Pathway level subtyping identifies a slow-cycling biological phenotype associated with poor clinical outcomes in colorectal cancer.

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.

MmCMS: mouse models' consensus molecular subtypes of colorectal cancer.

BACKGROUND: Colorectal cancer (CRC) primary tumours are molecularly classified into four consensus molecular subtypes (CMS1-4). Genetically engineered mouse models aim to faithfully mimic the complexity of human cancers and, when appropriately aligned, represent ideal pre-clinical systems to test new drug treatments. Despite its importance, dual-species classification has been limited by the lack of a reliable approach. Here we utilise, develop and test a set of options for human-to-mouse CMS classifications of CRC tissue. METHODS: Using transcriptional data from established collections of CRC tumours, including human (TCGA cohort; n = 577) and mouse (n = 57 across n = 8 genotypes) tumours with combinations of random forest and nearest template prediction algorithms, alongside gene ontology collections, we comprehensively assess the performance of a suite of new dual-species classifiers. RESULTS: We developed three approaches: MmCMS-A; a gene-level classifier, MmCMS-B; an ontology-level approach and MmCMS-C; a combined pathway system encompassing multiple biological and histological signalling cascades. Although all options could identify tumours associated with stromal-rich CMS4-like biology, MmCMS-A was unable to accurately classify the biology underpinning epithelial-like subtypes (CMS2/3) in mouse tumours. CONCLUSIONS: When applying human-based transcriptional classifiers to mouse tumour data, a pathway-level classifier, rather than an individual gene-level system, is optimal. Our R package enables researchers to select suitable mouse models of human CRC subtype for their experimental testing.

Epithelial TGFß engages growth-factor signalling to circumvent apoptosis and drive intestinal tumourigenesis with aggressive features.

The pro-tumourigenic role of epithelial TGFß signalling in colorectal cancer (CRC) is controversial. Here, we identify a cohort of born to be bad early-stage (T1) colorectal tumours, with aggressive features and a propensity to disseminate early, that are characterised by high epithelial cell-intrinsic TGFß signalling. In the presence of concurrent Apc and Kras mutations, activation of epithelial TGFß signalling rampantly accelerates tumourigenesis and share transcriptional signatures with those of the born to be bad T1 human tumours and predicts recurrence in stage II CRC. Mechanistically, epithelial TGFß signalling induces a growth-promoting EGFR-signalling module that synergises with mutant APC and KRAS to drive MAPK signalling that re-sensitise tumour cells to MEK and/or EGFR inhibitors. Together, we identify epithelial TGFß signalling both as a determinant of early dissemination and a potential therapeutic vulnerability of CRC's with born to be bad traits.

Biological Misinterpretation of Transcriptional Signatures in Tumor Samples Can Unknowingly Undermine Mechanistic Understanding and Faithful Alignment with Preclinical Data.

PURPOSE: Precise mechanism-based gene expression signatures (GES) have been developed in appropriate in vitro and in vivo model systems, to identify important cancer-related signaling processes. However, some GESs originally developed to represent specific disease processes, primarily with an epithelial cell focus, are being applied to heterogeneous tumor samples where the expression of the genes in the signature may no longer be epithelial-specific. Therefore, unknowingly, even small changes in tumor stroma percentage can directly influence GESs, undermining the intended mechanistic signaling. EXPERIMENTAL DESIGN: Using colorectal cancer as an exemplar, we deployed numerous orthogonal profiling methodologies, including laser capture microdissection, flow cytometry, bulk and multiregional biopsy clinical samples, single-cell RNA sequencing and finally spatial transcriptomics, to perform a comprehensive assessment of the potential for the most widely used GESs to be influenced, or confounded, by stromal content in tumor tissue. To complement this work, we generated a freely-available resource, ConfoundR; https://confoundr.qub.ac.uk/, that enables users to test the extent of stromal influence on an unlimited number of the genes/signatures simultaneously across colorectal, breast, pancreatic, ovarian and prostate cancer datasets. RESULTS: Findings presented here demonstrate the clear potential for misinterpretation of the meaning of GESs, due to widespread stromal influences, which in-turn can undermine faithful alignment between clinical samples and preclinical data/models, particularly cell lines and organoids, or tumor models not fully recapitulating the stromal and immune microenvironment. CONCLUSIONS: Efforts to faithfully align preclinical models of disease using phenotypically-designed GESs must ensure that the signatures themselves remain representative of the same biology when applied to clinical samples.

Activation of innate-adaptive immune machinery by poly(I:C) exposes a therapeutic vulnerability to prevent relapse in stroma-rich colon cancer.

OBJECTIVE: Stroma-rich tumours represent a poor prognostic subtype in stage II/III colon cancer (CC), with high relapse rates and limited response to standard adjuvant chemotherapy. DESIGN: To address the lack of efficacious therapeutic options for patients with stroma-rich CC, we stratified our human tumour cohorts according to stromal content, enabling identification of the biology underpinning relapse and potential therapeutic vulnerabilities specifically within stroma-rich tumours that could be exploited clinically. Following human tumour-based discovery and independent clinical validation, we use a series of in vitro and stroma-rich in vivo models to test and validate the therapeutic potential of elevating the biology associated with reduced relapse in human tumours. RESULTS: By performing our analyses specifically within the stroma-rich/high-fibroblast (HiFi) subtype of CC, we identify and validate the clinical value of a HiFi-specific prognostic signature (HPS), which stratifies tumours based on STAT1-related signalling (High-HPS v Low-HPS=HR 0.093, CI 0.019 to 0.466). Using in silico, in vitro and in vivo models, we demonstrate that the HPS is associated with antigen processing and presentation within discrete immune lineages in stroma-rich CC, downstream of double-stranded RNA and viral response signalling. Treatment with the TLR3 agonist poly(I:C) elevated the HPS signalling and antigen processing phenotype across in vitro and in vivo models. In an in vivo model of stroma-rich CC, poly(I:C) treatment significantly increased systemic cytotoxic T cell activity (p<0.05) and reduced liver metastases (p<0.0002). CONCLUSION: This study reveals new biological insight that offers a novel therapeutic option to reduce relapse rates in patients with the worst prognosis CC.

Molecular Subtyping Resource: a user-friendly tool for rapid biological discovery from transcriptional data.

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.

In-depth Clinical and Biological Exploration of DNA Damage Immune Response as a Biomarker for Oxaliplatin Use in Colorectal Cancer.

PURPOSE: The DNA damage immune response (DDIR) assay was developed in breast cancer based on biology associated with deficiencies in homologous recombination and Fanconi anemia pathways. A positive DDIR call identifies patients likely to respond to platinum-based chemotherapies in breast and esophageal cancers. In colorectal cancer, there is currently no biomarker to predict response to oxaliplatin. We tested the ability of the DDIR assay to predict response to oxaliplatin-based chemotherapy in colorectal cancer and characterized the biology in DDIR-positive colorectal cancer. EXPERIMENTAL DESIGN: Samples and clinical data were assessed according to DDIR status from patients who received either 5-fluorouracil (5-FU) or 5FUFA (bolus and infusion 5-FU with folinic acid) plus oxaliplatin (FOLFOX) within the FOCUS trial (n = 361, stage IV), or neoadjuvant FOLFOX in the FOxTROT trial (n = 97, stage II/III). Whole transcriptome, mutation, and IHC data of these samples were used to interrogate the biology of DDIR in colorectal cancer. RESULTS: Contrary to our hypothesis, DDIR-negative patients displayed a trend toward improved outcome for oxaliplatin-based chemotherapy compared with DDIR-positive patients. DDIR positivity was associated with microsatellite instability (MSI) and colorectal molecular subtype 1. Refinement of the DDIR signature, based on overlapping IFN-related chemokine signaling associated with DDIR positivity across colorectal cancer and breast cancer cohorts, further confirmed that the DDIR assay did not have predictive value for oxaliplatin-based chemotherapy in colorectal cancer. CONCLUSIONS: DDIR positivity does not predict improved response following oxaliplatin treatment in colorectal cancer. However, data presented here suggest the potential of the DDIR assay in identifying immune-rich tumors that may benefit from immune checkpoint blockade, beyond current use of MSI status.

Downregulation of PPARα during Experimental Left Ventricular Hypertrophy Is Critically Dependent on Nox2 NADPH Oxidase Signalling.

Pressure overload-induced left ventricular hypertrophy (LVH) is initially adaptive but ultimately promotes systolic dysfunction and chronic heart failure. Whilst underlying pathways are incompletely understood, increased reactive oxygen species generation from Nox2 NADPH oxidases, and metabolic remodelling, largely driven by PPARα downregulation, are separately implicated. Here, we investigated interaction between the two as a key regulator of LVH using in vitro, in vivo and transcriptomic approaches. Phenylephrine-induced H9c2 cardiomyoblast hypertrophy was associated with reduced PPARα expression and increased Nox2 expression and activity. Pressure overload-induced LVH and systolic dysfunction induced in wild-type mice by transverse aortic constriction (TAC) for 7 days, in association with Nox2 upregulation and PPARα downregulation, was enhanced in PPARα-/- mice and prevented in Nox2-/- mice. Detailed transcriptomic analysis revealed significantly altered expression of genes relating to PPARα, oxidative stress and hypertrophy pathways in wild-type hearts, which were unaltered in Nox2-/- hearts, whilst oxidative stress pathways remained dysregulated in PPARα-/- hearts following TAC. Network analysis indicated that Nox2 was essential for PPARα downregulation in this setting and identified preferential inflammatory pathway modulation and candidate cytokines as upstream Nox2-sensitive regulators of PPARα signalling. Together, these data suggest that Nox2 is a critical driver of PPARα downregulation leading to maladaptive LVH.

The Intricate Interplay between Epigenetic Events, Alternative Splicing and Noncoding RNA Deregulation in Colorectal Cancer.

Colorectal cancer (CRC) results from a transformation of colonic epithelial cells into adenocarcinoma cells due to genetic and epigenetic instabilities, alongside remodelling of the surrounding stromal tumour microenvironment. Epithelial-specific epigenetic variations escorting this process include chromatin remodelling, histone modifications and aberrant DNA methylation, which influence gene expression, alternative splicing and function of non-coding RNA. In this review, we first highlight epigenetic modulators, modifiers and mediators in CRC, then we elaborate on causes and consequences of epigenetic alterations in CRC pathogenesis alongside an appraisal of the complex feedback mechanisms realized through alternative splicing and non-coding RNA regulation. An emphasis in our review is put on how this intricate network of epigenetic and post-transcriptional gene regulation evolves during the initiation, progression and metastasis formation in CRC.

Exosomal lncRNAs and cancer: connecting the missing links.

Motivation: Extracellular vesicles (EVs), including exosomes and microvesicles, are potent and clinically valuable tools for early diagnosis, prognosis and potentially the targeted treatment of cancer. The content of EVs is closely related to the type and status of the EV-secreting cell. Circulating exosomes are a source of stable RNAs including mRNAs, microRNAs and long non-coding RNAs (lncRNAs). Results: This review outlines the links between EVs, lncRNAs and cancer. We highlight communication networks involving the tumor microenvironment, the immune system and metastasis. We show examples supporting the value of exosomal lncRNAs as cancer biomarkers and therapeutic targets. We demonstrate how a system biology approach can be used to model cell-cell communication via exosomal lncRNAs and to simulate effects of therapeutic interventions. In addition, we introduce algorithms and bioinformatics resources for the discovery of tumor-specific lncRNAs and tools that are applied to determine exosome content and lncRNA function. Finally, this review provides a comprehensive collection and guide to databases for exosomal lncRNAs. Supplementary information: Supplementary data are available at Bioinformatics online.

Computational and Experimental Identification of Tissue-Specific MicroRNA Targets.

In this chapter we discuss computational methods for the prediction of microRNA (miRNA) targets. More specifically, we consider machine learning-based approaches and explain why these methods have been relatively unsuccessful in reducing the number of false positive predictions. Further we suggest approaches designed to improve their performance by considering tissue-specific target regulation. We argue that the miRNA targetome differs depending on the tissue type and introduce a novel algorithm that predicts miRNA targets specifically for colorectal cancer. We discuss features of miRNAs and target sites that affect target recognition, and how next-generation sequencing data can support the identification of novel miRNAs, differentially expressed miRNAs and their tissue-specific mRNA targets. In addition, we introduce some experimental approaches for the validation of miRNA targets as well as web-based resources sharing predicted and validated miRNA target interactions.

RNA Systems Biology for Cancer: From Diagnosis to Therapy.

It is due to the advances in high-throughput omics data generation that RNA species have re-entered the focus of biomedical research. International collaborate efforts, like the ENCODE and GENCODE projects, have spawned thousands of previously unknown functional non-coding RNAs (ncRNAs) with various but primarily regulatory roles. Many of these are linked to the emergence and progression of human diseases. In particular, interdisciplinary studies integrating bioinformatics, systems biology, and biotechnological approaches have successfully characterized the role of ncRNAs in different human cancers. These efforts led to the identification of a new tool-kit for cancer diagnosis, monitoring, and treatment, which is now starting to enter and impact on clinical practice. This chapter is to elaborate on the state of the art in RNA systems biology, including a review and perspective on clinical applications toward an integrative RNA systems medicine approach. The focus is on the role of ncRNAs in cancer.

Naïve Bayes classifier predicts functional microRNA target interactions in colorectal cancer.

Alterations in the expression of miRNAs have been extensively characterized in several cancers, including human colorectal cancer (CRC). Recent publications provide evidence for tissue-specific miRNA target recognition. Several computational methods have been developed to predict miRNA targets; however, all of these methods assume a general pattern underlying these interactions and therefore tolerate reduced prediction accuracy and a significant number of false predictions. The motivation underlying the presented work was to unravel the relationship between miRNAs and their target mRNAs in CRC. We developed a novel computational algorithm for miRNA-target prediction in CRC using a Naïve Bayes classifier. The algorithm, which is referred to as CRCmiRTar, was trained with data from validated miRNA target interactions in CRC and other cancer entities. Furthermore, we identified a set of position-based, sequence, structural, and thermodynamic features that identify CRC-specific miRNA target interactions. Evaluation of the algorithm showed a significant improvement of performance with respect to AUC, and sensitivity, compared to other widely used algorithms based on machine learning. Based on miRNA and gene expression profiles in CRC tissues with similar clinical and pathological features, our classifier predicted 204 functional interactions, which involve 11 miRNAs and 41 mRNAs in this cancer entity. While the approach is here validated for CRC, the implementation of disease-specific miRNA target prediction algorithms can be easily adopted for other applications too. The identification of disease-specific miRNA target interactions may also facilitate the identification of potential drug targets.

MicroRNA-mRNA interactions in colorectal cancer and their role in tumor progression.

MicroRNAs (miRNA/miR) play an important role in gene regulatory networks through targeting mRNAs. They are involved in diverse biological processes such as cell proliferation, differentiation, angiogenesis, and apoptosis. Due to their pivotal effects on multiple genes and pathways, dysregulated miRNAs have been reported to be associated with different diseases, including colorectal cancer (CRC). Recent evidence indicates that aberrant miRNA expression is tightly linked with the initiation and progression of CRC. To elucidate the influence of miRNA regulation in CRC, it is critical to identify dysregulated miRNAs, their target mRNA genes and their involvement in gene regulatory and signaling networks. Various experimental and computational studies have been conducted to decipher the function of miRNAs involved in CRC. Experimental studies that are used for this purpose can be classified into two categories: direct/individual and indirect/high-throughput gene expression studies. Here we review miRNA target identification studies related to CRC with an emphasis on experimental data based on Luciferase reporter assays. Recent advances in determining the function of miRNAs and the signaling pathways they are involved in have also been summarized. The review helps bioinformaticians and biologists to find extensive information about downstream targets of dysregulated miRNAs, and their pro-/anti-CRC effects.