cells exhibiting all the characteristic features of claudin-low tumors, including low expression of tight and adherens junction genes, EMT traits, and stem cell-like characteristics. Claudin-low tumors are believed to be the most primitive breast malignancies, having arisen through transformation of an early epithelial precursor with inherent stemness properties and metaplastic features. Challenging this prevailing view, we propose that these aggressive tumors arise from cells committed to luminal differentiation, through a process driven by EMT inducers and combining malignant transformation and transdifferentiation.
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Notch signalling is important for development and tissue homeostasis and activated in many human cancers. Nevertheless, mutations in Notch pathway components are rare in solid tumours. ZEB1 is an activator of an epithelial-mesenchymal transition (EMT) and has crucial roles in tumour progression towards metastasis. ZEB1 and miR-200 family members repress expression of each other in a reciprocal feedback loop. Since miR-200 members target stem cell factors, ZEB1 indirectly induces stemness maintenance and associated drug resistance. Here, we link ZEB1 and its cancer promoting properties to Notch activation. We show that miR-200 members target Notch pathway components, such as Jagged1 (Jag1) and the mastermind-like coactivators Maml2 and Maml3, thereby mediating enhanced Notch activation by ZEB1. We further detected a coordinated upregulation of Jag1 and ZEB1, associated with reduced miR-200 expression in two aggressive types of human cancer, pancreatic adenocarcinoma and basal type of breast cancer. These findings explain increased Notch signalling in some types of cancers, where mutations in Notch pathway genes are rare. Moreover, they indicate an additional way how ZEB1 exerts its tumour progressing functions.
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Notch signalling is crucial for the correct development and growth of numerous organs and tissues, and when subverted it can cause cancer. Loss of miR-8/200 microRNAs (miRNAs) is commonly observed in advanced tumours and correlates with their invasion and acquisition of stem-like properties. Here, we show that this miRNA family controls Notch signalling activation in Drosophila and human cells. In an overexpression screen, we identified the Drosophila miR-8 as a potent inhibitor of Notch-induced overgrowth and tumour metastasis. Gain and loss of mir-8 provoked developmental defects reminiscent of impaired Notch signalling and we demonstrated that miR-8 directly inhibits Notch ligand Serrate. Likewise, miR-200c and miR-141 directly inhibited JAGGED1, impeding proliferation of human metastatic prostate cancer cells. It has been suggested that JAGGED1 may also be important for metastases. Although in metastatic cancer cells, JAGGED1 modestly regulated ZEB1, the miR-200c's target in invasion, studies in Drosophila revealed that only concurrent overexpression of Notch and Zfh1/ZEB1 induced tumour metastases. Together, these data define a new way to attenuate or boost Notch signalling that may have clinical interest.
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The activation of β-catenin signalling is a key step in intestinal tumorigenesis. Interplay between the β-catenin and Notch pathways during tumorigenesis has been reported, but the mechanisms involved and the role of Notch remain unclear. Notch status was analysed by studying expression of the Notch effector Hes1 and Notch ligands/receptors in human colorectal cancer (CRC) and mouse models of Apc mutation. A genetic approach was used, deleting the Apc and RBP-J or Atoh1 genes in murine intestine. CRC cell lines were used to analyse the control of Hes1 and Atoh1 by β-catenin signalling. Notch signalling was found to be activated downstream from β-catenin. It was rapidly induced and maintained throughout tumorigenesis. Hes1 induction was mediated by β-catenin and resulted from both the induction of the Notch ligand/receptor and Notch-independent control of the Hes1 promoter by β-catenin. Surprisingly, the strong phenotype of unrestricted proliferation and impaired differentiation induced by acute Apc deletion in the intestine was not rescued by conditional Notch inactivation. Hyperactivation of β-catenin signalling overrode the forced differention induced by Notch inhibition, through the downregulation of Atoh1, a key secretory determinant factor downstream of Notch. This process involves glycogen synthase k