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Most current therapies that target plasma membrane receptors function by antagonizing ligand binding or enzymatic activities. However, typical mammalian proteins comprise multiple domains that execute discrete but coordinated activities. Thus, inhibition of one domain often incompletely suppresses the function of a protein. Indeed, targeted protein degradation technologies, including proteolysis-targeting chimeras 1 (PROTACs), have highlighted clinically important advantages of target degradation over inhibition 2 . However, the generation of heterobifunctional compounds binding to two targets with high affinity is complex, particularly when oral bioavailability is required 3 . Here we describe the development of proteolysis-targeting antibodies (PROTABs) that tether cell-surface E3 ubiquitin ligases to transmembrane proteins, resulting in target degradation both in vitro and in vivo. Focusing on zinc- and ring finger 3 (ZNRF3), a Wnt-responsive ligase, we show that this approach can enable colorectal cancer-specific degradation. Notably, by examining a matrix of additional cell-surface E3 ubiquitin ligases and transmembrane receptors, we demonstrate that this technology is amendable for ‘on-demand’ degradation. Furthermore, we offer insights on the ground rules governing target degradation by engineering optimized antibody formats. In summary, this work describes a strategy for the rapid development of potent, bioavailable and tissue-selective degraders of cell-surface proteins. Membrane-bound E3 ubiquitin ligases RNF43 and ZNRF3 are overexpressed in colorectal cancer, and can be repurposed using proteolysis-targeting antibodies (PROTABs) to selectively degrade cell-surface receptors in tumours.

Cancers of various organs have been categorized into distinct subtypes after increasingly sophisticated taxonomies. Additionally, within a seemingly homogeneous subclass, individual cancers contain diverse tumour cell populations that vary in important cancer‐specific traits such as clonogenicity and invasive potential. Differences that exist between and within a given tumour type have hampered significantly both the proper selection of patients that might benefit from therapy, as well as the development of new targeted agents. In this review, we discuss the differences associated with organ‐specific cancer subtypes and the factors that contribute to intra‐tumour heterogeneity. It is of utmost importance to understand the biological causes that distinguish tumours as well as distinct tumour cell populations within malignancies, as these will ultimately point the way to more rational anti‐cancer treatments. EMBO reports advance online publication 12 July 2013; doi:10.1038/embor.2013.92 In this review Jan Paul Medema and colleagues discuss the differences associated with organ‐specific cancer subtypes and the factors that contribute to intra‐tumour heterogeneity. Understanding tumour heterogeneity is crucial for the development of more rational anti‐cancer treatments.

An international consortium of colorectal cancer researchers undertakes a large-scale data sharing project to achieve a consensus molecular classification of colorectal cancers. 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 (CMSs) with distinguishing features: CMS1 (microsatellite instability immune, 14%), hypermutated, microsatellite unstable and strong immune activation; CMS2 (canonical, 37%), epithelial, marked WNT and MYC signaling activation; CMS3 (metabolic, 13%), epithelial and 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 intratumoral 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.

During the past decade, a stem-cell-like subset of cancer cells has been identified in many malignancies. These cells, referred to as cancer stem cells (CSCs), are of particular interest because they are believed to be the clonogenic core of the tumour and therefore represent the cell population that drives growth and progression. Many efforts have been made to design therapies that specifically target the CSC population, since this was predicted to be the crucial population to eliminate. However, recent insights have complicated the initial elegant model, by showing a dominant role for the tumour microenvironment in determining CSC characteristics within a malignancy. This is particularly important since dedifferentiation of non-tumorigenic tumour cells towards CSCs can occur, and therefore the CSC population in a neoplasm is expected to vary over time. Moreover, evidence suggests that not all tumours are driven by rare CSCs, but might instead contain a large population of tumorigenic cells. Even though these results suggest that specific targeting of the CSC population might not be a useful therapeutic strategy, research into the hierarchical cellular organisation of malignancies has provided many important new insights in the biology of tumours. In this Personal View, we highlight how the CSC concept is developing and influences our thinking on future treatment for solid tumours, and recommend ways to design clinical trials to assess drugs that target malignant disease in a rational fashion.

Antibody-mediated inhibition of R-spondin-3 in colorectal tumours decreases tumour growth and promotes differentiation—these effects are associated with a decrease in expression of genes associated with stem-cell function. Stem cell function in colorectal tumours Frederic de Sauvage and colleagues have previously identified translocations in genes encoding the Wnt regulator R-spondins in some colorectal tumours. Here they demonstrate that antibody-mediated inhibition of R-spondin 3 (RSPO3) in such tumours decreases tumour growth and promotes differentiation. These effects are associated with a decrease in expression of genes associated with stem cell function. This work raises the possibility of targeting stem -cell properties within tumours as therapeutic approach in colorectal tumours. Colorectal cancer remains a major unmet medical need, prompting large-scale genomics efforts in the field to identify molecular drivers for which targeted therapies might be developed 1 , 2 , 3 . We previously reported the identification of recurrent translocations in R-spondin genes present in a subset of colorectal tumours 4 . Here we show that targeting RSPO3 in PTPRK-RSPO3 -fusion-positive human tumour xenografts inhibits tumour growth and promotes differentiation. Notably, genes expressed in the stem-cell compartment of the intestine were among those most sensitive to anti-RSPO3 treatment. This observation, combined with functional assays, suggests that a stem-cell compartment drives PTPRK-RSPO3 colorectal tumour growth and indicates that the therapeutic targeting of stem-cell properties within tumours may be a clinically relevant approach for the treatment of colorectal tumours.

OTULIN (OTU deubiquitinase with linear linkage specificity) removes linear polyubiquitin from proteins that have been modified by LUBAC (linear ubiquitin chain assembly complex) and is critical for preventing auto-inflammatory disease 1 , 2 and embryonic lethality during mouse development 3 . Here we show that OTULIN promotes rather than counteracts LUBAC activity by preventing its auto-ubiquitination with linear polyubiquitin. Thus, knock-in mice that express catalytically inactive OTULIN, either constitutively or selectively in endothelial cells, resembled LUBAC-deficient mice 4 and died midgestation as a result of cell death mediated by TNFR1 (tumour necrosis factor receptor 1) and the kinase activity of RIPK1 (receptor-interacting protein kinase 1). Inactivation of OTULIN in adult mice also caused pro-inflammatory cell death. Accordingly, embryonic lethality and adult auto-inflammation were prevented by the combined loss of cell death mediators: caspase 8 for apoptosis and RIPK3 for necroptosis. Unexpectedly, OTULIN mutant mice that lacked caspase 8 and RIPK3 died in the perinatal period, exhibiting enhanced production of type I interferon that was dependent on RIPK1. Collectively, our results indicate that OTULIN and LUBAC function in a linear pathway, and highlight a previously unrecognized interaction between linear ubiquitination, regulators of cell death, and induction of type I interferon. OTULIN, which removes ubiquitin chains deposited by LUBAC, promotes LUBAC activity by preventing its auto-ubiquitination, thereby supporting normal mouse embryo development and preventing pro-inflammatory cell death in adult mice.

Background Colon cancer stem cells are shown to be the self-renewing cells within a tumor that give rise to all lineages of more differentiated tumor cells. In this respect they are remarkably similar to their non-malignant counterparts that orchestrate the intestinal lining. This suggests that, despite the numerous genetic aberrations and morphological changes that have occurred during cancer initiation and progression, a remnant homeostatic regulation persists. Findings Using a number of human and mouse intestinal-derived organoid cultures from normal, adenoma and cancerous tissues, we show here that Notch signals coordinate self-renewal and lineage determination not only in normal, but also at the adenoma and carcinoma stage in both mice and humans. Moreover, the Wnt pathway, which carries activating mutations in virtually all colon cancers, is not as previously predicted constitutively active in adenomas and carcinomas, but still displays a heterogeneous activity pattern that determined stemness in all stages of disease. Conclusion These data for the first time provide a comprehensive overview of Wnt and Notch-mediated signaling in the different stages of the adenoma-carcinoma sequence and demonstrates that these morphogenic pathways, despite mutations, remain crucial determinants of both architecture and hierarchy in normal and malignant intestinal tissue.

Analysis of gene-expression profiles led to the identification of three molecularly distinct subtypes of colon cancer. One of these subtypes is new, and its identification is of clinical interest because it tends to have a particularly unfavorable prognosis and is refractory to existing targeted therapies. 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 1 . 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) 2 . 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.

Solid malignancies have been speculated to depend on cancer stem cells (CSCs) for expansion and relapse after therapy. Here we report on quantitative analyses of lineage tracing data from primary colon cancer xenograft tissue to assess CSC functionality in a human solid malignancy. The temporally obtained clone size distribution data support a model in which stem cell function in established cancers is not intrinsically, but is entirely spatiotemporally orchestrated. Functional stem cells that drive tumour expansion predominantly reside at the tumour edge, close to cancer-associated fibroblasts. Hence, stem cell properties change in time depending on the cell location. Furthermore, although chemotherapy enriches for cells with a CSC phenotype, in this context functional stem cell properties are also fully defined by the microenvironment. To conclude, we identified osteopontin as a key cancer-associated fibroblast-produced factor that drives in situ clonogenicity in colon cancer. Lenos et al. report that the spatiotemporal regulation of stem cell functionality is not intrinsically determined but environmentally defined during tumour growth and drug response in colon cancer.

Cancer stem cell activity may not merely be an intrinsic characteristic of a subset of cancer cells and could be regulated by environmental cues. High Wnt activity in human colorectal cancer designates a tumour-initiating population and is orchestrated by the microenvironment. Despite the presence of mutations in APC or β-catenin, which are believed to activate the Wnt signalling cascade constitutively, most colorectal cancers show cellular heterogeneity when β-catenin localization is analysed, indicating a more complex regulation of Wnt signalling. We explored this heterogeneity with a Wnt reporter construct and observed that high Wnt activity functionally designates the colon cancer stem cell (CSC) population. In adenocarcinomas, high activity of the Wnt pathway is observed preferentially in tumour cells located close to stromal myofibroblasts, indicating that Wnt activity and cancer stemness may be regulated by extrinsic cues. In agreement with this notion, myofibroblast-secreted factors, specifically hepatocyte growth factor, activate β-catenin-dependent transcription and subsequently CSC clonogenicity. More significantly, myofibroblast-secreted factors also restore the CSC phenotype in more differentiated tumour cells both in vitro and in vivo . We therefore propose that stemness of colon cancer cells is in part orchestrated by the microenvironment and is a much more dynamic quality than previously expected that can be defined by high Wnt activity.