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Colorectal cancer (CRC) is one of the leading causes of cancer-related deaths worldwide. Therefore, development of effective (chemo) preventive strategies is an intense field of study. CRC is a disease that develops in the course of many years from the first oncogenic hit causing a premalignant lesion to invasive growth and ultimately metastatic disease. The oncogenic mutations that underlie the different histopathological stages have been fairly well characterized [1]. Commonly the first hit is an inactivating mutation of the Apc gene in intestinal stem cells (ISCs). This leads to hyperactive Wnt signaling and an evolutionary advantage of the affected stem cell over neighboring wild type ISCs, eventually giving rise to premalignant lesions with uncontrolled cellular proliferation [2]. Although the initial oncogenic hits are known and the effects on ISC population dynamics have been elucidated, the cellular adaptation process following gain of a tumorigenic mutation remains largely elusive. However, this knowledge is crucial to develop effective chemopreventive strategies for cancer in a rational manner.

Colorectal cancer (CRC) is a heterogeneous disease posing a challenge for accurate classification and treatment of this malignancy. There is no common genetic molecular feature that would allow for the identification of patients at risk for developing recurrences and thus selecting patients who would benefit from more stringent therapies still poses a major clinical challenge. Recently, an international multicenter consortium (CRC Subtyping Consortium) was established aiming at the classification of CRC patients in biologically homogeneous CRC subtypes. Four consensus molecular subtypes (CMSs) were identified, of which the mesenchymal CMS4 presented with worse prognosis signifying the importance of identifying these patients. Despite the large number of samples analyzed and their clear association with unifying biological programs and clinical features, single-driver mutations could not be identified and patients are heterogeneous with regard to currently used clinical markers. We therefore set out to define the regulatory mechanisms underlying the distinct gene expression profiles using a network-based approach involving multiple molecular modalities such as gene expression, methylation levels and microRNA (miR) expression. The miR-200 family presented as the most powerful determinant of CMS4-specific gene expression, tuning the majority of genes differentially expressed in the poor prognosis subtype, including genes associated with the epithelial–mesenchymal transition program. Furthermore, our data show that two epigenetic marks, namely the methylation of the two miR-200 promoter regions, can identify tumors belonging to the mesenchymal subtype and is predictive of disease-free survival in CRC patients. Importantly, epigenetic silencing of the miR-200 family is also detected in epithelial CRC cell lines that belong to the mesenchymal CMS. We thus show that determining regulatory networks is a powerful strategy to define drivers of distinct cancer subtypes, which possess the ability to identify subtype affiliation and to shed light on biological behavior.Oncogene advance online publication, 9 May 2016; doi:10.1038/onc.2016.134.

Potentially carcinogenic compounds may cause cancer through direct DNA damage or through indirect cellular or physiological effects. To study possible carcinogens, the fields of endocrinology, genetics, epigenetics, medicine, environmental health, toxicology, pharmacology and oncology must be considered. Disruptive chemicals may also contribute to multiple stages of tumor development through effects on the tumor microenvironment. In turn, the tumor microenvironment consists of a complex interaction among blood vessels that feed the tumor, the extracellular matrix that provides structural and biochemical support, signaling molecules that send messages and soluble factors such as cytokines. The tumor microenvironment also consists of many host cellular effectors including multipotent stromal cells/mesenchymal stem cells, fibroblasts, endothelial cell precursors, antigen-presenting cells, lymphocytes and innate immune cells. Carcinogens can influence the tumor microenvironment through effects on epithelial cells, the most common origin of cancer, as well as on stromal cells, extracellular matrix components and immune cells. Here, we review how environmental exposures can perturb the tumor microenvironment. We suggest a role for disrupting chemicals such as nickel chloride, Bisphenol A, butyltins, methylmercury and paraquat as well as more traditional carcinogens, such as radiation, and pharmaceuticals, such as diabetes medications, in the disruption of the tumor microenvironment. Further studies interrogating the role of chemicals and their mixtures in dose-dependent effects on the tumor microenvironment could have important general mechanistic implications for the etiology and prevention of tumorigenesis.

Glucocorticoids (GCs) are powerful immunosuppressive agents that control genomic effects through GC receptor (GR)-dependent transcriptional changes. A common complication of GC therapy is insulin resistance, but the underlying molecular mechanism remains obscure. Evidence is increasing for rapid genomic-independent GC action on cellular physiology. Here, we generate a comprehensive description of nongenomic GC effects on insulin signaling using peptide arrays containing 1176 different kinase consensus substrates. Reduced kinase activities of the insulin receptor (INSR) and several downstream INSR signaling intermediates (i.e. p70S6k, AMP-activated protein kinase, glycogen synthase kinase-3, and Fyn) were detected in adipocytes and T lymphocytes due to short-term treatment with dexamethasone (DEX), a synthetic fluorinated GC. Western blot analysis confirmed suppressed phosphorylation of the INSR and a series of downstream INSR targets (i.e. INSR substrate-1, p70S6k, protein kinase B, phosphoinositide-dependent protein kinase, Fyn, and glycogen synthase kinase-3) after DEX treatment. DEX inhibited insulin signaling through a GR-dependent (RU486 sensitive) and transcription-independent (actinomycin D insensitive) mechanism. Overall, we postulate here a molecular mechanism for GC-induced insulin resistance based on nongenomic GR-dependent inhibition of insulin signaling.

Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use -- https://www.nature.com/authors/policies/license.html#terms 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.

Background & Aims Drugs that inhibit the erb-b2 receptor tyrosine kinase 2 (ERBB2 or HER2) are the standard treatment of patients with different types of cancer, including HER2-overexpressing gastroesophageal tumors. Unfortunately, cancer cells become resistant to these drugs, so overall these drugs provide little benefit to patients with these tumors. We investigated mechanisms that mediate resistance of esophageal adenocarcinoma (EAC) cells and patient-derived xenograft tumors to ERBB inhibitors. Methods We cultured primary tumor cells, isolated from EAC patient samples, and OE19 and OE33 EAC cell lines with trastuzumab (an inhibitor of HER2), with or without pertuzumab (which inhibits dimerization of HER2 with HER3) or a specific antibody against HER3 (anti-HER3). HER2 was knocked down by expression of small hairpin RNAs. In addition, cells were incubated with NRG1-β, a mediator of HER2–HER3 signaling, or A83-01, an inhibitor of transforming growth factor beta (TGFβ) signaling. Cells were analyzed for markers of the epithelial to mesenchymal transition (EMT) using flow cytometry, immunofluorescence, and quantitative reverse transcription polymerase chain reaction. We performed limiting dilution, transwell, and cell viability assays to study the functional effects of HER2 and HER3 inhibition and reactivation. We analyzed publicly available EAC gene expression datasets to correlate expression of ERBB genes with genes encoding epithelial and mesenchymal proteins. NOD.Cg- Prkdc scid Il2rg tm1Wjl /SzJ (NSG) mice were given subcutaneous injections of AMC-EAC-007B cells and also given injections of single or combined inhibitors; growth of these patient-derived xenograft tumors was quantified. Results EAC cells incubated with trastuzumab decreased expression of epithelial markers (CD24, CD29, and CDH1) and increased expression of mesenchymal markers (CXCR4, VIM, ZEB1, SNAI2, and CDH2), compared with cells not exposed to trastuzumab, indicating induction of EMT. Addition of NRG1-β to these cells restored their epithelial phenotype. Incubation of EAC cells with trastuzumab and pertuzumab accelerated the expression of EMT markers, compared with cells incubated with trastuzumab alone. EAC cells cultured for 2 months with a combination of trastuzumab and pertuzumab became resistant to chemotherapeutic agents (5-fluoruracil, carboplatin, cisplatin, eribulin, and paclitaxel), based on their continued viability, which was accompanied with an enhanced migratory capacity in transwell assays and clonogenicity in limiting dilution analyses. In comparisons of EAC gene expression patterns, we associated high expression of ERBB3 with an epithelial gene expression signature; expression of TGFβ correlated with expression of EMT-related genes, and we found an inverse correlation between expression of TGFB1 and ERBB3. EAC cells incubated with ERBB inhibitors began to secrete ligands for the TGFβ receptor and underwent EMT. Incubation of EAC cells with trastuzumab, followed by 10 days of incubation with the TGFβ receptor inhibitor in the presence of trastuzumab, caused cells to regain an epithelial phenotype. EAC patient-derived xenograft tumors grew more slowly in mice given the combination of trastuzumab, pertuzumab, and the TGFβ inhibitor than in mice given single agents or a combination of trastuzumab and pertuzumab. Tumors exposed to trastuzumab and pertuzumab expressed EMT markers and were poorly differentiated, whereas tumors exposed to the combination of trastuzumab, pertuzumab, and the TGFβ inhibitor expressed epithelial markers and were more differentiated. Conclusions EAC cells become resistant to trastuzumab and pertuzumab by activating TGFβ signaling, which induces EMT. Agents that block TGFβ signaling can increase the anti-tumor efficacies of trastuzumab and pertuzumab.

Recently we published two independent studies describing novel gene expression-based classifications of colorectal cancer (CRC). Notably, each study stratified CRC into a different number of subtypes: one reported 3 subtypes, whereas the second highlighted 5. Given that each ascribed clinical significance, distinctive biology, and therapeutic prognosis to the different subtypes, we sought to reconcile this apparent incongruity in subtype stratification of CRC, and to interrelate the results. To do so, we each evaluated the other's data sets and analytical methods and discovered that the subtypes and their classifiers are, in fact, clearly related to each other; indeed, the 5 subtype outcomes can be coalesced into the same three. In addition to presenting this clarification, we briefly discuss how both classification methods can be viewed within the broader literature on CRC subtypes, and potentially applied.

Correction to: Oncogene (2016) 35, 4179–4187; doi:10.1038/onc.2015.483; published online 11 January 2016 Since the online publication of this article, the authors would like to add a new co-author, Jordyn Walter. The institutional affiliation for Jordyn Walter is: Department of Biomedical Sciences, University of Minnesota Medical School, Duluth, MN, USA

CD44 marks stem cell-like cells in a number of tumour types, including colorectal cancer (CRC), while aberrant CD44 expression conveys increased tumourigenic, invasive, and metastatic potential. Previous data indicate that CD44 is a direct target of p53-mediated transcriptional repression in breast cancer. Since inactivating p53 mutations are frequent genetic events in CRC these could unleash expression of CD44. In the present study, we therefore explored the relation between p53 mutational status and CD44 expression in a cohort of 90 localized primary CRCs and studied the effect of radiation-induced p53 activation on CD44 expression. Interestingly, we observed that, in contrast to breast cancer, loss of function p53 mutations were not associated with elevated CD44 expression in colon cancer. Moreover, DNA-damage induced p53 activation did not result in repression of CD44 expression, neither in colon cancer cells nor in normal intestinal epithelial cells. Our data demonstrate that CD44 expression in normal and malignant intestinal epithelial cells is not regulated by p53, implying that regulation of this potentially important therapeutic target is tissue and cancer-type specific.

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.

Background: Colorectal cancer (CRC) is the third most common cancer worldwide. Accurately identifying stage II CRC patients at high risk of recurrence and stage III patients at low risk of recurrence are key unmet clinical needs. We previously identified KCNQ1 as a tumour suppressor gene of which loss of expression was associated with poor survival in patients with CRC liver metastases. The present study aimed to examine the prognostic value of KCNQ1 in stage II and III colon cancer patients. Methods: KCNQ1 mRNA expression was assessed in 90 stage II colon cancer patients (AMC-AJCCII-90) using microarray gene expression data. KCNQ1 protein expression was evaluated by immuno-histochemistry on tissue microarrays of 386 stage II and III colon cancer patients. Results: Low KCNQ1 mRNA expression in microsatellite stable (MSS) stage II colon cancers was associated with poor disease free survival (DFS) (HR 3.35; 95% CI 1.16-9.66; p Conclusion: We conclude that KCNQ1 is a strong prognostic biomarker for prediction of disease recurrence (HR∼4) and may aid stratification of patients with stage II MSS colon cancer and stage III MSI CRC for adjuvant chemotherapy. Because KCNQ1 protein expression is determined by immuno-histochemistry, this biomarker can be implemented in standard clinical care using existing workflows. Citation Format: Sjoerd H. den Uil, Veerle M.H. Coupe, Janneke F. Linnekamp, Evert van den Broek, Jeroen A.C.M. Goos, Pien M. Delis-van Diemen, Eric J.T. Belt, Nicole C.T. van Grieken, Patricia M. Scott, Louis Vermeulen, Jan Paul Medema, Herman Bril, Hein B.A.C. Stockmann, Robert T. Cormier, Gerrit A. Meijer, Remond J. Fijneman. KCNQ1 expression is a strong prognostic biomarker for disease recurrence in stage II and III colon cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3125.

Purpose: High expression of cancer stem cell (CSC) marker CD133 has been used as a predictor for prognosis in colorectal cancer (CRC), suggesting that enumeration of CSCs, using CD133, is predictive for disease progression. However, we showed recently that both CD133 mRNA and protein are not downregulated during differentiation of colon CSCs, pointing to an alternative reason for the prognostic value of CD133. We therefore set out to delineate the relation between CD133 expression and prognosis. Experimental Design: A CRC patient series was studied for expression of CD133 and other CSC markers by microarray and quantitative PCR analysis. In addition, several common mutations were analyzed to determine the relation with CD133 expression. Results: CD133 mRNA expression predicted relapse-free survival in our patient series, whereas several other CSC markers could not. Moreover, no correlation was found between expression of other CSC markers and CD133. Interestingly, high CD133 expression was related to mutations in K-Ras and B-Raf, and inhibition of mutant K-Ras or downstream mitogen-activated protein kinase kinase (MEK) signaling decreases CD133 expression. In addition, an activated K-Ras gene expression signature could predict CD133 expression in our patient set as well as data sets of other tumor types. Conclusion: CD133 expression is upregulated in CRC tumors that have a hyperactivated Ras–Raf–MEK–ERK pathway and is therefore related to mutations in K-Ras or B-Raf. As mutations in either gene have been related to poor prognosis, we conclude that CD133 expression is not indicative for CSC numbers but rather related to the mutation or activity status of the Ras–Raf pathway. Clin Cancer Res; 18(11); 3132–41. ©2012 AACR.

The identification of intestinal stem cells as well as their malignant counterparts, colon cancer stem cells, has undergone rapid development in recent years. Under physiological conditions, intestinal homeostasis is a carefully balanced and efficient interplay between stem cells, their progeny and the microenvironment. These interactions regulate the astonishingly rapid renewal of the intestinal epithelial layer, which consequently puts us at serious risk of developing cancer. Here we highlight the microenvironment-derived signals that regulate stem-cell fate and epithelial differentiation. As our understanding of normal intestinal crypt homeostasis grows, these developments may point towards new insights into the origin of cancer and the maintenance and regulation of cancer stem cells.

Tumors are being increasingly perceived as abnormal organs that, in many respects, recapitulate the outgrowth and differentiation patterns of normal tissues. In line with this idea is the observation that only a small fraction of tumor cells is capable of initiating a new tumor. Because of the features that these cells share with somatic stem cells, they have been termed cancer stem cells (CSC). Normal stem cells reside in a “stem cell niche” that maintains them in a stem-like state. Recent data suggest that CSCs also rely on a similar niche, dubbed the “CSC niche,” which controls their self-renewal and differentiation. Moreover, CSCs can be generated by the microenvironment through induction of CSC features in more differentiated tumor cells. In addition to a role in CSC maintenance, the microenvironment is hypothesized to be involved in metastasis by induction of the epithelial-mesenchymal transition, leading to dissemination and invasion of tumor cells. The localization of secondary tumors also seems to be orchestrated by the microenvironment, which is suggested to form a premetastatic niche. Thus, the microenvironment seems to be of crucial importance for primary tumor growth as well as metastasis formation. Combined with its role in the protection of CSCs against genotoxic insults, these data strongly put forward the niche as an important target for novel therapies. Cancer Res; 71(3); 634–9. ©2011 AACR.

SummaryGene 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.

How mutations lead to tumor formation is a central question in cancer research. Although cellular changes that follow the occurrence of common mutations are well characterized, much less is known about their effects on the population level. Now, two recent studies reveal in what way oncogenic aberrations alter stem cell dynamics to provide cells with an evolutionary advantage over their neighbors (Amoyel et al, 2014; Baker et al, 2014).

Colon cancer stem cells (CSC) can be identified with AC133, an antibody that detects an epitope on CD133. However, recent evidence suggests that expression of CD133 is not restricted to CSCs, but is also expressed on differentiated tumor cells. Intriguingly, we observed that detection of the AC133 epitope on the cell surface decreased upon differentiation of CSC in a manner that correlated with loss of clonogenicity. However, this event did not coincide with a change in CD133 promoter activity, mRNA, splice variant, protein expression, or even cell surface expression of CD133. In contrast, we noted that with CSC differentiation, a change occured in CD133 glycosylation. Thus, AC133 may detect a glycosylated epitope, or differential glycosylation may cause CD133 to be retained inside the cell. We found that AC133 could effectively detect CD133 glycosylation mutants or bacterially expressed unglycosylated CD133. Moreover, cell surface biotinylation experiments revealed that differentially glycosylated CD133 could be detected on the membrane of differentiated tumor cells. Taken together, our results argue that CD133 is a cell surface molecule that is expressed on both CSC and differentiated tumor cells, but is probably differentially folded as a result of differential glycosylation to mask specific epitopes. In summary, we conclude that AC133 can be used to detect cancer stem cells, but that results from the use of this antibody should be interpreted with caution. Cancer Res; 70(2); 719–29

Despite the presence of mutations in APC or beta-catenin, which are believed to activate the Wnt signalling cascade constitutively, most colorectal cancers show cellular heterogeneity when beta-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 beta-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.

KABISA is a computer-based program for training in diagnostic problems in (sub-) tropical regions. It challenges the individual student with a randomly generated case, for which he should try to find the diagnosis, asking questions, performing a physical examination, and ordering tests. The built-in tutor follows the student’s input with complex logical algorithms and mathematical computations, gives comments and support, and accepts the final diagnosis if sufficient evidence has been built up. Several problems arose with the development. In the first place, the evolution in the teaching of clinical logic is always ahead of the program, so regular updating of the computer logic is necessary. Secondly, the choice of MS Access as computer language has provoked problems of stability, especially the installation of an MSAccess runtime. Thirdly, and most importantly, scholars want proof of the added value of computer programs over classical teaching. Moreover, the concept of a pedagogical “game” is often regarded as childish. Finally, the planning and financing of an “open-ended” pedagogical project is questioned by deciders, as is the case with all operational research.

Colon carcinoma is one of the leading causes of death from cancer and is characterized by a heterogenic pool of cells with distinct differentiation patterns. Recently, it was reported that a population of undifferentiated cells from a primary tumor, so-called cancer stem cells (CSC), can reconstitute the original tumor on xenotransplantation. Here, we show that spheroid cultures of these colon CSCs contain expression of CD133, CD166, CD44, CD29, CD24, Lgr5, and nuclear β-catenin, which have all been suggested to mark the (cancer) stem cell population. More importantly, by using these spheroid cultures or freshly isolated tumor cells from multiple colon carcinomas, we now provide compelling evidence to indicate that the capacity to propagate a tumor with all differentiated progeny resides in a single CSC. Single-cell-cloned CSCs can form an adenocarcinoma on xenotransplantation but do not generate the stroma within these tumors. Moreover, they can self-renew and are capable of multilineage differentiation. Further analysis indicated that the lineage decision is dictated by phosphoinositide 3-kinase (PI3K) signaling in CSCs. These data support the hypothesis that tumor hierarchy can be traced back to a single CSC that contains multilineage differentiation capacity, and provides clues to the regulation of differentiation in colon cancers in vivo .

A novel paradigm in tumor biology suggests that cancer growth is driven by stem-like cells within a tumor. Here, we describe the identification and characterization of such cells from colon carcinomas using the stem cell marker CD133 that accounts around 2% of the cells in human colon cancer. The CD133(+) cells grow in vitro as undifferentiated tumor spheroids, and they are both necessary and sufficient to initiate tumor growth in immunodeficient mice. Xenografts resemble the original human tumor maintaining the rare subpopulation of tumorigenic CD133(+) cells. Further analysis revealed that the CD133(+) cells produce and utilize IL-4 to protect themselves from apoptosis. Consistently, treatment with IL-4Ralpha antagonist or anti-IL-4 neutralizing antibody strongly enhances the antitumor efficacy of standard chemotherapeutic drugs through selective sensitization of CD133(+) cells. Our data suggest that colon tumor growth is dictated by stem-like cells that are treatment resistant due to the autocrine production of IL-4.

Resolving the origin of intratumor heterogeneity has proven to be one of the central challenges in cancer research during recent years. Two theoretical models explaining the emergence of intratumor heterogeneity have come to dominate cancer biology literature: the clonal evolution model and the hierarchical/cancer stem cell model. Recently, a plastic model that combines elements of both the clonal and the hierarchical model has gained traction. Basically, this model proposes that cancer stem cells engage in bidirectional interconversion with non-stem cells, thereby providing the missing link between the 2 conventional models. Confirming bidirectional interconversion as a hallmark of cancer is a crucial step in understanding tumor heterogeneity and has important therapeutic implications. In this review, current methodologies and theoretical and empirical evidence regarding bidirectional interconversion will be discussed.

BACKGROUND Recent molecular profiling of colorectal cancer has identified three distinct subtypes: two good-prognosis subtypes characterised by chromosomal instability (Subtype 1) and microsatellite instability (Subtype 2), and a poor prognosis subtype defined by epithelial-mesenchymal transition and stemness (Subtype 3). A key challenge lies in the subsequent implementation of this classification system in the clinic, where the requirement for sufficient bulk tumour and high cost hampers the widespread use of genomic profiling. In this proof-of-concept study, we have adapted our molecular signatures to an automated immunohistochemistry (IHC)-based classifier which can be used a rapid screening tool, and validated the feasibility of this approach in a multi-centre study. PATIENTS AND METHODS A total of 1080 patients from four different centres were used in this study. Tumour microarrays (TMAs) from each patient were stained using immunohistochemistry for a panel of five stains in conjunction with microsatellite instability status (MSS). An automated image analysis pipeline was developed to quantitate and normalize all staining. A classifier to distinguish colorectal cancer subtypes was trained on 74 patients from a single centre and applied to the three remaining cohorts. RESULTS We have developed an automated IHC-based classification system which demonstrated 84% correlation with our genomic profiling system. The prognostic value of colorectal cancer subtyping was validated in three independent cohorts, taking into account age, sex and stage (Hazards Ratios with 95% Confidence Interval: 1.8 (1.2 - 2.6), 1.4 (1.1 - 1.7) and 1.2 (1.0 - 1.6)). We evaluated the predictive value of the subtyping system in a retrospective analysis of a late-stage clinical trial and demonstrated the benefit of adjuvant cetuximab in KRAS/BRAF wild type Subtype 1 patients (Hazards Ratio 0.6 (0.4-0.9)) but not in Subtype 3 patients. CONCLUSION In this proof-of-concept study we have demonstrated the effectiveness of an automated IHC-TMA classifier as a surrogate for genomic profiling. Using this approach, we have validated the prognostic value of colorectal subtyping in a multi-centre study, and shown the predictive value of subtyping for adjuvant cetuximab therapy. This approach has demonstrated high clinical potential as a rapid screening tool, in particular in retrospective examination of patient cohorts where only formalin-fixed paraffin-embedded (FFPE) tissue is available. Citation Format: Anne Trinh, Filipe De Sousa E Melo, Xin Wang, Joan H de Jong, Evelyn Fessler, Marnix Jansen, Gerrit KJ Hooijer, Jan Paul Medema, Florian Markowetz, Louis Vermeulen. Automated immunohistochemistry-based identification of molecular subtypes in colorectal cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-108. doi:10.1158/1538-7445.AM2015-LB-108

The outcome of pancreatic ductal adenocarcinoma (PDAC) remains dismal. Factors that contribute to the lethality of this disease include its intrinsic resistance to therapeutics, an aggressive growth pattern, and a large degree of both inter- as well as intratumor heterogeneity. All these features have been attributed (at least in part) to the presence and function of a population of pluripotent cells called cancer stem cells. Despite the alleged importance of these cells for clinical outcome, the methods that have been used to study these cells in PDAC are troubled by some serious caveats. Defining important quantitative parameters for these cells in an unbiased, marker-agnostic, way is called for and we aim to achieve this by unbiased tracing methods in patient derived xenografts. A panel of PDX-derived lines generated in our institute was labeled using the LeGO (Lentiviral Gene Ontology; Weber et al. 2012 Nat. Prot) system to enable tracking of single cell clones by color. The system utilizes three vectors coding for red, green, and blue fluorophores much like a television uses these colors to generate an almost infinite range of colors in the visible range. Nuances in color and intensity are generated by the many possible combinations of fluorophores, the different integration numbers of the genes coding for them, and the variations in expression levels determined by for instance the integration sites. The end result is a population of cells in which each cell is endowed with a unique color. This will allow detection of the offspring of such a cell by identifying clones within a structural unit in for instance a tumor, or in a culture dish, of similar color. The ability to give rise to such a structure is then, depending on the experimental context and outcome, considered a proxy for stem cell function. Several primary lines have proven amenable to LeGO-marking. These lines showed stable color patterns during culturing in vitro as analyzed by continuous FACS measurements, and tumors derived from marked cells did not show a difference to umarked tumors as assessed by gross histology and growth rate. We are currently testing the ability of these cells to faithfully occupy and mark structural units in PDAC, much like intestinal stem cells would in a lineage tracing experiment in the gut, and derive numbers for stem cell number as well as replacement rate. Experiments are currently being performed to assess the system's potential to measure parameters associated with stemness, and possibly also differentiation status of the original tumor, as the cells grow in vitro. (Radio)chemotherapy regimens are included to formally address the purported resistance of cancer stem cells to these treatments without having to rely on an a priori chosen marker. Furthermore, more detailed comparison should reveal use of LeGO-derived data to measure the intra- and intertumor heterogeneity in PDAC tumor biology in our panel of PDXs. Citation Format: Veronique Veenstra, Helene Damhofer, Tom van Leusden, Jan Kessler, Jan Paul Medema, Hanneke van Laarhoven, Louis Vermeulen, Maarten Bijlsma. Heterogeneity of pancreatic ductal adenocarcinoma visualized. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5177. doi:10.1158/1538-7445.AM2015-5177

Colon cancer is the fourth leading cause of cancer-related death worldwide. Extensive studies of the Wnt signal cascade have determined its role in colon cancer development and progress. Several targets of the Wnt-cascade like APCDD1, ASCL2, AXIN2 or DKK1 are thought to reduce Wnt activity by negative feedback. As a result, expression of these genes prevents pathway activation and silencing of these targets can cause proliferation of the tumor. Recently, we have shown that silencing of Wnt target genes by DNA-methylation predicts poor prognosis in colon cancer patients. Moreover, demethylation of those genes results in reexpression and is associated with reduced tumor growth in mice. Hence, treatment with demethylating agents can be beneficial for a subgroup of colon cancer patients with high methylation of Wnt target genes. The aim of this study is to clarify whether Wnt target gene methylation is an important driver in development of colon cancer and can be an effective target in colon cancer. To determine the role of Wnt target genes in colon cancer more extensively, we study the effect of selective expression of these genes via an inducible vector system in established colorectal cell lines and in patient derived cultures that display CpG island methylation on these genes. In that way, we can monitor the effect of induced overexpression on tumor growth, tumor invasion and sensitivity towards chemotherapeutic agents. As a control, baseline expression in the same cell population, but in an uninduced state will be used. Furthermore, we are also studying the effect of the demethylating agent decitabine on Wnt target gene methylation in a clinical trial in stage II/III colon cancer patients. Patients are pre-operatively treated with decitabine. Tumor material prior to decitabine treatment obtained during endoscopy is compared to resection material after treatment. Differences in methylation and expression of Wnt target genes are determined by respectively pyrosequencing and q-PCR. We hypothesize that reexpression of Wnt target genes via an inducible vector results in lower Wnt pathway activity through negative feedback. As a result, tumor growth and invasion will be reduced and might be more sensitive towards chemotherapeutics. To translate this into the clinic, demethylation of Wnt target genes by decitabine will hopefully give a similar effect in patients. Citation Format: Janneke Linnekamp, Raju Kandimalla, Louis Vermeulen, Hanneke van Laarhoven, Jan Paul Medema. Role of methylation of Wnt target genes in tumorigenesis and effect of re-expression with demethylating agent decitabine in colon cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5273. doi:10.1158/1538-7445.AM2015-5273

Background: Gene expression-based subtyping is widely accepted as a relevant source of disease stratification. Despite the widespread use, its translational and clinical utility is hampered by discrepant results, likely related to differences in data processing and algorithms applied to diverse patient cohorts, sample preparation methods, and gene expression platforms. In the absence of a clear methodological gold standard to perform such analyses, a more general framework that integrates and compares multiple strategies is needed to define common disease patterns in a principled, unbiased manner. Methods: We formed a consortium of 6 independent experts groups - each with a previously published CRC classifier, ranging from 3 to 6 subtypes - to understand similarities and differences of their subtyping systems. Sage Bionetworks functioned as neutral party to aggregate public and proprietary data (Synapse platform) and perform meta-analysis. Each group applied its CRC subtyping signature to the collection of data sets with gene expression (n = 4,151, predominantly stage II and III). Using the resulting subtype labels, we developed a network-based model and applied a Markov cluster algorithm to detect robust network substructures that would indicate recurring subtype patterns and therefore a consensus subtyping system. Correlative analyses using clinico-pathological, genomic and epigenomic features was performed to robustly characterize the identified subtypes. Results: This analytical framework revealed significant interconnectivity between the six independent classification systems, leading to the identification of four biologically distinct consensus molecular subtypes (CMS) enriched for key pathway traits: CMS1 (MSI Immune), hypermutated, microsatellite unstable, with strong immune activation; CMS2 (Canonical), epithelial, chromosomally unstable, with marked WNT and MYC signaling activation; CMS3 (Metabolic), epithelial, with evident metabolic dysregulation; and CMS4 (Mesenchymal), prominent TGFβ activation, angiogenesis, stromal invasion. Patients diagnosed with MSI Immune tumors had worse survival after relapse and those with mesenchymal tumors had increased risk of metastasis and worse overall survival. Discussion: We describe a novel methodological paradigm for deriving benchmarks of disease subtyping. Our work represents the first example of a community of experts identifying and advocating for a single reproducible model for cancer subtyping, effectively unifying previous classifiers. In the CRC domain, the uniformity afforded by this new classification system and its application to a large data set revealed important subtype-specific biological associations that were previously unnoticed or marginally significant, supporting a new taxonomy of the disease. Citation Format: Justin Guinney, Rodrigo Dienstmann, Xin Wang, Aurelien de Reynies, Andreas Schlicker, Charlotte Soneson, Laetitia Marisa, Paul Roepman, Gift Nyamundanda, Paolo Angelino, Brian Bot, Jeffrey S. Morris, Iris Simon, Sarah Gerster, Evelyn Fessler, Felipe de Sousa e Melo, Edoardo Missiaglia, Hena Ramay, David Barras, Krisztian Homicsko, Dipen Maru, Ganiraju Manyam, Bradley Broom, Valerie Boige, Ted Laderas, Ramon Salazar, Joe W. Gray, Josep Tabernero, Rene Bernards, Stephen Friend, Pierre Laurent-Puig, Jan P. Medema, Anguraj Sadanandam, Lodewyk Wessels, Mauro Delorenzi, Scott Kopetz, Louis Vermeulen, Sabine Tejpar. Consensus molecular subtyping through a community of experts advances unsupervised gene expression-based disease classification and facilitates clinical translation. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 603. doi:10.1158/1538-7445.AM2015-603

3511 Background: Recently, a number of independent groups reported novel molecular subtypes in colorectal cancer (CRC). A formal comparison across these classifiers is needed to reconcile findings ...

A remarkable feature of regenerative processes is their ability to halt proliferation once an organ’s structure has been restored. The Wnt signaling pathway is the major driving force for homeostatic self-renewal and regeneration in the mammalian intestine. The mechanisms that counterbalance Wnt-driven proliferation are poorly understood. We demonstrate here that YAP, a protein known for its powerful growth-inducing and oncogenic properties1-2, has an unexpected growth-suppressive function restricting Wnt signals during intestinal regeneration. Transgenic expression of YAP reduces Wnt target gene expression and results in the rapid loss of intestinal crypts. In addition, loss of YAP results in Wnt hypersensitivity during regeneration, leading to hyperplasia, expansion of intestinal stem cells (ISCs) and niche cells, and formation of ectopic crypts and microadenomas. We find that cytoplasmic YAP restricts elevated Wnt signaling independently of the APC/Axin/GSK3β complex partly by limiting the activity of Dishevelled (DVL). DVL signals in the nucleus of ISCs and its forced expression leads to enhanced Wnt signaling in crypts. YAP dampens Wnt signals by restricting DVL nuclear translocation during regenerative growth. Finally, we provide evidence that YAP is silenced in a subset of highly aggressive and undifferentiated human colorectal carcinomas (CRC) and its expression can restrict the growth of CRC xenografts. Collectively, our work describes a novel mechanistic paradigm for how proliferative signals are counterbalanced in regenerating tissues. Additionally, our findings have important implications for the targeting of YAP in human malignancies.

Aberrant regulation of Wnt signaling is a common theme seen across many tumor types. Decades of research have unraveled the epigenetic and genetic alterations that result in elevated Wnt pathway activity. More recently, it has become apparent that Wnt signaling levels identify stem-like tumor cells that are responsible for fueling tumor growth. As therapeutic targeting of these tumor stem cells is an intense area of investigation, a concise understanding on how Wnt activity relates to cancer stem cell traits is needed. This review attempts at summarizing the intricacies between Wnt signaling and cancer stem cell biology with a special emphasis on colorectal cancer.