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Model of cell type-specific regulation of ERK activity. ERK is regulated cell type-specific and cell-intrinsic via different strengths of feedback inhibition and feed-forward signalling from MEK to ERK. Dual-specificity phosphatases (DUSPs) are important regulators of ERK activity. β-catenin and KRAS G12V activities modulate cell fate decisions towards the generation of cells with high ERK activity, likely in part due to low expression of genes encoding DUSPs
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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...
Citations
... ERK levels are generally higher in cancer cells adjacent to stromal cells at the invasive front and lower in more central areas of cancer specimens. 173 ...
Rat sarcoma (RAS), as a frequently mutated oncogene, has been studied as an attractive target for treating RAS-driven cancers for over four decades. However, it is until the recent success of kirsten-RAS (KRAS)G12C inhibitor that RAS gets rid of the title "undruggable". It is worth noting that the therapeutic effect of KRASG12C inhibitors on different RAS allelic mutations or even different cancers with KRASG12C varies significantly. Thus, deep understanding of the characteristics of each allelic RAS mutation will be a prerequisite for developing new RAS inhibitors. In this review, the structural and biochemical features of different RAS mutations are summarized and compared. Besides, the pathological characteristics and treatment responses of different cancers carrying RAS mutations are listed based on clinical reports. In addition, the development of RAS inhibitors, either direct or indirect, that target the downstream components in RAS pathway is summarized as well. Hopefully, this review will broaden our knowledge on RAS-targeting strategies and trigger more intensive studies on exploiting new RAS allele-specific inhibitors.
... Signaling evaluations indicated a decrease in VEGFR2 and phAKT protein levels with MSI2 KD (Figure 3A,B), while phERK protein levels were not changed (Supplementary Figure S4B). This result is expected because our models involve KRASmut, and published papers show that KRASmut results in constitutive activation of ERK [41,42]. Next, we established cell lines with VEGFR2 OE to evaluate the effects of MSI2 on AKT signaling via VEGFR2 regulation. ...
Lung cancer is the most frequently diagnosed cancer type and the leading cause of cancer-related deaths worldwide. Non-small cell lung cancer (NSCLC) represents most of the diagnoses of lung cancer. Vascular endothelial growth factor receptor-2 (VEGFR2) is a member of the VEGF family of receptor tyrosine kinase proteins, which are expressed on both endothelial and tumor cells, are one of the key proteins contributing to cancer development, and are involved in drug resistance. We previously showed that Musashi-2 (MSI2) RNA-binding protein is associated with NSCLC progression by regulating several signaling pathways relevant to NSCLC. In this study, we performed Reverse Protein Phase Array (RPPA) analysis of murine lung cancer, which suggests that VEGFR2 protein is strongly positively regulated by MSI2. Next, we validated VEGFR2 protein regulation by MSI2 in several human lung adenocarcinoma cell line models. Additionally, we found that MSI2 affected AKT signaling via negative PTEN mRNA translation regulation. In silico prediction analysis suggested that both VEGFR2 and PTEN mRNAs have predicted binding sites for MSI2. We next performed RNA immunoprecipitation coupled with quantitative PCR, which confirmed that MSI2 directly binds to VEGFR2 and PTEN mRNAs, suggesting a direct regulation mechanism. Finally, MSI2 expression positively correlated with VEGFR2 and VEGF-A protein levels in human lung adenocarcinoma samples. We conclude that the MSI2/VEGFR2 axis contributes to lung adenocarcinoma progression and is worth further investigations and therapeutic targeting.
... Epithelial-to-mesenchymal transition marker Vimentin was mostly absent in HCT116 cells yet strongly expressed in LAN6. Apoptosis markers cleaved Caspase 3 (Kuida et al., 1996;Woo et al., 1998) and cleaved PARP (Cohausz and Althaus, 2009) were higher in CRC organoid line OT326 than OT227, as expected (Schütte et al., 2017;Brandt et al., 2019). Celltype markers LGR5, PROM1, CD24, EphB2, Krt20, and PTK7 all correlated with previously known RNA expression data of the cell lines compared (Fig. S6 A; Iorio et al., 2016). ...
Colorectal cancer progression is intrinsically linked to stepwise deregulation of the intestinal differentiation trajectory. In this process, sequential mutations of APC, KRAS, TP53, and SMAD4 enable oncogenic signaling and establish the hallmarks of cancer. Here, we use mass cytometry of isogenic human colon organoids and patient-derived cancer organoids to capture oncogenic signaling, cell phenotypes, and differentiation states in a high-dimensional single-cell map. We define a differentiation axis in all tumor progression states from normal to cancer. Our data show that colorectal cancer driver mutations shape the distribution of cells along the differentiation axis. In this regard, subsequent mutations can have stem cell promoting or restricting effects. Individual nodes of the cancer cell signaling network remain coupled to the differentiation state, regardless of the presence of driver mutations. We use single-cell RNA sequencing to link the (phospho-)protein signaling network to transcriptomic states with biological and clinical relevance. Our work highlights how oncogenes gradually shape signaling and transcriptomes during tumor progression.
... Signaling evaluation indicated decrease VEGFR2 and phAKT protein levels with MSI2 KD (Fig. 3A and B), while phERK protein level not changed ( Supplementary Fig.4B). This result is expected, because our models are KRASmut, and published papers show that KRASmut results in constitutive activation of ERK [46,47]. Next, we established cell lines with VEGFR2 OE to evaluate MSI2 effects AKT signaling via VEGFR2 regulation. ...
Lung cancer is the most frequently diagnosed cancer type and the leading cause of cancer-related deaths worldwide. Non-small cell lung cancer (NSCLC) represents most of the lung cancer. Vascular endothelial growth factor receptor-2 (VEGFR2) is a member of the VEGF family of receptor tyrosine kinase proteins, expressed on both endothelial and tumor cells which is one of the key proteins contributing to cancer development and involved in drug resistance. We previously showed that Musashi-2 (MSI2) RNA-binding protein is associated with NSCLC progression by regulating several signaling pathways relevant to NSCLC. In this study, we performed Reverse Protein Phase Array (RPPA) analysis of murine lung cancer which nominated VEGFR2 protein as strongly positively regulated by MSI2. Next, we validated VEGFR2 protein regulation by MSI2 in several human NSCLC cell line models. Additionally, we found that MSI2 affected AKT signaling via negative PTEN mRNA translation regulation. In silico prediction analysis suggested that both VEGFR2 and PTEN mRNAs have predicted binding sites for MSI2. We next performed RNA immunoprecipitation coupled with quantitative PCR which confirmed that MSI2 directly binds to VEGFR2 and PTEN mRNAs, suggesting direct regulation mechanism. Finally, MSI2 expression positively correlated with VEGFR2 and VEGF-A protein levels in human NSCLC samples. We conclude that MSI2/VEGFR2 axis contributes to NSCLC progression and is worth further investigations and therapeutic targeting.
... Lowering ERK activation by treatment with ERK or MEK (mitogen-activated protein kinase kinase) inhibitor counteracted BRAF V600E -induced organoid disintegration 14,16 . It is therefore presumed that maintaining ERK activation within a narrow threshold range to avoid engaging 5 tumor suppression is pivotal for mutant BRAF to exhibit the strongest transforming activity. ...
... However, despite being highly anticipated 16 , the existence of in vivo intrinsic fine-tuning of mutant BRAF-induced ERK activation has never been experimentally examined. Given that over 60 mutations have now been identified in BRAF 13,17 , theoretically, mutation-selection could be a way to achieve optimal ERK activation. ...
BRAFV600E mutation is a driver mutation in the serrated pathway to colorectal cancers. BRAFV600E drives tumorigenesis through constitutive downstream extracellular signal-regulated kinase (ERK) activation, but high-intensity ERK activation can also trigger tumor suppression. Whether and how oncogenic ERK signaling can be intrinsically adjusted to a “just-right” level optimal for tumorigenesis remains undetermined. In this study, we found that FAK (Focal adhesion kinase) expression was reduced in BRAFV600E-mutant adenomas/polyps in mice and patients. In Vill-Cre;BRAFV600E/+;Fakfl/fl mice, Fak deletion maximized BRAFV600E’s oncogenic activity and increased cecal tumor incidence to 100%. Mechanistically, our results showed that Fak loss, without jeopardizing BRAFV600E-induced ERK pathway transcriptional output, reduced EGFR (epidermal growth factor receptor)-dependent ERK phosphorylation. Reduction in ERK phosphorylation resulted in increased mRNA expression and stability of Lgr4, promoting intestinal stemness and cecal tumor formation. Together, our findings show that a “just-right” ERK signaling optimal for BRAFV600E-induced cecal tumor formation can be achieved via Fak loss-mediated downregulation of ERK phosphorylation.
... therefore, facilitate colorectal carcinogenesis. 28 Literature supports our finding that p38 activates MEK cascade signalling via RIT and RIN (p=0.01) and that ARMS mediated activation triggers MAPK cascade (p=0.01) increasing vulnerability to CRC. 27 MAPK pathway activation also results from phosphorylation of Frs2 (p=0.01) and from Trk activation (p=0.047). ...
Background:
Identification of gene targets and biological pathways involved in colorectal carcinoma (CRC) is essential for better management of patients. Our study aims to highlight common somatic mutations in colorectal carcinoma and to identify dysregulated pathways and gene enrichment based on KRAS and BRAF interaction network analysis.
Methods:
By using cancer browser tool in COSMIC database, mutation frequencies of the top 20 mutated genes listed for colorectal adenocarcinoma were identified. The most frequent variants of selected genes were explored with ClinVar database which led to identification of protein change along with its cytogenic location, variant type, variant length and the associated single nucleotide polymorphism (SNP). These identified SNPs were searched in Pakistani database using 1000genome in an attempt to identify common polymorphisms. Using the database ClinicalTrial.gov the number of clinical trials based upon these selected mutations was explored. Enrichment and protein interaction (PI) analysis of KRAS and BRAF was carried out to reveal significant biological pathways associated with these genes.
Results:
In cumulative data, among all variants about 57% of substitution mutations are observed to be G>A including mutations in KRAS, Tp53, SMAD4, PI3K and NRAS. The mutations of KRAS (c.35G>A), TP53 (c.524G>A) and APC (c.4348C>T) were found to be pathogenic with single nucleotide variation and variant length of 1bp. Searching 1000genome database revealed that 100 % of alleles found in East Asian population studied are 'C'(frequency=1). Significant biological pathways (<0.05) identified by our search include Trk receptor signalling mediated by the MAPK pathway, signalling to p38 via RIT and RIN, signalling to ERKs, Frs2-mediated activation, ARMS-mediated activation and prolonged ERK activation events.
Conclusions:
Our study highlights the role of genetic profiling in CRC, with emphasis on mutations which may define treatment outcome. Targeting several collateral pathways simultaneously may be further explored to improve colorectal cancer therapeutics.
... The results in this thesis show that a quantitative understanding of differences in signalling networks is key to understand some resistances and an essential tool to derive effective treatments. STASNet has been and is currently used to study the role of MAPK on stem cell differentiation depending on the number of X chromosomes (Sultana et al. manuscript and thesis in preparation), the role of PI3K/AKT signalling in pancreatic neuroendocrine cell lines (Simon et al, manuscript and thesis in preparation), the changes in signalling networks mediating radioresistance in head and neck cancer cell lines (Tim Rose, Master thesis), the effect of KRAS and NRAS mutations in inducible colon cancer cell lines (Mert Dikmen, Master thesis), the effect of RAS isoforms oncogenic mutations in the colon cancer cell line SW48 (Hood et al., 2019), the cell type specific differences in signalling of intestinal mouse organoids (Brandt et al., 2019), B-cell-receptor signalling in B-cell Lymphoma cell lines (Bertram Klinger, manuscript in preparation), and signalling in human patientderived colon cancer organoids (Bertram Klinger, manuscript in preparation). Overall, STASNet has proven a versatile toolbox for users less versed in programing and in multiple contexts, and should prove a useful tool to analyse data coming from new perturbation methods. ...
... Both technologies offer new ways of apprehending signalling networks, where 3D culture systems take into account inter-cellular interactions and single cell technology disentangles the resulting heterogeneity and cell type specificities. While perturbing signalling network in tumour patients will remain extremely challenging as only one treatment after another can be attempted in a changing tumor environment, ex vivo cultures such as organoids are an option to provide personnalised signalling analysis, as so called "avatar" (Brandt et al., 2019;Saez-Rodriguez and Blüthgen, 2020;Driehuis et al., 2020). The features of robustness and vulnerability learned from signalling models trained on cell line panels can be used to reduce the set of perturbations required in these avatars to inform a model rapidly and at reduced cost so as to predict efficient treatment options. ...
... Methods such as CyTOF (Bendall et al., 2011) or targeted phosphoproteomics (Osinalde et al., 2017) promise to increase the number of signalling readouts at a decreased cost. Combined, these approaches offer rich datasets and STASNet has already been used to study the specificities of the signalling network in the different cells types present in colon cancer organoids (Brandt et al., 2019), forecasting a potential use to study signalling networks closer to the patients. STASNet and other similar tools that disentangle perturbation responses should see increased use as richer and more complex data are generated. ...
Obwohl die Krebstherapie im letzten Jahrhundert große Fortschritte gemacht hat, bleibt die Resistenz gegen medikamentöse Behandlungen ein großes Hindernis im Kampf gegen den Krebs. In dieser Arbeit habe ich ein R-Paket namens STASNet entwickelt, das semi-quantitative Modelle der Signaltransduktion aus Signalisierungs-Störungsantwortdaten unter Verwendung von Least Square Modular Response Analysis-Modellen generiert. Um zu untersuchen, wie gut STASNet die Aktivität von Signalwegen quantifizieren kann, haben wir Perturbationsdaten von einem Paar isogener Darmkrebszelllinien mit und ohne SHP2-Knock-out, einem bekannten Resistenzmechanismus bei dieser Krebsart, verwendet. Ich habe dann untersucht die Resistenz gegen die MEK- und ALK-Hemmung beim Neuroblastom, einem pädiatrischen Krebs mit schlechter Prognose. Ein Wirkstoffscreening zeigte, dass der MEK-Inhibitor Selumetinib ein Panel von Neuroblastom-Zelllinien in drei sensitive und sechs resistente Zelllinien trennte, dass konnte nicht mit einzelnen molekularen Markern erklärt. STASNet-Modelle zeigten, dass die starke Resistenz gegen Selumetinib durch eine starke Rückkopplung von ERK auf MEK oder eine vielschichtige Rückkopplung sowohl auf MEK als auch auf IGF1R getrieben wurde. Aus dem Modell konnte eine kombinatorische Therapie abgeleitet werden, die auf MEK in Kombination mit entweder RAF oder IGF1R abzielt, je nach Art der in der Zelllinie vorhandenen Rückkopplungen. Schließlich ergab die Untersuchung der Wirkung von NF1-KO auf die Signalübertragung, dass der Verlust von NF1 den MAPK-Weg für die Liganden-induzierte Aktivierung hypersensibilisierte, aber das ERK-RAF-Rückkopplung störte. Die Erkenntnisse aus den in dieser Arbeit entwickelten Modellen werden somit dazu beitragen, personalisierte Kombinationen von Inhibitoren zu entwerfen, die als Zweitlinientherapie nach molekularer Untersuchung der Tumorreaktion auf die Erstbehandlung eingesetzt werden könnten.
... Mass cytometry was performed as described before 66 . In brief, we used the following preconjugated antibodies (Fluidigm) as per manufacturers recommendation: p-H2AX [S139] (1:100; 3147016A, 147-Sm), cPARP (1:100; 3143011A, 143Nd). ...
The human gastric epithelium forms highly organized gland structures with different subtypes of cells. The carcinogenic bacterium Helicobacter pylori can attach to gastric cells and subsequently translocate its virulence factor CagA, but the possible host cell tropism of H. pylori is currently unknown. Here, we report that H. pylori preferentially attaches to differentiated cells in the pit region of gastric units. Single-cell RNA-seq shows that organoid-derived monolayers recapitulate the pit region, while organoids capture the gland region of the gastric units. Using these models, we show that H. pylori preferentially attaches to highly differentiated pit cells, marked by high levels of GKN1, GKN2 and PSCA. Directed differentiation of host cells enable enrichment of the target cell population and confirm H. pylori preferential attachment and CagA translocation into these cells. Attachment is independent of MUC5AC or PSCA expression, and instead relies on bacterial TlpB-dependent chemotaxis towards host cell-released urea, which scales with host cell size.
... 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. ...
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.
... The modelling approach we utilized is based on the well-established modular response analysis (MRA) (Bruggeman et al., 2002;Kholodenko et al., 2002) that has been employed to model signaling networks in multiple cancer cell lines (Klinger et al., 2013;Dorel et al., 2018;Hood et al., 2019;Brandt et al., 2019). The method is implemented in the R package STeady-STate Analysis of Signalling Networks (STASNet) for a more efficient utilization of the approach. ...
Neuroendokrine Neoplasmen der Bauchspeicheldrüse (PanNEN) sind eine seltene und vielfältige Form von Krebs. PanNENs umfassen hochgradige PanNECs und NETG3 PanNETs, sowie die öfter diagnostizierten NETG1 und NETG2 PanNETs. Hochgradige PanNENs weisen eine schlechte Prognose auf, und sind histologisch schwierig zu diagnostizieren. In dieser Studie wird eine auf Methylierung basierende Klassifizierung vorgestellt, die PanNETs von PanNECs unterscheidet und die zugrunde liegende Komplexität in Bezug auf molekularen Merkmalen und den Ursprungszellen der PanNENs aufzeigt. Zuerst wurden PanNENs auf Grundlage ihrer Methylierungsprofile gruppiert, wodurch sich die PanNETs und PanNECs in zwei Gruppen A und B aufteilten. Während Tumore der Gruppe B häufig Veränderungen in KRAS, TP53 und SMAD4 aufweisen, umfasst das Mutationsspektrum von Gruppe A Veränderungen in klassischen PanNEN-Genen wie MEN1, DAXX, ATRX und VHL. Darüber hinaus ist Gruppe A durch chromosomale Aberrationen geprägt, während Gruppe B eine signifikante fokale Deletion des RB1-Lokus aufweist. Anhand von Methylierungsprofilen von alpha-, beta-, duktalen und azinären Pankreaszellen sowie von Expressionsmustern normaler Zelltyp Markergene innerhalb der Tumore folgt, dass die PanNETs alpha-, beta- und intermediär-ähnliche Tumore endokrinen Ursprungs sind, während die PanNECs azinäre Tumore vermutlich exokrinen Ursprungs sind. Ausprägung des Azinuszellenprofils und Expression des SOX9-Proteins in Gruppe B sind vergleichbar mit denen des duktalen Adenokarzinoms der Pankreas (PDAC), einer Tumorentität mit nachgewiesen exokrinen Zellursprung. Insgesamt ergeben die neuen Erkenntnisse dieser Arbeit ein umfassendes Profil, das PanNET- und PanNEC-Tumore genetisch und epigenetisch eindeutig charakterisiert. Weiterhin liefert diese Arbeit starke Beweise für die aufkommende, aber unbewiesene Theorie des exokrinen Ursprungs von PanNECs und somit einen neuen Ansatz für die Behandlung dieser seltenen, aber oft tödlichen Krankheit.
