Cancer Research Tumor and Stem Cell Biology Suppressing TGFb Signaling in Regenerating Epithelia in an Inflammatory Microenvironment Is Sufficient to Cause Invasive Intestinal Cancer Hiroko Oshima1, Mizuho Nakayama1,2, Tae-Su Han1,2, Kuniko Naoi1, Xiaoli Ju1, Yusuke Maeda1, Sylvie Robine3, Kiichiro Tsuchiya4, Toshiro Sato5, Hiroshi Sato6, Makoto Mark Taketo7, and Masanobu Oshima1 Abstract Genetic alterations in the TGFb signaling pathway in combination with oncogenic alterations lead to cancer development in the intestines. However, the mechanisms of TGFb signaling suppression in malignant progression of intestinal tumors have not yet been fully understood. We have examined ApcD716 Tgfbr2DIEC compound mutant mice that carry mutations in Apc and Tgfbr2 genes in the intestinal epithelial cells. We found inflammatory microenvironment only in the invasive intestinal adenocarcinomas but not in noninvasive benign polyps of the same mice. We thus treated simple Tgfbr2DIEC mice with dextran sodium sulfate (DSS) that causes ulcerative colitis. Importantly, these Tgfbr2DIEC mice developed invasive colon cancer associated with chronic inflammation. We also found that TGFb signaling is suppressed in human colitis–associated colon cancer cells. In the mouse invasive tumors, macrophages infiltrated and expressed MT1-MMP, causing MMP2 activation. These results suggest that inflammatory microenvironment contributes to submucosal invasion of TGFb signaling–repressed epithelial cells through activation of MMP2. We further found that regeneration was impaired in Tgfbr2DIEC mice for intestinal mucosa damaged by DSS treatment or X-ray irradiation, resulting in the expansion of undifferentiated epithelial cell population. Moreover, organoids of intestinal epithelial cells cultured from irradiated Tgfbr2DIEC mice formed "long crypts" in Matrigel, suggesting acquisition of an invasive phenotype into the extracellular matrix. These results, taken together, indicate that a simple genetic alteration in the TGFb signaling pathway in the inflamed and regenerating intestinal mucosa can cause invasive intestinal tumors. Such a mechanism may play a role in the colon carcinogenesis associated with inflammatory bowel disease in humans. Cancer Res; 75(4); 766–76. 2015 AACR. Introduction suppression of colon tumor development (5–7). These results indicate that activation of inflammatory pathways through PGE2, NF-kB, and Stat3 is required for intestinal tumorigenesis. However, it has not yet been elucidated what role the inflammatory responses play in the progression of benign intestinal tumors to invasive adenocarcinomas. Most intestinal adenomas are induced by APC mutations, resulting in the Wnt signaling activation, and tumors progress to adenocarcinomas by additional mutations such as those encoding RAS or transforming growth factor b (TGFb) type II receptor (TGFbRII; ref. 8). Mouse genetic studies have indicated that suppression of TGFb signaling accelerates development of malignant intestinal tumors in combination with mutations in Kras or Pten, although TGFb suppression alone does not cause tumorous changes (9, 10). These results indicate that suppression of the TGFb signaling is a key process involved in the malignant progression. The TGFb ligand binds TGFbRII, followed by the activation of TGFbRI. Activated TGFbRI then phosphorylates Smad2/3, which causes their binding with Smad4, and the Smad complex induces transcription of TGFb target genes (11). We have previously shown that immature myeloid cells (iMC) are recruited and express matrix metalloproteinase 2 (MMP2) at the invasion front of compound ApcD716 Smad4 knockout mouse intestinal adenocarcinomas, which contribute to their submucosal invasion (12, 13). Moreover, disruption of Tgfbr2, encoding TGFbRII, in mouse mammary tumor cells results in the recruitment of Accumulating evidence indicates that inflammatory responses play an important role in cancer development (1, 2). The disruption of genes encoding cyclooxygenase 2 (COX-2) or prostaglandin E2 (PGE2) receptor, EP2, in ApcD716 knockout mice results in significant suppression of intestinal polyposis (3, 4). Moreover, blocking transcription factors NF-kB and Stat3 in a chemically induced colitis-associated colon tumor model in mice causes 1 Division of Genetics, Cancer Research Institute, Kanazawa University, Kanazawa, Japan. 2Core Research for Evolutional Science and Technology (CREST), the Japan Science and Technology Agency, Tokyo, Japan. 3Equipe de Morphogenese et Signalisation cellulaires, Institut Curie, Paris, France. 4Department of Advanced Therapeutics for Gastrointestinal Disease, Graduate School Tokyo Medical and Dental University, Tokyo, Japan. 5Department of Gastroenterology, Keio University School of Medicine, Tokyo, Japan. 6Division of Virology and Oncology, Cancer Research Institute, Kanazawa University, Kanazawa, Japan. 7Department of Pharmacology, Kyoto University Graduate School of Medicine, Kyoto, Japan. Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). Corresponding Author: Masanobu Oshima, Division of Genetics, Cancer Research Institute, Kanazawa University, Kakuma-machi, Kanazawa, Ishikawa 920-1192, Japan. Phone: 81-76-264-6760; Fax: 81-76-234-4519; E-mail: oshimam@staff.kanazawa-u.ac.jp doi: 10.1158/0008-5472.CAN-14-2036 2015 American Association for Cancer Research. 766 Cancer Res; 75(4) February 15, 2015 Downloaded from cancerres.aacrjournals.org on April 12, 2017. © 2015 American Association for Cancer Research. Invasion by TGFb Blocking in Regenerating Inflamed Mucosa myeloid cells into tumor tissues, which promotes tumor metastasis through a process involving metalloproteinase activation (14). These results indicate that suppression of TGFb signaling generates a microenvironment that is critical for progression of intestinal tumors. However, it is not understood how the inflammatory responses affect the tumor progression induced by TGFb suppression. Moreover, it has not been elucidated whether TGFbsuppressed epithelial cells acquire an invasive phenotype in tumor tissues. We herein show that ulcerative colitis causes submucosal invasion of Tgfbr2-disrupted intestinal epithelia, leading to the development of invasive colon cancer. Moreover, TGFb signaling suppression in regenerating epithelia caused long crypt formation in Matrigel, which may reflect an increased capacity for invasion. We also found that TGFb signaling is suppressed in human colitis–associated colon cancer cells. These results provide a novel mechanism for the development of invasive colon cancer where TGFb signaling suppression, chronic inflammation, and the regeneration of epithelial cells are compounded. Materials and Methods Animal models Wild-type C57BL/6 mice were purchased from CLEA. ApcD716 mice, Tgfbr2flox/flox mice, and villin-CreER mice have been described previously (15–17). Tgfbr2flox/flox mice were obtained from Mouse Repository (NCI-Frederick, Strain Number: 01XN5, Frederick, MD). All animal experiments were carried out according to the protocol approved by the Committee on Animal Experimentation of Kanazawa University, Japan. Animal experiments ApcD716 Tgfbr2flox/flox villin-CreER mice were treated with tamoxifen at 4 mg/mouse once a week, from 5 weeks of age to generate ApcD716 Tgfbr2DIEC mice. Intestinal tumors of ApcD716 mice and ApcD716 Tgfbr2DIEC mice were examined at 15 weeks of age (n ¼ 4). For the dextran sodium sulfate (DSS) treatment experiments, Tgfbr2flox/flox villin-CreER mice were treated with tamoxifen at 4 mg/mouse for 3 consecutive days to generate Tgfbr2DIEC mice, and mice were treated with 2% DSS in drinking water (MP Biomedicals) for 5 days. After DSS treatment, Tgfbr2DIEC mice were treated with tamoxifen for 2 days, and were examined at 3 days, 4 weeks, 6 to 10 weeks (n ¼ 10 for each) or 40 weeks (n ¼ 2) after DSS treatment. For the X-ray irradiation experiments, C57BL/6 mice and Tgfbr2DIEC mice were irradiated with X-ray at 9 Gy (n ¼ 15 for each), and were examined for the intestine phenotype chronologically from days 0 to 6. For the azoxymethane/DSS colitis-associated colon tumor model, wild-type mice (n ¼ 10) were intraperitoneally injected with 10 mg/kg azoxymethane (Sigma), followed by treatment with 2.0% DSS (MP Biomedicals) in drinking water for 5 days (week 1). This cycle was repeated twice during weeks 4 and 7, and mice were euthanized at week 15. Tumor tissues were used for immunoblotting analysis and gelatin zymography. Histology and immunohistochemistry The tissue sections were stained with H&E or Masson's trichrome stain, or were processed for immunohistochemistry. Staining signals of immunohistochemistry were visualized using www.aacrjournals.org the Vectastain Elite Kit (Vector Laboratories). Antibodies against E-cadherin (R&D Systems), a-SMA (Sigma), F4/80 (Serotec), MT1-MMP (GeneTex), Ki67 (Life Technologies), GFP (Molecular Probes), Collagen type IV (Nichirei Biosciences), CD44 (Millipore), SOX7 (R&D Systems), phosphorylated Smad2 (P-Smad2) at Ser465/467 (Millipore) and b-catenin (Sigma) were used for immunohistochemistry. For fluorescence immunohistochemistry, Alexa Fluor 594 or Alexa Fluor 488 antibodies (Molecular Probes) were used as the secondary antibody. Approval for the projects using human tissue sections was obtained from the Tokyo Medical and Dental University Hospital Ethics Committee and Keio University Ethics Committee. Bone marrow transplantation Bone marrow (BM) cells were prepared from the femurs and tibias of green fluorescent protein (GFP) gene transgenic mice. Recipient mice were irradiated with 9 Gy of X-rays, followed by intravenous injection of 2  106 BM cells. Real-time RT-PCR Size-classified intestinal polyps and normal intestines of ApcD716 mice (n ¼ 5 for each) were used for RNA extraction. For DSS-treated mouse samples, normal colon tissues or invasive colon tumors at 3 days, 4 weeks or 6 to 10 weeks (n ¼ 10 for each) after DSS treatment were used for RNA extraction. The total RNAs were reverse-transcribed using the PrimeScript RT reagent kit (TaKaRa) and were PCR-amplified using the SYBR Premix ExTaqII (TaKaRa). The primers used for the real-time RT-PCR were purchased from TaKaRa. Immunoblotting analysis Tissues were homogenized in lysis buffer, and protein sample was separated in a 10% SDS-polyacrylamide gel. Antibodies against active form of b-catenin (dephosphorylated at Ser37 and Thr41; Millipore) and total b-catenin (Sigma), Stat3 (Cell Signaling Technology), and phosphorylated Stat3 at Tyr705 (Cell Signaling Technology) were used. An anti-b-actin antibody (Sigma) was used as the internal control. The ECL detection system (GE Healthcare) was used to detect the signals. Gelatin zymography Tissue sample was lysed in SDS sample buffer, incubated for 20 minutes at 37 C and separated in a 10% polyacrylamide gel containing 0.005% gelatin labeled with Alexa Fluor 670 (Abcam). After electrophoresis, gels were soaked in 2.5% Triton X-100 for 1 hour, then gelatinolysis was carried out by incubation at 37 C for 24 hours. The gel was monitored by an Odyssey infrared imaging system (LI-COR). Organoid culture The organoid culture using small intestinal epithelial cells was performed as described previously (18). Briefly, organoids were cultured in Matrigel with Advanced DMEM/F12 medium (Invitrogen) supplemented with 50 ng/mL EGF (Invitrogen), R-Spondin1 conditioned medium (a kind gift from Dr. Marc Leushacke, A STAR, Institute of Medical Biology, Singapore), and 100 ng/mL Noggin (Peprotech). The cultures were passaged once, and the crypt length was measured under a dissecting microscope. Organoid cell proliferation was detected using the Click-iT EdU Imaging System (Invitrogen). After EdU staining, organoids were Cancer Res; 75(4) February 15, 2015 Downloaded from cancerres.aacrjournals.org on April 12, 2017. © 2015 American Association for Cancer Research. 767 Oshima et al. Apc-mutant mice develop only benign adenomas (12, 19–21). Accordingly, the combination of Wnt signaling activation and TGFb signaling suppression is implicated in the malignant invasion of intestinal tumors. To further investigate the mechanism underlying the submucosal invasion of intestinal tumors, we constructed compound mutant mice carrying both ApcD716 and Tgfbr2 mutation in intestinal epithelial cells (ApcD716Tgfbr2DIEC). We confirmed that ApcD716 Tgfbr2DIEC mice developed adenocarcinomas with submucosal invasion, whereas simple ApcD716 mice had only noninvasive adenomas (Fig. 1A and Supplementary Fig. S1). Notably, only the polyps >1 mm in diameter showed submucosal invasion in the ApcD716 Tgfbr2DIEC mice (Fig. 1B and Supplementary Fig. S1B). Histologically, large invasive polyps in ApcD716 Tgfbr2DIEC mice were associated with increased stroma and macrophage infiltration, which was not found in the small noninvasive polyps (Fig. 1C). incubated with anti-CD44 antibody (Chemicon) followed by secondary antibody, Alexa Fluor 488 (Molecular Probes). The stained organoids were analyzed using a Zeiss 510 META laserscanning microscope (Zeiss). Statistical analyses The data were analyzed using an unpaired t test, and are presented as the means  standard deviation (SD). A value of P < 0.05 was considered as statistically significant. Results Size-dependent submucosal invasion of intestinal tumors It has been demonstrated that the disruption of Tgfbr2, Smad4, or Smad3 in Apc-mutant mice causes development of invasive adenocarcinomas in the intestine, whereas simple A Apc Δ716 B Apc Δ716 HE (high magnification) Invaded Not invaded E-cadherin/αSMA T T SM SM φ < 0.5 mm 0.5 – 1 mm F4/80 HE 2 mm < φ 1 – 2 mm MT1-MMP MT Invasive Non-invasive ApcD716 Tgfbr2DIEC tumors 2 mm < φ 1 – 2 mm Apc Δ716 Tgfbr2 ΔIEC C Size-classified polyps D Small intestine 7 6 5 4 3 2 1 * * * 400 500 300 300 200 100 IL1β * 300 * 400 200 100 * 200 100 IL6 768 Cancer Res; 75(4) February 15, 2015 60 50 40 30 20 10 * * 5 4 3 2 * 1 COX-2 * Colon 1 < φ < 2 mm φ < 1 mm Normal mucosa Relative expression level φ < 0.5 mm 0.5 – 1 mm Not invaded Invaded HE Apc Δ716 Tgfbr2 ΔIEC 350 300 250 200 150 100 50 *10 * Normal mucosa 2 mm < φ * 8 6 4 2 MT1-MMP 6 5 4 3 2 1 5 *4 * * * 25 20 3 15 2 10 1 5 Adam10 2 mm < φ * * 70 60 50 40 30 20 10 * Figure 1. Size-dependent submucosal invasion of intestinal tumors. A, representative D716 mouse benign photographs of Apc D716 adenomas (left) and Apc DIEC Tgfbr2 mouse invasive adenocarcinomas (right). H&E (top) and enlarged images of the boxed areas (middle), and fluorescence immunohistochemistry for E-cadherin (red) and aSMA (green; bottom). T, tumor; SM, submucosa. Arrowheads, submucosal invasion of tumor epithelial cells. Bars, 400 mm (top) and 200 mm (middle and bottom). B, size classification of intestinal tumors of D716 D716 mice (top) and Apc Apc DIEC Tgfbr2 mice (bottom) scored using "Swiss roll" histology sections. Each dot indicates an individual polyp. Different colors indicate independent mice. C, representative photographs of noninvasive (top) and invasive D716 polyps (bottom) of Apc DIEC Tgfbr2 ; mice. H&E staining, immunohistochemistry for F4/80 and MT1-MMP, and Masson trichrome (MT) staining (left to right). Insets, high-powered magnification. Bars, 200 mm (top) and 400 mm (bottom). D, expression levels of the indicated factors relative to the mean level of normal mucosa in size-classified small D716 intestinal and colon tumors of Apc mice (mean  SD).  , P < 0.05 compared with the normal mucosal level. Epiregulin Cancer Research Downloaded from cancerres.aacrjournals.org on April 12, 2017. © 2015 American Association for Cancer Research. Invasion by TGFb Blocking in Regenerating Inflamed Mucosa A DSS 5 days Tmx 3 days Tmx 2 days Examination at 4, 6, 10, 40 weeks Examination at 3 days B Wild-type + DSS C Tgfbr2DIEC + DSS 3 days 3 days 4 weeks 4 weeks Tgfbr2DIEC + DSS (10 weeks) H&E * * H&E MT * D Tgfbr2DIEC + DSS (40 weeks) Ce Co E Tgfbr2DIEC + DSS Apc Δ716 AOM/DSS polyps polyps 3 days 2 weeks 8 weeks (inv) NM Active β-catenin Total β-catenin β-Actin Ile FUC-associated colon cancer (p-Smad2) β-Catenin Sporadic Surface area Ki67 CAC Invaded tumor Figure 2. Submucosal invasion of TGFb signaling-suppressed cells by ulcerative colitis. A, schedule for tamoxifen and DSS treatment and examination of mice. B, representative DIEC photographs (H&E) of DSS-treated wild-type (left) and DSS-treated Tgfbr2 (right) mouse colons 3 days (top) and 4 weeks (down) after treatment. Arrows (top), ulcer lesions. Arrowheads (bottom right), submucosal invasion of epithelial cells.  , impaired regeneration of the normal gland structure. Bars, 200 mm. C, representative DIEC photographs of invasive tumors of a DSS-treated Tgfbr2 mouse 10 weeks after treatment. H&E (top), enlarged images of the boxed areas (bottom left), and Masson trichrome (MT) staining of serial section (bottom right). Arrows, location of muscularis mucosae.  , impaired regeneration of the normal gland structure. Bars, DIEC 500 mm (top and middle) and 200 mm (bottom). D, representative macroscopic photographs of invasive tumors developed in a DSS-treated Tgfbr2 mouse at 40 weeks after treatment (arrows, top left), and the H&E (top right) and immunohistochemistry for Ki67 (bottom). Arrows (bottom), the location of muscularis mucosae; and inset, enlarged image of the boxed area. Ce, cecum; Ile, ileum; Co, colon. Bars, 2 mm (top) and 400 mm (bottom). E, immunoblotting for active D716 DIEC and AOM/DSS-treated mice (left) and DSS-treated Tgfbr2 mice (right) b-catenin and total b-catenin in the intestinal mucosa and polyps in Apc at the indicated time points. NM, normal mucosa; and inv, invasive tumors. b-Actin was used as an internal control. F, representative immunohistochemical findings of p-Smad2 in ulcerative colitis–associated human colon cancer tissues (left, center), and the b-catenin in sporadic colon cancer (top right) and ulcerative colitis– associated colon cancer (UC; right bottom). Arrows (center), surface epithelial cells and invading tumor cells. Bars, 500 mm (left) and 50 mm (center and right). Membrane-type 1 matrix metalloproteinase (MT1-MP) expression was induced in the stroma of the large invasive tumors, and collagen fiber deposition was also found in the stroma by Masson trichrome staining. Moreover, expression of IL1b, IL6, and COX-2 was induced when polyp size increased beyond >1 mm in diameter in ApcD716 mouse small intestine, and expres- www.aacrjournals.org sion levels of MT1-MMP, Adam10, and epiregulin were also increased in the large polyps (Fig. 1D). The induction of these factors was also found in the colon polyps >2 mm in diameter. By a laser microdissection–based RT-PCR analysis, we found that these factors, except for Adam10, were predominantly expressed in the tumor stroma (Supplementary Fig. S2). Taken Cancer Res; 75(4) February 15, 2015 Downloaded from cancerres.aacrjournals.org on April 12, 2017. © 2015 American Association for Cancer Research. 769 Oshima et al. A B DSS-Tgfbr2 [GFP Tg-BMT] inv non-inv GFP inv GFP * 100 Relative expression level DIEC Invasive tumor Normal mucosa * 10,000 1,000 10 100 100 0.1 CCL2 non-inv F4/80/E-cad inv 10 1 1 1 1 0.1 * 10 10 10 1 100 1,000 * * CCL3 0.1 CCL4 0.1 CCL7 0.1 CCL8 Tgfbr2DIEC + DSS WT WT + DSS Normal 3 days(uc) 4 wks(rep) 3 days(uc) 8 wks(inv) C p-Stat3 Relative band intensities of p-Stat3/Stat3 Stat3 D Tgfbr2DIEC DSS 3 days DSS 6-10 weeks (inv) Wild-type DSS 3 days DSS 4 weeks Relative expression level 100 10 1,000 *ns 100 1,000 ns * * ns β-Actin 3 2 1 WT ns 1,000 100 100 10 10 1 1 0.1 0.1 * 0.1 * * 100 * 100 10 10 1 TNFα ns 10,000 1,000 10 1 0.1 Tgfbr2DIEC + DSS WT + DSS IL1β 0.01 IL6 0.01 1 1 0.1 CXCL1 0.01 CXCL2 0.1 COX-2 Figure 3. DIEC DIEC mice. A, representative photographs of immunohistochemistry for GFP in a DSS-treated Tgfbr2 Chronic inflammation in invasive tumors of Tgfbr2 mouse that had undergone BM transplantation from a GFP transgenic mouse (top). Enlarged images of invasive (bottom left) and noninvasive (bottom center) mucosa of the boxed areas (top). Fluorescent immunostaining for F4/80 (green) and E-cadherin (red) in an invasive tumor (bottom right). DIEC Bars, 400 mm (top) and 200 mm (bottom). B, relative expression levels of the indicated chemokines in the invasive tumors in DSS-treated Tgfbr2 mice to the mean level of wild-type mouse colons (mean  SD).  , P < 0.05. C, immunoblotting for phosphorylated Stat3 and total Stat3 in the colon mucosa of control DIEC wild-type mice (WT), DSS-treated wild-type and Tgfbr2 mice. b-Actin was used as an internal control. uc, ulcerative colitis; rep, repaired mucosa; inv, invasive tumor. The band intensities for pStat3/Stat3 relative to the mean level of wild-type mice (red bar) are shown in a bar graph (bottom). DIEC D, expression levels of cytokines, chemokines, and COX-2 in DSS-treated wild-type or Tgfbr2 mouse colonic mucosa relative to the mean levels of the repaired mucosa (green; mean  SD).  , P < 0.05. ns, not significant. together, these results indicate that the inflammatory microenvironment is generated by a tumor size-dependent mechanism in both the small intestinal and colon polyps. It is therefore possible that such microenvironment is required for submucosal invasion. Submucosal invasion by TGFb signaling suppression and ulcerative colitis We thus examined the role of inflammatory responses in TGFb signaling suppression-associated submucosal invasion using a colitis mouse model by treating Tgfbr2DIEC mice with DSS (Fig. 2A). The wild-type mice treated with DSS showed ulcerative colitis beginning 3 days after DSS treatment, and the colonic mucosa was repaired in 4 weeks after treatment (Fig. 2B). Although mucosal ulcers were repaired in the Tgfbr2DIEC mice in 4 weeks after DSS treatment, regeneration of the normal gland structure was significantly impaired (Fig. 2B, asterisk). Importantly, colonic epithelial cells invaded to the submucosa in Tgfbr2DIEC mice by the end of fourth week after treatment. These invading epithelial cells continued prolifer- 770 Cancer Res; 75(4) February 15, 2015 ation, and formed invasive tumors associated with deposition of collagen fibers by the 10 weeks after DSS treatment (Fig. 2C). Furthermore, large solid tumors were visible from outside of the colon and cecum of Tgfbr2DIEC mice by the 40th week after treatment (Fig. 2D). Epithelial cells of tumors were positive for Ki67, indicating that the tumor cells continued proliferation even at 40 weeks after DSS treatment. We confirmed that disruption of Tgfbr2 in normal intestinal mucosa did not cause any morphologic changes (Supplementary Fig. S3), which is consistent with previous reports (20, 22). The level of active b-catenin was significantly increased both in the ApcD716 mouse intestinal tumors and colitis-associated colon tumors that were chemically induced by treatment with azoxymethane and DSS, however, active b-catenin level was not increased in the invasive tumors of DSS-treated Tgfbr2DIEC mice (Fig. 2E). These results indicate that ulcerative colitis induces invasive tumors in the TGFb-suppressed mucosa without activation of the Wnt signaling. We next examined human inflammatory bowel disease (IBD)– related colon cancer. Nuclear localized P-Smad2 was found in the Cancer Research Downloaded from cancerres.aacrjournals.org on April 12, 2017. © 2015 American Association for Cancer Research. Invasion by TGFb Blocking in Regenerating Inflamed Mucosa Wild-type–repaired mucosa Tgfbr2DIEC-invasive tumors 5 2 4 1.5 3 1 2 0.5 1 0 0 MMP2 MMP9 E ten t La MMP ten tM 9 MP 2 MT1-MMP * La DSS-treated Tgfbr2DIEC N1 N2 N3 T1 T2 T3 AOM/DSS 2.5 C * 6 F4/80/MT1-MMP Wild-type (DSS-rep) 7 E-cadherin/MT1-MMP Wild-type (DSS-uc) * 3 9 8 7 6 5 4 3 2 1 0 ApcD716 Tgfbr2DIEC 8 3.5 B ApcD716 Relative expression level A N1 N2 A1 A2 C1 C2 I1 I2 R1 R2 T1 T2 Latent MMP9 Latent MMP9 Latent MMP2 Active MMP2 Latent MMP2 Active MMP2 40 kDa Collagen type IV Relative band intensities of active MMP2 40 kDa D 3 2.5 2 1.5 1 0.5 0 N1 N2 A1 A2 C1 C2 I1 I2 R1 R2 T1 T2 Figure 4. DIEC mouse invasive Expression of MT1-MMP and activation of MMP2 in invasive tumors. A, expression levels of indicated MMPs in the DSS-treated Tgfbr2  tumors (red) relative to the level of repaired mucosa of DSS-treated wild-type mice (green; mean  SD). , P < 0.05 versus wild-type level. B, fluorescent DIEC immunostaining of the invasive tumors in DSS-treated Tgfbr2 mice for E-cadherin (green), MT1-MMP (red; left), F4/80 (green), and MT1-MMP (red; right). Insets, enlarged images of the boxed areas. Bars, 200 mm. C, gelatin zymography of the normal mucosa (N1-N3) and invasive tumors (T1-T3) of three DIEC mice. Latent MMP9 and MMP2 were used as positive controls. D, immunostaining for collagen type IV in the invasive individual DSS-treated Tgfbr2 DIEC mice. White arrowheads, invading epithelial cells in submucosa; closed arrowheads, noninvading epithelial colon tumors of DSS-treated Tgfbr2 D716 cells. Bars, 50 mm. E, gelatin zymography of the normal intestinal mucosa (N1-N2) and intestinal adenomas (A1-A2) of Apc mice, invasive D716 DIEC Tgfbr2 mice, ulcerative colitis tissues of DSS-treated wild-type mice (I1-I2), repaired mucosa of DSS-treated adenocarcinomas (C1-C2) of Apc wild-type mice (R1-R2), and noninvasive colon tumors (T1-T2) of azoxymethane/DSS-treated wild-type mice. Coomassie Brilliant Blue staining of the 40-kDa bands is shown at the bottom of the zymography gels as a protein level control (C and E). Relative band intensities for active MMP2 to the D716 adenomas (red bar) are shown in a bar graph (E, bottom). mean level of Apc surface epithelial cells of ulcerative colitis, however, it was not detected in the invaded tumor cells, indicating that there was suppression of TGFb signaling (Fig. 2F). A loss of P-Smad2 staining in tumor cells was found in five out of eight cases (62.5%) of ulcerative colitis–associated colon cancer. Moreover, b-catenin accumulation was not detected in seven out of eight ulcerative colitis–associated tumors (87.5%). Accordingly, it is possible that suppression of TGFb signaling causes colon cancer invasion in human patients with IBD without Wnt signaling activation. Chronic inflammatory responses in the invasive tumors We found that BM-derived cells and macrophages infiltrated in the stroma of invasive colon tumors, but not in the noninvasive www.aacrjournals.org mucosa of DSS-treated Tgfbr2DIEC mice (Fig. 3A). Consistently, expression of monocyte-trophic chemokines (23), CCL2, CCL3, CCL4, CCL7, and CCL8, was increased significantly in the invasive colon tumors (Fig. 3B). Moreover, Stat3 was constitutively phosphorylated in the invasive tumors of DSS-treated Tgfbr2DIEC mice, whereas its level was decreased in the repaired mucosa of DSS-treated wild-type mice (Fig. 3C). These results indicate that inflammatory responses are chronically maintained in the invasive tumor tissues, even after the repair of DSS-induced ulcers. We thus determined the levels of inflammatory cytokines in both the invasive tumors and DSS-induced ulcerative colitis. Expression was significantly upregulated for TNFa, IL1b, IL6, CXCL1, CXCL2, and COX-2 by DSS-induced ulcerative colitis in both the wild-type and Tgfbr2DIEC mice (Fig. 3D, yellow and blue Cancer Res; 75(4) February 15, 2015 Downloaded from cancerres.aacrjournals.org on April 12, 2017. © 2015 American Association for Cancer Research. 771 Oshima et al. A CD44 E-cadherin / Ki67 SOX7 Tgfbr2DIEC Wild-type H&E C (%) 100 Relative SOX9 expression level Survival after irradiation B Wild-type 80 60 40 Tgfbr2DIEC 20 0 1 2 3 4 5 6 7 8 (days after irradiation) 12 ApcD716 polyp Tgfbr2DIEC- irradiated † * ns * 10 8 * 6 * 4 2 0 D Wild-type Tgfbr2DIEC 14 Day 0 Day 2 Day 4 E Apc Δ716 WT- irradiated Tgfbr2 ΔIEC- irradiated day 0 day 2 day 4 day 0 day 2 day 4 β-Catenin Normal polyp Active β-catenin Total β-catenin Figure 5. Impaired mucosal regeneration by suppression of TGFb signaling. A, representative photographs of X-ray– irradiated wild-type (top) and DIEC (bottom) mouse small Tgfbr2 intestines 6 days after irradiation. H&E staining, fluorescence immunostaining for E-cadherin (red), and Ki67 (green), immunohistochemistry for CD44 and SOX7 (left to right) are shown. White arrowheads, Ki67-positive cells. Arrows, CD44-positive epithelial cells; closed arrowheads, SOX7-positive epithelial cells. Bars, 200 mm (left) and 100 mm (center and right). B, survival DIEC curve of wild-type and Tgfbr2 mice after X-ray irradiation at 9 Gy. C, expression levels of SOX9 in X-ray– irradiated wild-type (gray bars) and DIEC Tgfbr2 (closed bars) mouse intestines relative to the mean level of nonirradiated wild-type mice (day 0; mean  SD).  , P < 0.05 versus day 0 level; dagger, P < 0.05. ns, not significant. D, immunohistochemical D716 staining for b-catenin in an Apc mouse intestinal polyp (left) and DIEC irradiated Tgfbr2 mouse small intestinal mucosa (right). White arrowheads, b-catenin nuclear accumulation. Bars, 25 mm. E, immunoblotting for active b-catenin and total b-catenin in the indicated intestinal tissues. b-Actin was used as an internal control. β-Actin circles, respectively), whereas the levels of these factors in wildtype mice decreased significantly after the mucosa was repaired (Fig. 3D, green circles). In the invasive tumors of the Tgfbr2DIEC mice, expression of TNFa, IL6, and CXCL2 remained high, whereas that of IL1b, CXCL1, and COX-2 decreased to the level of repaired mucosa (Fig. 3D, red circles). These results indicate that different types of inflammatory responses are induced in the invasive tumor tissues of the Tgfbr2DIEC mice compared with those in the mice with DSS-induced acute colitis. Expression of MT1-MMP and activation of MMP2 in invasive tumors To investigate the mechanism underlying chronic inflammation-associated invasion, we examined expression of MMPs that are important for submucosal invasion (24). It has been shown that MT1-MMP plays a key role in activation of MMP2 (25) and MT1-MMP is expressed in macrophages, regulating inflammatory responses (26, 27). The expression levels of MT1MMP, MMP2, and MMP9 were significantly increased in the invasive tumors of DSS-treated Tgfbr2DIEC mice (Fig. 4A). Fluorescence immunohistochemistry showed that the stromal cells of invasive tumors expressed MT1-MMP, and most MT1-MMP– 772 Cancer Res; 75(4) February 15, 2015 expressing cells in tumor stroma were F4/80-positive macrophages (Fig. 4B). Moreover, gelatin zymography analyses revealed that MMP2 was activated in the invasive tumor tissues, but not in the normal colonic mucosa of the same DSS-treated Tgfbr2DIEC mice (Fig. 4C). Consistently, the immunostaining signal for collagen type IV in the basement membrane was significantly decreased in the invading epithelial cells (Fig. 4D). These results suggest that chronic inflammation contributes to the submucosal invasion through macrophage-expressed MT1MMP, which leads to MMP2 activation, resulting in degradation of basement membrane. Because MT1-MMP expression was also induced in the ApcD716 benign adenomas (Fig. 1C and D), we further examined MMP activation in other mouse tumor models. As anticipated, MMP2 was activated in the invasive tumors of ApcD716 Tgfbr2DIEC mice (Fig. 4E). Notably, MMP2 activation was also found in the noninvasive benign tumors of ApcD716 mice and AOM/DSS-treated mice, although the band intensities were lower compared with those of the ApcD716 Tgfbr2DIEC mouse invasive tumors (Fig. 4E). These results suggest that MMP2 activation is already induced in benign intestinal tumors, and its activation level increases with the progression of the tumor. Accordingly, it is also possible that Cancer Research Downloaded from cancerres.aacrjournals.org on April 12, 2017. © 2015 American Association for Cancer Research. Invasion by TGFb Blocking in Regenerating Inflamed Mucosa A diation in Tgfbr2DIEC mice also showed a decrease in the proliferating crypt cell population, followed by increased numbers of proliferating undifferentiated cells (Supplementary Fig. S4). However, the extent of the injury was more severe in the Tgfbr2DIEC mice, and the regeneration of the normal mucosal structure was impaired in both the small intestine and colon (Fig. 5A and Supplementary Fig. S5). Because of such severe phenotypes, most irradiated Tgfbr2DIEC mice died by day 7 (Fig. 5B). Moreover, markers of undifferentiated epithelial cells, CD44 and SOX7, were expressed in the entire intestinal mucosa of the X-ray–irradiated Tgfbr2DIEC mice, whereas their expression was limited to the crypt bottom in the wild-type mice (Fig. 5A and Supplementary Fig. S5B). Consistently, an intestinal progenitor cell marker, SOX9 (29), was upregulated in the intestine by irradiation, and the SOX9 level was significantly higher in Tgfbr2DIEC mice than in wild-type mice (Fig. 5C). Moreover, the nuclear accumulation and stabilization of b-catenin was not found in the irradiated Tgfbr2DIEC mouse intestinal epithelia (Fig. 5D and E). Taken together, these results indicate that TGFb signaling is required for differentiation of regenerating epithelial cells, and that blocking the TGFb pathway causes expansion of the proliferating and undifferentiated epithelial cell population without activation of Wnt signaling. EdU/CD44/DAPI Tgfbr2DIEC Wild-type Bright field B 450 Crypt length (μm) 400 350 300 250 200 150 100 2 ΔIE C C fbr Tg 2 ΔIE fbr Tg fbr 2 ΔIE C /flo x Tg fbr 2 flox Tg fbr 2 flox /flo x /flo x 2 flox Cont Tg fbr Tg fbr Tg W ild -ty 2 ΔIE C pe 50 X ray–irradiated (9Gy) Figure 6. Acquisition of invasive phenotype by TGFb inhibition in regenerating epithelia. A, representative bright-field photographs of organoid cultures in Matrigel (left) and confocal images of organoids immunostained for EdU (red) and CD44 (green; right) of wild-type (top) and X-ray–irradiated DIEC Tgfbr2 (bottom) mouse intestinal epithelia. Arrowheads, EdU-negative and CD44 weak cells in wild-type organoid. Bars, 200 mm (left) and 50 mm (right). B, crypt lengths of organoids derived from wild-type and DIEC flox/flox nonirradiated Tgfbr2 mice (light blue), irradiated Tgfbr2 (control) DIEC mice (red). Red bar, 200-mm threshold, mice (blue), and irradiated Tgfbr2 and crypts longer than 200 mm were judged to be long crypts. suppression of TGFb signaling causes acquisition of invasiveness of epithelial cells in such microenvironment. Impaired mucosal regeneration by suppression of TGFb signaling Impaired mucosal regeneration from ulcer in Tgfbr2DIEC mice (Fig. 2B and C) suggested a role of TGFb signaling in regeneration of injured intestinal mucosa. To test this possibility, mice were irradiated with X-ray at 9 Gy. In the irradiated wild-type mice, the number of proliferating cells in the crypt decreased on days 1 to 3 after irradiation, followed by destruction of the mucosal structure by day 4 (Supplementary Fig. S4). At the same time, the undifferentiated cell population expanded, and normal crypt-villous structures were regenerated by day 6, which was consistent with a previous report (28). X-ray irra- www.aacrjournals.org Acquisition of invasive phenotype by blocking TGFb signaling in regenerating epithelial cells We next studied the effect of TGFb signaling suppression in regenerating mucosa on invasive phenotype. Organoid culture of the irradiated wild-type mouse-derived intestinal epithelial cells showed budding from cysts, forming mini-crypt structures (Fig. 6A), which was consistent with the original report (18). Importantly, intestinal epithelial cells derived from irradiated Tgfbr2DIEC mice formed gland-like long crypt structures in the Matrigel (Fig. 6A). We further examined the epithelial cell proliferation in the organoids by evaluating the EdU incorporation to nuclei, and examined the undifferentiated status by determining the CD44 expression. Notably, the expression of EdU and CD44 was found in the epithelial cells along the long crypts of Tgfbr2DIEC mouse-derived organoids, although that was detected only in budding crypts of wild-type organoids. These results suggest that the long crypts are comprised of proliferating undifferentiated epithelial cells. The proportion of long crypts >200 mm was significantly higher in the irradiated Tgfbr2DIEC mouse-derived intestinal epithelial cells (42%  13% of all crypts) compared with epithelial cells derived from irradiated Tgfbr2flox/flox control mice (3.9  4.5%; Fig. 6B). Such long crypt formation was not found also in organoids of nonirradiated Tgfbr2DIEC mouse-derived intestinal epithelial cells. It is possible that long crypt formation in Matrigel reflects "collective cell migration" in the extracellular matrix, which is one of strategies used by cancer cells for invasion (30). Therefore, it is conceivable that suppression of TGFb signaling in regenerating mucosa results in the acquisition of invasive phenotype, which leads to collective migration in the inflammatory microenvironment. Discussion Genome-wide analyses have indicated that accumulation of genetic alterations in oncogenic and tumor-suppressor pathways Cancer Res; 75(4) February 15, 2015 Downloaded from cancerres.aacrjournals.org on April 12, 2017. © 2015 American Association for Cancer Research. 773 Oshima et al. Figure 7. A schematic drawing of the TGFb signaling suppression-induced invasive tumor development in regenerating and inflamed mucosa (A) and in Wnt signaling–activated adenomas (B). is responsible for development of colon cancer (31). On the other hand, the nature and significance of the individual genetic alterations are not yet understood (32). In addition, relatively few mutations have been identified that are responsible for invasion and/or metastasis (8, 33), suggesting that microenvironment can promote malignant progression. We have herein demonstrated that simple genetic alterations in the TGFb pathway can lead to the development of invasive gastrointestinal cancers without additional genetic alterations when the mucosa is inflamed and regenerating from injury (Fig. 7A). It has been shown that suppression of TGFb signaling in the intestinal and mammary gland tumor cells induces chemokine expression, which recruits myeloid cells to the tumor microenvironment (12, 14). These myeloid cells express metalloproteinases, such as MT1-MMP, MMP2, and MMP9 that contribute to the invasion or metastasis of tumor cells. These results suggest that elaboration of an inflammatory microenvironment is critical for the malignant progression mediated by inhibition of the TGFb signaling. We also found that MMP2 is activated by macrophageexpressing MT1-MMP in the invasive tumors. However, we found that MMP2 is activated also in the benign intestinal tumor tissues with intact TGFb signaling. Accordingly, it is conceivable that the acquisition of an invasive phenotype by epithelial cells is further required for malignant progression where TGFb signaling is suppressed. Blocking TGFb signaling in the intestinal epithelial cells did not cause morphologic changes, indicating that TGFb signaling is not required for differentiation of normal intestinal stem/ progenitor cells (20, 22). However, we found that suppression of TGFb signaling in the injured intestinal mucosa blocked mucosal regeneration by suppressing differentiation, which caused the expansion of undifferentiated cell population. Accordingly, TGFb signaling is essential for regeneration from damaged mucosa in the gastrointestinal tract. Notably, intestinal epithelial cells derived from irradiated Tgfbr2DIEC mice 774 Cancer Res; 75(4) February 15, 2015 showed increased invasion in Matrigel, possibly caused by expansion of undifferentiated epithelial cell population. However, irradiation of Tgfbr2DIEC mice caused only dysplastic changes without tumor development, indicating that blocking TGFb signaling in regenerating epithelial cells alone is insufficient for induction of invasive tumors. Accordingly, it is required for the development of invasive tumors that both the inflammatory microenvironment where MT1-MMP is expressed and the regenerating epithelial cells with increased invasiveness by inhibition of TGFb signaling (Fig. 7A). Such a mechanism is possibly important for cancer development associated with IBD. In IBD lesions, the mucosa is continuously regenerating in a chronic inflammatory microenvironment, and the expression of MT1-MMP, together with inflammatory chemokines, is upregulated similar to that observed in Tgfbr2DIEC mouse tumors (Supplementary Fig. S6). Furthermore, we herein demonstrated that TGFb signaling is suppressed and Wnt signaling is not activated in more than 60% of ulcerative colitis–related colon cancer cells. Consistently, it has also been reported that human colitis–associated colon cancer does not follow the adenoma–carcinoma sequence, and mutations in b-catenin or APC are not common either (34). Accordingly, simple genetic alterations in the TGFb signaling pathway may cause the development of invasive tumors under IBD condition (Fig. 7A). On the other hand, compound mutant mice carrying mutations in Apc and TGFb pathway genes showed progression of invasive adenocarcinomas from Wnt-activated adenomas (19–21), indicating that the combination of Wnt activation and TGFb signaling suppression is sufficient for malignant progression. In the regenerating mucosa, stem cell population is expanded, with the signaling in the Wnt and Notch pathways activated (35). It is therefore possible that the activation of Wnt signaling is necessary for malignant progression in sporadic tumors where TGFb signaling is blocked without mucosa regeneration. Accordingly, combination of Wnt activation and TGFb suppression in the Cancer Research Downloaded from cancerres.aacrjournals.org on April 12, 2017. © 2015 American Association for Cancer Research. Invasion by TGFb Blocking in Regenerating Inflamed Mucosa MT1-MMP–expressing inflammatory microenvironment is sufficient for the induction of invasive adenocarcinoma in normal intestine (Fig. 7B). In conclusion, we have demonstrated that suppression of TGFb signaling in the regenerating epithelial cells results in suppression of epithelial differentiation and acquisition of invasive phenotype of epithelial cells. Chronic inflammation induces development of an MMP2-activating microenvironment. The cooperation between TGFb signaling suppression in the regenerating epithelia and the inflammatory microenvironment can cause invasive colon cancer development, which may explain mechanism of IBD-associated colon tumorigenesis. Therefore, controlling the inflammatory microenvironment may help an effective preventive or therapeutic strategy against the malignant progression of colon cancer. Disclosure of Potential Conflicts of Interest No potential conflicts of interest were disclosed. Authors' Contributions Conception and design: H. Oshima, M. Nakayama, H. Sato, M. Oshima Development of methodology: H. Oshima, M. Nakayama, H. Sato, M. Oshima Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): H. Oshima, M. Nakayama, T.-S. Han, K. Naoi, X. Ju, Y. Maeda, S. Robine, K. Tsuchiya, H. Sato, M. Oshima Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): H. Oshima, M. Nakayama, H. Sato, M.M. Taketo, M. Oshima Writing, review, and/or revision of the manuscript: H. Sato, M.M. Taketo, M. Oshima Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): H. Oshima, M. Nakayama, T. Sato, H. Sato, M. Oshima Study supervision: H. Sato, M. Oshima Acknowledgments The authors thank Manami Watanabe and Ayako Tsuda for technical assistance. Grant Support This work was supported by CREST, the Japan Science and Technology Agency, Japan (M. Oshima), Grants-in-Aid for Scientific Research on Innovative Areas from the Ministry of Education, Culture, Sports, Science and Technology of Japan #22114005 (M. Oshima), #24300325 (M. Oshima), and Takeda Science Foundation, Japan (H. Oshima and M. Oshima). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Received July 9, 2014; revised November 25, 2014; accepted December 1, 2014; published online February 16, 2015. References 1. Coussens LM, Werb Z. Inflammation and cancer. Nature 2002;420:860–7. 2. Elinav E, Nowarski R, Thaiss CA, Hu B, Jin C, Flavell RA. Inflammationinduced cancer: crosstalk between tumours, immune cells and microorganisms. Nat Rev Cancer 2013;13:759–71. 3. Oshima M, Dinchuk JE, Kargman SL, Oshima H, Hancock B, Kwong E, et al. 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Updated version Supplementary Material Cited articles Citing articles E-mail alerts Reprints and Subscriptions Permissions Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/75/4/766 Access the most recent supplemental material at: http://cancerres.aacrjournals.org/content/suppl/2015/03/18/75.4.766.DC1 http://cancerres.aacrjournals.org/content/suppl/2015/03/20/75.4.766.DC2 This article cites 34 articles, 7 of which you can access for free at: http://cancerres.aacrjournals.org/content/75/4/766.full.html#ref-list-1 This article has been cited by 2 HighWire-hosted articles. Access the articles at: /content/75/4/766.full.html#related-urls Sign up to receive free email-alerts related to this article or journal. To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at pubs@aacr.org. 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