Abstract
Colorectal cancer initiation and progression result from the accumulation of genetic and epigenetic alterations. Although aberrant gene expression and DNA methylation profiles are considered hallmarks of colorectal cancer development, the precise timing at which these are produced during tumor establishment remains elusive. Here we investigated the early transcriptional and epigenetic changes induced by adenomatous polyposis coli (Apc) inactivation in intestinal crypts. Hyperactivation of the Wnt pathway via Apc inactivation in crypt base columnar intestinal stem cells (ISC) led to their rapid accumulation driven by an impaired molecular commitment to differentiation, which was associated with discrete alterations in DNA methylation. Importantly, inhibiting the enzymes responsible for de novo DNA methylation restored the responsiveness of Apc-deficient intestinal organoids to stimuli regulating the proliferation-to-differentiation transition in ISC. This work reveals that early DNA methylation changes play critical roles in the establishment of the impaired fate decision program consecutive to Apc loss of function.
This study demonstrates the functional impact of changes in DNA methylation to determine the colorectal cancer cell phenotype following loss of Apc function.
Introduction
Although colorectal cancer remains one of the leading causes of cancer-related death in developed countries (1), the mechanisms involved in its initiation remain only partially understood. The vast majority (80%–90%) of colorectal cancers is initiated by the constitutive activation of the Wnt pathway due to inactivating mutations in the adenomatous polyposis coli (APC) tumor suppressor gene. Mouse models with germline or inducible Apc deletion recapitulate the early stages of tumorigenesis (2, 3). In such models, adenomatous growth occurs upon biallelic mutation of Apc in the stem cell compartment, but not in differentiated cells, which are short lived, highlighting the key role of stem cells in tumorigenesis (4). Intestinal stem cells are intercalated between postmitotic Paneth cells, which constitute the stem cell niche at the base of crypts (5). Apc loss of function in those cells causes the formation of hypertrofic/hyperproliferative intestinal crypts with impaired epithelial organization (6, 7). In both humans and mice, benign adenomas then progress toward malignant stages through the sequential accumulation of further genetic alterations (8–10). Beside those genetic alterations, the analysis of epigenetic profiles in colorectal cancer samples has initially revealed important remodeling of the DNA methylation profiles, distinguishing tumor tissues from their surrounding nontumoral mucosa (11). Indeed, cancer cells display a general genomic hypomethylation, although genomic regions associated with tumor suppressors are frequently hypermethylated, which leads to their long-term transcriptional silencing (12). Recently, part of the alterations associated with colorectal cancer was shown to be already present in the intestinal adenomas found in ApcMin mice, as compared with the surrounding nontransformed tissue (13). However, the extent and dynamics of DNA methylation changes occurring during early tumorigenesis, as well as whether they functionally contribute to tumor initiation, remain unclear. De novo patterns of methylation are established in unmethylated DNA regions by methyltransferases DNMT3A and DNMT3B, and then maintained through cell divisions by DNMT1 (14). Importantly, de novo methyltransferases are preferentially expressed in the epithelial proliferative crypt compartment and overexpressed in tumor samples (15), and their activity was reported to promote adenoma formation through the methylation of specific loci (16–18). Importantly though, all these comparisons have been based on heterogenous biological samples (i.e., cancer samples at various stages vs. nontransformed mucosa) in which the proportions of the different cell types are significantly altered, whereas transcriptomic programs and DNA methylation profiles can vary extensively during cell differentiation, including in the intestinal epithelium (15, 19–25). Here, we investigated the earliest consequences of the loss of Apc function specifically in the predominant tumor cells-of-origin expressing the leucine rich repeat containing G protein-coupled receptor 5 (Lgr5) crypt base columnar (CBC) stem cell marker, early after the induction of Apc deletion. We then exploited the organoid culture system to dissect further the functional role of DNA methylation variations during the early tumorigenic process.
Materials and Methods
Animal strains and procedures
All animal experiments were approved by the French Agriculture and Forestry Ministry. All the mice were on a C57BL/6/J genetic background and maintained in an SPF animal facility. Extended experimental procedures are described in the Supplementary Information.
Flow cytometry, FACS, and cell-cycle analyses
Single-cell suspensions of the most proximal 1/3 of freshly isolated small intestine were used for the cell-cycle analysis or FACS sorting of GFP+ cells directly in RLT+ lysis buffer (Qiagen) for subsequent gDNA/RNA extraction, using a FACSAria (Becton Dickinson). Flow cytometry analyses were performed by using the FlowJo software (FlowJo LLC).
Organoid formation assay and organotypic culture methods
Establishment of primary organotypic models from Lgr5-CreERT2-GFP+ cells or Villin-CreERT2 intestinal crypts, expansion and maintenance were performed as described previously (26). Extensive details on cell culture procedures, including passaging, are in the Supplementary Information. To induce the Cre-mediated recombination of Apc in vitro, cells were cultured during 3 days in a medium supplemented with 4-OH-tamoxifen (200 nmol/L) resuspended in ethanol. To inhibit the activity of DNA methyltransferases, medium was supplemented with nanaomycin A (5 μmol/L) reconstituted in DMSO or 5-azacytidine (200 nmol/L; Sigma-Aldrich) administered at the same time than 4-OH-tamoxifen treatment and then maintained all along the culture. Lentiviral-mediated transduction and antibiotic selection were performed as described previously (27), with minor modifications. Experimental procedures for the evaluation of growth kinetics, clonogenic potential, and responsiveness to R-spondin1 (Rspo1) depletion or bone morphogenetic protein (BMP) stimulation are detailed in the Supplementary Information. All primary models used in this study were routinely (once-a-month) tested using the Mycoalert Mycoplasma Detection Kit (Lonza) and appeared Mycoplasma-free until the end of the study. All experiments were performed within a period of culture shorter than 4 months from initial establishment of primary models. Mouse cell line authentication was not performed as we do not have appropriate benchmarks against which to check mouse primary models in this study.
gDNA and RNA methods
DNA and RNA of FACS-sorted eGFP+ cell and intestinal organoids were isolated using the Allprep DNA/RNA Micro (GFP+ cells) or Mini (organoids) Kit (Qiagen) according to the manufacturer's instructions and used for next-generation sequencing, RT-PCR, qRT-PCR, and McrBC-qPCR methylation assay.
Data access
The accession number for the sequencing data reported in this work is GEO: GSE123006.
Fluorescent IHC on paraffin-embedded tissue and organotypic cultures
Tissue dissection and IHC on 5-μm-thick sections of paraffin-embedded tissue and organotypic cultures were performed essentially as described previously (28). All experiments were performed on formalin-fixed tissues with epitope retrieval in boiling 10 mmol/L sodium citrate (pH 6.4).
Results
Apc loss of function leads to the expansion of the Lgr5+ stem cell compartment without increasing the rate of stem cell division
To investigate the cellular and molecular dynamics of the intestinal stem cell (ISC) compartment during early tumorigenesis, we used the Lgr5-CreERT2-ires-eGFP mouse line that allows specific identification and genetic manipulation of the intestinal CBC stem cell population (29). In this model, the variegated expression of the eGFP and the CreERT2 recombinase leads to two distinct populations of intestinal crypts, expressing or not the knocked-in allele under the control of the Lgr5 promoter. When in combination with ApcLoxP/+ or ApcLoxP/LoxP alleles, the administration of tamoxifen results in deletion of one or both Apc alleles in Lgr5-expressing ISCs. Such recombined ISCs will be called hereafter ApcHET and ApcKO, respectively, whereas nonrecombined ICSs will be named ApcWT. As early as 15 days after tamoxifen administration, the loss of a single Apc allele did not produce significant changes in the size of the eGFP+ stem/progenitor cell compartment, whereas the biallelic loss, resulting in Wnt activation in most of the GFP-expressing intestinal crypts, produced a mean 9-fold expansion of eGFP+ cells as compared with ApcWT controls (Fig. 1A and B). Consistently with this observation, a clonogenic assay performed by seeding FACS-sorted eGFP+ cells led to the development of significantly more multicellular organotypic structures from ApcKO eGFP+ cells as compared with ApcWT eGFP+ cells, thus confirming the increased proportion of eGFP+ cells endowed with stem ability in ApcKO crypts (Fig. 1C). Indeed, we observed an increase in the number of cells expressing the bona fide ISC marker Olfm4 in ApcKO crypts compared with their ApcWT counterparts (Fig. 1D), as well as cells expressing Sox9 (Fig. 1E), a marker shared by ISCs and Paneth cells in the intestinal epithelium. Moreover, quantification of eGFP+ ISCs on intestinal sections using the very restrictive anatomic criterion of a direct contact with the lyzozyme-expressing Paneth cell niche denoted an accumulation of ISCs following Apc deletion, associated with the concomitant increase of Paneth cell numbers in ApcKO crypts (Supplementary Fig. S1A and S1B). Together, these results indicate that Apc inactivation induces a rapid expansion of the ISC compartment.
The Wnt signaling pathway is considered as a major driving force of intestinal epithelial proliferation. Accordingly, deletion of its transcriptional effector Tcf7l2/Tcf4 causes proliferation arrest, whereas aberrant constitutive activation of the Wnt pathway following Apc loss-of-function results in the accumulation of proliferating cells and formation of hypertrophic crypts (30, 31). As expected, Apc-deficient ISCs gave rise to crypts with increased numbers of actively proliferative BrdU+ cells (Fig. 1F and G). To evaluate the direct contribution of ISCs proliferation in the expansion of Apc-deficient crypts, we assessed the cell-cycle status of ISCs following Apc deletion. Surprisingly, the incorporation of BrdU after a pulse of 2 hours (Fig. 1F) indicated a slight but significant reduction of BrdU+ eGFP+ cells upon deletion of Apc (Fig. 1H), instead of an increased proliferation of ApcKO ISCs. We confirmed that Apc deletion is not directly linked to increased proliferation rates in ICSs by flow cytometry quantification of the proportion of eGFP+ cells found in the S-phase using propidium iodide staining of cellular DNA (Fig. 1I). In addition, no significant difference was found between numbers of ApcKO and ApcWT BrdU+ cells when we only considered ISCs, defined as eGFP+ cells in direct contact with Lyz+ Paneth cells (Fig. 1J). Of note, the proportion of eGFP+ cells expressing markers of terminal differentiation into Paneth (Lyz+), enteroendocrine (Chga+), or tuft (Dclk1+) postmitotic cell lineages was slight (<3%) for both genotypes (Supplementary Fig. S1C–S1F). Moreover, analysis of the postmitotic tuft cell surface marker Siglec-F (Supplementary Fig. S1G), revealed a physiologic representation of tuft cells (∼0.4%; Supplementary Fig. S1H) within the epithelial samples, with minimal and comparable colocalization of this marker with eGFP in ApcWT and ApcKO sorted cells (Supplementary Fig. S1I). Together, these data ruled out the possibility that the increased proportion of nondividing cells in the total Lgr5-GFP+ ApcKO compartment may reflect a larger proportion of terminally differentiated cells with residual eGFP protein with our cell-sorting strategy. We thus concluded that Apc deletion causes the accumulation of eGFP+ cells with ISC features in ApcKO intestinal crypts, which cannot be ascribed to an augmented rate of cell division in ISCs.
ISC accumulation following Apc loss of function relies on an impaired molecular commitment toward differentiation
To identify the mechanisms underlying the rapid ISC accumulation following Apc loss of function, we next investigated the molecular consequences associated with Apc disruption, specifically in eGFP+ cells comprising ISCs and their most direct progeny in the transit-amplifying (TA) crypt compartment. The comparison of gene expression profiles of ApcWT and ApcHET FACS-sorted eGFP+ cells by RNA-seq showed that the first genetic hit in the Apc locus, that is, the loss of a single Apc allele (ApcHET), significantly altered the expression of more than 400 transcripts (FDR-adjusted P < 0.01) as compared with ApcWT eGFP+ cells (Fig. 2A, results listed in Supplementary Table S1).
Quantification of the residual loxP-flanked exon-15 showed an average 83% reduction of this exon in cells from ApcLoxP/LoxP;Lgr5-CreERT2 (ApcKO) mice (Supplementary Fig. S2A), therefore confirming the efficient loss of the full-length Apc transcript in the majority of sorted cells in our experimental setting. Remarkably, such a biallelic disruption of Apc, resulting in constitutive Wnt activation, caused dysregulation of approximately 5,000 transcripts (Fig. 2A) in ApcKO eGFP+ cells, highlighting a dramatic rewiring of gene expression in ISCs and their immediate progeny associated with the constitutive activation of Wnt signaling. Remarkably, a subset of genes was exclusively expressed in either one or the other genetic condition. Among these markers, the endoderm-specific SRY-box 17 (Sox17) transcription factor (Supplementary Fig. S2B) was undetectable in both ApcWT and ApcHET eGFP+ cells and expressed in ApcKO eGFP+ cells. IHC detection confirmed that this gene is ectopically expressed by scattered ApcKO eGFP+ cells within the ISC zone as early as day 6 postrecombination, and then intensely expressed in the transit amplification compartment of ApcKO crypts (Supplementary Fig. S2C).
To gain further insight into the changes occurring upon the disruption of Apc in ISCs, we performed gene set enrichment analyses (GSEA). The analyses on the representation of wide collections of gene sets related to chemical and genetic perturbations (c2.cgp.v6.1) and KEGG pathways (c2.cp.kegg.v6.1.symbols) in ApcWT and ApcKO gene expression profiles revealed an overlap with the results from other studies on the outcome of Apc inactivation in mouse models of early tumorigenesis and human adenomas (reported in Supplementary Tables S2 and S3; examples of sets related to early tumorigenesis are shown in Supplementary Fig. S2G). Consistently with Apc abrogation, GSEA interrogation of our data confirmed an exacerbated Wnt signaling in ApcKO eGFP+ cells, with an increased expression (negative correlation with ApcWT gene expression, NES WT versus KO = −1.57) of the set of genes representing β-catenin transcriptional targets (Fig. 2B). In line with the cell-cycle analyses of ISCs from ApcWT or ApcKO mice, indicating absence of increased cell proliferation (Fig. 1F–J), no preferential enrichment was found in ApcKO or ApcWT eGFP+ cell profiles for the set of genes associated with progression through the cell cycle (Fig. 2B). Concomitantly, the gene signature associated with ISC identity (32; Supplementary Table S4) showed a significant enrichment (NES WT vs. KO = −1.6) in the transcriptomic profiles of ApcKO eGFP+ cells (Fig 2B). Conversely, the signature associated with intestinal epithelial differentiation (33) was depleted in the gene expression profiles associated with ApcKO cells (Fig 2B; NES WT vs. KO = 2.95), whereas no significant enrichment was detected for the set of genes associated with progenitor cells from the transit-amplification compartment in one or the other conditions (Fig. 2B). Indeed, a subset of bona fide ISC markers, including Lgr5, Axin2, Musashi RNA Binding Protein 1 (Msi1), and others, was upregulated in ApcKO eGFP+ cells (Fig. 2C), whereas early markers of intestinal epithelial commitment (Atoh1, Spdef) and differentiation into enterocytes (Elf3, Krt20), goblet (Cdx2, Muc2), enteroendocrine (Insm1, Chga), tuft (Pou2f3, Dclk1) and microfold (Spi-B) epithelial cell lineages were downregulated (Fig. 2D). The most important exception was represented by markers associated with Paneth cell maturation, which is known to rely on Wnt signaling (7, 34). Indeed, we could observe the expression of transcription factors involved in lineage commitment in eGFP+ CBC cells and, more frequently, in eGFP+ cells immediately above the CBC cell zone in control mice (Fig. 2E–H). We found ApcWT-eGFP+ cells clearly expressing markers involved in lineage commitment such as Elf3 [an enterocyte specification marker (35)] and Cdx2 [a pan-epithelial marker involved in enteroendocrine cell commitment (36)] or, less frequently, Insm1 [enteroendocrine cells (37)] or Pou2f3 [tuft cells specification (38)]. Overall, the expression of these markers in the self-renewal compartment supports the early lineage specification occurring in ISCs and their daughter TA cells, which is impaired by the inactivation of the gate-keeper Apc gene.
We then decided to examine the possible involvement in these findings of the transcriptional regulator Myc, an important Wnt target gene described previously as a key mediator of the epithelial phenotype consequent to Apc loss (39, 40). As expected, the size of the Myc+ compartment expanded during time following Apc inactivation, as Myc was expressed by the increasing number of cells showing β-catenin translocation in ApcKO crypts compared with ApcWT control crypts (Supplementary Fig. S2D). However, constitutive activation of Wnt signaling in ApcKO Lgr5-GFP cells did not further increase the expression of Myc in individual FACS-sorted stem/progenitor cells (Supplementary Fig. S2E). Together, these observations suggest that the specific effects of Apc loss and exacerbated Wnt signaling found in ISCs do not rely on an altered level of Myc expression. Interestingly, the expression of the Myc paralogue Mycn, which is known to be preferentially expressed by differentiated cells in the intestinal epithelium (39), was significantly reduced in ApcKO cells (Supplementary Fig.S2F). Overall, these results revealed that the primary outcome of the oncogenic Wnt pathway hyperactivation in ApcKO ISCs consists in their impaired commitment toward differentiation, which explains their rapid accumulation in spite of the absence of increased ISC division rate.
Exacerbated Wnt activation is accompanied by rapid and defined alterations in the DNA methylation of the self-renewal compartment
We then investigated whether the impairment in the ability of Apc-deficient ISCs to commit toward differentiation is accompanied by rapid alterations of DNA methylation profiles. Some instructive changes in the DNA methylation patterns have been observed during homeostatic intestinal differentiation (15, 25). Moreover, alterations in the DNA methylation patterns have been reported in advanced stages of colorectal cancer and, more recently, in early adenomas (11–13, 41, 42). However, whether these alterations are immediate consequences of the oncogenic loss of Apc function remained unclear. We therefore performed reduced representation bisulfite sequencing (RRBS) on FACS-sorted ApcWT, ApcHET, and ApcKO eGFP+ cells. Hierarchical clustering of the methylation scores provided fair discrimination of the ApcKO from the ApcWT and ApcHET samples, indicating a specific impact of Apc inactivation on the methylome (Fig. 3A). Overall, RRBS analyses revealed significant discrete rearrangements of the DNA methylation profiles in ApcHET and ApcKO eGFP+ cells as compared with ApcWT controls (Fig. 3B). We identified 58 differentially methylated regions (DMR) in ApcHET eGFP+ cells compared with eGFP+ cells from control mice, and 790 DMRs in ApcKO eGFP+ cells (detailed in Supplementary Table S5). In line with the notion of a general DNA hypomethylation in colorectal cancer samples (11, 12), 75% of the DMRs were hypomethylated in ApcKO cells (Fig. 3C). These DMRs were reproducible between replicates, highlighting the consistent effect exerted by Apc loss on the DNA methylation profiles (Fig. 3D). Both hypomethylated and hypermethylated DMRs identified by RRBS were generally distant from gene transcription start sites (TSS), with only 57 of 790 DMRs located within 2,000 bps from the most proximal TSSs (Fig. 3E), and are preferentially located within intronic and intergenic regions (Supplementary Fig. S3A). This is in accordance with previous findings obtained by comparing the DNA methylation in adenoma samples and in ISCs (42). Importantly, the DNA methylation changes occurring 15 days after Apc deletion remained focal and less extensive than the general remodeling described in colon cancer (11, 41). For instance, repeated elements belonging to endogenous retrotransposon families, such as intrascisternal A particles (IAP), are severely methylated in normal cells and frequently hypomethylated in cancer cells (43, 44). However, the methylation of these regions was not affected 15 days following Wnt constitutive activation (Fig. 3F). This result was confirmed experimentally for IAPs by qPCR on genomic DNA fragments obtained after digestion with the McrBC methylation-sensitive restriction enzyme (Supplementary Fig. S3B). Taken together, these observations indicate that the DNA methylation status of a defined subset of genetic loci changes very rapidly at tumor initiation following loss of Apc function.
To investigate the direct involvement of altered DNA methylation in the impaired ability of ISCs to commit toward differentiation, we first interrogated the RRBS profiles for the enrichment of epithelial stem and postmitotic signatures (Supplementary Table S4), by analyzing the methylation of promoter regions (encompassing −1,000 bps to +500 bps from the TSS). GSEA results did not show global tendencies in the methylation of the TSSs associated to genes belonging to those sets (Fig. 3G). Even restricting the analysis to the list of TSSs associated with the identified DMRs (Supplementary Table S5) did not show preferential hypermethylation of genes associated with postmitotic commitment (Fig. 3G), therefore indicating that altered DNA methylation of loci directly associated with stemness/maturation does not represent an instructive mechanism for the imbalanced fate program dictated by Apc loss. As ISC identity and commitment are regulated by opposite gradients of Wnt and BMP/TGFβ signaling along the crypts-villus axis (a schematic representation is provided in Supplementary Fig. S3C), we focused our attention on these two signaling pathways. Indeed, secretion of Wnt ligands and BMP/TGFβ inhibitors cooperate to the formation of a stem-permissive niche in both homeostatic crypts and adenomas (45–47). Thus, forcing BMP inhibition concomitantly to Wnt constitutive activation leads to the expansion, or even to the ectopic formation of an adenoma stem cell compartment (48, 49), whereas increasing secretion of BMP ligands along the villus axis was shown to promote epithelial differentiation (46). Combining the data from RRBS and RNA-seq analyses revealed an altered extent of methylation and expression of four loci associated to components of the Wnt pathway (Axin2, Nfatc2, Prkca, Vangl1) and three loci associated with the TGFβ/BMP signaling pathway (Inhbb, Bmp7, Smad6) in ApcKO eGFP+ cells (Supplementary Fig. S3D and S3E). The Smad6 locus, coding for an intracellular inhibitor of the BMP signaling pathway, was used for experimental validation using gDNA and RNA samples from an independent cohort of ApcWT and ApcKO eGFP+ cell samples from intestinal epithelia. McrBC-qPCR and RT-PCR analyses confirmed the hypomethylation (Supplementary Fig. S3F) and increased expression (Supplementary Fig. S3G) of Smad6 in ApcKO ISCs. Overall, these observations suggested that rapid, defined, changes in the methylation patterns of specific loci may be implicated in impaired cell fate decisions at tumor initiation.
Inhibition of de novo DNA methyltransferases partially compensates the consequences of constitutive Wnt signaling after Apc inactivation in intestinal organoids
We next evaluated the contribution of DNA methylation changes in the altered behavior of ApcKO ISCs. We first reassessed the expression patterns of de novo methyltransferase genes Dnmt3a and Dnmt3b in the normal and ApcKO epithelium. RNA-seq data showed no significant differences in the expression of Dnmt3a between ApcWT and ApcKO Lgr5-GFP cells (Fig. 4A), whereas the expression of Dnmt3b was slightly but significantly increased upon Apc-loss in those cells (Fig. 4C), probably reflecting ISC accumulation within the compartment. Specific immunostainings confirmed that these enzymes are preferentially expressed in the normal intestinal crypts, with Dnmt3a signal spanning from ISCs to TA cells at crypt/villus border, whereas Dnmt3b staining was accentuated in ISCs and immediate daughter cells in the normal crypt (Fig. 4B and D, top panels). Those patterns were clearly expanded upon Apc deletion in transgenic crypts, in accordance with the outgrowth of cells displaying β-catenin translocation (Fig. 4B and D, middle panels). Furthermore, these enzymes were expressed in virtually all ApcKO epithelial cells in well-developed adenomas from ApcΔ14/+ mice harboring a germline heterozygous mutation on Apc, and spontaneously developing clonal adenomas from stem cells in which the remaining wild-type Apc allele has been mutated or silenced (Fig. 4B and D, bottom panels). Having shown that the dynamics of de novo Dnmt patterns coincide with the expansion of the intestinal self-renewal compartment, we then investigated whether the precocious changes in DNA methylation that we observed in ICSs following constitutive Wnt activation are functionally involved in such an expansion at the expense of homeostatic commitment to differentiation. To this end, we assessed the outcome of de novo methyltransferases inhibition in intestinal organotypic cultures (organoids) derived from the intestinal epithelium of ApcLoxP/LoxP;VillinCreERT2 mice. Such organoids are normal (ApcWT) until they are treated with tamoxifen, which results in the deletion of both Apc alleles (ApcKO) in all epithelial cells. qRT-PCR quantification confirmed a substantial reduction (>400-fold change) in the LoxP-flanked exon-15 upon treatment with 4-OH-tamoxifen, therefore validating the effective abrogation of the functional full-length Apc transcript (Supplementary Fig. S4A). Indeed, we constantly observed a switch toward a spheroid morphology (Supplementary Fig. S4B), an increased proportion of actively proliferating (Ki67+) cells (Supplementary Fig. S4C) consistent with increased global crypt proliferation in mice (see Fig. 1G), overall confirming the Wnt oncogenic activation in ApcKO intestinal organoids. All those phenotypic changes were associated to the accumulation of cells expressing the ISC maker Sox9 (Fig. 4E) in ApcKO organoids, which is consistent with the increased stemness described in Fig. 1.
ApcLoxP/LoxP;VillinCreERT2 organoids were lentivirally transduced with shRNAs directed against Dnmt3a and Dnmt3b transcripts or, as a control, with lentiviral vectors containing length-paired nontarget sequences. IHC detection confirmed the reduced amount of nuclear Dnmt3a and Dnmt3b enzymes (Supplementary Fig. S4D and quantification in Supplementary Fig. S4E and S4F). The knockdown of the two enzymes did not affect the general rate of proliferation of ApcWT models (total average of 59.9% ± 7.1 vs. 51.2% ± 5.70 of Ki67+ cells in replicates from two NT-control and two double-sh clones, respectively, Fig. 4F). As expected, after Apc deletion, the general extent of cell proliferation was increased in control organoids expressing nontarget shRNAs (total average of 77.1% ± 2.5 in two clones). In sharp contrast, the combined knockdown of Dnmt3a/Dnmt3b reduced the general proliferation of ApcKO organoids to an extent comparable with ApcWT organoids (total average of 53.9% ± 3.4 in two clones, Fig. 4F), supporting the notion of a requirement of de novo methyltransferase function in the phenotype resulting from Apc loss-of-function. Compared with ApcKO organoids transduced with nontarget shRNA, ApcKO organoids with Dnmt3a/Dnmt3b knockdown also displayed a reduced sphere formation ability in 3/3 replicates when replating was performed by dissociating a number of organoids equivalent to an identical number of cells (Fig. 4G), therefore suggesting a decreased stemness, as defined by the ability of a single cell to proliferate and generate a multicellular organoid, of ApcKO organoid cells with reduced Dnmt3a/b expression. To confirm these findings with an independent approach, we tested the effect of de novo methyltransferases inhibition by treating organoids with the specific Dnmt3b inhibitor nanaomycin A, or with the demethylating agent 5-azacytidine (50). Remarkably, the administration of either inhibitor concomitantly with the deletion of Apc resulted in reduced proliferation rates in three independent organotypic models (Fig. 4H), at extents comparable with those of isogenic ApcWT organoids, in which cell division rate was not affected by the presence of inhibitors, although increased cell-death was observed early after administration of 5-azacytidine. Moreover, when we examined the expression of Wnt-related genes (Axin2, Nfatc2, Prkca, and Vangl1) and BMP-related genes (Smad6, Inhbb, and Bmp7) that were differentially methylated and expressed in ApcKO CBC (see Supplementary Fig. S3D and S3E), we found that those genes were also differentially expressed in ApcKO organoids compared with ApcWT controls (Fig. 4I), thus confirming the relevance of organotypic models in the study of homeostatic disruption upon Apc inactivation. Taken together, these results indicate that the activity of de novo methyltransferases is required for the increase in the proportion of actively proliferating cells able to form growing organoids following Apc loss of function.
De novo DNA methyltransferase activity contributes in altering Wnt and BMP signaling, driving ISC accumulation upon Apc inactivation
Next, we sought to test whether de novo methyltransferase activity is implicated in the responsiveness of ApcKO epithelial cells to environmental Wnt and BMP stimuli, regulating the homeostatic choice between self-renewal and differentiation. Intestinal organoids are routinely maintained in presence of recombinant Rspo1, which cooperates with Wnt signals to define ISC properties (51), and becomes dispensable following constitutive Wnt activation (26, 52). Indeed, we observed a loss of spheroid formation ability and reduction of spheroid size in ApcKO organoids with combined Dnmt3a and Dnmt3b knockdown. Strikingly, these effects were further accentuated upon withdrawal of Rspo1 from the culture medium (Fig. 5A and B). This confirmed the reduction in the sphere-formation ability and proved the recovered dependence to Rspo1 upon de novo Dnmts knockdown in ApcKO organoids. We then assessed the responsiveness of ApcKO organoids to the stimuli that modulate the BMP signaling. Stimulation of organoid cultures with recombinant BMP2 after withdrawal of the BMP inhibitor Noggin from the medium dramatically increased the formation of crypt-like structures in Dnmt3a/Dnmt3b shRNA-expressing ApcKO organoids as compared with the nontarget shRNA control ApcKO organoids (Fig. 5C). This indicated a restoration of the ability of epithelial cells to morphologically self-organize the formation of distinct crypt and differentiated compartments. Indeed, control individually seeded ApcKO cells were largely more prone to form spheroids than nanaomycin A–treated cells in the presence of rBMP2 stimulation (Fig. 5D), supporting the recovered responsiveness to differentiation stimuli orchestrated by BMP signaling upon de novo Dnmt inhibition. As in the case of FACS-sorted eGFP+ cells from ApcWT and ApcKO crypts (Supplementary Fig. S3E), ApcKO organoid cultures displayed an upregulation of the BMP inhibitor Smad6 mRNA as compared with ApcWT organoids. Moreover, treatment with 5-azacytidine reduced the expression level of this gene in 2/2 independent ApcKO organoid cultures (Supplementary Fig. S5A) and nanaomycin A exerted comparable effect in 2/3 independent cultures (Supplementary Fig. S5B). To further elucidate the responsiveness to BMP signaling as involved in the Apc phenotype, we therefore decided to test the implication of the Alk-pSmad axis physiologically regulated by Smad6 during homeostasis. Strikingly, BMP inhibition resulting from the addition of the Alk-pSmad inhibitor LDN193189 abolished the rescue exerted by Dnmt3b inhibition mediated by nanaomycin A treatment, as demonstrated by the size (Fig. 5E and F, quantification from two independent models) and proliferative rate (Supplementary Fig S5C) of spheroids compared with controls and nanaomycin A–treated organoids. Together, these results confirmed that de novo DNA methylation occurring upon Apc inactivation is critically involved in the increase in the proportion of actively proliferating cells within the intestinal epithelium by dictating a reduced responsiveness of ISCs to the different stimuli regulating the proliferation-to-differentiation balance (summarized in Fig. 5G).
Discussion
We investigated the early consequences of oncogenic Apc loss-of-function, the most common genetic initiating event in colorectal cancer, on the behavior of ISCs. Importantly, we show that this tumor suppressor does not operate by negatively controlling the rate of cell division of intestinal stem cells but rather represents a critical regulator of the ability of ISCs to engage toward differentiation. In line with the rapid accumulation of ISCs observed early upon Apc deletion, the analysis of ISC gene expression profiles clearly confirmed the impaired ability of stem cells and their progeny to commit toward homeostatic differentiation, with a reduced expression of several markers for the different postmitotic cell lineages. Recently, single-cell RNA-seq analyses from purified Lgr5+ cells revealed a relatively homogeneous stem cell population mixed with rare secretory cells, likely reflecting early fate commitment decision in some progenitor cells (53). Indeed, we show that some markers of lineage commitment are expressed in the self-renewal compartment, supporting a role of Apc in controlling early homeostatic fate decision in ISCs. Moreover, transcriptomic profiles provided us with a list of early markers of Wnt oncogenic activation that are exclusively expressed in ApcKO cells, as we show in the case of Sox17, which is undetectable in the ApcWT epithelium, including in ISCs, and could be used to trace aberrant Wnt activation.
Of note, the overactivation of Wnt signaling in ISCs led to an increased number of Myc-expressing cells without increasing the cellular expression of Myc, a key mediator of the phenotype associated with Apc loss of function in intestinal epithelium (40), whose expression coincides with β-catenin signaling in both normal and pretumoral crypts. This finding allowed us to conclude that Myc is expressed in cells with active Wnt signaling and, reminiscently to its role during development (39), cooperates to the establishment of a crypt-like phenotype in the ApcKO epithelium, but its role in ISCs is not exacerbated upon oncogenic Wnt overactivation.
Alterations in DNA methylation have extensively been proven in advanced tumor stages, and were suspected to play a pivotal role during intestinal tumorigenesis (54, 55). However, the confounding cellular heterogeneity of the tissues used for comparison between tumor and healthy profiles has hampered precise conclusions about its actual contribution to cancer initiation. The RRBS profiling of ISCs and early progenitors provided evidences that the oncogenic Wnt activation does not trigger an immediate extensive remodeling of the DNA methylation landscape in the ISC compartment. Instead, Apc disruption rapidly produces discrete DNA methylation changes, indicating that focal remodeling of DNA methylation profiles initiates as early as the first oncogenic event, that is, loss of Apc function, before becoming more generalized in later stages of the disease. The characterization of CpG-rich genomic regions methylation showed that these changes are rarely found in the close proximity to gene TSSs. Of note, methylation of non-CpG-rich regions, as well as other epigenetic marks, may also contribute to the transcriptional and functional dysregulation observed in ApcKO ISCs. However, we show that genes implicated in the signaling pathways governing intestinal cell fate decisions represent preferential targets for altered DNA methylation during early tumorigenesis. The short timing in our experimental design rules out the possibility that these alterations represent the result of a clonal selection, and rather suggests the existence of a specific program associated with the loss of Apc function and Wnt constitutive activation.
Several studies have reported the impact of DNA methylation in controlling cell differentiation (15, 19–25), and the activity of de novo DNMTs was shown to regulate hematopoietic multipotency and stemness both in homeostasis and cancer (56). The specific role of DNMT3A and DNMT3B in intestinal homeostasis awaits further elucidation. However, the fact that these factors are expressed in the ISC compartment, together with the expansion of their pattern of expression in early lesions prompted us to investigate their functional implication in ISCs during intestinal tumorigenesis. Importantly, we show that Dnmt3a/Dnmt3b knockdown/inhibition in ApcKO intestinal organotypic cultures reduces the proportion of actively proliferating cells to a homeostatic level comparable with ApcWT organoids, therefore restraining the uncontrolled expansion of ApcKO organoids. Moreover, functional organotypic assays suggest that the activity of de novo methyltransferases may contribute to ISC accumulation by impairing their responsiveness to exogenous stimuli controlling the homeostatic balance between self-renewal and differentiation, such as Wnt and BMP signaling upon Apc inactivation. BMP signaling has been recently shown to play a crucial role in the commitment of Lgr5+ ISCs by repressing the signature associated with stemness without affecting Wnt signaling, therefore preventing ISC outgrowth during homeostasis and regeneration (47). However, we show that Wnt constitutive activation rapidly impairs the responsiveness of ISCs to BMP signals, hence demonstrating an interaction between these pathways in early oncogenesis. More investigation is needed to identify specific and overlapping genomic targets of the two de novo DNMTs in the Lgr5+ compartment both during homeostasis and tumorigenesis, as recently accomplished in hematopoietic and epidermal stem cells (56, 57). This question might be addressed by combining conditional Dnmt3(a/b)Flox and ApcFlox alleles with the Lgr5-CreERT2-Ires-EGFP model. Overall, our findings establish a critical effector role for DNA methylation in ISCs at the onset of the tumor phenotype resulting from Apc disruption and paves the way toward the design of novel strategies suitable to target the tumor stem cell compartment.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Authors' Contributions
Conception and design: M. Weber, P. Jay
Development of methodology: M. Bruschi, L. Garnier, M. Weber, F. Gerbe
Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): M. Bruschi, L. Garnier, E. Cleroux, A. Giordano, F. Gerbe, P. Jay
Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): M. Bruschi, L. Garnier, M. Dumas, A.F. Bardet, T. Kergrohen, M. Weber, F. Gerbe, P. Jay
Writing, review, and/or revision of the manuscript: M. Bruschi, S. Quesada, M. Weber, F. Gerbe, P. Jay
Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): M. Bruschi, M. Weber, P. Jay
Study supervision: M. Weber, P. Jay
Other (performed experiments): P. Cesses
Acknowledgments
This work was supported by ITMO Cancer (Plan Cancer 2009-2013 EPIG201311, to P. Jay and M. Weber), SIRIC Montpellier Cancer (grant INCa_Inserm_DGOS_12553 to P. Jay), ARC (SL220110603456 to P. Jay), ANR (ANR-14-CE14-0025-01 and ANR-17-CE15-0016-01 to P. Jay), INCa (2014-174 and INCA_2018-158 to P. Jay), the Labex EpiGenMed (an “Investissements d'avenir” program ANR-10-LABX-12-01 to P. Jay), the PJ team is “Equipe Labellisée Ligue contre le Cancer,” and the European Research Council (Consolidator grant no. 615371 to M. Weber). M. Bruschi was supported by Ligue Nationale contre le Cancer. We acknowledge M. Zenati, S. Barahoui, and E. Sidot for contribution to the collection of data, J. Pannequin and T. Bouschet for reagents, protocols, and advices, C. Perret (Cochin Institute, France) for sharing the Apc-mutated mice, the team of S. Fre (Curie Institute, France) for protocols of organotypic culture, F. Gallardo and D. Greuet in the iExplore facility for maintenance of mouse colonies, and C. Duperray in the Montpellier Ressources Imagerie (MRI) facility.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.