bims-instec 2023-01-15 papers

bims-instec

Biomed News

on Intestinal stem cells and chemoresistance in colon cancer and intestinal regeneration

Issue of 2023–01–15
twenty-one papers selected by
Maria-Virginia Giolito, Free University of Brussels

Senescent cells perturb intestinal stem cell differentiation through Ptk7 induced noncanonical Wnt and YAP signaling.
Enteroendocrine cells protect the stem cell niche by regulating crypt metabolism in response to nutrients.
A Comprehensive Biomarker Analysis of Microsatellite Unstable/Mismatch Repair Deficient Colorectal Cancer Cohort Treated with Immunotherapy.
Heat stress disrupts intestinal stem cell migration and differentiation along the crypt-villus axis through FAK signaling.
Lentiviral in situ targeting of stem cells in unperturbed intestinal epithelium.
Synthetic lethal interaction with BCL-XL blockade deepens response to cetuximab in patient-derived models of metastatic colorectal cancer.
General transcription factor TAF4 antagonizes epigenetic silencing by Polycomb to maintain intestine stem cell functions.
The complex network of transcription factors, immune checkpoint inhibitors and stemness features in colorectal cancer: A recent update.
Comprehensive analysis of RNA-binding protein SRSF2-dependent alternative splicing signature in malignant proliferation of colorectal carcinoma.
Gut immune cells and intestinal niche imprinting.
Protocol to establish a mouse model for hepatic metastasis of colorectal cancer.
ONECUT2 regulates RANKL-dependent enterocyte and microfold cell differentiation in the small intestine; a multi-omics study.
Transcriptomic Changes Associated with ERBB2 Overexpression in Colorectal Cancer Implicate a Potential Role of the Wnt Signaling Pathway in Tumorigenesis.
Intravital imaging of Wnt/β-catenin and ATF2-dependent signalling pathways during tumour cell invasion and metastasis.
Optimized protocol for the generation of an orthotopic colon cancer mouse model and metastasis.
The intestinal microenvironment shapes macrophage and dendritic cell identity and function.
Integrated clinical and genomic analysis identifies driver events and molecular evolution of colitis-associated cancers.
Multistain deep learning for prediction of prognosis and therapy response in colorectal cancer.
SPOCK1 and POSTN are valuable prognostic biomarkers and correlate with tumor immune infiltrates in colorectal cancer.
Arginine limitation drives a directed codon-dependent DNA sequence evolution response in colorectal cancer cells.
An immune, stroma, and epithelial-mesenchymal transition-related signature for predicting recurrence and chemotherapy benefit in stage II-III colorectal cancer.

Nat Commun. 2023 Jan 11. 14(1): 156

Senescent cells perturb intestinal stem cell differentiation through Ptk7 induced noncanonical Wnt and YAP signaling.

Jina Yun, Simon Hansen, Otto Morris, David T Madden, Clare Peters Libeu, Arjun J Kumar, Cameron Wehrfritz, Aaron H Nile, Yingnan Zhang, Lijuan Zhou, Yuxin Liang, Zora Modrusan, Michelle B Chen, Christopher C Overall, David Garfield, Judith Campisi, Birgit Schilling, Rami N Hannoush, Heinrich Jasper.

Cellular senescence and the senescence-associated secretory phenotype (SASP) are implicated in aging and age-related disease, and SASP-related inflammation is thought to contribute to tissue dysfunction in aging and diseased animals. However, whether and how SASP factors influence the regenerative capacity of tissues remains unclear. Here, using intestinal organoids as a model of tissue regeneration, we show that SASP factors released by senescent fibroblasts deregulate stem cell activity and differentiation and ultimately impair crypt formation. We identify the secreted N-terminal domain of Ptk7 as a key component of the SASP that activates non-canonical Wnt / Ca2+ signaling through FZD7 in intestinal stem cells (ISCs). Changes in cytosolic [Ca2+] elicited by Ptk7 promote nuclear translocation of YAP and induce expression of YAP/TEAD target genes, impairing symmetry breaking and stem cell differentiation. Our study discovers secreted Ptk7 as a factor released by senescent cells and provides insight into the mechanism by which cellular senescence contributes to tissue dysfunction in aging and disease.

DOI: https://doi.org/10.1038/s41467-022-35487-9
Cell Mol Gastroenterol Hepatol. 2023 Jan 03. pii: S2352-345X(22)00267-3. [Epub ahead of print]

Enteroendocrine cells protect the stem cell niche by regulating crypt metabolism in response to nutrients.

Heather A McCauley, Anne Marie Riedman, Jacob R Enriquez, Xinghao Zhang, Miki Watanabe-Chailland, J Guillermo Sanchez, Daniel O Kechele, Emily F Paul, Kayle Riley, Courtney Burger, Richard A Lang, James M Wells.

   BACKGROUND AND AIMS: The intestinal stem cell niche is exquisitely sensitive to changes in diet, with high fat diet, caloric restriction, and fasting resulting in altered crypt metabolism and intestinal stem cell function. Unlike cells on the villus, cells in the crypt are not immediately exposed to the dynamically changing contents of the lumen. We hypothesized that enteroendocrine cells (EECs), which sense environmental cues and in response release hormones and metabolites, are essential for relaying the luminal and nutritional status of the animal to cells deep in the crypt.
METHODS: We used the tamoxifen-inducible VillinCreERT2 mouse model to deplete EECs (Neurog3fl/fl) from adult intestinal epithelium and we generated human intestinal organoids from wild-type and NEUROG3-null human pluripotent stem cells. We used indirect calorimetry, 1H-NMR metabolomics, mitochondrial live imaging, and the Seahorse bioanalyzer to assess metabolism. Intestinal stem cell activity was measured by proliferation and enteroid-forming capacity. Transcriptional changes were assessed using 10X Genomics single-cell sequencing.
RESULTS: Loss of EECs resulted in increased energy expenditure in mice, an abundance of active mitochondria, and a shift of crypt metabolism to fatty acid oxidation. Crypts from mouse and human intestinal organoids lacking EECs displayed increased intestinal stem cell activity and failed to activate phospho-S6 ribosomal protein, a marker for activity of the master metabolic regulator mammalian target of rapamycin (mTOR). These phenotypes were similar to those observed when control mice were deprived of nutrients.
CONCLUSIONS: EECs are essential regulators of crypt metabolism. Depletion of EECs recapitulated a fasting metabolic phenotype despite normal levels of ingested nutrients. These data suggest that EECs are required to relay nutritional information to the stem cell niche and are essential regulators of intestinal metabolism.

Keywords:  enteroendocrine cells; intestinal metabolism; intestinal stem cell; mitochondria

DOI:  https://doi.org/10.1016/j.jcmgh.2022.12.016
Int J Mol Sci. 2022 Dec 21. pii: 118. [Epub ahead of print]24(1):

A Comprehensive Biomarker Analysis of Microsatellite Unstable/Mismatch Repair Deficient Colorectal Cancer Cohort Treated with Immunotherapy.

Elena Élez, Núria Mulet-Margalef, Miriam Sanso, Fiorella Ruiz-Pace, Francesco M Mancuso, Raquel Comas, Javier Ros, Guillem Argilés, Giulia Martini, Enrique Sanz-Garcia, Iosune Baraibar, Francesc Salvà, Alba Noguerido, Jose Luis Cuadra-Urteaga, Roberta Fasani, Ariadna Garcia, Jose Jimenez, Susana Aguilar, Stefania Landolfi, Javier Hernández-Losa, Irene Braña, Paolo Nuciforo, Rodrigo Dienstmann, Josep Tabernero, Ramon Salazar, Ana Vivancos.

  The search for immunotherapy biomarkers in Microsatellite Instability High/Deficient Mismatch Repair system (MSI-H/dMMR) metastatic colorectal cancer (mCRC) is an unmet need. Sixteen patients with mCRC and MSI-H/dMMR (determined by either immunohistochemistry or polymerase chain reaction) treated with PD-1/PD-L1 inhibitors at our institution were included. According to whether the progression-free survival with PD-1/PD-L1 inhibitors was longer than 6 months or shorter, patients were clustered into the IT-responder group (n: 9 patients) or IT-resistant group (n: 7 patients), respectively. In order to evaluate determinants of benefit with PD-1/PD-L1 inhibitors, we performed multimodal analysis including genomics (through NGS panel tumour-only with 431 genes) and the immune microenvironment (using CD3, CD8, FOXP3 and PD-L1 antibodies). The following mutations were more frequent in IT-resistant compared with IT-responder groups: B2M (4/7 versus 2/9), CTNNB1 (2/7 versus 0/9), and biallelic PTEN (3/7 versus 1/9). Biallelic ARID1A mutations were found exclusively in the IT-responder group (4/9 patients). Tumour mutational burden did not correlate with immunotherapy benefit, neither the rate of indels in homopolymeric regions. Of note, biallelic ARID1A mutated tumours had the highest immune infiltration and PD-L1 scores, contrary to tumours with CTNNB1 mutation. Immune microenvironment analysis showed higher densities of different T cell subpopulations and PD-L1 expression in IT-responders. Misdiagnosis of MSI-H/dMMR inferred by discordances between immunohistochemistry and polymerase chain reaction was only found in the IT-resistant population (3/7 patients). Biallelic ARID1A mutations and Wnt signalling activation through CTNNB1 mutation were associated with high and low T cell immune infiltrates, respectively, and deserve special attention as determinants of response to PD-1/PD-L1 inhibitors. The non-MSI-H phenotype in dMMR is associated with poor benefit to immunotherapy. Our results suggest that mechanisms of resistance to immunotherapy are multi-factorial.

Keywords:  MSI-H/dMMR; biomarkers; colorectal cancer; immunotherapy

DOI:  https://doi.org/10.3390/ijms24010118
Biochim Biophys Acta Mol Cell Res. 2023 Jan 09. pii: S0167-4889(23)00002-2. [Epub ahead of print] 119431

Heat stress disrupts intestinal stem cell migration and differentiation along the crypt-villus axis through FAK signaling.

Geng-Xiu Zan, Ying-Chao Qin, Wen-Wen Xie, Chun-Qi Gao, Hui-Chao Yan, Xiu-Qi Wang, Jia-Yi Zhou.

  During heat stress (HS), the intestinal epithelium suffers damage due to imbalance of tissue homeostasis. However, the specific mechanism by which intestinal stem cells (ISCs) migrate and differentiate along the crypt-villus axis to heal lesions upon insult is unclear. In our study, C57BL/6 mice and IPEC-J2 cells were subjected to normal ambient conditions (25 °C for 7 days in vivo and 37 °C for 18 h in vitro) or 41 °C. The results showed that HS impaired intestinal morphology and barrier function. The numbers of ISCs (SOX9+ cells), mitotic cells (PCNA+ cells), and differentiated cells (Paneth cells marked by lysozyme, absorptive cells marked by Villin, goblet cells marked by Mucin2, enteroendocrine cells marked by Chromogranin A, and tuft cells marked by DCAMKL1) were reduced under high temperature. Importantly, BrdU incorporation confirmed the decreased migration ability of jejunal epithelial cells exposed to 41 °C. Furthermore, intestinal organoids (IOs) expanded from jejunal crypt cells in the HS group exhibited greater growth disadvantages. Mechanistically, the occurrence of these phenotypes was accompanied by FAK/paxillin/F-actin signaling disruption in the jejunum. The fact that the FAK agonist ZINC40099027 reversed the HS-triggered inhibition of IPEC-J2 cell differentiation and migration further confirmed the dominant role of FAK in response to high-temperature conditions. Overall, the present investigation is the first to reveal a major role of FAK/paxillin/F-actin signaling in HS-induced ISC migration and differentiation along the crypt-villus axis, which indicates a new therapeutic target for intestinal epithelial regeneration after heat injuries.

Keywords:  Differentiation; FAK; Heat stress; Intestinal stem cell; Migration

DOI:  https://doi.org/10.1016/j.bbamcr.2023.119431
BMC Biol. 2023 Jan 11. 21(1): 6

Lentiviral in situ targeting of stem cells in unperturbed intestinal epithelium.

George B Garside, Madeline Sandoval, Slobodan Beronja, K Lenhard Rudolph.

   BACKGROUND: Methods for the long-term in situ transduction of the unperturbed murine intestinal epithelium have not been developed in past research. Such a method could speed up functional studies and screens to identify genetic factors influencing intestinal epithelium biology. Here, we developed an efficient method achieving this long-sought goal.
RESULTS: We used ultrasound-guided microinjections to transduce the embryonic endoderm at day 8 (E8.0) in utero. The injection procedure can be completed in 20 min and had a 100% survival rate. By injecting a small volume (0.1-0.2 μl) of concentrated virus, single shRNA constructs as well as lentiviral libraries can successfully be transduced. The new method stably and reproducibly targets adult intestinal epithelium, as well as other endoderm-derived organs such as the lungs, pancreas, liver, stomach, and bladder. Postnatal analysis of young adult mice indicates that single transduced cells at E8.0 gave rise to crypt fields that were comprised of 20-30 neighbouring crypts per crypt-field at 90 days after birth. Lentiviral targeting of ApcMin/+ mutant and wildtype mice revealed that heterozygous loss of Apc function suppresses the developmental normal growth pattern of intestinal crypt fields. This suppression of crypt field sizes did not involve a reduction of the crypt number per field, indicating that heterozygous Apc loss impaired the growth of individual crypts within the fields. Lentiviral-mediated shRNA knockdown of p53 led to an approximately 20% increase of individual crypts per field in both Apc+/+ and ApcMin/+ mice, associating with an increase in crypt size in ApcMin/+ mice but a slight reduction in crypt size in Apc+/+ mice. Overall, p53 knockdown rescued the reduction in crypt field size in Apc-mutant mice but had no effect on crypt field size in wildtype mice.
CONCLUSIONS: This study develops a novel technique enabling robust and reproducible in vivo targeting of intestinal stem cells in situ in the unperturbed intestinal epithelium across different regions of the intestine. In vivo somatic gene editing and genetic screening of lentiviral libraries has the potential to speed up discoveries and mechanistic understanding of genetic pathways controlling the biology of the intestinal epithelium during development and postnatal life. The here developed method enables such approaches.

Keywords:  APC; Clonality; Crypt formation; Endoderm; Genetic engineering; Intestinal stem cells; Intestine; Microinjection; Stable in vivo targeting; Transduction

DOI:  https://doi.org/10.1186/s12915-022-01466-1
Clin Cancer Res. 2023 Jan 09. pii: CCR-22-2550. [Epub ahead of print]

Synthetic lethal interaction with BCL-XL blockade deepens response to cetuximab in patient-derived models of metastatic colorectal cancer.

Simonetta M Leto, Martina Ferri, Francesco Sassi, Eugenia R Zanella, Francesca Cottino, Valentina Vurchio, Irene Catalano, Alessandro Ferrero, Caterina C Zingaretti, Caterina Marchiò, Elena Grassi, Livio Trusolino, Andrea Bertotti.

PURPOSE: Approximately 20% of patients with RAS wild-type metastatic colorectal cancer (mCRC) experience objective responses to the anti-EGFR antibody cetuximab, but disease eradication is seldom achieved. The extent of tumor shrinkage correlates with long-term outcome. We aimed to find rational combinations that potentiate cetuximab efficacy by disrupting adaptive dependencies on anti-apoptotic molecules (BCL2, BCL-XL, MCL1).
EXPERIMENTAL DESIGN: Experiments were conducted in patient-derived xenografts (PDXs) and organoids (PDXOs). Apoptotic priming was analyzed by BH3 profiling. Pro- and anti-apoptotic protein complexes were evaluated by co-immunoprecipitation and electroluminescence sandwich assays. The effect of combination therapies was assessed by caspase activation in PDXOs and by monitoring PDX growth.
RESULTS: A population trial in 314 PDX cohorts, established from as many patients, identified 46 models (14.6%) with appreciable (>50% tumor shrinkage) but incomplete response to cetuximab. From these models, 14 PDXOs were derived. Cetuximab primed cells for apoptosis, but only concomitant blockade of BCL-XL precipitated cell death. Mechanistically, exposure to cetuximab induced upregulation of the pro-apoptotic protein BIM and its sequestration by BCL-XL. Inhibition of BCL-XL resulted in displacement of BIM, which was not buffered by MCL1 and thereby became competent to induce apoptosis. In 5 PDX models, combination of cetuximab and a selective BCL-XL inhibitor triggered apoptosis and led to more pronounced tumor regressions and longer time to relapse after treatment discontinuation than cetuximab alone.
CONCLUSIONS: In mCRC tumors that respond to cetuximab, antibody treatment confers a synthetic-lethal dependency on BCL-XL. Targeting this dependency unleashes apoptosis and increases the depth of response to cetuximab.

DOI: https://doi.org/10.1158/1078-0432.CCR-22-2550
Cell Death Differ. 2023 Jan 13.

General transcription factor TAF4 antagonizes epigenetic silencing by Polycomb to maintain intestine stem cell functions.

Susanna Säisä-Borreill, Guillaume Davidson, Thomas Kleiber, Andréa Thevenot, Elisabeth Martin, Stanislas Mondot, Hervé Blottière, Alexandra Helleux, Gabrielle Mengus, Michelina Plateroti, Isabelle Duluc, Irwin Davidson, Jean-Noel Freund.

Taf4 (TATA-box binding protein-associated factor 4) is a subunit of the general transcription factor TFIID, a component of the RNA polymerase II pre-initiation complex that interacts with tissue-specific transcription factors to regulate gene expression. Properly regulated gene expression is particularly important in the intestinal epithelium that is constantly renewed from stem cells. Tissue-specific inactivation of Taf4 in murine intestinal epithelium during embryogenesis compromised gut morphogenesis and the emergence of adult-type stem cells. In adults, Taf4 loss impacted the stem cell compartment and associated Paneth cells in the stem cell niche, epithelial turnover and differentiation of mature cells, thus exacerbating the response to inflammatory challenge. Taf4 inactivation ex vivo in enteroids prevented budding formation and maintenance and caused broad chromatin remodeling and a strong reduction in the numbers of stem and progenitor cells with a concomitant increase in an undifferentiated cell population that displayed high activity of the Ezh2 and Suz12 components of Polycomb Repressive Complex 2 (PRC2). Treatment of Taf4-mutant enteroids with a specific Ezh2 inhibitor restored buddings, cell proliferation and the stem/progenitor compartment. Taf4 loss also led to increased PRC2 activity in cells of adult crypts associated with modification of the immune/inflammatory microenvironment that potentiated Apc-driven tumorigenesis. Our results reveal a novel function of Taf4 in antagonizing PRC2-mediated repression of the stem cell gene expression program to assure normal development, homeostasis, and immune-microenvironment of the intestinal epithelium.

DOI: https://doi.org/10.1038/s41418-022-01109-6
Semin Cancer Biol. 2023 Jan 05. pii: S1044-579X(23)00001-9. [Epub ahead of print]

The complex network of transcription factors, immune checkpoint inhibitors and stemness features in colorectal cancer: A recent update.

Maysaloun Merhi, Fareed Ahmad, Nassiba Taib, Varghese Inchakalody, Shahab Uddin, Alaaeldin Shablak, Said Dermime.

  Cancer immunity is regulated by several mechanisms that include co-stimulatory and/or co-inhibitory molecules known as immune checkpoints expressed by the immune cells. In colorectal cancer (CRC), CTLA-4, LAG3, TIM-3 and PD-1 are the major co-inhibitory checkpoints involved in tumor development and progression. On the other hand, the deregulation of transcription factors and cancer stem cells activity plays a major role in the development of drug resistance and in the spread of metastatic disease in CRC. In this review, we describe how the modulation of such transcription factors affects the response of CRC to therapies. We also focus on the role of cancer stem cells in tumor metastasis and chemoresistance and discuss both preclinical and clinical approaches for targeting stem cells to prevent their tumorigenic effect. Finally, we provide an update on the clinical applications of immune checkpoint inhibitors in CRC and discuss the regulatory effects of transcription factors on the expression of the immune inhibitory checkpoints with specific focus on the PD-1 and PD-L1 molecules.

Keywords:  Cancer Stem Cells; Colorectal cancer; Immune Checkpoint Inhibitors; Transcription factors

DOI:  https://doi.org/10.1016/j.semcancer.2023.01.001
J Biol Chem. 2023 Jan 06. pii: S0021-9258(23)00008-X. [Epub ahead of print] 102876

Comprehensive analysis of RNA-binding protein SRSF2-dependent alternative splicing signature in malignant proliferation of colorectal carcinoma.

Weizhen Liu, Dongfang Li, Ting Lu, Haosheng Zhang, Zhengxin Chen, Qinli Ruan, Zihui Zheng, Linlin Chen, Jun Guo.

  Aberrant expression of the splicing factor SRSF2 can lead to tumorigenesis, but its molecular mechanism in colorectal cancer is currently unknown. Herein, we found SRSF2 to be highly expressed in human colorectal cancer (CRC) samples when compared to normal tissues. Both in vitro and in vivo, SRSF2 significantly accelerated the proliferation of colon cancer cells. Using RNA-seq, we screened and identified 33 alternative splicing (AS) events regulated by SRSF2. Knockdown of SLMAP-L or CETN3-S splice isoform could suppress the cell growth of colon cancer cells, predicting their role in malignant proliferation of colon cancer cells. Mechanistically, the in vivo CLIP assay demonstrated the direct binding of the RRM domain of the SRSF2 protein to SLMAP and CETN3 pre-mRNAs. SRSF2 activated the inclusion of SLMAP alternative exon 24 by binding to constitutive exon 25, while SRSF2 facilitated the exclusion of CETN3 alternative exon 5 by binding to neighboring exon 6. Knockdown of SRSF2, its splicing targets SLMAP-L, or CETN3-S caused colon cancer cells to arrest in the G1 phase of the cell cycle. Rescue of SLMAP-L or CETN3-S splice isoform in SRSF2 knockdown colon cancer cells could effectively reverse the inhibition of cell proliferation due to SRSF2 knockdown by mediating cell cycle progression. Importantly, the percentage of SLMAP exon 24 inclusion increased and CETN3 exon 5 inclusion decreased in CRC samples compared to paired normal samples. Collectively, our findings identify that SRSF2 dysregulates colorectal carcinoma proliferation at the molecular level of splicing regulation, and reveal potential splicing targets in CRC patients.

Keywords:  SRSF2; alternative splicing; colorectal cancer; proliferation

DOI:  https://doi.org/10.1016/j.jbc.2023.102876
Semin Cell Dev Biol. 2023 Jan 10. pii: S1084-9521(23)00006-X. [Epub ahead of print]

Gut immune cells and intestinal niche imprinting.

Claudia A Rivera, Ana-Maria Lennon-Duménil.

  The intestine comprises the largest proportion of immune cells in the body. It is continuously exposed to new antigens and immune stimuli from the diet, microbiota but also from intestinal pathogens. In this review, we describe the main populations of immune cells present along the intestine, both from the innate and adaptive immune system. We later discuss how intestinal niches significantly impact the phenotype and function of gut immune populations at steady state and upon infection.

Keywords:  Epithelial cells; Immune cells; Imprinting; Intestine; Niche; Retinoic acid

DOI:  https://doi.org/10.1016/j.semcdb.2023.01.006
STAR Protoc. 2023 Jan 12. pii: S2666-1667(22)00903-0. [Epub ahead of print]4(1): 102023

Protocol to establish a mouse model for hepatic metastasis of colorectal cancer.

Yajuan Zhang, Ming Zhao, Weiwei Yang.

  Hepatic metastasis is the leading cause of colorectal-cancer (CRC)-associated death. Here we describe an optimized protocol to establish a more clinically relevant mouse model of CRC metastasis. We detail steps for subcutaneous transplantation of luciferase-expressing CRC cells and subsequent orthotopic transplantation of subcutaneous tumor tissues. This mouse model allows CRC cells to form tumors within the intestinal tract and metastasize to the liver, thereby providing the approach to assess hepatic metastasis of CRC in vivo. For complete details on the use and execution of this protocol, please refer to Zhang et al. (2021).1.

Keywords:  Cancer; Model Organisms

DOI:  https://doi.org/10.1016/j.xpro.2022.102023
Nucleic Acids Res. 2023 Jan 10. pii: gkac1236. [Epub ahead of print]

ONECUT2 regulates RANKL-dependent enterocyte and microfold cell differentiation in the small intestine; a multi-omics study.

Maria V Luna Velez, Hannah K Neikes, Rebecca R Snabel, Yarah Quint, Chen Qian, Aniek Martens, Gert Jan C Veenstra, Michael R Freeman, Simon J van Heeringen, Michiel Vermeulen.

Microfold (M) cells reside in the intestinal epithelium of Peyer's patches (PP). Their unique ability to take up and transport antigens from the intestinal lumen to the underlying lymphoid tissue is key in the regulation of the gut-associated immune response. Here, we applied a multi-omics approach to investigate the molecular mechanisms that drive M cell differentiation in mouse small intestinal organoids. We generated a comprehensive profile of chromatin accessibility changes and transcription factor dynamics during in vitro M cell differentiation, allowing us to uncover numerous cell type-specific regulatory elements and associated transcription factors. By using single-cell RNA sequencing, we identified an enterocyte and M cell precursor population. We used our newly developed computational tool SCEPIA to link precursor cell-specific gene expression to transcription factor motif activity in cis-regulatory elements, uncovering high expression of and motif activity for the transcription factor ONECUT2. Subsequent in vitro and in vivo perturbation experiments revealed that ONECUT2 acts downstream of the RANK/RANKL signalling axis to support enterocyte differentiation, thereby restricting M cell lineage specification. This study sheds new light on the mechanism regulating cell fate balance in the PP, and it provides a powerful blueprint for investigation of cell fate switches in the intestinal epithelium.

DOI: https://doi.org/10.1093/nar/gkac1236
Cancers (Basel). 2022 Dec 26. pii: 130. [Epub ahead of print]15(1):

Transcriptomic Changes Associated with ERBB2 Overexpression in Colorectal Cancer Implicate a Potential Role of the Wnt Signaling Pathway in Tumorigenesis.

Eman A Abdul Razzaq, Khuloud Bajbouj, Amal Bouzid, Noura Alkhayyal, Rifat Hamoudi, Riyad Bendardaf.

  Colorectal cancer (CRC) remains the third most common cause of cancer mortality worldwide. Precision medicine using OMICs guided by transcriptomic profiling has improved disease diagnosis and prognosis by identifying many CRC targets. One such target that has been actively pursued is an erbb2 receptor tyrosine kinase 2 (ERBB2) (Human Epidermal Growth Factor Receptor 2 (HER2)), which is overexpressed in around 3-5% of patients with CRC worldwide. Despite targeted therapies against HER2 showing significant improvement in disease outcomes in multiple clinical trials, to date, no HER2-based treatment has been clinically approved for CRC. In this study we performed whole transcriptome ribonucleic acid (RNA) sequencing on 11 HER2+ and 3 HER2- CRC patients with advanced stages II, III and IV of the disease. In addition, transcriptomic profiling was carried out on CRC cell lines (HCT116 and HT29) and normal colon cell lines (CCD841 and CCD33), ectopically overexpressing ERBB2. Our analysis revealed transcriptomic changes involving many genes in both CRC cell lines overexpressing ERBB2 and in HER2+ patients, compared to normal colon cell lines and HER2- patients, respectively. Gene Set Enrichment Analysis indicated a role for HER2 in regulating CRC pathogenesis, with Wnt/β-catenin signaling being mediated via a HER2-dependent regulatory pathway impacting expression of the homeobox gene NK2 homeobox 5 (NKX2-5). Results from this study thus identified putative targets that are co-expressed with HER2 in CRC warranting further investigation into their role in CRC pathogenesis.

Keywords:  ERBB2; HER2; NKX2-5; RNA-seq; Wnt signaling; colorectal cancer; whole transcriptomic analysis

DOI:  https://doi.org/10.3390/cancers15010130
J Cell Sci. 2023 Jan 09. pii: jcs.260285. [Epub ahead of print]

Intravital imaging of Wnt/β-catenin and ATF2-dependent signalling pathways during tumour cell invasion and metastasis.

Konstantin Stoletov, Saray Sanchez, Irantzu Gorroño, Miriam Rabano, Maria Vivanco, Robert Kypta, John D Lewis.

  Wnt signalling is implicated as a driver of tumour cell metastasis, but less is known about which branches of Wnt signalling are involved and when they act in the metastatic cascade. Using a unique intravital imaging platform and fluorescent reporters, we visualised Wnt/β-catenin/TCF/LEF-dependent and ATF2-dependent signalling activities during human cancer cell invasion, intravasation and metastatic lesion formation in the chick embryo host. We found that cancer cells readily shifted between states of low and high canonical Wnt activity. Cancer cells that displayed low Wnt canonical activity showed higher invasion and intravasation potential in primary tumours and in metastatic lesions. In contrast, cancer cells showing low ATF2-dependent activity were significantly less invasive both at the front of primary tumours and in metastatic lesions. Simultaneous visualisation of both these reporters using a double reporter cell line confirmed their complementary activities in primary tumours and metastatic lesions. These findings may inform the development of therapies that target different branches of Wnt signalling at specific stages of metastasis.

Keywords:   in vivo imaging; Cancer metastasis; Wnt

DOI:  https://doi.org/10.1242/jcs.260285
STAR Protoc. 2023 Jan 12. pii: S2666-1667(22)00902-9. [Epub ahead of print]4(1): 102022

Optimized protocol for the generation of an orthotopic colon cancer mouse model and metastasis.

Sophie Richon, Olivier Zajac, Carlos Perez Gonzalez, Danijela Matic Vignjevic.

  The microenvironment plays an essential role in tumor development and metastatic progression. Here, we describe a simple and rapid protocol to generate tumors in mice using colon cancer cell lines or tumoroids in the correct microenvironment, colonic mucosa. We also detail steps for monitoring the growth of the primary tumor in real time using colonoscopy or in vivo imaging system, as well as monitoring metastasis development. Finally, we describe tissue collection and sample preparation for subsequent immunohistochemistry analysis.

Keywords:  Cancer; Cell Biology; Health Sciences; Microscopy; Model Organisms; Organoids

DOI:  https://doi.org/10.1016/j.xpro.2022.102022
Immunol Lett. 2023 Jan 07. pii: S0165-2478(23)00003-2. [Epub ahead of print]253 41-53

The intestinal microenvironment shapes macrophage and dendritic cell identity and function.

Alessandra A Filardy, Jesuino R M Ferreira, Rafael M Rezende, Brian L Kelsall, Rafael P Oliveira.

  The gut comprises the largest body interface with the environment and is continuously exposed to nutrients, food antigens, and commensal microbes, as well as to harmful pathogens. Subsets of both macrophages and dendritic cells (DCs) are present throughout the intestinal tract, where they primarily inhabit the gut-associate lymphoid tissue (GALT), such as Peyer's patches and isolated lymphoid follicles. In addition to their role in taking up and presenting antigens, macrophages and DCs possess extensive functional plasticity and these cells play complementary roles in maintaining immune homeostasis in the gut by preventing aberrant immune responses to harmless antigens and microbes and by promoting host defense against pathogens. The ability of macrophages and DCs to induce either inflammation or tolerance is partially lineage imprinted, but can also be dictated by their activation state, which in turn is determined by their specific microenvironment. These cells express several surface and intracellular receptors that detect danger signals, nutrients, and hormones, which can affect their activation state. DCs and macrophages play a fundamental role in regulating T cells and their effector functions. Thus, modulation of intestinal mucosa immunity by targeting antigen presenting cells can provide a promising approach for controlling pathological inflammation. In this review, we provide an overview on the characteristics, functions, and origins of intestinal macrophages and DCs, highlighting the intestinal microenvironmental factors that influence their functions during homeostasis. Unraveling the mechanisms by which macrophages and DCs regulate intestinal immunity will deepen our understanding on how the immune system integrates endogenous and exogenous signals in order to maintain the host's homeostasis.

Keywords:  Colon; Diet-derived antigens; Environmental immunomodulators; Microbiota; Small intestine

DOI:  https://doi.org/10.1016/j.imlet.2023.01.003
Nat Commun. 2023 Jan 07. 14(1): 110

Integrated clinical and genomic analysis identifies driver events and molecular evolution of colitis-associated cancers.

Walid K Chatila, Henry Walch, Jaclyn F Hechtman, Sydney M Moyer, Valeria Sgambati, David M Faleck, Amitabh Srivastava, Laura Tang, Jamal Benhamida, Dorina Ismailgeci, Carl Campos, Fan Wu, Qing Chang, Efsevia Vakiani, Elisa de Stanchina, Martin R Weiser, Maria Widmar, Rhonda K Yantiss, Manish A Shah, Adam J Bass, Zsofia K Stadler, Lior H Katz, Ingo K Mellinghoff, Nilay S Sethi, Nikolaus Schultz, Karuna Ganesh, David Kelsen, Rona Yaeger.

Inflammation has long been recognized to contribute to cancer development, particularly across the gastrointestinal tract. Patients with inflammatory bowel disease have an increased risk for bowel cancers, and it has been posited that a field of genetic changes may underlie this risk. Here, we define the clinical features, genomic landscape, and germline alterations in 174 patients with colitis-associated cancers and sequenced 29 synchronous or isolated dysplasia. TP53 alterations, an early and highly recurrent event in colitis-associated cancers, occur in half of dysplasia, largely as convergent evolution of independent events. Wnt pathway alterations are infrequent, and our data suggest transcriptional rewiring away from Wnt. Sequencing of multiple dysplasia/cancer lesions from mouse models and patients demonstrates rare shared alterations between lesions. These findings suggest neoplastic bowel lesions developing in a background of inflammation experience lineage plasticity away from Wnt activation early during tumorigenesis and largely occur as genetically independent events.

DOI: https://doi.org/10.1038/s41467-022-35592-9
Nat Med. 2023 Jan 09.

Multistain deep learning for prediction of prognosis and therapy response in colorectal cancer.

Sebastian Foersch, Christina Glasner, Ann-Christin Woerl, Markus Eckstein, Daniel-Christoph Wagner, Stefan Schulz, Franziska Kellers, Aurélie Fernandez, Konstantina Tserea, Michael Kloth, Arndt Hartmann, Achim Heintz, Wilko Weichert, Wilfried Roth, Carol Geppert, Jakob Nikolas Kather, Moritz Jesinghaus.

Although it has long been known that the immune cell composition has a strong prognostic and predictive value in colorectal cancer (CRC), scoring systems such as the immunoscore (IS) or quantification of intraepithelial lymphocytes are only slowly being adopted into clinical routine use and have their limitations. To address this we established and evaluated a multistain deep learning model (MSDLM) utilizing artificial intelligence (AI) to determine the AImmunoscore (AIS) in more than 1,000 patients with CRC. Our model had high prognostic capabilities and outperformed other clinical, molecular and immune cell-based parameters. It could also be used to predict the response to neoadjuvant therapy in patients with rectal cancer. Using an explainable AI approach, we confirmed that the MSDLM's decisions were based on established cellular patterns of anti-tumor immunity. Hence, the AIS could provide clinicians with a valuable decision-making tool based on the tumor immune microenvironment.

DOI: https://doi.org/10.1038/s41591-022-02134-1
BMC Gastroenterol. 2023 Jan 07. 23(1): 4

SPOCK1 and POSTN are valuable prognostic biomarkers and correlate with tumor immune infiltrates in colorectal cancer.

Caiqin Gan, Mengting Li, Yuanyuan Lu, Ganjing Peng, Wenjie Li, Haizhou Wang, Yanan Peng, Qian Hu, Wanhui Wei, Fan Wang, Lan Liu, Qiu Zhao.

   BACKGROUND: Immune cells and stromal cells in the tumor microenvironment play a vital role in the progression of colorectal cancer (CRC). The study aimed to screen valuable prognostic biomarkers in CRC based on stromal and immune scores.
METHOD: The ESTIMATE algorithm was used to calculate the immune and stromal scores of CRC samples in TCGA. Then samples were divided into high and low score groups based on the median value of the scores. Differentially expressed genes (DEGs) associated with immune and stromal scores were screened. WGCNA and univariate COX regression analysis were performed to further identify key prognostic genes. Analysis of scRNA-seq for CRC was used for verifying the main source of the key genes. The prognostic value of they was validated based on The Gene Expression Profiling Interactive Analysis and GSE17536 dataset. TIMER and CIBERSORT algorithms were applied to analyze the correlations among key genes and tumor-infiltrating immune cells. Several pairs of colon cancer tissue were used to be proven.
RESULT: 1314 upregulated and 4 downregulated genes were identified, which were significantly enriched in immune-related biological processes and pathways. Among these DEGs, SPOCK1 and POSTN were identified as key prognostic genes and mainly expressed in cancer-associated fibroblasts for CRC. High expression of SPCOK1 and POSTN was associated with advanced clinical stage, T stage, N stage, and poor prognosis of CRC. The results from CIBERSORT and TIMER revealed that SPOCK1 and POSTN were associated with tumor-infiltrating immune cells, especially macrophages and neutrophils. Meanwhile, in several pairs of human colorectal tissue samples, SPOK1 and POSTN were found to be significantly overexpressed in colorectal tissue compared with para-cancer tissue, and macrophage surface markers CD68 (co-expressed by M1 and M2 macrophages) and CD206 (M2-specific macrophage expression) were also overexpressed in cancer tissue. Besides, SPOCK1 and POSTN expression were positively correlated with the expression of immune checkpoints.
CONCLUSION: Collectively, our results indicate that SPOCK1 and POSTN associated with CAF may be novel prognostic biomarkers in CRC and correlate with immune infiltrates.

Keywords:  Cancer-associated fibroblasts; Colorectal cancer; POSTN; SPOCK1; Tumor microenvironment; Tumor-infiltrating immune cells

DOI:  https://doi.org/10.1186/s12876-022-02621-2
Sci Adv. 2023 Jan 06. 9(1): eade9120

Arginine limitation drives a directed codon-dependent DNA sequence evolution response in colorectal cancer cells.

Dennis J Hsu, Jenny Gao, Norihiro Yamaguchi, Alexandra Pinzaru, Qiushuang Wu, Nandan Mandayam, Maria Liberti, Søren Heissel, Hanan Alwaseem, Saeed Tavazoie, Sohail F Tavazoie.

Utilization of specific codons varies between organisms. Cancer represents a model for understanding DNA sequence evolution and could reveal causal factors underlying codon evolution. We found that across human cancer, arginine codons are frequently mutated to other codons. Moreover, arginine limitation-a feature of tumor microenvironments-is sufficient to induce arginine codon-switching mutations in human colon cancer cells. Such DNA codon switching events encode mutant proteins with arginine residue substitutions. Mechanistically, arginine limitation caused rapid reduction of arginine transfer RNAs and the stalling of ribosomes over arginine codons. Such selective pressure against arginine codon translation induced an adaptive proteomic shift toward low-arginine codon-containing genes, including specific amino acid transporters, and caused mutational evolution away from arginine codons-reducing translational bottlenecks that occurred during arginine starvation. Thus, environmental availability of a specific amino acid can influence DNA sequence evolution away from its cognate codons and generate altered proteins.

DOI: https://doi.org/10.1126/sciadv.ade9120
Cancer Med. 2023 Jan 11.

An immune, stroma, and epithelial-mesenchymal transition-related signature for predicting recurrence and chemotherapy benefit in stage II-III colorectal cancer.

Du Cai, Wei Wang, Min-Er Zhong, Dejun Fan, Xuanhui Liu, Cheng-Hang Li, Ze-Ping Huang, Qiqi Zhu, Min-Yi Lv, Chuling Hu, Xin Duan, Xiao-Jian Wu, Feng Gao.

   BACKGROUND: Debates exist on the treatment decision of the stage II/III colorectal cancer (CRC) due to the insufficiency of the current TNM stage-based risk stratification system. Epithelial-mesenchymal transition (EMT) and tumor microenvironment (TME) have both been linked to CRC progression in recent studies. We propose to improve the prognosis prediction of CRC by integrating TME and EMT.
METHODS: In total, 2382 CRC patients from seven datasets and one in-house cohort were collected, and 1640 stage II/III CRC patients with complete survival information and gene expression profiles were retained and divided into a training cohort and three independent validation cohorts. Integrated analysis of 398 immune, stroma, and epithelial-mesenchymal transition (ISE)-related genes identified an ISE signature independently associated with the recurrence of CRC. The underlying biological mechanism of the ISE signature and its influence on adjuvant chemotherapy was further explored.
RESULTS: We constructed a 26-gene signature which was significantly associated with poor outcome in Training cohort (p < 0.001, HR [95%CI] = 4.42 [3.25-6.01]) and three independent validation cohorts (Validation cohort-1: p < 0.01, HR [95%CI] = 1.70 [1.15-2.51]; Validation cohort-2: p < 0.001, HR [95% CI] = 2.30 [1.67-3.16]; Validation cohort-3: p < 0.01, HR [95% CI] = 2.42 [1.25-4.70]). After adjusting for known clinicopathological factors, multivariate cox analysis confirmed the ISE signature's independent prognostic value. Subgroup analysis found that stage III patients with low ISE score might benefit from adjuvant chemotherapy (p < 0.001, HR [95%CI] = 0.15 [0.04-0.55]). Hypergeometric test and enrichment analysis revealed that low-risk group was enriched in thr immune pathway while high-risk group was associated with the EMT pathway and CMS4 subtype.
CONCLUSION: We proposed an ISE signature for robustly predicting the recurrence of stage II/III CRC and help treatment decision by identifying patients who will not benefit from current standard treatment.

Keywords:  colorectal cancer; epithelial-mesenchymal transition; immune; prognosis; stroma

DOI:  https://doi.org/10.1002/cam4.5534