bims-instec Biomed News
on Intestinal stem cells and chemoresistance in colon cancer and intestinal regeneration
Issue of 2022‒11‒06
29 papers selected by
Maria-Virginia Giolito
Free University of Brussels

  1. EMBO Mol Med. 2022 Nov 02. e16194
      The majority of colorectal cancers (CRCs) present with early mutations in tumor suppressor gene APC. APC mutations result in oncogenic activation of the Wnt pathway, which is associated with hyperproliferation, cytoskeletal remodeling, and a global increase in mRNA translation. To compensate for the increased biosynthetic demand, cancer cells critically depend on protein chaperones to maintain proteostasis, although their function in CRC remains largely unexplored. In order to investigate the role of molecular chaperones in driving CRC initiation, we captured the transcriptomic profiles of murine wild type and Apc-mutant organoids during active transformation. We discovered a strong transcriptional upregulation of Hspb1, which encodes small heat shock protein 25 (HSP25). We reveal an indispensable role for HSP25 in facilitating Apc-driven transformation, using both in vitro organoid cultures and mouse models, and demonstrate that chemical inhibition of HSP25 using brivudine reduces the development of premalignant adenomas. These findings uncover a hitherto unknown vulnerability in intestinal transformation that could be exploited for the development of chemopreventive strategies in high-risk individuals.
    Keywords:  Apc mutations; Wnt signaling; colorectal cancer; heat shock proteins; intestinal stem cells
  2. J Vis Exp. 2022 Oct 11.
      The intestinal epithelium regenerates every 5-7 days, and is controlled by the intestinal epithelial stem cell (IESC) population located at the bottom of the crypt region. IESCs include active stem cells, which self-renew and differentiate into various epithelial cell types, and quiescent stem cells, which serve as the reserve stem cells in the case of injury. Regeneration of the intestinal epithelium is controlled by the self-renewing and differentiating capabilities of these active IESCs. In addition, the balance of the crypt stem cell population and maintenance of the stem cell niche are essential for intestinal regeneration. Organoid culture is an important and attractive approach to studying proteins, signaling molecules, and environmental cues that regulate stem cell survival and functions. This model is less expensive, less time-consuming, and more manipulatable than animal models. Organoids also mimic the tissue microenvironment, providing in vivo relevance. The present protocol describes the isolation of colonic crypts, embedding these isolated crypt cells into a three-dimensional gel matrix system and culturing crypt cells to form colonic organoids capable of self-organization, proliferation, self-renewal, and differentiation. This model allows one to manipulate the environment-knocking out specific proteins such as claudin-7, activating/deactivating signaling pathways, etc.-to study how these effects influence the functioning of colonic stem cells. Specifically, the role of tight junction protein claudin-7 in colonic stem cell function was examined. Claudin-7 is vital for maintaining intestinal homeostasis and barrier function and integrity. Knockout of claudin-7 in mice induces an inflammatory bowel disease-like phenotype exhibiting intestinal inflammation, epithelial hyperplasia, weight loss, mucosal ulcerations, epithelial cell sloughing, and adenomas. Previously, it was reported that claudin-7 is required for intestinal epithelial stem cell functions in the small intestine. In this protocol, a colonic organoid culture system is established to study the role of claudin-7 in the large intestine.
  3. Cell Regen. 2022 Nov 02. 11(1): 39
      Intestinal organoids, derived from intestinal stem cell self-organization, recapitulate the tissue structures and behaviors of the intestinal epithelium, which hold great potential for the study of developmental biology, disease modeling, and regenerative medicine. The intestinal epithelium is exposed to dynamic mechanical forces which exert profound effects on gut development. However, the conventional intestinal organoid culture system neglects the key role of mechanical microenvironments but relies solely on biological factors. Here, we show that adding cyclic stretch to intestinal organoid cultures remarkably up-regulates the signature gene expression and proliferation of intestinal stem cells. Furthermore, mechanical stretching stimulates the expansion of SOX9+ progenitors by activating the Wnt/β-Catenin signaling. These data demonstrate that the incorporation of mechanical stretch boosts the stemness of intestinal stem cells, thus benefiting organoid growth. Our findings have provided a way to optimize an organoid generation system through understanding cross-talk between biological and mechanical factors, paving the way for the application of mechanical forces in organoid-based models.
    Keywords:  Intestinal organoid; Lgr5+ stem cell; Mechanical stretching; Regeneration; Wnt/β-catenin signaling
  4. Commun Biol. 2022 Oct 31. 5(1): 1159
      Despite the fact that 5-fluorouracil (5-FU) is the backbone for chemotherapy in colorectal cancer (CRC), the response rates in patients is limited to 50%. The mechanisms underlying 5-FU toxicity are debated, limiting the development of strategies to improve its efficacy. How fundamental aspects of cancer, such as driver mutations and phenotypic heterogeneity, relate to the 5-FU response remains obscure. This largely relies on the limited number of studies performed in pre-clinical models able to recapitulate the key features of CRC. Here, we analyzed the 5-FU response in patient-derived organoids that reproduce the different stages of CRC. We find that 5-FU induces pyrimidine imbalance, which leads to DNA damage and cell death in the actively proliferating cancer cells deficient in p53. Importantly, p53-deficiency leads to cell death due to impaired cell cycle arrest. Moreover, we find that targeting the Warburg effect in KRASG12D glycolytic tumor organoids enhances 5-FU toxicity by further altering the nucleotide pool and, importantly, without affecting non-transformed WT cells. Thus, p53 emerges as an important factor in determining the 5-FU response, and targeting cancer metabolism in combination with replication stress-inducing chemotherapies emerges as a promising strategy for CRC treatment.
  5. Annu Rev Pathol. 2022 Nov 02.
      Reprogrammed metabolism is a hallmark of colorectal cancer (CRC). CRC cells are geared toward rapid proliferation, requiring nutrients and the removal of cellular waste in nutrient-poor environments. Intestinal stem cells (ISCs), the primary cell of origin for CRCs, must adapt their metabolism along the adenoma-carcinoma sequence to the unique features of their complex microenvironment that include interactions with intestinal epithelial cells, immune cells, stromal cells, commensal microbes, and dietary components. Emerging evidence implicates modifiable risk factors related to the environment, such as diet, as important in CRC pathogenesis. Here, we focus on describing the metabolism of ISCs, diets that influence CRC initiation, CRC genetics and metabolism, and the tumor microenvironment. The mechanistic links between environmental factors, metabolic adaptations, and the tumor microenvironment in enhancing or supporting CRC tumorigenesis are becoming better understood. Thus, greater knowledge of CRC metabolism holds promise for improved prevention and treatment. Expected final online publication date for the Annual Review of Pathology: Mechanisms of Disease, Volume 18 is January 2023. Please see for revised estimates.
  6. Mol Nutr Food Res. 2022 Oct 30. e2200168
      SCOPE: Proliferation and differentiation of intestinal stem cells (ISCs) are crucial for functional restoration after injury, which can be regulated by nutritional molecules. Aspartate is implicated in maintaining intestinal barrier after injury, but underlying mechanisms remain elusive. Here, this study seeks to investigate if aspartate alleviates colonic epithelial damage by regulating ISC function, and to elucidate its mechanisms.METHODS AND RESULTS: Eight-week-old male C57BL/6 mice supplement with or without 1% L-aspartate are subjected to drinking water or 2.5% DSS to induce colitis. In this study, aspartate administration alleviates the severity of colitis, as indicated by reduced body weight loss, colon shortening, and inhibited pro-inflammatory cytokine expression in DSS-challenged mice. Additionally, aspartate promotes colonic epithelial cell proliferation and differentiation after DSS-induced damage in mice. Pretreatment with aspartate not only enhances ISC proliferation but also induces ISC differentiation toward enterocytes and goblet cells, which prevent TNF-α-induced colonoid damage. Mechanistically, aspartate ameliorates DSS/TNF-α-induced perturbation of mitochondrial metabolism and maintains mitochondrial dynamics in colonic epithelium and colonoids. Moreover, aspartate-mediated ISC proliferation and differentiation are primarily dependent on mitochondrial fusion rather than fission.
    CONCLUSIONS: The findings indicate that aspartate promotes ISC proliferation and differentiation to alleviate colonic epithelial damage by regulation of mitochondrial metabolism and dynamics.
    Keywords:  aspartate; colonoids; intestinal epithelial homeostasis; intestinal stem cell; mitochondrial dynamics
  7. Oncogene. 2022 Nov 01.
      Colorectal cancer causes >900,000 deaths every year and a deeper understanding of the molecular mechanisms underlying this disease will contribute to improve its clinical management and survival. Myosin Vb (MYO5B) regulates intracellular vesicle trafficking, and inactivation of this myosin disrupts the polarization and differentiation of intestinal epithelial cells causing microvillous inclusion disease (MVID), a rare congenital disorder characterized by intractable life-threatening diarrhea. Here, we show that the loss Myosin Vb interfered with the differentiation/polarization of colorectal cancer cells. Although modulation of Myosin Vb expression did not affect the proliferation of colon cancer cells, MYO5B inactivation increased their migration, invasion, and metastatic potential. Moreover, Myo5b inactivation in an intestine-specific knockout mouse model caused a >15-fold increase in the number of azoxymethane-initiated small intestinal tumors. Consistently, reduced expression of Myosin Vb in a cohort of 155 primary colorectal tumors was associated with shorter patient survival. In conclusion, we show here that loss of Myosin Vb reduces polarization/differentiation of colon cancer cells while enhancing their metastatic potential, demonstrating a tumor suppressor function for this myosin. Moreover, reduced expression of Myosin Vb in primary tumors identifies a subset of poor prognosis colorectal cancer patients that could benefit from more aggressive therapeutic regimens.
  8. J Gastroenterol. 2022 Nov 03.
      Mutations in Bone Morphogenetic Protein (BMP) Receptor (BMPR)1A and SMAD4 are detected in 50% of juvenile polyposis syndrome (JPS) patients, who develop stroma-rich hamartomatous polyps. The established role of stromal cells in regulating BMP activity in the intestine implies a role for stromal cells in polyp development. We used conditional Cre-LoxP mice to investigate how specific loss of BMPR1A in endothelial cells, fibroblasts, or myofibroblasts/smooth muscle cells affects intestinal homeostasis. Selective loss of BMPR1A in fibroblasts causes severe histological changes in the intestines with a significant increase in stromal cell content and epithelial cell hyperproliferation, leading to numerous serrated polyps. This phenotype suggests that crucial changes occur in the fibroblast secretome that influences polyp development. Analyses of publicly available RNA expression databases identified CXCL12 as a potential candidate. RNAscope in situ hybridization showed an evident increase of Cxcl12-expressing fibroblasts. In vitro, stimulation of fibroblasts with BMPs resulted in downregulation of CXCL12, while inhibition of the BMP pathway resulted in gradual upregulation of CXCL12 over time. Moreover, neutralization of CXCL12 in vivo in the fibroblast-specific BMPR1A KO mice resulted in a significant decrease in polyp formation. Finally, in CRC patient specimens, mRNA-expression data showed that patients with high GREMLIN1 and CXCL12 expression had a significantly poorer overall survival. Significantly higher GREMLIN1, NOGGIN, and CXCL12 expression were detected in the Consensus Molecular Subtype 4 (CMS4) colorectal cancers, which are thought to arise from serrated polyps. Taken together, these data imply that fibroblast-specific BMP signaling-CXCL12 interaction could have a role in the etiology of serrated polyp formation.
    Keywords:  BMPR1A; CXCL12; Colorectal cancer; Consensus molecular subtypes 4; Fibroblasts
  9. BMC Cancer. 2022 Oct 29. 22(1): 1107
      BACKGROUND: AZD0156 is an oral inhibitor of ATM, a serine threonine kinase that plays a key role in DNA damage response (DDR) associated with double-strand breaks. Topoisomerase-I inhibitor irinotecan is used clinically to treat colorectal cancer (CRC), often in combination with 5-fluorouracil (5FU). AZD0156 in combination with irinotecan and 5FU was evaluated in preclinical models of CRC to determine whether low doses of AZD0156 enhance the cytotoxicity of irinotecan in chemotherapy regimens used in the clinic.METHODS: Anti-proliferative effects of single-agent AZD0156, the active metabolite of irinotecan (SN38), and combination therapy were evaluated in 12 CRC cell lines. Additional assessment with clonogenic assay, cell cycle analysis, and immunoblotting were performed in 4 selected cell lines. Four colorectal cancer patient derived xenograft (PDX) models were treated with AZD0156, irinotecan, or 5FU alone and in combination for assessment of tumor growth inhibition (TGI). Immunofluorescence was performed on tumor tissues. The DDR mutation profile was compared across in vitro and in vivo models.
    RESULTS: Enhanced effects on cellular proliferation and regrowth were observed with the combination of AZD0156 and SN38 in select models. In cell cycle analysis of these models, increased G2/M arrest was observed with combination treatment over either single agent. Immunoblotting results suggest an increase in DDR associated with irinotecan therapy, with a reduced effect noted when combined with AZD0156, which is more pronounced in some models. Increased TGI was observed with the combination of AZD0156 and irinotecan as compared to single-agent therapy in some PDX models. The DDR mutation profile was variable across models.
    CONCLUSIONS: AZD0156 and irinotecan provide a rational and active combination in preclinical colorectal cancer models. Variability across in vivo and in vitro results may be related to the variable DDR mutation profiles of the models evaluated. Further understanding of the implications of individual DDR mutation profiles may help better identify patients more likely to benefit from treatment with the combination of AZD0156 and irinotecan in the clinical setting.
    Keywords:  ATM; AZD0156; Colorectal cancer; DNA damage repair; Irinotecan
  10. Int J Nanomedicine. 2022 ;17 5049-5061
      Background: Transgenic C57BL/6-APC(Min/+) spontaneous cancer mouse model and the Azoxymethane (AOM)/Dextran Sulfate Sodium (DSS) chemically induced orthotopic colorectal cancer mouse model represented distinct pathogenesis of colorectal cancers. Our previous study revealed that the combination of Rapamycin liposomes (Rapa/Lps) and 5-Fluorouracil (5-FU) has anti-colorectal cancer effects. However, the therapeutic efficacy of Rapa/Lps and 5-FU in other colorectal cancer mice models is yet to be thoroughly explored. The purpose of this study was to investigate the anti-tumor effect of Rapa/Lps combined with 5-FU in vivo and in vitro.Methods: In this study, we evaluated the effect of Rapa/Lps and 5-FU on APC (Min/+) mice and AOM/DSS-induced colorectal cancer mice. The small intestine, colorectum, serum, and plasma of mice in each group were collected following sacrifice to record the number of tumors. HE staining was utilized for observing pathological damage to intestine tissues. Tube formation assay, Transwell assay, wound healing assay, Western Blot were used to explore the anti-angiogenesis effect of drugs in HUVECs.
    Results: As expected, Rapa/Lps and 5-FU significantly suppressed tumor formation, decreased the number of tumors, and tumor load both in two mouse models, and had no influence on mouse weight. Mechanically, the anti-tumor effect of the drug also was associated in inhibiting angiogenesis and proliferation. Furthermore, we found that Rapa/Lps obviously inhibited HUVECs tube formation and migration.
    Conclusion: Altogether, we revealed the Rapa/Lps synergism with 5-FU decreased colon and small intestinal tumorigenesis in AOM/DSS-treated and APC (Min/+) mice, respectively, and correlated with anti-angiogenesis.
    Keywords:  5-fluorouracil; AOM/DSS; APC (Min/+) mice; angiogenesis; colorectal cancer; rapamycin liposomes
  11. Front Oncol. 2022 ;12 906260
      Chemoresistance in colorectal cancer initiating cells (CICs) involves the sustained activation of multiple drug resistance (MDR) and WNT/β-catenin signaling pathways, as well as of alternatively spliced-isoforms of CD44 containing variable exon-6 (CD44v6). In spite of its importance, mechanisms underlying the sustained activity of WNT/β-catenin signaling have remained elusive. The presence of binding elements of the β-catenin-interacting transcription factor TCF4 in the MDR1 and CD44 promoters suggests that crosstalk between WNT/β-catenin/TCF4-activation and the expression of the CD44v6 isoform mediated by FOLFOX, a first-line chemotherapeutic agent for colorectal cancer, could be a fundamental mechanism of FOLFOX resistance. Our results identify that FOLFOX treatment induced WNT3A secretion, which stimulated a positive feedback loop coupling β-catenin signaling and CD44v6 splicing. In conjunction with FOLFOX induced WNT3A signal, specific CD44v6 variants produced by alternative splicing subsequently enhance the late wave of WNT/β-catenin activation to facilitate cell cycle progression. Moreover, we revealed that FOLFOX-mediated sustained WNT signal requires the formation of a CD44v6-LRP6-signalosome in caveolin microdomains, which leads to increased FOLFOX efflux. FOLFOX-resistance in colorectal CICs occurs in the absence of tumor-suppressor disabled-2 (DAB2), an inhibitor of WNT/β-catenin signaling. Conversely, in sensitive cells, DAB2 inhibition of WNT-signaling requires interaction with a clathrin containing CD44v6-LRP6-signalosome. Furthermore, full-length CD44v6, once internalized through the caveolin-signalosome, is translocated to the nucleus where in complex with TCF4, it binds to β-catenin/TCF4-regulated MDR1, or to CD44 promoters, which leads to FOLFOX-resistance and CD44v6 transcription through transcriptional-reprogramming. These findings provide evidence that targeting CD44v6-mediated LRP6/β-catenin-signaling and drug efflux may represent a novel approach to overcome FOLFOX resistance and inhibit tumor progression in colorectal CICs. Thus, sustained drug resistance in colorectal CICs is mediated by overexpression of CD44v6, which is both a functional biomarker and a therapeutic target in colorectal cancer.
    Keywords:  CD44v6; MDR1; WNT3A; cancer initiating cells (CICs); colon rectal cancer (CRC)
  12. Cancer Discov. 2022 Nov 04. OF1
      Genetic and epigenetic modifications in colorectal cancer cells occur independently with plastic intratumor variation.
  13. STAR Protoc. 2022 Dec 16. 3(4): 101712
      Immunotherapy has revolutionized cancer treatment, but preclinical models are required to understand immunotherapy resistance mechanisms underlying patient relapse. This protocol describes how to generate an acquired resistance humanized in vivo model to immunotherapies in patient-derived xenografts (PDX). We detail steps to inject human CD34+ cells into NSG mice, followed by generation of immunoresistant PDX in humanized mice. This approach recapitulates the human immune system, allowing investigators to generate preclinical resistance models to different immunotherapies for identifying the resistant phenotype. For complete details on the use and execution of this protocol, please refer to Martínez-Sabadell et al., 2022 and Arenas et al. (2021).
    Keywords:  Cancer; Cell isolation; Health sciences; Immunology; Model organisms; Stem cells
  14. Biofabrication. 2022 Nov 02. 15(1):
      We describe the development of a high-throughput bioprinted colorectal cancer (CRC) spheroid platform with high levels of automation, information content, and low cell number requirement. This is achieved via the formulation of a hydrogel bioink with a compressive Young's modulus that is commensurate with that of colonic tissue (1-3 kPa), which supports exponential growth of spheroids from a wide range of CRC cell lines. The resulting spheroids display tight cell-cell junctions, bioink matrix-cell interactions and necrotic hypoxic cores. By combining high content light microscopy imaging and processing with rapid multiwell plate bioprinting, dose-response profiles are generated from CRC spheroids challenged with oxaliplatin (OX) and fluorouracil (5FU), as well as radiotherapy. Bioprinted CRC spheroids are shown to exhibit high levels of chemoresistance relative to cell monolayers, and OX was found to be significantly less effective against tumour spheroids than in monolayer culture, when compared to 5FU.
    Keywords:  bioprinting; cancer; colorectal; spheroid
  15. BMC Med Genomics. 2022 Nov 01. 15(1): 229
      BACKGROUND: Colorectal cancer is common among obese individuals. The purpose of the current study was to determine changes in DNA methylation status and mRNA expression of thyroid hormone receptor beta (THRB), as a tumor suppressor, and thyroid hormone inactivating enzyme, type 3 deiodinase (DIO3) genes, in human epithelial colon tissues of healthy obese individuals.METHODS: Colon biopsies were analyzed by methylation sensitive-high resolution melting (MS-HRM) to investigate promoter methylation of DIO3 and THRB, and by quantitative real-time polymerase chain reaction to assay expression of DIO3 and THRB mRNA on eighteen obese and twenty-one normal-weight healthy men.
    RESULTS: There was no significant difference in mean methylation levels at the THRB promoter region between the two groups. Nevertheless, obesity decreased THRB expression levels, significantly (P < 0.05; fold change: 0.19). Furthermore, obesity attenuated DNA methylation (P < 0.001) and enhanced mRNA expression of DIO3 (P < 0.05; fold change: 3).
    CONCLUSIONS: Our findings suggest that obesity may alter expression of THRB and DIO3 genes through epigenetic mechanism. Alterations of THRB and DIO3 expressions may predispose colon epithelium of obese patients to neoplastic transformation.
    Keywords:  Colorectal cancer; DIO3; DNA methylation; Obesity; THRB
  16. Brain Behav Immun. 2022 Oct 31. pii: S0889-1591(22)00420-2. [Epub ahead of print]
      Almost 2/3rds of stroke survivors exhibit vascular cognitive impairment and a third of stroke patients will develop dementia 1-3 years after stroke. These dire consequences underscore the need for effective stroke therapies. In addition to its damaging effects on the brain, stroke rapidly dysregulates the intestinal epithelium, resulting in elevated blood levels of inflammatory cytokines and toxic gut metabolites due to a 'leaky' gut. We tested whether repairing the gut via intestinal epithelial stem cell (IESC) transplants would also improve stroke recovery. Organoids containing IESCs derived from young rats transplanted into older rats after stroke were incorporated into the gut, restored stroke-induced gut dysmorphology and decreased gut permeability, and reduced circulating levels of endotoxin LPS and the inflammatory cytokine IL-17A. Remarkably, IESC transplants also improved stroke-induced acute (4d) sensory-motor disability and chronic (30d) cognitive-affective function. Moreover, IESCs from older animals displayed senescent features and were not therapeutic for stroke. These data underscore the gut as a critical therapeutic target for stroke and demonstrate the effectiveness of gut stem cell therapy.
    Keywords:  IL17; Intestinal epithelial stem cells; Lgr5+; Stroke; Transplant; organoids; post stroke cognitive impairment
  17. Clin Exp Med. 2022 Nov 01.
      Forkhead Box M1 (FOXM1)-a key cell cycle regulator is a member of the Forkhead transcription factor family. It plays a key role in embryogenesis and cell proliferation and has been strongly linked to various solid tumors. We sought to understand the regulation of FOXM1 in colorectal cancer (CRC), as well as if and to what extent other clinicopathological characteristics are associated with FOXM1. The investigation comprised 98 CRC samples and normal tissues (controls). All colon cancer patients had a colonoscopy and targeted biopsy. All rectal cancer patients had a CT and MRI. Real-time PCR, Immunohistochemistry, and Western blotting were used to evaluate FOXM1 expression, and the findings were analyzed using SPSS (v.26). FOXM1 mRNA and protein expression were substantially upregulated in tumor tissues, with the majority of these proteins localized in nucleo-cytoplasm. Elevated protein levels of FOXM1 were strongly correlated with lower education level, larger tumor size, lymph node status, lymphovascular invasion (LVI), perineural invasion (PNI), lymph node metastasis (LNM), tumor invasion depth (subserosal and serosal invasion), late stage (III and IV), localization (nucleo-cytoplasmic), intensity (strong) and recurrence. Based on survival analysis, FOXM1 overexpression and nucleo-cytoplasmic localization were associated with shorter disease-free survival while stage and PNI were linked to poorer overall and disease-free survival. According to the results of the Cox regression analysis, stage and PNI were significant predictors of prognosis in CRC patients. FOXM1 expression was elevated in CRC and was linked to reduced disease-free survival. These findings support prior reports and hence FOXM1 can be an important prognostic marker for CRC and a promising therapeutic target. Additionally, we found a link between poor disease-free survival and FOXM1's nucleo-cytoplasmic localization. However, since the sample size of this study was small, further research is needed to validate our findings.
    Keywords:  Colorectal cancer; Immunohistochemistry; Kashmir real-time PCR; Western blotting
  18. Front Oncol. 2022 ;12 959407
      Cancer therapy failure is a fundamental challenge in cancer treatment. One of the most common reasons for therapy failure is the development of acquired resistance of cancer cells. DNA-damaging agents are frequently used in first-line chemotherapy regimens and DNA damage response, and DNA repair pathways are significantly involved in the mechanisms of chemoresistance. MRE11, a part of the MRN complex involved in double-strand break (DSB) repair, is connected to colorectal cancer (CRC) patients' prognosis. Our previous results showed that single-nucleotide polymorphisms (SNPs) in the 3' untranslated region (3'UTR) microRNA (miRNA) binding sites of MRE11 gene are associated with decreased cancer risk but with shorter survival of CRC patients, which implies the role of miRNA regulation in CRC. The therapy of colorectal cancer utilizes oxaliplatin (oxalato(trans-l-1,2-diaminocyclohexane)platinum), which is often compromised by chemoresistance development. There is, therefore, a crucial clinical need to understand the cellular processes associated with drug resistance and improve treatment responses by applying efficient combination therapies. The main aim of this study was to investigate the effect of miRNAs on the oxaliplatin therapy response of CRC patients. By the in silico analysis, miR-140 was predicted to target MRE11 and modulate CRC prognosis. The lower expression of miR-140 was associated with the metastatic phenotype (p < 0.05) and poor progression-free survival (odds ratio (OR) = 0.4, p < 0.05). In the in vitro analysis, we used miRNA mimics to increase the level of miR-140 in the CRC cell line. This resulted in decreased proliferation of CRC cells (p < 0.05). Increased levels of miR-140 also led to increased sensitivity of cancer cells to oxaliplatin (p < 0.05) and to the accumulation of DNA damage. Our results, both in vitro and in vivo, suggest that miR-140 may act as a tumor suppressor and plays an important role in DSB DNA repair and, consequently, CRC therapy response.
    Keywords:  DNA damage; DNA repair; MRE11; colorecal cancer; miR-140; miRNA; oxaliplatin; therapy response
  19. FEBS J. 2022 Oct 30.
      The nuclear RNA surveillance mechanism is essential for cancer cell survival and is ensured by the RNA nuclear exosome including some co-factors, such as the RNA helicase MTR4. Recent studies suggest an involvement of DNA repair proteins such as APE1, a major endodeoxyribonuclease of Base Excision Repair (BER), in RNA metabolism and RNA decay of oxidized and abasic RNA. Cisplatin (CDDP) and 5-fluorouracil (5-FU) are commonly used for the treatment of solid tumors. Whether APE1 is involved in the elimination of CDDP- or 5-FU-damaged RNA is unknown, as is its possible interaction with the nuclear exosome complex. Here, by using different human cancer cell models, we demonstrated that: i) APE1 is involved in the elimination of damaged-RNA, upon CDDP- and 5-FU-treatments, in a MTR4-independent manner; ii) the interaction between APE1 and MTR4 is stimulated by CDDP- and 5-FU-treatments through lysine residues in the APE1 N-terminal region and is, in part, mediated by nucleic acids; and iii) APE1- and MTR4-depletion lead to the generation of R-loop formation causing the activation of the DNA damage response (DDR) pathway through the ATM-p53-p21 axis. Our data demonstrate a role of MTR4 in DDR underpinning the function of APE1 in controlling the RNA quality upon genotoxic treatments with possible implications in chemoresistance.
    Keywords:  5-FU; APE1; CDDP; MTR4; RNA
  20. Front Microbiol. 2022 ;13 932294
      Abdominal irradiation (IR) destroys the intestinal mucosal barrier, leading to severe intestinal infection. There is an urgent need to find safe and effective treatments to reduce IR-induced intestinal injury. In this study, we reported that metformin protected mice from abdominal IR-induced intestinal injury by improving the composition and diversity of intestinal flora. The elimination of intestinal microbiota (Abx) abrogated the protective effects of metformin on irradiated mice. We further characterized that treatment of metformin increased the murine intestinal abundance of Lactobacillus, which mediated the radioprotective effect. The administration of Lactobacillus or fecal microbiota transplantation (FMT) into Abx mice considerably lessened IR-induced intestinal damage and restored the radioprotective function of metformin in Abx mice. In addition, applying the murine intestinal organoid model, we demonstrated that IR inhibited the formation of intestinal organoids, and metformin alone bore no protective effect on organoids after IR. However, a combination of metformin and Lactobacillus or Lactobacillus alone displayed a strong radioprotection on the organoid formation. We demonstrated that metformin/Lactobacillus activated the farnesoid X receptor (FXR) signaling in intestinal epithelial cells and hence upregulated tight junction proteins and mucins in intestinal epithelia, increased the number of goblet cells, and augmented the mucus layer thickness to maintain the integrity of intestinal epithelial barrier, which eventually contributed to reduced radiation intestinal injury. In addition, we found that Lactobacillus abundance was significantly increased in the intestine of patients receiving metformin while undergoing abdominal radiotherapy and the abundance was negatively correlated with the diarrhea duration of patients. In conclusion, our results demonstrate that metformin possesses a protective effect on IR-induced intestinal injury by upregulating the abundance of Lactobacillus in the intestine.
    Keywords:  Lactobacillus; farnesoid X receptor (FXR); intestinal barrier; irradiation-induced intestinal injury; metformin; microbiota
  21. Transl Oncol. 2022 Oct 31. pii: S1936-5233(22)00234-0. [Epub ahead of print]27 101575
      Chimeric antigen receptor (CAR) T-cell therapy is emerging as an effective cancer treatment, such as for hematological malignancies, however its effectiveness as an approach to treat solid tumors, such as in colorectal cancer (CRC), remains to be better developed. One area of intense development has been in the identification and characterization of novel cancer-related ligand receptors for CAR design and evaluation. It is known that the CD6 receptors CD166 and CD318 are highly expressed in CRC, and several CAR-Ts have also been explored in preclinical and clinical studies for the treatment of CRC, with promising safety and efficacy findings. Here, we constructed a CAR based on the extracellular domain of CD6 and demonstrate its cytotoxic effect in target positive human CRC cell lines. Unexpectedly, we found that CD6-CAR-T cells targeted CD166 instead of CD318. Furthermore, CD6-CAR-T cells show robust cytotoxicity to CD166-positive cell lines in a dose-dependent manner with cytokine IFN-γ significantly released. Particularly, CD6-CAR-T cells show potent cytotoxicity targeting CRC cancer stem cells (CSCs), highlighting that CD6-CAR-T is a promising approach for the therapy of CRC.
    Keywords:  CD166; CD318; Cancer stem cell; Chimeric antigen receptor; Colorectal cancer
  22. STAR Protoc. 2022 Dec 16. 3(4): 101795
      Single-cell techniques have revolutionized biology; however, the required sample processing inherently implies the loss of spatial localization. Here, using an approach called photoconversion of areas to dissect micro-environments (PADME), we detail steps to isolate live single cells from a primary breast tumor while retaining spatial information by combining cell photolabeling and FACS (fluorescence-activated cell sorting). These live cells can be subsequently used for myriad techniques, from flow cytometry to single-cell RNA sequencing or other single cell "omics" approach. For complete details on the use and execution of this protocol, please refer to Baldominos et al. (2022).
    Keywords:  Biotechnology and bioengineering; Cancer; Cell isolation; Flow cytometry/Mass cytometry; Immunology; Microscopy; Molecular/Chemical probes; Single cell
  23. Front Immunol. 2022 ;13 1011590
      Background: Current stratification systems for tumor prognostic prediction and immunotherapeutic efficacy evaluation are less satisfying in colorectal cancer (CRC). As infiltrating immune cells in tumor microenvironment (TME) played a key role in tumor progression and responses to immune checkpoint blockade (ICB) therapy, we want to construct an immune-related scoring system with detailed immune profiles to stratify CRC patients.Methods: We developed a scoring system based on immune-related signatures and validated its ability to predict prognosis and immunotherapeutic outcomes in CRC. CD45+ cells from CRC patients were sorted to investigate detailed immune profiles of the stratification system using mass cytometry. A single-cell RNA sequencing dataset was used to analyze transcriptomic profiles.
    Results: We constructed an immune-related signature score (IRScore) based on 54 recurrence-free survival (RFS)-related immune signatures to stratify CRC patients. We revealed that IRScore was positively correlated with RFS and favorable outcomes in ICB treatment. Moreover, we depicted a detailed immune profile in TME using mass cytometry and identified that CD103+CD39+ T cells, characterized by an exhaustive, cytotoxic and proliferative phenotype, were enriched in CRC patients with high IRScore. As a beneficial immune signature, CD103+CD39+ T cells could predict prognosis and responses to ICB therapy in CRC.
    Conclusions: All the analyses above revealed that IRScore could be a valuable tool for predicting prognosis and facilitating the development of new therapeutic strategies in CRC, and CD103+CD39+ T cells were one of defined immune signatures in IRScore, which might be a key factor for antitumor immunity.
    Keywords:  colorectal cancer; high-dimensional single-cell analysis; immune cell diversity; immunotherapy; prognosis
  24. Cancer Sci. 2022 Oct 30.
      Functional reprogramming of tumor-associated macrophages (TAMs) is crucial to their potent tumor-supportive capacity. However, the molecular mechanism behind the reprogramming process remains poorly understood. Here, we identify engulfment and cell motility protein 1 (ELMO1) as a crucial player for TAM reprogramming in colorectal cancer (CRC). The expression of ELMO1 in stromal but not epithelial tumor cells was positively associated with advanced clinical stage and poor disease-free survival in CRC. An increase in ELMO1 expression was specifically found in TAMs, but not in other multiple non-malignant stromal cells. Gain- and loss-of-function assays indicated ELMO1 reprogrammed macrophages to a TAM-like phenotype via Rac1 activation. In turn, ELMO1-reprogrammed macrophages were shown to not only facilitate the malignant behaviors of CRC cells but exhibited potent phagocytosis of tumor cells. Taken together, our work underscores the importance of ELMO1 in determining functional reprogramming of TAMs and may provide new insights on potential therapeutic strategies against CRC.
    Keywords:  Colorectal cancer; ELMO1; TAM reprogramming; Tumor-associated macrophages
  25. BMC Cancer. 2022 Oct 31. 22(1): 1113
      BACKGROUND: Overall survival of advanced colorectal cancer (CRC) patients remains poor, and gene expression analysis could potentially complement detection of clinically relevant mutations to personalize CRC treatments.METHODS: We performed RNA sequencing of formalin-fixed, paraffin-embedded (FFPE) cancer tissue samples of 23 CRC patients and interpreted the data obtained using bioinformatic method Oncobox for expression-based rating of targeted therapeutics. Oncobox ranks cancer drugs according to the efficiency score calculated using target genes expression and molecular pathway activation data. The patients had primary and metastatic CRC with metastases in liver, peritoneum, brain, adrenal gland, lymph nodes and ovary. Two patients had mutations in NRAS, seven others had mutated KRAS gene. Patients were treated by aflibercept, bevacizumab, bortezomib, cabozantinib, cetuximab, crizotinib, denosumab, panitumumab and regorafenib as monotherapy or in combination with chemotherapy, and information on the success of totally 39 lines of therapy was collected.
    RESULTS: Oncobox drug efficiency score was effective biomarker that could predict treatment outcomes in the experimental cohort (AUC 0.77 for all lines of therapy and 0.91 for the first line after tumor sampling). Separately for bevacizumab, it was effective in the experimental cohort (AUC 0.87) and in 3 independent literature CRC datasets, n = 107 (AUC 0.84-0.94). It also predicted progression-free survival in univariate (Hazard ratio 0.14) and multivariate (Hazard ratio 0.066) analyses. Difference in AUC scores evidences importance of using recent biosamples for the prediction quality.
    CONCLUSION: Our results suggest that RNA sequencing analysis of tumor FFPE materials may be helpful for personalizing prescriptions of targeted therapeutics in CRC.
    Keywords:  Bioinformatics; Biomarkers detection; Colorectal cancer; Molecular diagnostics; Oncobox; Personalized medicine; RNA sequencing; Second opinion system; Targeted therapy; Transcriptomics
  26. Thyroid. 2022 Nov 02.
      BACKGROUND: The gut is a target organ of thyroid hormone that exerts its action via the nuclear thyroid hormone receptor 1 (TR1) expressed in intestinal epithelial cells. Thyroid hormones are partially metabolized via hepatic sulfation and glucuronidation resulting in the production of conjugated iodothyronines. Gut microbiota play an important role in peripheral thyroid hormone metabolism as they produce and secrete enzymes with deconjugation activity (β-glucuronidase and sulfatase), via which TH can re-enter the enterohepatic circulation.SUMMARY: Intestinal epithelium homeostasis (the finely tuned balance between cell proliferation and differentiation) is controlled by the crosstalk between T3 and TRα1 and the presence of specific TH transporters and TH-activating and inactivating enzymes. Patients and experimental murine models with a dominant-negative mutation in the TR exhibit gross abnormalities in the morphology of the intestinal epithelium and suffer from severe symptoms of a dysfunctional gastrointestinal tract. Over the past decade, gut microbiota has been identified as an essential factor in health and disease, depending on its compositional and functional profile. This has led to a renewed interest in the so-called gut-thyroid axis. Disruption of gut microbial homeostasis (dysbiosis) is associated with autoimmune thyroid disease (AITD), including Hashimoto's thyroiditis (HT), Graves' disease (GD), and Graves' orbitopathy (GO). These studies reviewed here provide new insights into the gut microbiota roles in thyroid disease pathogenesis and may be an initial step toward microbiota-based therapies in AITD. However, it should be noted that cause-effect mechanisms remain to be proven, for which prospective cohort studies, randomized clinical trials, and experimental studies are needed.
    CONCLUSION: This review aims to provide a comprehensive insight into the interplay between thyroid hormone metabolism and gut homeostasis.
  27. Front Genet. 2022 ;13 1043297
      Background: Recent studies demonstrate that N6-methyladenosine (m6A) methylation plays a crucial role in colorectal cancer (CRC). Therefore, we conducted a comprehensive analysis to assess the m6A modification patterns and identify m6A-modified genes in patients with CRC recurrence. Methods: The m6A modification patterns were comprehensively evaluated by the NMF algorithm based on the levels of 27 m6A regulators, and tumor microenvironment (TME) cell-infiltrating characteristics of these modification patterns were systematically assessed by ssGSEA and CIBERSORT algorithms. The principal component analysis algorithm based on the m6A scoring scheme was used to explore the m6A modification patterns of individual tumors with immune responses. The weighted correlation network analysis and univariable and multivariable Cox regression analyses were applied to identify m6A-modified gene signatures. The single-cell expression dataset of CRC samples was used to explore the tumor microenvironment affected by these signatures. Results: Three distinct m6A modification patterns with significant recurrence-free survival (RFS) were identified in 804 CRC patients. The TME characterization revealed that the m6A modification pattern with longer RFS exhibited robust immune responses. CRC patients were divided into high- and low-score subgroups according to the m6A score individually, which was obtained from the m6A-related signature genes. The patients with low m6A scores had both longer RFS and overall survival (OS) with altered immune cell infiltration. Notably, m6A-modified genes showed significant differences related to the prognosis of CRC patients in the meta-GEO cohort and TCGA cohort. Single-cell expression indicated that ALVRL1 was centrally distributed in endothelial tip cells and stromal cells. Conclusion: The m6A modification plays an indispensable role in the formation of TME diversity and complexity. Importantly, the signatures (TOP2A, LRRC58, HAUS6, SMC4, ACVRL1, and KPNB1) were identified as m6A-modified genes associated with CRC recurrence, thereby serving as a promising predictive biomarker or therapeutic target for patients with CRC recurrence.
    Keywords:  colorectal cancer; m6A methylation modification; overall survival; recurrence; tumor immune microenvironment