bims-instec 2023-02-19 papers

bims-instec

Biomed News

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

Issue of 2023–02–19
eleven papers selected by
Maria-Virginia Giolito, Free University of Brussels

STAT3 regulates 5-Fu resistance in human colorectal cancer cells by promoting Mcl-1-dependent cytoprotective autophagy.
Efficacious Combination Drug Treatment for Colorectal Cancer that Overcomes Resistance to KRAS G12C Inhibitors.
γδ T cells and their clinical application in colon cancer.
Re-evaluating the Role of Deep Crypt Secretory Cells in Intestinal Homeostasis.
Physiological hypoxia improves growth and functional differentiation of human intestinal epithelial organoids.
In colon cancer cells fascin1 regulates adherens junction remodeling.
Differential miRNA Expression Contributes to Emergence of Multiple Cancer Stem Cell Subpopulations in Human Colorectal Cancer.
Epithelial Gab1 calibrates RIPK3-dependent necroptosis to prevent intestinal inflammation.
SATB2 loss in inflammatory bowel disease-associated small intestinal metaplasia of the distal colon.
Preliminary clinical study of personalized neoantigen vaccine therapy for microsatellite stability (MSS)-advanced colorectal cancer.
Crosstalk between Protein Kinase C α and Transforming Growth Factor β Signaling Mediated by Runx2 in Intestinal Epithelial Cells.

Cancer Sci. 2023 Feb 14.

STAT3 regulates 5-Fu resistance in human colorectal cancer cells by promoting Mcl-1-dependent cytoprotective autophagy.

Yuanyi Yue, Qiang Zhang, Zhengrong Sun, Xueqing Wang.

  Chemoresistance to 5-Fluorouracil (5-Fu)-based chemotherapy is one of the primary reasons for the failure of colorectal cancer (CRC) management. STAT3 can mediate tumor drug resistance through a variety of diverse mechanisms. Nonetheless, the underlying mechanisms of STAT3-induced 5-Fu resistance in CRC are still poorly understood. Here, we aimed to investigate the potential mechanism(s) of STAT3-induced 5-Fu resistance in CRC. Quantitative RT-PCR and Western blot were used to test the expression of STAT3 and Mcl-1 in chemosensitive and chemoresistant CRC tissues and cell lines. After overexpression or knockdown of STAT3 or Mcl-1, and/or treated with or without 5-Fu or chloroquine (CQ), we tested cell viability, inhibitory concentration 50% (IC50 ) value of 5-FU, cell apoptosis, proliferation, migration, and autophagy. STAT3 and Mcl-1 were significantly upregulated in the chemoresistant CRC tissues and cell lines, and STAT3 positively regulated Mcl-1. Functional studies demonstrated that STAT3 promoted 5-Fu resistance in CRC. Mechanistically, STAT3 triggered autophagy via Mcl-1 to induce cancer chemoresistance. Our results show that STAT3 regulates 5-Fu resistance in CRC by promoting Mcl-1-dependent cytoprotective autophagy. Our results provide a novel role of STAT3 and may offer a new approach for managing CRC 5-Fu resistance.

Keywords:  5-Fluorouracil; Colorectal cancer; Cytoprotective autophagy; Mcl-1; STAT3

DOI:  https://doi.org/10.1111/cas.15761
Mol Cancer Ther. 2023 Feb 10. pii: MCT-22-0411. [Epub ahead of print]

Efficacious Combination Drug Treatment for Colorectal Cancer that Overcomes Resistance to KRAS G12C Inhibitors.

Hiroyuki Matsubara, Hiroyuki Miyoshi, Fumihiko Kakizaki, Tomonori Morimoto, Kenji Kawada, Takehito Yamamoto, Kazutaka Obama, Yoshiharu Sakai, Makoto Mark Taketo.

Recent advances in combinatorial chemistry led to the discovery of inhibitors targeting the KRAS G12C mutant protein. However, efficacy of its monotherapy on colorectal cancer (CRC) is limited. Thus, effective combination drugs should be explored for applicable CRC patients to fully benefit from the KRAS G12C inhibitor treatment. Here we employed a patient-derived CRC stem cell (PD-CRC-SC) spheroid culture and demonstrated that three-drug combination of inhibitors against KRAS G12C, EGFR, and FGFR synergistically suppressed the growth of CRC cells carrying the KRAS G12C mutation. Likewise, combination of KRAS G12C and SHP2 inhibitors was also effective. Importantly, activation of the phosphatidylinositol 3-kinase (PI3K)/AKT pathway in heregulin-responsive CRC cells canceled out the effect of KRAS G12C inhibition, which was largely overcome by PI3K inhibitors. These results reveal that evaluating efficacy of combination therapies with PD-CRC-SC spheroids can be a promising strategy to find the best regimen for individual CRC patients.

DOI: https://doi.org/10.1158/1535-7163.MCT-22-0411
Front Immunol. 2023 ;14 1098847

γδ T cells and their clinical application in colon cancer.

Anna Maria Corsale, Marta Di Simone, Elena Lo Presti, Francesco Dieli, Serena Meraviglia.

  In recent years, research has focused on colorectal cancer to implement modern treatment approaches to improve patient survival. In this new era, γδ T cells constitute a new and promising candidate to treat many types of cancer because of their potent killing activity and their ability to recognize tumor antigens independently of HLA molecules. Here, we focus on the roles that γδ T cells play in antitumor immunity, especially in colorectal cancer. Furthermore, we provide an overview of small-scale clinical trials in patients with colorectal cancer employing either in vivo activation or adoptive transfer of ex vivo expanded γδ T cells and suggest possible combinatorial approaches to treat colon cancer.

Keywords:  MHC- unrestricted activation; colon rectal cancer; gamma delta T cells; immunotherapy; tumor

DOI:  https://doi.org/10.3389/fimmu.2023.1098847
Cell Mol Gastroenterol Hepatol. 2023 Feb 10. pii: S2352-345X(23)00005-X. [Epub ahead of print]

Re-evaluating the Role of Deep Crypt Secretory Cells in Intestinal Homeostasis.

Huw Mccarthy, Simon Keely.

DOI: https://doi.org/10.1016/j.jcmgh.2023.01.005
Front Immunol. 2023 ;14 1095812

Physiological hypoxia improves growth and functional differentiation of human intestinal epithelial organoids.

Gunnar Andreas Walaas, Shreya Gopalakrishnan, Ingunn Bakke, Helene Kolstad Skovdahl, Arnar Flatberg, Ann Elisabet Østvik, Arne Kristian Sandvik, Torunn Bruland.

   Background: The epithelium in the colonic mucosa is implicated in the pathophysiology of various diseases, including inflammatory bowel diseases and colorectal cancer. Intestinal epithelial organoids from the colon (colonoids) can be used for disease modeling and personalized drug screening. Colonoids are usually cultured at 18-21% oxygen without accounting for the physiological hypoxia in the colonic epithelium (3% to <1% oxygen). We hypothesize that recapitulating the in vivo physiological oxygen environment (i.e., physioxia) will enhance the translational value of colonoids as pre-clinical models. Here we evaluate whether human colonoids can be established and cultured in physioxia and compare growth, differentiation, and immunological responses at 2% and 20% oxygen.
Methods: Growth from single cells to differentiated colonoids was monitored by brightfield images and evaluated with a linear mixed model. Cell composition was identified by immunofluorescence staining of cell markers and single-cell RNA-sequencing (scRNA-seq). Enrichment analysis was used to identify transcriptomic differences within cell populations. Pro-inflammatory stimuli induced chemokines and Neutrophil gelatinase-associated lipocalin (NGAL) release were analyzed by Multiplex profiling and ELISA. Direct response to a lower oxygen level was analyzed by enrichment analysis of bulk RNA sequencing data.
Results: Colonoids established in a 2% oxygen environment acquired a significantly larger cell mass compared to a 20% oxygen environment. No differences in expression of cell markers for cells with proliferation potential (KI67 positive), goblet cells (MUC2 positive), absorptive cells (MUC2 negative, CK20 positive) and enteroendocrine cells (CGA positive) were found between colonoids cultured in 2% and 20% oxygen. However, the scRNA-seq analysis identified differences in the transcriptome within stem-, progenitor- and differentiated cell clusters. Both colonoids grown at 2% and 20% oxygen secreted CXCL2, CXCL5, CXCL10, CXCL12, CX3CL1 and CCL25, and NGAL upon TNF + poly(I:C) treatment, but there appeared to be a tendency towards lower pro-inflammatory response in 2% oxygen. Reducing the oxygen environment from 20% to 2% in differentiated colonoids altered the expression of genes related to differentiation, metabolism, mucus lining, and immune networks.
Conclusions: Our results suggest that colonoids studies can and should be performed in physioxia when the resemblance to in vivo conditions is important.

Keywords:  chemokines (cytokines); differentiation; inflammatory bowel disease; intestinal epithelial cells (IECs); oxygen; proliferation; single-cell RNA-sequencing (scRNAseq); transcriptome

DOI:  https://doi.org/10.3389/fimmu.2023.1095812
FASEB J. 2023 03;37(3): e22786

In colon cancer cells fascin1 regulates adherens junction remodeling.

Amin Esmaeilniakooshkghazi, Eric Pham, Sudeep P George, Afzal Ahrorov, Fabian R Villagomez, Michael Byington, Srijita Mukhopadhyay, Srinivas Patnaik, Jacinta C Conrad, Monali Naik, Saathvika Ravi, Niall Tebbutt, Jennifer Mooi, Camilla M Reehorst, John M Mariadason, Seema Khurana.

  Adherens junctions (AJs) are a defining feature of all epithelial cells. They regulate epithelial tissue architecture and integrity, and their dysregulation is a key step in tumor metastasis. AJ remodeling is crucial for cancer progression, and it plays a key role in tumor cell survival, growth, and dissemination. Few studies have examined AJ remodeling in cancer cells consequently, it remains poorly understood and unleveraged in the treatment of metastatic carcinomas. Fascin1 is an actin-bundling protein that is absent from the normal epithelium but its expression in colon cancer is linked to metastasis and increased mortality. Here, we provide the molecular mechanism of AJ remodeling in colon cancer cells and identify for the first time, fascin1's function in AJ remodeling. We show that in colon cancer cells fascin1 remodels junctional actin and actomyosin contractility which makes AJs less stable but more dynamic. By remodeling AJs fascin1 drives mechanoactivation of WNT/β-catenin signaling and generates "collective plasticity" which influences the behavior of cells during cell migration. The impact of mechanical inputs on WNT/β-catenin activation in cancer cells remains poorly understood. Our findings highlight the role of AJ remodeling and mechanosensitive WNT/β-catenin signaling in the growth and dissemination of colorectal carcinomas.

Keywords:  Fascin1; WNT signaling; adherens junctions; bidirectional migration; carcinomas; collective migration; mechanotransduction

DOI:  https://doi.org/10.1096/fj.202201454R
bioRxiv. 2023 Feb 06. pii: 2023.02.06.527341. [Epub ahead of print]

Differential miRNA Expression Contributes to Emergence of Multiple Cancer Stem Cell Subpopulations in Human Colorectal Cancer.

Victoria A Stark, Caroline O B Facey, Lynn Opdenaker, Jeremy Z Fields, Bruce M Boman.

One reason for lack of efficacy in cancer therapeutics is tumor heterogeneity. We hypothesize that tumor heterogeneity arises due to emergence of multiple cancer stem cell (CSC) subpopulations because miRNAs regulate expression of stem cell genes in CSCs. Our goal was to determine if: i ) multiple CSC subpopulations exist in a human CRC cell population, and ii ) miRNAs are differentially expressed in the different CSC subpopulations. We discovered that at least four different CSC populations (ALDH1, CD166, LGR5, LRIG1) exist in the HT29 cell line. CSC subpopulations were quantified using co-staining for multiple stem cell markers, isolated using FACS, and analyzed by NanoString miRNA profiling. The miRNA expression pattern in each CSC subpopulation was analyzed relative to miRNA expression patterns in other CSC subpopulations. Messenger RNAs predicted to be targeted by the upregulated miRNAs in each CSC subpopulation were: 1) identified using bioinformatics analyses, and 2) classified according to their predicted functions using David functional annotation analyses. We found multiple CSC subpopulations with a unique miRNA signature in each CSC subpopulation. Notably, the miRNAs expressed within one CSC subpopulation are predicted to target and downregulate the CSC genes and pathways that establish the other CSC subpopulations. Moreover, mRNAs predicted to be targeted by miRNAs in the different CSC subpopulations have different cellular functional classifications. That different CSC subpopulations express miRNAs that are predicted to target CSC genes expressed in other CSC subpopulations provides a mechanism that might explain the co-existence of multiple CSC subpopulations, tumor heterogeneity, and cancer therapy resistance.

DOI: https://doi.org/10.1101/2023.02.06.527341
JCI Insight. 2023 Feb 16. pii: e162701. [Epub ahead of print]

Epithelial Gab1 calibrates RIPK3-dependent necroptosis to prevent intestinal inflammation.

Jiaqi Xu, Shihao Li, Wei Jin, Hui Zhou, Tingting Zhong, Xiaoqing Cheng, Yujuan Fu, Peng Xiao, Hongqiang Cheng, Di Wang, Yuehai Ke, Zhinong Jiang, Xue Zhang.

  As a hallmark for inflammatory bowel disease (IBD), elevated intestinal epithelial cell (IEC) death compromises the gut barrier, activating inflammatory response and triggering more IEC death. However, the precise intracellular machinery that prevents IEC death and break this vicious feedback remains largely unknown. Here, we report that Gab1 expression is decreased in patients with IBD and inversely correlated with IBD severity. Gab1 deficiency in intestinal epithelial cells accounts for the exacerbated colitis induced by dextran sodium sulfate (DSS) owing to sensitizing IECs to RIPK3-mediated necroptosis, which irreversibly disrupted homeostasis of the epithelial barrier and promoted intestinal inflammation. Mechanistically, Gab1 negatively regulates necroptosis signaling through inhibiting the formation of RIPK1/RIPK3 complex in response to TNF-α. Importantly, administration of RIPK3 inhibitor reveals a curative effect in epithelial Gab1-deficient mice. Further analysis indicates mice with Gab1 deletion are prone to inflammation associated colorectal tumorigenesis. Collectively, our study defines a protective role for Gab1 in colitis and colitis-driven colorectal cancer through negatively regulating RIPK3-dependent necroptosis, in which may serve as an important target to fine-tune necroptosis and intestinal inflammation-related disease.

Keywords:  Gastroenterology; Inflammation; Inflammatory bowel disease; Molecular pathology; Signal transduction

DOI:  https://doi.org/10.1172/jci.insight.162701
bioRxiv. 2023 Feb 03. pii: 2023.02.01.526729. [Epub ahead of print]

SATB2 loss in inflammatory bowel disease-associated small intestinal metaplasia of the distal colon.

Maged Zeineldin, Tatianna C Larman.

Epithelial metaplasia is a common adaptation to chronic inflammatory processes and can be associated with increased risk of dysplasia and cancer. The distal colon of patients with inflammatory bowel disease (IBD) commonly shows crypt architectural distortion and Paneth cell metaplasia (PCM), and IBD patients also carry increased risk of colitis-associated dysplasia and cancer (CAC). Loss of SATB2 expression (Special AT-rich binding 2 protein, a colon-restricted chromatin remodeler) has recently been shown to distinguish colitis-associated dysplasia and CAC from sporadic disease. Here we report non-diffuse heterogeneous patterns of SATB2 loss across non-dysplastic distal colon biopsies from IBD patients (n=20). This cohort was specifically curated to include biopsies with well-developed histologic features of villiform growth and PCM. Notably, CDX2 was strongly expressed and P53 showed a wild-type immunolabeling pattern across our non-dysplastic cohort, regardless of SATB2 immunolabeling pattern. Our findings fit with recent murine studies in which colon-specific Satb2 deletion resulted in histologic conversion of colonic mucosa to small intestinal-like mucosa, including emergence of villi and Paneth cells. Taken together, we show that SATB2 loss is associated with a preneoplastic metaplastic response to chronic injury in human IBD and chronic colitis, reframing PCM more broadly as small intestinal metaplasia. We propose that inflammation-associated SATB2 loss mediates a remodeled chromatin landscape permissive for dysplasia and CAC.

DOI: https://doi.org/10.1101/2023.02.01.526729
Cancer Immunol Immunother. 2023 Feb 16.

Preliminary clinical study of personalized neoantigen vaccine therapy for microsatellite stability (MSS)-advanced colorectal cancer.

Yao-Jun Yu, Na Shan, Li-Yi Li, Yue-Sheng Zhu, Li-Miao Lin, Chen-Chen Mao, Ting-Ting Hu, Xiang-Yang Xue, Xiao-Ping Su, Xian Shen, Zhen-Zhai Cai.

  Immunotherapy based on immune checkpoint inhibitors (ICIs) has provided revolutionary results in treating various cancers. However, its efficacy in colorectal cancer (CRC), especially in microsatellite stability-CRC, is limited. This study aimed to observe the efficacy of personalized neoantigen vaccine in treating MSS-CRC patients with recurrence or metastasis after surgery and chemotherapy. Candidate neoantigens were analyzed from whole-exome and RNA sequencing of tumor tissues. The safety and immune response were assessed through adverse events and ELISpot. The clinical response was evaluated by progression-free survival (PFS), imaging examination, clinical tumor marker detection, circulating tumor DNA (ctDNA) sequencing. Changes in health-related quality of life were measured by the FACT-C scale. A total of six MSS-CRC patients with recurrence or metastasis after surgery and chemotherapy were administered with personalized neoantigen vaccines. Neoantigen-specific immune response was observed in 66.67% of the vaccinated patients. Four patients remained progression-free up to the completion of clinical trial. They also had a significantly longer progression-free survival time than the other two patients without neoantigen-specific immune response (19 vs. 11 months). Changes in health-related quality of life improved for almost all patients after the vaccine treatment. Our results shown that personalized neoantigen vaccine therapy is likely to be a safe, feasible and effective strategy for MSS-CRC patients with postoperative recurrence or metastasis.

Keywords:  Colorectal cancer; Immunotherapy; MSS; Neoantigen; Personalized vaccine

DOI:  https://doi.org/10.1007/s00262-023-03386-7
J Biol Chem. 2023 Feb 13. pii: S0021-9258(23)00149-7. [Epub ahead of print] 103017

Crosstalk between Protein Kinase C α and Transforming Growth Factor β Signaling Mediated by Runx2 in Intestinal Epithelial Cells.

Xinyue Li, Navneet Kaur, Mustafa E Albahrani, Adam R Karpf, Adrian R Black, Jennifer D Black.

  Tight coordination of growth regulatory signaling is required for intestinal epithelial homeostasis. Protein kinase C α (PKCα) and transforming growth factor β (TGFβ) are negative regulators of proliferation with tumor suppressor properties in the intestine. Here, we identify novel crosstalk between PKCα and TGFβ signaling. RNA-Seq analysis of non-transformed intestinal crypt-like cells and colorectal cancer cells identified TGFβ receptor 1 (TGFβR1) as a target of PKCα signaling. Quantitative RT-PCR and immunoblot analysis confirmed that PKCα positively regulates TGFβR1 mRNA and protein expression in these cells. As with other PKCα targets, effects on TGFβR1 were dependent on Ras-ERK signaling. Nascent RNA and promoter-reporter analysis indicated that PKCα induces TGFβR1 transcription, and Runx2 was identified as an essential mediator of the effect. PKCα promoted ERK-mediated activating phosphorylation of Runx2, which preceded transcriptional activation of the TGFβR1 gene and induction of Runx2 expression. Thus, we have identified a novel PKCα→ERK→Runx2→TGFβR1 signaling axis. In further support of a link between PKCα and TGFβ signaling, PKCα knockdown reduced the ability of TGFβ to induce SMAD2 phosphorylation and cell cycle arrest, and inhibition of TGFβR1 activity decreased PKCα-induced upregulation of p21Cip1 and p27Kip1 in intestinal cells. The physiological relevance of these findings is also supported by TCGA data showing correlation between PKCα, Runx2, and TGFβR1 mRNA expression in human colorectal cancer. PKCα also regulated TGFβR1 in endometrial cancer cells and PKCα, Runx2, and TGFβR1 expression correlates in uterine tumors, indicating that crosstalk between PKCα and TGFβ signaling may be a common mechanism in diverse epithelial tissues.

Keywords:  ERK; PKCα; Runx2; TGFβ; TGFβR1; colon cancer; growth arrest; intestinal epithelium; transcription

DOI:  https://doi.org/10.1016/j.jbc.2023.103017