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KRAS-mutant colorectal cancers are resistant to therapeutics, presenting a significant problem for ∼40% of cases. Rapalogs, which inhibit mTORC1 and thus protein synthesis, are significantly less potent in KRAS-mutant colorectal cancer. Using Kras-mutant mouse models and mouse- and patient-derived organoids, we demonstrate that KRAS with G12D mutation fundamentally rewires translation to increase both bulk and mRNA-specific translation initiation. This occurs via the MNK/eIF4E pathway culminating in sustained expression of c-MYC. By genetic and small-molecule targeting of this pathway, we acutely sensitize KRASG12D models to rapamycin via suppression of c-MYC. We show that 45% of colorectal cancers have high signaling through mTORC1 and the MNKs, with this signature correlating with a 3.5-year shorter cancer-specific survival in a subset of patients. This work provides a c-MYC–dependent cotargeting strategy with remarkable potency in multiple Kras-mutant mouse models and metastatic human organoids and identifies a patient population that may benefit from its clinical application.Significance:KRAS mutation and elevated c-MYC are widespread in many tumors but remain predominantly untargetable. We find that mutant KRAS modulates translation, culminating in increased expression of c-MYC. We describe an effective strategy targeting mTORC1 and MNK in KRAS-mutant mouse and human models, pathways that are also commonly co-upregulated in colorectal cancer.This article is highlighted in the In This Issue feature, p. 995

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The transcription factor AP4 mediates epithelial–mesenchymal transition (EMT) in colorectal cancer but its control in this setting is not fully understood. Here, we report the definition of a double-negative feedback loop involving AP4 and miR-15a/16-1 that regulates EMT and metastatic progression. In colorectal cancer cells, AP4 was downregulated by DNA damage in a p53-dependent manner. AP4 downregulation by p53 was mediated indirectly by the tumor-suppressive microRNAs miR-15a and miR-16-1, which targeted the 3′ untranslated region (3′-UTR) of AP4 mRNA, induced mesenchymal–epithelial transition (MET), and inhibited colorectal cancer cell migration and invasion. The downregulation of AP4 was necessary for induction of MET and cell cycle arrest by miR-15a/16-1. In tumor xenoplants, ectopic miR-15a/16-1 suppressed formation of lung metastases. Furthermore, AP4 directly suppressed expression of miR-15a/16-1. In clinical specimens of colorectal cancer, miR-15a levels inversely correlated with AP4 protein levels shown previously to correlate with distant metastasis and poor survival. In summary, our results define a double-negative feedback loop involving miR-15a/16-1 and AP4 that stabilizes epithelial and mesenchymal states, respectively, which may determine metastatic prowess. Cancer Res; 74(2); 532–42. ©2013 AACR.

PDF file - 43K, Supplementary Table 1. Oligonucleotides used for cloning and site-directed mutagenesis Supplementary Table 2. Oligonucleotides used for qChIP analyses Supplementary Table 3. Antibodies used for Western blot (WB) and immunofluorescence (IF) analysis Supplementary Table 4. Oligonucleotides used for qPCR analyses Supplementary Table 5. Sub-analysis using cases with the most prominent miR-15a expression levels.

PDF file - 519K, Supplementary Figure 1 Western blot analysis of AP4 protein expression in SW480 cells after transfection of the indicated siRNA for 48 hours. Supplementary Figure 2 In silico analysis of the 3�-UTR of EMT transcription factors. Supplementary Figure 3 Ectopic AP4 overcomes a G1-cell cycle arrest induced by miR-15a/16-1. Supplementary Figure 4 Cell proliferation of SW620-Luc2 cells

Patient derived organoids closely resemble the biology of tissues and tumors. They are enabling ex vivo modeling of human diseases and dissecting key features of tumor biology like anatomical diversity or inter- and intra-tumoral heterogeneity. In the last years, organoids were established as models for drug discovery and explored to guide clinical decision making. In this review, we discuss the recent developments in organoid based research, elaborating on the developments in colorectal cancer as a prime example. We focus our review on the role of organoids to decode cancer cell dynamics and tumor microenvironmental complexity with the underlying bi-directional crosstalk. Additionally, advancements in the development of living biobanks, screening approaches, organoid based precision medicine and challenges of co-clinical trials are highlighted. We discuss ongoing efforts to overcome challenges that the field faces and indicate potential future directions.

Colorectal cancer (CRC) is among the deadliest cancers worldwide, with metastasis being the main cause of patient mortality. During CRC progression the complex tumor ecosystem changes in its composition at virtually every stage. However, clonal dynamics and associated niche-dependencies at these stages are unknown. Hence, it is of importance to utilize models that faithfully recapitulate human CRC to define its clonal dynamics. We used an optical barcoding approach in mouse-derived organoids (MDOs) that revealed niche-dependent clonal selection. Our findings highlight that clonal selection is controlled by a site-specific niche, which critically contributes to cancer heterogeneity and has implications for therapeutic intervention.

Modelling human colon cancer has long been the ambition of researchers and oncologists with the aim to better replicate disease progression and treatment response. Advances in our understanding of genetics, stem cell biology, tumour microenvironment and immunology have prepared the groundwork for recent major advances. In the last two years the field has seen the progression of: using patient derived organoids (alone and in co-culture) as predictors of treatment response; molecular stratification of tumours that predict outcome and treatment response; mouse models of metastatic disease; and transplant models that can be used to de-risk clinical trials. We will discuss these advances in this review.

Oncogenic KRAS mutations and inactivation of the APC tumor suppressor co-occur in colorectal cancer (CRC). Despite efforts to target mutant KRAS directly, most therapeutic approaches focus on downstream pathways, albeit with limited efficacy. Moreover, mutant KRAS alters the basal metabolism of cancer cells, increasing glutamine utilization to support proliferation. We show that concomitant mutation of Apc and Kras in the mouse intestinal epithelium profoundly rewires metabolism, increasing glutamine consumption. Furthermore, SLC7A5, a glutamine antiporter, is critical for colorectal tumorigenesis in models of both early- and late-stage metastatic disease. Mechanistically, SLC7A5 maintains intracellular amino acid levels following KRAS activation through transcriptional and metabolic reprogramming. This supports the increased demand for bulk protein synthesis that underpins the enhanced proliferation of KRAS-mutant cells. Moreover, targeting protein synthesis, via inhibition of the mTORC1 regulator, together with Slc7a5 deletion abrogates the growth of established Kras-mutant tumors. Together, these data suggest SLC7A5 as an attractive target for therapy-resistant KRAS-mutant CRC.

The WNT pathway is a pleiotropic signaling pathway that controls developmental processes, tissue homeostasis, and cancer. The WNT pathway is commonly mutated in many cancers, leading to widespread research into the role of WNT signaling in carcinogenesis. Understanding which cancers are reliant upon WNT activation and which components of the WNT signaling pathway are mutated is paramount to advancing therapeutic strategies. In addition, building holistic insights into the role of WNT signaling in not only tumor cells but also the tumor microenvironment is a vital area of research and may be a promising therapeutic strategy in multiple immunologically inert cancers. Novel compounds aimed at modulating the WNT signaling pathway using diverse mechanisms are currently under investigation in preclinical/early clinical studies. Here, we review how the WNT pathway is activated in multiple cancers and discuss current strategies to target aberrant WNT signaling.

Neutrophils have been implicated in poor outcomes in cancer and severe inflammation. We found that neutrophils expressing intermediate levels of Ly6G (Ly6GInt) were present in mouse cancer models and more abundant in those with high rates of spontaneous metastasis. Maturation, age, tissue localization and functional capacity all drive neutrophil heterogeneity. Recent studies have proposed various markers to distinguish between these heterogeneous sub-populations; however, these markers are limited to specific models of inflammation and cancer. Here, we identify and define Ly6G expression level as a robust and reliable marker to distinguish neutrophils at different stages of maturation. Ly6GInt neutrophils were bona fide ‘immature neutrophils’ with reduced immune regulatory and adhesion capacity. Whereas the bone marrow is a more recognised site of granulopoiesis, the spleen also produces neutrophils in homeostasis and cancer. Strikingly, neutrophils matured faster in the spleen than in the bone marrow with unique transcriptional profiles. We propose that developmental origin is critical in neutrophil identity and postulate that neutrophils that develop in the spleen supplement the bone marrow by providing an intermediate more mature reserve before emergency haematopoiesis.

The c-MYC oncogene is activated in ~50% of all tumors and its product, the c-MYC transcription factor, regulates numerous processes, which contribute to tumor initiation and progression. Therefore, the genome-wide characterization of c-MYC targets and their role in different tumor entities is a recurrent theme in cancer research. Recently, next-generation sequencing (NGS) has become a powerful tool to analyze mRNA and miRNA expression, as well as DNA binding of proteins in a genome-wide manner with an extremely high resolution and coverage. Since the c-MYC transcription factor regulates mRNA and miRNA expression by binding to specific DNA elements in the vicinity of promoters, NGS can be used to generate integrated representations of c-MYC-mediated regulations of gene transcription and chromatin modifications. Here, we provide protocols and examples of NGS-based analyses of c-MYC-regulated mRNA and miRNA expression, as well as of DNA binding by c-MYC. Furthermore, we describe the validation of single c-MYC targets identified by NGS . Taken together, these approaches allow an accelerated and comprehensive analysis of c-MYC function in numerous cellular contexts. Ultimately, these analyses will further illuminate the role of this important oncogene.

KRAS-mutant colorectal cancers are resistant to therapeutics, presenting a significant problem for ∼40% of cases. Rapalogs, which inhibit mTORC1 and thus protein synthesis, are significantly less potent in KRAS-mutant colorectal cancer. Using Kras-mutant mouse models and mouse- and patient-derived organoids, we demonstrate that KRAS with G12D mutation fundamentally rewires translation to increase both bulk and mRNA-specific translation initiation. This occurs via the MNK/eIF4E pathway culminating in sustained expression of c-MYC. By genetic and small-molecule targeting of this pathway, we acutely sensitize KRASG12D models to rapamycin via suppression of c-MYC. We show that 45% of colorectal cancers have high signaling through mTORC1 and the MNKs, with this signature correlating with a 3.5-year shorter cancer-specific survival in a subset of patients. This work provides a c-MYC–dependent cotargeting strategy with remarkable potency in multiple Kras-mutant mouse models and metastatic human organoids and identifies a patient population that may benefit from its clinical application. Significance: KRAS mutation and elevated c-MYC are widespread in many tumors but remain predominantly untargetable. We find that mutant KRAS modulates translation, culminating in increased expression of c-MYC. We describe an effective strategy targeting mTORC1 and MNK in KRAS-mutant mouse and human models, pathways that are also commonly co-upregulated in colorectal cancer. This article is highlighted in the In This Issue feature, p. 995

Evasion of apoptosis is a hallmark of cancer, which is frequently mediated by upregulation of the antiapoptotic BCL-2 family proteins. In colorectal cancer (CRC), previous work has highlighted differential antiapoptotic protein dependencies determined by the stage of the disease. While intestinal stem cells (ISCs) require BCL-2 for adenoma outgrowth and survival during transformation, ISC-specific MCL1 deletion results in disturbed intestinal homeostasis, eventually contributing to tumorigenesis. Colon cancer stem cells (CSCs), however, no longer require BCL-2 and depend mainly on BCL-XL for their survival. We therefore hypothesized that a shift in antiapoptotic protein reliance occurs in ISCs as the disease progresses from normal to adenoma to carcinoma. By targeting antiapoptotic proteins with specific BH3 mimetics in organoid models of CRC progression, we found that BCL-2 is essential only during ISC transformation while MCL1 inhibition did not affect adenoma outgrowth. BCL-XL, on the other hand, was crucial for stem cell survival throughout the adenoma-to-carcinoma sequence. Furthermore, we identified that the limited window of BCL-2 reliance is a result of its downregulation by miR-17-5p, a microRNA that is upregulated upon APC-mutation driven transformation. Here we show that BCL-XL inhibition effectively impairs adenoma outgrowth in vivo and enhances the efficacy of chemotherapy. In line with this dependency, expression of BCL-XL, but not BCL-2 or MCL1, directly correlated to the outcome of chemotherapy-treated CRC patients. Our results provide insights to enable the rational use of BH3 mimetics in CRC management, particularly underlining the therapeutic potential of BCL-XL targeting mimetics in both early and late-stage disease.

Cancer-associated fibroblasts (CAFs) promote tumor malignancy, but the precise transcriptional mechanisms regulating the acquisition of the CAF phenotype are not well understood. We show that the upregulation of SOX2 is central to this process, which is repressed by protein kinase Cζ (PKCζ). PKCζ deficiency activates the reprogramming of colonic fibroblasts to generate a predominant SOX2-dependent CAF population expressing the WNT regulator Sfrp2 as its top biomarker. SOX2 directly binds the Sfrp1/2 promoters, and the inactivation of Sox2 or Sfrp1/2 in CAFs impaired the induction of migration and invasion of colon cancer cells, as well as their tumorigenicity in vivo. Importantly, recurrence-free and overall survival of colorectal cancer (CRC) patients negatively correlates with stromal PKCζ levels. Also, SOX2 expression in the stroma is associated with CRC T invasion and worse prognosis of recurrence-free survival. Therefore, the PKCζ-SOX2 axis emerges as a critical step in the control of CAF pro-tumorigenic potential.

Summary Competitive cell interactions play a crucial role in quality control during development and homeostasis. Here, we show that cancer cells use such interactions to actively eliminate wild-type intestine cells in enteroid monolayers and organoids. This apoptosis-dependent process boosts proliferation of intestinal cancer cells. The remaining wild-type population activates markers of primitive epithelia and transits to a fetal-like state. Prevention of this cell-state transition avoids elimination of wild-type cells and, importantly, limits the proliferation of cancer cells. Jun N-terminal kinase (JNK) signaling is activated in competing cells and is required for cell-state change and elimination of wild-type cells. Thus, cell competition drives growth of cancer cells by active out-competition of wild-type cells through forced cell death and cell-state change in a JNK-dependent manner., Graphical abstract, Highlights • In organoids, wild-type intestine cells are actively eliminated by cancer cells • Cell competition boosts proliferation of intestinal cancer cells • Remaining wild-type cells adopt a fetal-like state • JNK signaling in wild-type cells drives cell competition, Interactions between cells helps coordinated decision making in tissues. Krotenberg Garcia et al. show that intestinal cancer cells use competitive cell interactions to actively eliminate surrounding wild-type cells. These interactions drive oncogenic growth in organoids.

KRAS-mutant colorectal cancers (CRC) are resistant to therapeutics, presenting a significant problem for ~40% of cases. Rapalogs, which inhibit mTORC1 and thus protein synthesis, are significantly less potent in KRAS-mutant CRC. Using Kras-mutant mouse models and mouse- and patient-derived organoids we demonstrate that KRAS with G12D mutation fundamentally rewires translation to increase both bulk and mRNA-specific translation initiation. This occurs via the MNK/eIF4E pathway culminating in sustained expression of c-MYC. By genetic and small molecule targeting of this pathway, we acutely sensitize KRAS(G12D) models to rapamycin via suppression of c-MYC. We show that 45% of CRCs have high signaling through mTORC1 and the MNKs, with this signature correlating with a 3.5-year shorter cancer-specific survival in a subset of patients. This work provides a c-MYC-dependent co-targeting strategy with remarkable potency in multiple Kras-mutant mouse models and metastatic human organoids and identifies a patient population who may benefit from its clinical application.

Oncogenic KRAS mutations and inactivation of the APC tumor suppressor co-occur in colorectal cancer (CRC). Despite efforts to target mutant KRAS directly, most therapeutic approaches focus on downstream pathways, albeit with limited efficacy. Moreover, mutant KRAS alters the basal metabolism of cancer cells, increasing glutamine utilization to support proliferation. We show that concomitant mutation of Apc and Kras in the mouse intestinal epithelium profoundly rewires metabolism, increasing glutamine consumption. Furthermore, SLC7A5, a glutamine antiporter, is critical for colorectal tumorigenesis in models of both early- and late-stage metastatic disease. Mechanistically, SLC7A5 maintains intracellular amino acid levels following KRAS activation through transcriptional and metabolic reprogramming. This supports the increased demand for bulk protein synthesis that underpins the enhanced proliferation of KRAS-mutant cells. Moreover, targeting protein synthesis, via inhibition of the mTORC1 regulator, together with Slc7a5 deletion abrogates the growth of established Kras-mutant tumors. Together, these data suggest SLC7A5 as an attractive target for therapy-resistant KRAS-mutant CRC.

Vascular dysfunction contributes to the pro-oncogenic tumor microenvironment and impedes the delivery of therapeutics. Normalizing of the tumor vasculature has therefore become a potential therapeutic objective. We previously reported that the secreted glycoprotein, leucine-rich α-2-glycoprotein 1 (LRG1), contributes to the formation of pathogenic neovascularization. Here we show that in mouse models of cancer, Lrg1 is induced in tumor endothelial cells. We demonstrate that the expression of LRG1 impacts on tumor progression as Lrg1 deletion or treatment with a LRG1 function-blocking antibody inhibited tumor growth and improved survival. Inhibition of LRG1 increased endothelial cell pericyte coverage and improved vascular function resulting in significantly enhanced efficacy of cisplatin chemotherapy, adoptive T-cell therapy and immune checkpoint inhibition (anti-PD1) therapy. With immunotherapy, LRG1 inhibition led to a significant shift in the tumor microenvironment from being predominantly immune silent (cold) to immune active (hot). LRG1 therefore drives vascular abnormalization and its inhibition represents a novel and effective means of improving the efficacy of cancer therapeutics.

Summary Background A poorly functioning tumor vasculature is pro-oncogenic and may impede the delivery of therapeutics. Normalizing the vasculature, therefore, may be beneficial. We previously reported that the secreted glycoprotein leucine-rich α-2-glycoprotein 1 (LRG1) contributes to pathogenic neovascularization. Here, we investigate whether LRG1 in tumors is vasculopathic and whether its inhibition has therapeutic utility. Methods Tumor growth and vascular structure were analyzed in subcutaneous and genetically engineered mouse models in wild-type and Lrg1 knockout mice. The effects of LRG1 antibody blockade as monotherapy, or in combination with co-therapies, on vascular function, tumor growth, and infiltrated lymphocytes were investigated. Findings In mouse models of cancer, Lrg1 expression was induced in tumor endothelial cells, consistent with an increase in protein expression in human cancers. The expression of LRG1 affected tumor progression as Lrg1 gene deletion, or treatment with a LRG1 function-blocking antibody, inhibited tumor growth and improved survival. Inhibition of LRG1 increased endothelial cell pericyte coverage and improved vascular function, resulting in enhanced efficacy of cisplatin chemotherapy, adoptive T cell therapy, and immune checkpoint inhibition (anti-PD1) therapy. With immunotherapy, LRG1 inhibition led to a significant shift in the tumor microenvironment from being predominantly immune silent to immune active. Conclusions LRG1 drives vascular abnormalization, and its inhibition represents a novel and effective means of improving the efficacy of cancer therapeutics. Funding Wellcome Trust (206413/B/17/Z), UKRI/MRC (G1000466, MR/N006410/1, MC/PC/14118, and MR/L008742/1), BHF (PG/16/50/32182), Health and Care Research Wales (CA05), CRUK (C42412/A24416 and A17196), ERC (ColonCan 311301 and AngioMature 787181), and DFG (CRC1366).

Different thresholds of Wnt signalling are thought to drive stem cell maintenance, regeneration, differentiation and cancer. However, the principle that oncogenic Wnt signalling could be specifically targeted remains controversial. Here we examine the requirement of BCL9/9l, constituents of the Wnt-enhanceosome, for intestinal transformation following loss of the tumour suppressor APC. Although required for Lgr5+ intestinal stem cells and regeneration, Bcl9/9l deletion has no impact upon normal intestinal homeostasis. Loss of BCL9/9l suppressed many features of acute APC loss and subsequent Wnt pathway deregulation in vivo. This resulted in a level of Wnt pathway activation that favoured tumour initiation in the proximal small intestine (SI) and blocked tumour growth in the colon. Furthermore, Bcl9/9l deletion completely abrogated β-catenin driven intestinal and hepatocellular transformation. We speculate these results support the just-right hypothesis of Wnt-driven tumour formation. Importantly, loss of BCL9/9l is particularly effective at blocking colonic tumourigenesis and mutations that most resemble those that occur in human cancer.

Tumours depend on altered rates of protein synthesis for growth and survival, which suggests that mechanisms controlling mRNA translation may be exploitable for therapy. Here, we show that loss of APC, which occurs almost universally in colorectal tumours, strongly enhances the dependence on the translation initiation factor eIF2B5. Depletion of eIF2B5 induces an integrated stress response and enhances translation of MYC via an internal ribosomal entry site. This perturbs cellular amino acid and nucleotide pools, strains energy resources and causes MYC-dependent apoptosis. eIF2B5 limits MYC expression and prevents apoptosis in APC-deficient murine and patient-derived organoids and in APC-deficient murine intestinal epithelia in vivo. Conversely, the high MYC levels present in APC-deficient cells induce phosphorylation of eIF2α via the kinases GCN2 and PKR. Pharmacological inhibition of GCN2 phenocopies eIF2B5 depletion and has therapeutic efficacy in tumour organoids, which demonstrates that a negative MYC–eIF2α feedback loop constitutes a targetable vulnerability of colorectal tumours.

Background: Molecular subtyping of colon cancer (CC) has repeatedly identified a poor-prognostic group of patients, characterized by high levels of stroma (particularly fibroblast) in the tumor microenvironment. To date, the benefit of standard 5FU-based adjuvant chemotherapy in these high-fibroblast (HiFi) patients has been unclear. Given TGF-β signaling is associated with HiFi tumors, a number of recent clinical trials have focussed on targeting TGF-β in these patients. While these efforts are ongoing, we set out to identify novel therapeutically-relevant biological signaling within HiFi tumors. Methods: Untreated stage II (n=215) and stage II/III (n=258) tumors were assigned a fibroblast score, using single-sample gene set enrichment analysis, enabling stratification into HiFi and LoFi groups based on their histology and transcriptome. Supervised stratification, based on relapse-free survival, within the HiFi group allowed for in silico discovery, interrogation and independent validation of the HiFi-specific biology underpinning relapse. Upstream regulators of these processes were identified as potential therapeutic targets, and assessed in an in vitro co-culture model, to confirm mechanistic signaling, and an in vivo HiFi model, to confirm efficacy. Results: We confirmed the poor prognosis of the HiFi group (p = 0.008), followed by discovery and independent validation of the prognostic value of STAT1-related signaling in stratifying HiFi tumors based on disease relapse (HR 0.2 (0.1-0.5) and 0.09 (0.02-0.47)). This signaling was significantly associated with activation of antigen processing and presentation in specific immune lineages (p < 0.001). In line with the upstream regulator analysis, treatment with poly I:C (a TLR3 agonist) increased STAT1-related signaling and antigen processing in an in vitro macrophage-stromal co-culture system. Conclusions: We have found that increased levels of STAT1-related signaling, resulting in antigen processing and presentation in specific subclasses of immune cells, is associated with reduced risk of recurrence in the otherwise poor-prognostic HiFi subtype of CC. Using in silico and in vitro methods, we demonstrate that poly I:C is a potential therapeutic option for patients with stromal-rich tumors. Results from ongoing in vivo validation in a HiFi mouse model will provide preclinical evidence of the utility of poly I:C in this setting and support a phase II clinical trial. Citation Format: Amy M. McCorry, Niamh A. Leonard, Rene Jackstadt, Dustin J. Flanagan, Owen J. Sansom, Tim Maughan, Simon Leedham, Emma M. Kerr, Aideen E. Ryan, Mark Lawler, Philip D. Dunne, ACRCelerate and S:CORT consortia. STAT1-related antigen processing and presentation dictates prognosis in the fibroblast-rich subtype of stage II/III colon cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3867.

221 Background: 5-year survival of patients with resectable colorectal liver metastases is 25-40%. Mechanisms of disease progression are heterogenous and do not follow a clearly defined pathway from genotype to phenotype. In stage I-III colorectal cancer (CRC), patients with high tumor stroma exhibit poor prognosis, while those with high immune cell infiltrate do well following resection. We hypothesise that stromally-dense phenotypes lead to T cell exclusion, myeloid cell accumulation and aggressive metastatic progression. Here, we examine relationships between histological tumor phenotype, cellular infiltrate and outcomes in metastatic CRC. Methods: A unique cohort of synchronously resected primary CRC and matched liver metastases (n = 46) were assessed for immune cell infiltration (CD3, CD4, CD8, CD68, CD66b), inflammatory signalling (CXCR2, PDL-1, MMP9) and hypoxia (CAIX) using immunohistochemistry. Tumors were phenotypically subtyped using immune infiltrate (Klintrup-Makinen Grade (KM)), stromal invasion (tumor-stroma percentage (TSP)) and proliferation (Ki67). Results: Phenotypic subtype of primary tumors was predictive of metastatic subtype (rho = 0.522, p = 0.003). Immune phenotypes were associated with good prognosis and stromal phenotypes with poor prognosis (p = 0.004). Infiltration of macrophages and granulocytes associated with poor outcomes (p = 0.018) and increased CXCR2 expression (p = 0.03) at both sites. Increased CXCR2+ cells and macrophages at both sites associated with stromal phenotype (p = 0.02), tumour budding (p = 0.002), low KM grade (p = 0.05) and poor prognosis (p = 0.002). Macrophage and MMP9 levels increased in metastases compared to primary tumour, but no changes were seen in lymphocyte infiltration, CXCR2 and CD66b. Conclusions: Density of immune cell infiltrate, in the primary and metastatic niche, conferred good prognosis. In contrast, stromal, myeloid rich tumors convey poor prognosis. This clinically relevant and histologically efficient process permits segregation of disease and supports further study of relationships in the tumour microenvironment of CRC in the context of chemotherapy to better target therapeutics to individual patients.

Background: Frequent disease relapse and a lack of effective therapies result in a very poor outcome in pancreatic ductal adenocarcinoma (PDAC) patients. Thus, identification of prognostic biomarkers and possible therapeutic targets is essential. Besides their function in cell–cell adhesion, desmogleins may play a role in tumour progression and invasion that has not been investigated in PDAC to date. This study evaluated desmoglein expression as a biomarker in PDAC. Methods: Using immunohistochemistry, we examined desmoglein 1 (DSG1), desmoglein 2 (DSG2) and desmoglein 3 (DSG3) expression in the tumour tissue of 165 resected PDAC cases. Expression levels were correlated to the patients' clinicopathological parameters and postoperative survival times. We confirmed these results in two independent gene expression data sets. Results: A total of 36% of the tumours showed high DSG3 expression that correlated significantly with shorter patient survival (P=0.011) and poor tumour differentiation (P

The transcription factor AP4 mediates epithelial–mesenchymal transition (EMT) in colorectal cancer but its control in this setting is not fully understood. Here, we report the definition of a double-negative feedback loop involving AP4 and miR-15a/16-1 that regulates EMT and metastatic progression. In colorectal cancer cells, AP4 was downregulated by DNA damage in a p53-dependent manner. AP4 downregulation by p53 was mediated indirectly by the tumor-suppressive microRNAs miR-15a and miR-16-1, which targeted the 3′ untranslated region (3′-UTR) of AP4 mRNA, induced mesenchymal–epithelial transition (MET), and inhibited colorectal cancer cell migration and invasion. The downregulation of AP4 was necessary for induction of MET and cell cycle arrest by miR-15a/16-1. In tumor xenoplants, ectopic miR-15a/16-1 suppressed formation of lung metastases. Furthermore, AP4 directly suppressed expression of miR-15a/16-1. In clinical specimens of colorectal cancer, miR-15a levels inversely correlated with AP4 protein levels shown previously to correlate with distant metastasis and poor survival. In summary, our results define a double-negative feedback loop involving miR-15a/16-1 and AP4 that stabilizes epithelial and mesenchymal states, respectively, which may determine metastatic prowess. Cancer Res; 74(2); 532–42. ©2013 AACR.

Adaptive cellular responses are often required during wound repair. Following disruption of the intestinal epithelium, wound‐associated epithelial (WAE) cells form the initial barrier over the wound. Our goal was to determine the critical factor that promotes WAE cell differentiation. Using an adaptation of our in vitro primary epithelial cell culture system, we found that prostaglandin E2 (PGE 2) signaling through one of its receptors, Ptger4, was sufficient to drive a differentiation state morphologically and transcriptionally similar to in vivo WAE cells. WAE cell differentiation was a permanent state and dominant over enterocyte differentiation in plasticity experiments. WAE cell differentiation was triggered by nuclear β‐catenin signaling independent of canonical Wnt signaling. Creation of WAE cells via the PGE 2‐Ptger4 pathway was required in vivo, as mice with loss of Ptger4 in the intestinal epithelium did not produce WAE cells and exhibited impaired wound repair. Our results demonstrate a mechanism by which WAE cells are formed by PGE 2 and suggest a process of adaptive cellular reprogramming of the intestinal epithelium that occurs to ensure proper repair to injury.

Summary The metastatic process of colorectal cancer (CRC) is not fully understood and effective therapies are lacking. We show that activation of NOTCH1 signaling in the murine intestinal epithelium leads to highly penetrant metastasis (100% metastasis; with >80% liver metastases) in KrasG12D-driven serrated cancer. Transcriptional profiling reveals that epithelial NOTCH1 signaling creates a tumor microenvironment (TME) reminiscent of poorly prognostic human CRC subtypes (CMS4 and CRIS-B), and drives metastasis through transforming growth factor (TGF) β-dependent neutrophil recruitment. Importantly, inhibition of this recruitment with clinically relevant therapeutic agents blocks metastasis. We propose that NOTCH1 signaling is key to CRC progression and should be exploited clinically., Graphical Abstract, Highlights • Epithelial NOTCH1 signaling drives metastasis in serrated CRC • Poor-prognosis CRC subtypes CMS4/CRIS-B are controlled by NOTCH1 • TGF-β-mediated neutrophil infiltration is critical for NOTCH1-driven metastasis • Neutrophil targeting provides therapeutic opportunity in metastatic CRC, In a genetically engineered mouse model, Jackstadt et al. show that NOTCH1 activation drives metastasis in KRASG12D-driven serrated colorectal cancer (CRC) through TGFβ-dependent neutrophil recruitment. Thus, targeting neutrophil recruitment is a potential therapeutic approach in metastatic CRC.

Here, we show that expression of ZNF281/ZBP-99 is controlled by SNAIL and miR-34a/b/c in a coherent feed-forward loop: the epithelial–mesenchymal transition (EMT) inducing factor SNAIL directly induces ZNF281 transcription and represses miR-34a/b/c, thereby alleviating ZNF281 mRNA from direct down-regulation by miR-34. Furthermore, p53 activation resulted in a miR-34a-dependent repression of ZNF281. Ectopic ZNF281 expression in colorectal cancer (CRC) cells induced EMT by directly activating SNAIL, and was associated with increased migration/invasion and enhanced β-catenin activity. Furthermore, ZNF281 induced the stemness markers LGR5 and CD133, and increased sphere formation. Conversely, experimental down-regulation of ZNF281 resulted in mesenchymal–epithelial transition (MET) and inhibition of migration/invasion, sphere formation and lung metastases in mice. Ectopic c-MYC induced ZNF281 protein expression in a SNAIL-dependent manner. Experimental inactivation of ZNF281 prevented EMT induced by c-MYC or SNAIL. In primary CRC samples, expression of ZNF281 increased during tumour progression and correlated with recurrence. Taken together, these results identify ZNF281 as a component of EMT-regulating networks, which contribute to metastasis formation in CRC.

The c-Kit receptor tyrosine kinase is commonly over-expressed in different types of cancer. p53 activation is known to result in the down-regulation of c-Kit. However, the underlying mechanism has remained unknown. Here, we show that the p53-induced miR-34 microRNA family mediates repression of c-Kit by p53 via a conserved seed-matching sequence in the c-Kit 3'-UTR. Ectopic miR-34a resulted in a decrease in Erk signaling and transformation, which was dependent on the down-regulation of c-Kit expression. Furthermore, ectopic expression of c-Kit conferred resistance of colorectal cancer (CRC) cells to treatment with 5-fluorouracil (5-FU), whereas ectopic miR-34a sensitized the cells to 5-FU. After stimulation with c-Kit ligand/stem cell factor (SCF) Colo320 CRC cells displayed increased migration/invasion, whereas ectopic miR-34a inhibited SCF-induced migration/invasion. Activation of a conditional c-Kit allele induced several stemness markers in DLD-1 CRC cells. In primary CRC samples elevated c-Kit expression also showed a positive correlation with markers of stemness, such as Lgr5, CD44, OLFM4, BMI-1 and β-catenin. On the contrary, activation of a conditional miR-34a allele in DLD-1 cells diminished the expression of c-Kit and several stemness markers (CD44, Lgr5 and BMI-1) and suppressed sphere formation. MiR-34a also suppressed enhanced sphere-formation after exposure to SCF. Taken together, our data establish c-Kit as a new direct target of miR-34 and demonstrate that this regulation interferes with several c-Kit-mediated effects on cancer cells. Therefore, this regulation may be potentially relevant for future diagnostic and therapeutic approaches.

A chronic inflammatory microenvironment favors tumor progression through molecular mechanisms that are still incompletely defined. In inflammation-induced skin cancers, IL-1 receptor- or caspase-1–deficient mice, or mice specifically deficient for the inflammasome adaptor protein ASC (apoptosis-associated speck-like protein containing a CARD) in myeloid cells, had reduced tumor incidence, pointing to a role for IL-1 signaling and inflammasome activation in tumor development. However, mice fully deficient for ASC were not protected, and mice specifically deficient for ASC in keratinocytes developed more tumors than controls, suggesting that, in contrast to its proinflammatory role in myeloid cells, ASC acts as a tumor-suppressor in keratinocytes. Accordingly, ASC protein expression was lost in human cutaneous squamous cell carcinoma, but not in psoriatic skin lesions. Stimulation of primary mouse keratinocytes or the human keratinocyte cell line HaCaT with UVB induced an ASC-dependent phosphorylation of p53 and expression of p53 target genes. In HaCaT cells, ASC interacted with p53 at the endogenous level upon UVB irradiation. Thus, ASC in different tissues may influence tumor growth in opposite directions: it has a proinflammatory role in infiltrating cells that favors tumor development, but it also limits keratinocyte proliferation in response to noxious stimuli, possibly through p53 activation, which helps suppressing tumors.

Members of the miR-34 family are induced by the tumor suppressor p53 and are known to inhibit epithelial-to-mesenchymal transition (EMT) and therefore presumably suppress the early phases of metastasis. Here, we determined that exposure of human colorectal cancer (CRC) cells to the cytokine IL-6 activates the oncogenic STAT3 transcription factor, which directly represses the MIR34A gene via a conserved STAT3-binding site in the first intron. Repression of MIR34A was required for IL-6-induced EMT and invasion. Furthermore, we identified the IL-6 receptor (IL-6R), which mediates IL-6-dependent STAT3 activation, as a conserved, direct miR-34a target. The resulting IL-6R/STAT3/miR-34a feedback loop was present in primary colorectal tumors as well as CRC, breast, and prostate cancer cell lines and associated with a mesenchymal phenotype. An active IL-6R/STAT3/miR-34a loop was necessary for EMT, invasion, and metastasis of CRC cell lines and was associated with nodal and distant metastasis in CRC patient samples. p53 activation in CRC cells interfered with IL-6-induced invasion and migration via miR-34a-dependent downregulation of IL6R expression. In Mir34a-deficient mice, colitis-associated intestinal tumors displayed upregulation of p-STAT3, IL-6R, and SNAIL and progressed to invasive carcinomas, which was not observed in WT animals. Collectively, our data indicate that p53-dependent expression of miR-34a suppresses tumor progression by inhibiting a IL-6R/STAT3/miR-34a feedback loop.

In the past ten years microRNAs (miRNAs) have been widely implicated as components of tumor suppressive and oncogenic pathways. Also the proto-typic oncogene c-MYC has been connected to miRNAs. The c-MYC gene is activated in approximately half of all tumors, and its product, the c-MYC transcription factor, regulates numerous processes e.g. cell cycle progression, metabolism, epithelial–mesenchymal transition (EMT), metastasis, stemness, and angiogenesis, thereby facilitating tumor initiation and progression. c-MYC target-genes, which mediate these functions of c-MYC, represent a complex network of protein- and non-coding RNAs, including numerous miRNAs. For example, c-MYC directly regulates expression of the miR-17–92 cluster, miR-34a, miR-15a/16-1 and miR-9. Moreover, the expression and activity of c-MYC itself are under the control of miRNAs. Here, we survey how these networks mediate and regulate c-MYC functions. In the future, miRNAs connected to c-MYC may be used for diagnostic and therapeutic approaches. This article is part of a Special Issue entitled: Myc proteins in cell biology and pathology.