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The analysis of stem cell hierarchies in human cancers has been hampered by the impossibility of identifying or tracking tumor cell populations in an intact environment. To overcome this limitation, we devised a strategy based on editing the genomes of patient‐derived tumor organoids using CRISPR/Cas9 technology to integrate reporter cassettes at desired marker genes. As proof of concept, we engineered human colorectal cancer (CRC) organoids that carry EGFP and lineage‐tracing cassettes knocked in the LGR5 locus. Analysis of LGR5‐EGFP + cells isolated from organoid‐derived xenografts demonstrated that these cells express a gene program similar to that of normal intestinal stem cells and that they propagate the disease to recipient mice very efficiently. Lineage‐tracing experiments showed that LGR5+ CRC cells self‐renew and generate progeny over long time periods that undergo differentiation toward mucosecreting‐ and absorptive‐like phenotypes. These genetic experiments confirm that human CRCs adopt a hierarchical organization reminiscent of that of the normal colonic epithelium. The strategy described herein may have broad applications to study cell heterogeneity in human tumors.

Colorectal cancers (CRCs) are composed of an amalgam of cells with distinct genotypes and phenotypes. Here, we reveal a previously unappreciated heterogeneity in the biosynthetic capacities of CRC cells. We discover that the majority of ribosomal DNA transcription and protein synthesis in CRCs occurs in a limited subset of tumor cells that localize in defined niches. The rest of the tumor cells undergo an irreversible loss of their biosynthetic capacities as a consequence of differentiation. Cancer cells within the biosynthetic domains are characterized by elevated levels of the RNA polymerase I subunit A (POLR1A). Genetic ablation of POLR1A-high cell population imposes an irreversible growth arrest on CRCs. We show that elevated biosynthesis defines stemness in both LGR5(+) and LGR5(−) tumor cells. Therefore, a common architecture in CRCs is a simple cell hierarchy based on the differential capacity to transcribe ribosomal DNA and synthesize proteins.

DNA methylation and chromatin remodeling are frequently implicated in the silencing of genes involved in carcinogenesis. Long Range Epigenetic Silencing (LRES) is a mechanism of gene inactivation that affects multiple contiguous CpG islands and has been described in different human cancer types. However, it is unknown whether there is a coordinated regulation of the genes embedded in these regions in normal cells and in early stages of tumor progression. To better characterize the molecular events associated with the regulation and remodeling of these regions we analyzed two regions undergoing LRES in human colon cancer in the mouse model. We demonstrate that LRES also occurs in murine cancer in vivo and mimics the molecular features of the human phenomenon, namely, downregulation of gene expression, acquisition of inactive histone marks, and DNA hypermethylation of specific CpG islands. The genes embedded in these regions showed a dynamic and autonomous regulation during mouse intestinal cell differentiation, indicating that, in the framework considered here, the coordinated regulation in LRES is restricted to cancer. Unexpectedly, benign adenomas in Apc(Min/+) mice showed overexpression of most of the genes affected by LRES in cancer, which suggests that the repressive remodeling of the region is a late event. Chromatin immunoprecipitation analysis of the transcriptional insulator CTCF in mouse colon cancer cells revealed disrupted chromatin domain boundaries as compared with normal cells. Malignant regression of cancer cells by in vitro differentiation resulted in partial reversion of LRES and gain of CTCF binding. We conclude that genes in LRES regions are plastically regulated in cell differentiation and hyperproliferation, but are constrained to a coordinated repression by abolishing boundaries and the autonomous regulation of chromatin domains in cancer cells.

Murine epidermal stem cells undergo alternate cycles of dormancy and activation, fuelling tissue renewal. However, only a subset of stem cells becomes active during each round of morphogenesis, indicating that stem cells coexist in heterogeneous responsive states. Using a circadian-clock reporter-mouse model, here we show that the dormant hair-follicle stem cell niche contains coexisting populations of cells at opposite phases of the clock, which are differentially predisposed to respond to homeostatic cues. The core clock protein Bmal1 modulates the expression of stem cell regulatory genes in an oscillatory manner, to create populations that are either predisposed, or less prone, to activation. Disrupting this clock equilibrium, through deletion of Bmal1 (also known as Arntl) or Per1/2, resulted in a progressive accumulation or depletion of dormant stem cells, respectively. Stem cell arrhythmia also led to premature epidermal ageing, and a reduction in the development of squamous tumours. Our results indicate that the circadian clock fine-tunes the temporal behaviour of epidermal stem cells, and that its perturbation affects homeostasis and the predisposition to tumorigenesis.

Eph receptor tyrosine kinases and their ligands, the ephrins, play key roles in the regulation of migration and cell adhesion during development, thereby influencing cell fate, morphogenesis and organogenesis. Recent findings suggest that Eph signalling also controls the architecture and physiology of different tissues in the adult body under normal and pathological conditions such as cancer. A prime example is the intestinal epithelium where EphB-ephrinB interactions regulate both cell positioning and tumor progression. Here, we will review recent advances on the role of Eph-ephrin signalling in the intestine and other organs.

Mouse models of intestinal crypt cell differentiation and tumorigenesis have been used to characterize the molecular mechanisms underlying both processes. DNA methylation is a key epigenetic mark and plays an important role in cell identity and differentiation programs and cancer. To get insights into the dynamics of cell differentiation and malignant transformation we have compared the DNA methylation profiles along the mouse small intestine crypt and early stages of tumorigenesis. Genome-scale analysis of DNA methylation together with microarray gene expression have been applied to compare intestinal crypt stem cells (EphB2high), differentiated cells (EphB2negative), ApcMin/+ adenomas and the corresponding non-tumor adjacent tissue, together with small and large intestine samples and the colon cancer cell line CT26. Compared with late stages, small intestine crypt differentiation and early stages of tumorigenesis display few and relatively small changes in DNA methylation. Hypermethylated loci are largely shared by the two processes and affect the proximities of promoter and enhancer regions, with enrichment in genes associated with the intestinal stem cell signature and the PRC2 complex. The hypermethylation is progressive, with minute levels in differentiated cells, as compared with intestinal stem cells, and reaching full methylation in advanced stages. Hypomethylation shows different signatures in differentiation and cancer and is already present in the non-tumor tissue adjacent to the adenomas in ApcMin/+ mice, but at lower levels than advanced cancers. This study provides a reference framework to decipher the mechanisms driving mouse intestinal tumorigenesis and also the human counterpart. MF and EC were supported by respective FPI fellowships from the Ministerio de Economía y Competitividad. AMS held an AECC postdoctoral fellowship. AD-V was supported in part by a contract PTC2011-1091 from Ministerio de Economía y Competitividad. This work was supported by grants from the Ministerio de Economía y Competitividad/n(SAF2011/23638) to MAP; and an ERC grant to EB.

Whereas the series of genetic events leading to colorectal cancer (CRC) have been well established, the precise functions that these alterations play in tumor progression and how they disrupt intestinal homeostasis remain poorly characterized. Activation of the Wnt/Wg signaling pathway by a mutation in the gene APC is the most common trigger for CRC, inducing benign lesions that progress to carcinomas due to the accumulation of other genetic alterations. Among those, Ras mutations drive tumour progression in CRC, as well as in most epithelial cancers. As mammalian and Drosophila's intestines share many similarities, we decided to explore the alterations induced in the Drosophila midgut by the combined activation of the Wnt signaling pathway with gain of function of Ras signaling in the intestinal stem cells. Here we show that compound Apc-Ras clones, but not clones bearing the individual mutations, expand as aggressive intestinal tumor-like outgrowths. These lesions reproduce many of the human CRC hallmarks such as increased proliferation, blockade of cell differentiation and cell polarity and disrupted organ architecture. This process is followed by expression of tumoral markers present in human lesions. Finally, a metabolic behavioral assay shows that these flies suffer a progressive deterioration in intestinal homeostasis, providing a simple readout that could be used in screens for tumor modifiers or therapeutic compounds. Taken together, our results illustrate the conservation of the mechanisms of CRC tumorigenesis in Drosophila, providing an excellent model system to unravel the events that, upon mutation in Apc and Ras, lead to CRC initiation and progression. © 2014 Martorell et al., This work was supported by the MICINN (BFU2010-16016 and BFU2011-23479) to A.C.

© 2014 The Authors. Activating mutations in Wnt and EGFR/Ras signaling pathways are common in colorectal cancer (CRC). Remarkably, clonal coactivation of these pathways in the adult Drosophila midgut induces >tumor-like> overgrowths. Here, we show that, in these clones and in CRC cell lines, Dpp/TGF-β acts as a tumor suppressor. Moreover, we discover that the Iroquois/IRX-family-protein Mirror downregulates the transcription of core components of the Dpp pathway, reducing its tumor suppressor activity. We also show that this genetic interaction is conserved in human CRC cells, where the Iro/IRX proteins IRX3 and IRX5 diminish the response to TGF-β. IRX3 and IRX5 are upregulated in human adenomas, and their levels correlate inversely with the gene expression signature of response to TGF-β. In addition, Irx5 expression confers a growth advantage in the presence of TGF-β, but is selected against in its absence. Together, our results identify a set of Iro/IRX proteins as conserved negative regulators of Dpp/TGF-β activity. We propose that during the characteristic adenoma-to-carcinoma transition of human CRC, the activity of IRX proteins could reduce the sensitivity to the cytostatic effect of TGF-β, conferring a growth advantage to tumor cells prior to the acquisition of mutations in TGF-β pathway components., O.M. is a recipient of a FPU fellowship from the MEC, and F.M.B holds an IRB/PhD La Caixa fellowship. This work was supported by the MICINN (BFU2010-16016 and BFU2011-23479) to A.C.

Zinc-dependent metalloproteases can mediate the shedding of the extracellular domain of many unrelated transmembrane proteins from the cell surface. In most instances, this process, also known as ectodomain shedding, is regulated via protein kinase C (PKC). The tumor necrosis factor alpha-converting enzyme (TACE) was the first protease involved in regulated protein ectodomain shedding identified. Although TACE belongs to the family of metalloprotease-disintegrins, few members of this family have been shown to participate in regulated ectodomain shedding. In fact, the phenotype of tace-/- cells and that of Chinese hamster ovary cell mutants defective in ectodomain shedding points to the existence of a common PKC-activated ectodomain shedding system, whose proteolytic component is TACE, that acts on a variety of transmembrane proteins. Examples of these proteins include the Alzheimer's disease-related protein beta-amyloid precursor protein (betaAPP) and the transmembrane growth factors protransforming growth factor-alpha (pro-TGF-alpha) and, as shown in this report, proheparin-binding epidermal growth factor-like growth factor (pro-HB-EGF). Here we show that the mercurial compound 4-aminophenylmercuric acetate (APMA), frequently used to activate in vitro recombinant matrix metalloproteases, is an activator of the shedding of betaAPP, pro-HB-EGF, and pro-TGF-alpha. Treatment of tace-/- cells or Chinese hamster ovary shedding-defective mutants with APMA activates the cleavage of pro-TGF-alpha but not that of pro-HB-EGF or betaAPP, indicating that APMA activates TACE and also a previously unacknowledged proteolytic activity specific for pro-TGF-alpha. Characterization of this proteolytic activity indicates that it acts on pro-TGF-alpha located at the cell surface and that it is a metalloprotease active in cells defective in furin activity. In summary, treatment of shedding-defective cell lines with APMA unveils the existence of a metalloprotease activity alternative to TACE with the ability to specifically shed the ectodomain of pro-TGF-alpha.

In general, plasma membrane integral proteins, such as the membrane-anchored growth factor proTGF-alpha, are assumed to be transported to the cell surface via a nonregulated, constitutive pathway. proTGF-alpha C-terminal mutants are retained in an early secretory compartment. Here, using a two-hybrid screen, we identify two TACIPs (proTGF-alpha cytoplasmic domain-interacting proteins) that contain PDZ domains and do not interact with proTGF-alpha C-terminal mutants. The binding specificity of one of them, TACIP18 (previously identified and named Syntenin or mda-9), coincides with that of the component that possibly mediates the normal trafficking of proTGF-alpha. TACIP18 colocalizes and interacts specifically with immature, intracellular forms of proTGF-alpha. Therefore, it appears that the interaction of TACIP18 with proTGF-alpha in the early secretory pathway is necessary for the targeting of the latter to the cell surface.

The extracellular domain of a heterogeneous group of transmembrane proteins can be proteolytically released from the cell surface, a process known as protein ectodomain shedding. Despite the biomedical importance of several substrates of the shedding system, such as the beta-amyloid precursor protein (betaAPP), little is known about the regulation of protein ectodomain shedding, and the only protease known to be involved is the metalloprotease disintegrin, tumor necrosis factor-alpha converting enzyme (TACE). Here, we show that previously described pro-transforming growth factor-alpha shedding-defective cell mutants (M2 cells), known to be defective in ectodomain shedding of several molecules, that include betaAPP, fail to shed the ectodomain of pro-TNF-alpha. The target of the mutation is a component required for TACE activity, since transfection of TACE into M2 cells has no effect on the shedding of pro-TNF-alpha and somatic cell fusions between M2 cells and TACE null cells recover the ability to shed pro-TNF-alpha, pro-transforming growth factor-alpha, and betaAPP. Furthermore, we show that TACE is also necessary for the shedding of betaAPP since TACE null cells show defective betaAPP shedding. Biochemical evidence shows that the component that controls TACE is different from protein kinase C, the only known activator of protein ectodomain shedding, and that this component does not affect biosynthesis or processing of TACE or other metalloprotease disintegrins. The component mutated in M2 cells is likely to control only a subset of metalloprotease disintegrins involved in regulated ectodomain shedding, since Notch processing, a process known to be dependent on the activity of another metalloprotease disintegrin, Kuzbanian, is normal in M2 cells.

Here we describe the isolation of stem cells of the human colonic epithelium. Differential cell surface abundance of ephrin type-B receptor 2 (EPHB2) allows the purification of different cell types from human colon mucosa biopsies. The highest EPHB2 surface levels correspond to epithelial colonic cells with the longest telomeres and elevated expression of intestinal stem cell (ISC) marker genes. Moreover, using culturing conditions that recreate the ISC niche, a substantial proportion of EPHB2-high cells can be expanded in vitro as an undifferentiated and multipotent population. [KEYWORDS: Cell Differentiation/physiology, Cell Proliferation, Colon/ cytology, Flow Cytometry, Humans, Immunohistochemistry, Intestinal Mucosa/ cytology, Microscopy, Confocal, Multipotent Stem Cells/cytology/ physiology, Real-Time Polymerase Chain Reaction, Receptor, EphB2/ metabolism, Telomere/metabolism]

Protein ectodomain shedding is a specialized type of regulated proteolysis that releases the extracellular domain of transmembrane proteins. The metalloprotease disintegrin tumor necrosis factor-α-converting enzyme (TACE) has been convincingly shown to play a central role in ectodomain shedding, but despite its broad interest, very little is known about the mechanisms that regulate its activity. An analysis of the biosynthesis of TACE in mutant cell lines that have a gross defect in ectodomain shedding (M1 and M2) shows a defective removal of the prodomain that keeps TACE in an inactive form. Using LoVo, a cell line that lacks of active furin, and α1-Antitrypsin Portland, a protein inhibitor of proprotein convertases, we show that TACE is normally processed by furin and other proprotein convertases. The defect in MI and M2 cells is due to a blockade of the exit of TACE from the endoplasmic reticulum. The processing of other zinc-dependent metalloproteases, previously suggested to participate in activated ectodomain shedding is normal in the mutant cells, indicating that the component mutated is highly specific for TACE. In summary, the characterization of shedding-defective somatic cell mutants unveils the existence of a specific mechanism that directs the proteolytic activation of TACE through the control of its exit from the ER., This work was supported by grants from the Spanish Comisión Interministerial de Ciencia y Tecnología (SAF2000-0203), Fundació La Marató de TV3, Fondo de Investigación Sanitaria (PI021003) and the EMBO Young Investigator Program (to J. A.), and predoctoral fellowships from the Fundació “la Caixa” and from the Fundació per a la Recerca i Docència dels Hospitals Vall d’Hebron (to M. B.-P. and S. R.-P., respectively).

Publisher Summary Certain cell-surface proteins can release their biologically active extracellular domains to the pericellular milieu through limited proteolysis; however, the broad recognition of this type of proteolysis as a general mechanism that regulates the function of virtually all types of cell surface proteins has been relatively recent. Initially, general terms such as “cleavage and release”, “processing”, or “cleavage-secretion” were used by different authors. Currently, the most frequently used term to designate this type of proteolysis is “ectodomain shedding.” Shedding affects type I, type II, and even GPI-bound membrane-anchored proteins that function as receptors, ligands, cell adhesion molecules, or ectoenzymes. This heterogeneity in the proteins seems to indicate that many different mechanisms are involved in ectodomain shedding. However, as the shedding of different proteins was analyzed, common features were found. The shedding of many proteins could be enhanced by phorbol esters and could be blocked by compounds derived from hydroxamic acid. Phorbol esters are well-characterized activators of protein kinase C (PKC), while hydroxamate-based compounds are known to inhibit Zn-dependent metalloproteases of the metzincin superfamily, indicating that metzincins regulated by PKC are responsible for the shedding of many proteins. This chapter describes the shedding characteristics of many different proteins, emphasizing recent findings that support the view that, despite the variety of proteins shed, there are common components necessary for protein ectodomain shedding.

Although the number of cell-surface transmembrane molecules known to release their extracellular domain to the cell media has grown greatly during past years, the protease(s) involved have remained elusive until recently. The analysis of ectodomain shedding of proteins as diverse as membrane-anchored growth factors, such as pro-transforming growth factor a (proTGF-a) and pro-tumour necrosis factor a (proTNF-ol), growth factor receptors, ectoenzymes, cell adhesion molecules and the /j amyloid precursor protein (/jAPP) has revealed several common features. The shedding of most proteins occurs at a fixed distance from the plasma membrane, can be activated via protein kinase C (PKC) and can be prevented by hydroxamic acid-based metalloprotease inhibitors initially developed to block the shedding of proTNF-x [l]. The structural diversity of proteins that undergo protein ectodomain shedding initially suggested the existence of several proteases involved, each endowed with a restricted specificity. However, Chinese hamster ovary (CHO) cell mutants selected for a lack of proTGF-x shedding (M1 and M2 cells) showed a general defect in protein ectodomain shedding, indicating the existence of at least a common component necessary for the shedding of multiple transmembrane proteins [2,3]. The first protease shown to be involved in a shedding event acts on proTNF-z and belongs to the family of metalloprotease disintegrins (also known as the ADAM family), a family of modular transmembrane Znzf-dependent metalloproteases that contain a disintegrin domain. Metalloprotease disintegrins are involved in sperm-egg fusion, muscle fusion in C2C12 myoblasts and the regulation of neural cell fate at different stages of neurogenesis in Ilrosophila (metalloprotease disintegrins are reviewed in [4,5]). The substrates of most members of the metallopro

Transforming growth factor alpha (TGF-(alpha)) is synthesized as a precursor transmembrane molecule (proTGF-(alpha)) whose ectodomain is shed from the cell surface generating mature, soluble, growth factor. In agreement with recent reports, here we show that the structural determinant that targets proTGF-(alpha) to the cell surface maps to the very C-terminal cytoplasmic amino acid, valine. The primary localization of proTGF-(alpha) C-terminal mutants is a perinuclear area that colocalizes with ER markers. Since the ectodomain shedding machinery that acts on proTGF-(alpha) is known to be located at the cell surface, deficient transport provides an explanation for the previously reported lack of PKC activated ectodomain shedding of proTGF-(alpha) C-terminal mutants. The transport of wild-type proTGF-(alpha) to the cell surface was found to be mediated by a mechanism that includes a specific component saturable by wild-type proTGF-(alpha) but not by cell surface transmembrane proteins whose trafficking is independent of their cytoplasmic tail such as betaglycan. C-terminal valines are likely to be a general determinant of the subcellular location of cell surface transmembrane proteins since the maturation and trafficking of MT1-MMP C-terminal mutants are severely impaired. Our data suggest the existence of a targeting mechanism that acts on cell surface transmembrane molecules as diverse as proTGF-(alpha) and MT1-MMP and that the interaction with such a mechanism depends on the identity of the C-terminal amino acid of the targeted molecules.

A frequent complication in colorectal cancer (CRC) is regeneration of the tumor after therapy. Here, we report that a gene signature specific for adult intestinal stem cells (ISCs) predicts disease relapse in CRC patients. ISCs are marked by high expression of the EphB2 receptor, which becomes gradually silenced as cells differentiate. Using EphB2 and the ISC marker Lgr5, we have FACS-purified and profiled mouse ISCs, crypt proliferative progenitors, and late transient amplifying cells to define a gene program specific for normal ISCs. Furthermore, we discovered that ISC-specific genes identify a stem-like cell population positioned at the bottom of tumor structures reminiscent of crypts. EphB2 sorted ISC-like tumor cells display robust tumor-initiating capacity in immunodeficient mice as well as long-term self-renewal potential. Taken together, our data suggest that the ISC program defines a cancer stem cell niche within colorectal tumors and plays a central role in CRC relapse. This work has been supported by grants to E.B. from ERC (FP7-EU) and MICINN (Plan Nacional I+D+i and Consolider programs) and by grants PS09/00965 (MICINN) and NanoCoMets (CIBERBBN) to R.M