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Transgenic BRAFV600E, but not KRASG12V disrupts organoids due to high ERK activity. a Simplified representations of transgenes and the RAS-ERK and Wnt/β-catenin pathways, indicating relative positions of the KRAS and BRAF proto-oncogenes. b Organoid survival 4 days after induction of oncogenic KRASG12V or BRAFV600E. Organoids are counted immediately after passaging, and fractions of surviving organoids were calculated at day 4. Control organoids comprise of mixed non-induced cultures of KRASG12V and BRAFV600E lines. c Electron microscopy reveals loss of epithelial integrity after BRAFV600E induction. Images of the intestinal organoid epithelium, 24 h after induction of control FLUC, KRASG12V or BRAFV600E transgenes. Detailed views (right) represent a zoom into areas marked by red boxes in the overviews (left). Detailed views show apical surfaces of adjacent enterocytes with brush border. Red arrows mark desmosomes. Intercellular vacuoles, most visible in the KRASG12V model (marked by *) are likely fixation-induced artefacts, see ref. ²⁷. Scale bars are 10 µm in the overview panels and 500 nm in the detailed view panels. d Quantification of ERK phosphorylation in organoids, 24 h after induction of control, BRAF or KRAS transgenes, using a capillary protein analysis. e Quantification of organoid survival, 4 days after inhibition of EGFR, MEK, ERK and/or induction of BRAFV600E, as in panel (b). Error bars in panels (b), (d) and (e) denote standard deviations. Data shown in panels (b), (d), and (e) are available as a Source Data file

Transgenic BRAFV600E, but not KRASG12V disrupts organoids due to high ERK activity. a Simplified representations of transgenes and the RAS-ERK and Wnt/β-catenin pathways, indicating relative positions of the KRAS and BRAF proto-oncogenes. b Organoid survival 4 days after induction of oncogenic KRASG12V or BRAFV600E. Organoids are counted immediately after passaging, and fractions of surviving organoids were calculated at day 4. Control organoids comprise of mixed non-induced cultures of KRASG12V and BRAFV600E lines. c Electron microscopy reveals loss of epithelial integrity after BRAFV600E induction. Images of the intestinal organoid epithelium, 24 h after induction of control FLUC, KRASG12V or BRAFV600E transgenes. Detailed views (right) represent a zoom into areas marked by red boxes in the overviews (left). Detailed views show apical surfaces of adjacent enterocytes with brush border. Red arrows mark desmosomes. Intercellular vacuoles, most visible in the KRASG12V model (marked by *) are likely fixation-induced artefacts, see ref. ²⁷. Scale bars are 10 µm in the overview panels and 500 nm in the detailed view panels. d Quantification of ERK phosphorylation in organoids, 24 h after induction of control, BRAF or KRAS transgenes, using a capillary protein analysis. e Quantification of organoid survival, 4 days after inhibition of EGFR, MEK, ERK and/or induction of BRAFV600E, as in panel (b). Error bars in panels (b), (d) and (e) denote standard deviations. Data shown in panels (b), (d), and (e) are available as a Source Data file

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Article
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Oncogenic mutations in KRAS or BRAF are frequent in colorectal cancer and activate the ERK kinase. Here, we find graded ERK phosphorylation correlating with cell differentiation in patient-derived colorectal cancer organoids with and without KRAS mutations. Using reporters, single cell transcriptomics and mass cytometry, we observe cell type-specif...

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... In intestinal stem cells (ISCs), loss of APC function mutations drives intestinal adenomas by enhancing intracellular Wnt signaling [16]. Brandt et al. found that oncogenic KRAS, together with β-Catenin, favoured the expansion of crypt cells with high ERK activity [17]. At present, molecular targets of colon cancer comprise EGFR, VEGF, ERBB2, BRAF, KRAS, PD-1, CTLA-4, NTRK etc. ...
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Studies of both, microbiota and target therapy associated with gene mutations in colorectal cancer, (CRC) have attracted increasing attention. However, only a few of them analyzed the combined effects on CRC. we analyzed differences in intestinal microbiota of 44 colorectal cancer patients and 20 healthy controls (HC) using 16S rRNA gene sequencing of fecal samples. For 39 of the CRC patients, targeted Next Generation Sequencing (NGS) was carried out at formalin fixed paraffin embedded (FFPE) samples to identify somatic mutation profiles. Compared to the HC group, the microbial diversity of CRC patients was significantly lower. In the CRC group, we found a microbiome that was significantly enriched for strains of Bifidobacterium, Bacteroides, and Megasphaera whereas in the HC group the abundance of Collinsella, Faecalibacterium, and Agathobacter strains was higher. Among the mutations detected in the CRC group, the APC gene had the highest mutation rate (77%, 30/39). We found that the KRAS mutant type was closely associated with Faecalibacterium, Roseburia, Megamonas, Lachnoclostridium, and Harryflintia. Notably, Spearman correlation analysis showed that KRAS mutations were negatively correlated with the existence of Bifidobacterium and positively correlated with Faecalibacterium. By employing 16S rRNA gene sequencing, we identified more unique features of microbiota profiles in CRC patients. For the first time, our study showed that gene mutations could directly be linked to the microbiota composition of CRC patients. We hypothesize that the effect of a targeted colorectal cancer therapy is also closely related to the colorectal flora, however, this requires further investigation.
... Improved surface rendering tools such as Imaris 9.5 can be applied to serial confocal fluorescence images to enhance visualization and quantification capacity of cells expressing markers of proliferation and self-renewal in primary and secondary CRC tumor organoids ( Figure 6E). In addition, extensive analysis of cellular heterogeneity can be conducted on tumor organoids by assessing the abundance and distribution various markers, using either cytometry approaches (fluorescence, CyTOF) or single-cell genomics upon completing steps -32-34 (Brandt et al., 2019;Chen et al., 2018;Gonzalez-Exposito et al., 2019). ...
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Organoids can enable the study of solid tumors initiated from a single cancer stem cell (CSC) ex vivo. We describe a serial tumor organoid plating protocol using primary colorectal cancer (CRC) tissues as a rapid and cost-efficient approach to evaluate the impact of therapeutic interventions on CSC functions. We detail the isolation of primary colorectal CSCs, organoid embedding, serial passaging, and CSC-related analytical techniques. For complete details on the use and execution of this protocol, please refer to Masibag et al. (2021) and Bergin et al. (2021).
... Our description of the wiring of the RAS/MAPK pathway in neuroblastoma will support the design of clinical trials using combinatorial treatments to prevent or overcome therapy resistance. In addition, the framework described here could be used to analyse signalling in tumours of individual patients While it will be technically challenging to assess signalling network responses in tumour patients, ex vivo cultures-so-called avatars-could be an option [45,46]. We envision that learning features of robustness and vulnerability of tumours from signalling models on cell line panels might greatly reduce the required set of perturbations in those avatars that are sufficient to inform a model, and allow reliable stratification and prediction of treatment options. ...
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Very high risk neuroblastoma is characterised by increased MAPK signalling, and targeting MAPK signalling is a promising therapeutic strategy. We used a deeply characterised panel of neuroblastoma cell lines and found that the sensitivity to MEK inhibitors varied drastically between these cell lines. By generating quantitative perturbation data and mathematical modelling, we determined potential resistance mechanisms. We found that negative feedbacks within MAPK signalling and via the IGF receptor mediate re-activation of MAPK signalling upon treatment in resistant cell lines. By using cell-line specific models, we predict that combinations of MEK inhibitors with RAF or IGFR inhibitors can overcome resistance, and tested these predictions experimentally. In addition, phospho-proteomic profiling confirmed the cell-specific feedback effects and synergy of MEK and IGFR targeted treatment. Our study shows that a quantitative understanding of signalling and feedback mechanisms facilitated by models can help to develop and optimise therapeutic strategies. Our findings should be considered for the planning of future clinical trials introducing MEKi in the treatment of neuroblastoma.
... Biosensing capabilities can be linked directly to hydrogel matrices, as in Leight et al.'s use of matrix-tethered peptides to quantify dynamic changes in melanoma matrix metalloproteinase activity and cell motility [30]. Alternatively, fluorescent reporter genes can be employed for single-cell lineage tracing [31] or identifying dynamic pathway activation mechanisms via fluorescent genetic switches [32]. ...
Article
Bioengineered in vitro models have advanced from 2D cultures and simple 3D cell aggregates to more complex organoids and organ-on-a-chip platforms. This shift has been substantial in cancer research; while simple systems remain in use, multi-tissue type tumor and tissue chips and patient-derived tumor organoids have grown rapidly. These more advanced models offer new tools to cancer researchers based on human tumor physiology and the potential for interactions with nontumor tissue physiology while avoiding critical differences between human and animal biology. In this focused review, the authors discuss the importance of organoid and organ-on-a-chip platforms, with a particular focus on modeling cancer, to highlight oncology-focused in vitro model platform technologies that improve upon the simple 2D cultures and 3D spheroid models of the past.
... The extracellular signal-regulated kinases (ERKs) is frequently hyperactivated in many types of cancer, which is usually induced by activating mutations of the K-Ras that accounts for approximately 25% of NSCLC patients (Chatterjee et al., 2017). ERK can activate a series of transcription factors that orchestrate a complex cellular response, which is often considered as pro-proliferative (Brandt et al., 2019). To date, direct inhibition of K-Ras has not yet been successful in clinical therapy, whereas inhibitors targeting ERK, such as binimetinib and trametinib, are approved to treat melanoma (Roskoski, 2020) and are under clinical trial for treatment of lung cancer (Heigener et al., 2015). ...
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Aberrant activation of the Ras–ERK signaling pathway drives many important cancer phenotypes, and several inhibitors targeting such pathways are under investigation and/or approved by the FDA as single- or multi-agent therapy for patients with melanoma and non–small-cell lung cancer (NSCLC). Here, we show that betulinic acid (BA), a natural pentacyclic triterpenoid, inhibits cell proliferation, and induces apoptosis and protective autophagy in NSCLC cells. Thus, the cancer cell killing activity of BA is enhanced by autophagy inhibition. Mitogen-activated protein kinases, and especially ERK that facilitates cancer cell survival, are also activated by BA treatment. As such, in the presence of ERK inhibitors (ERKi), lung cancer cells are much more sensitive to BA. However, the dual treatment of BA and ERKi results in increased protective autophagy and AKT phosphorylation. Accordingly, inhibition of AKT has a highly synergistic anticancer effect with co-treatment of BA and ERKi. Notably, autophagy inhibition by hydroxychloroquine (HCQ) increases the response of lung cancer cells to BA in combination with ERKi. In vivo, the three-drug combination (BA, ERKi, and HCQ), resulted in superior therapeutic efficacy than single or dual treatments in the xenograft mouse model. Thus, our study provides a combined therapy strategy that is a highly effective treatment for patients with NSCLC.
... Several alterations in signaling pathway activity were also common across conditions. TGFB1 treatment and spheroids were associated with higher levels of ERK activity, which have both been linked to stemness in epithelial 34 and carcinoma cells 35 . In EGF-free media, paracrine/autocrine signaling is established, maintaining ERK activity in stem cell populations of intestinal organoids 35 . ...
... TGFB1 treatment and spheroids were associated with higher levels of ERK activity, which have both been linked to stemness in epithelial 34 and carcinoma cells 35 . In EGF-free media, paracrine/autocrine signaling is established, maintaining ERK activity in stem cell populations of intestinal organoids 35 . While these mechanisms may contribute to stemness in OSE spheroids or those treated with TGFB1, increased ERK activity was not enhanced following Snail overexpression or Brca1 loss. ...
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The ovarian surface epithelium (OSE) is a monolayer of epithelial cells surrounding the ovary that ruptures during each ovulation to allow release of the oocyte. This wound is quickly repaired, but mechanisms promoting repair are poorly understood. The contribution of tissue-resident stem cells in the homeostasis of several epithelial tissues is widely accepted, but their involvement in OSE is unclear. We show that traits associated with stem cells can be increased following exposure to the cytokine TGFB1, overexpression of the transcription factor Snai1, or deletion of Brca1. We find that stemness is often linked to mesenchymal-associated gene expression and higher activation of ERK signalling, but is not consistently dependent on their activation. Expression profiles of these populations are extremely context specific, suggesting that stemness may not be associated with a single, distinct population, but rather is a heterogeneous cell state that may emerge from diverse environmental cues. These findings support that the OSE may not require distinct stem cells for long-term maintenance, and may instead achieve this through transient dedifferentiation into a stem-like state.
... Expectedly, oncogenic mutations inducing the hyperactivation of both pathways perturb intestinal homeostasis and result in intestinal malignancies. Of note are the oncogenic mutations of K-Ras (KRAS) within the EGFR-MAPK/ERK pathway which has shown to be involved in CRC development (105,106). Resveratrol has been found to possess a broad-spectrum of health benefits including anti-cancer activities (107) and findings by Saud et al. specify that resveratrol acts directly to suppress KRAS expression (108). Using a conditional knockout model of APC in mice supplemented with resveratrol, the authors determined that resveratrol inhibits tumor growth and proliferation which is accompanied by a reduction in LGR5, KRAS and nuclear β-catenin expression. ...
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Maintenance of intestinal homeostasis requires the integration of immunological and molecular processes together with environmental, diet, metabolic and microbial cues. Key to this homeostasis is the proper functioning of epithelial cells originating from intestinal stem cells (ISCs). While local factors and numerous molecular pathways govern the ISC niche, the conduit through which these processes work in concordance is the aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor, whose role in immunoregulation is critical at barrier surfaces. In this review, we discuss how AhR signaling is emerging as one of the critical regulators of molecular pathways involved in epithelial cell renewal. In addition, we examine the putative contribution of specific AhR ligands to ISC stemness and epithelial cell fate.
... A recurring idea within the large body of according network inference methods is to conceive the system as ordinary differential equations and describe edges in the directed network by the entries of an inferred Jacobian matrix Tegner et al., 2003;Kholodenko, 2007;. Such methods have been successfully applied to describe various types of regulatory networks in different organisms (Ciofani et al., 2012;Arrieta-Ortiz et al., 2015;Lorenz et al., 2009;Brandt et al., 2019). They are continuously improved, e.g. to reduce the effect of noise, incorporate heterogeneous data sets, or allow for the analysis of single cell data (Greenfield et al., 2013;Kang et al., 2015; and have thus become a standard research tool. ...
... A well-established method amongst these is the Modular Response Analysis (MRA) . It has been applied in a range of contexts (Santos et al. 2007;Stelniec-Klotz et al. 2012;Halasz et al. 2016;Speth et al. 2017;Brandt et al. 2019;Jimenez-Dominguez et al. 2020) and its many extensions ) turned it into an established tool of network inference. Yet until now, the identifiability of interaction strengths within the MRA formalism has not been sufficiently analysed. ...
Thesis
Hochdurchsatzverfahren quantifizieren eine Vielzahl zellulärer Komponenten, können aber selten deren Interaktionen beschreiben. Daher wurden in den letzten 20 Jahren verschiedenste Netzwerk-Rekonstruktionsmethoden entwickelt. Insbesondere Perturbationsdaten erlauben dabei Rückschlüsse über funktionelle Mechanismen in der Genregulierung, Signal Transduktion, intra-zellulärer Kommunikation und anderen Prozessen zu ziehen. Dennoch bleibt Netzwerkinferenz ein ungelöstes Problem, weil die meisten Methoden auf ungeeigneten Annahmen basieren und die Identifizierbarkeit von Netzwerkkanten nicht aufklären. Diesbezüglich beschreibt diese Dissertation eine neue Rekonstruktionsmethode, die auf einfachen Annahmen von Perturbationsausbreitung basiert. Damit ist sie in verschiedensten Zusammenhängen anwendbar und übertrifft andere Methoden in Standard-Benchmarks. Für MAPK und PI3K Signalwege in einer Adenokarzinom-Zellline generiert sie plausible Netzwerkhypothesen, die unterschiedliche Sensitivitäten von PI3K-Mutanten gegenüber verschiedener Inhibitoren überzeugend erklären. Weiterhin wird gezeigt, dass sich Netzwerk-Identifizierbarkeit durch ein intuitives Max-Flow Problem beschreiben lässt. Dieses analytische Resultat erlaubt effektive, identifizierbare Netzwerke zu ermitteln und das experimentelle Design aufwändiger Perturbationsexperimente zu optimieren. Umfangreiche Tests zeigen, dass der Ansatz im Vergleich zu zufällig generierten Perturbationssequenzen die Anzahl der für volle Identifizierbarkeit notwendigen Perturbationen auf unter ein Drittel senkt. Schließlich beschreibt die Dissertation eine mathematische Weiterentwicklung der Modular Response Analysis. Es wird gezeigt, dass sich das Problem als analytisch lösbare orthogonale Regression approximieren lässt. Dies erlaubt eine drastische Reduzierung des nummerischen Aufwands, womit sich deutlich größere Netzwerke rekonstruieren und neueste Hochdurchsatz-Perturbationsdaten auswerten lassen.
... Although patients with colon cancer have not benefited from immunotherapy, several studies have shown that colon tumors with high mutational burden may be potential targets of immune checkpoint inhibitors [20,21]. Inhibition of MEK upregulates IFN-gammamediated human leukocyte antigen (HLA) and programmed death-1 receptor (PD-L1) expression in melanoma, colorectal, and breast cancers [22,23]. The product of HLA genes-MHC protein-can also regulate the immune system [24]. ...
Article
The heterogeneity of colon cancer tumors suggests that therapeutics targeting specific molecules may be effective in only a few patients. It is therefore necessary to explore gene mutations in colon cancer. In this study, we obtained colon cancer samples from The Cancer Genome Atlas, and the International Cancer Genome Consortium. We evaluated the landscape of somatic mutations in colon cancer and found that KRAS mutations, particularly rs121913529, were frequent and had prognostic value. Using ESTIMATE analysis, we observed that the KRAS-mutated group had higher tumor purity, lower immune score, and lower stromal score than the wild-type group. Through single-sample Gene Set Enrichment Analysis and Gene Set Enrichment Analysis, we found that KRAS mutations negatively correlated with enrichment levels of tumor infiltrating lymphocytes, inflammation, and cytolytic activities. HLA gene expression and checkpoint-related genes were also lower in the KRAS-mutated group. Finally, we found 24 immune-related genes that differed in expression between the KRAS-mutated and wild-type samples, which may provide clues to the mechanism of KRAS-related immune alteration. Our findings are indicative of the prognostic and predictive value of KRAS and illustrate the relationship between KRAS mutations and immune activity in colon cancer.
... ERK1/2 are activated through phosphorylation by upstream MAPK/ERK kinases MAPK/ERK kinase 1 and 2 (MEK1/2), which are activated through phosphorylation by upstream Raf serine/ threonine protein kinases (Raf-1, B-Raf, and A-Raf) (18,19). The activity of the Raf/MEK/ERK signaling pathway plays a redundant role in regulating fundamental biological processes, such as proliferation, survival, metastasis, and differentiation in many types of cancer, including cervical cancer (20)(21)(22)(23). However, the potential links between HK2 and the Raf/MEK/ERK signaling pathway, which mediates cervical carcinoma initiation and growth, remain less known. ...
Article
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Hexokinase 2 (HK2) is a member of the hexokinases (HK) that has been reported to be a key regulator during glucose metabolism linked to malignant growth in many types of cancers. In this study, stimulation of HK2 expression was observed in squamous cervical cancer (SCC) tissues, and HK2 expression promoted the proliferation of cervical cancer cells in vitro and tumor formation in vivo by accelerating cell cycle progression, upregulating cyclin A1, and downregulating p27 expression. Moreover, transcriptome sequencing analysis revealed that MAPK3 (ERK1) was upregulated in HK2-overexpressing HeLa cells. Further experiments found that the protein levels of p-Raf, p-MEK1/2, ERK1/2, and p-ERK1/2 were increased in HK2 over-expressing SiHa and HeLa cells. When ERK1/2 and p-ERK1/2 expression was blocked by an inhibitor (FR180204), reduced cyclin A1 expression was observed in HK2 over-expressing cells, with induced p27 expression and inhibited cell growth. Therefore, our data demonstrated that HK2 promoted the proliferation of cervical cancer cells by upregulating cyclin A1 and down-regulating p27 expression through the Raf/MEK/ERK signaling pathway.