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. Here were report the isolation and characterization of two cancer stem cell subtypes from the SW480 CRC cell line. We find these cancer stem cells are oncogenic versions of the normal Crypt Base Columnar (CBC) and Regenerative Stem Cell (RSC) populations from intestinal crypts and that their gene signatures are consistent with the "Admixture" and other CRC classification systems. Using publicly available single cell RNA sequencing (scRNAseq) data from CRC patients, we determine that RSC and CBC cancer stem cells are commonly co-present in human CRC. To characterize influences on the tumor microenvironment, we develop subtype-specific xenograft models and we define their tumor microenvironments at high resolution via scRNAseq. RSCs create differentiated, inflammatory, slow growing tumors. CBCs create proliferative, undifferentiated, invasive tumors. With this enhanced resolution, we unify current CRC patient classification schema with TME phenotypes and organization.

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, ß-CATENIN. Because TCF7L1 plays an important role in regulating pluripotency, we began to characterize the protein complex associated with TCF7L1 when bound to chromatin in hESCs using rapid immunoprecipitation of endogenous proteins (RIME).  Data are available via ProteomeXchange with identifier PXD047582. These data identify known and new partners of TCF7L1 on chromatin and provide novel insights into how TCF7L1 and pluripotency itself might be regulated.

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. In response to standard chemotherapy and TGF-ßR1 inhibition, we observe heterogeneous responses between pVMT derived from 6 patient biopsies, with the pVMT recapitulating tumor growth, histological features, metabolic heterogeneity, and drug responses of actual CRC tumors. Our results suggest that a translational infrastructure providing rapid information from patient-derived tumor cells in the pVMT, as established in this study, will support efforts to improve patient outcomes.

Disruption of the circadian clock drives Apc 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 Apc-driven CRC pathogenesis in vivo. Using an intestinal organoid model, we demonstrate that clock disruption promotes transformation by driving Apc loss of heterozygosity, which hyperactivates Wnt signaling. This up-regulates c-Myc, a known Wnt target, which drives heightened glycolytic metabolism. Using patient-derived organoids, we show that circadian rhythms are lost in human tumors. Last, we identify that variance between core clock and Wnt pathway genes significantly predicts the survival of patients with CRC. Overall, our findings demonstrate a previously unidentified mechanistic link between clock disruption and CRC, which has important implications for young onset cancer prevention.

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. Phosphorylation of any one, or even any two, of the three sites does not result in the level of SUFU release seen when all three sites are phosphorylated. Tumor-derived mutations in R100 and S105, residues bordering S102, also diminish SUFU binding, collectively defining a novel evolutionarily conserved SUFU affinity-modulating region. In cultured mammalian cells, GLI1 variants containing phosphomimetic substitutions of S102, S116, and S130 displayed an increased ability to drive transcription. We conclude that multisite phosphorylation of GLI1 by ERK2 or other MAP kinases weakens GLI1-SUFU binding, thereby facilitating GLI1 activation and contributing to both physiological and pathological crosstalk.

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. We demonstrate MOSAICA's multiplexing scalability in detecting 10-plex targets in fixed colorectal cancer cells using combinatorial labeling of five fluorophores with facile error-detection and removal of autofluorescence. MOSAICA's analysis is strongly correlated with sequencing data (Pearson's r = 0.96) and was further benchmarked using RNAscopeTM and LGC StellarisTM. We further apply MOSAICA for multiplexed analysis of clinical melanoma Formalin-Fixed Paraffin-Embedded (FFPE) tissues. We finally demonstrate simultaneous co-detection of protein and mRNA in cancer cells.

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

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. We determine that Wnt-reduced cancer cells exhibit a more aggressive disease phenotype, including increased mobility in vitro and disruptive invasion into mucosa and smooth muscle in an orthotopic mouse model. RNA sequencing reveals that interference with Wnt signaling leads to an upregulation of gene programs that favor cell migration and invasion and a downregulation of inflammation signatures in the tumor microenvironment. We identify a set of upregulated genes common among the Wnt perturbations that are predictive of poor patient outcomes in early-invasive colon cancer. Our findings suggest that while targeting Wnt signaling may reduce tumor burden, an inadvertent side effect is the emergence of invasive cancer. IMPLICATIONS: Decreased Wnt signaling in colon tumors leads to a more aggressive disease phenotype due to an upregulation of gene programs favoring cell migration in the tumor and downregulation of inflammation programs in the tumor microenvironment; these impacts must be carefully considered in developing Wnt-targeting therapies.

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 (surfactant-assisted one-pot sample processing at the standard volume coupled with MS) for convenient robust proteome profiling of 50-1000 mammalian cells. Building upon our recent development of SOPs-MS for label-free single-cell proteomics at a low µL volume, we have systematically evaluated its processing volume at 10-200 µL using 100 human cells. The processing volume of 50 µL that is in the range of volume for standard proteomics sample preparation has been selected for easy sample handling with a benchtop micropipette. SOPs-MS allows for reliable label-free quantification of ∼1200-2700 protein groups from 50 to 1000 MCF10A cells. When applied to small subpopulations of mouse colon crypt cells, SOPs-MS has revealed protein signatures between distinct subpopulation cells with identification of ∼1500-2500 protein groups for each subpopulation. SOPs-MS may pave the way for routine deep proteome profiling of small numbers of cells and low-input samples.

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. Here we have validated this microphysiological system (MPS) platform for the study of colorectal cancer (CRC), the second leading cause of cancer-related deaths, by showing that gene expression, tumor heterogeneity, and treatment responses in the VMT more closely model CRC tumor clinicopathology than current standard drug screening modalities, including 2-dimensional monolayer culture and 3-dimensional spheroids.

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 in vivo via decreasing intracellular alpha-ketoglutarate (aKG) levels. aKG supplementation is sufficient to rescue low-glutamine induced stemness and Wnt hyperactivation. Mechanistically, we found that aKG promotes hypomethylation of DNA and histone H3K4me3, leading to an upregulation of differentiation-associated genes and downregulation of Wnt target genes, respectively. Using CRC patient-derived organoids and several in vivo CRC tumour models, we show that aKG supplementation suppresses Wnt signaling and promotes cellular differentiation, thereby significantly restricting tumour growth and extending survival. Together, our results reveal how metabolic microenvironment impacts Wnt signaling and identify aKG as a potent antineoplastic metabolite for potential differentiation therapy for CRC patients.

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. Here we show that alternative mRNA splicing and polyadenylation dominate changes in the transcriptome as stem cells differentiate into progenitors. In contrast, as progenitors differentiate into mature cell types, changes in mRNA levels dominate the transcriptome. RNA processing targets regulators of cell cycle, RNA, cell adhesion, SUMOylation, and Wnt and Notch signaling. Additionally, global proteome profiling detected >2,800 proteins and revealed RNA:protein patterns of abundance and correlation. Paired together, these data highlight new potentials for autocrine and feedback regulation and provide new insights into cell state transitions in the crypt.

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

Canonical Wnt/ß-catenin signaling is frequently dysregulated in myeloid leukemias and is implicated in leukemogenesis. Nuclear-localized ß-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 ß-catenin even where cytosolic ß-catenin is abundant. Control of the subcellular localization of ß-catenin therefore represents an additional mechanism regulating Wnt signaling in hematopoietic cells. To investigate the factors mediating the nuclear-localization of ß-catenin, we carried out the first nuclear/cytoplasmic proteomic analysis of the ß-catenin interactome in myeloid leukemia cells and identified putative novel ß-catenin interactors. Comparison of interacting factors between Wnt-responsive cells (high nuclear ß-catenin) versus Wnt-unresponsive cells (low nuclear ß-catenin) suggested the transcriptional partner, LEF-1, could direct the nuclear-localization of ß-catenin. The relative levels of nuclear LEF-1 and ß-catenin were tightly correlated in both cell lines and in primary AML blasts. Furthermore, LEF-1 knockdown perturbed ß-catenin nuclear-localization and transcriptional activation in Wnt-responsive cells. Conversely, LEF-1 overexpression was able to promote both nuclear-localization and ß-catenin-dependent transcriptional responses in previously Wnt-unresponsive cells. This is the first ß-catenin interactome study in hematopoietic cells and reveals LEF-1 as a mediator of nuclear ß- catenin level in human myeloid leukemia.

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. We explored the role of transcriptional regulators of WNT signaling, the TCF/LEFs. As in mouse ESCs, TCF7L1 is the predominant family member expressed in hESCs. Genome-wide, it binds a gene cohort involved in primitive streak formation at gastrulation, including NODAL, BMP4 and WNT3 Comparing TCF7L1-bound sites with those bound by the WNT signaling effector ß-catenin indicates that TCF7L1 acts largely on the WNT signaling pathway. TCF7L1 overlaps less with the pluripotency regulators OCT4 and NANOG than in mouse ESCs. Gain- and loss-of-function studies indicate that TCF7L1 suppresses gene cohorts expressed in the primitive streak. Interestingly, we find that BMP4, another driver of hESC differentiation, downregulates TCF7L1, providing a mechanism of BMP and WNT pathway intersection. Together, our studies indicate that TCF7L1 plays a major role in maintaining hESC pluripotency, which has implications for human development during gastrulation.

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

The highly conserved Wnt signalling pathway plays an important role in embryonic development and disease pathogenesis, most notably cancer. The 'canonical' or ß-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, ß-catenin, into the nucleus. ß-Catenin then forms a complex with T-cell factor (TCF)/lymphoid enhancer binding factor (LEF) transcription factors to regulate multiple gene programmes. These programmes play roles in cell proliferation, migration, vasculogenesis, survival and metabolism. Mutations in Wnt signalling pathway components lead to constitutively active Wnt signalling that drives aberrant expression of these programmes and development of cancer. It has been a longstanding and challenging goal to develop therapies that can interfere with the TCF/LEF-ß-catenin transcriptional complex. This review will focus on the (i) structural considerations for targeting the TCF/LEF-ß-catenin and co-regulatory complexes in the nucleus, (ii) current molecules that directly target TCF/LEF-ß-catenin activity and (iii) ideas for targeting newly discovered components of the TCF/LEF-ß-catenin complex and/or downstream gene programmes regulated by these complexes. LINKED ARTICLES: This article is part of a themed section on WNT Signalling: Mechanisms and Therapeutic Opportunities. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v174.24/issuetoc.

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. To understand this pattern, we developed a reaction-diffusion model that incorporates Wnt signaling, a pathway known to upregulate PDK1 and Warburg metabolism. Partial interference with Wnt alters the size and intensity of the spotted pattern in tumors and in the model. The model predicts that Wnt inhibition should trigger an increase in proteins that enhance the range of Wnt ligand diffusion. Not only was this prediction validated in xenograft tumors but similar patterns also emerge in radiochemotherapy-treated colorectal cancer. The model also predicts that inhibitors that target glycolysis or Wnt signaling in combination should synergize and be more effective than each treatment individually. We validated this prediction in 3D colon tumor spheroids.

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 (PDK1) gene transcription. Additional metabolism genes are sensitive to Wnt signaling and exhibit correlative expression with PDK1. Whether these genes are also regulated at the transcriptional level, and therefore a part of a core metabolic gene program targeted by oncogenic WNT signaling, is not known. RESULTS: Here, we identify monocarboxylate transporter 1 (MCT-1; encoded by SLC16A1) as a direct target gene supporting Wnt-driven Warburg metabolism. We identify and validate Wnt response elements (WREs) in the proximal SLC16A1 promoter and show that they mediate sensitivity to Wnt inhibition via dominant-negative LEF-1 (dnLEF-1) expression and the small molecule Wnt inhibitor XAV939. We also show that WREs function in an independent and additive manner with c-Myc, the only other known oncogenic regulator of SLC16A1 transcription. MCT-1 can export lactate, the byproduct of Warburg metabolism, and it is the essential transporter of pyruvate as well as a glycolysis-targeting cancer drug, 3-bromopyruvate (3-BP). Using sulforhodamine B (SRB) assays to follow cell proliferation, we tested a panel of colon cancer cell lines for sensitivity to 3-BP. We observe that all cell lines are highly sensitive and that reduction of Wnt signaling by XAV939 treatment does not synergize with 3-BP, but instead is protective and promotes rapid recovery. CONCLUSIONS: We conclude that MCT-1 is part of a core Wnt signaling gene program for glycolysis in colon cancer and that modulation of this program could play an important role in shaping sensitivity to drugs that target cancer metabolism.

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. Each isoform regulates the expression of distinct sets of genes and recruits, colocalizes, and competes in a distinct fashion with the Wnt/ß-catenin mediator T-cell factor 4 (TCF4) at CTTTG motifs as well as at AP-1 motifs (TGAXTCA). Protein binding microarrays (PBMs) show that HNF4α and TCF4 share some but not all binding motifs and that single nucleotide polymorphisms (SNPs) in sites bound by both HNF4α and TCF4 can alter binding affinity in vitro, suggesting that they could play a role in cancer susceptibility in vivo. Thus, the HNF4α isoforms play distinct roles in colon cancer, which could be due to differential interactions with the Wnt/ß-catenin/TCF4 and AP-1 pathways.

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. We have previously shown that the LEF1 5' untranslated region recruits a complex of proteins to its IRES, including the translation initiation factor eIF4A. In this report, we use two small molecule inhibitors, PP242 (dual mTOR (mammalian target of rapamycin) kinase inhibitor) and hippuristanol (eIF4A inhibitor), to define IRES regulation via a Bcr-Abl-mTOR-eIF4A axis in CML cell lines and primary patient leukaemias. We found that LEF1 and other IRESs are uniquely sensitive to the activities of Bcr-Abl/mTOR. Most notably, we discovered that eIF4A, an RNA helicase, elicits potent non-canonical effects on the LEF1 IRES. Hippuristanol inhibition of eIF4A stalls translation of IRES mRNA and triggers dissociation from polyribosomes. We propose that a combination drug strategy which targets mTOR and IRES-driven translation disrupts key factors that contribute to growth and proliferation in CML.