bims-instec 2025-01-05 papers

bims-instec

Biomed News

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

Issue of 2025–01–05
nineteen papers selected by
Maria-Virginia Giolito, Université Catholique de Louvain

Enterocyte-like differentiation defines metabolic gene signatures of CMS3 colorectal cancers and provides therapeutic vulnerability.
STAT1 regulates immune-mediated intestinal stem cell proliferation and epithelial regeneration.
GPLD1+ cancer stem cells contribute to chemotherapy resistance and tumor relapse in intestinal cancer.
Nicotine enhances the stemness and tumorigenicity in intestinal stem cells via Hippo-YAP/TAZ and Notch signal pathway.
A tunable human intestinal organoid system achieves controlled balance between self-renewal and differentiation.
PSAT1 promotes the progression of colorectal cancer by regulating Hippo-YAP/TAZ-ID1 axis via AMOT.
Knockdown of RFC3 enhances the sensitivity of colon cancer cells to oxaliplatin by inducing ferroptosis.
Glutamic-pyruvic transaminase 1 deficiency-mediated metabolic reprogramming facilitates colorectal adenoma-carcinoma progression.
THBS2 + cancer-associated fibroblasts promote EMT leading to oxaliplatin resistance via COL8A1-mediated PI3K/AKT activation in colorectal cancer.
Intestinal organoids: The path towards clinical application.
Gasdermin D regulates the activation of EGFR in colorectal cancer.
Fibroblasts modulate epithelial cell behavior within the proliferative niche and differentiated cell zone within a human colonic crypt model.
Loss of Mist1 alters the characteristics of Paneth cells and impacts the function of intestinal stem cells in physiological conditions and after radiation injury.
Nucleus-translocated GCLM promotes chemoresistance in colorectal cancer through a moonlighting function.
TYMS Enhances Colorectal Cell Antioxidant Capacity Via the KEAP1-NRF2 Pathway to Resist Ferroptosis.
Eicosapentaenoic acid enhances intestinal stem cell-mediated colonic epithelial regeneration by activating the LSD1-WNT signaling pathway.
Co-evolution of atypical BRAF and KRAS mutations in colorectal tumorigenesis.
Enrichment of Cancer-Associated Fibroblasts, Macrophages, and Up-Regulated TNF-α Signaling in the Tumor Microenvironment of CMS4 Colorectal Peritoneal Metastasis.
Single-cell sequencing analysis reveals cetuximab resistance mechanism and salvage strategy in colorectal cancer.

Nat Commun. 2025 Jan 02. 16(1): 264

Enterocyte-like differentiation defines metabolic gene signatures of CMS3 colorectal cancers and provides therapeutic vulnerability.

Arezo Torang, Aleksandar B Kirov, Veerle Lammers, Kate Cameron, Valérie M Wouters, Rene F Jackstadt, Tamsin R M Lannagan, Joan H de Jong, Jan Koster, Owen Sansom, Jan Paul Medema.

Colorectal cancer (CRC) is stratified into four consensus molecular subtypes (CMS1-4). CMS3 represents the metabolic subtype, but its wiring remains largely undefined. To identify the underlying tumorigenesis of CMS3, organoids derived from 16 genetically engineered mouse models are analyzed. Upon in vitro Cre-recombinase activation, transformation is established and transcriptional profiling reveals that distinct CMSs (CMS2-4) are modeled with different organoids. CMS3-like, metabolic signature-positive, organoids are induced by KRAS mutations. Interestingly, metabolic signatures are subsequently shown to result from enterocyte-like differentiation both in organoids and human cancers. Further analysis reveals carbamoyl-phosphate synthase 1 (CPS1) and sucrase-isomaltase (SI) as signature proteins. More importantly, CPS1 is crucial for de novo pyrimidine synthesis in CMS3 and its inhibition targets proliferation and stemness, facilitating enterocyte-like differentiation, while CMS2 and CMS4 models are not affected. Our data point to an enterocyte-like differentiation of CMS3 CRCs and reveal a selective vulnerability of this subtype through CPS1 inhibition.

DOI: https://doi.org/10.1038/s41467-024-55574-3
Nat Commun. 2025 Jan 02. 16(1): 138

STAT1 regulates immune-mediated intestinal stem cell proliferation and epithelial regeneration.

Shuichiro Takashima, Roshan Sharma, Winston Chang, Marco Calafiore, Ya-Yuan Fu, Suze A Jansen, Takahiro Ito, Anastasiya Egorova, Jason Kuttiyara, Viktor Arnhold, Jessica Sharrock, Endi Santosa, Ojasvi Chaudhary, Heather Geiger, Hiromi Iwasaki, Chen Liu, Joseph Sun, Nicolas Robine, Linas Mazutis, Caroline A Lindemans, Alan M Hanash.

The role of the immune system in regulating tissue stem cells remains poorly understood, as does the relationship between immune-mediated tissue damage and regeneration. Graft vs. host disease (GVHD) occurring after allogeneic bone marrow transplantation (allo-BMT) involves immune-mediated damage to the intestinal epithelium and its stem cell compartment. To assess impacts of T-cell-driven injury on distinct epithelial constituents, we have performed single cell RNA sequencing on intestinal crypts following experimental BMT. Intestinal stem cells (ISCs) from GVHD mice have exhibited global transcriptomic changes associated with a substantial Interferon-γ response and upregulation of STAT1. To determine its role in crypt function, STAT1 has been deleted within murine intestinal epithelium. Following allo-BMT, STAT1 deficiency has resulted in reduced epithelial proliferation and impaired ISC recovery. Similarly, epithelial Interferon-γ receptor deletion has also attenuated proliferation and ISC recovery post-transplant. Investigating the mechanistic basis underlying this epithelial response, ISC STAT1 expression in GVHD has been found to correlate with upregulation of ISC c-Myc. Furthermore, activated T cells have stimulated Interferon-γ-dependent epithelial regeneration in co-cultured organoids, and Interferon-γ has directly induced STAT1-dependent c-Myc expression and ISC proliferation. These findings illustrate immunologic regulation of a core tissue stem cell program after damage and support a role for Interferon-γ as a direct contributor to epithelial regeneration.

DOI: https://doi.org/10.1038/s41467-024-55227-5
J Biochem. 2025 Jan 02. pii: mvae082. [Epub ahead of print]

GPLD1+ cancer stem cells contribute to chemotherapy resistance and tumor relapse in intestinal cancer.

Taisuke Mizoo, Takeru Oka, Osamu Sugahara, Takafumi Minato, Tsunaki Higa, Keiichi I Nakayama.

  Cancer stem cells (CSCs) play a central role in cancer progression, therapy resistance, and disease recurrence. With the use of a quadruple-mutant mouse intestinal cancer organoid model and single-cell RNA-sequencing analysis, we have now identified glycosylphosphatidylinositol-specific phospholipase D1 (GPLD1), an enzyme that catalyzes the cleavage of glycosylphosphatidylinositol (GPI) anchors of membrane proteins, as a marker of slowly cycling CSCs. Ablation of Gpld1+ cells in combination with 5-fluorouracil treatment greatly attenuated cell viability in and regrowth of the intestinal cancer organoids. In addition, we identified serine protease 8 (PRSS8) as a key substrate of GPLD1 in human colorectal cancer cells. GPLD1 cleaves the GPI anchor of PRSS8 and thereby mediates release of the protease from the plasma membrane, resulting in the activation of Wnt signaling and promotion of the epithelial-mesenchymal transition (EMT) in the cancer cells. Pharmacological inhibition of GPLD1 suppressed Wnt signaling activity and EMT in association with upregulation of the amount of functional PRSS8 at the plasma membrane. Our findings suggest that targeting of GPLD1 in colorectal cancer might contribute to a new therapeutic strategy that is based on suppression of Wnt signaling and EMT-related cancer progression driven by CSCs.

Keywords:  Gpld1; Prss8; cancer stem cell; chemotherapy resistance; intestinal cancer

DOI:  https://doi.org/10.1093/jb/mvae082
Elife. 2025 Jan 03. pii: RP95267. [Epub ahead of print]13

Nicotine enhances the stemness and tumorigenicity in intestinal stem cells via Hippo-YAP/TAZ and Notch signal pathway.

Ryosuke Isotani, Masaki Igarashi, Masaomi Miura, Kyoko Naruse, Satoshi Kuranami, Manami Katoh, Seitaro Nomura, Toshimasa Yamauchi.

  Cigarette smoking is a well-known risk factor inducing the development and progression of various diseases. Nicotine (NIC) is the major constituent of cigarette smoke. However, knowledge of the mechanism underlying the NIC-regulated stem cell functions is limited. In this study, we demonstrate that NIC increases the abundance and proliferative activity of murine intestinal stem cells (ISCs) in vivo and ex vivo. Moreover, NIC induces Yes-associated protein (YAP) /Transcriptional coactivator with PDZ-binding motif (TAZ) and Notch signaling in ISCs via α7-nicotinic acetylcholine receptor (nAchR) and protein kinase C (PKC) activation; this effect was not detected in Paneth cells. The inhibition of Notch signaling by dibenzazepine (DBZ) nullified the effects of NIC on ISCs. NIC enhances in vivo tumor formation from ISCs after loss of the tumor suppressor gene Apc, DBZ inhibited NIC-induced tumor growth. Hence, this study identifies a NIC-triggered pathway regulating the stemness and tumorigenicity of ISCs and suggests the use of DBZ as a potential therapeutic strategy for treating intestinal tumors.

Keywords:  Notch; YAP/TAZ; intestinal stem cell; mouse; nAChR; nicotine; paneth cell; regenerative medicine; stem cells

DOI:  https://doi.org/10.7554/eLife.95267
Nat Commun. 2025 Jan 02. 16(1): 315

A tunable human intestinal organoid system achieves controlled balance between self-renewal and differentiation.

Li Yang, Xulei Wang, Xingyu Zhou, Hongyu Chen, Sentao Song, Liling Deng, Yao Yao, Xiaolei Yin.

A balance between stem cell self-renewal and differentiation is required to maintain concurrent proliferation and cellular diversification in organoids; however, this has proven difficult in homogeneous cultures devoid of in vivo spatial niche gradients for adult stem cell-derived organoids. In this study, we leverage a combination of small molecule pathway modulators to enhance the stemness of organoid stem cells, thereby amplifying their differentiation potential and subsequently increasing cellular diversity within human intestinal organoids without the need for artificial spatial or temporal signaling gradients. Moreover, we demonstrate that this balance between self-renewal and differentiation can be effectively and reversibly shifted from secretory cell differentiation to the enterocyte lineage with enhanced proliferation using BET inhibitors, or unidirectional differentiation towards specific intestinal cell types by manipulating in vivo niche signals such as Wnt, Notch, and BMP. As a result, we establish an optimized human small intestinal organoid (hSIO) system characterized by high proliferative capacity and increased cell diversity under a single culture condition. This optimization facilitates the scalability and utility of the organoid system in high-throughput applications.

DOI: https://doi.org/10.1038/s41467-024-55567-2
Mol Cell Biochem. 2024 Dec 31.

PSAT1 promotes the progression of colorectal cancer by regulating Hippo-YAP/TAZ-ID1 axis via AMOT.

Minshan Tang, Kai Song, Danning Xie, Xinyu Yuan, Yaxuan Wang, Zhiyang Li, Xiansheng Lu, Liang Guo, Xiaotong Zhu, Le Xiong, Wenqian Zhou, Jie Lin.

  Colorectal cancer (CRC) ranks third for morbidity and second for mortality among all digestive malignant tumors worldwide, but its pathogenesis remains not entirely clear. Bioinformatic analyses were performed to find out important biomarkers for CRC. For validation, reverse transcription-quantitative PCR, western blotting, and immunohistochemistry were performed. Then, cell transfection, gain- and loss-of-function assays, immunofluorescence, cell line RNA-sequencing and analyses, and in vivo tumorigenesis assay were also performed to further explore the mechanism. We prioritized phosphoserine aminotransferase 1 (PSAT1) as an important biomarker in CRC. PSAT1 expression was gradually up-regulated as the CRC disease progresses and may relate to poor prognosis. PSAT1 promoted the malignant behaviors of CRC cells. Although PSAT1 is an enzyme essential to serine biosynthesis, an exogenous supplement of serine did not completely rescue the malignant behaviors in PSAT1-knockdown CRC cells. Interestingly, PSAT1 inhibited the Hippo tumor-suppressor pathway by promoting the nucleus-localization of YAP/TAZ and increasing the expression of ID1 in CRC cells. Furthermore, AMOT, a vascular-related molecule that molecularly interacts with YAP/TAZ, was up-regulated upon PSAT1 knockdown in CRC cells. Knocking down AMOT partially rescued the inhibition of proliferation and the reduced nuclear localization of YAP/TAZ caused by PSAT1 knockdown in CRC cells. Moreover, PSAT1 was closely related to vascular-related pathways, in which AMOT might act as a mediator. Finally, PSAT1 promoted CRC proliferation by negatively regulating AMOT in vivo. PSAT1 could enhance the progression of colorectal cancer by regulating Hippo-YAP/TAZ-ID1 axis via AMOT, which is independent of the metabolic function of PSAT1.

Keywords:  AMOT; Colorectal cancer; Hippo pathway; PSAT1; Serine synthesis; Tumor progression

DOI:  https://doi.org/10.1007/s11010-024-05194-8
Fundam Clin Pharmacol. 2025 Feb;39(1): e13044

Knockdown of RFC3 enhances the sensitivity of colon cancer cells to oxaliplatin by inducing ferroptosis.

Youyi Wu, Tingting Chen, Songsong Wu, Yiwei Huang, Fuyao Li.

   BACKGROUND: The development of resistance to oxaliplatin is a multifaceted process, often involving modifications in drug transport, DNA repair mechanisms, and the ability of cells to evade drug-induced apoptosis.
OBJECTIVE: To evaluate whether knocking down RFC3 promotes the sensitivity of colorectal cancer (CRC) cells to oxaliplatin, potentially offering a new approach to combat drug resistance.
METHODS: siRNA-mediated knockdown of RFC3 was employed in colorectal cancer cell lines to assess the impact on oxaliplatin responsiveness. Cell viability assays, clonogenic survival assays, and flow cytometry were conducted to evaluate the effects on cell growth and apoptosis. Additionally, immunoblot analysis was used to scrutinize modifications in the expression of pivotal protein expression in the Wnt/β-catenin/GPX4 axis.
RESULTS: RFC3 is highly expressed in CRC tissues and associated with prognosis. Knocking down RFC3 enhances the sensitivity of CRC cells to oxaliplatin. Additionally, the reduction of RFC3 promotes the susceptibility of chemoresistant tumor cells to oxaliplatin by inducing ferroptosis. Furthermore, the knockdown of RFC3 disrupts the Wnt/β-catenin/GPX4 axis.
CONCLUSION: Depletion of RFC3 enhances the sensitivity of CRC cells to oxaliplatin via inducing ferroptosis.

Keywords:  Ferroptosis; RFC3; Wnt/β‐catenin/GPX4; colorectal cancer (CRC); oxaliplatin

DOI:  https://doi.org/10.1111/fcp.13044
Sci Transl Med. 2025 Jan;17(779): eadp9805

Glutamic-pyruvic transaminase 1 deficiency-mediated metabolic reprogramming facilitates colorectal adenoma-carcinoma progression.

Li Xiong, Xin Yang, Huashan Liu, Xianrui Wu, Tanxing Cai, Ming Yuan, Liang Huang, Chi Zhou, Xiaobin Zheng, Wenxin Li, Ziwei Zeng, Shujuan Li, Ping Lan, Liang Kang, Zhenxing Liang.

The tumorigenesis of colorectal cancer (CRC) often follows the normal-adenoma-carcinoma (N-A-C) sequence. However, the molecular mechanisms underlying colorectal adenoma carcinogenesis remain largely unknown. Here, we analyzed transcriptomic profile changes in normal, advanced adenoma, and carcinoma tissues from patients with CRC, revealing that glutamic-pyruvic transaminase 1 (GPT1) in colorectal tissues was down-regulated during the N-A-C process and correlated with poor CRC prognosis. Mechanistically, GPT1 was transcriptionally activated by Krüppel-like factor 4 (KLF4). GPT1 reprogrammed metabolism and suppressed CRC tumorigenesis in cells and mouse models not only through enzyme-dependent α-ketoglutarate (α-KG) production and WNT signaling inhibition but also through enzyme-independent disruption of the folate cycle through binding with methylenetetrahydrofolate dehydrogenase 1-like (MTHFD1L). Furthermore, we identified poliumoside as a GPT1 activator that restrained CRC progression in cells, patient-derived CRC organoids, and patient-derived xenograft (PDX) models of CRC. Our study uncovers a role for GPT1 in CRC tumorigenesis and shows that poliumoside is a potential drug for the prevention and treatment of CRC.

DOI: https://doi.org/10.1126/scitranslmed.adp9805
Mol Cancer. 2024 Dec 28. 23(1): 282

THBS2 + cancer-associated fibroblasts promote EMT leading to oxaliplatin resistance via COL8A1-mediated PI3K/AKT activation in colorectal cancer.

Xing Zhou, Jiashu Han, Anning Zuo, Yuhao Ba, Shutong Liu, Hui Xu, Yuyuan Zhang, Siyuan Weng, Zhaokai Zhou, Long Liu, Peng Luo, Quan Cheng, Chuhan Zhang, Yukang Chen, Dan Shan, Benyu Liu, Shuaixi Yang, Xinwei Han, Jinhai Deng, Zaoqu Liu.

  Cancer-associated fibroblasts (CAFs) exert multiple tumor-promoting functions and are key contributors to drug resistance. The mechanisms by which specific subsets of CAFs facilitate oxaliplatin resistance in colorectal cancer (CRC) have not been fully explored. This study found that THBS2 is positively associated with CAF activation, epithelial-mesenchymal transition (EMT), and chemoresistance at the pan-cancer level. Together with single-cell RNA sequencing and spatial transcriptomics analyses, we identified THBS2 specifically derived from subsets of CAFs, termed THBS2 + CAFs, which could promote oxaliplatin resistance by interacting with malignant cells via the collagen pathway in CRC. Mechanistically, COL8A1 specifically secreted from THBS2 + CAFs directly interacts with the ITGB1 receptor on resistant malignant cells, activating the PI3K-AKT signaling pathway and promoting EMT, ultimately leading to oxaliplatin resistance in CRC. Moreover, elevated COL8A1 promotes EMT and contributes to CRC oxaliplatin resistance, which can be mitigated by ITGB1 knockdown or AKT inhibitor. Collectively, these results highlight the crucial role of THBS2 + CAFs in promoting oxaliplatin resistance of CRC by activating EMT and provide a rationale for a novel strategy to overcome oxaliplatin resistance in CRC.

Keywords:  COL8A1; Cancer-associated fibroblasts; Colorectal cancer; EMT; Oxaliplatin resistance

DOI:  https://doi.org/10.1186/s12943-024-02180-y
Eur J Cell Biol. 2024 Dec 25. pii: S0171-9335(24)00091-8. [Epub ahead of print]104(1): 151474

Intestinal organoids: The path towards clinical application.

Hady Yuki Sugihara, Ryuichi Okamoto, Tomohiro Mizutani.

  Organoids have revolutionized the whole field of biology with their ability to model complex three-dimensional human organs in vitro. Intestinal organoids were especially consequential as the first successful long-term culture of intestinal stem cells, which raised hopes for translational medical applications. Despite significant contributions to basic research, challenges remain to develop intestinal organoids into clinical tools for diagnosis, prognosis, and therapy. In this review, we outline the current state of translational research involving adult stem cell and pluripotent stem cell derived intestinal organoids, highlighting the advances and limitations in disease modeling, drug-screening, personalized medicine, and stem cell therapy. Preclinical studies have demonstrated a remarkable functional recapitulation of infectious and genetic diseases, and there is mounting evidence for the reliability of intestinal organoids as a patient-specific avatar. Breakthroughs now allow the generation of structurally and cellularly complex intestinal models to better capture a wider range of intestinal pathophysiology. As the field develops and evolves, there is a need for standardized frameworks for generation, culture, storage, and analysis of intestinal organoids to ensure reproducibility, comparability, and interpretability of these preclinical and clinical studies to ultimately enable clinical translation.

Keywords:  Clinical application; Drug screening; Intestinal organoids; Personalized medicine; Regenerative medicine; Tissue engineering

DOI:  https://doi.org/10.1016/j.ejcb.2024.151474
J Transl Med. 2024 Dec 31. 22(1): 1170

Gasdermin D regulates the activation of EGFR in colorectal cancer.

Ying Li, Jiayao Chen, Huijun Liang, Qindan Du, Jingjie Shen, Xiaoying Wang.

   BACKGROUND: Gasdermin D (GSDMD) is a key effector molecule that activates pyroptosis through its N terminal domain (GSDMD-NT). However, the roles of GSDMD in colorectal cancer (CRC) have not been fully explored. The role of the full-length GSDMD (GSDMD-FL) is also not clear. In this study, we observed that GSDMD modulates CRC progression through other mechanisms in addition to activating GSDMD-NT.
METHODS: Clinical CRC samples and human-derived CRC cell lines were used in this study. GSDMD expression was evaluated by RT-qPCR, Western blot and immunohistochemical (IHC) analysis. GSDMD knockdown and overexpression stable cell lines were established by Lentiviral transduction. CCK-8 assay, flow cytometry analysis for cell cycle, Transwell assay, and cell scratch assay were performed in vitro to explore the impact of GSDMD on CRC progression. Mouse subcutaneous transplantation tumor models were constructed to assess the role of GSDMD in vivo. Intestinal epithelial cell (IEC)-specific knockout of Gsdmd mice (GsdmdΔIEC) was used to evaluate the effect of GSDMD on intestinal adenoma formation in AOM-DSS and Apcmin/+ mouse models. RNA sequencing was performed to explore the regulatory pathways associated with the role of GSDMD in CRC cells. Co-Immunoprecipitation (CO-IP), Western blot and immunofluorescence (IF) were conducted to investigate the interactions between GSDMD and EGFR. Exogenous addition of Gefitinib was used to evaluate the effect of GSDMD on autophosphorylation of EGFR at the Tyr1068 site.
RESULTS: GSDMD was highly expressed in clinical CRC tissues and human-derived CRC cell lines. GSDMD knockdown inhibited the viability, cell cycle changes, invasion ability and migration ability of CRC cell lines in vitro and vivo, whereas GSDMD overexpression had the opposite effects. Intestinal adenoma development was reduced in GsdmdΔIEC mice in both AOM-DSS and Apcmin/+ mouse models. GSDMD-FL interacted with EGFR and promoted CRC progression by inducing autophosphorylation of EGFR at the Tyr1068 site, subsequently activating ERK1/2. Exogenous Gefitinib abrogated the tumorigenic properties of GSDMD.
CONCLUSIONS: GSDMD-FL promotes CRC progression by inducing EGFR autophosphorylation at the Tyr1068 site, subsequently activating the downstream ERK1/2. Inhibition of GSDMD is a potential strategy for the treatment of colorectal cancer.

Keywords:  CRC; EGFR Tyr1068; ERK1/2; GSDMD-FL

DOI:  https://doi.org/10.1186/s12967-024-05984-0
Front Bioeng Biotechnol. 2024 ;12 1506976

Fibroblasts modulate epithelial cell behavior within the proliferative niche and differentiated cell zone within a human colonic crypt model.

Angelo Massaro, Cecilia Villegas Novoa, Yuli Wang, Nancy L Allbritton.

  Colonic epithelium is situated above a layer of fibroblasts that provide supportive factors for stem cells at the crypt base and promote differentiation of cells in the upper crypt and luminal surface. To study the fibroblast-epithelial cell interactions, an in vitro crypt model was formed on a shaped collagen scaffold with primary epithelial cells growing above a layer of primary colonic fibroblasts. The crypts possessed a basal stem cell niche populated with proliferative cells and a differentiated, nondividing cell zone at the luminal crypt end. The presence of fibroblasts enhanced cell differentiation and accelerated the rate at which a high resistance epithelial cell layer formed relative to cultures without fibroblasts. The fibroblasts modulated cell proliferation within crypts increasing the number of crypts populated with proliferative cells but decreasing the total number of proliferative cells in each crypt. Bulk-RNA sequencing revealed 41 genes that were significantly upregulated and 190 genes that were significantly downregulated in cocultured epithelium relative to epithelium cultured without fibroblasts. This epithelium-fibroblast crypt model suggests bidirectional communication between the two cell types and has the potential to serve as a model to investigate fibroblast-epithelial cell interactions in health and disease.

Keywords:  colon crypt; epithelial cells; large intestine; microphysiological system; organ-on-chip; pericryptal fibroblasts

DOI:  https://doi.org/10.3389/fbioe.2024.1506976
J Pathol. 2025 Jan 03.

Loss of Mist1 alters the characteristics of Paneth cells and impacts the function of intestinal stem cells in physiological conditions and after radiation injury.

Yujun Huang, Siyu Tu, Zhenni Xu, Lu Xu, Xi Wang, Hefei Tian, Qican He, Lingxiao Huang, Xudan Lei, Shubin Wang, Mingyue Qu, Dengqun Liu.

  Intestinal stem cells (ISCs) and Paneth cells (PCs) reside at the bottom of the crypts of Lieberkühn in the small intestine. Recent studies have shown that the transcription factor Mist1, also named BHLHA15, plays an important role in the maturation of PCs. Since there is an intimate interaction between PCs and ISCs, we speculated that the loss of Mist1 could impact these two neighboring cell types. Here, we report that mice lacking Mist1 had fewer but larger PCs with shrunken secretory granules, accompanied by an increase in goblet cells and tuft cells. Mist1 loss significantly decreased the number of proliferative crypt cells, especially columnar basal cells (CBCs). In addition, Mist1-deficient enteroids needed supplemental Wnt3a to support their growth. Results from RNA sequencing (RNA-seq) demonstrated an apparent deficiency of innate immunity in Mist1-knockout mice. Intriguingly, Mist1 loss increased the survival rate of mice subjected to whole abdominal irradiation (WAI). Moreover, radiation injury was ameliorated in Mist1-knockout mice compared with their wild-type littermates based on histological analysis and enteroid culture, which might be a consequence of increased contents of the endoplasmic reticulum (ER) and the increased activity of mTORC1 in Paneth cells. In summary, our data uncover that Mist1 plays an important functional role in PCs and regulates the maintenance of ISCs and their response to radiation injury. © 2024 The Pathological Society of Great Britain and Ireland.

Keywords:  Mist1; Paneth cell; intestinal stem cell; organoid; radiation

DOI:  https://doi.org/10.1002/path.6360
Nat Commun. 2025 Jan 02. 16(1): 263

Nucleus-translocated GCLM promotes chemoresistance in colorectal cancer through a moonlighting function.

Jin-Fei Lin, Ze-Xian Liu, Dong-Liang Chen, Ren-Ze Huang, Fen Cao, Kai Yu, Ting Li, Hai-Yu Mo, Hui Sheng, Zhi-Bing Liang, Kun Liao, Yi Han, Shan-Shan Li, Zhao-Lei Zeng, Song Gao, Huai-Qiang Ju, Rui-Hua Xu.

Metabolic enzymes perform moonlighting functions during tumor progression, including the modulation of chemoresistance. However, the underlying mechanisms of these functions remain elusive. Here, utilizing a metabolic clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 knockout library screen, we observe that the loss of glutamate-cysteine ligase modifier subunit (GCLM), a rate-limiting enzyme in glutathione biosynthesis, noticeably increases the sensitivity of colorectal cancer (CRC) cells to platinum-based chemotherapy. Mechanistically, we unveil a noncanonical mechanism through which nuclear GCLM competitively interacts with NF-kappa-B (NF-κB)-repressing factor (NKRF), to promote NF-κB activity and facilitate chemoresistance. In response to platinum drug treatment, GCLM is phosphorylated by P38 MAPK at T17, resulting in its recognition by importin a5 and subsequent nuclear translocation. Furthermore, elevated expression of nuclear GCLM and phospho-GCLM correlate with an unfavorable prognosis and poor benefit from standard chemotherapy. Overall, our work highlights the essential nonmetabolic role and posttranslational regulatory mechanism of GCLM in enhancing NF-κB activity and subsequent chemoresistance.

DOI: https://doi.org/10.1038/s41467-024-55568-1
J Cancer. 2025 ;16(2): 417-429

TYMS Enhances Colorectal Cell Antioxidant Capacity Via the KEAP1-NRF2 Pathway to Resist Ferroptosis.

Jingtian Chen, Wei Wu, Lingxiao Wang, Jingjing Zhang, Xin Che, Liqin Zhai, Zhenxiang Zhao, Yaoping Li.

  Purpose: Thymidylate synthase (TYMS) is a key regulatory enzyme in DNA synthesis. We identified the biological effect and molecular mechanisms of TYMS in colorectal cancer (CRC). Methods: We employed western blot and immunohistochemistry for the assessment of TYMS expression in CRC samples. MTT and colony assay were carried out to illuminate the effect of TYMS on the proliferation of CRC cells. Xenograft models were constructed to evaluate the consequences of TYMS overexpression on CRC in vivo. Metabolomics was utilized to analyze the alterations in cellular molecular metabolites subsequent to TYMS overexpression. The impact of TYMS on NRF2 localization and KEAP1 expression was explored by means of western blot. The expression levels of GSH, ROS, MDA, and PTGS2 mRNA were measured to assess ferroptosis. Results: TYMS expression in CRC tumor tissues was upregulated compared to adjacent non-cancerous tissues. Cells overexpressing TYMS displayed enhanced proliferative capabilities. Metabolomic analysis revealed that overexpression of TYMS was associated with elevated levels of GSH within cells and a decrease in the lipid peroxidation product, 4-hydroxyhexenal. ROS detection assays further demonstrated a significant enhancement in cellular antioxidant capacity due to TYMS overexpression. Overexpression of TYMS downregulated KEAP1 expression and promoted NRF2 translocation into the nucleus. Consequently, transcription of downstream antioxidant genes was upregulated, enhancing cellular antioxidant capacity, reducing ROS levels, diminishing lipid peroxidation products, and heightening resistance to ferroptosis induced by erastin. Additionally, our study indicated that the TYMS inhibitor 5-fluorouracil (5-FU) exhibited favorable drug synergism with erastin. Conclusion: TYMS was overexpressed in CRC, which was correlated with poor prognosis of CRC patients. TYMS enhanced the antioxidant capacity of CRC cells via the KEAP1-NRF2 pathway, thereby increasing resistance to erastin-induced ferroptosis.

Keywords:  Colorectal cancer; Kelch-like ECH-associated protein 1; Nuclear factor erythroid 2-related factor 2; ROS; Thymidylate synthase; ferroptosis

DOI:  https://doi.org/10.7150/jca.102931
J Adv Res. 2024 Dec 30. pii: S2090-1232(24)00628-3. [Epub ahead of print]

Eicosapentaenoic acid enhances intestinal stem cell-mediated colonic epithelial regeneration by activating the LSD1-WNT signaling pathway.

Dan Wang, Nianbang Wu, Pei Li, Xiaojuan Zhang, Wenshuai Xie, Shunkang Li, Ding Wang, Yanling Kuang, Shaokui Chen, Yulan Liu.

   INTRODUCTION: Inflammatory bowel disease (IBD) is often associated with impaired proliferation and differentiation of intestinal stem cells (ISCs). Eicosapentaenoic acid (EPA), which is predominantly found in fish oil, has been recognized for its intestinal health benefits, although the potential mechanisms are not well understood.
OBJECTIVES: This study aimed to investigate the regulatory role and mechanism of EPA in colonic epithelial regeneration, specifically from the perspective of ISCs.
METHODS: Wild-type mice whose diet was supplemented with 5% EPA-enriched fish oil were subjected to dextran sulfate sodium (DSS) to induce colitis. We utilized intestinal organoids, ISC-specific lysine-specific demethylase 1 (LSD1) knockout mice, and WNT inhibitor-treated mice to explore how EPA influences ISC proliferation and differentiation. ISC proliferation, differentiation and apoptosis were assessed using tdTomato and propidium iodide tracer testing, histological analyses, and immunofluorescence staining.
RESULTS: EPA treatment significantly mitigated the symptoms of DSS-induced acute colitis, as evidenced by lower body weight loss and decreased disease activity index, histological scores and proinflammatory cytokine levels. Additionally, EPA increased the numbers of proliferative cells, absorptive cells, goblet cells, and enteroendocrine cells, which enhanced the regeneration of intestinal epithelium. Pretreatment with EPA increased ISC proliferation and differentiation, and protected against TNF-α-induced cell death in intestinal organoids. Mechanistically, EPA upregulated G protein-coupled receptor 120 (GPR120) to induce LSD1 expression, which facilitated ISC proliferation and differentiation in organoids. ISC-specific ablation of LSD1 negated the protective effect of EPA on DSS-induced colitis in mice. Moreover, EPA administration activated the WNT signaling pathway downstream of LSD1 in ISCs, while inhibiting WNT signaling abolished the beneficial effects of EPA.
CONCLUSIONS: These findings demonstrate that EPA promotes ISC proliferation and differentiation, thereby enhancing colonic epithelial regeneration through the activation of LSD1-WNT signaling. Consequently, dietary supplementation with EPA represents a promising alternative therapeutic strategy for managing IBD.

Keywords:  EPA; Intestinal stem cell; Lysine-specific demethylase 1; WNT signaling pathway

DOI:  https://doi.org/10.1016/j.jare.2024.12.050
Mol Cancer Res. 2024 Jan 03.

Co-evolution of atypical BRAF and KRAS mutations in colorectal tumorigenesis.

Connor E Woolley, Enric Domingo, Juan Fernandez-Tajes, Kathryn A F Pennel, Patricia Roxburgh, Joanne Edwards, Susan D Richman, Tim S Maughan, David J Kerr, Ignacio Soriano, Ian P M Tomlinson.

BRAF mutations in colorectal cancer (CRC) comprise three functional classes: Class 1 (V600E) with strong constitutive activation, Class 2 with pathogenic kinase activity lower than Class 1, and Class 3 which paradoxically lacks kinase activity. Non-Class 1 mutations associate with better prognosis, microsatellite stability, distal tumour location and better anti-EGFR response. Analysis of 13 CRC cohorts (n=6,605 tumours) compared Class 1 (n=709, 10.7% of CRCs), Class 2 (n=31, 0.47%) and Class 3 (n=81, 1.22%) mutations. Class 2- and Class 3-mutant CRCs frequently co-occurred with additional Ras pathway mutations (29.0% and 45.7% respectively vs 2.40% in Class 1, p<0.001), often at atypical sites (KRAS non-codon 12/13/61, NRAS, or NF1). Ras pathway activation was highest in Class 1 and lowest in Class 3, with greater distal expression of EGFR ligands (AREG/EREG) supporting weaker BRAF driver mutations. Unlike Class 1 mutants, Class 3 tumours resembled chromosomally-unstable CRCs in mutation burdens, signatures, driver mutations and transcriptional subtypes, while Class 2 mutants displayed intermediate characteristics. Atypical BRAF mutations were associated with longer overall survival than Class 1 (HR=0.25, p=0.011), but lost this advantage in cancers with additional Ras mutation (HR=0.93, p=0.86). This study supports the suggestion that Class 3 BRAF mutations amplify existing Ras signalling in a two-mutation model and that enhancement of weak/atypical Ras mutations may suffice for tumorigenesis, with potentially clinically-important heterogeneity in the Class 2/3 sub-group. Implications: The heterogeneous nature of BRAF-mutant CRCs, particularly among Class 2/3 mutations which frequently harbour additional Ras mutations, highlights the necessity of comprehensive molecular profiling.

DOI: https://doi.org/10.1158/1541-7786.MCR-24-0464
Cancer Med. 2025 Jan;14(1): e70521

Enrichment of Cancer-Associated Fibroblasts, Macrophages, and Up-Regulated TNF-α Signaling in the Tumor Microenvironment of CMS4 Colorectal Peritoneal Metastasis.

Eirik Høye, Chakravarthi Kanduri, Annette Torgunrud, Susanne Lorenz, Bjørn Edwin, Stein G Larsen, Åsmund A Fretland, Vegar J Dagenborg, Kjersti Flatmark, Christin Lund-Andersen.

BACKGROUND: Metastatic colorectal cancer (mCRC) is the main cause of CRC mortality, with limited treatment options. Although immunotherapy has benefited some cancer patients, mCRC typically lacks the molecular features that respond to this treatment. However, recent studies indicate that the immune microenvironment of mCRC may be modified to enhance the effect of immune checkpoint inhibitors. This study aimed to explore the metastatic tumor microenvironment (TME) by comparing cell populations in colorectal liver (CLM), lung (mLu), and peritoneal (PM) metastases.
METHODS: RNA isolated from 20 CLM, 15 mLu, and 35 PM samples was subjected to mRNA sequencing and explored through TME deconvolution tools, consensus molecular subtyping (CMS), and differential gene expression and gene set enrichment analysis, with respect to the metastatic sites. Clinical data and KRAS/BRAF hotspot mutation status were also obtained for all the cases.
RESULTS: The cell type fractions in the TME were relatively similar between the metastatic sites, except for cancer-associated fibroblasts (CAFs), B cells, endothelial cells, and CD4+ T cells. Notably, PM showed enrichment for CAFs and endothelial cells, consistent with distinct pathways associated with metastatic growth and progression in the peritoneal cavity. PM with the mesenchymal subtype, CMS4, had increased CAFs, endothelial cells, and macrophages, along with up-regulated genes related to TNF-α signaling via NF-κB, EMT, and angiogenesis.
CONCLUSIONS: Tumor samples from different metastatic sites exhibited a broadly similar TME in terms of immune cell composition, with some intriguing differences. Targeting CAF-associated pathways, macrophages, and TNF-α signaling through NR4A could represent potential novel therapeutic approaches in CMS4 PM.

DOI: https://doi.org/10.1002/cam4.70521
Clin Transl Med. 2025 Jan;15(1): e70151

Single-cell sequencing analysis reveals cetuximab resistance mechanism and salvage strategy in colorectal cancer.

Shiyun Chen, Zhaoli Tan, Xiaojie Wu, Yanli Lin, Xiang Li, Yumeng Cui, Weiling Man, Fang Pang, Yanghua Li, Faliang Shi, Lu Han, Miaomiao Gou, Li Zhou, Zhikuan Wang, Youliang Wang, Guanghai Dai.

DOI: https://doi.org/10.1002/ctm2.70151