Mass spectrometry imaging can produce large amounts of complex spectral and spatial data. Such data sets are often analyzed with unsupervised machine learning approaches, which aim at reducing their ...complexity and facilitating their interpretation. However, choices made during data processing can impact the overall interpretation of these analyses. This work investigates the impact of the choices made at the peak selection step, which often occurs early in the data processing pipeline. The discussion is done in terms of visualization and interpretation of the results of two commonly used unsupervised approaches: t-distributed stochastic neighbor embedding and k-means clustering, which differ in nature and complexity. Criteria considered for peak selection include those based on hypotheses (exemplified herein in the analysis of metabolic alterations in genetically engineered mouse models of human colorectal cancer), particular molecular classes, and ion intensity. The results suggest that the choices made at the peak selection step have a significant impact in the visual interpretation of the results of either dimensionality reduction or clustering techniques and consequently in any downstream analysis that relies on these. Of particular significance, the results of this work show that while using the most abundant ions can result in interesting structure-related segmentation patterns that correlate well with histological features, using a smaller number of ions specifically selected based on prior knowledge about the biochemistry of the tissues under investigation can result in an easier-to-interpret, potentially more valuable, hypothesis-confirming result. Findings presented will help researchers understand and better utilize unsupervised machine learning approaches to mine high-dimensionality data.
Ral GTPases are RAS effector molecules and by implication a potential therapeutic target for RAS mutant cancer. However, very little is known about their roles in stem cells and tissue homeostasis. ...Using Drosophila, we identified expression of RalA in intestinal stem cells (ISCs) and progenitor cells of the fly midgut. RalA was required within ISCs for efficient regeneration downstream of Wnt signaling. Within the murine intestine, genetic deletion of either mammalian ortholog, Rala or Ralb, reduced ISC function and Lgr5 positivity, drove hypersensitivity to Wnt inhibition, and impaired tissue regeneration following damage. Ablation of both genes resulted in rapid crypt death. Mechanistically, RALA and RALB were required for efficient internalization of the Wnt receptor Frizzled-7. Together, we identify a conserved role for RAL GTPases in the promotion of optimal Wnt signaling, which defines ISC number and regenerative potential.
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•RAL GTPases are widely expressed in the intestinal epithelium•RAL GTPases activate Wnt signaling through internalization of Wnt receptors•RAL GTPase signaling is required for optimal stem cell numbers•RAL GTPases drive regeneration of fruit fly and mouse intestine following damage
RAL GTPases are central players in intestinal biology. RALs are required to activate canonical Wnt signaling in Drosophila, mice, and human cell lines. Within intestinal stem cells, RALs are essential to sustain tissue homeostasis and regeneration. RAL small GTPases modulate Wnt pathway activity through internalization of cell-surface Wnt receptors.
Wnt pathway deregulation is a common characteristic of many cancers. Only colorectal cancer predominantly harbours mutations in APC, whereas other cancer types (hepatocellular carcinoma, solid ...pseudopapillary tumours of the pancreas) have activating mutations in β‐catenin (CTNNB1). We have compared the dynamics and the potency of β‐catenin mutations in vivo. Within the murine small intestine (SI), an activating mutation of β‐catenin took much longer to achieve Wnt deregulation and acquire a crypt‐progenitor cell (CPC) phenotype than Apc or Gsk3 loss. Within the colon, a single activating mutation of β‐catenin was unable to drive Wnt deregulation or induce the CPC phenotype. This ability of β‐catenin mutation to differentially transform the SI versus the colon correlated with higher expression of E‐cadherin and a higher number of E‐cadherin:β‐catenin complexes at the membrane. Reduction in E‐cadherin synergised with an activating mutation of β‐catenin resulting in a rapid CPC phenotype within the SI and colon. Thus, there is a threshold of β‐catenin that is required to drive transformation, and E‐cadherin can act as a buffer to sequester mutated β‐catenin.
Synopsis
In contrast to other Wnt‐driven malignancies colorectal cancer is usually linked to APC loss, and very rarely to β‐catenin activating mutations. Different E‐cadherin levels can explain the variation in transforming potential of β‐catenin mutations in different tumors.
Activating β‐catenin mutations in the mouse small intestine but not the colon lead to Wnt‐activation.
Increased E‐cadherin levels can buffer mutated β‐catenin in the colon epithelium.
β‐catenin activating mutations are linked to human cancers that show reduced levels of E‐cadherin.
In contrast to other Wnt‐driven malignancies colorectal cancer is usually linked to APC loss, and very rarely to β‐catenin activating mutations. Different E‐cadherin levels can explain the variation in transforming potential of β‐catenin mutations in different tumors.
-mutant colorectal cancers are resistant to therapeutics, presenting a significant problem for ∼40% of cases. Rapalogs, which inhibit mTORC1 and thus protein synthesis, are significantly less potent ...in
-mutant colorectal cancer. Using
-mutant mouse models and mouse- and patient-derived organoids, we demonstrate that KRAS with G12D mutation fundamentally rewires translation to increase both bulk and mRNA-specific translation initiation. This occurs via the MNK/eIF4E pathway culminating in sustained expression of c-MYC. By genetic and small-molecule targeting of this pathway, we acutely sensitize KRAS
models to rapamycin via suppression of c-MYC. We show that 45% of colorectal cancers have high signaling through mTORC1 and the MNKs, with this signature correlating with a 3.5-year shorter cancer-specific survival in a subset of patients. This work provides a c-MYC-dependent cotargeting strategy with remarkable potency in multiple
-mutant mouse models and metastatic human organoids and identifies a patient population that may benefit from its clinical application. SIGNIFICANCE:
mutation and elevated c-MYC are widespread in many tumors but remain predominantly untargetable. We find that mutant
modulates translation, culminating in increased expression of c-MYC. We describe an effective strategy targeting mTORC1 and MNK in
-mutant mouse and human models, pathways that are also commonly co-upregulated in colorectal cancer.
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Molecular stratification using gene-level transcriptional data has identified subtypes with distinctive genotypic and phenotypic traits, as exemplified by the consensus molecular subtypes (CMS) in ...colorectal cancer (CRC). Here, rather than gene-level data, we make use of gene ontology and biological activation state information for initial molecular class discovery. In doing so, we defined three pathway-derived subtypes (PDS) in CRC: PDS1 tumors, which are canonical/LGR5
stem-rich, highly proliferative and display good prognosis; PDS2 tumors, which are regenerative/ANXA1
stem-rich, with elevated stromal and immune tumor microenvironmental lineages; and PDS3 tumors, which represent a previously overlooked slow-cycling subset of tumors within CMS2 with reduced stem populations and increased differentiated lineages, particularly enterocytes and enteroendocrine cells, yet display the worst prognosis in locally advanced disease. These PDS3 phenotypic traits are evident across numerous bulk and single-cell datasets, and demark a series of subtle biological states that are currently under-represented in pre-clinical models and are not identified using existing subtyping classifiers.
Generation of transcriptional data has dramatically increased in the past decade, driving the development of analytical algorithms that enable interrogation of the biology underpinning the profiled ...samples. However, these resources require users to have expertise in data wrangling and analytics, reducing opportunities for biological discovery by 'wet-lab' users with a limited programming skillset. Although commercial solutions exist, costs for software access can be prohibitive for academic research groups. To address these challenges, we have developed an open source and user-friendly data analysis platform for on-the-fly bioinformatic interrogation of transcriptional data derived from human or mouse tissue, called Molecular Subtyping Resource (MouSR). This internet-accessible analytical tool, https://mousr.qub.ac.uk/, enables users to easily interrogate their data using an intuitive 'point-and-click' interface, which includes a suite of molecular characterisation options including quality control, differential gene expression, gene set enrichment and microenvironmental cell population analyses from RNA sequencing. The MouSR online tool provides a unique freely available option for users to perform rapid transcriptomic analyses and comprehensive interrogation of the signalling underpinning transcriptional datasets, which alleviates a major bottleneck for biological discovery. This article has an associated First Person interview with the first author of the paper.
Loss of the tumour suppressor PTEN is frequent in human melanoma, results in MAPK activation, suppresses senescence and mediates metastatic behaviour. How PTEN loss mediates these effects is unknown. ...Here we show that loss of PTEN in epithelial and melanocytic cell lines induces the nuclear localization and transcriptional activation of β-catenin independent of the PI3K-AKT-GSK3β axis. The absence of PTEN leads to caveolin-1 (CAV1)-dependent β-catenin transcriptional modulation in vitro, cooperates with NRAS(Q61K) to initiate melanomagenesis in vivo and induces efficient metastasis formation associated with E-cadherin internalization. The CAV1-β-catenin axis is mediated by a feedback loop in which β-catenin represses transcription of miR-199a-5p and miR-203, which suppress the levels of CAV1 mRNA in melanoma cells. These data reveal a mechanism by which loss of PTEN increases CAV1-mediated dissociation of β-catenin from membranous E-cadherin, which may promote senescence bypass and metastasis.
Recent studies have suggested increased plasticity of differentiated cells within the intestine to act both as intestinal stem cells (ISCs) and tumour-initiating cells. However, little is known of ...the processes that regulate this plasticity. Our previous work has shown that activating mutations of Kras or the NF-κB pathway can drive dedifferentiation of intestinal cells lacking Apc. To investigate this process further, we profiled both cells undergoing dedifferentiation in vitro and tumours generated from these cells in vivo by gene expression analysis. Remarkably, no clear differences were observed in the tumours; however, during dedifferentiation in vitro we found a marked upregulation of TGFβ signalling, a pathway commonly mutated in colorectal cancer (CRC). Genetic inactivation of TGFβ type 1 receptor (Tgfbr1/Alk5) enhanced the ability of Kras
mutation to drive dedifferentiation and markedly accelerated tumourigenesis. Mechanistically this is associated with a marked activation of MAPK signalling. Tumourigenesis from differentiated compartments is potently inhibited by MEK inhibition. Taken together, we show that tumours arising in differentiated compartments will be exposed to different suppressive signals, for example, TGFβ and blockade of these makes tumourigenesis more efficient from this compartment.
Nitric Oxide (NO) and soluble adhesion molecules are promising biomarkers, which predict endothelial dysfunction in sickle cell disease (SCD). Several studies have investigated the relationship ...between NO (as well as its metabolites) and endothelial adhesion molecules in SCD. However, these studies were done mainly in the developed world, and it is difficult to extrapolate the findings to SCD populations in other geographical regions such as Africa due to significant disparities in the results. The aim of the current study was to determine the correlation between levels of nitric oxide metabolites (NOx) and adhesion molecules in SCD patients in a tertiary hospital in Ghana. A case control cross-sectional study involving 100 SCD (made up of HbSS and HbSC patients) and 60 healthy controls was conducted. Concentrations of NOx and soluble endothelial adhesion molecules (ICAM-1, VCAM-1 and E-selectin) were measured in all the study participants (n = 160) by the Griess reagent system and enzyme-linked immunosorbent assay (ELISA). Correlation analysis was performed to determine a possible link between the variables. Levels of soluble adhesion molecules were higher in the HbSS patients. Correlation of NOx with ICAM-1 almost approached significance (r = 0.565,
= 0.058) in the HbSS patients. There were no correlations between NOx and E-selectin in both HbSS and HbSC patients. There were no significant correlations between NOx and VCAM-1 in all the study participants (
> 0.05). Of the soluble adhesion molecules, ICAM-1 showed a significant positive correlation with VCAM-1 in the HbSC patients. There were no significant differences between the adhesion molecules and the age of participants in the various study groups. Whether or not a significant correlation exists between NOx and soluble adhesion molecules may not depend on the sickle cell genotype. The expression of adhesion molecules may not depend on age.
Nitric oxide (NO) plays a fundamental role in maintaining normal vasomotor tone. Recent clinical and experimental data suggest that NO may play a role in the pathogenesis and therapy of sickle cell ...disease (SCD). The aim of this study was to determine NO metabolites (NOx) in SCD patients at steady state and in vaso-occlusive crisis (VOC), as well as those with hemolytic clinical sub-phenotype that includes leg ulcers and priapism.
This was a case-control cross-sectional study conducted on a total of 694 subjects including 148 comparison group HbAA, 208 HbSS SCD patients in steady state, 82 HbSC SCD patients in steady state, 156 HbSS SCD patients in VOC, 34 HbSC SCD patients in VOC, 34 HbSS SCD patients in post VOC, 21 HbSS SCD patients with leg ulcer and 11 HbSS SCD patients with priapism, with age ranging from 15 to 65 years. Laboratory diagnosis of SCD was done at the Sickle Cell Clinic of the Korle-Bu Teaching Hospital. Plasma nitric oxide metabolites were measured using Griess reagent system by ELISA method.
Mean NOx of 59.66±0.75 µMol/L in the comparison group was significantly different from those in steady state (
=0.02). During VOC, there was a significant reduction in mean NOx levels to 6.08±0.81 µMol/L (
<0.001). Mean NOx levels were however, significantly higher (50.97±1.68 µMol/L) (
<0.001) in the immediate postcrisis period. The mean NOx levels in the leg ulcer (21.70±1.18 µMol/L) (
<0.001) and priapism (28.97±1.27 µMol/L) (
<0.001) patients were significantly low as compared to the SCD patients in the steady state and comparison group.
This study presents the first report on plasma NOx levels in SCD complication in Ghanaian SCD patients and confirms reduced plasma NOx levels in SCD patients in general.
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