Sonic hedgehog inhibitors prevent colitis-associated cancer via orchestrated mechanisms of IL-6/gp130 inhibition, 15-PGDH induction, Bcl-2 abrogation, and tumorsphere inhibition

Abstract

Sonic hedgehog (SHH) signaling is essential in normal development of the gastrointestinal (GI) tract, whereas aberrantly activated SHH is implicated in GI cancers because it facilitates carcinogenesis by redirecting stem cells. Since colitis-associated cancer (CAC) is associated with inflammatory bowel diseases, in which SHH and IL-6 signaling, inflammation propagation, and cancer stem cell (CSC) activation have been implicated, we hypothesized that SHH inhibitors may prevent CAC by blocking the above SHH-related carcinogenic pathways. In the intestinal epithelial cells IEC-6 and colon cancer cells HCT-116, IL-6 expression and its signaling were assessed with SHH inhibitors and levels of other inflammatory mediators, proliferation, apoptosis, tumorsphere formation, and tumorigenesis were also measured. CAC was induced in C57BL/6 mice by administration of azoxymethane followed by dextran sodium sulfate administration. SHH inhibitors were administered by oral gavage and the mice were sacrificed at 16 weeks. TNF-α-stimulated IEC-6 cells exhibited increased levels of proinflammatory cytokines and enzymes, whereas SHH inhibitors suppressed TNF-α-induced inflammatory signaling, especially IL-6/IL-6R/gp130 signaling. SHH inhibitors significantly induced apoptosis, inhibited cell proliferation, suppressed tumorsphere formation, and reduced stemness factors. In the mouse model, SHH inhibitors significantly reduced tumor incidence and multiplicity, decreased the expression of IL-6, TNF-α, COX-2, STAT3, and NF-κB, and significantly induced apoptosis. In colosphere xenografts, SHH inhibitor significantly suppressed tumorigenesis by inhibiting tumorsphere formation. Taken together, our data suggest that administration of SHH inhibitors could be an effective strategy to prevent colitis-induced colorectal carcinogenesis, mainly by targeting IL-6 signaling, ablating CSCs, and suppressing oncogenic inflammation, achieving chemoquiescence ultimately.

Keywords: 15-PGDH; anti-inflammation; cancer stem cell; colitis associated cancer (CAC); sonic hedgehog (SHH).

Figure 1. Blocking the SHH pathway suppresses the IL-6/STAT3 pathway in vitro

(A) IEC-6 cells were pretreated with SHH inhibitors for 1 h and then stimulated with 50 ng/ml TNF-α for 6 h. Expression of mRNAs for IL-6 and its receptors was quantified by RT-PCR and expressed relative to that of GAPDH. SHH inhibitors decreased the levels of TNF-α-induced IL-6 and mRNAs for its receptors IL-6R and gp130. (B) IL-6 (red) and gp130 (green) were detected by multiple immunofluorescence labeling and confocal laser scanning microscopy (×400). Sections were counterstained with DAPI (blue). The bottom panel shows a merged image. (C) IEC-6 cells were transiently transfected with luciferase reporter plasmid containing IL-6 promoter elements. After 24 h, cells were pretreated with SHH inhibitors for 1 h, and then stimulated with 50 ng/ml TNF-α for 6 h. Luciferase activity was then determined and normalized to β-galactosidase activity to account for differences in transfection efficiency. (D) IL-6 concentration in conditioned medium was analyzed by ELISA. (E) Western blot analysis showed the effect of treatment with SHH inhibitors for 3 h on the levels of TNF-α–induced STAT3, ERK, and Akt. β-actin was used as an equal loading control. (F) Regulation of STAT3-responsive genes including SOCS3, Grb2, and Gab1 was determined by RT-PCR. All data are representative of at least three independent experiments.

Figure 2. SHH inhibitor inhibits TNF-α–induced proinflammatory cytokines and inactivates NF-κB transcription factor

(A) Changes in the expression of mRNAs for proinflammatory cytokines and enzymes including COX-2, TNF-α, iNOS, IL-8, and MCP-1 were evaluated by RT-PCR. IEC-6 cells were pretreated with the indicated concentrations of SHH inhibitors for 1 h, and then stimulated with 50 ng/mL of TNF-α for 6 h. (B) IEC-6 cells were transiently transfected with luciferase reporter plasmid containing Cox-2 promoter elements. After 24 h, cells were pretreated with SHH inhibitors for 1 h, and then stimulated with 50 ng/ml TNF-α for 6 h. Luciferase activity was then determined and normalized to β-galactosidase activity to account for differences in transfection efficiency. Three independent assays were performed and the data shown are the mean ± SD. (C) Effect of SHH inhibitors on TNF-α–induced phosphorylation of IκBα. IEC-6 cells were pretreated with SHH inhibitors for 1 h followed by 50 ng/mL of TNF-α for 3 h. Nuclear localization of NF-κB was determined by Western blot analysis. (D) Transcription factor activity of NF-kB-p65 was analyzed by ELISA.

Figure 3. SHH inhibitors provoke apoptosis and a change in cell cycle in HCT-116 cells

(A) HCT-116 cells were treated with the indicated concentrations of SHH inhibitors for 24 h, and their viability was determined by the MTT assay. The results are presented relative to the viability of untreated cells (control), which was considered as 1, and are means ± SD. *p < 0.01 in comparison with untreated cells. (B) Western blots showing a significant increase in apoptotic proteins (cleaved caspase-3, cleaved caspase-8, and cleaved PARP) after treatment with SHH inhibitors for 24 h. (C) HCT-116 cells were treated with SHH inhibitors for 24 h and analyzed by flow cytometry with FITC–Annexin V and propidium iodide (PI) staining to verify the induction of apoptosis. (D) Apoptosis protein array analysis of HCT-116 cells (untreated or treated with 15 μg/ml cerulenin or 50 μM cyclopamine for 24 h). (E) Western blots showing a significant decrease in anti-apoptotic proteins (Bcl-2, survivin, and XIAP). (F) The expression of the cell cycle markers CDK4, Cyclin D1, Cyclin E, and p21 was assessed by Western blot analysis after treatment with an SHH inhibitor. Data are means ± SD, *p < 0.05, **p < 0.01 in comparison with untreated control.

Figure 4. SHH inhibitors suppress HCT-116 cell migration and proliferation

(A) Wound healing assays were carried out in 6-well plates. A line of cells in a confluent monolayer was damaged by scratching. Cell motility was examined under a light microscope (×40 magnification) for the indicated durations (24 h). (B) SHH inhibitor treatment decreased phosphorylation of β-catenin, but did not change the levels of GSK-3 (C) β-Catenin expression in different animal groups was assessed by using immunofluorescence staining. (D) The percentage of BrdU-positive cells was significantly lower in the presence of SHH inhibitors than in the control group.

Figure 5. Effects of SHH inhibitors on stemness and sphere formation

(A) Blocking the SHH pathway inhibited the expression of the colon cancer stem cell markers CD44, Nanog, Oct-4A, c-Kit, and ABCG2. HCT-116 cells were treated with SHH inhibitors for 24 h and cell extracts were analyzed by Western blotting. (B) SHH inhibitors attenuated tumorsphere formation and survival in a dose-dependent manner (upper part). Secondary tumorspheres were grown in serum-free medium (as described in Methods) for 7 days in the presence or absence of SHH inhibitors. The total number of tumorspheres formed from 500 single cells is shown in the lower part. (C) Effects of SHH inhibitors on the colony formation ability of tumorsphere cells under anchorage-independent conditions were assessed by soft agar assays. A representative photograph of a colony is shown on the left. Statistical analysis is shown on the right. All data are representative of three independent experiments.

Figure 6. SHH inhibitors prevent AOM-initiated, DSS-promoted colitis-associated cancer

(A) Overview of the experimental protocol of the AOM+DSS model (left panel) and representative gross appearance of mouse colons (right panel). Group 1; control, Group 2; AOM + DSS, Group 3; AOM + DSS + cerulenin 10 mg/kg, Group 4; AOM + DSS + cerulenin 30 mg/kg, Group 5; AOM + DSS + itraconazole 100 mg/kg. Black arrows indicate tumor location in colon tissue (right panel). (B) Tumor incidence (left), multiplicity (middle), and size (right). Data are means ± SD (n = 9 in each group). Inhibition of the SHH pathway decreased the expression of proinflammatory cytokines, inactivated the NF-κB and STAT3 pathways and promoted apoptosis in a colitis-associated colon cancer mouse model. (C) The levels of IL-6 and TNF-α mRNAs were determined by RT-PCR. (D) SHH inhibitors inactivated NF-κB and STAT3. Nuclear extracts were analyzed by Western blotting (n = 4). (E) Localization and expression levels of NF-κB and F4/80 were analyzed by immunohistochemistry in different groups (see Supplementary Figure 5A and 5B for immunohistochemical staining of NF-κB p65 and F4/80, respectively). (F) Effect of SHH inhibitors on the expression of COX-2 and 15-PGDH. The mean expression level is shown for each group. (G) TUNEL staining was performed to compare apoptosis in different groups (×200 magnification). Apoptotic index is shown for each group. *p < 0.01 vs. Group 2 (lower left). Equal amounts of total protein extracted from colon tissues were subjected to Western blot analysis using antibodies against Bcl-2, XIAP, and cleaved caspase-3. Data are means ± SD (lower right).

Figure 7. SHH inhibitors significantly inhibit cancer stem cell activation

(A) The overall scheme of the use of SHH inhibitors to prevent colitis-associated colon cancer. (B) Mean tumor weights in the control group and groups treated with SHH inhibitors (cerulenin or cyclopamine). Both SHH inhibitors significantly inhibited tumorigenesis (p < 0.05). (C) Xenograft tumors were subjected to H & E, DAPI, and TUNEL assay. The SHH inhibitors significantly decreased the numbers of tumor nests and living tumor cells, and significantly increased the number of TUNEL-positive cells, *p < 0.01 (×100 magnification). (D) A scheme of the preventive actions of SHH inhibitors against colitis-associated cancer: 1) significant inhibition of IL-6 signaling through STAT3 inactivation as well as TNF-α attenuation through NF-κB repression, 2) significant inhibition of β-catenin–dependent cell proliferation and significant induction of apoptosis, 3) preservation of 15-hydroxyprostaglandin dehydrogenase (15-PGDH) to counteract oncogenic signaling of COX-2, and 4) ablation of cancer stem cells resulting in significant inhibition of tumorsphere formation.