{"id":618993,"date":"2024-06-19T20:00:00","date_gmt":"2024-06-20T00:00:00","guid":{"rendered":"https:\/\/platohealth.ai\/non-stem-cell-lineages-as-an-alternative-origin-of-intestinal-tumorigenesis-in-the-context-of-inflammation-nature-genetics\/"},"modified":"2024-06-21T12:34:14","modified_gmt":"2024-06-21T16:34:14","slug":"non-stem-cell-lineages-as-an-alternative-origin-of-intestinal-tumorigenesis-in-the-context-of-inflammation-nature-genetics","status":"publish","type":"post","link":"https:\/\/platohealth.ai\/non-stem-cell-lineages-as-an-alternative-origin-of-intestinal-tumorigenesis-in-the-context-of-inflammation-nature-genetics\/","title":{"rendered":"Non-stem cell lineages as an alternative origin of intestinal tumorigenesis in the context of inflammation – Nature Genetics","gt_translate_keys":[{"key":"rendered","format":"text"}]},"content":{"rendered":"
We first reported on the ability of PCs to re-enter the cell cycle and dedifferentiate upon irradiation and inflammation to acquire stem cell-like features and contribute to the tissue regenerative response11<\/a>,12<\/a>,13<\/a><\/sup>. Consequently, we questioned whether PCs could be the origin of intestinal cancer in the context of inflammation. We bred mice carrying lox<\/i> alleles at the tumor suppressors and oncogenes most frequently mutated along the adenoma-to-carcinoma sequence, namely Apc<\/i>14<\/a><\/sup>, Kras<\/i>15<\/a><\/sup> and Trp53<\/i> (encoding p53)16<\/a><\/sup>, each combined with Cre specific for Lgr5<\/i>+<\/sup> ISCs (Lgr5<\/i>CreERT2-EGFP<\/sup>)17<\/a><\/sup> or for PCs (Lyz1<\/i>CreERT2<\/sup>)18<\/a><\/sup>. Following Cre activation by tamoxifen, dextran sulfate sodium (DSS) was administered through drinking water to model inflammation (Fig. 1a<\/a>). In the absence of DSS-induced inflammation, PC-specific single gene mutations did not give rise to intestinal tumors. By contrast, loss of Apc<\/i> in Lgr5<\/i>+<\/sup> ISCs transformed crypts into \u03b2-cateninhi<\/sup> foci that grew into multiple adenomas 4\u20136\u2009weeks after Cre induction (Fig. 1b<\/a>). When single gene mutations were combined with DSS administration, Apc<\/i> loss in PCs resulted in increased nuclear and cytoplasmic \u03b2-catenin expression eventually leading to the formation of PC-derived adenomas (Fig. 1c<\/a>). Of note, Paneth-specific Kras<\/i> or Trp53<\/i> mutations did not result in tumor formation even in the presence of the inflammatory stimulus (Fig. 1d<\/a>). However, the compound loss of Apc<\/i> and oncogenic activation of Kras<\/i> in PCs resulted in a striking increase in tumor multiplicity (6.1-fold) even in the absence of DSS (6.9-fold) (Fig. 1e<\/a>). The combination of Apc<\/i> and Trp53<\/i> mutations in PCs also led to an increase in tumor multiplicity upon DSS administration (1.6-fold), although to a lesser extent when compared with the compound Apc<\/i>\/Kras<\/i>-mutant genotype, possibly indicating a distinct mechanism underlying tumor onset in these mice. Indeed, phospho-histone H2A.X (Ser139) immunohistochemistry (IHC) analysis confirmed an increase in DNA damage and chromosomal instability in Trp53<\/i>-mutant tumors (Extended Data Fig. 1a<\/a>). Targeting all three genes in PCs resulted in a very aggressive phenotype with high tumor multiplicity (10.1-fold) in the absence of the inflammatory stimulus (Fig. 1e<\/a>). When compared with Apc<\/i>-driven tumors that originated in PCs, the histology of adenomas from mice in which two or three genes were targeted revealed a progressive increase in dysplasia and invasive morphology (Extended Data Fig. 1b<\/a>). The distribution of adenomas along the small intestine also followed distinct patterns with a prevalence of duodenal tumors in compound Apc<\/i>\/Kras<\/i> tumors regardless of DSS (Extended Data Fig. 2a<\/a>).<\/p>\n a<\/b>, Cre\u2013lox<\/i> strategy aimed at the targeting of Apc<\/i>, Kras<\/i> and Trp53<\/i> mutations in ISCs (Lgr5<\/i>+<\/sup> ISCs) and PCs (Lyz1<\/i>+<\/sup> PCs). w\/wo indicates the presence (with) or absence (without) of DSS. b<\/b>,c<\/b>, \u03b2-Catenin IHC analysis of intestinal tumors initiated from Lgr5<\/i>+<\/sup> ISCs (b<\/b>) and PCs (c<\/b>). Asterisks indicate Lgr5<\/i>+<\/sup> ISCs and PCs with enhanced cytoplasmic and nuclear \u03b2-catenin accumulation; tumor foci and adenomas are indicated by dashed lines. d<\/b>,e<\/b>, Tumor multiplicity was calculated according to tumor-bearing mice (d<\/b>) and by tumor number per genotype (e<\/b>) in the presence\/absence of DSS and based on Swiss roll counts. Error bars denote s.d. P<\/i> values denote one-way ANOVA and Tukey\u2019s post hoc tests for group comparisons. f<\/b>, Lineage-tracing analysis of PCs, labeled using yellow fluorescent protein (YFP), at different stages of tumor initiation and progression. g<\/b>\u2013j<\/b>, LYZ1 (g<\/b>) and DCLK1 (i<\/b>) IHC analysis of Lgr5<\/i>+<\/sup> ISC-derived (left) and PC-derived (right) adenomas, and quantification of number of Lyz1<\/i>+<\/i><\/sup> (h<\/b>) and Dclk1<\/i>+<\/sup> (j<\/b>) tumor cells. P<\/i> values depict one-way ANOVA and Tukey\u2019s post hoc tests for group comparisons. k<\/b>, Lyz1<\/i>, Dclk1<\/i> and Axin2<\/i> quantitative PCR expression analysis across different adenoma genotypes. P<\/i> values represent one-way ANOVA and Tukey\u2019s post hoc tests for group comparisons.<\/p>\n Source data<\/a><\/p>\n<\/div>\n<\/div>\n To validate the PC origin of the observed intestinal tumors, we bred Lyz1<\/i>CreERT2<\/sup> mice with R26LSL-tdTomato<\/sup> or R26LSL-YFP<\/sup> reporters and traced their lineage upon tamoxifen-driven targeting of the Apc, Kras<\/i> and Trp53<\/i> mutations. As shown in Fig. 1f<\/a> and Extended Data Fig. 2b<\/a>, this confirmed the PC origin of the corresponding tumors by capturing the process from microscopic lesions to adenoma formation.<\/p>\n Overall, these results demonstrate that PCs can initiate intestinal adenomas upon genetic ablation of Apc<\/i> in the context of inflammation. In combination with Apc<\/i> loss, activation of oncogenic Kras<\/i> or loss of Trp53<\/i> function rescues the need for an inflammatory stimulus and results in increased PC-derived tumor multiplicities and progression to a malignant phenotype.<\/p>\n Next, we characterized lineage-specific markers in PC- and ISC-originated tumors using IHC. Of note, although cells expressing the PC marker lysozyme (LYZ1) were notable in Lgr5<\/i>-derived tumors (Lgr5<\/i>\/Apc<\/i>: 30.0%\u2009\u00b1\u200918.5% positive tumor cells), they were almost absent in adenomas that originated from PCs (Lyz1<\/i>\/Apc<\/i>: 0.48%\u2009\u00b1\u20091.16%) (Fig. 1g,h<\/a>). The opposite was observed for DCLK1 (doublecortin-like kinase 1), a Tuft19<\/a><\/sup> and tumor stem cell marker20<\/a>,21<\/a><\/sup>, that was more frequently detected among PC-derived adenomas (Lyz1<\/i>\/Apc<\/i>: 54.1%\u2009\u00b1\u200910.5%) when compared with Lgr5<\/i>-derived tumors (Lgr5<\/i>\/Apc<\/i>: 15.6%\u2009\u00b1\u200915.7%) (Fig. 1i,j<\/a>). Other lineage-specific markers for enteroendocrine (CHGcA), goblet (MUC2) and stem cells (OLFM4) showed variable levels without clear-cut differences among tumors with different cells-of-origin (Extended Data Fig. 2c<\/a>). The increased Dclk1<\/i> expression in PC-derived tumors is of interest in view of its association with increased immune and stromal infiltration in colon cancer22<\/a><\/sup>.<\/p>\n To confirm these results at the transcriptional level, expression levels of Lyz1<\/i> and Dclk1<\/i> genes were analyzed by quantitative PCR with reverse transcription (Fig. 1k<\/a>). Indeed, Lyz1<\/i> expression was lower in Paneth-derived tumors (Lgr5<\/i>\/Apc<\/i> versus Lyz1<\/i>\/Apc<\/i>: log2<\/sub>-transformed fold change\u2009=\u20092.64, P<\/i>\u2009=\u20097.5\u2009\u00d7\u200910\u22124<\/sup>) when compared with Lgr5<\/i>-derived tumors. Dclk1<\/i> expression was very low and variable at the RNA level, and did not show significant differences across the groups.<\/p>\n To assess the relative activation of the WNT signaling pathway among the different tumor groups, we measured expression levels of Axin2<\/i>, a well-established WNT downstream target. Axin2<\/i> expression was higher in Lgr5<\/i>-derived tumors compared with PC-derived tumors (Lgr5<\/i>\/Apc<\/i> versus Lyz1<\/i>\/Apc<\/i>: log2<\/sub>-transformed fold change\u2009=\u20092.12, P<\/i>\u2009=\u20090.017) (Fig. 1k<\/a>). Moreover, both Kras<\/i> oncogenic activation and inflammation gradually increased Axin2<\/i> levels in PC-derived tumors, in agreement with the previously reported synergism between Apc<\/i> and Kras<\/i> mutations in activation of the WNT pathway23<\/a><\/sup>.<\/p>\n Thus, upon tumorigenesis, PCs dedifferentiate to a state that hampers secretory differentiation leading to specific patterns of tumor histology and gene expression distinct from that of Lgr5<\/i>-derived tumors.<\/p>\n To elucidate the mechanisms that underlie the conversion of PCs into cells-of-origin of small intestinal tumors in the context of inflammation and\/or of specific genetic hits, we combined single-cell RNA sequencing (scRNA-seq) analysis with lineage tracing. To this end, we induced the Apc<\/i>, Kras<\/i> and Trp53<\/i> genetic mutations in R26LSL-tdTomato<\/sup>\/Lyz1<\/i>CreERT2<\/sup> (or R26LSL-YFP<\/sup>) reporter strains in the presence or absence of DSS (Fig. 2a<\/a>). Subsequently, cells were harvested from the intestinal epithelium, purified by FACS and transcriptionally profiled by scRNA-seq (Methods<\/a> and Extended Data Fig. 3<\/a>). After preprocessing, we obtained the transcriptomes of 23.231 epithelial cells from 32 mice, distributed over the different lineages of the intestinal epithelium (Fig. 2b<\/a>). Close examination of cells positive for the reporter genes revealed novel clusters of PCs that arise upon DSS administration and specific gene mutations, but were not observed among PCs under homeostatic conditions (PC cluster 1\u20134, Fig. 2c<\/a>).<\/p>\n a<\/b>, Schematics of the experimental approach, adapted from ref. 63<\/a><\/sup>, Springer Nature Limited. After genetic targeting of PCs, intestinal crypts were extracted, and the isolated cells were labeled with hashing antibodies and sorted according to three different strategies: epithelium, PC-enriched and PC-traced cells. b<\/b>, UMAP embedding of the different cell clusters or lineages (left), annotated according to the expression of canonical marker genes (right). EE, enteroendocrine cells; EP, enterocytes progenitors; TA, transit-amplifying cells. c<\/b>, Bar plot of the distribution of traced cells across the different mouse genotypes and experimental conditions. d<\/b>, Violin plots representing marker genes of the newly identified Paneth-derived cell clusters (PC cluster 1\u20134). e<\/b>, Association analysis of the RSC signature with PC cluster 1\u20134. The P<\/i> value denotes the result of one-way ANOVA. f<\/b>, RNA in situ hybridizations of the Clu<\/i> gene in small tumors derived from PCs upon compound targeting of Apc<\/i> and Kras<\/i> mutations. g<\/b>, Gene sets variation analysis among refs. 30<\/a>,64<\/a><\/sup> and the current study. EMT, epithelila-to-mesenchymal transition.<\/p>\n Source data<\/a><\/p>\n<\/div>\n<\/div>\n To characterize the novel PC-derived states, we performed differential expression analysis and identified cluster-specific markers (Fig. 2d<\/a> and Supplementary Table 1<\/a>). Whereas PC cluster 1 appeared at low frequency across different genotypes, PC cluster 2 arises directly upon exposure to the inflammatory stimulus. Both PC cluster 1 and cluster 2 are characterized by increased expression of two markers of radio-resistant and secretory progenitors with self-renewal capacity during regeneration, namely Krt19<\/i> (ref. 24<\/a><\/sup>) and Atoh1<\/i> (ref. 25<\/a><\/sup>), whereas increased expression of Reg3b<\/i>, known for its protective role in the development of colitis and ileitis26<\/a><\/sup>, and Cdkn1a<\/i> (encoding p21), a marker of terminal differentiation in the intestine27<\/a><\/sup>, was observed in cluster 2 compared with cluster 1.<\/p>\n PC cluster 3 became apparent in mice carrying Apc<\/i> mutations (7.23%\u2009\u00b1\u20095.77% of traced cells) alone and in combination with DSS treatment (16.40%\u2009\u00b1\u20092.10% of traced cells), and in double- and triple-mutant (AP, AK and AKP) mice, although not in mice carrying single Kras<\/i> or Trp53<\/i> mutations. PC cluster 3 showed increased expression of Gif<\/i> (gastric intrinsic factor), Cd81<\/i>, a tetraspanin family member known to mark the response to gamma-irradiation and correlated with the expression of ISC- and proliferation genes28<\/a><\/sup>, and Prom1<\/i> (also known as CD133<\/i>), a well-established colon cancer stem cell marker29<\/a><\/sup>.<\/p>\n PC cluster 4 consisted of cells from mice in which double (AK) and triple (AKP) mutations were targeted to PCs (23.25%\u2009\u00b1\u200911.23% and 52.26%\u2009\u00b1\u200928.23% of traced cells, respectively). Increased expression of Anxa2<\/i> (encoding annexin 2), a functional marker of inflammatory response, and Clu<\/i> (clusterin), previously shown to earmark revival stem cells (RSCs) upon gamma-irradiation30<\/a><\/sup>, feature in PC cluster 4. Accordingly, evaluation of the RSC signature showed elevated expression among the PC clusters (Fig. 2e<\/a>), and in situ hybridization analysis in PC-derived tumors from mice carrying compound mutations (AK and AKP) (Fig. 2f<\/a> and Extended Data Fig. 4a,b<\/a>) confirmed increased Clu<\/i> expression. Finally, pathway analysis revealed the similarities between the PC-derived cluster 4 and RSCs, both earmarked by the activation of Yap1 signaling and specific inflammatory pathways (Fig. 2g<\/a>). Compared with RSCs, PC-derived and Apc\/Kras-<\/i>mutant cells from cluster 4 showed increased levels of TGF\u03b2 and WNT signaling (Extended Data Fig. 4c<\/a>).<\/p>\n Thus, upon genetic targeting or inflammatory stimulus, PCs escape their homeostatic identity and acquire distinct cellular features, as shown by scRNA-seq and FACS analysis (Extended Data Fig. 4d,e<\/a>). Of note, DSS treatment led to lower expression of Lgr5<\/i> and Ascl2<\/i> in stem cells, as well as a lower association of the ISC signature, confirming our and others\u2019 previous observations that resident stem cells lose their multipotency upon acute inflammation13<\/a><\/sup> (Extended Data Fig. 4f<\/a>).<\/p>\n Collectively, these findings demonstrate that PCs efficiently dedifferentiate upon genetic targeting or inflammatory stimulus leading to distinct cellular identities. During tumorigenesis driven by Apc\/Kras<\/i>, PCs share features with the YAP1-dependent RSC identity, and further activate TGF\u03b2 and WNT signaling in their conversion to bona fide tumor cells.<\/p>\n To investigate the consequences of cell-of-origin identity on the transcriptional profile of the resulting intestinal tumors, we performed bulk RNA sequencing (RNA-seq) of macroscopically dissected lesions originating from ISCs and PCs (Fig. 3a<\/a>). Principal component analysis (PCA) revealed that the major variance component (61%) was attributed to differences in the cell-of-origin, whereas the impact of genotype or inflammatory stimulus became notable in the second component of variation (10%) (Fig. 3b<\/a>). Differential expression analysis between tumors derived from PCs and ISCs in the same genetic and inflammatory context (Apc<\/i> and DSS) revealed tumor signatures specific for each cell-of-origin (Supplementary Table 2<\/a>).<\/p>\n<\/a><\/div>\n
PCs dedifferentiate into revival stem cells upon enhanced WNT signaling activation<\/h3>\n
<\/a><\/div>\n
Transcriptomic comparison of Paneth- and Lgr5<\/i>-derived tumors reveals a dichotomy in stem cell phenotypes<\/h3>\n