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Analysis of the transcriptomic changes in murine small and large intestinal organoids after the sudden expression of BRAFV600E and/or p53R172H

ABSTRACT: BRAFV600E confers poor prognosis and a distinct molecular type of colorectal cancer, which is often associated with TP53 alterations. In order to understand how BRAFV600E and p53R172H promote tumorigenesis in the intestinal epithelium, we generated murine organoids harboring conditional BraffloxV600E and/or Trp53LSL-R172H alleles and conducted RNA sequencing analysis after sudden oncogene induction. As expected from their phenotypes, organoids expressing BRAFV600E, either singly or in combination with p53R172H, displayed strong changes in their transcriptome, while induction of mutant p53 alone had only little impact on gene expression. Our transcriptome analyses revealed there are profound differences in the transcriptomes of SI and COL organoids, in their oncogene naïve ground state and upon BRAFV600E expression. We observed many transcripts positively associated with proliferation, invasion and metastasis to be upregulated in both organoid types expressing BRAFV600E or BRAFV600E together with p53R172H. However, several of them were only differentially regulated in COL organoids, or displayed a more pronounced fold change in COL than in SI organoids. Interestingly, COL organoids more closely recapitulate human CRC patterns and p53R172H cooperates with mutant BRAFV600E in the regulation of several transcripts.

ORGANISM(S): Mus musculus

PROVIDER: GSE132551 | GEO | 2021/06/30

REPOSITORIES: GEO

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Project description:The endodermal lining of the adult gastro-intestinal tract harbors stem cells that are responsible for the day-to-day regeneration of the epithelium. Stem cells residing in the pyloric glands of the stomach and in the small intestinal crypts differ in their differentiation program and in the gene repertoire that they express. Both types of stem cells have been shown to grow from single cells into 3D structures (organoids) in vitro. We show that single adult Lgr5-positive stem cells, isolated from small intestinal organoids, require Cdx2 to maintain their intestinal identity and are converted cell-autonomously into pyloric stem cells in the absence of this transcription factor. Clonal descendants of Cdx2null small intestinal stem cells enter the gastric differentiation program instead of producing intestinal derivatives. Conversely, forced expression of Cdx2 in gastric organoids results in their intestinalization. The intestinal genetic program is thus critically dependent on the single transcription factor encoding gene Cdx2. Small intestinal crypts and stomach glands were isolated from Cdx2-/fl / Lgr5-EGFP-CreERT2 mice and cultured for a week in order to generate small intestinal (SI) and stomach (Sto) in vitro organoids. The Lgr5-CreERT2 enzyme activity has been induced by overnight 4-hydroxytamoxifen induction. Tamoxifen treated and untreated Lgr5-EGFPhi SI and Sto stem cells were FACS sorted and seeded back into ENRWfg (Sto med) culture conditions in order to generate Cdx2-/fl small intestinal (Control SI), Cdx2null small intestinal (Cdx2null SI) and Cdx2-/fl stomach (Control Sto) clonal organoids. Cdx2-/fl SI organoids and Cdx2-/fl Sto organoids have been also cultured in ENR (SI med) to induce differentiation. After some passages of clonal organoid expansion, RNA was isolated from Control SI, Cdx2null SI and Control Sto Lgr5-EGFPhi FACS sorted stem cell populations and from smal intestinal and stomach organoids cultured in different conditions and hybridized on Affymetrix Mouse Gene ST 1.1 arrays.

Project description: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. To investigate the effect of single and combined mutations in colon and small intestine of mouse models, we have generated a large set of organoids across 16 different genotypes zooming in on microsatellite stable CRC (CMS2-4). This set of organoids includes villinCreER Apcfl/fl (A), villinCreER Apcfl/fl Trp53fl/fl (AP), villinCreER Apcfl/fl Trp53R172H/fl (AP2), villinCreER Apcfl/fl KrasG12D/+ Trp53fl/fl (AKP), villinCreER Apcfl/fl KrasG12D/+ Trp53fl/fl Smad4fl/+ (AKPS), villinCreER KrasG12D/+ (K), villinCreER KrasG12D/+ Trp53fl/fl (KP), villinCreER KrasG12D/+ Trp53fl/fl Rosa26N1ind/+ (KPN), villinCreER BrafV600E/+ Trp53fl/fl Rosa26N1ind/+ (BPN), villinCreER BrafV600E/+ Trp53fl/fl Rosa26N1ind/+ Alk5fl/fl (BPNA2), villinCreER Apcfl/fl KrasG12D/+ (AK), villinCreER Apcfl/fl KrasG12D/+ Trp53R172H/fl (AKP2), villinCreER Apcfl/fl KrasG12D/+ Trp53fl/fl Rosa26N1ind/+ (AKPN), villinCreER Apcfl/fl Trp53fl/fl Rosa26N1ind/+ (APN), villinCreER BrafV600E/+ Trp53fl/fl (BP), and villinCreER KrasG12D/+ Trp53fl/fl Rosa26N1ind/+ Alk5fl/fl (KPNA2).

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Analysis of the transcriptomic changes in murine small and large intestinal organoids after the sudden expression of BRAFV600E and/or p53R172H

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