bims-instec 2021-10-31 papers

Exp Cell Res. 2021 Oct 21. pii: S0014-4827(21)00446-8. [Epub ahead of print]409(1): 112892

CPT2 downregulation triggers stemness and oxaliplatin resistance in colorectal cancer via activating the ROS/Wnt/β-catenin-induced glycolytic metabolism.

Hui Li, JuHui Chen, Jie Liu, Yiqin Lai, Sha Huang, Liang Zheng, Nanfeng Fan.

Carnitine palmitoyltransferase 2 (CPT2) has been demonstrated to act as a tumor promotor or suppressor in different types of cancers. However, little is known about the effect of CPT2 on colorectal cancer (CRC). In the present study, we analyzed CPT2 expression in CRC tissues and cells. CPT2 was overexpressed in CRC cell lines (SW480 and RKO), and its effects and molecular mechanism on the proliferation, glycolysis, stemness, and oxaliplatin sensitivity were investigated. The xenograft experiment was used to confirm the influence of CPT2 on CRC tumorigenesis in vivo. We found that CPT2 expression was significantly downregulated in CRC patients, and its lower expression was associated with the poor prognosis, large tumor size, advanced TNM stage, and poor histological grade differentiation of patients. Upregulation of CPT2 significantly inhibited the proliferation, glycolytic metabolism, cancer stem cell properties, and oxaliplatin resistance in CRC cells. Also, the increase of CPT2 inhibited tumorigenesis, stemness and glycolysis, while enhanced oxaliplatin sensitivity in mouse models. Mechanistically, CPT2 functioned via suppressing the activation of Wnt/β-catenin pathway through repressing ROS production. In conclusion, our results demonstrated that CPT2 was decreased in CRC, and CPT2 downregulation could trigger stemness and oxaliplatin resistance in CRC via activating the ROS/Wnt/β-catenin-induced glycolytic metabolism. This study indicates that CPT2 is a potential therapeutic target for CRC.

Keywords: CPT2; Colorectal cancer; Oxaliplatin resistance; Stemness; Wnt/β-catenin

DOI: https://doi.org/10.1016/j.yexcr.2021.112892

Cell Mol Gastroenterol Hepatol. 2021 Oct 23. pii: S2352-345X(21)00224-1. [Epub ahead of print]

TNFα Induces LGR5+ Stem Cell Dysfunction In Patients With Crohn's Disease.

Chnasu Lee, Minae An, Je-Gun Joung, Woong-Yang Park, Dong Kyung Chang, Young-Ho Kim, Sung Noh Hong.

BACKGROUND & AIMS: Tumor necrosis factor-alpha (TNFα) is considered a major tissue damage-promoting effector in Crohn's disease (CD) pathogenesis. Patient-derived intestinal organoid (enteroid) recapitulates the disease-specific characteristics of the intestinal epithelium. This study aimed to evaluate the intestinal epithelial responses to TNFα in enteroids derived from healthy controls and compare them with those of CD patient-derived enteroids.
METHODS: Human enteroids derived from patients with CD and controls were treated with TNFα (30 ng/ml), and cell viability and gene expression patterns were evaluated.
RESULTS: TNFα induced MLKL-mediated necroptotic cell death, which was more pronounced in CD patient-derived enteroids than in control enteroids. Immunohistochemistry and RNA-sequencing revealed that treatment with TNFα caused expansion of the intestinal stem cell (ISC) populations. However, expanded ISC subpopulations were differed in control and CD patient-derived enteroids; LGR5+ active ISCs in control enteroids and reserve ISCs, like BMI1+ cells, in CD patient-derived enteroids. In single-cell RNA-sequencing, LGR5+ ISC-enriched cell cluster showed strong expression of TNFRSF1B (TNFR2) and Cox-PGE2 activation. In TNFα-treated CD patient-derived enteroids, exogenous PGE2 (10 nM) induced the expansion of the LGR5+ ISC population and improved organoid-forming efficiency, viability, and wound healing.
CONCLUSIONS: TNFα increases necroptosis of differentiated cells and induces the expansion of LGR5+ ISCs. In CD patient-derived enteroids, TNFα causes LGR5+ stem cell dysfunction (expansion failure) and exogenous PGE2 treatment restored the functions of LGR5+ stem cells. Therefore, PGE2 can be used to promote mucosal healing in patients with CD.

Keywords: Crohn’s disease; intestinal organoid; intestinal stem cell; prostaglandin E2; tumor necrosis factor-alpha

DOI: https://doi.org/10.1016/j.jcmgh.2021.10.010

STAR Protoc. 2021 Dec 17. 2(4): 100880

FACS-based protocol to assess cytotoxicity and clonogenic potential of colorectal cancer stem cells using a Wnt/β-catenin signaling pathway reporter.

Simone Di Franco, Le Zhang, Miriam Gaggianesi, Melania Lo Iacono, Jan Paul Medema, Giorgio Stassi.

Cancer stem cells (CSCs) play a key role in tumor initiation and progression. A real-time tool to evaluate the activation of CSC-specific signaling pathways is crucial for the study of this cancer cell subset. Here, we present a protocol to monitor, in vitro, the activation of Wnt/β-catenin signaling pathway, which is considered a functional biomarker for colorectal CSCs (CR-CSCs). This flow-cytometry-based protocol allows it to isolate CR-CSCs and to evaluate their cytotoxicity upon anti-tumor treatments. For complete details on the use and execution of this protocol, please refer to Di Franco et al. (2021).

Keywords: Cancer; Cell Biology; Cell isolation; Cell-based Assays; Flow Cytometry/Mass Cytometry; Molecular/Chemical Probes; Signal Transduction; Stem Cells

DOI: https://doi.org/10.1016/j.xpro.2021.100880

Front Oncol. 2021 ;11 748730

Adipocytic Glutamine Synthetase Upregulation via Altered Histone Methylation Promotes 5FU Chemoresistance in Peritoneal Carcinomatosis of Colorectal Cancer.

Xuan Zhang, Qing Li, Aibei Du, Yifei Li, Qing Shi, Yanrong Chen, Yang Zhao, Bin Wang, Feng Pan.

The development of resistance to 5-fluorouracil (5FU) chemotherapy is a major handicap for sustained effective treatment in peritoneal carcinomatosis (PC) of colorectal cancer (CRC). Metabolic reprogramming of adipocytes, a component of the tumor microenvironment and the main composition of peritoneum, plays a significant role in drug resistance of PC, with the mechanisms being not fully understood. By performing metabolomics analysis, we identified glutamine (Gln), an important amino acid, inducing resistance to 5FU-triggered tumor suppression of CRC-PC through activating mTOR pathway. Noteworthily, genetic overexpression of glutamine synthetase (GS) in adipocytes increased chemoresistance to 5FU in vitro and in vivo while this effect was reversed by pharmacological blockage of GS. Next, we showed that methionine metabolism were enhanced in amino acid omitted from CRC-PC of GS transgenic (TgGS) mice, increasing intracellular levels of S-carboxymethy-L-cys. Moreover, loss of dimethylation at lysine 4 of histone H3 (H3k4me2) was found in adipocytes in vitro, which may lead to increased expression of GS. Furthermore, biochemical inhibition of lysine specific demethylase 1 (LSD1) restored H3k4me2, thereby reducing GS-induced chemoresistance to 5FU. Our findings indicate that GS upregulation-induced excessive of Gln in adipocytes via altered histone methylation is potential mediator of resistance to 5FU chemotherapy in patients with CRC-PC.

Keywords: chemoresistance; colorectal cancer; glutamine synthetase; histone methylation; peritoneal carcinomatosis

DOI: https://doi.org/10.3389/fonc.2021.748730

Biol Open. 2021 Oct 25. pii: bio.058904. [Epub ahead of print]

Src family kinases inhibit differentiation of intestinal epithelial cells through the Hippo effector YAP1.

Sepideh Fallah, Jean-François Beaulieu.

Intestinal cell lineage differentiation is a tightly regulated mechanism that involves several intracellular signaling pathways affecting the expression of a variety of transcription factors, which ultimately regulate cell specific gene expression. Absorptive and goblet cells are the two main epithelial cell types of the intestine. Previous studies from our group using an shRNA knockdown approach have shown that YAP1, one of the main Hippo pathway effectors, inhibits the differentiation of these two cell types. In the present study, we show that YAP1 activity is regulated by Src family kinases (SFKs) in these cells. Inhibition of SFKs led to a sharp reduction in YAP1 expression at the protein level, an increase in CDX2 and the P1 forms of HNF4α and of absorptive and goblet cell differentiation specific markers. Interestingly, in Caco-2/15 cells which express both YAP1 and its paralog TAZ, TAZ was not reduced by the inhibition of SFKs and its specific knockdown rather impaired absorptive cell differentiation indicating that YAP1 and TAZ are not always interchangeable for regulating cell functions.

Keywords: CDX2; Differentiation; HNF1; HNF4; Intestinal cell; Src family kinases; TAZ; YAP1

DOI: https://doi.org/10.1242/bio.058904