Distinct Roles of HES1 in Normal Stem Cells and Tumor Stem-Like

Published OnlineFirst May 23, 2017; DOI: 10.1158/0008-5472.CAN-16-3192 Cancer Tumor and Stem Cell Biology Research

Distinct Roles of HES1 in Normal Stem Cells and Tumor Stem-like Cells of the Intestine Norihiro Goto1, Taro Ueo1, Akihisa Fukuda1, Kenji Kawada2, Yoshiharu Sakai2, Hiroyuki Miyoshi3, Makoto Mark Taketo3, Tsutomu Chiba1, and Hiroshi Seno1

Abstract

þ Cancer stem cells (CSC) have attracted attention as thera- homeostasis. Furthermore, in Lgr5 NSC, deletion of Hes1 peutic targets; however, CSC-targeting therapy may disrupt and b-catenin stabilization limited tumor formation and pro- þ þ normaltissuehomeostasisbecausemanyCSCmoleculesare longed host survival. Notably, in Lgr5 or Dclk1 tumor also expressed by normal stem cells (NSC). Here, we demon- stem cells derived from established intestinal tumors, Hes1 strate that NSC-specific and CSC-specific roles of the stem cell deletion triggered immediate apoptosis, reducing tumor bur- transcription factor Hes1 in the intestine enable the feasibility den. Our results show how Hes1 plays different roles in NSCs of a specific cancer therapy. Hes1 expression was upregulated in and CSCs, in which Hes1 disruption leads to tumor regression NSCs and intestinal tumors. Lineage-tracing experiments in without perturbing normal stem cell homeostasis, preclinically þ adult mouse intestine revealed that Hes1 deletion in Lgr5 or validating Hes1 as a cancer therapeutic target. Cancer Res; 77(13); þ Bmi1 NSCs resulted in loss of self-renewal but did not perturb 1–13. 2017 AACR.

Introduction However, this result is not directly applicable to adult ISCs. Because early fetal and adult ISCs are regulated by different Tumor stem cells (TSC), capable of self-renewing and giving mechanisms (15, 16), an unknown compensation may exist in rise to progeny cells in the tumor, have attracted attention as the developmental phase. The role of Hes1 in adult ISCs needs promising targets for antitumor therapeutics (1–3). However, clarification to explore the possibility of Hes1-targeting tumor TSC-targeting therapy may disrupt normal tissue homeostasis therapy and to provide further insight into stem cell biology. because most of the TSC markers are also expressed by normal To address these issues, we examined the role of Hes1 in the stem cells (NSC; refs. 4–8).Toaddressthisconcern,two NSCs of small intestine in adult mice. Hes1 deletion in NSCs led strategies can be employed: the identification of TSC-specific to the loss of self-renewal capacity but did not disrupt homeo- markers (9) and the identification of functional genes that act stasis. We investigated the role of Hes1 in intestinal tumors. differently in NSCs and TSCs. Testing the feasibility of the latter Notably, we found that Hes1 deletion in the TSCs of established approach, by targeting both NSCs and TSCs, requires proper in intestinal tumors led to tumor regression by inducing immediate vivo models. apoptosis. We propose that Hes1 may be a novel tumor-specific Hes1 is a basic helix-loop-helix transcription factor and therapeutic target that does not perturb intestinal homeostasis. represses transcription by active and passive repression mechanisms (10). Because Hes1 protein negatively regulates its expression by binding to its own promoter, Hes1 expression oscillates in Materials and Methods many cells, which plays an important role in the self-renewal of Animal models stem cells (11). Although Notch signaling is important in the Lgr5–EGFP-IRES-CreERT2 mice (JAX strain 008875), Bmi1- maintenance of intestinal stem cells (ISC; refs. 12, 13), the CreER mice (JAX strain 010531), ApcMin mice (JAX strain downstream target required for ISC self-renewal remains elusive. 002020), and Rosa26-LacZ mice (JAX strain 003309) were Previously, we showed that the maintenance of ISCs is not affected obtained from The Jackson Laboratory. Dclk1-CreERT2-IRES- after the prenatal triple deletion of Hes1, Hes3, and Hes5 (14). EGFP mice and Ctnnb1lox(ex3) mice were generated as described fl previously (9, 17). Hes1 ox mice were kindly gifted by Dr. Ryoi- chiro Kageyama (Institute for Virus Research, Kyoto University, 1Department of Gastroenterology and Hepatology, Kyoto University Graduate Kyoto, Japan; ref. 18). Mice were maintained on a C57BL/6 School of Medicine, Sakyo-ku, Kyoto, Japan. 2Department of Surgery, Kyoto background. For induction of Cre-mediated recombination, 3 University Graduate School of Medicine, Sakyo-ku, Kyoto, Japan. Department 200 mL of 20 mg/mL tamoxifen (Sigma-Aldrich) in corn oil (for of Pharmacology, Kyoto University Graduate School of Medicine, Sakyo-ku, a single injection), or 100 mL of 20 mg/mL tamoxifen either twice a Kyoto, Japan. day over 2 consecutive days or once a day over four consecutive Note: Supplementary data for this article are available at Cancer Research days (for intensive induction) was intraperitoneally injected. For Online (http://cancerres.aacrjournals.org/). experiments using normal intestinal tissue, 8-week-old mice Corresponding Author: Hiroshi Seno, Graduate School of Medicine, Kyoto were used. For experiments using established intestinal tumors, University, 54 Kawara-cho, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan. Phone: 6-month-old mice were used. All experiments involving mice 817-5751-4319; Fax: 817-5751-4303; E-mail: [email protected] were approved by the animal research committee of Kyoto Uni- doi: 10.1158/0008-5472.CAN-16-3192 versity (Kyoto, Japan) and performed in accordance with Japanese 2017 American Association for Cancer Research. government regulations.

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Human subjects culture. For spheroid culture of intestinal tumors, advanced Surgically resected specimens were obtained from 20 colorectal DMEM/F-12 (Invitrogen) supplemented with 50 ng/mL mouse cancer patients at Kyoto University Hospital. Analyses for human EGF, 100 ng/mL Noggin (all from PeproTech), penicillin/strep- subjects were approved by the ethical committee of Kyoto Uni- tomycin, 10 mmol/L HEPES, GlutaMAX, and 1B27 (all from versity Hospital and conducted in accordance with the Declara- Invitrogen) was added to each well. For in vitro induction of Cre- tion of Helsinki. Written informed consent was obtained from all mediated recombination, 1 mmol/L 4-hydroxytamoxifen (4-OHT; subjects. Sigma) was added to the cultures.

þ Histologic analyses and immunostaining Lgr5 cell sorting For histologic analyses, mouse organs were isolated and fixed For sorting experiments, isolated crypts/glands units or tumor overnight in 4% paraformaldehyde, embedded in paraffin, and cell aggregates were additionally dissociated with TrypLE Express sectioned at a thickness of 5 mm. Sections were then deparaffi- (Invitrogen) for 20 minutes at 37C. Dissociated cells were passed nized, rehydrated, and stained with hematoxylin and eosin through a 30-mm cell strainer and sorted by FACSAria II (BD (H&E). For immunohistochemical analyses, sections were incu- Biosciences). Single cells were gated by forward scatter pulse width bated with primary antibody overnight at 4 C and washed with and side scatter pulse width. Dead cells were excluded by labeling PBS. Washed sections were incubated with biotinylated secondary with 7-AAD. The data were analyzed using FlowJo software antibody for 1 hour at room temperature. Sections were then (version 10, TreeStar). incubated with avidin biotin–peroxidase complex (Vector Labo- ratories), labeled with peroxidase, and colored with diaminoben- Statistical analysis zidine substrate (Dako). For immunofluorescence, sections were All values are presented as mean SEM unless otherwise stated. incubated with primary antibody overnight at 4 C and washed The two-tailed Student t test was used for statistical analysis of fl with PBS. Washed sections were incubated with uorescence- continuous data. The log-rank test was used for statistical analysis conjugated secondary antibody (Invitrogen) for 1 hour at room of Kaplan–Meier survival curves. P values of <0.05 were consid- temperature. Primary antibodies are listed in Supplementary ered to be significant. Table S1. Results qRT-PCR Total RNA was extracted from tissues or cells using the RNeasy Hes1 expression is upregulated in intestinal stem cells Micro Kit (Qiagen). Single-strand cDNA was synthesized using a To confirm the pattern of Hes1 expression, we performed IHC Min Transcriptor First Strand cDNA Synthesis Kit (Roche Applied analysis of Hes1 using samples of Apc mice and human tissues Science). qRT-PCR was performed using SYBR Green I Master with colon cancer. Consistent with previous reports (14, 21–23), (Roche Applied Science) and LightCycler 480 (Roche Applied in the normal intestine, Hes1 was expressed in the crypt base Science). Values are expressed as arbitrary units relative to columnar cells and transit-amplifying (TA) cells but not in Paneth GAPDH. Primers were listed in Supplementary Table S1. cells or cells of the villi (Supplementary Fig. S1A and S1B). Hes1 was ubiquitously expressed in murine intestinal tumors and b-Galactosidase (LacZ) staining human colon cancers (Supplementary Fig. S1A). We sorted Freshly isolated small intestine was incubated in ice-cold fix- GFP-positive cells from intestinal epithelial cells of Lgr5-EGFP- ative solution (PBS containing 4% paraformaldehyde, 5 mmol/L IRES-CreERT2 mice and performed qRT-PCR. FACS distinguished high EGTA, 2 mmol/L MgCl2, 0.2% glutaraldehyde, and 0.02% NP-40) a population with high expression of Lgr5-GFP (Lgr5-GFP ) for 1 hour at 4C. After washing twice in PBS for 20 minutes, and another with low expression of Lgr5-GFP (Lgr5-GFP-low), tissues were incubated with LacZ substrate (PBS containing which have been reported to correspond to stem cells and their 5 mmol/L K3Fe(CN)6, 5 mmol/L K4Fe(CN)6, 2 mmol/L MgCl2, immediate TA daughters, respectively (Supplementary Fig. S1C; 0.02% NP-40, 0.1% sodium deoxycholate, and 1 mg/mL X- ref. 20). The expression of Axin2 mRNA, which reflects Wnt/ high galactosidase) overnight at room temperature. After washing twice b-catenin activity, was significantly higher in the Lgr5-GFP in PBS for 20 minutes, tissues were fixed overnight in 4% para- population; Hes1 expression was also significantly higher in the high formaldehyde in PBS at 4C. Paraformaldehyde was removed, and Lgr5-GFP population (Supplementary Fig. S1D). The expres- the stained tissues were transferred to tissue cassettes. sion of Notch1 and Notch2 was significantly higher in the Lgr5- GFPhigh population; however, the expression of other Notch high Spheroid culture effectors, Hes3 and Hes5, was not upregulated in the Lgr5-GFP Conditioned medium of L-cell line secreting Wnt3a, R-spondin population (Supplementary Fig. S1E). Wnt/b-catenin and Notch 3, and Noggin (L-WRN CM) was created as described previously signaling has been reported to regulate Hes1 expression, respec- (19). L-WRN (ATCC; CRL-3276) was generated by H. Miyoshi tively (10, 21, 24); these data imply that both Notch and Wnt/ (2013) as described previously (14) and passaged for fewer than 6 b-catenin signaling may be important for the upregulation of months. Spheroid cultures were established as described previ- Hes1 in NSCs. The high expression of Hes1 in NSCs prompted us ously (19, 20). In short, crypts/glands units or tumor cell aggre- to examine the role of Hes1 in NSCs. gates were isolated and embedded in Matrigel (BD Biosciences). þ For spheroid culture of normal epithelium, 50% L-WRN CM Hes1 deletion in Lgr5 NSCs results in loss of self-renewal but supplemented with 10 mmol/L Y-27632 (Tocris Bioscience) does not perturb homeostasis and 10 mmol/L SB431542 (Tocris Bioscience) was added to each To investigate the role of Hes1 in NSCs, we deleted Hes1 in þ þ well. CHIR99021 (Focus Biomolecules) and valproic acid (Tocris Lgr5 NSCs and performed lineage tracing using Lgr5CreERT2/ ; fl fl þ Bioscience) were added to the medium for Lgr5-GFPhigh cell Hes1 ox/ ox; Rosa26LacZ/ mice. After administering 4 mg

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þ þ þ þ fl fl tamoxifen, LacZ-labeled blue cells appeared in crypt base colum- Lgr5CreERT2/ ; Hes1 / and Lgr5CreERT2/ ; Hes1 ox/ ox mice; this nar cells at day 1, proliferated in the crypts at day 3, and gave rise to approach circumvents mosaic reporter expression. Then, we cul- progeny cells that appeared as blue stripes from the crypts to the tured Lgr5-GFPhigh cells in Matrigel, in 50% L-WRN CM, villi at day 5 (Fig. 1A). At day 7, these blue stripes started to CHIR99021, and valproic acid, which facilitates homogenous disappear in the crypts, and only a few blue cells were detected on Lgr5-GFPhigh stem cell culture, with 4-OHT induction for 48 hours þ the tip of the villi (Fig. 1A). Provided that Lgr5 NSCs divide every from the start of the culture (Fig. 2A). Compared with that of the 24 hours, and epithelial cells are renewed every 4 to 5 days (25), control, colony formation was almost completely abrogated in þ this indicates that Hes1-deleted Lgr5 NSCs can give rise to Hes1-deleted Lgr5-GFPhigh single cells (Fig. 2B and C), showing progeny cells but cannot self-renew. that Hes1 is required for the self-renewal of Lgr5-GFPhigh stem þ To further validate these results, we maximized the induction cells. We also assessed the effect of Hes1 deletion in Lgr5 cells on efficiency by administering 2 mg tamoxifen twice a day over 2 established spheroids (Fig. 2D). Spheroids grown in 50% L-WRN consecutive days. Using this intensive induction, we also evalu- CM were enriched for stem and/or progenitor cells (19). The Lgr5 ated the effect of Hes1 deletion in NSCs on intestinal homeostasis. expression level in the spheroids decreased when cultured without þ First, we sorted Lgr5 NSCs 2 days after the last injection and CHIR99021 and valproic acid (Fig. 2E), suggesting the presence of confirmed the downregulation of Hes1 expression in Hes1-delet- Lgr5 TA cells in these cultures. Three days after induction, the ed Lgr5 NSCs (Supplementary Fig. S2A). Surprisingly, Hes3 and growth of established spheroids, cultured in 50% L-WRN CM, was þ Hes5 expression was not upregulated (Supplementary Fig. S2A), not affected, and Lgr5 cells were detected (Fig. 2F and G); these þ suggesting that, unlike the prenatal deletion of Hes1, Hes3 and Lgr5 cells were likely generated from the Lgr5 TA cells. These Hes5 do not compensate for Hes1 deficiency in adult NSCs. Three data demonstrate that deletion of Hes1 in NSCs does not affect days after the last injection, IHC revealed that the expression of intestinal homeostasis. þ Hes1 was almost lost in the LacZ-labeled crypts of Lgr5CreERT2/ ; fl fl þ þ Hes1 ox/ ox; Rosa26LacZ/ mice (Fig. 1B and C), confirming that Hes1 deletion in Bmi1 NSCs results in loss of self-renewal but recombination of the floxed Hes1 allele occurred efficiently. does not perturb homeostasis þ Cleaved caspase-3 was not expressed in LacZ-labeled crypts of Bmi1 is expressed in NSCs of the intestine (6); Bmi1 cells serve þ fl fl þ þ Lgr5CreERT2/ ; Hes1 ox/ ox; Rosa26LacZ/ mice (Supplementary Fig. as reserve stem cells in cases of injury or Lgr5 cell ablation (26). þ S2B), thereby excluding the possibility that Hes1-deleted Lgr5 We assessed whether Hes1 is important in the maintenance of NSCs or their daughter TA cells were lost due to immediate these NSCs that can serve as reserve stem cells. We deleted Hes1 in þ þ apoptosis. Five days after the last injection, the proportion of Bmi1 NSCs and performed lineage tracing using Bmi1CreER/ ; þ fl fl þ LacZ crypt–villi axes in the proximal intestine did not differ Hes1 ox/ ox; Rosa26LacZ/ mice. After injection with 4 mg tamox- þ þ þ þ þ between Lgr5CreERT2/ ; Hes1 / ; Rosa26LacZ/ and Lgr5CreERT2/ ; ifen, LacZ-labeled blue cells appeared four or five cells above the fl fl þ Hes1 ox/ ox; Rosa26LacZ/ mice (Supplementary Fig. S2C and S2D). base of the crypt at day 1 and gave rise to progeny cells that Fourteen days after the last injection, most of the epithelial cells arranged into blue stripes, reaching from the crypts to the villi, at þ in proximal small intestine, where Lgr5 NSCs are abundantly day 3 (Fig. 3A). From days 5 to 7, blue stripes started to disappear þ þ þ þ detected, were labeled blue in Lgr5CreERT2/ ; Hes1 / ; Rosa26LacZ/ in some crypts, and only a few blue cells were detected on the tip of control mice (Fig. 1D and E; Supplementary Fig. S2D). This the villi (Fig. 3A). indicates that, with this intensive induction, recombination We maximized the induction efficiency by administering 2 mg þ occurred in most of the Lgr5 cells in proximal small intestine. tamoxifen over 4 consecutive days. IHC was used to confirm þ fl fl þ Conversely, in Lgr5CreERT2/ ; Hes1 ox/ ox; Rosa26LacZ/ mice, most that recombination of the floxed Hes1 allele occurred efficiently of the LacZ-labeled blue cells were depleted and repopulated with (Fig. 3B and C). Ten days after the last injection, most of the LacZ- LacZ cells (Fig. 1D and E; Supplementary Fig. S2D). These results labeled blue cells were depleted and repopulated with LacZ cells þ þ fl fl þ further indicate that Hes1-deleted Lgr5 cells cannot self-renew. in Bmi1CreER/ ; Hes1 ox/ ox; Rosa26LacZ/ mice compared with þ þ fl þ þ The repopulated LacZ intestinal epithelium of Lgr5CreERT2/ ; those in Bmi1CreER/ ; Hes1 ox/ ; Rosa26LacZ/ control mice fl fl þ þ Hes1 ox/ ox; Rosa26LacZ/ mice did not display obvious morpho- (Fig. 3D and E). The number of Hes1 cells did not differ between þ fl þ þ þ logic abnormalities (Fig. 1E). Immunohistochemical analysis Bmi1CreER/ ; Hes1 ox/ ; Rosa26LacZ/ mice and Bmi1CreER/ ; þ fl fl þ revealed newly generated LacZ Lgr5 cells at the base of Hes1 ox/ ox; Rosa26LacZ/ mice at day 10 (Fig. 3C and E). These þ þ the crypts and LacZ Hes1 cells in the crypts of Lgr5CreERT2/ ; data demonstrate that, regardless of the stem cell markers tested, fl fl þ þ Hes1 ox/ ox; Rosa26LacZ/ mice (Fig. 1E). The number of Lgr5 cells Hes1 is necessary for the self-renewal of NSCs; however, Hes1 þ þ þ þ and Hes1 cells did not differ between Lgr5CreERT2/ ; Hes1 / ; deletion in NSCs does not perturb intestinal homeostasis. þ þ fl fl Rosa26LacZ/ control mice and Lgr5CreERT2/ ; Hes1 ox/ ox; þ þ Rosa26LacZ/ mice at day 14 (Fig. 1C). Although Lgr5 NSCs Concomitant Hes1 deletion in b-catenin–stabilized NSCs þ cannot self-renew after the deletion of Hes1, Lgr5 NSCs were abrogates tumor formation newly generated, likely from some of the Lgr5 reserve stem cells To investigate the role of Hes1 in tumorigenesis, we stabilized þ (e.g., Bmi1 cells), as previously reported (26). Furthermore, we b-catenin for intestinal tumor formation and deleted Hes1 þ þ þ fl fl also evaluated the effect of Hes1 deletion on the proliferative in Lgr5 cells using Lgr5CreERT2/ ; Ctnnb1lox(ex3)/ ; Hes1 ox/ ox mice. capacity and secretory cell differentiation before (at day 5) and Twenty-eight days after injection with 2 mg tamoxifen over after (at day 14) replacement with LacZ cells; however, no 4 consecutive days, we found that only microadenomas were þ þ fl fl significant difference was found (Supplementary Fig. S3A-C). formed in Lgr5CreERT2/ ; Ctnnb1lox(ex3)/ ; Hes1 ox/ ox mice, whereas Lgr5-EGFP-IRES-CreERT2 mice have mosaic expression of the large tumors were formed throughout the intestine in þ þ þ þ þ reporter (27); there remains some unlabeled Lgr5 cells that Lgr5CreERT2/ ; Ctnnb1lox(ex3)/ ;Hes1 / control mice (Fig. 4A). escape the induction. To address this issue, we sorted single IHC revealed the expression of Hes1 and nuclear accumulation þ þ Lgr5-GFPhigh cells from the normal intestinal epithelia of of b-catenin in the large tumors of Lgr5CreERT2/ ; Ctnnb1lox(ex3)/ ;

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Figure 1. Hes1 deletion in Lgr5þ NSCs results in loss of self-renewal, but does not perturb intestinal homeostasis. A, Macroscopic images and H&E staining of LacZ staining in the small intestine in Lgr5CreERT2/þ; Hes1flox/flox; Rosa26LacZ/þ mice at day 1, 3, 5, and 7 after tamoxifen induction. B, Immunostaining of Hes1 in Lgr5CreERT2/þ; Hes1þ/þ; Rosa26LacZ/þ and Lgr5CreERT2/þ; Hes1flox/flox; Rosa26LacZ/þ mice at day 3 after intensive induction. C, Quantification of Hes1þ cells per crypt at day 3 after intensive induction and Lgr5-GFPþ cells and Hes1þ cells per crypt at day 14 after intensive induction. n ¼ 20–30, each. , P < 0.05. D, Macroscopic images of LacZ staining in the small intestine in Lgr5CreERT2/þ; Hes1þ/þ; Rosa26LacZ/þ and Lgr5CreERT2/þ; Hes1flox/flox; Rosa26LacZ/þ mice at day 14 after intensive induction. E, H&E staining and immunostaining of Lgr5-GFP and Hes1 in Lgr5CreERT2/þ;Hes1þ/þ; Rosa26LacZ/þ and Lgr5CreERT2/þ; Hes1flox/flox; Rosa26LacZ/þ mice at day 14 after intensive induction. Scale bar, 20 mm(A, B,andE).

þ þ þ þ Hes1 / mice and microadenomas of Lgr5CreERT2/ ; mice were formed only from Lgr5 cells that escaped deletion þ fl fl Ctnnb1lox(ex3)/ ;Hes1ox/ ox mice (Fig. 4A). These results suggest of Hes1 but had not escaped b-catenin stabilization. Because þ þ fl fl þ that microadenomas in Lgr5CreERT2/ ; Ctnnb1lox(ex3)/ ;Hes1 ox/ ox of the strongly suppressed tumor formation, Lgr5CreERT2/ ;

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A B

D E

Figure 2. Spheroid formation was abrogated in Hes1-deleted Lgr5-GFPhigh single cells. A, Scheme of Hes1 deletion in Lgr5-GFPhigh single cells. B and C, The number of spheroids formed from 1,000 Lgr5-GFPhigh single cells, sorted from Lgr5CreERT2/þ;Hes1þ/þ and Lgr5CreERT2/þ; Hes1flox/flox mice, at day 6 (B); images were acquired at days 0 and 6 (C). n ¼ 11–13, each. D, Scheme of Hes1 deletion in Lgr5þ NSCs of established spheroids. E, qRT-PCR analysis of Lgr5 in spheroids cultured in 50% L-WRN CM, CHIR99021, and valproic acid and in spheroids cultured in 50% L-WRN CM. n ¼ 4, each. F and G, Time course images (F) and diameter (G) of spheroids generated from Lgr5CreERT2/þ; Hes1þ/þ and Lgr5CreERT2/þ; Hes1flox/flox mice after induction. n ¼ 20, each. Scale bar, 400 mm(C and F); 200 mm (black boxes in F).

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Figure 3. Hes1 deletion in Bmi1þ NSCs results in loss of self-renewal, but does not perturb intestinal homeostasis. A, Macroscopic images and H&E staining of LacZ staining of the small intestine in Bmi1CreERT/þ;Hes1flox/flox; Rosa26LacZ/þ mice at day 1, 3, 5, and 7 after tamoxifen induction. B, Immunostaining of Hes1 in Bmi1CreERT/þ; Hes1flox/þ; Rosa26LacZ/þ and Bmi1CreERT2/þ; Hes1flox/flox; Rosa26LacZ/þ mice 3 days after intensive induction. C, Quantification of Hes1þ cells per crypt at day 3 and day 10 after intensive induction. n ¼ 20, each. , P < 0.05. D, Macroscopic images of LacZ staining in the small intestine in Bmi1CreERT/þ;Hes1flox/þ; Rosa26LacZ/þ and Bmi1CreERT/þ; Hes1flox/flox; Rosa26LacZ/þ mice 10 days after intensive induction. E, H&E staining and immunostaining of Hes1 in Bmi1CreERT/þ; Hes1flox/þ; Rosa26LacZ/þ and Bmi1CreERT/þ; Hes1flox/flox; Rosa26LacZ/þ mice 10 days after intensive induction. Scale bar, 20 mm(A, B,andE).

þ fl fl Ctnnb1lox(ex3)/ ; Hes1 ox/ ox mice survived significantly longer size of a few nascent adenomas gradually increased over time, than the control mice (median survival time: 96 days vs. resulting in the formation of some microadenomas and a few 27 days, respectively; Fig. 4B). We analyzed the number and size macroadenomas at 15 weeks (Fig. 4C). IHC revealed the þ þ fl fl of adenomas in Lgr5CreERT2/ ; Ctnnb1lox(ex3)/ ;Hes1 ox/ ox mice at expression of Hes1 in microadenomas, macroadenomas, and þ þ different times after the administration of tamoxifen. We found the normal intestinal epithelia of Lgr5CreERT2/ ; Ctnnb1lox(ex3)/ ; fl fl that the total number of adenomas did not increase, but the Hes1 ox/ ox mice (Fig. 4D), reaffirming that tumors were formed

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P < 0.0001

Days

>1,000 µm 200–1,000 µm <200 µm

Figure 4. Concomitant Hes1 deletion in b-catenin–stabilized NSCs abrogates tumor formation. A, Macroscopic images of the duodenum, H&E staining, and immunostaining of b-catenin and Hes1 in Lgr5CreERT2/þ; Ctnnb1lox(ex3)/þ; Hes1þ/þ and Lgr5CreERT2/þ; Ctnnb1lox(ex3)/þ; Hes1flox/flox mice at day 28 after tamoxifen induction. Arrowheads, microadenoma. B, The log-rank test revealed a significantly longer survival of Lgr5CreERT2/þ; Ctnnb1lox(ex3)/þ; Hes1flox/flox mice compared with Lgr5CreERT2/þ; Ctnnb1lox(ex3)/þ; Hes1þ/þ control mice after tamoxifen induction (P < 0.0001). MST, median survival time. n ¼ 15, each. C, Time course of change in size and number of intestinal tumors of Lgr5CreERT2/þ; Ctnnb1lox(ex3)/þ; Hes1flox/flox mice after tamoxifen induction. D, Immunostaining of Hes1 in macroadenoma, microadenoma, and normal mucosa of Lgr5CreERT2/þ; Ctnnb1lox(ex3)/þ; Hes1flox/flox mice at week 15 after tamoxifen induction. E and F, Macroscopic images (E) and H&E staining (F) of LacZ staining in the small intestine in Lgr5CreERT2/þ; Ctnnb1lox(ex3)/þ; Hes1þ/þ; Rosa26LacZ/þ and Lgr5CreERT2/þ; Ctnnb1lox(ex3)/þ; Hes1flox/flox; Rosa26LacZ/þ mice at day 28 after tamoxifen induction. Scale bar, 50 mm(A, D,andF).

þ fl fl þ only from Lgr5 cells that had escaped Hes1 deletion. We Hes1 ox/ ox; Rosa26LacZ/ mice, whereas LacZ-labeled blue intes- þ þ þ þ þ þ performed lineage tracing using Lgr5CreERT2/ ; Ctnnb1lox(ex3)/ ; tinal tumors formed in Lgr5CreERT2/ ; Ctnnb1lox(ex3)/ ; Hes1 / ; þ þ þ þ þ þ Hes1 / ; Rosa26LacZ/ and Lgr5CreERT2/ ; Ctnnb1lox(ex3)/ ; Rosa26LacZ/ control mice (Fig. 4E and F). These results confirm fl fl þ Hes1 ox/ ox; Rosa26LacZ/ mice. Twenty-eight days after induc- that b-catenin stabilization cannot offset the inability of Hes1- þ tion, most of the LacZ-labeled blue cells were depleted and repo- deleted Lgr5 NSCs to self-renew, which leads to substantial þ þ pulated by LacZ epithelium in Lgr5CreERT2/ ; Ctnnb1lox(ex3)/ ; suppression of intestinal tumor formation.

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þ þ fl fl þ Hes1 deletion in Lgr5 TSCs of established intestinal tumors significantly reduced in Lgr5CreERT2/ ; Hes1 ox/ ox; ApcMin/ mice results in tumor regression by inducing immediate apoptosis 5 days after intensive induction (Fig. 5E). IHC revealed that the þ We have shown that concomitant Hes1 deletion in b-catenin– number of Lgr5 cells was unaltered in the normal intestinal stabilized NSCs abrogates tumor formation; however, to recapit- epithelia, but significantly reduced in the remaining few intestinal þ fl fl þ ulate Hes1-targeting tumor therapy, it is necessary to examine the tumors of Lgr5CreERT2/ ; Hes1 ox/ ox; ApcMin/ mice at day 5 (Fig. effect of Hes1 deletion in established intestinal tumors. It has been 5F). These results suggest that, in contrast with the normal þ shown that Lgr5 marks TSCs in the intestinal tumors of Apc- intestinal epithelia, no new Lgr5 cells were generated after Hes1 deficient mice (5). To investigate the role of Hes1 in the TSCs of deletion in the intestinal tumors. þ established intestinal tumors, we deleted Hes1 in Lgr5 TSCs of To observe the time course of the same intestinal tumors after the intestinal tumors and performed lineage tracing using Hes1 deletion, we generated spheroids (19) from the intestinal þ fl fl þ þ þ þ þ þ þ Lgr5CreERT2/ ; Hes1 ox/ ox; ApcMin/ ; Rosa26LacZ/ mice. Five days tumors of Lgr5CreERT2/ ; Hes1 / ; ApcMin/ and Lgr5CreERT2/ ; fl fl þ after 4 mg tamoxifen administration, normal intestinal epithelia Hes1 ox/ ox; ApcMin/ mice, using a medium containing EGF and were intact and labeled blue; however, the intestinal tumors were Noggin; we then compared the spheroid growth after 4-OHT þ fl fl injured and devoid of blue cells in Lgr5CreERT2/ ; Hes1 ox/ ox; administration for 24 hours (Supplementary Fig. S5A). The þ þ þ þ fl fl þ ApcMin/ ; Rosa26LacZ/ mice (Fig. 5A). In the Lgr5CreERT2/ ; spheroids from Lgr5CreERT2/ ; Hes1 ox/ ox; ApcMin/ mice ceased to þ þ þ þ þ Hes1 / ; ApcMin/ ; Rosa26LacZ/ control mice, the normal intesti- expand, and Lgr5-GFP cells were depleted at day 3 (Supplemen- nal epithelia and the intestinal tumors were labeled blue (Sup- tary Fig. S5B and S5C), in contrast with the unaltered growth of plementary Fig. S4A and S4B). We maximized induction efficien- established spheroids generated from normal intestinal epithelia cy by administering 2 mg tamoxifen over 4 consecutive days. (Fig. 2D–G). These results demonstrate that Hes1 deletion in þ The intestinal tumors were more severely injured at day 5 Lgr5 cells specifically suppresses tumor growth. (Fig. 5A); the blue cells of the normal intestinal epithelia and þ the injured intestinal tumors were depleted at day 10 in Hes1 deletion in Dclk1 TSCs of established intestinal tumors þ fl fl þ þ Lgr5CreERT2/ ; Hes1 ox/ ox; ApcMin/ ; Rosa26LacZ/ mice (Fig. 5A). results in tumor regression by inducing immediate apoptosis þ To further evaluate the effect of Hes1 deletion in Lgr5 TSCs, we We have recently identified Dclk1 as a TSC-specific marker in followed the time course of LacZ-labeled blue cells in the intes- the intestine by murine lineage-tracing experiments; we proposed þ þ tinal tumors after a single 4 mg injection of tamoxifen in that Lgr5 Dclk1 cells are the bona fide TSCs in the intestinal þ fl fl þ þ Lgr5CreERT2/ ; Hes1 ox/ ox; ApcMin/ ; Rosa26LacZ/ mice. Blue cells tumors (9). To further confirm that Hes1 deletion in TSCs leads to þ in the tumors were present at day 1 and depleted by day 3 (Fig. tumor regression, we deleted Hes1 in Dclk1 TSCs of established 5B). At day 1, IHC showed cleaved caspase-3 expression in Hes1- intestinal tumors and performed lineage tracing using þ þ fl fl þ þ deleted Lgr5 TSCs (Fig. 5B). The proportion of cleaved caspase- Dclk1CreERT2/ ; Hes1 ox/ ox; ApcMin/ ; Rosa26LacZ/ mice. Five days þ þ 3 cells in Lgr5 cells was significantly higher in the intestinal after administering 4 mg tamoxifen, the intestinal tumors of þ fl fl þ þ fl fl þ þ tumors of Lgr5CreERT2/ ; Hes1 ox/ ox; ApcMin/ mice than in those of Dclk1CreERT2/ ; Hes1 ox/ ox; ApcMin/ ; Rosa26LacZ/ mice were þ þ þ þ Lgr5CreERT2/ ; Hes1 / ; ApcMin/ mice (15% vs. 2.7%, respective- injured and devoid of blue cells (Fig. 6A), which contrasted the þ þ þ þ ly), whereas cleaved caspase-3 expression was not detected in blue intestinal tumors in Dclk1CreERT2/ ; Hes1 / ; ApcMin/ ; þ þ þ Lgr5 cells of the normal intestinal epithelia of Lgr5CreERT2/ ; Rosa26LacZ/ control mice (Fig. 6A). Next, we followed the time fl fl þ þ Hes1 ox/ ox; ApcMin/ mice at day 1 (Fig. 5C). To further assess the course of LacZ-labeled Hes1-deleted Dclk1 cells after induction. þ þ behaviors of Lgr5 NSCs and Lgr5 TSCs after the deletion of Blue cells were present in the intestinal tumors at day 1 and þ þ þ Hes1, we sorted Lgr5 NSCs and Lgr5 TSCs from Lgr5CreERT2/ ; depleted by day 3 (Fig. 6B). IHC revealed cleaved caspase-3 fl fl þ þ Hes1 ox/ ox; ApcMin/ mice at day 1 after a single 4 mg injection of expression in Dclk1 cells at day 1 (Fig. 6C), suggesting that þ tamoxifen and used qRT-PCR to compare the levels of mRNA deletion of Hes1 in Dclk1 TSCs also induces immediate expression with those in the control mice. Hes1 expression was apoptosis. þ þ downregulated in Lgr5 NSCs and Lgr5 TSCs at day 1 after We maximized induction efficiency by administering 2 mg induction (Fig. 5D; Supplementary Fig. S4C). Of note, the expres- tamoxifen over 4 consecutive days and analyzed the number of þ þ þ þ sion of antiapoptotic gene Bcl2 was significantly downregulated, intestinal tumors in Dclk1CreERT2/ ; Hes1 / ; ApcMin/ and þ fl fl þ and the expression of proapoptotic gene Bax had a tendency to be Dclk1CreERT2/ ; Hes1 ox/ ox; ApcMin/ mice. Compared with the upregulated in Hes1-deleted TSCs, whereas the expression levels number of intestinal tumors in the control mice, the number of þ of Bax and Bcl2 were not altered in Hes1-deleted NSCs (Fig. 5D; intestinal tumors was significantly reduced in Dclk1CreERT2/ ; fl fl þ Supplementary Fig. S4C). Previous studies showed that Hes1 Hes1 ox/ ox; ApcMin/ mice 5 days after intensive induction (Fig. directly suppresses Pten (28, 29), which upregulates p53 activity 6D). These results demonstrate that, irrespective of stem cell (30) and downregulates the PI3K–Akt pathway (31) to induce markers analyzed, Hes1 deletion in the TSCs leads to tumor apoptosis. Interestingly, Pten was upregulated in the Hes1-deleted regression by inducing immediate apoptosis in the TSCs. TSCs, but not in the Hes1-deleted NSCs at day 1 (Fig. 5D; Supplementary Fig. S4C). These results demonstrate that Hes1 þ deletion results in immediate apoptosis of Lgr5 TSCs, but not Discussion þ Lgr5 NSCs. In this study, we focused on the specific role of Hes1 in the NSCs þ To evaluate whether Hes1 deletion in Lgr5 TSCs can be used as and TSCs of the intestine and showed that the different roles of a tumor-specific treatment, we analyzed the number of intestinal Hes1 can enable stem cell–targeting tumor therapy. We first þ þ þ þ tumors in Lgr5CreERT2/ ; Hes1 / ; ApcMin/ control mice and demonstrated that the deletion of Hes1 in the NSCs results in þ fl fl þ Lgr5CreERT2/ ; Hes1 ox/ ox; ApcMin/ mice after administering 2 mg loss of self-renewal but does not perturb homeostasis. The Notch þ tamoxifen over 4 consecutive days. Compared with Lgr5CreERT2/ ; and Wnt/b-catenin pathways play a crucial role in the mainte- þ þ þ Hes1 / ; ApcMin/ mice, the number of intestinal tumors was nance of NSCs (12, 13, 32). Hes1 is a well-known Notch effector

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(10), and its expression is also directly regulated by the Wnt/ perturb homeostasis either. It is possible that adjacent TA cells b-catenin pathway (21, 24). Our study showed that although the compensate for the loss of reserve stem cells, because Hes1 expression of other Notch effectors, Hes3 and Hes5, was not deletion, which results in loss of self-renewal, has milder influ- upregulated, the expression of Hes1 was upregulated in the NSCs. ences on the adjacent TA cells than does ablation. Further studies Although the interaction between Notch and Wnt/b-catenin path- are required to corroborate this speculation. ways is complex and context dependent (13, 21, 33, 34), their role Several reports have shown the potential of Hes1 as a target in the maintenance of NSCs may be dependent on their common for antitumor therapeutics. Hes1 knockdown suppresses cell downstream target, Hes1. proliferation in T-cell acute lymphoblastic leukemia and colon Previously, we employed the Villin-Cre mice to show that cancer cell lines (29, 37, 38), and Hes1 overexpression induces prenatal Hes1 deletion induces transient secretary differentia- self-renewal properties in colon cancer cell lines (23). Our tion, which is compensated by Hes3 and Hes5, and that the previous study showed that when Hes1 is deleted prenatally, maintenance of NSCs is unaffected even after the prenatal the characteristics of ApcMin tumors mimic those of normal triple deletion of Hes1, Hes3, and Hes5 (14). We speculate intestinal epithelia, such as differentiation and low prolifer- that other Notch effectors (e.g., Hey1, Hey2, and HeyL) had ative capacity (14). However, it is possible that compensation compensated in the maintenance of NSCs. Recombination in in the developmental phase permitted atypical tumor forma- Villin-Cre mice occurs early in the developmental stage (35) tion; additionally, we did not examine apoptosis or immediate when the intestinal epithelium is generated from progenitors changes after postnatal Hes1 deletion. Therefore, in this study, þ other than Lgr5 cells (15, 16); these progenitors may tran- we performed TSC-specific Hes1 deletion, which indicated that siently permit Hes1 deficiency and provide time for the com- the deletion of Hes1 in the TSCs of established tumors leads pensation. In contrast, this study showed that Hes1 deficiency to tumor regression by inducing immediate apoptosis. These was not compensated in adult NSCs, indicating a novel role of findings provide strong evidence for Hes1-targeting tumor þ Hes1 in the maintenance of adult NSCs, which was masked in therapy. The dramatic change in Hes1-deleted Lgr5 TSCs can our previous analysis of prenatal Hes1 deletion. However, Lgr5- be attributed to the abrupt upregulation of Pten. Hes1 directly EGFP-IRES-CreERT2 mice have mosaic expression of the report- suppresses Pten (28, 29), and Pten upregulation induces apo- er (27); we cannot exclude the possibility that Hes1-expressing ptosis via direct upregulation of p53 activity (30) and down- þ þ unlabeled Lgr5 cells had replaced Hes1-deleted Lgr5 cells by regulation of the PI3K–Akt pathways, such as the PI3K–Akt– neutral drift dynamics (36) in vivo. To address this, we sorted mTOR and PI3K–Akt–GSK3b pathways (29, 31). Provided that þ Lgr5-GFPhigh cells and showed that Hes1-deleted Lgr5 stem Hes1 knockdown in the wild-type and mutant p53 colon cells cannot self-renew in vitro. Hes1 deletion using stem cell– cancer cell lines increases apoptosis (29), multiple pathways, specific reporter mice without the mosaic problem (27) could such as Pten-p53 and Pten-Akt, may be involved in inducing further corroborate the role of Hes1 in vivo.Incontrastto apoptosis in Hes1-deleted TSCs. þ prenatal Hes1 deletion, Hes1 deletion in adult NSCs did not In contrast to normal intestinal epithelia, Lgr5 TSCs were not increase the number of secretory cells (e.g., goblet cells, enter- generated from the surrounding tumor cells after deletion of þ oendocrine cells, and Paneth cells). This was probably because Hes1. The loss of Lgr5 TSCs after Hes1 deletion can be attributed the depletion of Hes1-deleted NSCs did not provide sufficient to the following two factors. First, the deletion of Hes1 in the TSCs time for the differentiation shift. results in changes that are more severe than those in NSCs þ After Hes1 deletion in Lgr5 cells, normal intestinal epithelia (immediate apoptosis in the former compared with loss of were replaced by wild-type epithelia with normal Hes1 expression self-renewal in the latter), and so the surrounding cells could not þ in the crypts. The intact epithelium following Hes1 deletion in compensate for the loss of Lgr5 TSCs due to apoptosis. Second, þ þ Lgr5 NSCs could be explained by the previous report showing because the population of Lgr5 cells expanded in the intestinal þ that ablation of Lgr5 NSCs itself does not disrupt homeostasis tumors (2.9% in the normal intestine compared with 13% in the þ because of compensation by reserve stem cells (26). To address intestinal tumors; Fig. 5F), it is possible that the Lgr5 cells in the þ the issue, we deleted Hes1 in Bmi1 NSCs, which have the intestinal tumors consist of both stem cells and progenitor cells. potential capacity of reserve stem cells (26). It has been shown The first factor may be more plausible because Hes1 deletion in þ þ that, in contrast to the ablation of Lgr5 NSCs, the ablation of Dclk1 TSCs also leads to tumor regression by inducing imme- þ Bmi1 NSCs leads to crypt loss, perturbing the intestinal homeo- diate apoptosis. Using murine lineage-tracing experiments, we þ stasis (6). We showed that Hes1 deletion in Bmi1 cells does not have recently shown that Dclk1 marks TSCs in the intestine and

Figure 5. Hes1 deletion in Lgr5þ TSCs of established intestinal tumors results in tumor regression by inducing immediate apoptosis. A, Macroscopic image of LacZ staining in the small intestine in Lgr5CreERT2/þ; Hes1flox/flox; ApcMin/þ; Rosa26LacZ/þ mice at day 5 after tamoxifen induction revealed injured intestinal tumors devoid of blue cells (red circles). H&E staining at day 5 after intensive induction revealed severely injured intestinal tumor (black circle), and macroscopic image of LacZ staining at day 10 after intensive induction revealed tumor regression. B, H&E staining of Lgr5CreERT2/þ; Hes1flox/flox; ApcMin/þ; Rosa26LacZ/þ mice at day 1 and day 3 after tamoxifen induction. Immunofluorescent staining of cleaved caspase-3 and Lgr5-GFP in Lgr5CreERT2/þ; Hes1flox/flox; ApcMin/þ mice at day 1 after tamoxifen induction. C, Proportion of cleaved caspase-3þ cells in Lgr5-GFPþ cells in the normal intestine and intestinal tumors of Lgr5CreERT2/þ; Hes1þ/þ; ApcMin/þ and Lgr5CreERT2/þ; Hes1flox/flox; ApcMin/þ mice at day 1 after tamoxifen induction. , P < 0.05. D, qRT-PCR of Hes1, Bcl2, Bax,andPten in sorted Lgr5þ cells from intestinal tumors of Lgr5CreERT2/þ;Hes1þ/þ; ApcMin/þ and Lgr5CreERT2/þ; Hes1flox/flox; ApcMin/þ mice at day 1 after tamoxifen induction. n ¼ 3, each. , P < 0.05. E, Macroscopic images and the number of intestinal tumors in Lgr5CreERT2/þ;Hes1þ/þ; ApcMin/þ and Lgr5CreERT2/þ; Hes1flox/flox; ApcMin/þ mice at day 5 after intensive tamoxifen induction. n ¼ 7–11, each. , P < 0.05. F, Immunostaining of Lgr5-GFP in Lgr5CreERT2/þ; Hes1þ/þ; ApcMin/þ and Lgr5CreERT2/þ; Hes1flox/flox; ApcMin/þ mice at day 5 after intensive induction. Proportion of Lgr5-GFPþ cells in normal intestinal epithelia and intestinal tumors in Lgr5CreERT2/þ; Hes1þ/þ; ApcMin/þ and Lgr5CreERT2/þ; Hes1flox/flox; ApcMin/þ mice at day 5 after intensive induction. , P < 0.05. Scale bars, 100 mm(A); 20 mm(B); and 200 mm(F).

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Figure 6. Hes1 deletion in Dclk1þ TSCs of established intestinal tumors results in tumor regression by inducing immediate apoptosis. A, Macroscopic images and H&E staining of LacZ staining in the small intestine in Dclk1CreERT2/þ; Hes1þ/þ; ApcMin/þ; Rosa26LacZ/þ and Dclk1CreERT2/þ; Hes1flox/flox; ApcMin/þ; Rosa26LacZ/þ mice at day 5 after tamoxifen induction. Red circles, intestinal tumors; black circle, injured tumor. B, H&E staining of LacZ staining in Dclk1CreERT2/þ; Hes1flox/flox; ApcMin/þ; Rosa26LacZ/þ mice at day 1 and day 3 after tamoxifen induction. C, Immunofluorescent staining of cleaved caspase-3 and Dclk1 in Dclk1CreERT2/þ; Hes1flox/flox; ApcMin/þ mice at day 1 after tamoxifen induction. D, Macroscopic images and the number of intestinal tumors of Dclk1CreERT2/þ; Hes1þ/þ; ApcMin/þ and Dclk1CreERT2/þ; Hes1flox/flox; ApcMin/þ mice at day 5 after intensive tamoxifen induction. n ¼ 7–8, each. , P < 0.05. Scale bar, 50 mm(A–C).

þ þ proposed that Lgr5 Dclk1 cells are the bona fide TSCs in Inhibition of Hes1 by small molecules, and in vivo delivery of intestinal tumors (9). In this study, we showed that Hes1 deletion Hes1 siRNA, are two approaches that can be employed to target þ þ in Dclk1 cells as well as in Lgr5 cells leads to tumor regression, Hes1 for therapeutic purposes. Several compounds can inhibit þ þ implying that Hes1 deletion in Lgr5 Dclk1 cells, which account Hes1 activity (38–40). Agalloside, which directly binds to for only a small subset of the tumor cells, may be sufficient for Hes1, has been recently identified as a naturally occurring Hes1 tumor regression. dimer inhibitor (39). Furthermore, siRNA delivery systems are

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Figure 7. Hes1 can be a tumor-speciﬁc therapeutic target. A, Scheme of Hes1 deletion in NSCs. Hes1-deleted NSCs cannot self-renew, but Hes1þ NSCs are B newly generated; mucosa remains intact. B, Scheme of Hes1 deletion in TSCs. Hes1-deleted TSCs undergo immediate apoptosis, which leads to tumor regression.

being developed for cancer treatment and have already entered Administrative, technical, or material support (i.e., reporting or organizing clinical trials (41). The advance of these technologies may data, constructing databases): K. Kawada, H. Miyoshi, T. Chiba realize the potential of Hes1-targeting tumor stem cell therapy Study supervision: A. Fukuda, H. Seno in the future. In summary, using several murine models with different NSC Acknowledgments flox and TSC markers, we have shown the novel role of Hes1 in the The authors thank Ryoichiro Kageyama for kindly providing Hes1 mice maintenance of NSCs and TSCs in the adult intestine. Hes1 and Tetsuo Sudo for kindly providing the Hes1 antibody. deletion in the NSCs leads to loss of self-renewal but does not disrupt homeostasis (Fig. 7A). Conversely, Hes1 deletion in the Grant Support TSCs leads to tumor regression by inducing immediate apoptosis This work was supported in part by Grants-in-Aid KAKENHI (Fig. 7B). On the basis of the different roles of Hes1 in the NSCs (25112707, 26293173, 15J03212, 16K09394, 16K15216, and 16K15427) and a research program of the Project for Development of and TSCs, we propose that Hes1 can be used as a novel tumor- fi Innovative Research on Cancer Therapeutics (P-Direct) from the Ministry speci c therapeutic target that does not perturb intestinal of Education, Culture, Sports, Science, and Technology, and the Japan homeostasis. Society for the Promotion of Science. This work was also supported by Health Labour Sciences Research Grants from the Ministry of Health, Disclosure of Potential Conflicts of Interest Labour, and Welfare (the development of innovative therapeutic drug for fl No potential conflicts of interest were disclosed. the intractable in ammatory bowel disease), the Kobayashi Foundation for Cancer Research, the Naito Foundation, Princess Takamatsu Cancer Authors' Contributions Research Fund13-24514, the Mochida Foundation, and the Japanese Society of Gastroenterology. Conception and design: N. Goto, T. Ueo, H. Seno The costs of publication of this article were defrayed in part by the Development of methodology: N. Goto, T. Ueo payment of page charges. This article must therefore be hereby marked Acquisition of data (provided animals, acquired and managed patients, advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate provided facilities, etc.): N. Goto this fact. Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): N. Goto, M.M. Taketo Writing, review, and/or revision of the manuscript: N. Goto, T. Ueo, Received November 29, 2016; revised March 17, 2017; accepted May 8, 2017; A. Fukuda, M.M. Taketo, T. Chiba, H. Seno published OnlineFirst May 23, 2017.

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Distinct Roles of HES1 in Normal Stem Cells and Tumor Stem-like Cells of the Intestine

Norihiro Goto, Taro Ueo, Akihisa Fukuda, et al.

Cancer Res Published OnlineFirst May 23, 2017.

Updated version Access the most recent version of this article at: doi:10.1158/0008-5472.CAN-16-3192

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