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RNA sequencing of long-term label-retaining colon cancer stem cells identifies novel regulators of quiescence.

ABSTRACT: Recent data suggest that therapy-resistant quiescent cancer stem cells (qCSCs) are the source of relapse in colon cancer. Here, using colon cancer patient-derived organoids and xenografts, we identify rare long-term label-retaining qCSCs that can re-enter the cell cycle to generate new tumors. RNA sequencing analyses demonstrated that these cells display the molecular hallmarks of quiescent tissue stem cells, including expression of p53 signaling genes, and are enriched for transcripts common to damage-induced quiescent revival stem cells of the regenerating intestine. In addition, we identify negative regulators of cell cycle, downstream of p53, that we show are indicators of poor prognosis and may be targeted for qCSC abolition in both p53 wild-type and mutant tumors. These data support the temporal inhibition of downstream targets of p53 signaling, in combination with standard-of-care treatments, for the elimination of qCSCs and prevention of relapse in colon cancer.

SUBMITTER: Regan JL

PROVIDER: S-EPMC8185225 | biostudies-literature | 2021 Jun

REPOSITORIES: biostudies-literature

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RNA sequencing of long-term label-retaining colon cancer stem cells identifies novel regulators of quiescence.

Regan Joseph L JL Schumacher Dirk D Staudte Stephanie S Steffen Andreas A Lesche Ralf R Toedling Joern J Jourdan Thibaud T Haybaeck Johannes J Mumberg Dominik D Henderson David D Győrffy Balázs B Regenbrecht Christian R A CRA Keilholz Ulrich U Schäfer Reinhold R Lange Martin M

iScience 20210524 6

Recent data suggest that therapy-resistant quiescent cancer stem cells (qCSCs) are the source of relapse in colon cancer. Here, using colon cancer patient-derived organoids and xenografts, we identify rare long-term label-retaining qCSCs that can re-enter the cell cycle to generate new tumors. RNA sequencing analyses demonstrated that these cells display the molecular hallmarks of quiescent tissue stem cells, including expression of p53 signaling genes, and are enriched for transcripts common to ...[more]

PMID: 34142064

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Project description:BackgroundSessile serrated adenomas/polyps (SSA/Ps) may account for 20-30% of colon cancers. Although large SSA/Ps are generally recognized phenotypically, small (<1 cm) or dysplastic SSA/Ps are difficult to differentiate from hyperplastic or small adenomatous polyps by endoscopy and histopathology. Our aim was to define the comprehensive gene expression phenotype of SSA/Ps to better define this cancer precursor.ResultsRNA sequencing was performed on 5' capped RNA from seven SSA/Ps collected from patients with the serrated polyposis syndrome (SPS) versus eight controls. Highly expressed genes were analyzed by qPCR in additional SSA/Ps, adenomas and controls. The cellular localization and level of gene products were examined by immunohistochemistry in syndromic and sporadic SSA/Ps, adenomatous and hyperplastic polyps and controls. We identified 1,294 differentially expressed annotated genes, with 106 increased ≥10-fold, in SSA/Ps compared to controls. Comparing these genes with an array dataset for adenomatous polyps identified 30 protein coding genes uniquely expressed ≥10-fold in SSA/Ps. Biological pathways altered in SSA/Ps included mucosal integrity, cell adhesion, and cell development. Marked increased expression of MUC17, the cell junction protein genes VSIG1 and GJB5, and the antiapoptotic gene REG4 were found in SSA/Ps, relative to controls and adenomas, were verified by qPCR analysis of additional SSA/Ps (n = 21) and adenomas (n = 10). Immunohistochemical staining of syndromic (n≥11) and sporadic SSA/Ps (n≥17), adenomatous (n≥13) and hyperplastic (n≥10) polyps plus controls (n≥16) identified unique expression patterns for VSIG1 and MUC17 in SSA/Ps.ConclusionA subset of genes and pathways are uniquely increased in SSA/Ps, compared to adenomatous polyps, thus supporting the concept that cancer develops by different pathways in these phenotypically distinct polyps with markedly different gene expression profiles. Immunostaining for a subset of these genes differentiates both syndromic and sporadic SSA/Ps from adenomatous and hyperplastic polyps.

Project description:The epithelial lining of the small intestine is continuously renewed from a small number of stem cells including Lgr5 expressing crypt base cells that have been shown to be a rapidly cycling stem cell population in homeostasis. Alternative markers of intestinal stem cells have variously identified populations as rapidly cycling or quiescent with proposed roles for the latter as a parallel or reserve stem cell population. However, the exact nature of quiescent crypt cells remains unknown. Here by applying novel mouse models that permit their isolation and characterisation as label-retaining cells (LRCs), and for the first time performing lineage tracing from them, we show quiescent cells to be committed secretory precursors that are capable of recall to the stem cell state. We reveal LRCs to be a small subset of the rapidly cycling Lgr5 expressing population committed along the Paneth-enteroendocrine lineage. Significantly, after injury and subsequent regeneration they are clonogenic, as shown by both lineage tracing and organoid growth demonstrating that they can be recalled to the stem cell pool. These findings in establishing quiescent cells as an effective clonogenic reserve during injury provides a motivation for investigating their role in the maintenance of cancer growth following adjuvant treatment. Six age and sex (female) matched Cyp1a1-H2B-YFP mice ten days post βNF induction were used comparing three cell populations from each. Following epithelial isolation, single cell preparation and staining with anti-CD24 anitbody and UEA-1 lectin, 30,000 cells were flow sorted for each population: Paneth cells (Paneth) were defined as CD24+/UEA+, YFP-LRCs (YFPpos) as CD24+/UEA-/YFP+ and LCCs (YFPneg) as CD24+/UEA-/YFP-. Microarray expression comparison was then performed to identify unique markers of each population. RNA amplification and hybridisation was performed at the Paterson Institute, Manchester, UK, Microarray Facility using Nugen Ovation Pico WTA System for amplification and then hybridisation to a Mouse Exon 1.0 ST Array. Arrays were scanned using an Affymetrix GeneChip scanner 3000 running GCOS software.

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RNA sequencing of long-term label-retaining colon cancer stem cells identifies novel regulators of quiescence.

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RNA sequencing of long-term label-retaining colon cancer stem cells identifies novel regulators of quiescence.

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