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 surpris- ingly 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. Ó2018 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 Ó2018 American Association for Cancer Research. most frequently mutated genes in colon cancers (14), and loss of 4878 Cancer Res; 78(17) September 1, 2018 Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 2018 American Association for Cancer Research. Published OnlineFirst July 9, 2018; DOI: 10.1158/0008-5472.CAN-18-0043 SMAD4 Suppresses WNT-Driven Dedifferentiation in the Gut 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