bims-instec Biomed News
on Intestinal stem cells and chemoresistance in colon cancer and intestinal regeneration
Issue of 2022‒12‒18
sixteen papers selected by
Maria-Virginia Giolito
Free University of Brussels

  1. Acta Biochim Biophys Sin (Shanghai). 2022 Dec 25. 55(3): 1-11
      Neoadjuvant therapy (NAT) for advanced colorectal cancer (ACRC) is a kind of well-evidenced therapy, yet a portion of ACRC patients have poor therapeutic response. To date, no suitable biomarker used for assessing NAT efficacy has been reported. Here, we collect 72 colonoscopy biopsy tissue specimens from ACRC patients before undergoing NAT and investigate the relationship between HOXA13 expression and NAT efficacy. The results show that HOXA13 expression in pretreated tumor specimens is negatively associated with tumor regression ( P<0.001) and progression-free survival ( P<0.05) in ACRC patients who underwent NAT. Silencing of HOXA13 or its regulator HOTTIP significantly enhances the chemosensitivity of colorectal cancer (CRC) cells, leading to an increase in cell apoptosis and the DNA damage response (DDR) to chemotherapeutic drug treatment. In contrast, HOXA13 overexpression causes a significant increase in chemoresistance in CRC cells. In summary, we find that the HOTTIP/HOXA13 axis is involved in regulating chemotherapeutic sensitivity in CRC cells by modulating the DDR and that HOXA13 serves as a promising marker for NAT efficacy prediction in ACRC patients.
    Keywords:  DNA damage response; HOTTIP; HOXA13; colorectal cancer; neoadjuvant therapy
  2. Cell Mol Gastroenterol Hepatol. 2022 Dec 08. pii: S2352-345X(22)00249-1. [Epub ahead of print]
      BACKGROUND & AIMS: Although trimethylation of histone H3 lysine 27 (H3K27me3) by Polycomb Repressive Complex 2 is required for intestinal function, the role of the antagonistic process - H3K27me3 demethylation - in the intestine remains unknown. The aim of this study was to determine the contribution of H3K27me3 demethylases to intestinal homeostasis.METHODS: An inducible mouse model was employed to simultaneously ablate the two known H3K27me3 demethylases, lysine (K)-specific demethylase 6A (Kdm6a) and lysine (K)-specific demethylase 6B (Kdm6b), from the intestinal epithelium. Mice were analyzed at acute and prolonged timepoints following Kdm6a/b ablation. Cellular proliferation and differentiation were measured using immunohistochemistry, while RNA-Sequencing and chromatin immunoprecipitation followed by sequencing for H3K27me3 were employed to identify gene expression and chromatin changes following Kdm6a/b loss. Intestinal epithelial renewal was evaluated using a radiation-induced injury model, while Paneth cell homeostasis was measured via immunohistochemistry, immunoblot, and transmission electron microscopy.
    RESULTS: We did not detect any effect of Kdm6a/b ablation on intestinal cell proliferation or differentiation towards secretory cell lineages. Acute and prolonged Kdm6a/b loss perturbed expression of gene signatures belonging to multiple cell lineages (adjusted p-value<0.05), and a set of 72 genes were identified as being downregulated with an associated increase in H3K27me3 levels following Kdm6a/b ablation (FDR<0.05). Following prolonged Kdm6a/b loss, dysregulation of the Paneth cell gene signature was associated with perturbed MMP7 localization (p<0.0001) and expression.
    CONCLUSIONS: Although KDM6A/B may not regulate intestinal cell differentiation, both enzymes are required to support the full transcriptomic and epigenomic landscape of the intestinal epithelium and the expression of key Paneth cell genes.
    Keywords:  ChIP-Seq; KDM6A; KDM6B; Lgr5(+) ISCs; PRC2; Paneth cells
  3. Cell Mol Gastroenterol Hepatol. 2022 Dec 11. pii: S2352-345X(22)00252-1. [Epub ahead of print]
      BACKGROUND & AIMS: Glycoprotein (GP)96 is an endoplasmic reticulum (ER)-resident master chaperone for cell surface receptors including the Wnt co-receptors LRP5/6. Intestinal epithelial cells (IEC)-specific deletion of Gp96 is embryonically lethal. However, the role of GP96 in adult intestinal tissue and especially within the intestinal stem cell (ISC) niche has not been studied so far. Here, we investigated how GP96-loss interferes with intestinal homeostasis by compromising viability, proliferation and differentiation of IEC.METHODS: Tamoxifen was used to induce Cre-mediated deletion of Gp96 in GP96-VillincreERT2 mice and intestinal organoids. With H&E- and immunofluorescence staining we assessed alterations in intestinal morphology and the presence and localization of IEC-types. Real-time PCR and Western blot analysis were performed to explore the molecular mechanisms underlying the severe phenotype of Gp96 KO mice and organoids.
    RESULTS: IEC-specific deletion of Gp96 in adult mice resulted in a rapid degeneration of the stem cell niche, followed by a complete eradication of the epithelial layer and death within few days. These effects were due to severe defects in ISC renewal and premature ISC differentiation, which resulted from defective Wnt and Notch signaling. Furthermore, depletion of GP96 led to massive induction of ER stress. While effects on ISC renewal and adequate differentiation were partly reversed upon activation of Wnt/Notch signaling, viability could not be restored, indicating that reduced viability was mediated by other mechanisms.
    CONCLUSIONS: Our work demonstrates that GP96 plays a fundamental role in regulating ISC fate and epithelial regeneration and is therefore indispensable for maintaining intestinal epithelial homeostasis.
    Keywords:  ER stress; Intestinal stem cells; LRP6; Notch signaling; Wnt signaling
  4. Cell Rep. 2022 Dec 13. pii: S2211-1247(22)01684-9. [Epub ahead of print]41(11): 111796
      Intestinal epithelial replenishment is fueled by continuously dividing intestinal stem cells (ISCs) resident at the crypt niche. However, the cell type(s) enabling replenishment upon damage and subsequent loss of whole crypts remain largely unclear. Using Set domain-containing protein 4 (Setd4), we identify a small population with reserve stem cell characteristics in the mouse intestine. Upon irradiation-induced injury, Setd4-expressing (Setd4+) cells survive radiation exposure and then activate to produce Sca-1-expressing cell types to restore the epithelial wall and regenerate crypts de novo via crypt fission. Setd4+ cells are confirmed to originate from the early fetal period, subsequently contributing to the development of embryonic gut and the establishment of postnatal crypts. Setd4+ cells are therefore represented as both originators and key regenerators of the intestine.
    Keywords:  CP: Developmental biology; CP: Stem cell research; Setd4; crypt fission; crypt genesis; embryonic gut development; homeostasis; intestinal stem cells; postnatal gut development; regeneration; reserve stem cells
  5. Cell Death Dis. 2022 Dec 16. 13(12): 1049
      BAF53A, an important subunit of the SWI/SNF epigenetic chromatin regulatory complex, has been implicated as the driver of diverse cancers. However, the role of BAF53A in colorectal cancer (CRC) remains poorly understood. Here, we examined the expression of BAF53A in CRC samples and observed that BAF53A was significantly upregulated in CRC tissues compared with paired adjacent normal tissues. In vitro and in vivo studies suggested that ectopic expression of BAF53A promoted colorectal cancer cell proliferation, colony formation, and tumorigenesis, whereas knockdown of BAF53A hindered these cellular functions. DUSP5 (dual-specificity phosphatase 5), an ERK1/2-specific endogenous phosphatase, was expressed at low levels in CRC. We found a negative correlation between BAF53A and DUSP5 expression in a set of CRC samples. Mechanistic studies revealed that P63 was a potential transcription repressor of DUSP5. BAF53A could interact with P63, decreasing the DUSP5 expression level and subsequently promoting ERK1/2 phosphorylation. Thus, our study provides insights into the applicability of the BAF53A-DUSP5-ERK1/2 axis as a potential therapeutic target in CRC.
  6. Int J Mol Sci. 2022 Dec 06. pii: 15364. [Epub ahead of print]23(23):
      The mammalian intestinal epithelium contains more immune cells than any other tissue, and this is largely because of its constant exposure to pathogens. Macrophages are crucial for maintaining intestinal homeostasis, but they also play a central role in chronic pathologies of the digestive system. We developed a versatile microwell-based intestinal organoid-macrophage co-culture system that enables us to recapitulate features of intestinal inflammation. This microwell-based platform facilitates the controlled positioning of cells in different configurations, continuous in situ monitoring of cell interactions, and high-throughput downstream applications. Using this novel system, we compared the inflammatory response when intestinal organoids were co-cultured with macrophages versus when intestinal organoids were treated with the pro-inflammatory cytokine TNF-α. Furthermore, we demonstrated that the tissue-specific response differs according to the physical distance between the organoids and the macrophages and that the intestinal organoids show an immunomodulatory competence. Our novel microwell-based intestinal organoid model incorporating acellular and cellular components of the immune system can pave the way to unravel unknown mechanisms related to intestinal homeostasis and disorders.
    Keywords:  co-culture; immune system; intestinal organoids; macrophages; microwells
  7. J Cancer Res Ther. 2022 Dec;18(Supplement): S420-S427
      Background: CDX2, a nuclear protein, is essential for the proliferation and development of intestinal epithelial cells and is frequently down-regulated during tumorigenesis. We have evaluated the pattern of CDX2 expression in all stages of colorectal cancer (CRC) and its association with prognosis.Methods: We performed CDX2 staining by immunohistochemistry (IHC) on the available biopsy blocks of patients of CRC registered in our institute from January 2014 to January 2018. CDX2 scoring was done using the semi-quantitative method.
    Results: A total of 286 patients were registered during the study period, of which only 110 biopsy blocks were available for staining. Of 110 patients, 77 (70%) had colon cancer and 33 (30%) had rectal cancer. The median age was 54.2 years, with 62 (56.4%) being male and 48 (43.6%) female with a male to female ratio of 1.3:1. In the study cohort, 33 (30%) patients had stage II disease, 30 (27.3%) had stage III, and 47 (42.7%) had stage IV. Seventy-three (66.4%) were positive for CDX2 and 37 (33.4%) were negative. Loss of CDX2 expression was significantly associated with advanced stage, rectal site, poor grade of differentiation, and presence of lymphovascular invasion. With a median follow-up of 16 months, progression-free survival (PFS) at 2 years was 30% for CDX2-negative patients compared to 67% for CDX2-positive patients (P = 0.009), whereas the overall survival (OS) at 2 years was 46% for CDX2-negative versus 77% for CDX2-positive patients (P = 0.01).
    Conclusion: Loss of CDX2 expression is associated with advanced stage, higher tumor grade, presence of LVSI, worse PFS, and OS and thereby functions as a poor prognostic factor in CRC.
    Keywords:  CDX2; colorectal cancer; pattern; prognosis; survival
  8. Immunity. 2022 Dec 13. pii: S1074-7613(22)00596-9. [Epub ahead of print]55(12): 2228-2230
      Diverse intestinal components (e.g., gut-associated neurons, immune cells, gut microbes, and epithelium) are intimately intertwined with each other to maintain homeostasis in the gut. In a recent issue of Cell, Zhang et al. (2022) and Yang et al. (2022) present complementary studies uncovering interactions between nociceptor neurons, gut epithelium, and the microbiome to protect intestinal tissue from inflammation.
  9. Cancer Res. 2022 Dec 16. pii: CAN-22-1837. [Epub ahead of print]
      Immunotherapy can elicit robust anticancer responses in the clinic. However, a large proportion of patients with colorectal cancer (CRC) do not benefit from treatment. While previous studies have shown that hydrogen sulfide (H2S) is involved in CRC development and immune escape, further insights into the mechanisms and related molecules are needed to identify approaches to reverse the tumor supportive functions of H2S. Here, we observed significantly increased H2S levels in CRC tissues. Decreasing H2S levels by using CBS+/- mice or feeding mice a sulfur amino acid-restricted diet (SARD) led to a marked decrease in differentiated CD4+CD25+Foxp3+ Tregs and an increase in the CD8+ T cell/Treg ratio. Endogenous or exogenous H2S depletion enhanced the efficacy of anti-PD-L1 and anti-CTLA-4 treatment. H2S promoted Treg activation through persulfidation of ENO1 at cysteine 119. Furthermore, H2S inhibited the migration of CD8+ T cells by increasing the expression of AAK-1 via ELK4 persulfidation at cysteine 25. Overall, reducing H2S levels engenders a favorable immune microenvironment in CRC by decreasing the persulfidation of ENO1 in Tregs and ELK4 in CD8+ T cells. SARD represents a potential dietary approach to promote responses to immunotherapies in CRC.
  10. Am J Transl Res. 2022 ;14(11): 7758-7770
      OBJECTIVE: The molecular mechanisms underlying tumor progression and drug resistance in colorectal cancer remain to be fully understood. Recent studies have reported a pro-tumorigenic role of an amino acid oxidase named interleukin-4-induced-1 (IL4I1). Here, we investigate the role and molecular mechanism of IL4I1 in colorectal cancer.METHODS: We employed bioinformatics analysis and experimental validation by using clinical samples and a variety of cell-based assays, including western blot, Transwell assay, patient-derived organoid culture, Immunofluorescence assay, T cell cytotoxicity assay, and flow cytometry.
    RESULTS: Bioinformatics analysis showed a higher IL4I1 expression in colorectal cancer tissues than in normal tissues. In vitro overexpression of IL4I1 enhanced the proliferation, migration, and invasion of colorectal cancer cells. In addition, deprivation of Tryptophan (Trp) in cultural medium diminished the oncogenic effect of IL4I1. Furthermore, we observed a positive correlation of IL4I1 and AHR expression in the TCGA database of colorectal cancer. We also detected an enhanced cytoplasmic expression and nuclear translocation of Aryl hydrocarbon receptor (AHR). Moreover, IL4I1 overexpression suppressed the cytolytic killing of tumor cells and enhanced T cell exhaustion. Finally, in the organoid culture model, we found that immunotherapy and SR-1 combination treatment could induce higher level of apoptosis than did the immunotherapy or SR-1 treatment alone.
    CONCLUSION: we demonstrated that IL4I1 facilitated colorectal cancer progression and immunosuppression through tryptophan metabolism dependent on AHR activation.
    Keywords:  AHR; IL4I1; T cell exhaustion; colorectal cancer; immunosuppression; pro-tumorigenic
  11. Cancer Res. 2022 Dec 13. pii: CAN-22-2145. [Epub ahead of print]
      Metabolic reprogramming can contribute to colorectal cancer (CRC) progression and therapy resistance. Identification of key regulators of CRC metabolism could provide new approaches to improve treatment and reduce recurrence. Here, we demonstrate a critical role for the COP9 signalosome subunit CSN6 in rewiring nucleotide metabolism in CRC. Transcriptomic analysis of CRC patient samples revealed a correlation between CSN6 expression and purine and pyrimidine metabolism. A colitis-associated colorectal cancer model established that Csn6 intestinal conditional deletion decreased tumor development and altered nucleotide metabolism. CSN6 knockdown increased the chemosensitivity of CRC cells in vitro and in vivo, which could be partially reversed with nucleoside supplementation. Isotope metabolite tracing showed that CSN6 loss reduced de novo nucleotide synthesis. Mechanistically, CSN6 upregulated purine and pyrimidine biosynthesis by increasing expression of PHGDH, a key enzyme in the serine synthesis pathway. CSN6 inhibited β-Trcp-mediated DDX5 polyubiquitination and degradation, which in turn promoted DDX5-mediated PHGDH mRNA stabilization, leading to metabolic reprogramming and CRC progression. Butyrate treatment decreased CSN6 expression and improved chemotherapy efficacy. These findings unravel the oncogenic role of CSN6 in regulating nucleotide metabolism and chemosensitivity in CRC.
  12. Int J Mol Sci. 2022 Dec 03. pii: 15232. [Epub ahead of print]23(23):
      Colorectal cancer is a serious threat to human health. Poor prognosis and frequently reported drug resistance urges research into novel biomarkers and mechanisms to aid in the understanding of the development and progression of colorectal cancer and to optimise therapeutic strategies. In the current study, we investigated the roles of a putative tumour suppressor, EPLIN, in colorectal cancer. Our clinical colorectal cancer cohort and online databases revealed a downregulation of EPLIN in colorectal cancer tissues compared with normal tissues. The reduced expression of EPLIN was associated with poor clinical outcomes of patients. In vitro cellular function assays showed that EPLIN elicited an inhibitory effect on cellular growth, adhesion, migration and invasion. Utilising a protein microarray on protein samples from normal and tumour patient tissues suggested HSP60, Her2 and other signalling events were novel potential interacting partners of EPLIN. It was further revealed that EPLIN and HSP60 were negative regulators of Her2 in colorectal cancer cells. The clinical cohort also demonstrated that expression of HSP60 and Her2 affected clinical outcomes, but most interestingly the combination of EPLIN, HSP60 and Her2 was able to identify patients with the most unfavourable clinical outcome by independently predicting patient overall survival and disease free survival. Furthermore, EPLIN and HSP60 exhibited potential to regulate cellular response to chemotherapeutic and EGFR/Her2 targeted therapeutic agents. In conclusion, EPLIN is an important prognostic factor for patients with colon cancer and reduced EPLIN in CRC contributes to aggressive traits of CRC cells and their responses to chemotherapeutic drugs. Collectively, EPLIN is a pivotal factor for the development and progression of colorectal cancer and has important clinical and therapeutic values in this cancer type.
    Keywords:  EPLIN; HSP60; Her2; colorectal cancer; drug resistance
  13. NPJ Aging. 2022 Dec 16. 8(1): 16
      Intestinal epithelial cells are critical for gastrointestinal homeostasis. However, their function declines during aging. The aging-related loss of organ performance is largely driven by the increase in senescent cells. To date, the hallmarks and molecular mechanisms related to cellular senescence are not fully understood. Microtubules control epithelial functions, and we identified microtubule stabilization as a phenotypic marker of senescent intestinal epithelial cells. The senescence inducer determined the pathway to microtubule stabilization. Specifically, enhanced microtubule stability was associated with α-tubulin hyperacetylation or increased abundance of the microtubule-binding protein tau. We show further that overexpression of MAPT, which encodes tau, augmented microtubule stability in intestinal epithelial cells. Notably, pharmacological microtubule stabilization was sufficient to induce cellular senescence. Taken together, this study provides new insights into the molecular mechanisms that control epithelial cell homeostasis. Our results support the concept that microtubule stability serves as a critical cue to trigger intestinal epithelial cell senescence.
  14. EMBO J. 2022 Dec 14. e111673
      Adenosine N6-methylation (m6A) and N6,2'-O-dimethylation (m6Am) are regulatory modifications of eukaryotic mRNAs. m6Am formation is catalyzed by the methyl transferase phosphorylated CTD-interacting factor 1 (PCIF1); however, the pathophysiological functions of this RNA modification and PCIF1 in cancers are unclear. Here, we show that PCIF1 expression is upregulated in colorectal cancer (CRC) and negatively correlates with patient survival. CRISPR/Cas9-mediated depletion of PCIF1 in human CRC cells leads to loss of cell migration, invasion, and colony formation in vitro and loss of tumor growth in athymic mice. Pcif1 knockout in murine CRC cells inhibits tumor growth in immunocompetent mice and enhances the effects of anti-PD-1 antibody treatment by decreasing intratumoral TGF-β levels and increasing intratumoral IFN-γ, TNF-α levels, and tumor-infiltrating natural killer cells. We further show that PCIF1 modulates CRC growth and response to anti-PD-1 in a context-dependent mechanism with PCIF1 directly targeting FOS, IFITM3, and STAT1 via m6Am modifications. PCIF1 stabilizes FOS mRNA, which in turn leads to FOS-dependent TGF-β regulation and tumor growth. While during immunotherapy, Pcif1-Fos-TGF-β, as well as Pcif1-Stat1/Ifitm3-IFN-γ axes, contributes to the resistance of anti-PD-1 therapy. Collectively, our findings reveal a role of PCIF1 in promoting CRC tumorigenesis and resistance to anti-PD-1 therapy, supporting that the combination of PCIF1 inhibition with anti-PD-1 treatment is a potential therapeutic strategy to enhance CRC response to immunotherapy. Finally, we developed a lipid nanoparticles (LNPs) and chemically modified small interfering RNAs (CMsiRNAs)-based strategy to silence PCIF1 in vivo and found that this treatment significantly reduced tumor growth in mice. Our results therefore provide a proof-of-concept for tumor growth suppression using LNP-CMsiRNA to silence target genes in cancer.
    Keywords:  NK cells; PCIF1; anti-PD-1 treatment; colorectal carcinoma; m6Am methylation
  15. Pharmacol Ther. 2022 Dec 13. pii: S0163-7258(22)00226-1. [Epub ahead of print] 108332
      Despite significant advances in the screening, diagnosis, and treatment of colorectal cancer (CRC) immune checkpoint inhibitors (ICIs) continue to have limited utility outside of microsatellite-high disease. Given the durable response to immunotherapy seen across malignancies, increasing CRC response rates to ICI therapy is an active area of clinical research. An increasing body of work has demonstrated that tumor-derived extracellular vesicles (TEVs) are key modulators in tumor signaling and the determinants of the tumor microenvironment. Pre-clinical models have shown that TEVs are directly involved in antigen presentation and are involved in radiation-induced DNA damage signaling. Both direct and indirect modifications of these TEVs can alter CRC immunogenicity and ICI treatment response, making them attractive targets for potential therapeutic development. In addition, modified TEVs can be developed using several different mechanisms, with varied cargo including micro-RNAs and small peptide molecules. Recent work has shown strong pre-clinical evidence of injected modified TEV-induced ICI activity, with knockdown of the micro-RNA miR-424 in TEVs improving CRC immunogenicity and increasing anti-PD-1 activity in mouse models. Clinical trials are ongoing in the evaluation of modified TEVs in cancer therapy, but they appear to be a promising therapeutic target in CRC.
    Keywords:  Colorectal cancer; Immunotherapy; Tumor-derived extracellular vesicles; micro-RNA
  16. J Am Coll Surg. 2023 Jan 01. 236(1): 126-134
      INTRODUCTION: Infiltrating tumor border configuration (ITBC) portends a poor prognosis compared with pushing tumor border configuration (PTBC) in colorectal cancer. The tumor and its surrounding immune microenvironment of tumor border configuration is not well-characterized. We aim to elucidate the differences in expression of molecular markers between the 2 groups using tissue microarray (TMA).STUDY DESIGN: Immunohistochemistry was performed on TMAs of surgical pathology specimens obtained from colorectal cancer patients consecutively operated at our institution from 2004 to 2015. TMAs were stained for immune cells (CD8, FOXP3, LAG3, PU1, CD163, and PDL1); HLA II, beta 2 microglobulin, and HC10 on tumor cells; BRAFV600E mutation; and DNA mismatch repair proteins (MMR) status. Patients who received neoadjuvant therapy were excluded.
    RESULTS: There were 646 tumors with ITBC and 310 tumors with PTBC. There was a significantly lower expression (p < 0.05) of immune components, namely CD8, FOXP3, LAG3, PU1, PDL1 immune cells, and Beta-2 Microglobulin on tumor cells in the tumors with ITBC compared with PTBC, except CD163 immune cells, and HC10 and HLAII on tumor cells. Tumors with ITBC were less likely to be associated with BRAFV600E mutations and deficient MMR proteins (p < 0.001). On analyzing MMR-proficient tumors separately, we could not find any difference in the expression of any molecular marker (including BRAF), except a lower expression of PDL1 immune cells in tumors with ITBC (p < 0.001).
    CONCLUSIONS: Colorectal tumors with ITBC are associated with a generalized low immune microenvironment and low rates of BRAFV600E mutation compared with tumors with PTBC. However, the molecular expression of tumor border configuration seems confounded by the MMR molecular signature. MMR-proficient colorectal tumors with ITBC are associated with a lower expression of only PDL1 immune cells among all immune markers examined.