Colorectal Cancer Stem Cell Subtypes Orchestrate Distinct Tumor Microenvironments.

Several classification systems have been developed to define tumor subtypes in colorectal cancer (CRC). One system proposes that tumor heterogeneity derives in part from distinct cancer stem cell populations that co-exist as admixtures of varying proportions. However, the lack of single cell resolution has prohibited a definitive identification of these types of stem cells and therefore any understanding of how each influence tumor phenotypes.

Characterization of a chromatin-associated TCF7L1 complex in human embryonic stem cells.

Human embryonic stem cells (hESCs) resemble the pluripotent epiblast cells found in the early postimplantation human embryo and represent the "primed" state of pluripotency. One factor that helps primed pluripotent cells retain pluripotency and prepare genes for differentiation is the transcription factor TCF7L1, a member of a small family of proteins known as T cell factors/Lymphoid enhancer factors (TCF/LEF) that act as downstream components of the WNT signaling pathway. Transcriptional output of the WNT pathway is regulated, in part, by the activity of TCF/LEFs in conjunction with another component of the WNT pathway, β-CATENIN.

A human vascularized microtumor model of patient-derived colorectal cancer recapitulates clinical disease.

Accurately modeling tumor biology and testing novel therapies on patient-derived cells is critically important to developing therapeutic regimens personalized to a patient's specific disease. The vascularized microtumor (VMT), or "tumor-on-a-chip," is a physiologic preclinical cancer model that incorporates key features of the native human tumor microenvironment within a transparent microfluidic platform, allowing rapid drug screening in vitro. Herein we optimize methods for generating patient-derived VMT (pVMT) using fresh colorectal cancer (CRC) biopsies and surgical resections to test drug sensitivities at the individual patient level.

Disruption of the circadian clock drives loss of heterozygosity to accelerate colorectal cancer.

An alarming rise in young onset colorectal cancer (CRC) has been reported; however, the underlying molecular mechanism remains undefined. Suspected risk factors of young onset CRC include environmental aspects, such as lifestyle and dietary factors, which are known to affect the circadian clock. We find that both genetic disruption and environmental disruption of the circadian clock accelerate driven CRC pathogenesis in vivo.

ERK2 MAP kinase regulates SUFU binding by multisite phosphorylation of GLI1.

Crosstalk between the Hedgehog and MAPK signaling pathways occurs in several types of cancer and contributes to clinical resistance to Hedgehog pathway inhibitors. Here we show that MAP kinase-mediated phosphorylation weakens the binding of the GLI1 transcription factor to its negative regulator SUFU. ERK2 phosphorylates GLI1 on three evolutionarily conserved target sites (S102, S116, and S130) located near the high-affinity binding site for SUFU; these phosphorylations cooperate to weaken the affinity of GLI1-SUFU binding by over 25-fold.

Spatial transcriptomics using combinatorial fluorescence spectral and lifetime encoding, imaging and analysis.

Multiplexed mRNA profiling in the spatial context provides new information enabling basic research and clinical applications. Unfortunately, existing spatial transcriptomics methods are limited due to either low multiplexing or complexity. Here, we introduce a spatialomics technology, termed Multi Omic Single-scan Assay with Integrated Combinatorial Analysis (MOSAICA), that integrates in situ labeling of mRNA and protein markers in cells or tissues with combinatorial fluorescence spectral and lifetime encoded probes, spectral and time-resolved fluorescence imaging, and machine learning-based decoding.

Disruption of β-Catenin-Dependent Wnt Signaling in Colon Cancer Cells Remodels the Microenvironment to Promote Tumor Invasion.

Unlabelled: The recent classification of colon cancer into molecular subtypes revealed that patients with the poorest prognosis harbor tumors with the lowest levels of Wnt signaling. This is contrary to the general understanding that overactive Wnt signaling promotes tumor progression from early initiation stages through to the later stages including invasion and metastasis. Here, we directly test this assumption by reducing the activity of ß-catenin-dependent Wnt signaling in colon cancer cell lines at either an upstream or downstream step in the pathway.

Facile One-Pot Nanoproteomics for Label-Free Proteome Profiling of 50-1000 Mammalian Cells.

Recent advances in sample preparation enable label-free mass spectrometry (MS)-based proteome profiling of small numbers of mammalian cells. However, specific devices are often required to downscale sample processing volume from the standard 50-200 μL to sub-μL for effective nanoproteomics, which greatly impedes the implementation of current nanoproteomics methods by the proteomics research community. Herein, we report a facile one-pot nanoproteomics method termed SOPs-MS (urfactant-assisted ne-ot sample processing at the tandard volume coupled with MS) for convenient robust proteome profiling of 50-1000 mammalian cells.

An in vitro vascularized micro-tumor model of human colorectal cancer recapitulates in vivo responses to standard-of-care therapy.

Around 95% of anti-cancer drugs that show promise during preclinical study fail to gain FDA-approval for clinical use. This failure of the preclinical pipeline highlights the need for improved, physiologically-relevant in vitro models that can better serve as reliable drug-screening and disease modeling tools. The vascularized micro-tumor (VMT) is a novel three-dimensional model system (tumor-on-a-chip) that recapitulates the complex human tumor microenvironment, including perfused vasculature, within a transparent microfluidic device, allowing real-time study of drug responses and tumor-stromal interactions.

Publisher Correction: Transcriptomic and proteomic signatures of stemness and differentiation in the colon crypt.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

α-Ketoglutarate attenuates Wnt signaling and drives differentiation in colorectal cancer.

Genetic-driven deregulation of the Wnt pathway is crucial but not sufficient for colorectal cancer (CRC) tumourigenesis. Here, we show that environmental glutamine restriction further augments Wnt signaling in APC mutant intestinal organoids to promote stemness and leads to adenocarcinoma formation via decreasing intracellular alpha-ketoglutarate (aKG) levels. aKG supplementation is sufficient to rescue low-glutamine induced stemness and Wnt hyperactivation.

Transcriptomic and proteomic signatures of stemness and differentiation in the colon crypt.

Intestinal stem cells are non-quiescent, dividing epithelial cells that rapidly differentiate into progenitor cells of the absorptive and secretory cell lineages. The kinetics of this process is rapid such that the epithelium is replaced weekly. To determine how the transcriptome and proteome keep pace with rapid differentiation, we developed a new cell sorting method to purify mouse colon epithelial cells.

LEF-1 drives aberrant β-catenin nuclear localization in myeloid leukemia cells.

Canonical Wnt/β-catenin signaling is frequently dysregulated in myeloid leukemias and is implicated in leukemogenesis. Nuclear-localized β-catenin is indicative of active Wnt signaling and is frequently observed in acute myeloid leukemia (AML) patients; however, some patients exhibit little or no nuclear β-catenin even where cytosolic β-catenin is abundant. Control of the subcellular localization of β-catenin therefore represents an additional mechanism regulating Wnt signaling in hematopoietic cells.

TCF7L1 suppresses primitive streak gene expression to support human embryonic stem cell pluripotency.

Human embryonic stem cells (hESCs) are exquisitely sensitive to WNT ligands, which rapidly cause differentiation. Therefore, hESC self-renewal requires robust mechanisms to keep the cells in a WNT inactive but responsive state. How they achieve this is largely unknown.

Inhibition of nuclear Wnt signalling: challenges of an elusive target for cancer therapy.

Unlabelled: The highly conserved Wnt signalling pathway plays an important role in embryonic development and disease pathogenesis, most notably cancer. The 'canonical' or β-catenin-dependent Wnt signal initiates at the cell plasma membrane with the binding of Wnt proteins to Frizzled:LRP5/LRP6 receptor complexes and is mediated by the translocation of the transcription co-activator protein, β-catenin, into the nucleus. β-Catenin then forms a complex with T-cell factor (TCF)/lymphoid enhancer binding factor (LEF) transcription factors to regulate multiple gene programmes.

Mathematical modeling links Wnt signaling to emergent patterns of metabolism in colon cancer.

Cell-intrinsic metabolic reprogramming is a hallmark of cancer that provides anabolic support to cell proliferation. How reprogramming influences tumor heterogeneity or drug sensitivities is not well understood. Here, we report a self-organizing spatial pattern of glycolysis in xenograft colon tumors where pyruvate dehydrogenase kinase (PDK1), a negative regulator of oxidative phosphorylation, is highly active in clusters of cells arranged in a spotted array.

Lactate/pyruvate transporter MCT-1 is a direct Wnt target that confers sensitivity to 3-bromopyruvate in colon cancer.

Background: There is increasing evidence that oncogenic Wnt signaling directs metabolic reprogramming of cancer cells to favor aerobic glycolysis or Warburg metabolism. In colon cancer, this reprogramming is due to direct regulation of pyruvate dehydrogenase kinase 1 () gene transcription. Additional metabolism genes are sensitive to Wnt signaling and exhibit correlative expression with PDK1.

Cancer: leaping the E-cadherin hurdle.

Aberrant activation of the Wnt signaling pathway is a common cause of colon cancer and other tumor types, accomplishing many of the hallmarks of cancer including sustained proliferative signaling, replicative immortality, reprogrammed metabolism, angiogenesis, and invasion. Yet, the dominant mutation that leads to chronic Wnt signaling in colon cancer is quite different from the spectrum of mutations that activate Wnt signaling in other tumor types. In this issue of , Huels (2015) focus on the influential role E-cadherin plays in shaping these differences.

Differential Effects of Hepatocyte Nuclear Factor 4α Isoforms on Tumor Growth and T-Cell Factor 4/AP-1 Interactions in Human Colorectal Cancer Cells.

The nuclear receptor hepatocyte nuclear factor 4α (HNF4α) is tumor suppressive in the liver but amplified in colon cancer, suggesting that it also might be oncogenic. To investigate whether this discrepancy is due to different HNF4α isoforms derived from its two promoters (P1 and P2), we generated Tet-On-inducible human colon cancer (HCT116) cell lines that express either the P1-driven (HNF4α2) or P2-driven (HNF4α8) isoform and analyzed them for tumor growth and global changes in gene expression (transcriptome sequencing [RNA-seq] and chromatin immunoprecipitation sequencing [ChIP-seq]). The results show that while HNF4α2 acts as a tumor suppressor in the HCT116 tumor xenograft model, HNF4α8 does not.

The TCF C-clamp DNA binding domain expands the Wnt transcriptome via alternative target recognition.

LEF/TCFs direct the final step in Wnt/β-catenin signalling by recruiting β-catenin to genes for activation of transcription. Ancient, non-vertebrate TCFs contain two DNA binding domains, a High Mobility Group box for recognition of the Wnt Response Element (WRE; 5'-CTTTGWWS-3') and the C-clamp domain for recognition of the GC-rich Helper motif (5'-RCCGCC-3'). Two vertebrate TCFs (TCF-1/TCF7 and TCF-4/TCF7L2) use the C-clamp as an alternatively spliced domain to regulate cell-cycle progression, but how the C-clamp influences TCF binding and activity genome-wide is not known.

A novel Bcr-Abl-mTOR-eIF4A axis regulates IRES-mediated translation of LEF-1.

Internal ribosome entry sites (IRESs) in cellular mRNAs direct expression of growth-promoting factors through an alternative translation mechanism that has yet to be fully defined. Lymphoid enhancer factor-1 (LEF-1), a Wnt-mediating transcription factor important for cell survival and metastasis in cancer, is produced via IRES-directed translation, and its mRNA is frequently upregulated in malignancies, including chronic myeloid leukaemia (CML). In this study, we determined that LEF1 expression is regulated by Bcr-Abl, the oncogenic protein that drives haematopoietic cell transformation to CML.

Predicting mechanism of biphasic growth factor action on tumor growth using a multi-species model with feedback control.

A large number of growth factors and drugs are known to act in a biphasic manner: at lower concentrations they cause increased division of target cells, whereas at higher concentrations the mitogenic effect is inhibited. Often, the molecular details of the mitogenic effect of the growth factor are known, whereas the inhibitory effect is not. Hepatoctyte Growth Factor, HGF, has recently been recognized as a strong mitogen that is present in the microenvironment of solid tumors.

Disulfide bond requirements for active Wnt ligands.

Secreted Wnt lipoproteins are cysteine-rich and lipid-modified morphogens that bind to the Frizzled (FZD) receptor and LDL receptor-related protein 6 (LRP6). Wnt engages FZD through protruding thumb and index finger domains, which are each assembled from paired β strands secured by disulfide bonds and grasp two sides of the FZD ectodomain. The importance of Wnt disulfide bonds has been assumed but uncharacterized.

Wnt signaling directs a metabolic program of glycolysis and angiogenesis in colon cancer.

Much of the mechanism by which Wnt signaling drives proliferation during oncogenesis is attributed to its regulation of the cell cycle. Here, we show how Wnt/β-catenin signaling directs another hallmark of tumorigenesis, namely Warburg metabolism. Using biochemical assays and fluorescence lifetime imaging microscopy (FLIM) to probe metabolism in vitro and in living tumors, we observe that interference with Wnt signaling in colon cancer cells reduces glycolytic metabolism and results in small, poorly perfused tumors.

A three-dimensional in vitro model of tumor cell intravasation.

Metastasis is the cause of over 90% of all human cancer deaths. Early steps in the metastatic process include: the formation of new blood vessels, the initiation of epithelial-mesenchymal transition (EMT), and the mobilization of tumor cells into the circulation. There are ongoing efforts to replicate the physiological landscape of human tumor tissue using three-dimensional in vitro culture models; however, few systems are able to capture the full range of authentic, complex in vivo events such as neovascularization and intravasation.