Tpl2 regulates intestinal myofibroblast HGF release to suppress colitis-associated tumorigenesis

Abstract

The tumor microenvironment plays a significant role in colitis-associated cancer (CAC). Intestinal myofibroblasts (IMFs) are cells in the intestinal lamina propria secreting factors that are known to modulate carcinogenesis; however, the physiological role of IMFs and signaling pathways influencing CAC have remained unknown. Tumor progression locus 2 (Tpl2) is a MAPK that regulates inflammatory and oncogenic pathways. In this study we addressed the role of Tpl2 in CAC using complete and tissue-specific ablation of Tpl2 in mutant mice. Tpl2-deficient mice did not exhibit significant differences in inflammatory burdens following azoxymethane (AOM)/dextran sodium sulfate (DSS) administration compared with wild-type mice; however, the mutant mice developed significantly increased numbers and sizes of tumors, associated with enhanced epithelial proliferation and decreased apoptosis. Cell-specific ablation of Tpl2 in IMFs, but not in intestinal epithelial or myeloid cells, conferred a similar susceptibility to adenocarcinoma formation. Tpl2-deficient IMFs upregulated HGF production and became less sensitive to the negative regulation of HGF by TGF-β3. In vivo inhibition of HGF-mediated c-Met activation blocked early, enhanced colon dysplasia in Tpl2-deficient mice, indicating that Tpl2 normally suppresses the HGF/c-Met pathway. These findings establish a mesenchyme-specific role for Tpl2 in the regulation of HGF production and suppression of epithelial tumorigenesis.

Figure 1. Tpl2^D/D mice display increased tumorigenesis upon AOM/DSS administration.

Western blot of Tpl2 expression on days 0 and 15 of the AOM/DSS regime (A) and densitometric analysis (B) of both isoforms of Tpl2 protein (52 kDa and 58 kDa) relative to β-actin. Data represent mean ± SEM. n = 5; **P < 0.01. Tpl2^D/D mice and wild-type littermate controls were subjected to the AOM/DSS model of colitis-associated colon carcinogenesis. Body weight changes (C) and survival rates (D) were monitored during the course of the experimental procedure. (E) Disease index was measured on day 13 after initial AOM injection. Colon length (F), number of tumors per mouse (G), and size of tumors (H) measured at the end of the experimental protocol. The data correspond to one representative experiment of three (n = 6), except in the case of survival curves and size distribution graphs, which are cumulative from 3 experiments. Data represent mean ± SEM. *P < 0.05, **P < 0.01. (I) Representative images of formalin-fixed and H&E-stained colon sections at the end of the experimental protocol (day 60). Scale bars: 200 μm. (J) Representative images of formalin-fixed and H&E-stained colon sections early (day 15) during the experimental protocol. Scale bars: 50 μm. (K) Dysplasia incidence was measured at an early time point, 5 days after the end of the first DSS cycle. The data shown correspond to the average of 4 experiments performed (n = 6). Data represent mean ± SEM. **P < 0.01.

Figure 2. Tpl2^D/D mice display enhanced proliferation and decreased apoptosis, associated with tumorigenic protein and gene expression signatures early during the disease.

(A) Formalin-fixed, paraffin-embedded colon sections from wild-type and Tpl2^D/D on day 15 after AOM/DSS initiation were stained with BrdU and TUNEL assay kits for assessment of proliferation and apoptosis, respectively. Representative images are presented. Scale bars: 50 μm. Quantification of BrdU-positive cells per crypt (B) and TUNEL-positive cells per field (C) was performed using at least 20 random crypts and 10 random fields, respectively. Data represent mean ± SEM. n = 6; ***P < 0.001. (D) Western blot analysis of whole colon lysates from WT and Tpl2^D/D mice on day 8 after AOM/DSS administration. Data represent one of 3 experiments performed. (E) qRT-PCR of genes of interest in whole colon of WT and Tpl2^D/D mice on day 8 after AOM/DSS administration relative to untreated controls. Gene expression was normalized to B2m levels. Data represent mean ± SEM of 6 mice per genotype (2 mice from each of 3 individual experiments). *P < 0.05, **P < 0.01, ***P < 0.001. (F) Representative images from immunostaining of colon tissue samples (n = 6) on days 15 and 60 of the experimental protocol with antibodies against F4/80-, Gr-1–, and CD4-positive cells. Scale bars: 20 μm.

Figure 3. Tpl2^IMFko mice, but not Tpl2^myelko or Tpl2^IECko mice, exhibit increased susceptibility to CAC.

Tpl2^fl/fl mice were crossed with LysM-Cre, villin-Cre and ColVI-Cre mice in order to accomplish cell-specific deletion of Tpl2 in myeloid cells (Tpl2^myelko), IECs (Tpl2^IECko), and intestinal myofibroblasts (Tpl2^IMFko), respectively. (A) Western blot analysis in protein lysate from thioglycolate elicited peritoneal macrophages (TEPMs), IECs, and IMFs was used in each case to verify efficient deletion of Tpl2 protein. (B) These mice were subjected to the AOM/DSS protocol of CAC. Tumor multiplicity was measured in Tpl2^myelko, Tpl2^IECko, and Tpl2^IMFko mice in comparison to Tpl2^fl/fl littermate controls on day 60 after AOM/DSS administration. Data represent mean ± SEM from one of 2 (for Tpl2^myelko mice) or 3 (for Tpl2^IECko and Tpl2^IMFko) experiments performed. n = 5; **P < 0.01. (C) Size distribution of colonic tumors in Tpl2^myelko, Tpl2^IECko, and Tpl2^IMFko mice and their respective littermate controls at the end of the experimental protocol. The data shown are cumulative of 2 (for Tpl2^myelko mice) or 3 (for Tpl2^IECko and Tpl2^IMFko) experiments performed. (D) Representative images from H&E staining of colon tissues from Tpl2^myelko, Tpl2^IECko, and Tpl2^IMFko mice and their respective littermate controls on day 60 of the regime. Scale bars: 200 μm.

Figure 4. Tpl2^IMFko mice display a pro-tumorigenic phenotype similar to Tpl2^D/D mice.

(A) Representative images from H&E, BrdU and TUNEL staining of colon tissue slides from Tpl2^IMFko and Tpl2^fl/fl mice on day 15 after AOM injection. Scale bars: 50 μm. (B) Dysplasia incidence was measured 15 days after the end of the AOM injection. The data correspond to the average of 4 experiments (n = 5). Data represent mean ± SEM. *P < 0.05. (C and D) Proliferation and apoptosis as determined by measuring BrdU-positive cells per crypt (C) and TUNEL-positive cells per field (D) in colon tissue from Tpl2^IMFko and Tpl2^fl/fl mice on day 15 after AOM injection. At least 20 random crypts and 10 random fields were used, respectively. Data represent mean ± SEM. n = 6; *P < 0.05. (E) qRT-PCR of colon tissue on day 15 of the experimental procedure relative to samples from untreated controls. Gene expression was normalized to B2m levels. Data represent mean ± SEM of 6 mice per genotype (2 mice from each of 3 individual experiments). *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 5. Tpl2 ablation results in increased HGF expression in IMFs and subsequent activation of the c-Met signaling pathway in IECs.

(A) Hgf gene expression levels in colons from wild-type and Tpl2^D/D mice on days 0 and 8 of the AOM/DSS experimental protocol. Gene expression was normalized to B2m levels. Data represent mean ± SEM of 6 mice per genotype (2 mice from each of 3 individual experiments). **P < 0.01. (B) Western blot analysis of colon lysates from the same mice for p-Met and p-Akt. β-Actin was used as a loading control. This experiment represents one of 3. (C) HGF gene expression levels in colons from Tpl2^fl/fl and Tpl2^IMFko mice on days 0 and 15 after initial injection with AOM. Gene expression was normalized to B2m levels. Data represent mean ± SEM of 6 mice per genotype (2 mice from each of 3 individual experiments). *P < 0.05, **P < 0.01. (D) Western blot analysis of colon lysates from the same mice for p-Met and p-Akt. β-Actin was used as a loading control. This experiment represents one of 3 performed. (E) Western blot analysis from epithelial cells (IECs) and stroma, isolated from wild-type and Tpl2^D/D mice, on days 0 and 8 of the AOM/DSS experimental protocol. β-Actin was used as a loading control. This experiment represents one of 3 performed. (F) Hgf gene expression levels in epithelial cells (IECs) and stroma, isolated from wild-type and Tpl2^D/D mice on day 8 of the AOM/DSS experimental protocol. Gene expression was normalized to B2m levels. Data represent mean ± SEM of 6 mice per genotype (3 mice from each of 2 individual experiments).**P < 0.01.

Figure 6. Tpl2 in IMFs modulates spontaneous and TGF-β3–regulated HGF production.

(A) HGF ELISA in supernatants from wild-type and Tpl2^D/D primary IMF culture before and after addition of IL-1β (10 ng/ml), TNF (10 ng/ml), and TGF-β3 (10 ng/ml) for 24 hours. Data represent mean ± SEM of one of 3 experiments performed in triplicate. *P < 0.05, **P < 0.01. (B) IMFs were treated with IL-1β (10 ng/ml), TNF (10 ng/ml), and TGF-β3 (10 ng/ml) for 0, 15, and 30 minutes. Cell lysates were subjected to Western blot for p-ERK, p-JNK, and p-p38. One representative experiment of 3 is shown.

Figure 7. In vivo inhibition of HGF-driven c-Met activation blocks enhanced dysplasia in Tpl2^D/D mice.

(A) Diagram of PHA-665752 administration during the first 15 days of the AOM/DSS model. Tpl2^D/D and wild-type littermate controls (5 mice per group) received 4 daily i.v. injections of PHA-665752 at a concentration of 25 mg/kg during the first DSS cycle of the CAC protocol. (B) Body weight changes were measured throughout the regime, and (C) colon length was measured at the end of the protocol. Data represent mean ± SEM from one of 2 experiments performed. *P < 0.05, **P < 0.01, Tpl2^D/D mice that were injected with the inhibitor versus those that received control DMSO. n = 5. (D) Colon tissue slides were stained with H&E for the assessment of dysplasia index. Staining against BrdU and TUNEL-positive cells was used to determine the effect of the inhibitor in proliferation and apoptosis, respectively. Representative images from one of 2 experiments are shown. Scale bars: 50 μm. Arrows indicate TUNEL-positive cells (E) H&E-stained sections were scored for dysplasia. Data represent mean ± SEM from one of 2 experiments performed. n = 5. Quantification of BrdU-positive cells per crypt (F) and TUNEL-positive cells per field (G) in colon tissue from wild-type and Tpl2^D/D mice with and without treatment with the c-Met inhibitor on day 15 after AOM injection. At least 20 random crypts and 10 random fields were used, respectively. Data represent mean ± SEM. n = 6; *P < 0.05, **P < 0.01.