SMAD4 Suppresses WNT-Driven Dedifferentiation and Oncogenesis in the Differentiated Gut Epithelium Ansu O

Published OnlineFirst July 9, 2018; DOI: 10.1158/0008-5472.CAN-18-0043

Cancer Molecular Cell Biology Research

SMAD4 Suppresses WNT-Driven Dedifferentiation and Oncogenesis in the Differentiated Gut Epithelium Ansu O. Perekatt1,2, Pooja P. Shah1, Shannon Cheung1, Nidhi Jariwala2,3, Alex Wu1, Vishal Gandhi1, Namit Kumar1, Qiang Feng4, Neeket Patel1, Lei Chen1, Shilpy Joshi2, Anbo Zhou1, M. Mark Taketo5, Jinchuan Xing1, Eileen White2, Nan Gao2,4, Michael L. Gatza1,2,3, and Michael P. Verzi1,2

Abstract

The cell of origin of colon cancer is typically thought to be the WNT pathway led to dedifferentiation and rapid adenoma the resident somatic stem cells, which are immortal and escape formation in differentiated tissue. Transcriptional profiling the continual cellular turnover characteristic of the intestinal revealed acquisition of stem cell characteristics, and colabeling epithelium. However, recent studies have identified certain indicated that cells expressing differentiated enterocyte conditions in which differentiated cells can acquire stem-like markers entered the cell cycle and reexpressed stem cell properties and give rise to tumors. Defining the origins of genes upon simultaneous loss of SMAD4 and activation of tumors will inform cancer prevention efforts as well as cancer the WNT pathway. These results indicate that SMAD4 therapies, as cancers with distinct origins often respond dif- functions to maintain differentiated enterocytes in the ferently to treatments. We report here a new condition in presence of oncogenic WNT signaling, thus preventing which tumors arise from the differentiated intestinal epithe- dedifferentiation and tumor formation in the differentiated lium. Inactivation of the differentiation-promoting transcrip- intestinal epithelium. tion factor SMAD4 in the intestinal epithelium was surprisingly well tolerated in the short term. However, after several Significance: This work identifies a mechanism through months, adenomas developed with characteristics of activated which differentiated cells prevent tumor formation by suppres- WNT signaling. Simultaneous loss of SMAD4 and activation of sing oncogenic plasticity. Cancer Res; 78(17); 4878–90. 2018 AACR.

Introduction cancer—a model elegantly supported by in vivo mouse studies (1). However, histologic evidence from human patients with colon The intestinal epithelium is strictly compartmentalized—with cancer supports the notion that dysplastic cells arise from differ- stem and proliferative cells nestled into the intestinal wall in the entiated cells (2). Such a scenario requires the differentiated cells crypts of Lieberkuhn€ and differentiated cells lining the lumen on to acquire stem-like properties for oncogenesis (3, 4). The tumor villi in the small intestine or mucosal surface in the colon. Cells cell of origin often dictates the epigenetic status of the tumor and continuously transit from the crypt to the mucosal surface, where can inﬂuence treatment strategies (5, 6). they are shed into the lumen. Stem cells and their niche-support- Recent studies have shown that combinations of genetic altera- ing Paneth cells are the only cell types that remain anchored to the tions can trigger stem cell activity from differentiated intestinal crypt base and escape this cellular transit. This pattern of move- epithelial cells in mice. These include simultaneous mutations in ment prevents accumulation of oncogenic mutations in differen- APC and KRAS as well as simultaneous activation of the WNT and tiated cells, as differentiated cells will be lost into the lumen. NF-kB pathways (7, 8). The potential for plasticity in normal Hence, stem cells are thought to be the cell of origin of colon differentiated intestinal epithelial cells is also supported by experiments showing that upon loss of endogenous crypt base þ columnar (Lgr5 ) stem cells, the progenitor cell populations of 1Department of Genetics, Human Genetics Institute of New Jersey, Rutgers, The distinct intestinal lineages can dedifferentiate and acquire the fate þ State University of New Jersey, Piscataway, New Jersey. 2Rutgers Cancer of Lgr5 cells, which are the actively cycling stem cells of the Institute of New Jersey, New Brunswick, New Jersey. 3Department of Radiation intestine (9). These studies have shown that cells previously Oncology, Robert Wood Johnson Medical School, New Brunswick, New Jersey. referred to as "reserve" or "þ4" cell populations include enter- 4Department of Biological Sciences, Rutgers University, Newark, New Jersey, 5 oendocrine progenitor cells that can dedifferentiate into active New Jersey. Division of Experimental Therapeutics, Graduate School of Med- – þ icine, Kyoto University, Sakyo Kyoto, Japan. stem cells (10 13). In these dedifferentiation models, Lgr5 stem cells are typically replaced by neighboring progenitor cells in the Note: Supplementary data for this article are available at Cancer Research crypt, rather than cells luminally positioned on the more differ- Online (http://cancerres.aacrjournals.org/). entiated villus. Corresponding Author: MichaelP.Verzi,Rutgers,TheStateUniversityofNew SMAD4 is a transcriptional effector of the BMP- and TGFb- Jersey,Piscataway,NJ08854.Phone:848-445-9578; E-mail: [email protected] signaling pathways. The Cancer Genome Atlas (TCGA) research doi: 10.1158/0008-5472.CAN-18-0043 network has identiﬁed mutations in SMAD4 to be among the 2018 American Association for Cancer Research. most frequently mutated genes in colon cancers (14), and loss of

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þ Smad4 heterozygosity in the Apcmin/ mouse colon tumor back- Microscopy and imaging ground promotes invasive progression of colon adenomas (15). Nikon Eclipse E800 microscope and Retiga 1300CCD BMP signaling is required to maintain the differentiated state (Q-Imaging) cameras were used for bright field microscopy. For in the intestinal epithelium (16–18), as expression of BMP fluorescent microscopy, Zeiss Axiovert 200M microscope and inhibitors beginning at embryonic stages leads to emergence of Retiga-SRV CCD (Q-Imaging) QC imaging were used for crypt-like structures in the villi of the adult tissue. However, image acquisition. Keynote, the apple software, was used for the mechanisms and subsequent events leading to the loss of adjustments of sharpness and contrast and applied equally for differentiation in the intestinal epithelium are incompletely comparative images. ImageJ software was used for merging fluo- understood, particularly whether adult-onset disruption of BMP rescent images. signaling would lead to tumorigenesis from differentiated tissues. The following study expands our understanding of differenti- Villi and crypt cultures ated-cell-derived tumorigenesis by demonstrating that simulta- Villi from the proximal half of the duodenum were isolated in neous loss of SMAD4 and activation of the WNT pathway triggers the following manner with extreme care to avoid contamination stem cell properties and adenoma formation in the differentiated from crypts. Mice of the indicated genotypes were treated with epithelium of the adult intestine. tamoxifen for 4 consecutive days. The proximal half of the duodenum was harvested on day 10 after the first tamoxifen injection, flushed with cold PBS, opened longitudinally and laid Materials and Methods on clean 100 mm petri plate with villi facing up. Two histologic Mouse models glass slides were used to isolate the villi by scraping: one to hold fl fl The Villin-CreERT2 transgene (19), Smad4 ox/ ox (20), and Catn- down the intestine, and the other to scrape. Only two villi-yielding þ blox(ex3)/ (21) alleles were integrated to generate the conditional scrapes were carried out (to avoid crypts). The villi collected were compound mutants and controls. The Lgr5-EGFP-ires-CreERT2 washed with PBS and isolated from smaller debris using a 70-mm allele (22) was used for mosaic Cre induction. Tamoxifen was filter with cold PBS to wash the villi and filter out any crypts if administered at 0.05 g/kg mice, intraperitoneally, for 4 consecu- present. Fifty villi in 25 mL of Matrigel (Corning, 356230) were tive days to induce Cre recombination. All the experiments con- plated per well in a 48-well plate. The villi were cultured in ENR ducted had the approval of the Rutgers Institutional Animal Care media (23) with 2 mg/mL primocin. Crypts were isolated and Use Committee. from proximal duodenum and cultured in BME-R1 Matrigel (Trevigen, Cat#3433-005-R1) according to previously described Histology and immunohistochemistry methods (23). Intestines were fixed with 4% paraformaldehyde overnight at 4 C and washed in PBS prior to dehydration and paraffin RNA isolation and analysis embedding. Mice were injected with 100 mg of EdU (Thermo RNA was extracted from jejunal epithelia of the indicated Fisher Scientific, cat #C10337) 1 hour prior to sacrificing when genotypes in triplicates following 4 consecutive days of tamoxifen the tissues were prepared for EdU labeling of proliferating cells. injection. Uninjected mice served as control. After flushing the Five-micron sections were prepared from paraffin for histologic freshly harvested jejunum with cold PBS, the epithelia were analysis. For immunostaining, antigen retrieval was performed dissociated from underlying mesenchyme by incubating with with 10 mmol/L sodium citrate buffer (pH6) in a pressure 3 mmol/L EDTA/PBS at 4C as described previously (24) and cooker. Tissues were permeabilized with 0.5% triton. Antibo- filtered through 70-mm filter to isolate the crypts. The crypts were dies used in the study were Ki-67 (Abcam, ab16667, 1:300), washed with PBS and pelleted to remove excess PBS prior to Smad4 (Santa Cruz Biotechnology, SC-7966, 1:500), OLFM4 addition of TRIzol for RNA extraction. Sequencing was performed (Cell Signaling Technology, 39141, 1:2,000), Sox9 (Millipore, using Illumina's TruSeq RNA Library Prep kit v2 and data pro- AB5535, 1:2,000), BrdUrd (AbD Serotec, MCA2060GA, 1:500), cessed with version 2.2.1 of Cufflinks suite of tools (25). CuffDiff lysozyme (DAKO, A 0099, 1:2,000), Keratin 20 (Cell Signaling was used to call significant differentially expressed genes between Technology, D13063, 1:400), CHGA (SC-376827, 1:1,000, and the control and the Smad4KO, as well as the control and double DCAMKL (Abcam, ab37994, 1:1,000 (IHC-P), 1:200 (IHC-F), mutant. We used quartile normalization and per-condition dis- b-catenin (Santa Cruz Biotechnology, SC-7199, 1:250; Cell persion. CuffNorm was used to calculate fragments per kilobase of Signaling Technology, 9587, 1:1,000). Goat serum for blocking transcript per million (FPKM) values using quartile normaliza- and secondary antibodies were used according to the tion. These results were used for further downstream analysis. manufacturer's protocol (ABC kit, Vector Labs, PK4001), With the R package cummeRbund (version 2.18.0), the CuffDiff and development of the color reaction after addition of 3, results were analyzed for significant differentially expressed genes, 0 3 -diaminobenzidine (Amresco, 0430) was monitored under defined as having an alpha value of 0.05 or lower. GO term a light microscope. Colabeling for Goblet cells and prolifera- analysis was performed with DAVID (version 6.8). Using FPKM tion markers was performed by periodic-acid-schiff (PAS) values calculated by CuffNorm, gene set enrichment analysis staining followed by Ki-67 immunohistochemistry. Alkaline (GSEA) was used to examine differentially expressed genes using phosphatase staining kit II (Stegment, cat# 00-0055) was the signal-to-noise metric, as described (26). All data have been used to mark enterocytes, followed by Ki-67 immunohis- deposited in GEO (GSE102171). tochemistry. Click-it reagent kit (Thermo Fisher Scientific, Cat# C10337) was used to visualize the EdU-positive proliferating TCGA data analysis cells after fluorescent IHC for the various differentiation mar- Colorectal adenocarcinoma (COAD) samples from the TCGA kers, KRT20 (1:200), DCAMKL (1:200) ChGA (1:200), and project were used to investigate the relationship between SMAD4 OLFM4 (1:200). and b-catenin signaling in human tumors. TCGA COAD Illumina

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HiSeq RSEM normalized gene expression (n ¼ 434) and reverse columnar stem cells, suggested the stem cell population was phase protein array (RPPA; n ¼ 493) data were acquired from the unaltered. Consistent with these observations, the Smad4 knock- Broad Institute GDAC Firehose website (https://gdac.broadinsti out mice remained viable and appeared healthy during the period tute.org/) on February 23, 2018; the data set was filtered to include of observation for over 6 months. Thus, mice are viable upon only primary COAD samples (n ¼ 379). For protein-based anal- losing SMAD4 in the mature intestinal epithelium and exhibit yses, each protein of interest was compared with SMAD4 by a overtly normal intestinal homeostasis, indicating that SMAD4 is Spearman rank correlation. To assess the relationship between surprisingly dispensable in the adult intestinal epithelium, at least SMAD4 mRNA levels and WNT signaling, each individual gene as over a span of several months. well as the combined mean expression of the 67 genes identified Although normal cell lineage distributions and tissue mor- by analyses of the Smad4KO;b-cateninGOF mouse model was ana- phology were observed in the Smad4f/f; Villin-CreERT2 intestinal lyzed by a Spearman rank correlation. The mean expression of the epithelium at 4 days after tamoxifen induction, notable differ- 67-gene WNT signature (Supplementary Tables S1 and S2) as well ences were observed in the transcriptome of these tissues. RNA- as specific significant genes are shown in the resulting heat maps. seq was performed in 3 biological replicates from jejunal epithe- All resulting P values and correlations are reported in Supple- lium of control and Smad4KO mice. Six hundred and forty-one mentary Tables S1 and S2. A list of all samples is presented in genes were defined as differentially regulated (q < 0.05), with 343 Supplementary Tables S3 and S4. transcripts increasing and 298 transcripts decreasing in the mutant including Smad4 transcripts (Fig. 1B). Canonical readouts of the Histologic quantification SMAD4 signaling pathway also exhibited markedly reduced tran- At least 30 villi per biological replicate were used to evaluate script levels, including negative feedback regulators Smad6 and the appearance EdU-positive (proliferative) or OLFM4-positive Smad7, and members of the ID family of transcription factors (Fig. (stem cells) over time in the villi of Smad4KO; b-cateninGOF 1C). These findings confirmed disruption of SMAD4 function in following tamoxifen induction. Between 296 and 334 cells our model. Gene ontology analysis revealed that transcripts expressing the differentiation markers were counted reduced in the Smad4-mutant tissues were associated with prop- to determine the number of differentiated cells coexpressing erties of differentiated intestinal epithelial cells such as lipid differentiation and proliferation markers in the villi of metabolism, oxidative phosphorylation, and localization to the Smad4KO;b-cateninGOF mice at 7 days after tamoxifen injection. brush border (Fig. 1D). Other transcriptomic signatures that were The proximal two thirds of the duodenum was used in all the disrupted in the Smad4-mutant epithelium included decreased above cases. Ectopic crypts were quantified by taking into levels of transcripts associated with differentiation and increased account the fraction of villi that display ectopic crypts in the levels of transcripts associated with the WNT signaling pathway differentiated compartment along the various regions of intes- (Fig. 1E–G). tine. In the Lgr5-Cre model, each SMAD4-negative adenoma Thus, while Smad4 loss has no immediate consequence on was scored for whether the underlying crypts were SMAD4 intestinal epithelial morphology or cellular distributions, there is positive or SMAD4 negative. Adenomas were counted on one a shift at the transcriptomic level, with reduced expression of swiss-roll preparation of the proximal two thirds of the duo- BMP/TGFb signaling target genes, and elevated expression of denum per biological replicate. signature genes of the WNT pathway (33).

Prolonged Smad4 loss in the adult intestinal epithelium results Results in adenoma development Smad4 inactivation is surprisingly tolerated in the adult Intestinal neoplasia is observed when Smad4 is deleted in T intestinal epithelium lymphocytes (32), and loss of heterozygosity in mice with germ- Smad4 is a critical transcriptional effector of TGFb/BMP sig- line mutations in Smad4 results in duodenal polyps within 9 naling and a tumor suppressor (27). Loss-of-function mutations months of age (29). However, the epithelial-specific function of in SMAD4 are found in juvenile polyposis syndrome in humans Smad4 in suppressing tumors upon adult-onset loss is unclear. To (28). Functional studies in mice implicating Smad4 function in determine if the observed molecular alterations in Smad4KO the intestine were often conducted using heterozygous germ line intestinal epithelium can lead to tumorigenesis, we sacrificed mutations (15, 29, 30), mosaic mutations (31) or T-cell–specific tamoxifen-injected Smad4f/f;Vil-CreERT2 mice within a period 6 Smad4 deletion (32). To study SMAD4 function specifically in the to 9 months after SMAD4 loss (Fig. 2A). Four of 6 mice with the adult intestinal epithelium, we used the Villin-CreERT2 transgenic Smad4KO intestinal epithelium developed tumors, which were model (19), which allows inactivation of floxed alleles of Smad4 primarily observed in the small intestine (Fig. 2B) and histolog- (20) in the intestinal epithelium upon tamoxifen injection. Four ically resembled conventional adenomas (Fig. 2C and D). Tumors days after the onset of tamoxifen treatment of Smad4f/f;Vil-CreERT2 featured highly glandular structures, nuclear b-catenin, increased mice, SMAD4 protein expression in the intestinal epithelium was expression of OLFM4, a marker for crypt-base columnar stem observed by immunohistochemistry and immunoblotting and cells, and SOX9, a marker enriched in crypts (Fig. 2E)—all found to be below the level of detection; qRT-PCR and RNA-seq properties of conventional adenomas. Thus, while acute SMAD4 corroborated severe reduction of Smad4 transcripts after tamox- loss is well tolerated (Fig. 1), prolonged deficiency of SMAD4 in ifen treatment (Fig. 1A and B; Supplementary Fig. S1). Histologic the intestinal epithelium leads to adenomas (Fig. 2). analysis (Fig. 1A) revealed an overtly normal intestinal epithelium without notable alterations in the various differentiated lineages: SMAD4 suppresses WNT-driven ectopic crypt formation in the enterocytes (alkaline phosphatase activity), tuft cells (DCAMKL), differentiated villus epithelium goblet cells (PAS), enteroendocrine cells (ChgA), and Paneth cells The high expression of nuclear b-catenin in the tumors of the (lysozyme). Immunostaining for OLFM4, a marker of crypt base Smad4KO epithelium (Fig. 2E) and the elevated WNT gene

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Figure 1. Smad4 inactivation in the intestinal epithelium results in no immediate overt phenotype. A, SMAD4 immunostaining in control and Villin-CreERT2;Smad4KO intestine confirms epithelial-specific loss of Smad4 at 4 days after the induction of the knockout by tamoxifen treatment. Alkaline phosphatase and PAS staining shows no alterations in the differentiation of enterocytes or goblet cells, respectively. Immunostains for lysozyme, DCAMKL, and CHGA, the respective markers for Paneth, tuft, and enteroendocrine cells showed no differences in the mutant. IHC for OLFM4 shows preservation of stem cells in the Smad4KO intestinal epithelium. Representative of two independent biological replicates each. Scale, 50 mm. B and C, Smad4 inactivation is confirmed by loss of Smad4 transcripts in RNA-seq data (three biological replicates each; B) and downregulation of canonical SMAD4 target genes (C). D, The downregulated transcripts in Smad4KO intestinal epithelium are enriched in GO terms associated with intestinal differentiated cell functions. E–G, GSEA analysis also indicates a shift from differentiated cell transcript expression to transcripts associated with proliferation (43) and WNT signaling (27). expression signature upon acute SMAD4 loss (Fig. 1) suggested with SMAD4 mRNA expression (Fig. 2F); 56/67 genes in the that further elevation of the WNT pathway might be required WNT signature were negatively correlated, with 34/67 being for tumor development. We explored the relevance of this statistically significant (Supplementary Table S1). These data possibility by examining RNA expression data (n ¼ 379) from collectively demonstrate a significant inverse relationship the TCGA project using COAD samples. The WNT signature between SMAD4 and b-catenin/WNT signaling in human colon that we find elevated upon loss of SMAD4 in our mouse model tumors. To further test the possibility that WNT pathway (Fig. 1G) was inversely correlated (P ¼ 3.1E05; r ¼0.21) activation would facilitate tumor formation in the Smad4

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Figure 2. Adenomas develop after prolonged SMAD4 loss from the adult intestinal epithelium. A, Schema for monitoring the effects of Villin-CreERT2;Smad4 deletion in the intestinal epithelium. B, Size and location of the tumors in Smad4KO mice. C, Gross morphology of small intestinal tumors in Smad4KO mice. D and E, Representative images of immunohistological evaluation of Smad4KO intestinal tumors show characteristics of conventional adenomas with regards to the presence of stem cells (OLFM4; n ¼ 4), nuclear b-catenin (n ¼ 5), and SOX9 expression (n ¼ 4). Scale, 50 mm. F, SMAD4 expression in human COAD samples from TCGA Illumina HiSeq database (n ¼ 379) is represented as a wave plot. Relative expression values ranged between 7andþ4 across 379 samples. Mean expression of the 67-gene WNT signature was compared with SMAD4 mRNA expression (P ¼ 3.1E–05, r ¼0.21, Spearman correlation rank coefﬁcient). H&E, hematoxylin and eosin.

mutant, we challenged Smad4KO mice with elevation of WNT permits deletion of the third exon of Ctnnb1, which encodes signaling using a single allele of an inducible b-catenin– phosphorylation sites that target b-catenin for degradation activating mutation, Catnblox(ex3), in the intestinal epithelium. (21). We chose this model, instead of other available b-catenin þ The heterozygous Catnblox(ex3)/ gain-of-function (GOF) allele transgenic models, because it allows expression of the

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stabilized b-catenin from its endogenous locus and efficiently functions typical of differentiated cells were decreased, while promotes proliferation in the intestinal epithelium genes with elevated transcripts were associated with secretory (Supplementary Fig. S2). Thus, tamoxifen treatment of processes (Fig. 5A). We also noted an increased expression of þ Smad4f/f;Catnblox(ex3)/ ;Vil-CreERT2 mice results in concomitant intestinal stem cell signature genes in the double mutants, con- SMAD4 loss and WNT activation (referred to throughout as sistent with reports indicating that loss of SMAD4 (35) or acti- Smad4KO;b-cateninGOF). Remarkably, 14 days after tamoxifen vation of the WNT pathway promotes expansion of stem cells treatment, ectopic, crypt-like structures are observed in the villi (Fig. 5B). We next queried the expression data for gene signatures of these double-mutant mice (Fig. 3A). The ectopic crypt-like that have been observed in previous models of villus-resident structures appearing in Smad4KO;b-cateninGOF-mutant mice proliferation. Upon loss of the cell-cycle suppressor Rb, villus cells were similar to structures observed previously upon fetal intro- have been observed to proliferate (36, 37). Changes in the duction of BMP-signaling antagonists (16, 18), but with dras- transcriptome in Rb-mutant mice were correlated to those tically reduced latency. We first saw the proliferative marker observed in our Smad4KO;b-cateninGOF model (Fig. 5C). It has Ki-67 in cells with the morphologic appearance of enterocytes also been shown that activation of the NF-kB pathway, in con- in the villi at 4 days after initiation of tamoxifen treatment junction with elevated WNT signaling, can trigger ectopic crypt (Fig. 3B). By 7 days, clusters of proliferative cells were invag- formation in villi (7). Interestingly, transcriptomic data from mice inating toward the villus mesenchyme, and by 10 days, these in which the NF-kB signaling pathway is perturbed (38) also structures nestled more deeply into the villus body and resem- correlated with changes observed in our Smad4KO;b-cateninGOF bled crypts. These cup-shaped structures continued to elongate model (Fig. 5D), though the correlation was less robust. The and/or branch for another week, at which time the mice became relevance of these observed relationships could be corroborated moribund and were humanely euthanized. Immunohisto- in human patient samples. RPPA data from the TCGA project chemical analyses revealed markers of stem cells (OLFM4), were used to assess the relationship between SMAD4 and Paneth cells (lysozyme), and SOX9-expressing cells in the b-cateninproteinexpressionin493tumorsamplesanddeter- ectopic crypts (Fig. 3C). Ectopic crypts were most frequently mined, consistent with our mouse model, that expression of found in the proximal intestine but were also present through- these proteins are inversely correlated in human COAD (Spear- out the small intestine and colon (Fig. 3D)—the ectopic crypts man correlation; P ¼ 4.96E25, r ¼4.42E01;Fig.5E).We in the small intestine being more perceptible owing to the further determined that both the phosphorylated (S536) p65 presence of villi (Fig. 3E). Importantly, ectopic crypts are not subunit of NF-kB(P ¼ 8.02E15, r ¼3.40E01) and phos- observed in the villi of control, Smad4KO,orb-cateninGOF single- phorylated (S807, S811) Rb (P ¼ 3.23E13, r ¼3.20E01) mutant mice alone (Supplementary Fig. S3). were negatively correlated with SMAD4 expression (Fig. 5E; As the ectopic crypts advanced to 14 days post-tamoxifen Supplementary Table S2). treatment, cells of all the differentiated lineages could be Together, these findings suggest that activation of b-catenin observed, suggesting that stem cells residing within ectopic crypts in the absence of SMAD4 may share common mechanisms are multipotent (Supplementary Fig. S4). We further assayed for used by Rb-loss or NF-kB activation to drive proliferation crypt-like properties of these villus-resident structures by in the differentiated epithelium, and thus contributes to a culturing the villi ex vivo (Fig. 4A), in conditions mimicking the growing appreciation of the proliferative capacity in differen- endogenous crypt environment (Sato, 2009). Villi isolated from tiated tissues. the Smad4KO;b-cateninGOF mice gave rise to organoids (Fig. 4B). Organoid precursors within the villi were visible within 4 days SMAD4 loss permits induction of WNT-driven adenomas in of culture, developed into organoids within 7 days of culture differentiated intestinal tissues (Fig. 4C and D), and resembled wild-type organoids grown in Induction of the Smad4KO;b-cateninGOF mutations in the intes- WNT-conditioned media in their propensity to form spheres (34). tine in a pan-epithelial manner with Villin-CreERT2 renders the Notably, villi from control, Smad4KO,orb-cateninGOF single- mice moribund within weeks, as virtually all epithelial cells mutant mice did not form organoids (0 of 300 villi) in Matrigel, become mutant. To monitor the progress of ectopic crypts over consistent with lack of ectopic crypts in the villi of these time, we induced these mutations in a mosaic manner using mutants (Fig. 4C). These organoids could be subcultured the Lgr5-CreERT2-IRES-GFP expression cassette (22), which þ repeatedly (Fig. 4D). Thus, combined inactivation of SMAD4 induces recombinase activity in 30% of the Lgr5 stem cells, and activation of b-catenin lead to the appearance of functional, as we have observed in our previous studies (24). Thus, tamoxifen þ ectopic crypts that express characteristics and functions of injection of Smad4f/f;Catnblox(ex3)/ ;Lgr5-CreERT2-IRES-GFP mice normal intestinal crypts, including the ability to give rise to results in Smad4KO;b-cateninGOF mutations in only about one organoid cultures. third of the intestinal crypts, while the majority of crypts retain the wild-type protein expression for Smad4 and b-catenin. The Transcriptomic changes upon SMAD4 loss and b-catenin reduced numbers of cells harboring mutations in this model activation suggest avenues toward ectopic crypt formation should allow the mouse to survive longer than the Villin-CreERT2 To better appreciate the potential mechanisms through which model. The mosaic mutation was tolerated in the mice for up to 4 concomitant loss of SMAD4 and activation of b-catenin triggers weeks. Histology revealed adenoma development in both small ectopic crypt formation, we investigated transcriptomic changes and large intestine (Fig. 6A). SMAD4 immunohistochemistry in the double-mutant mice compared with controls at 4 days after indicated that while the villus-resident adenomas lacked the onset of tamoxifen treatment (at the onset of ectopic prolif- SMAD4 expression, the normal-looking villi, and the majority eration in the villus). A total of 1,678 genes were downregulated, of the crypts retained Smad4 expression (Fig. 6A and B). This while 1,005 transcripts were upregulated in the double mutant observation suggests that despite induction of the mutation in (q < 0.05). Gene ontology analysis revealed that genes ascribed the crypt stem cells, SMAD4-mutant cells within the stem cell

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Figure 3. Smad4KO;b-cateninGOF compound-mutant villi exhibit proliferative cell clusters that develop into ectopic crypts. A, Immunohistochemistry for the proliferative marker Ki-67 revealed increased proliferation and ectopic crypts in the Villin-CreERT2;Smad4KO;b-cateninGOF intestinal villi. Scale, 100 mm. B, Development of ectopic crypts in the villi of Smad4KO;b-cateninGOF over time, which begins as isolated Ki-67þ cells at 4 days (4 days) after the onset of tamoxifen treatment, followed by formation of proliferative clusters (7 days) that invaginate into cup-like structures (9 days) that eventually resemble crypts (day 14). Scale, 10 mm. C, Ectopic crypts express crypt markers: lysozyme (14 days), OLFM4 (7 to 9 days), and SOX9 (7 days). Scale, 10 mm. Representative of two independent biological replicates. D, Quantiﬁcation of ectopic crypts along the intestinal epithelium shows highest density of ectopic crypts in the proximal intestine. E, Schematic depicting ectopic crypt formation from the villi and its similarities with the endogenous crypt compartment.

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Figure 4. Smad4KO;b-cateninGOF villi form organoids ex vivo. A, Schematic of preparation of villi for ex vivo culture showing serial filtration of scraped villi with 70-mm filter to yield villi without crypts. B, Quantification of the villi-derived organoids (three replicates of mice for each genotype). Neither Villin-CreERT2;Smad4KO nor Villin-CreERT2;b-cateninGOF villi form organoids ex vivo, indicating that the acquisition of stem cell activity is unique to the double-mutant villi. C, Villi from the various mutants after 7 days of culture in Matrigel shows organoid formation only from the Smad4KO;b-cateninGOF villi. (N ¼ 3). Scale, 50 mm. D, The passaged villi-derived organoids display spherical morphology, with or without budding structures. Scale, 50 mm.

compartment were replaced by adjacent SMAD4-positive stem surface of the differentiated colon epithelium (Fig. 6A), suggest- cells over time. Notably, the villus-resident adenomas appeared to ing that Smad4 suppresses WNT-driven adenomas in the dif- be discontinuous from the normal crypt base, as an average 46 ferentiated cells. Developing adenomas resident in the differ- SMAD4-negative adenomas were observed in villi overlaying entiated compartments of the small and large intestine morphologically normal, SMAD4-positive crypts (per duodenal expressed characteristics of adenomatous polyps (Fig. 6C); sample, SD ¼ 5.65, n ¼ 2), whereas SMAD4-negative crypts were however, elevated levels of WNT signaling did not appear to in contact with adenomas in only 2.5 adenomas per mouse (SD ¼ rise to the levels observed in aged SMAD4KO mice, based upon 0.7). Analogous structures were also observed in the luminal b-catenin immunoreactivity (Fig. 2E; Supplementary Fig. S5).

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Figure 5. GO term and GSEA of genes differentially expressed in the Smad4KO;b-cateninGOF-mutant epithelium. A, DAVID analysis of genes differentially expressed in the double-mutant mice shows loss of differentiation characteristics and a gain of transcripts associated with secretory functions. B–D, GSEA analyses revealed correlations between the transcriptome changes observed in the Villin-CreERT2; Smad4KO;b-cateninGOF-mutant epithelium with those observed in intestinal stem cells (B), upon loss of Rb (C), or upon alteration of the NF-kB signaling pathway (D). E, SMAD4 protein expression was queried in human COAD tumor samples from TCGA RPPA data set (n ¼ 493). Pairwise correlation was analyzed between SMAD4 and CTNNB1 (b-catenin), NF-kB (p65 subunit, phospho-S536), and Rb (phospho-S807/S811) protein expression level. A signiﬁcant, negative correlation was observed between SMAD4 and CTNNB1 (b-catenin, P ¼ 4.96E–25), NF-kB (p65 subunit, phospho-S536, P ¼ 8.02E–15), and Rb (phospho-S807/S811, P ¼ 3.23E–13), as represented in the heat map.

Notably, the location of adenomas within the villi of Smad4f/f; base (1). This suggests that SMAD4 plays an active role in þ Catnblox(ex3)/ ;Lgr5-CreERT2 mutants contrasts with most intes- suppressing WNT-driven adenoma formation within the dif- tinal tumor models in which the lesions expand from the crypt ferentiated intestinal epithelium.

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SMAD4 Suppresses WNT-Driven Dedifferentiation in the Gut

Figure 6. Development of villi-resident adenomas in the Smad4KO;b-cateninGOF mosaic mutant. A, SMAD4-negative adenomas emerge within the differentiated compartments of the small intestine and colon upon simultaneous inactivation of Smad4 and activation of b-catenin. B, Serial sections (a0-k0) spanning 110 mm are representative of forming adenomas that are discontinuous of the endogenous crypt base. Scale bar,100 mm, Representative of 5 independent biological replicates. C, Villus-resident adenomas show hyperplasia [hematoxylin and eosin (H&E) and BrdU] and cells expressing CD44 and Lgr5-driven GFP.

Villus enterocytes are the origin of ectopic crypts, with whereas OLFM4-positive cells did not appear until 24 hours later, proliferation preceding expression of stem cell markers and at a much lower frequency (Fig. 7A). This trend continued at 6 Villi in the normal intestinal epithelium harbor only postmi- days after initiation of tamoxifen treatment, at which time totic, differentiated cells. Because stem cells can give rise to all of OLFM4-positive cells were observed in most duodenal villi but þ the cell types in the intestinal epithelium, we sought to determine still were outnumbered by EdU cells (Fig. 7B). These results whether the dedifferentiating cells in Smad4KO;b-cateninGOF- suggest that villus cells reenter the cell cycle before acquiring stem mutant villi first reenter cell cycle or first express markers of stem cell properties. cell fate. We investigated the kinetics with which villus cells The initial appearance of proliferating cells in the either incorporate a marker of DNA replication (EdU) or express Smad4KO;b-cateninGOF villi occurred without any apparent the stem cell marker OLFM4. EdU labeling was done with a changes in differentiation of intestinal lineages. To pinpoint brief, 1 hour pulse chase to assess cells recently in S-phase. the identity of villus cells first expressing proliferative markers, EdU-labeled cells first appeared in the villi at 4 days after the dual staining for proliferation and differentiation markers for onset of tamoxifen treatment of the Smad4KO;b-cateninGOF mice, the various intestinal lineages was conducted. Of each lineage

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Figure 7. Proliferation is initiated in villus cells expressing enterocyte markers and precedes acquisition of stem cell markers. A, Costaining for EdU and OLFM4 reveals the appearance of proliferative cells (EdUþ) at 4 days, whereas immunoreactivity for the stem cell marker, OLFM4, is seen only 5 days after induction of the Smad4KO;b-cateninGOF mutation. B, Quantiﬁcation of EdU and OLFM4-positive cells over time reveals that proliferative cells precede the appearance of stem cells on the mutant villi (4 and 6 days, N ¼ 2; 5 days, N ¼ 1). Scale bar, 10 mm. C, Costaining for proliferation markers (EdU and Ki-67) and various lineage markers (KRT20, OLFM4, AP) on Smad4KO;b-cateninGOF villus epithelium shows proliferation (Ki-67 or EdU) or stem cell markers (OLFM4) in the enterocytes (KRT20 or AP) at 7 days after tamoxifen injection (top). N ¼ 2. No other lineage markers (lysozyme for Paneth cells; CHGA for enteroendocrine cells, and DCAMKL for tuft cells) coexpressed with the proliferative markers in the villi, suggesting that the dedifferentiating cells arise from enterocytes. Scale, 10 mm (zoom-in) and 50 mm (zoom-out). D, Quantiﬁcation of proliferating cells that coexpress various lineage markers shows that all the proliferating cells in the villi express the enterocyte markers, KRT20, and alkaline phosphatase. A small population (0.67%) of proliferating cells also expressed CHGA, the enteroendocrine marker (N ¼ 2 biological replicates each).

interrogated, only cells expressing enterocyte markers (alkaline markers indicates that cells committed or partially committed phosphatase or KRT20), were found coexpressing Ki-67 or to the enterocyte lineage were particularly capable of contrib- taking up the uridine analog, EdU (Fig. 7C and D). The uting to villus-resident mitosis. Conversely, markers of tuft coexpression of proliferation markers with enterocyte lineage (DCAMKL), enteroendocrine (CHGA), Paneth (lysozyme), or

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SMAD4 Suppresses WNT-Driven Dedifferentiation in the Gut

goblet cells (PAS) did not colocalize with proliferation markers transform upon simultaneous activation of WNT and KRAS (9). In (Fig. 7C). Thus, loss of SMAD4 allows villus-resident enter- light of these findings, our study results suggest that SMAD4 ocyte-like cells to undergo proliferation upon elevation of may mediate a specific role in blocking WNT-KRAS-mediated WNT signaling. Formally, lineage-specific Cre drivers would oncogenesis from enterocytes. be required to demonstrate that these nonenterocyte cell How does SMAD4 block adenoma formation in differentiated lineages are not the source of ectopic crypts, whereas enter- tissues? SMAD4 has recently been shown to suppress stem cell ocytes are the definitive source. Still, these costaining results renewal in the crypt base (35, 42); its loss in differentiated tissues, suggest that SMAD4 protects against WNT-driven dedifferen- upon exposure to stem cell–promoting WNT signaling, could thus tiation of enterocytes. create conditions favorable for the stem cell fate, consistent with our observations in the mutant transcriptome (Fig. 5B). We also Discussion observe increased expression of genes functioning in extracellular exosome formation (Fig. 5A), which could play a role in carving a Elegant studies in mice have demonstrated the potential of stem cell niche in the villus. Perturbations of NF-kB signaling (7) active WNT signaling to drive oncogenesis from stem cells, but not or loss of the cell-cycle inhibitor Rb (36) can also lead to prolif- in early progenitor or differentiated intestinal epithelial cells (1). eration in the villus, triggering transcriptomic changes that cor- Histologic evidence from human patients with colon cancer, relate with the transcriptomic changes we observe in our mutants however, suggests that dysplastic cells arise from differentiated (Fig. 5C and D), and which are also correlated with SMAD4-low cells (2). Our studies find that active WNT signaling can drive human tumors (Fig. 5E). Future studies can investigate whether dedifferentiation and adenoma formation in differentiated intes- these pathways might work upstream, downstream, or in parallel tinal epithelial cells upon concomitant SMAD4 loss in the adult. with BMP/SMAD4 signaling to regulate dedifferentiation of enter- SMAD4 can serve as the transcriptional effector of both BMP ocytes and will broaden our understanding of the potential cells of and TGFb signaling. Although our findings do not distinguish origin of colorectal cancer. which ligand SMAD4 functions through to suppress dedifferentiation, recent work would support BMP ligands as the suppres- fl sors of dedifferentiation. Loss of Tgfbr1 was recently shown to Disclosure of Potential Con icts of Interest fl accentuate dedifferentiation driven by APC-loss and KRAS acti- No potential con icts of interest were disclosed. vation; however, Tgfbr1 loss in conjunction with activation of WNT signaling does not lead to dedifferentiation (39). Notably, it Authors' Contributions is the expression of BMP antagonists, GREM1 and NOGGIN, that Conception and design: A.O. Perekatt, N. Patel, M.L. Gatza, M.P. Verzi Development of methodology: A.O. Perekatt, A. Wu, N. Patel, J. Xing have been demonstrated to trigger ectopic crypt formation Acquisition of data (provided animals, acquired and managed patients, (16, 18). Suppression of hedgehog signaling also triggers ectopic provided facilities, etc.): A.O. Perekatt, P.P. Shah, V. Gandhi, Q. Feng, N. Patel, crypt formation in the developing mouse, in a mechanism pro- L. Chen, S. Joshi, N. Gao posed to operate via BMP signaling. Therefore, taken together with Analysis and interpretation of data (e.g., statistical analysis, biostatistics, our work, these findings point to BMP signaling rather than TGFb computational analysis): A.O. Perekatt, P.P. Shah, S. Cheung, N. Jariwala, signaling, in a SMAD4-dependent mechanism, as the primary N. Kumar, Q. Feng, L. Chen, S. Joshi, A. Zhou, J. Xing, M.L. Gatza, M.P. Verzi Writing, review, and/or revision of the manuscript: A.O. Perekatt, P.P. Shah, suppressor of dedifferentiation in the intestinal epithelium. Our S. Cheung, N. Jariwala, J. Xing, M.L. Gatza, M.P. Verzi finding also refines the mechanism to a cell-autonomous role for Administrative, technical, or material support (i.e., reporting or organizing SMAD4 in the epithelium. Disentangling how the cells are inter- data, constructing databases): P.P. Shah, S. Cheung, Q. Feng, M.M. Taketo preting the BMP versus TGFb ligands to suppress dedifferentiation Study supervision: A.O. Perekatt, J. Xing, N. Gao, M.L. Gatza, M.P. Verzi will be an important next step in understanding how the epithe- Other (participate in conducting the experiment): A. Wu fi lium protects against oncogenesis. Other (provided scienti c input): E. White In this study, we observe that enterocytes, but not other differentiated lineages, reacquire stem cell marker expression and the Acknowledgments This research has been funded by grants from the NIH, NCI (R01CA190558), ability to reenter the cell cycle upon loss of SMAD4 and gain of and the New Jersey Health Foundation (PC46-16) to M.P. Verzi, the New Jersey WNT signaling. Certainly, other intestinal lineages have been Commission on Cancer Research (DFHS13PPC034) to A.O. Perekatt, the Aresty shown to be capable of acquiring stem cell activity upon targeted Undergraduate Research Program (N. Patel, S. Cheung, and P.P. Shah), loss of endogenous stem cells or upon conditions of tissue the Department of Life Sciences Summer Undergraduate Research Fellowship regeneration, including tuft cells, Paneth cells, and secretory (P.P. Shah), MacMillan Cancer Research grants to (P.P. Shah and S. Cheung), the precursor cells (11–13, 40, 41). We hypothesize that the SMAD4 National Cancer Institute of the National Institute of Health (CA166228), and the V Foundation for Cancer Research (V2016-013) to M.L. Gatza. M.P. Verzi, phenotype is specific to the enterocyte lineage in that it plays fi E. White, and M.L. Gatza are members of the Cancer Institute of New Jersey speci c roles in promoting enterocyte differentiation, whereas (P30CA072720), which provided core resources for sequencing. The work was other differentiation-promoting factors, unique to these other also supported by an Initiative for Multidisciplinary Research Teams award from lineages, may require suppression in order for dedifferentiation of Rutgers University, Newark, NJ (N. Gao and M.P. Verzi). these lineages. Enterocytes have been shown to have the capacity þ to dedifferentiate upon ablation of the Lgr5 stem cell popula- Received January 5, 2018; revised April 26, 2018; accepted July 2, 2018; tion, but the same study demonstrated that enterocytes do not published first July 9, 2018.

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SMAD4 Suppresses WNT-Driven Dedifferentiation and Oncogenesis in the Differentiated Gut Epithelium

Ansu O. Perekatt, Pooja P. Shah, Shannon Cheung, et al.

Cancer Res 2018;78:4878-4890. Published OnlineFirst July 9, 2018.

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