Fig 8 - available from: Nature Communications
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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
Source publication
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
... It is used widely in immunology research to quantify surface proteins and classify immune cells [Spitzer and Nolan, 2016;Bendall et al., 2011;Horowitz et al., 2013;Giesen et al., 2014;Georg et al., 2022]. Mass cytometry is also increasingly used to study intracellular signalling pathways by measuring phospho-protein abundance, providing insights into diverse cellular processes such as the differentiation pathways of colorectal cancer [Brandt et al., 2019;Sell et al., 2023], organoid heterogeneity [Sufi et al., 2021], acute myeloid leukaemia stem/progenitor cells [Han et al., 2015] and prediction of drug sensitivity in breast cancer [Tognetti et al., 2021]. While the distributions of surface proteins typi-cally show a bimodal pattern, those of intracellular signalling markers show a unimodal distribution with rather small quantitative shifts in response to perturbations. ...
High dimensional mass cytometry is confounded by unwanted covariance due to variations in cell size and staining efficiency, making analysis and interpretation challenging.
We present RUCova, a novel method designed to address confounding factors in mass cytometry data. RUCova removes unwanted covariance using multivariate linear regression on Surrogates of Unwanted Covariance (SUCs), and Principal Component Analysis (PCA). We exemplify the use of RUCova and show that it effectively removes unwanted covariance while preserving genuine biological signals. Our results demonstrate the efficacy of RUCova in elucidating complex data patterns, facilitating the identification of activated signalling pathways, and improving the classification of important cell populations. By providing a robust framework for data normalization and interpretation, RUCova enhances the accuracy and reliability of mass cytometry analyses, contributing to advancements in our understanding of cellular biology and disease mechanisms. The R package is available on https://github.com/molsysbio/RUCova . Detailed documentation, data, and the code required to reproduce the results are available on https://doi.org/10.5281/zenodo.10913464 . Supplementary material: Available at bioRxiv.
... Additional examination of the kinase cascade uncovered that RAF1 serves as the upstream kinase responsible for phosphorylating MEK1 at Ser222 and MEK2 at Ser218, thereby controlling the functionality of MEK. This process enables the transmission of MAPK signaling from RAS, RAF, MEK, and ultimately to ERK [11][12][13][14]. The RAF-MEK-ERK signaling pathway is strongly associated with the development of different types of cancers, and several drugs that target important components of this pathway have been effectively commercialized [13,[15][16][17][18]. ...
Objective
This study aimed to investigate TCF19′s role in lung cancer development, specifically its involvement in the RAF/MEK/ERK signaling pathway.
Methods
Lung cancer tissue analysis revealed significant TCF19 overexpression. In vitro experiments using A549 and Hop62 cells with TCF19 overexpression demonstrated enhanced cell growth. Transgenic mouse models confirmed TCF19′s role in primary tumor development. Transcriptome sequencing identified altered gene expression profiles, linking TCF19 to RAF/MEK/ERK pathway activation. Functional assays elucidated underlying mechanisms, revealing increased phosphorylation of Raf1, MEK1/2, and ERK1/2. Inhibiting RAF1 or ERK through shRaf1 or ERK inhibitor reduced cell cycle-related proteins and inhibited TCF19-overexpressing cell growth.
Results
TCF19 was identified as an oncogene in lung carcinoma, specifically impacting the RAF/MEK/ERK pathway. Elevated TCF19 levels in lung cancer suggest targeting TCF19 or its associated pathways as a promising strategy for disease management.
Conclusion
This study unveils TCF19′s oncogenic role in lung cancer, emphasizing its modulation of the RAF/MEK/ERK pathway and presenting a potential therapeutic target for TCF19-overexpressing lung cancers.
... Several methods to reconstruct population specific networks from single cell data have been proposed before (Rukhlenko et al., 2022;Brandt et al., 2019;Kumar et al., 2020). However, these methods use the single cell nature of the data only to cluster cells in distinct groups, and then aggregate the cells within one population. ...
Motivation
Signal transduction networks regulate a multitude of essential biological processes and are frequently aberrated in diseases such as cancer. Developing a mechanistic understanding of such networks is essential to understand disease or cell population specific signaling and to design effective treatment strategies. Typically, such networks are computationally reconstructed based on systematic perturbation experiments, followed by quantification of signaling protein activity. Recent technological advances now allow for the quantification of the activity of many (signaling) proteins simultaneously in single cells. This makes it feasible to reconstruct signaling networks from single cell data.
Results
Here we introduce single cell Comparative Network Reconstruction (scCNR) to derive signal transduction networks by exploiting the heterogeneity of single cell (phospho)protein measurements. scCNR treats stochastic variation in total protein abundances as natural perturbation experiments, whose effects propagate through the network. scCNR reconstructs cell population specific networks of the same underlying topology for cells from diverse populations. We extensively validated scCNR on simulated single cell data, and we applied it to a dataset of EGFR-inhibitor treated keratinocytes to recover signaling differences downstream of EGFR and in protein interactions associated with proliferation. scCNR will help to unravel the mechanistic signaling differences between cell populations by making use of single-cell data, and will subsequently guide the development of well-informed treatment strategies.
Availability and implementation
scCNR is available as a python module at https://github.com/ibivu/scmra . Additionally, code to reproduce all figures is available at https://github.com/tstohn/scmra_analysis .
Supplementary information
Supplementary information and data are available at Bioinformatics online.
... What's more, here we identified that KRAS genotype affects the activation of downstream ERK signaling pathway and KRAS p.G12D mutation-induced activation of ERK signaling is stronger than KRAS p.G13D and KRAS WT which is similar with a previous report that BRAF Article https://doi.org/10.1038/s41467-024-45572-w V600E could induce stronger ERK activity than KRAS p.G12V mutation 38 . ...
Fusobacterium nucleatum (F. nucleatum) promotes intestinal tumor growth and its relative abundance varies greatly among patients with CRC, suggesting the presence of unknown, individual-specific effectors in F. nucleatum-dependent carcinogenesis. Here, we identify that F. nucleatum is enriched preferentially in KRAS p.G12D mutant CRC tumor tissues and contributes to colorectal tumorigenesis in Villin-Cre/KrasG12D+/- mice. Additionally, Parabacteroides distasonis (P. distasonis) competes with F. nucleatum in the G12D mouse model and human CRC tissues with the KRAS mutation. Orally gavaged P. distasonis in mice alleviates the F. nucleatum-dependent CRC progression. F. nucleatum invades intestinal epithelial cells and binds to DHX15, a protein of RNA helicase family expressed on CRC tumor cells, mechanistically involving ERK/STAT3 signaling. Knock out of Dhx15 in Villin-Cre/KrasG12D+/- mice attenuates the CRC phenotype. These findings reveal that the oncogenic effect of F. nucleatum depends on somatic genetics and gut microbial ecology and indicate that personalized modulation of the gut microbiota may provide a more targeted strategy for CRC treatment.
... Our study suggests that the Ku70-ERK signaling pathway is tumor suppressive, which is in contrast to the observation that Ras/Raf mutations, which are common in colorectal cancer, drive aberrant activation of downstream ERK-MAPK signaling. However, in colon cancer, activation of ERK can occur in a cell type-specific manner (79). We observed that the expression of the gene encoding Ku70 is decreased in certain cell types of the epithelial and stromal compartments from the patients with Crohn's disease and colorectal cancer, suggesting that Ku70 might function in a cell type-specific manner during the development of intestinal inflammation and cancer. ...
The innate immune response contributes to the development or attenuation of acute and chronic diseases, including cancer. Microbial DNA and mislocalized DNA from damaged host cells can activate different host responses that shape disease outcomes. Here, we show that mice and humans lacking a single allele of the DNA repair protein Ku70 had increased susceptibility to the development of intestinal cancer. Mechanistically, Ku70 translocates from the nucleus into the cytoplasm where it binds to cytosolic DNA and interacts with the GTPase Ras and the kinase Raf, forming a tripartite protein complex and docking at Rab5 ⁺ Rab7 ⁺ early-late endosomes. This Ku70-Ras-Raf signalosome activates the MEK-ERK pathways, leading to impaired activation of cell cycle proteins Cdc25A and CDK1, reducing cell proliferation and tumorigenesis. We also identified the domains of Ku70, Ras, and Raf involved in activating the Ku70 signaling pathway. Therapeutics targeting components of the Ku70 signalosome could improve the treatment outcomes in cancer.
... Additional examination of the kinase cascade uncovered that RAF1 serves as the upstream kinase responsible for phosphorylating MEK1 at Ser222 and MEK2 at Ser218, thereby controlling the functionality of MEK. This process enables the transmission of MAPK signaling from RAS, RAF, MEK, and ultimately to ERK [8][9][10][11] . The RAF-MEK-ERK signaling pathway is strongly associated with the development of different types of cancers, and several drugs that target important components of this pathway have been effectively commercialized [10,[12][13][14][15] . ...
Objective
This study aimed to investigate the role of TCF19 in lung cancer, focusing on its impact on the development and progression of tumors. Specifically, the objective was to elucidate the molecular mechanisms underlying TCF19-mediated effects, with a particular emphasis on its involvement in the RAF/MEK/ERK signaling pathway.
Methods
The research involved the analysis of lung cancer tissues to assess the expression levels of TCF19. In vitro experiments were conducted using lung cancer cells (A549 and Hop62) with TCF19 overexpression. Transgenic mouse models were employed to study the in vivo effects of TCF19 on the development of primary tumors. Transcriptome sequencing was performed to identify alterations in gene expression profiles, and further experiments were carried out to investigate the activation status of the RAF/MEK/ERK pathway. Functional assays, including cell cycle progression and the levels of cell cycle-associated proteins, were conducted to understand the underlying mechanisms.
Results
The research findings demonstrated significant overexpression of TCF19 in lung cancer tissues. In vitro experiments revealed that TCF19 overexpression stimulated the growth of lung cancer cells and facilitated the development of primary tumors in transgenic mice. Mechanistically, TCF19 overexpression was associated with an elevation in the Ras and MAPK signaling pathways, as indicated by increased phosphorylation of Raf1, MEK1/2, and ERK1/2 in A549 and Hop62 cells. However, the inhibition of RAF1 or ERK, either through shRaf1 or ERK inhibitor, led to a reduction in cell cycle-related proteins and inhibited cell growth in TCF19-overexpressing cells.
Conclusion
In conclusion, this study identified TCF19 as an oncogene in lung carcinoma. The research highlighted its specific impact on the RAF/MEK/ERK signaling pathway, offering insights into a novel aspect of the molecular cascade involved in lung cancer development. Targeting TCF19 or its associated signaling pathways may present a promising avenue for the management of lung cancer characterized by elevated TCF19 levels.
... To assess the impact of Braf V600E gene dosage, intestinal tissues were sampled from Braf +/V600E , Braf fl/V600E , Braf V600E/V600E or control mice at a time point 3 days post-induction recombination. As previously reported, we found that Braf V600E/+ promotes proliferation in the intestinal crypt (BrdU + ), indeed to a greater degree than Kras G12D/+ at this time point (Supplementary Fig. 1a, b) 27,28 . In line with the phenotypes observed in KRAS mutant intestine, we find that altering allelic balance in favour of oncogenic Braf, either through breeding to homozygosity (Braf V600E/V600E ), or through conditional deletion of the wild-type allele (Braf fl/V600E ) leads to significant increase in crypt cell proliferation and loss of Lysozyme-positive Paneth cells ( Supplementary Fig. 1a, b). ...
Oncogenic KRAS mutations are well-described functionally and are known to drive tumorigenesis. Recent reports describe a significant prevalence of KRAS allelic imbalances or gene dosage changes in human cancers, including loss of the wild-type allele in KRAS mutant cancers. However, the role of wild-type KRAS in tumorigenesis and therapeutic response remains elusive. We report an in vivo murine model of colorectal cancer featuring deletion of wild-type Kras in the context of oncogenic Kras. Deletion of wild-type Kras exacerbates oncogenic KRAS signalling through MAPK and thus drives tumour initiation. Absence of wild-type Kras potentiates the oncogenic effect of KRASG12D, while incidentally inducing sensitivity to inhibition of MEK1/2. Importantly, loss of the wild-type allele in aggressive models of KRASG12D-driven CRC significantly alters tumour progression, and suppresses metastasis through modulation of the immune microenvironment. This study highlights the critical role for wild-type Kras upon tumour initiation, progression and therapeutic response in Kras mutant CRC.
... Reporters also vary in the timescale of ERK activity changes they can detect, with FRET reporters showing the fastest responses, followed closely by translocation-based reporters, and degradation reporters being the slowest. While rapid reporter responses are needed to accurately distinguish closely grouped pulses of ERK activity, the slow responses of a degradation-based reporter can be very useful for measuring the integrated activity of ERK over time [31,45,46]. ...
Extracellular signal-regulated kinase (ERK) has long been studied as a key driver of both essential cellular processes and disease. A persistent question has been how this single pathway is able to direct multiple cell behaviors, including growth, proliferation, and death. Modern biosensor studies have revealed that the temporal pattern of ERK activity is highly variable and heterogeneous, and critically, that these dynamic differences modulate cell fate. This two-part review discusses the current understanding of dynamic activity in the ERK pathway, how it regulates cellular decisions, and how these cell fates lead to tissue regulation and pathology. In part 1, we cover the optogenetic and live-cell imaging technologies that first revealed the dynamic nature of ERK, as well as current challenges in biosensor data analysis. We also discuss advances in mathematical models for the mechanisms of ERK dynamics, including receptor-level regulation, negative feedback, cooperativity, and paracrine signaling. While hurdles still remain, it is clear that higher temporal and spatial resolution provide mechanistic insights into pathway circuitry. Exciting new algorithms and advanced computational tools enable quantitative measurements of single-cell ERK activation, which in turn inform better models of pathway behavior. However, the fact that current models still cannot fully recapitulate the diversity of ERK responses calls for a deeper understanding of network structure and signal transduction in general.
... 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.
