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Nox1 and Npy1r mark regional colon stem cell populations that serve as cancer origins in vivo [scRNA_seq]

ABSTRACT: Current colorectal cancer (CRC) mouse models lack either colon specificity, limiting progression towards more advanced disease, or preclude evaluation of resident stem cells as cancer origins. Here, we report identification of Nox1 and Npy1r as cell-surface markers enriched in Lgr5+ stem cells predominantly within the caecum and exclusively within the mid-distal colorectum respectively. Selective dysregulation of Wnt signalling in Nox1+ or Npy1r+ stem cells using new CreERT2 mouse lines drives colon cancer initiation, predominantly within the caecum and the rectum respectively, establishing these stem cell populations as important sources of colon cancer. Selective conditional activation of Wnt signalling and oncogenic K-RAS in combination with loss of Trp53 in these stem cell compartments resulted in development of advanced, invasive cancers. This study establishes the new CreERT2 drivers as valuable tools for studying stem cell contributions to colon cancer.

ORGANISM(S): Mus musculus

PROVIDER: GSE303434 | GEO | 2025/07/29

REPOSITORIES: GEO

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Project description:NOX1 gene is member of the NADPH-dependent superoxide producing enzyme family. It generates reactive oxygen species (ROS) in regulated manner in response to growth factors, cytokines and calcium signal. Low levels of ROS play role in many processes including cell proliferation, inflammatory response and mitogenesis. NOX1 over-expression in colon cancer cell lines as well as surgical samples suggests a role in colon cancer. We identified unique target sequences of siRNAs for post-transcriptional silencing of NOX1. Gene specific and scrambled (control) sequence carrying U6 promoter/shRNA cassettes were cloned into GFP expression vector. Stable clones were selected based on NOX1 expression after transient transfection of HT-29 cells. Clone 6A and 6C showed 70-80% decrease in NOX1 expression. The cells were expanded and analyzed further. We measured decreased ROS production, cell proliferation and G1/S block of the cell cycle. HT-29 parental cells, clone SA (scrambled) and clone 6A (gene specific) were utilized to establish xenografts in athymic mice to investigate the effect of NOX1 silencing on tumor growth and gene expression in vivo. Significant decrease was measured in the volume of forming tumors. The stable clones and corresponding xenografts were analyzed on microarray. The microarray data was validated with real-time PCR and western-blot analysis demonstrating the inhibition of genes important in cell cycle regulation and angiogenesis. The results of this study show silencing NOX1 levels of generated ROS decreased, the tumor formation attenuated in vivo suggesting NOX1 may be a therapeutic target for colon cancer. Experiment Overall Design: We have found that NOX1 over-expression in colon cancer cell lines as well as surgical samples suggests a role in colon cancer. We identified unique target sequences of siRNAs for post-transcriptional silencing of NOX1. Gene specific and scrambled (control) sequences carrying the U6 promoter/shRNA cassettes were cloned into a GFP expression vector. Stable clones were selected based on NOX1 expression after transient transfection of HT-29 cells. HT-29 parental cells, clone SA (scrambled) and clone 6A (gene specific) were utilized to establish xenografts in athymic mice to investigate the effect of NOX1 silencing on tumor growth and gene expression in vivo. A significant decrease was measured in the volume of tumors formed. The stable clones and corresponding xenografts were analyzed on microarray. The microarray data was validated with real-time PCR and western-blot analysis demonstrating the inhibition of genes important in cell cycle regulation and angiogenesis. The results of this study show silencing NOX1 levels of generated ROS decreased, the tumor formation attenuated in vivo suggesting NOX1 may be a therapeutic target for colon cancer.

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Nox1 and Npy1r mark regional colon stem cell populations that serve as cancer origins in vivo [scRNA_seq]

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