bims-instec Biomed News
on Intestinal stem cells and chemoresistance in colon cancer and intestinal regeneration
Issue of 2022–05–15
nineteen papers selected by
Maria-Virginia Giolito, IRFAC/UMR-S1113 INSERM
  1. Autophagic State Confers Facultative Stem Cell Capacity in The Intestinal Epithelium.
  2. The ErbB3 Receptor Restricts Bmi1 to Regulate Paneth Cells.
  3. Refining colorectal cancer classification and clinical stratification through a single-cell atlas.
  4. The long noncoding RNA uc.230/CUG-binding protein 1 axis sustains intestinal epithelial homeostasis and response to tissue injury.
  5. Transcriptional integration of distinct microbial and nutritional signals by the small intestinal epithelium.
  6. Autophagy in PDGFRα+ mesenchymal cells is essential for intestinal stem cell survival.
  7. Colorectal Cancer Growth Is Reduced by the Ketone Body β-Hydroxybutyrate.
  8. N6-Methyladenosine (m6 A) Modifies Regenerative Transcripts in the Intestinal Epithelium.
  9. Paracrine signalling between intestinal epithelial and tumour cells induces a regenerative programme.
  10. Functional MYO5B Loss Alters Lipid Metabolic Pathways in Intestinal Progenitor Cells.
  11. 5-fluorouracil treatment of patient-derived scaffolds from colorectal cancer reveal clinically critical information.
  12. A permeability assay for mouse intestinal organoids.
  13. Variability exposes hidden buffering in the network specifying intestinal cell fate.
  14. Signaling Pathways Associated with miR-106b mediated Radioresistance in the Treatment of Colorectal Cancer.
  15. Biomarkers of Response and Resistance to Immunotherapy in Microsatellite Stable Colorectal Cancer: Toward a New Personalized Medicine.
  16. Overcoming Therapy Resistance in Colon Cancer by Drug Repurposing.
  17. P-cadherin regulates intestinal epithelial repair, but is dispensable for colitis associated colon cancer development.
  18. P-Cadherin Regulates Intestinal Epithelial Cell Migration and Mucosal Repair, but Is Dispensable for Colitis Associated Colon Cancer.
  19. Consensus molecular subtype differences linking colon adenocarcinoma and obesity revealed by a cohort transcriptomic analysis.
  1. FASEB J. 2022 May;36 Suppl 1
    Autophagic State Confers Facultative Stem Cell Capacity in The Intestinal Epithelium.
    Louis R Parham, Nicolette Johnson, Jeeyoon Na, Alena Klochkova, Patrick Williams, Ning Li, Kelly A Whelan, Christopher J Lengner, Kathryn E Hamilton.
       BACKGROUND: The intestinal epithelium is a highly proliferative tissue that undergoes complete turnover in 3 to 5 days. The presence of intestinal stem cells expressing the canonical Wnt target gene Lgr5, known as active intestinal stem cells (a-ISCs), are responsible for generating daughter cells that differentiate into specialized epithelial cells to carryout various functions in the intestine. The proliferative nature of the intestinal epithelium renders it susceptible to DNA damage-inducing injury, such as high-dose irradiation and chemotherapy. Although a-ISCs and early daughter cells are killed by these insults, a radio-resistant population of facultative intestinal stem cells (f-ISCs) can regenerate lost a-ISCs to restore homeostasis following injury. The functional characteristics of f-ISCs are becoming increasingly understood, however, whether any epithelial cell can act as an f-ISC is less clear. One reason for this ambiguity is a heavy reliance on Cre-driven reporter mouse models with low recombination efficiency. Our objective was to find a functional marker to identify f-ISCs based on cellular state rather than gene expression. The autophagy pathway has been shown to protect against DNA damage and irradiation-induced apoptosis in the intestinal epithelium. Furthermore, recent studies demonstrate a requirement for the autophagy pathway during cellular de-differentiation in both gastric chief and pancreatic acinar cells following metaplasia-inducing injury. Given that autophagy plays roles in both radio-resistance and cellular plasticity, two integral features of f-ISCs, we hypothesized that autophagic activity could serve to identify cells with f-ISC capacity.
    METHODS AND RESULTS: Using the autophagic vesicle tracer dye CytoID combined with fluorescence-activated cell sorting, we demonstrate that intestinal epithelial cells with high levels of CytoID exhibit increased organoid-formation efficiency (OFE) compared to CytoID 'low' cells. Single cell sequencing reveals that the CytoID high population is largely composed of secretory cells including Paneth, Goblet, Tuft, and Enteroendocrine cells, whereas the CytoID low population consisted mainly of a-ISCs and absorptive Enterocytes. Using reporter mice and antibodies against cell surface receptors, we observe that CytoID can identify cells with high OFE within Enteroendocrine, Paneth, and Goblet cell lineages. Finally, we demonstrate that autophagy is required for the enhanced organoid-formation observed in CytoID high cells by plating these cells in the presence of the lysosomal inhibitor Bafilomycin A1.
    CONCLUSIONS: Our new data support the notion that epithelial cells in a high autophagic state are biased to secretory lineages and that autophagy status functions as a lineage agnostic marker of f-ISCs. Furthermore, our data suggests that autophagy is required for organoid formation in these lineages.
    DOI:  https://doi.org/10.1096/fasebj.2022.36.S1.R3062
  2. FASEB J. 2022 May;36 Suppl 1
    The ErbB3 Receptor Restricts Bmi1 to Regulate Paneth Cells.
    Stephanie J King, Jonathan Hsieh, Dana Almohazey, Mark R Frey.
      ErbB3 is a RTK family member known to regulate proliferation and growth in the intestinal epithelium. Previous work by our lab determined that targeted Erbb3 deletion from the intestinal epithelium resulted in precocious Paneth cell development and an expanded population in mice. This was associated with increased expression of intestinal stem cell (ISC) markers. Conversely, ileal enteroids treated with the ErbB3 ligand neuregulin (NRG)-1β had significantly reduced expression of ISC markers, including Lgr5 and Bmi1. These data suggest that ErbB3 restricts secretory cell differentiation and stemness in the intestinal crypt. However, the underlying mechanisms are largely unknown. BMI1 is a proto-oncogene and member of the PRC complex. Bmi1+ intestinal epithelial cells represent a slow-cycling ISC population that can regenerate the epithelium following injury and loss of Lgr5+ ISCs. Recent evidence determined that Bmi1+ ISCs are enriched in enteroendocrine markers and can give rise to goblet cells, suggesting BMI1 may be critical for differentiation of secretory lineages. We hypothesized that ErbB3 restricts secretory cell differentiation through regulation of BMI1.
    AIMS: To define ErbB3-dependent regulation of Bmi1 and determine if ErbB3 restriction of Paneth cell development and differentiation is through suppression of Bmi1.
    METHODS: Mucosal scrapings were collected from Vil-Cre;Erbb3flox/flox mice and Erbb3flox/flox littermate controls for protein and gene expression analysis. Ileal enteroids and HT-29 colorectal adenocarcinoma cells were treated with vehicle DMSO, the PI3K inhibitor LY294002 (10 μM), the MEK inhibitor U0126 (5 μM), the BMI1 inhibitor PTC-209 (1 μM), and/or recombinant NRG-1β (10 ng/mL). Enteroids and cell monolayers were harvested and prepared for gene expression analysis. RT-qPCR for Bmi1 and Lyz1 was performed relative to Hprt as a loading control. Statistical analysis by one-way ANOVA was conducted using Prism 9.
    RESULTS: Consistent with previous data, BMI1 protein and gene expression were significantly increased in small and large intestinal mucosal scrapings from Vil-Cre;Erbb3flox/flox mice versus littermate controls. To determine if Bmi1 expression is sensitive to PI3K/Akt or MAPK signaling downstream of ErbB3, HT-29 cells were treated with inhibitors followed by NRG-1β after 1 hour, then harvested 24 hours later. PI3K and MAPK inhibition significantly increased Bmi1 expression. NRG-1β treatment was able to overcome MAPK inhibition to reduce BMI1 expression. Similarly, in ileal enteroids, PI3K and MAPK blockade increased Bmi1 expression and NRG-1β treatment reversed this effect. To determine whether BMI1 regulates expression of the Paneth cell marker LYZ1, HT-29 cells were treated with the BMI1 inhibitor PTC-209. BMI1 inhibition decreased LYZ1 expression.
    CONCLUSIONS: Our data demonstrate that ErbB3 regulates Bmi1 expression through PI3K/Akt and MAPK signaling in both human and mouse intestinal cells. Furthermore, BMI1 activity promotes LYZ1 expression. Together, these results support our hypothesis that ErbB3 regulates secretory cell differentiation through BMI1 in the intestinal epithelium.
    DOI:  https://doi.org/10.1096/fasebj.2022.36.S1.R4075
  3. Genome Biol. 2022 May 11. 23(1): 113
    Refining colorectal cancer classification and clinical stratification through a single-cell atlas.
    Ateeq M Khaliq, Cihat Erdogan, Zeyneb Kurt, Sultan Sevgi Turgut, Miles W Grunvald, Tim Rand, Sonal Khare, Jeffrey A Borgia, Dana M Hayden, Sam G Pappas, Henry R Govekar, Audrey E Kam, Jochen Reiser, Kiran Turaga, Milan Radovich, Yong Zang, Yingjie Qiu, Yunlong Liu, Melissa L Fishel, Anita Turk, Vineet Gupta, Ram Al-Sabti, Janakiraman Subramanian, Timothy M Kuzel, Anguraj Sadanandam, Levi Waldron, Arif Hussain, Mohammad Saleem, Bassel El-Rayes, Ameen A Salahudeen, Ashiq Masood.
       BACKGROUND: Colorectal cancer (CRC) consensus molecular subtypes (CMS) have different immunological, stromal cell, and clinicopathological characteristics. Single-cell characterization of CMS subtype tumor microenvironments is required to elucidate mechanisms of tumor and stroma cell contributions to pathogenesis which may advance subtype-specific therapeutic development. We interrogate racially diverse human CRC samples and analyze multiple independent external cohorts for a total of 487,829 single cells enabling high-resolution depiction of the cellular diversity and heterogeneity within the tumor and microenvironmental cells.
    RESULTS: Tumor cells recapitulate individual CMS subgroups yet exhibit significant intratumoral CMS heterogeneity. Both CMS1 microsatellite instability (MSI-H) CRCs and microsatellite stable (MSS) CRC demonstrate similar pathway activations at the tumor epithelial level. However, CD8+ cytotoxic T cell phenotype infiltration in MSI-H CRCs may explain why these tumors respond to immune checkpoint inhibitors. Cellular transcriptomic profiles in CRC exist in a tumor immune stromal continuum in contrast to discrete subtypes proposed by studies utilizing bulk transcriptomics. We note a dichotomy in tumor microenvironments across CMS subgroups exists by which patients with high cancer-associated fibroblasts (CAFs) and C1Q+TAM content exhibit poor outcomes, providing a higher level of personalization and precision than would distinct subtypes. Additionally, we discover CAF subtypes known to be associated with immunotherapy resistance.
    CONCLUSIONS: Distinct CAFs and C1Q+ TAMs are sufficient to explain CMS predictive ability and a simpler signature based on these cellular phenotypes could stratify CRC patient prognosis with greater precision. Therapeutically targeting specific CAF subtypes and C1Q + TAMs may promote immunotherapy responses in CRC patients.
    Keywords:  CMS classification; Cancer-associated fibroblast; Colorectal cancer; Immunotherapy; Single-cell analysis; Stromal signatures
    DOI:  https://doi.org/10.1186/s13059-022-02677-z
  4. FASEB J. 2022 May;36 Suppl 1
    The long noncoding RNA uc.230/CUG-binding protein 1 axis sustains intestinal epithelial homeostasis and response to tissue injury.
    Tingxi Yu, Sudhakar Kalakonda, Xiangzheng Liu, Naomi Han, Hee K Chung, Lan Xiao, Rao N Jaladanki, Tong-Chuan He, Jean-Pierre Raufman, Jian-Ying Wang.
       BACKGROUND & AIMS: The mammalian intestinal epithelium is colonized by complex microbiota and exposed to a wide variety of luminal noxious substances. Intestinal epithelial integrity is commonly disrupted in patients with critical disorders, but the exact underlying mechanisms are unclear. Long noncoding RNAs transcribed from ultraconserved regions (T-UCRs) control different cell functions and are involved in gut pathologies. Here, we investigated the role of T-UCRs in intestinal epithelial homeostasis and identified T-UCR uc.230 as a major regulator of epithelial renewal, apoptosis, and barrier function.
    METHODS: Intestinal mucosal tissues were collected from mice following dextran sodium sulfate (DSS) treatment or fasting, and from patients with ulcerative colitis and Crohn's disease. We isolated primary enterocytes from the small intestine of mice and developed intestinal organoids. Levels of uc.230 were silenced in intestinal epithelial cells (IECs) and organoids by transfection with small interfering RNAs or elevated using a plasmid vector that overexpressed uc.230. Association of uc.230 with miR-503 was determined by biotinylated RNA pulldown assays.
    RESULTS: Compared with controls, intestinal mucosal tissues from mice with DSS-induced colitis or fasted for 48 h and from patients with ulcerative colitis had increased levels of uc.230. The uc.230 content in Crohn's disease tissues was the same as in control tissue. Silencing uc.230 inhibited the growth of IECs and intestinal organoids and resulted in epithelial barrier dysfunction. Silencing uc.230 also increased IEC vulnerability to apoptotic cell death, whereas increasing uc.230 levels protected IECs against apoptosis. Mechanistic studies revealed that uc.230 increased CUG-binding protein 1 (CUGBP1) by acting as a natural decoy RNA for miR-503, which interacts with Cugbp1 mRNA and represses its translation.
    CONCLUSIONS: Our results indicate that uc.230 sustains intestinal mucosal homeostasis by promoting epithelial renewal and barrier function and protects IECs against apoptosis at least in part by serving as a natural sponge for miR-503, thereby preserving CUGBP1 expression.
    DOI:  https://doi.org/10.1096/fasebj.2022.36.S1.0R837
  5. Cell Mol Gastroenterol Hepatol. 2022 May 06. pii: S2352-345X(22)00081-9. [Epub ahead of print]
    Transcriptional integration of distinct microbial and nutritional signals by the small intestinal epithelium.
    Colin R Lickwar, James M Davison, Cecelia Kelly, Gilberto Padilla Mercado, Jia Wen, Briana R Davis, Matthew C Tillman, Ivana Semova, Sarah F Andres, Goncalo Vale, Jeffrey G McDonald, John F Rawls.
       BACKGROUND & AIMS: The intestine constantly interprets and adapts to complex combinations of dietary and microbial stimuli. However, the transcriptional strategies by which the intestinal epithelium integrates these coincident sources of information remain unresolved. We recently found that microbiota colonization suppresses epithelial activity of HNF4 nuclear receptor transcription factors, but their integrative regulation was unknown.
    METHODS: We compared adult mice reared germ-free (GF) or conventionalized with a microbiota (CV) either fed normally or after a single high-fat meal (HFM). Preparations of unsorted jejunal intestinal epithelial cells (IEC) were queried using lipidomics and genomewide assays for RNA-seq, and ChIP-seq for the activating histone mark H3K27ac and HNF4A.
    RESULTS: Analysis of lipid classes, genes, and regulatory regions identified distinct nutritional and microbial responses, but also simultaneous influence of both stimuli. H3K27ac sites preferentially increased by HFM in the presence of microbes neighbor lipid anabolism and proliferation genes, were previously identified intestinal stem cell (ISC) regulatory regions, and were not HNF4A targets. In contrast, H3K27ac sites preferentially increased by HFM in the absence of microbes neighbor targets of the energy homeostasis regulator PPARA, neighbored fatty acid oxidation (FAO) genes, were previously identified enterocyte regulatory regions, and were HNF4A bound.
    CONCLUSIONS: HNF4A supports a differentiated enterocyte and FAO program in GF mice, and that suppression of HNF4A by the combination of microbes and HFM may result in preferential activation of IEC proliferation programs. This identifies potential transcriptional mechanisms for intestinal adaptation to multiple signals and how microbiota may modulate intestinal lipid absorption, epithelial cell renewal and systemic energy balance.
    Keywords:  Chromatin; Intestine; Lipid Metabolism; Microbiome
    DOI:  https://doi.org/10.1016/j.jcmgh.2022.04.013
  6. Proc Natl Acad Sci U S A. 2022 May 24. 119(21): e2202016119
    Autophagy in PDGFRα+ mesenchymal cells is essential for intestinal stem cell survival.
    Yang Yang, Maria Gomez, Timothy Marsh, Laura Poillet-Perez, Akshada Sawant, Lei Chen, Noel R Park, S RaElle Jackson, Zhixian Hu, Noa Alon, Chen Liu, Jayanta Debnath, Jun-Lin Guan, Shawn Davidson, Michael Verzi, Eileen White.
      SignificanceAutophagy defects are a risk factor for inflammatory bowel diseases (IBDs), but the mechanism remains unknown. We show here that conditional whole-body deletion of Atg5 or Fip200, but not Atg7, is lethal due to loss of ileum stem cells and barrier function likely caused by different kinetics of autophagy loss, which was rescued by slow deletion. Specific autophagy loss in PDGFRα+ mesenchymal cells (PMCs) resulted in loss of Wnt signaling responsible for failed stem cell renewal. We also observed depletion of aspartate and nucleotides throughout the ileum. Our results illustrate that autophagy is required for PMC metabolism and survival necessary to sustain intestinal stem cells and mouse survival, and failure to maintain PMCs through autophagy contributes to IBD.
    Keywords:  IBD; autophagy; intestine; metabolism; stem cells
    DOI:  https://doi.org/10.1073/pnas.2202016119
  7. Cancer Discov. 2022 May 13. OF1
    Colorectal Cancer Growth Is Reduced by the Ketone Body β-Hydroxybutyrate.
      The ketone body β-hydroxybutyrate (BHB) mediates the growth inhibitory effects of the ketogenic diet (KD).
    DOI:  https://doi.org/10.1158/2159-8290.CD-RW2022-084
  8. FASEB J. 2022 May;36 Suppl 1
    N6-Methyladenosine (m6 A) Modifies Regenerative Transcripts in the Intestinal Epithelium.
    Charles H Danan, Katharina E Hayer, Lauren A Simon, Matthew D Weitzman, Kathryn E Hamilton.
       BACKGROUND: The intestinal epithelium forms a critical barrier between foreign antigens in the intestinal lumen and immune cells in the underlying mucosa. This barrier is compromised in inflammatory bowel disease (IBD), resulting in inflammation and epithelial cell death. Improved understanding of intestinal epithelial regeneration would identify novel therapeutic targets to enhance epithelial barrier repair in diseases such as IBD. After injury, tissues such as the blood, skeletal muscle, and central nervous system activate a regenerative program that is partially regulated by N6-methyladeonsine (m6 A) modification of RNA. However, few data exist regarding the role of m6 A within the intestinal epithelium. The goal of this study was to profile m6 A-modification transcriptome-wide in the regenerating intestinal epithelium. We hypothesize that after injury, m6 A promotes regeneration within the intestinal epithelium by modifying transcripts important in epithelial repair.
    METHODS: Wildtype mice were given dextran sodium sulfate (DSS) in drinking water to induce intestinal inflammation and epithelial cell loss. Control mice were provided untreated water. DSS was followed by a two-week washout period to allow for regenerating epithelium to appear adjacent to areas of epithelial ulceration. After DSS washout, regenerating intestinal epithelium was isolated by flow cytometry, RNA was extracted, and m6 A-modified transcripts were immunoprecipitated and sequenced (m6 A-seq).
    RESULTS: m6 A-seq yielded ~5500 total m6 A-modified sites within the regenerating intestinal epithelium. This corresponded to 314 m6 A-modified mRNAs that were unique to the regenerating as compared to the homeostatic epithelium. These unique transcripts were enriched for pathways involved in RNA transcription and processing, cell-cell junctions, and Hippo pathway signaling, all of which have previously been implicated in intestinal epithelial regeneration.
    CONCLUSIONS: . Our data support the hypothesis that m6 A modifies many transcripts with known roles in intestinal epithelial regeneration. Ongoing studies are evaluating how m6 A modification contributes to regulation of these transcripts and whether m6 A identifies transcripts with previously unknown roles in regeneration. We are also evaluating whether m6 A is required for optimal regeneration following epithelial injury.
    DOI:  https://doi.org/10.1096/fasebj.2022.36.S1.R2812
  9. Elife. 2022 May 11. pii: e76541. [Epub ahead of print]11
    Paracrine signalling between intestinal epithelial and tumour cells induces a regenerative programme.
    Guillaume Jacquemin, Annabelle Wurmser, Mathilde Huyghe, Wenjie Sun, Zeinab Homayed, Candice Merle, Meghan Perkins, Fairouz Qasrawi, Sophie Richon, Florent Dingli, Guillaume Arras, Damarys Loew, Danijela Vignjevic, Julie Pannequin, Silvia Fre.
      Tumours are complex ecosystems composed of different types of cells that communicate and influence each other. While the critical role of stromal cells in affecting tumour growth is well established, the impact of mutant cancer cells on healthy surrounding tissues remains poorly defined. Here, using mouse intestinal organoids, we uncover a paracrine mechanism by which intestinal cancer cells reactivate foetal and regenerative YAP-associated transcriptional programmes in neighbouring wildtype epithelial cells, rendering them adapted to thrive in the tumour context. We identify the glycoprotein thrombospondin-1 (THBS1) as the essential factor that mediates non-cell-autonomous morphological and transcriptional responses. Importantly, Thbs1 is associated with bad prognosis in several human cancers. This study reveals the THBS1-YAP axis as the mechanistic link mediating paracrine interactions between epithelial cells in intestinal tumours.
    Keywords:  YAP signalling; cancer biology; colon cancer; mouse; organoids
    DOI:  https://doi.org/10.7554/eLife.76541
  10. FASEB J. 2022 May;36 Suppl 1
    Functional MYO5B Loss Alters Lipid Metabolic Pathways in Intestinal Progenitor Cells.
    Izumi Kaji.
       BACKGROUND AND AIMS: Differentiation of intestinal epithelial cells involves cell division inhibition, cell lineage choice, and brush border elaboration. Myosin Vb (MYO5B) is a motor protein that is critical for cell polarization and membrane protein trafficking in intestinal epithelial cells and its deficiency causes both hyperproliferation and microvillus defects. Recently, we reported that a bioactive phospholipid, lysophosphatidic acid (LPA), can promote cell differentiation and nutrient absorption in tamoxifen-induced MYO5B knockout mice in vivo and in enteroids. Our RNA-sequencing data with KEGG pathway and GO enrichment analysis indicated that MYO5B loss disrupts stem cell characteristics, cell lineage differentiation, and energy metabolic pathways. We hypothesize that the alteration of metabolic pathways in progenitor cells causes the disruption of stem cell function.
    METHODS: Adult Villin-CreERT2 ;Myo5bflox/flox or littermate control (Myo5bflox/flox ) mice received a single dose of tamoxifen and the intestinal tissues were analyzed 4 days after the tamoxifen injection. Fresh frozen sections of jejunum were analyzed by imaging mass spectrometry (IMS). Immunostaining for key metabolic enzymes were utilized to support the RNA-seq and IMS observations.
    RESULTS: Using RNA-sequencing, MYO5B deficient epithelial cells showed significant decreases in transcripts for enzymes that mediate fatty acid ß-oxidation compared to control mouse jejunum. Instead, Acsl3, Acat2, and Hmgcs2 were significantly upregulated, proteins that are important for lipogenesis, cholesterol ester synthesis, and ketogenesis, respectively. Consistent with these results, IMS demonstrated that MYO5B deficient tissues accumulated long-chain fatty acids, phosphatidylinositol, cholesterol derivatives, and nucleotide products in the mucosa (Figure 1). Immunostaining signals of HMGCS2 in control mouse jejunum is limited to the crypt stem cells, whereas MYO5B-deficient intestine showed expanded HMGCS2 expression in the proliferative cell zone. Interestingly, systemic LPA treatment in MYO5B-deficient mice further increased the expression of HMGCS2 in both proliferating and differentiated epithelial cells (Figure 2). These observations suggest that MYO5B loss induces a starvation-like phenotype in proliferative epithelial cells and that LPA treatment increased ketone body production as alternative energy fuels, resulting in the promotion of epithelial cell differentiation.
    CONCLUSION: MYO5B loss impairs fatty acid oxidation in the intestinal progenitor cells, which likely leads to cell differentiation deficits. Alteration of metabolic pathways might be a therapeutic target for epithelial disfunction seen in diarrheal diseases.
    DOI:  https://doi.org/10.1096/fasebj.2022.36.S1.R3291
  11. J Transl Med. 2022 May 13. 20(1): 209
    5-fluorouracil treatment of patient-derived scaffolds from colorectal cancer reveal clinically critical information.
    Simona Salerno, Anders Ståhlberg, André Holdfeldt, Elinor Bexe Lindskog, Göran Landberg.
       BACKGROUND: Colorectal cancer is a commonly diagnosed cancer worldwide. Unfortunately, many patients do not respond to standard chemotherapy treatments and develop disease relapse and metastases. Besides cancer cell specific genetic changes, heterogeneity in the tumor microenvironment contribute to the clinical presentation of the disease and can potentially also influence drug resistance. By using a recently developed patient-derived scaffold method monitoring how a standardized reporter cancer cell line adapts to various microenvironments treated with chemotherapy, we wanted to clarify how individual patient specific microenvironments influence the chemotherapy response in colorectal cancer.
    METHODS: Surgically resected colorectal cancer specimens from 89 patients were decellularized to produce patient-derived scaffold, which were seeded with HT29 cells, cultured for 3 weeks, and treated with 5-fluorouracil. Gene expression changes of adapted and treated HT29 cells were monitored by qPCR and compared with clinical parameters including disease-free survival.
    RESULTS: The effects of 5-fluorouracil treatment varied between different patient-derived scaffold, but generally induced a reduced expression of proliferation genes and increased expression of pluripotency and epithelial-to-mesenchymal transition genes. Interestingly, patient-derived scaffold cultures obtained from patients with disease recurrences showed a significantly less pronounced anti-proliferative effect of 5-fluorouracil and more pronounced increase of pluripotency, with MKI67 and POU5F1 being among the most significant genes linked to disease relapse in colorectal cancer.
    CONCLUSIONS: Colorectal patient-derived scaffold can decode clinically relevant tumor microenvironmental influence of 5-fluorouracil treatment effects opening up for optimized precision medicine in colorectal cancer treatment.
    Keywords:  5-fluorouracil; Colorectal cancer; Decellularized matrix; Drug screening; Patient-derived scaffold
    DOI:  https://doi.org/10.1186/s12967-022-03423-6
  12. STAR Protoc. 2022 Jun 17. 3(2): 101365
    A permeability assay for mouse intestinal organoids.
    Stijn Aaron den Daas, Ugo Soffientini, Shilpa Chokshi, Gautam Mehta.
      Here, we describe an assay for intestinal permeability in mouse intestinal organoids, although this may also be adapted for other species. Propidium iodide (PI) does not penetrate intact biological membranes and thus cannot enter the lumen of intact organoids. Passage of PI within the lumen can be induced by tight junction disruption or epithelial cell death. This technique measures PI-stained extruded dead cells within the organoid lumen to analyze the effect of insults, toxins, or treatments on intestinal organoid permeability.
    Keywords:  Health Sciences; Model Organisms; Organoids
    DOI:  https://doi.org/10.1016/j.xpro.2022.101365
  13. Nat Rev Genet. 2022 May 10.
    Variability exposes hidden buffering in the network specifying intestinal cell fate.
    Elizabeth Mason.
      
    DOI:  https://doi.org/10.1038/s41576-022-00496-3
  14. FASEB J. 2022 May;36 Suppl 1
    Signaling Pathways Associated with miR-106b mediated Radioresistance in the Treatment of Colorectal Cancer.
    Susana Rubio-Guevara, Karyn Olascuaga-Castillo, Elena Cáceres-Andonaire, Dan Altamirano-Sarmiento, Olga Caballero-Aquiño, Elena Mantilla-Rodríguez, Julio Hilario-Vargas, Maxim Berezovski, José Andrés Morgado-Díaz.
      Tumor radioresistance, or the lack of control of certain tumors with this treatment, can result in locoregional recurrence; therefore, there is great interest in understanding the underlying biology and developing strategies to overcome this problem. MicroRNAs (miRNAs, miRs) are small non-coding RNAs that regulate gene expression at the post-transcriptional level and participate in cancer invasion, progression, metastasis, and therapeutic resistance. Emerging evidence indicates that miRNAs play a critical role in modulating key cellular pathways that mediate response to radiation, influencing radiosensitivity of cancer cells through interaction with other biological processes such as cell cycle checkpoints, apoptosis, autophagy, epithelialmesenchymal transition, and cancer stem cells. Today, most studies on patient data report different, results on the miRNAs evaluated for each tumor type, highlighting miR-106b whose overexpression can determine radioresistance both in vitro and in vivo by inhibiting apoptosis and promotion of cell proliferation. The objective of this study was to find the signaling pathways involved with miR-106b-mediated radioresistance.
    METHODS: CRC gene expression data sets were collected from the public database, The Cancer Genome Atlas (TCGA). In addition, web-based tools were used to explore TCGA data, specifically that developed by the Memorial Sloan Kettering Cancer Center (MSK) cBioPortal for Cancer Genomics. The public site cBioPortal is hosted at the MSK Molecular Oncology Center, in which the term "colorectal cancer" was searched and 12 studies were selected, creating a single combined study which has 4341 patients and 4488 samples, within which the search for miR-106b was carried out and the signaling pathways involved with the expression mediated by this miRNA were obtained.
    RESULTS: The following signaling pathways involved with miR-106b were obtained: WNT, TP53, TGF-Beta, RTK-RAS, PI3K, NRF2, NOTCH, MYC, HIPPO, and its influence on the cell cycle was also noted.
    CONCLUSION: miRNAs have been shown to play an important role in the regulation of CRC radio resistance, by controlling various signaling pathways, including cell cycle, proliferation, apoptosis, and DNA damage repair. Furthermore, these results are consistent with recent data that have shown that selective modulation of miRNA activity can improve the response to radiotherapy, providing an innovative antitumor approach based on miRNA-related gene therapy. Therefore, miRNAs could also serve as targets for the development of new therapeutic strategies to overcome radiation resistance in CRC.
    DOI:  https://doi.org/10.1096/fasebj.2022.36.S1.R4918
  15. Cancers (Basel). 2022 Apr 29. pii: 2241. [Epub ahead of print]14(9):
    Biomarkers of Response and Resistance to Immunotherapy in Microsatellite Stable Colorectal Cancer: Toward a New Personalized Medicine.
    Nicolas Huyghe, Elena Benidovskaya, Philippe Stevens, Marc Van den Eynde.
      Immune Checkpoint Inhibitors (ICIs) are well recognized as a major immune treatment modality for multiple types of solid cancers. However, for colorectal cancer (CRC), ICIs are only approved for the treatment of Mismatch-Repair-Deficient and Microsatellite Instability-High (dMMR/MSI-H) tumors. For the vast majority of CRC, that are not dMMR/MSI-H, ICIs alone provide limited to no clinical benefit. This discrepancy of response between CRC and other solid cancers suggests that CRC may be inherently resistant to ICIs alone. In translational research, efforts are underway to thoroughly characterize the immune microenvironment of CRC to better understand the mechanisms behind this resistance and to find new biomarkers of response. In the clinic, trials are being set up to study biomarkers along with treatments targeting newly discovered immune checkpoint molecules or treatments combining ICIs with other existing therapies to improve response in MSS CRC. In this review, we will focus on the characteristics of response and resistance to ICIs in CRC, and discuss promising biomarkers studied in recent clinical trials combining ICIs with other therapies.
    Keywords:  Immune Checkpoint Inhibitors; biomarker; colorectal cancer; immune checkpoint resistance; immune microenvironment; immunotherapy
    DOI:  https://doi.org/10.3390/cancers14092241
  16. Cancers (Basel). 2022 Apr 23. pii: 2105. [Epub ahead of print]14(9):
    Overcoming Therapy Resistance in Colon Cancer by Drug Repurposing.
    Talal El Zarif, Marcel Yibirin, Diana De Oliveira-Gomes, Marc Machaalani, Rashad Nawfal, Gianfranco Bittar, Hisham F Bahmad, Nizar Bitar.
      Colorectal cancer (CRC) is the third most common cancer in the world. Despite improvement in standardized screening methods and the development of promising therapies, the 5-year survival rates are as low as 10% in the metastatic setting. The increasing life expectancy of the general population, higher rates of obesity, poor diet, and comorbidities contribute to the increasing trends in incidence. Drug repurposing offers an affordable solution to achieve new indications for previously approved drugs that could play a protagonist or adjuvant role in the treatment of CRC with the advantage of treating underlying comorbidities and decreasing chemotherapy toxicity. This review elaborates on the current data that supports drug repurposing as a feasible option for patients with CRC with a focus on the evidence and mechanism of action promising repurposed candidates that are widely used, including but not limited to anti-malarial, anti-helminthic, anti-inflammatory, anti-hypertensive, anti-hyperlipidemic, and anti-diabetic agents.
    Keywords:  colorectal cancer; drug repurposing; in silico drug screens; therapy resistance
    DOI:  https://doi.org/10.3390/cancers14092105
  17. FASEB J. 2022 May;36 Suppl 1
    P-cadherin regulates intestinal epithelial repair, but is dispensable for colitis associated colon cancer development.
    Nayden G Naydenov, Susana Lechuga, Ajaykumar Zalavadia, Emina Huang, Andrei I Ivanov.
      Recurrent chronic mucosal inflammation, which is characteristics for inflammatory bowel diseases (IBD), triggers significant changes in the intestinal epithelial homeostasis. These changes include leakiness of the gut barrier, formation of mucosal wounds and, in most severe cases, oncogenic transformation of colonic epithelium resulting in colitis-associated colon cancer (CAC). Altered structure and dynamics of epithelial junctions is a hallmark of intestinal inflammation, mediating epithelial barrier injury and repair. P-cadherin (gene name: CDH3) is an important component of adherens junctions (AJ), which is poorly expressed in normal intestinal epithelium, but could be induced in inflamed and injured mucosa. The goal of this his study was to investigate the roles of P-cadherin in regulating intestinal inflammation, mucosal repair and CAC. P-cadherin expression was markedly induced in the colonic epithelium of human IBD patients and CAC tissues. Roles of P-cadherin in intestinal inflammation and tumorigenesis in vivo were investigated using a mouse strain with total P-cadherin knockout. Dextran sulfate sodium (DSS)-induced colitis was utilized to study mucosal inflammation, whereas CAC was established using a classical azoxymetane (AOM)/DSS model. Severity of acute DSS colitis was not affected by P-cadherin knockout, however, P-cadherin null mice exhibited faster recovery after DSS withdrawal indicating accelerated repair of injured mucosa. No significant differences in the number and size of colonic tumors was observed in P-cadherin null and control mice after AOM/DSS induced CAC. Consistently, CRISPR/Cas9-mediated knockout of P-cadherin in SK-CO15 and HCA7 human colonic epithelial cell lines accelerated epithelial wound healing without affecting cell proliferation. The faster migration of P-cadherin depleted cells was associated with diminished cell-matrix adhesions and increased cell spreading. Loss of P-cadherin resulted in activation of Src kinase and the pro-migratory phenotype of P-cadherin depleted cell was reversed by pharmacological inhibition of Src. Furthermore, accelerated epithelial wound healing caused by loss of P-cadherin was prevented by Rac1 inhibition. Our findings highlight P-cadherin as a negative regulator of intestinal epithelial would heling in vitro and mucosal repair in vivo. By contrast, this AJ protein appears to be dispensable for intestinal epithelial cell proliferation and CAC development.
    DOI:  https://doi.org/10.1096/fasebj.2022.36.S1.R4111
  18. Cells. 2022 Apr 27. pii: 1467. [Epub ahead of print]11(9):
    P-Cadherin Regulates Intestinal Epithelial Cell Migration and Mucosal Repair, but Is Dispensable for Colitis Associated Colon Cancer.
    Nayden G Naydenov, Susana Lechuga, Ajay Zalavadia, Pranab K Mukherjee, Ilyssa O Gordon, David Skvasik, Petra Vidovic, Emina Huang, Florian Rieder, Andrei I Ivanov.
      Recurrent chronic mucosal inflammation, a characteristic of inflammatory bowel diseases (IBD), perturbs the intestinal epithelial homeostasis resulting in formation of mucosal wounds and, in most severe cases, leads to colitis-associated colon cancer (CAC). The altered structure of epithelial cell-cell adhesions is a hallmark of intestinal inflammation contributing to epithelial injury, repair, and tumorigenesis. P-cadherin is an important adhesion protein, poorly expressed in normal intestinal epithelial cells (IEC) but upregulated in inflamed and injured mucosa. The goal of this study was to investigate the roles of P-cadherin in regulating intestinal inflammation and CAC. P-cadherin expression was markedly induced in the colonic epithelium of human IBD patients and CAC tissues. The roles of P-cadherin were investigated in P-cadherin null mice using dextran sulfate sodium (DSS)-induced colitis and an azoxymethane (AOM)/DSS induced CAC. Although P-cadherin knockout did not affect the severity of acute DSS colitis, P-cadherin null mice exhibited faster recovery after colitis. No significant differences in the number of colonic tumors were observed in P-cadherin null and control mice. Consistently, the CRISPR/Cas9-mediated knockout of P-cadherin in human IEC accelerated epithelial wound healing without affecting cell proliferation. The accelerated migration of P-cadherin depleted IEC was driven by activation of Src kinases, Rac1 GTPase and myosin II motors and was accompanied by transcriptional reprogramming of the cells. Our findings highlight P-cadherin as a negative regulator of IEC motility in vitro and mucosal repair in vivo. In contrast, this protein is dispensable for IEC proliferation and CAC development.
    Keywords:  cadherins; cell migration; cell-cell adhesions; colon cancer; epithelial cells; mucosal restitution
    DOI:  https://doi.org/10.3390/cells11091467
  19. PLoS One. 2022 ;17(5): e0268436
    Consensus molecular subtype differences linking colon adenocarcinoma and obesity revealed by a cohort transcriptomic analysis.
    Michael W Greene, Peter T Abraham, Peyton C Kuhlers, Elizabeth A Lipke, Martin J Heslin, Stanley T Wijaya, Ifeoluwa Odeniyi.
      Colorectal cancer (CRC) is the third-leading cause of cancer-related deaths in the United States and worldwide. Obesity-a worldwide public health concern-is a known risk factor for cancer including CRC. However, the mechanisms underlying the link between CRC and obesity have yet to be fully elucidated in part because of the molecular heterogeneity of CRC. We hypothesized that obesity modulates CRC in a consensus molecular subtype (CMS)-dependent manner. RNA-seq data and associated tumor and patient characteristics including body weight and height data for 232 patients were obtained from The Cancer Genomic Atlas-Colon Adenocarcinoma (TCGA-COAD) database. Tumor samples were classified into the four CMSs with the CMScaller R package; body mass index (BMI) was calculated and categorized as normal, overweight, and obese. We observed a significant difference in CMS categorization between BMI categories. Differentially expressed genes (DEGs) between obese and overweight samples and normal samples differed across the CMSs, and associated prognostic analyses indicated that the DEGs had differing associations on survival. Using Gene Set Enrichment Analysis, we found differences in Hallmark gene set enrichment between obese and overweight samples and normal samples across the CMSs. We constructed Protein-Protein Interaction networks and observed differences in obesity-regulated hub genes for each CMS. Finally, we analyzed and found differences in predicted drug sensitivity between obese and overweight samples and normal samples across the CMSs. Our findings support that obesity impacts the CRC tumor transcriptome in a CMS-specific manner. The possible associations reported here are preliminary and will require validation using in vitro and animal models to examine the CMS-dependence of the genes and pathways. Once validated the obesity-linked genes and pathways may represent new therapeutic targets to treat colon cancer in a CMS-dependent manner.
    DOI:  https://doi.org/10.1371/journal.pone.0268436
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