Colorectal cancer (CRC) is a frequently lethal disease with heterogeneous outcomes and drug responses. To resolve inconsistencies among the reported gene expression–based CRC classifications and facilitate clinical translation, we formed an international consortium dedicated to large-scale data sharing and analytics across expert groups. We show marked interconnectivity between six independent classification systems coalescing into four consensus molecular subtypes (CMS) with distinguishing features: CMS1 (MSI Immune, 14%), hypermutated, microsatellite unstable, strong immune activation; CMS2 (Canonical, 37%), epithelial, chromosomally unstable, marked WNT and MYC signaling activation; CMS3 (Metabolic, 13%), epithelial, evident metabolic dysregulation; and CMS4 (Mesenchymal, 23%), prominent transforming growth factor β activation, stromal invasion, and angiogenesis. Samples with mixed features (13%) possibly represent a transition phenotype or intra-tumoral heterogeneity. We consider the CMS groups the most robust classification system currently available for CRC – with clear biological interpretability – and the basis for future clinical stratification and subtype–based targeted interventions.

Colon cancer is a clinically diverse disease. This heterogeneity makes it difficult to determine which patients will benefit most from adjuvant therapy and impedes the development of new targeted agents. More insight into the biological diversity of colon cancers, especially in relation to clinical features, is therefore needed. We demonstrate, using an unsupervised classification strategy involving over 1,100 individuals with colon cancer, that three main molecularly distinct subtypes can be recognized. Two subtypes have been previously identified and are well characterized (chromosomal-instable and microsatellite-instable cancers). The third subtype is largely microsatellite stable and contains relatively more CpG island methylator phenotype-positive carcinomas but cannot be identified on the basis of characteristic mutations. We provide evidence that this subtype relates to sessile-serrated adenomas, which show highly similar gene expression profiles, including upregulation of genes involved in matrix remodeling and epithelial-mesenchymal transition. The identification of this subtype is crucial, as it has a very unfavorable prognosis and, moreover, is refractory to epidermal growth factor receptor-targeted therapy.

Immune cells promote the initial metastatic dissemination of carcinoma cells from primary tumors. In contrast to their well-studied functions in the initial stages of metastasis, the specific roles of immunocytes in facilitating progression through the critical later steps of the invasion-metastasis cascade remain poorly understood. Here we define novel functions of neutrophils in promoting intraluminal survival and extravasation at sites of metastatic dissemination. We show that CD11b+/Ly6G+ neutrophils enhance metastasis formation via two distinct mechanisms. First, neutrophils inhibit natural killer cell function, which leads to a significant increase in the intraluminal survival time of tumor cells. Thereafter, neutrophils operate to facilitate extravasation of tumor cells through the secretion of IL-1β and matrix metalloproteinases. These results identify neutrophils as key regulators of intraluminal survival and extravasation through their crosstalk with host cells and disseminating carcinoma cells.

PERK 1,2 . By contrast, the effects of CDDO on CHOP were not blunted by any individual EIF2α kinase deficiency (Extended Data Fig. 1g) , possibly owing to the limited specificity of related compounds 11 . The comparative interrogation of CHOP regulators following three distinct cellular insults allowed us to differentiate global regulators of CHOP biology (Extended Data Fig. 1h-m) from such selectively operating in the context of CCCP-induced mitochondrial depolarization (Extended Data Figs. 2-4). In particular, stringent filtering for genes that prominently scored with CCCP, but not TM or CDDO, highlighted the transcriptional regulators TAF4 and GABPB1, glycolysis factors SLC2A1 and TPI1, and RNA binding proteins RBM27 and CLUH (KIAA0664). Moreover, the signature contained the mitochondrial proteins ATP5IF1 (ATPIF1) and OMA1. Most strikingly, it revealed a strong requirement for HRI (EIF2AK1) and the scarcely studied protein DELE1 (KIAA0141) (Fig. 1a, Extended Data Fig. 5a-b). Cellular dynamics of DELE1Given the scant knowledge on DELE1 and the unexpected involvement of HRI, we first sought to validate their requirement in a panel of cell systems including non-transformed cells, and indeed could confirm their importance in all cases (Extended Data Fig. 5c). Furthermore, CHOP induction also depended on DELE1 and HRI for other types of mitochondrial stress, including inhibition of complex V (oligomycin), TRAP1 (GTPP), and genetic ablation of LONP1 (Extended Data Fig. 5d-f). Failure to induce CHOP after stimulation with CCCP was preceded by a defect in EIF2α phosphorylation in HRI-or DELE1-deficient cells, suggesting that like HRI, DELE1 operates upstream of this event (Extended Data Fig. 5g). Strikingly, expression of HRI in DELE1 knockout cells was able to partially restore CHOP induction, whereas DELE1 expression in HRI-deficient cells was unproductive (Fig. 2a, Extended Data Fig. 6a-b). This indicated that DELE1 requires HRI to trigger CHOP but not vice versa, suggesting that DELE1 may act upstream of both EIF2α and HRI. Given that DELE1 is a mitochondrial protein 12 (Extended Data Fig. 6c), whereas HRI resides in the cytoplasm, we next wondered whether the activity of DELE1 towards HRI might be regulated by its localization. To test this hypothesis, we investigated if artificially rerouting DELE1 to the cytosol would bypass the need for a mitochondrial insult to provoke CHOP expression. Indeed, expression of a DELE1 mutant lacking the mitochondrial targeting sequence (DELE1 ∆MTS ) yielded a predominantly cytoplasmic protein that readily induced CHOP expression independently of CCCP (Fig. 2b-c, Extended Data Fig. 6d-e). This constitutively active version of DELE1 still required HRI to induce CHOP, underscoring its likely role as an activator of HRI. Based on these findings, we asked whether the activity of wild-type DELE1 might be regulated via a similar mechanism. Indeed, while DELE1 localized to mitochondria in unperturbed cells, it could be detected in the cytosol upon CCCP treatment (Fig. 2d). We did not ob...

Cancer Research UK, Academy of Medical Sciences, Health Foundation, EU, ERC, NIHR, Wellcome Trust, Dutch Cancer Society, Dutch Digestive Foundation.

Gene signatures derived from cancer stem cells (CSCs) predict tumor recurrence for many forms of cancer. Here, we derived a gene signature for colorectal CSCs defined by high Wnt signaling activity, which in agreement with previous observations predicts poor prognosis. Surprisingly, however, we found that elevated expression of Wnt targets was actually associated with good prognosis, while patient tumors with low expression of Wnt target genes segregated with immature stem cell signatures. We discovered that several Wnt target genes, including ASCL2 and LGR5, become silenced by CpG island methylation during progression of tumorigenesis, and that their re-expression was associated with reduced tumor growth. Taken together, our data show that promoter methylation of Wnt target genes is a strong predictor for recurrence of colorectal cancer, and suggest that CSC gene signatures, rather than reflecting CSC numbers, may reflect differentiation status of the malignant tissue.

Colorectal cancer (CRC) is a highly heterogeneous disease both from a molecular and clinical perspective. Several distinct molecular entities, such as microsatellite instability (MSI), have been defined that make up biologically distinct subgroups with their own clinical course. Recent data indicated that CRC can be best segregated into four groups called consensus molecular subtypes (CMS1-4), each of which has a unique biology and gene expression pattern. In order to develop improved, subtype-specific therapies and to gain insight into the molecular wiring and origin of these subtypes, reliable models are needed. This study was designed to determine the heterogeneity and identify the presence of CMSs in a large panel of CRC cell lines, primary cultures and patient-derived xenografts (PDX). We provide a repository encompassing this heterogeneity and moreover describe that a large part of the models can be robustly assigned to one of the four CMSs, independent of the stromal contribution. We subsequently validate our CMS stratification by functional analysis which for instance shows mesenchymal enrichment in CMS4 and metabolic dysregulation in CMS3. Finally, we observe a clear difference in sensitivity to chemotherapy-induced apoptosis, specifically between CMS2 and CMS4. This relates to the in vivo efficacy of chemotherapy, which delays outgrowth of CMS2, but not CMS4 xenografts. Combined our data indicate that molecular subtypes are faithfully modelled in CRC cell cultures and PDXs, representing tumour cell intrinsic and stable features. This repository provides researchers with a platform to study CRC using the existing heterogeneity.

Purpose: Recent transcriptomic analyses have identified four distinct molecular subtypes of colorectal cancer with evident clinical relevance. However, the requirement for sufficient quantities of bulk tumor and difficulties in obtaining high-quality genome-wide transcriptome data from formalin-fixed paraffinembedded tissue are obstacles toward widespread adoption of this taxonomy. Here, we develop an immunohistochemistrybased classifier to validate the prognostic and predictive value of molecular colorectal cancer subtyping in a multicenter study.Experimental Design: Tissue microarrays from 1,076 patients with colorectal cancer from four different cohorts were stained for five markers (CDX2, FRMD6, HTR2B, ZEB1, and KER) by immunohistochemistry and assessed for microsatellite instability. An automated classification system was trained on one cohort using quantitative image analysis or semiquantitative pathologist scoring of the cores as input and applied to three independent clinical cohorts.Results: This classifier demonstrated 87% concordance with the gold-standard transcriptome-based classification. Application to three validation datasets confirmed the poor prognosis of the mesenchymal-like molecular colorectal cancer subtype. In addition, retrospective analysis demonstrated the benefit of adding cetuximab to bevacizumab and chemotherapy in patients with RAS wild-type metastatic cancers of the canonical epithelial-like subtypes.Conclusions: This study shows that a practical and robust immunohistochemical assay can be employed to identify molecular colorectal cancer subtypes and uncover subtypespecific therapeutic benefit. Finally, the described tool is available online for rapid classification of colorectal cancer samples, both in the format of an automated image analysis pipeline to score tumor core staining, and as a classifier based on semiquantitative pathology scoring.