PI3K/AKT signaling is essential for communication between tissue infiltrating mast cells, macrophages, and epithelial cells in colitis-induced cancer

Abstract

Purpose

To understand signaling pathways that shape inflamed tissue and predispose to cancer is critical for effective prevention and therapy of chronic inflammatory diseases. We have explored PI3K activity in human inflammatory bowel diseases (IBD) and mouse colitis models.

Experimental Design

We performed immunostaining of phosphorylated AKT (pAKT) and unbiased high throughput image acquisition and quantitative analysis of samples of non-inflamed normal colon, colitis, dysplasia, and colorectal cancer (CRC). Mechanistic insights were gained from ex vivo studies of cell interactions, the Piroxicam / IL-10^−/− mouse model of progressive colitis, and use of the PI3K inhibitor LY294002.

Results

Progressive increase in densities of pAKT-positive tumor-associated macrophages (TAMs) and increase in densities of mast cells (MCs) in the colonic submucosa were noted with colitis and progression to dysplasia and cancer. MCs recruited macrophages in ex vivo migration assays, and both MCs and TAMs promoted invasion of cancer cells. Pre-treatment of MCs with LY294002 blocked recruitment of TAMs. LY294002 inhibited MC and TAM-mediated tumor invasion, and in mice, blocked stromal PI3K, colitis, and cancer.

Conclusion

The PI3K / AKT pathway is active in cells infiltrating inflamed human colon tissue. This pathway sustains the recruitment of inflammatory cells through a positive feed back loop. The PI3K / AKT pathway is essential for tumor invasion and the malignant features of the Piroxicam / IL-10^−/− mouse model. LY294002 targets the PI3K pathway and hinders progressive colitis. These findings indicate that colitis and progression to cancer are dependent on stromal PI3K and sensitive to treatment with LY294002.

Introduction

Epithelial cells react to inflammation by increased mitotic activity, crypt architectural distortion, and ulcers, characteristics that in the chronic setting predispose to cellular transformation. Patients with ulcerative colitis or Crohn’s colitis who have persistent chronic inflammation of the colon for more than 8 to 10 years are in a very high risk of developing metastatic colorectal cancer (CRC). Approximately 25 to 30% of patients with a history of pan-colitis for more than 30 years develop colorectal cancer, because of prolonged exposure of the colon to chronic inflammation (1–3). It is suggested that tissue infiltrating pro-inflammatory cells drive the neoplastic changes in the inflamed colon leading to CRC.

In the Piroxicam / IL-10 mouse model, PI3K mediates activation of Akt and β-catenin in epithelial stem cells resulting in mitosis and crypt architectural changes that predispose to colitis (4). Furthermore, PI3K’s targeted ablation is protective (4, 5). The PI3K pathway is also etiologically linked with sporadic colorectal cancer (CRC), contributing to epithelial cell survival and proliferation (6, 7). Activated PI3K drives the transformation of well-differentiated epithelial cells to a less differentiated and more malignant phenotype (8–10).

MCs play a central role in the inflammatory response associated with cancer (11). PI3K-driven pathways control all receptor-mediated activation, differentiation, survival, and homing of MCs to their target tissues, and PI3K-deficient mice are devoid of MCs (12). In addition, a range of chemoattractants activating G protein-coupled receptors (GPCRs), receptor tyrosine kinases (RTKs) and Toll-like / IL-1 receptors (TLR/IL1Rs) initiate tumor inflammation by activating PI3K in TAMs (13). Knowledge of the cellular source of PI3K activity in healthy, inflamed, and tumor tissues is therefore important for understanding how PI3K activity causes colitis and predisposes to cancer. This knowledge will lead to understanding the mode of action of PI3K targeting drugs that are currently being tested for prevention and treatment of cancer (14).

We used paraffin-embedded human tissue specimens as well as fresh surgical tissue to study the tissue distribution of PI3K-active cells in the course of progression of colitis to CRC. We investigated interactions between two major pro-inflammatory tissue-infiltrating cells with known dependence on PI3K, MCs and TAMs. Furthermore, we looked at their contribution to tumor growth and invasion. LY294002 is a chemical inhibitor of PI3K that has been used to control experimental colitis and colon cancer (4, 15). LY294002 was used to interrupt PI3K activity and gain mechanistic insight. We report that in contrast to mouse tissue, in inflamed human colonic tissue, PI3K activity is most abundant amongst proinflamamtory cells within the stroma. PI3K activity and phosphorylation of AKT underlie both the escalation of inflammation as well as the proliferation and invasion of epithelial cells. These processes are interrupted by LY294002. Our observations emphasize the value of targeting stromal PI3K activity for effective prevention of colitis and therapy of CRC.

Methods

Tissue and tumor specimens

Paraffin-embedded specimens of normal, non-inflamed colon from 8 patients who had surgery for non-malignant lesions like colonic AVM or diverticular disease were used as controls [normal group]. Additionally, surgical specimens from 12 UC patients with active colitis (colitis group), 7 UC patients with active colitis and dysplasia (dysplasia group), and 7 UC patients with colitis and invasive colorectal cancer (cancer group) were obtained from Rush University Medical Center, Chicago. All procedures were approved by Rush University Medical Center Institutional Review Boards.

Mice

IL-10^−/− mice and C57LB6 mice were obtained from Jackson laboratories. Mice were maintained under specific pathogen-free conditions at Northwestern University Animal Care Facility, and the Animal Care Usage Committee of Northwestern University approved all experiments. IL-10^−/− mice (6 weeks old) were transferred to conventional housing and allowed 1 week to acclimate. Two groups were formed with 10 mice per group, called control and treatment group. Both groups of mice received 60 mg/kg body weight of Piroxicam from Day 0 to Day 7 and 80 mg/kg body weight Piroxicam from Day 8 to Day 14. From Day 15 to Day 35, the treatment group received 50 mg/kg LY294002 intraperitoneal (i.p) injections dissolved in 20% dimethyl sulfoxide (Sigma) every other day. The untreated group received dimethyl sulfoxide from Day 15 to Day 35, as described in an earlier study (4). Mice at 56 days post-commencement of Piroxicam treatment were sacrificed and used for histological evaluation.

Cell culture

LAD-2 cells were grown in stempro medium (Sigma Aldrich) with 100 ng/ml Stem Cell Factor (SCF) at 37°C. LAD-2 conditioned media (CM) was prepared as described earlier (16, 17). Gut-derived murine mast cells (GMMC) were grown from the gut of C57LB6 mice (18).

Conditioned medium

For the production of conditioned medium, 2×10⁶/ml LAD-2 MC or GMMCs or 1.5×10⁶/750 µl TILs were treated with carrier DMSO or 10 µM LY294002 for 1 hour, washed 4 times with serum-free medium and kept in fresh culture for 1 week (LAD-2 MC or GMMCs) and 72 hours (TILs) at 37°C and 5% CO₂. The conditioned medium was removed, filtered using 0.22-µm filters, and used for medium transfer experiments.

Immunofluorescence and Immunohistochemistry

4 µm thick paraffin-embedded sections were treated with Target retrieval solution (Dako, Carpinteria, CA) and incubated with anti-Tryptase (Neomarkers, Fremont, CA), anti-CD68 (Dako), anti–P-AKT^T308 (Cell Signaling Technology, Davers, MA), or anti-BrdU (Accurate Chemical, Westbury, NY), followed by anti- rabbit or mouse-horseradish peroxidase–labeled polymer (Dako), DAB substrate (Dako) and counterstained with hematoxylin (for immunohistochemistry) and anti-mouse Alexaflor-594 and anti-rabbit Alexaflor-488 followed by DAPI (Invitrogen) (for immunofluorescence). 10 µm frozen sections were fixed in cold methanol, blocked with 1% bovine serum albumin, incubated with anti-MAC-1, alpha M chain (Santacruz Biotech) and anti–P-AKT^T308 (Cell Signaling Technology, Davers, MA) followed by anti-mouse Alexaflor-594 (Molecular probes, Eugene, OR) and anti-rabbit Alexaflor-488 (Molecular probes, Eugene, OR) and DAPI (Invitrogen). Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay was performed as per manufacturer’s instruction (Millipore, Temecula, CA). Chloroacetate esterase (CAE) staining was performed as described earlier (18).

Tissuegnostics

TissueGnostics Tissue/Cell High Throughput Imaging and Analysis System and a semi-automated image acquisition microscope was used to acquire 200X magnification brightfield and fluorescence images throughout the section for all imaging experiments. Images were stitched in Adobe Photoshop Program and analyzed using ImageJ software.

Immunoblotting

Whole-cell extracts were prepared in RIPA buffer [10mM Tris-Cl [pH 7.5], 500mM NaCl, 0.1 % SDS, 1% NP-40, 1% Sodium deoxycholate, 2mM EDTA, and 1% protease, phosphatase I and II inhibitor cocktail (Sigma)]. Proteins (30ug) were separated by SDS-PAGE and transferred following standard protocols. Immunoreactive proteins were detected with antibodies to phospho-AKT T308, phospho-AKT S473, total AKT (Cell Signaling) and beta-actin (Sigma) using the HRP-conjugated secondary antibodies and SuperSignal chemiluminescent reagent (Thermo Scientific).

β- hexososaminidase release (mast cell degranulation) assay

For mouse degranulation, GMMCs were stimulated overnight using mouse anti-DNP IgE 1ug/ml concentration. On the next day, cells were harvested, excess IgE was washed with Tyrode buffer and treated for 120 minutes either with 10 µM LY294002 or the carrier and subsequently challenged with DNP-BSA from Sigma at 100ng/ml for 30 min. The supernatant was collected and stored at 4°C and the pellet was lyzed with 0.1% TritonX. The 20 µl of supernatant or pellet lysate were incubated with 1 mM 4-nitrophenyl N-acetyl-β-D-glucosaminide (PNAG) for 60 min at 37°C and the reaction was stopped with 200 µl Carbonate buffer (0.1 M, pH 10). β-hexosaminidase release in the supernatant was measured at 405 absorbance and interpreted as the % of total cellular (lysate + supernatant) β-hexosaminidase. For β-hexosaminindase release experiments with LAD-2 mast cells, a mast cell degranulation spectrophotometric kit from Millipore was used.

Macrophage Migration assay

Blood mononuclear cells were prepared using Ficoll-Paque from GE Healthcare. CD11b⁺ cells were isolated using biotinylated anti-CD11b, alpha M chain (BD Biosciences), streptavidin magnetic beads (Miltenyi Biotech) and LS column (Miltenyi Biotic) and purity checked using flow cytometry. For the migration assay, CD11b⁺ cells were resuspended in serum-free RPMI at 10⁶/ml concentration in 22.5 µl and seeded in triplicates in the top wells of 5 micron uncoated 96-well Chemo TX system (Neuro Probe, Gaithersburg, MD). The bottom well was loaded with 29 µl of Stempro medium without (negative control) or with 100 ng/ml SCF (positive control), or LAD-2 mast cell conditioned medium without or with 10µm LY294002. Alternatively, LAD-2 mast cells were pretreated with 10 µM LY294002 and washed prior to conditioning medium. After 3 hours of incubation at 37°C, the migrated CD11b⁺ cells were counted with trypan blue on hemocytometer. Assays were in triplicate.

Colon cancer epithelial cell Proliferation assay

1 × 10⁴ colon cancer epithelial HT-29 cells were seeded in triplicates in 100ul per well in a 96-well plate with McCoy’s 5A medium for 24 hours at 37°C and 5% CO₂. On the next day, the medium was removed and 100 µl of either McCoy's 5A or Stempro with 100 ng/ml SCF or LAD-2 mast cell conditioned medium or LAD-2 mast cell conditioned medium with 10 µM LY294002 or conditioned medium obtained after 10 µM LY294002 treatment of LAD-2 mast cells were added and incubated for 24, 48 and 72 hours at 37°C and 5% CO₂. 0.5mCurie of [³H] Thymidine was added to each well and incubated for 6 hours after 24, 48 and 72 hours time points. HT-29 cell proliferation was measured using a scintillation counter (LKB RackBeta; Wallac). Mouse CT44 cells were cultured in DMEM medium and used in a similar setup to the HT-29 proliferation assay.

Colon cancer epithelial cell invasion assay

3×10⁴ HT-29 colon cancer epithelial cells were seeded in triplicates in 500 µl per well of McCoy's 5A in the top wells (inserts) in a 24-well (12) insert plate (BD Biocoat). The bottom well contained the same media as used for the migration assay. After 48 hours at 37°C and 5% CO₂, the non-invaded cells were removed. After fixing the membrane and staining with Diff quick, invading cells were counted using a brightfield microscope.

To obtain tumor-infiltrating leukocytes, surgical colon cancer tissue samples from 3 patients bearing UC-associated colon cancer were washed with (DMEM with 0.5% penicillin/streptomycin, 10 µg/mL gentamycin sulfate), minced with surgical blades, digested using (750 U/mL type IV collagenase, Worthington Biochemical; 500 U/mg hyaluronidase, Sigma; 0.1 µg/mL DNase, Sigma) and subjected to Percoll gradient centrifugation (40–80%). The interphase was collected for invasion assays(16) and incubated in complete RPMI 1640 with 10% FBS with or without 10 µM LY294002 at 37°C, followed by two washes with complete RPMI. Immunohistochemical staining revealed the MCs % (41.75 ± 2.394) and TAMs % (31.25 ± 2.562) in the TIL population (data not shown). In setup 1, 3 × 10⁴ CFSE-labeled HT-29 cells were seeded in triplicates in the top wells (inserts) of an invasion assay plate, and in the lower chamber conditioned medium from 1.5 × 10⁶ TILs was plated. In setup 2, 3 × 10⁴ CFSE labeled HT-29 cells were seeded either alone, or in 1:1 ratio with TILs untreated or treated with 10 µM LY294002 in the top wells, while the bottom wells were filled with McCoy’s 5A. After 48 hours at 37°C and 5% CO₂, tumor invasion was manually recorded and quantified as above. Alternatively, 3 × 10⁴ CT44 cells were seeded in top wells in serum free DMEM in triplicates, while in bottom wells, conditioned medium obtained from untreated or LY294002 pre-treated primary mouse mast cells or carrier medium was added in the invasion assay chamber (BD biocoat).

Statistical analysis

All experiments were repeated 3 times and 10 mice were used in each group. Comparison of groups was assessed using the Student t test or ANOVA, where appropriate. For multiple comparisons, data was analyzed using ANOVA. P values lower than 0.05 were considered statistically significant.

Results

Bone marrow-derived pAKT-positive cells progressively increase in colitis, dysplasia, and colon cancer

To understand the spatial distribution and kinetics of PI3K activity in situ during progression from colitis to cancer, human surgical specimens were separated into four groups according to their histopathological and clinical findings, namely 1) no colitis no dysplasia (designated “normal” in this study), 2) ulcerative colitis without dysplasia (colitis), 3) ulcerative colitis with dysplasia (dysplasia) and 4) ulcerative colitis with invasive colorectal cancer (invasive cancer) (Figure 1A and Supplementary Table 1–4). The study was distributed according to mucosal and submucosal findings (Figure 1B and 1C). For mucosal tissue, data was analyzed from the muscularis mucosa extending to the lumen, including epithelium, lamina propria and the muscularis mucosa itself. Tissue underneath muscularis mucosa was considered submucosal (Figure 1). pAKT⁺ cells were detected by immunohistology in mucosa (Figure 2A, 2B and 2F) and submucosa (Figure 2C and 2G). The mean frequencies of epithelial pAKT⁺ cells in mucosa did not show significant differences when comparing normal (0.59 ± 0.23) to colitis (0.74 ± 0.13) to dysplasia (0.69 ± 0.13) and to invasive cancer (1.10 ± 0.17)(Figure 2A). The frequency of stromal pAKT⁺ cells infiltrating the mucosa in all cases outnumbered pAKT⁺ epithelial cells (compare Figure 2A and 2B). Significant increases in pAKT⁺ cells were detected in the stroma of the mucosa when progressed from “normal” (2.33 ± 0.65) to colitis tissue (6.83 ± 1.12, *P<0.05), but thereafter plateaued (Figure 2B) with no significant differences from colitis to dysplasia (5.81 ± 1.27) to invasive cancer (8.27 ± 1.48). In contrast, the frequency of pAKT⁺ cells steadily and significantly increased in the submucosa with each transition from “normal” (2.33 ± 0.65), to colitis (6.56 ± 0.80, *P<0.05), to dysplasia (16.19 ± 4.70, *P<0.05) and finally to cancer (37.87 ± 7.39, *P<0.05), with cancer having the highest density of pAKT⁺ cells (Figure 2C).

Figure 1.

Categorization of patient tissue specimens using Hematoxylin and Eosin staining. (A) TissueGnostics acquired stitched images of Normal, Colitis, Dysplasia and Invasive cancer. (B) H&E staining of mucosal tissue of Normal, Colitis (small arrow indicates ulcer), Dysplasia (small arrow indicates dysplastic crypts) and Invasive cancer. (C) H&E staining of submucosal tissue of Normal, Colitis, Dysplasia and Invasive cancer (small arrow indicates invaded crypts). Big arrows show mucosal and submucosal tissue. Scale bar = 50 µm.

Figure 2.

pAKT⁺ macrophages progressively infiltrate the colonic submucosa with progression to colitis and cancer. Bar graphs shows quantification of % mean ± standard error of positively stained cells of pAKT per total nuclei in (A) mucosal crypt-epithelium (B) mucosal stroma and (C) submucosa of normal and diseased human colonic tissue; quantification of % mean ± standard error of pAKT and CD68 double positive cells per total nuclei in (D) mucosa and (E) submucosa. Representative immunohistochemical staining depiction for pAKT⁺ cells in healthy and diseased human colonic (F) mucosa and (G) submucosa and double immunofluorescent staining of CD68⁺ and pAKT⁺ cells in health and diseased human colonic mucosa (H) and submucosa (I). Scale bar = 50 µm; * P < 0.05 represents the result of ANOVA.

To validate the presence of TAMs in tissue samples, we stained histologic sections with antibodies to pan-macrophage antigen CD11b (Mac1) (19) and CD68 (20, 21). Based on mouse modeling it has been suggested that chemoattractants, growth factors, and pathogen-associated molecular patterns initiate tumor inflammation by activating PI3K in CD11b⁺ myeloid cells (13). To test this notion, we did double immunofluorescence staining for pAKT and macrophage markers (Figure 2D and 2E; Supplementary Figure 3A and 3B). There was abundant co-localization of pAKT with CD68 (Figure 2D and 2E). Densities of pAKT⁺ CD68⁺ TAMs increased in the mucosa when disease progressed from normal (2.33 ± 0.56) to colitis (4.77 ± 0.96), but did not increase further in dysplasia (5.81 ± 1.27) and cancer (6.27 ± 1.14) (Figure 2D, 2H). By contrast, the densities of pAKT⁺CD68⁺ TAMs increased steadily from normal tissue (1.56 ± 0.44) to colitis (4.83 ± 0.67), dysplasia (12.58 ± 3.83) and cancer (34.54 ± 4.56) in the submucosa (Figure 2E, 2I). Total CD68⁺ cell densities (inclusive of all pAKT±) also increased progressively in the submucosa from normal (2.13 ± 0.45), colitis (6.18 ± 0.84), dysplasia (18.81 ± 4.23) to cancer (38.62 ± 4.51) (data not shown). TAM identity was validated by additional immunostainings for total Mac1+ and Mac1+pAKT+ cells (data not shown).

Mast cells increase with progression to colitis and to cancer and recruit TAMs in a PI3K-dependent manner

MCs are sentinel cells that are activated early in the process of intestinal carcinogenesis, and contribute to cancer initiation (11). In mouse models of cancer, MCs orchestrate further inflammatory reactions by mobilizing TAMs (18, 22, 23). Previously we provided evidence for MC recruitment of TAMs in human pancreatic cancer (16). To relate tissue MC densities to mobilization of TAMs in colitis progression to cancer we stained paraffin-embedded tissues for MC-tryptase (Figure 3). Images from 50 fields of vision were recorded for quantification by Tissuegnostics high-throughput imaging microscope for each sample of mucosa and submucosa. MCs were detected in mucosa and submucosa (Figure 3A–3D) from samples with colitis, dysplasia, and cancer. Relative densities and sub-tissue distributions of MC mirrored that of pAKT⁺ cells. In other words, MC frequencies in mucosa increased significantly from normal colon (4.75 ± 0.56) to colitis (14.17 ± 1.82, *P<0.05), but did not increase further as the disease progressed from colitis to invasive cancer (colitis: 14.17 ± 1.82; dysplasia: 15.54 ± 3.07; invasive cancer: 19.44 ± 3.74) (Figure 3A). In contrast, mean MC frequencies in sub-mucosa increased steadily as the disease progressed from normal (6.34 ± 0.99) to colitis (12.35 ± 1.86, *P<0.05), to dysplasia (33.54 ± 8.55, *P<0.05) and to invasive cancer (59.99 ± 7.09, *P<0.05) (Figure 3B).

Figure 3.

Tryptase⁺ mast cell densities progressively increase in the colonic submucosa with progression to colitis and cancer. Bar graphs show quantification of % mean ± standard error of positively stained cells of human mast cell tryptase per total nuclei in (A) mucosa and (B) submucosa of normal and diseased human colonic tissue. Representative immunohistochemical staining depiction for Tryptase positive mast cells in healthy and diseased human colonic (C) mucosa and (D) submucosa. Scale bar = 50 µm; * P < 0.05 represents the result ANOVA.

MCs orchestrate secondary inflammatory reactions by recruiting other bone marrow-derived cells (11, 24) such as TAMs that critically contribute to CRC progression (25). We postulated that PI3K activity in MC is needed for their chemotactic potential. To test this hypothesis, we pre-incubated human LAD2-MC in the presence or absence of LY294002, and tested inhibition of PI3K activity by Western-blot analysis of phospho-proteins. Proteins separated by gel electrophoresis and transferred to membrane were reacted with antibodies to phospho-AKT T308, phospho-AKT S473, total AKT (Cell Signaling) and beta-actin (Sigma). This analysis showed that pre-incubation of MC for 1 hour with 10 µM of LY294002, hindered T308-phosphorylation of AKT by 1.74 ± 0.15 fold and S473 by 4.01 ± 0.38 fold. (Figure 4A and 4B). Next we treated LAD2-MC with 10 µM LY294002 and washed the cells before putting them back into culture to collect conditioned medium. The chemotactic activity of conditioned media was then tested by measuring migration of CD11b⁺ macrophages freshly prepared from human peripheral blood mononuclear cells (PBMC) through Chemo TX system. To assay migration of macrophages we used the Chemo TX 5-µm pore size migration assay system. CD11b⁺ macrophages were loaded in the top chamber and the conditioned mediums from LAD2-MC were loaded in the bottom chamber. There was a significant migration of the CD11b⁺ macrophages to the bottom chamber containing conditioned medium from untreated MC as compared to similar setups where regular non-conditioned medium was used for comparison (*P<0.05) (Figure 4D). Pre-treatment of LAD2-MC with 10 µM LY294002 abrogated the bioactivity of the conditioned medium in this assay and thus CD11b⁺ macrophage migration (*P<0.05, Figure 4D and Supplementary Figure 2). PI3K activity is essential for differentiation of MCs, as well as their long-term survival and function (12). Mature MCs produce various biologically active mediators, which are released either by secretion or by degranulation. In particular, it has been reported that in mouse models of cancer, inhibiting MC degranulation abrogates tumor-promoting properties of MCs (26). Hence, we decided to test the impact of different concentrations (5 and 10 µM) of LY294002 on MC degranulation, and for this purpose we used human LAD2-MC. Treatment of LAD2-MC with LY294002 inhibited degranulation - the β-hexososaminidase release (%) in carrier-treated/control (72.38 ± 5.78) was reduced after 5 µM LY294002 (47.76 ± 6.43, *P<0.05) and 10 µM LY294002 treatment (39.82± 4.39, *P<0.05) (Figure 4E).

Figure 4.

LY294002 treatment attenuates mast cell degranulation and mast cells associated macrophage migration, HT-29 tumor proliferation and invasion. (A) Immunoblot of total and phosphorylated AKT in LAD-2 mast cells and TILs untreated and treated with 10 µM LY294002. Bar graphs indicate quantitation of the (B) ratio of LAD-2 MC band intensity with LAD-2 MC treated with 10µM LY294002 and (C) ratio of TILs band intensity with TILs treated with 10 µM LY294002, indicating fold inhibition of phosphorylation by LY294002 using ImageJ software. (D) Mean ± standard error CD11b migration in response to Stempro + SCF (control/ LAD-2 base growth medium), CM (LAD-2 conditioned medium), CM + 10 µM LY294002 (CM with 10 µM LY294002) and 10 µM LY pretreated CM (CM obtained after treatment of LAD-2 cells with 10 µM LY294002). (E) % β-hexosaminidase release from LAD-2 mast cells before and after treatment of 5 or 10 µM LY294002. Mean ± standard error HT-29 proliferation (F) post 24 hrs, (G) post 48 hrs and (H) post 72 hrs of treatment of McCoy’s 5A (negative control), Stempro + SCF (internal control for LAD-2 CM), CM (LAD-2 CM), CM+ 10 µM LY294002 (CM with 10 µM LY294002) and 10 µM LY pretreated CM (CM obtained after treatment of LAD-2 cells with 10 µM LY294002). (I) Quantification of mean ± standard error invaded HT-29 cells per well or chamber in response to Stempro + SCF (internal control for LAD-2 CM), LAD-2 CM, LAD-2 CM + 10 µM LY294002 (CM with 10 µM LY294002) and 10 µM LY pretreated CM (CM obtained after treatment of LAD-2 cells with 10 µM LY294002). (J) Quantitation of mean ± statndard error invaded HT-29 cells per well in the experimental setup-1 (HT-29 in top chamber and CM from TILs with or without 10 µM LY294002 pretreatment in bottom well) & (K) Quantitation of mean ± standard error invaded HT-29 cells per well in the experimental setup-2 (HT-29 in 1:1 coculture with TILs with or without 10µM LY294002 pretreatment in top chamber). * P < 0.05 represents the result of ANOVA.

LY294002 inhibits MC tumor promoting properties

Next, we hypothesized that MC produce soluble factors that enhance the proliferation and invasion of epithelial cancer cells (16) and investigated the role of PI3K in the proliferative response of HT-29 colon cancer cells to human LAD2-MC (27). LAD2-MC conditioned medium enhanced the rate of proliferation of HT-29 cells progressively at 24, 48 and 72 hours (*P<0.05) (Figure 4F–4H). Next we tested the effect of inhibition of PI3K by treating LAD2-MC with 10 µM LY294002 and preparing conditioned medium from washed cells. Pre-incubation with LY294002 significantly reduced the ability of the LAD2 conditioned medium to stimulate proliferation of HT-29 cells, measured at three separate time-points (*P<0.05, Figure 4F–H). We then tested the response of HT-29 cells against direct treatment with the PI3K inhibitor. LY294002 has direct inhibitory effects on the tumor cells. However, the direct inhibitory effect of LY294002 was less significant at 24 and 48-hour time point in comparison with the LY294002 pre-treated LAD-2 conditioned medium, whereas at 72 hours similar trend was seen, but data was not significant (Figure 4F–H). These observations suggest that PI3K activity in MC contributes to tumor proliferation and its inhibition by LY294002 is a critical event in suppression of tumor growth.

We further investigated the possibility that PI3K activity and phosphorylation of AKT in MC contributes to tumor invasion. To test this hypothesis, we performed in vitro invasion assays with the HT-29 colon cancer cells in the presence or absence of LAD2 conditioned medium. Since, LY294002-treated LAD2-CM attenuates HT-29 proliferation by 40% at 48 hours, we normalized the HT-29 invaded cell count (reduced the cell number by 40% in Control/Stempto+SCF and LAD-2CM groups for analysis and graphical representation). There was a significant increase in mean HT-29 cell invasion/well in Matrigel in response to LAD-2 MC conditioned medium (64.80 ± 6.92, *P<0.05) in comparison with the control (11.40 ± 1.03, *P<0.05) (Figure 4I; Supplementary Figure 3A). Invasion was attenuated when the conditioned medium was obtained from LAD2-MC that had been previously treated with 10 µM LY294002 as described above (17.67 ± 1.45, *P<0.05, Figure 4I; Supplementary Figure 3A). As with the proliferation response, LY294002 had direct inhibitory effect on tumor cell invasion. However, even in the presence of this inhibitor, LAD2 conditioned medium elicited a significant invasion response in the tumor cells (38.67 ± 4.91, *P<0.05, Figure 4I). These observations strongly suggest that MCs promote tumor invasion and that this property of MCs is partially PI3K-dependent. Thus, release of tumor promoting agents by MCs and potential contribution of MCs to tumor growth and invasion were blocked by LY294002.

Next, we measured the ability of tumor infiltrating leukocytes (TILs) isolated from CRC tumors to promote tumor cell invasion. TILs were checked for phosphorylation at the Threonine 308 residue (pAKT-T308) and the Serine 473 residue of AKT (pAKT-S473). 10µM LY294002 treatment significantly attenuated pAKT-T308 1.62 ± 0.05 fold and pAKT-S473 3.53 ± 0.17 fold in comparison with carrier-treated pAKT-T308 and pAKT-S473 (*P<0.05, Figure 4A and 4C). By pretreating the TILs with LY294002 we tested the dependence of tumor invasion promoting activity on PI3K. To address this question, we isolated TILs from fresh surgical specimens derived from tumors of colitis-associated colon cancer patients. Two different setups were used. In the first setup we added tumor cells in the top chamber and conditioned medium from the TILs (carrier or 10 µM LY294002 pretreated) in the bottom chamber. In the second setup, we plated the bottom well with conditioned medium derived from a co-culture of the TILs with the tumor cells, again pretreated with carrier or 10 µM LY294002. After normalization of HT-29 cell counts in Stempro+SCF, TILs CM and HT-29+TILs study groups, we found mean invasion of HT-29 cells into matrigel was significantly enhanced by the TILs in both experimental setups (76.80 ± 5.67 for setup-1 and 133.80 ± 7.99 for setup-2, *P<0.05, Figure 4J and 4K; Supplementary Figure 3B). Pretreatment of the TILs with 10 µM LY294002 significantly inhibited tumor invasion relative to treatment with CM from carrier-treated TILs or co-culture with carrier-treated TILs, respectively (26.67 ± 6.93 for setup-1 and 57.00 ± 4.72 for setup-2, *P<0.05, Figure 4J and 4K; Supplementary Figure 3B). These results are compatible with those obtained with MC conditioned medium and demonstrate that the PI3K inhibitor LY294002 inhibits production of mediators of tumor invasion by TILs, including TAMs and MCs.

LY294002 treatment inhibits mast cells, colitis, and cancer development in the IL-10^−/− piroxicam mouse model

To further validate our in vitro observations and to see if PI3K/AKT play central roles in the in vivo progression of colonic inflammation into colon cancer, we treated cancer-prone colitis mice with LY294002. IL-10^−/− mice, when treated with Piroxicam, develop colitis with ulcers, followed by invasive cancer by day 56 (mean invasive lesions 2.30 ± 0.26, Figure 5A and 5F) (4). LY294002 treatment reduced the incidence of invasive cancer in this model (0.100 ± 0.10, *P<0.05, Figure 5A and 5F), reduced frequency of Bromodeoxyuridine (BrdU) positive cells (*P<0.05, Figure 5B and 5G), increased apoptosis as measured by TUNEL (*P<0.05, Figure 5C and 5H), and reduced the pAKT levels within the crypt-epithelium (*P<0.05, Figure 5D and 5I) and stroma (*P<0.05, Figure 5E and 5J).

Figure 5.

LY294002 attenuates development of cancer in IL-10^−/− mice treated with Piroxicam. Graphical representation of the frequencies of (A) invasive lesions, (B) % BRDU⁺, (C) %TUNEL⁺, (D) %pAkt⁺ epithelial and (E) %pAkt⁺ stromal cells per total nuclei in the colon of untreated and LY294002 treated Il-10^−/− mice. (F) Histological evaluation of IL-10^−/− and IL-10^−/− mice treated with LY294002. H & E staining at 100X & 200X magnification of IL-10^−/− mice 56 days post Piroxicam ± LY294002 treatment; arrow indicates invaded colonic crypts. (G) BrdU staining on colon of IL-10^−/− or IL-10^−/− mice treated with LY294002 (H) TUNEL staining on colon of IL-10^−/− or IL-10^−/− treated with LY294002 (I) pAkt staining in colonic mucosal epithelium of IL-10^−/− or IL-10^−/− mice treated with LY294002. (J) pAKT staining in colonic stroma of IL-10^−/− or IL-10^−/− mice treated with LY294002, arrows indicate pAKT⁺ cells. Scale bar = 50 µm, * P < 0.05 represents the result of Student t test.

We had reported earlier a causative role for focal mastocytosis and pre-neoplasia in the mouse intestine (11, 18). Thus, we used CAE staining to study the in vivo impact of LY294002 on MCs infiltrating the gut tissue. CAE is a cytochemical staining that stains MCs and granulocytes (28). We found that LY294002 treatment inhibited the mean frequencies of tissue-infiltrating CAE⁺ cells (0.262 ± 0.06) in comparison with control untreated mice (0.98 ± 0.09, *P<0.05, Figure 6A and 6F). Moreover, LY294002 treatment significantly attenuated MC degranulation in situ (purple MCs) (% mean 30.12 ± 2.98), found predominantly in the submucosa (site of invasion) of the non-LY294002 treated mice (85.02 ± 1.57, *P<0.05, Figure 6B and 6G).

Figure 6.

LY294002 attenuates inflammation and mast cell degranulation in IL-10^−/− mice in vivo, and tumor cell proliferation and invasion ex vivo. (A) Quantification of % mean ± standard error CAE⁺ cells per total nuclei in LY294002 untreated and treated IL-10^−/− mice. (B) Quantitation of % mean ± standard error in vivo mast cell degranulation per total mast cells in the colon of the LY294002 untreated or treated IL-10^−/− mice (% in situ degranulation = total purple mast cells X 100 / total mast cells in LY294002 untreated or treated IL-10^−/−). (C) Quantitation of % in vitro β-hexosaminidase release from GMMCs after treatment of carrier or 5 µM or 10 µM LY294002. (D) Quantitation of mean ± standard error CT44 mouse colon cancer proliferation at 24 hour time point in response to carrier or 10 µM LY294002 treated GMMC conditioned medium. (E) Quantification of mean ± standard error CT44 cell invasion/well in response to carrier or 10 µM LY294002 treated GMMC conditioned medium. (F) CAE staining at 100X & 200X magnification respectively of IL-10^−/− mice 56 days post Piroxicam ± LY294002 treatment, small arrow indicate CAE positive cells. (G) Toluidine blue staining at 100X & 1000X magnification respectively of IL-10^−/− mice 56 days post Piroxicam ± LY294002 treatment, large black arrow indicates magnified, purple degranulating or blue non-degranulating mast cells. (H) CT44 mouse colon cancer cell invasion in response to conditioned medium obtained either carrier or LY294002 treated GMMCs. Scale bar in (F) and upper panel of (G) = 50 µm, Scale bar for 1000X magnification for G = 20 µm, * P < 0.05 represents the result of Student t test.

We used in vitro assays to validate inhibition of degranulation in gut derived primary mouse mast cells by LY294002. The β-hexososaminidase release (%) in carrier-treated GMMCs (33.75 ± 0.49) dropped after 5 µM LY294002 (11.28 ± 0.47, *P<0.05) and 10 µM LY294002 (6.86 ± 0.39, *P<0.05, Figure 6C) treatment. Next, we used the pretreated conditioned medium (with 10 µM LY294002 or carrier) obtained from mouse mast cells to study the effect of PI3K inhibition on mouse MCs in the context of CT44 mouse colon cancer proliferation and invasion. We found that, LY294002 pretreated conditioned medium significantly attenuated the mean CT44 cell proliferation counts (8580.00 ± 1009) in comparison to conditioned medium obtained from carrier-treated mouse MCs at 24 hours time points (12910.00 ± 678.20, *P<0.05, Figure 6D). However, at 48 and 72 hour time points there was no significant difference between the two groups (data not shown). Similarly, the mean CT44 cell invasion/well after normalization (CT44 invasion count number in carrier-treated study group only was normalized by reducing 33.53% since at 24 hours the CT44 proliferation was attenuated by 33.53%) was significantly attenuated by 10 µM LY294002 pretreated conditioned medium (303.70 ± 16.70) in comparison with conditioned medium from carrier-pretreated mouse mast cells (516.70 ± 45.18, *P<0.05, Figure 6E and 6H). These observations show that PI3K and phosphorylation of AKT are critical for mast cell functions that promote cancer and LY294002 inhibits these functions.

Discussion

Inflammation plays a pivotal role in the initiation and progression of colon cancer (11, 29). Chronic inflammation in UC patients increases the risk of rapidly progressing CRC (30, 31). It is known that PI3K activity significantly rises in CRC and is associated with poor prognosis (32, 33). Mouse models of UC have shown that PI3K activity is critical for progression to cancer (4, 18, 34). However, much of what is known is focused on the role of PI3K signaling in tumor cells; furthermore, the relevance to inflammation-driven colon cancer in humans remains unclear (4, 18, 34). By in situ staining of human surgical specimens we found that PI3K activity overlapped abundantly with tissue infiltrating MCs and macrophages and tumor infiltrating cells. Increasing densities of PI3K-stained cells as well as MCs and TAMs in the submucosa of colon tissues were noted in patients with ulcerative colitis (UC) as inflammation progressed to cancer. These reached a peak in the submucosal areas of pre-neoplasia and cancer. Earlier studies have demonstrated the presence of MC and TAMs in the sub-epithelial region of the mucosa in colitis (35, 36) and the stromal tumor front in CRC (37, 38). It is in these locales that MCs and TAMs have been shown to promote tumor growth via angiogenesis and pro-inflammatory growth factors (11, 25). Using in vitro experiments with human-derived cell lines we found that MCs readily mobilize macrophages and that conditioned medium from both promoted invasion of tumor cells into matrigel. LY294002 attenuated macrophage migration and tumor invasion. These results were validated and extended in the mouse model of experimental colitis and colon cancer, Piroxicam-treated IL-10^−/−. LY294002 treatment of the mice significantly inhibited PI3K activity and reduced infiltrating MC and TAMs, epithelial proliferation, and progression to cancer. Also, in vivo LY294002 treatment of mice attenuated MC degranulation in the colonic tissue and more specifically in the submucosa, and in vitro pre-treatment of gut-derived mouse MCs with LY294002 reduced epithelial cell proliferation and invasion in response to MC conditioned medium. These results indicate that PI3K activity in the tumor stroma and submucosa contributes towards cancer progression. MCs and macrophages escalate inflammation that predisposes to cancer, while cancer-associated inflammation promotes tumor proliferation and invasion. In pre-neoplasia, increased PI3K activity of epithelial cells is more likely to be caused by inflammation than mutation. Our observations suggest that the tissue and tumor microenvironments are the primary targets of action of LY294002. Both MCs and TAMs are inhibited by treatment with LY294002.

The current study provides for the first time insight into the spatial distribution of PI3K activity, MC and TAMs in human colon during progression from chronic inflammation to dysplasia and cancer. Our observations build upon previous reports on the prevalence of PI3K activity in human tumors and infiltration by MCs and TAMs (9, 32, 39–42). Much of the attention to the role of PI3K in pre-neoplasia and cancer in the GI tract has focused on gut epithelial cells. In particular, previous reports based on mouse modeling suggest that progression from colitis to cancer is associated with PI3K activity in crypt epithelial cells. In the IL-10 deficient mouse model, PI3K is required for induction of colitis, while PI3K’s targeted genetic ablation (4, 5) or treatment of mice with the broad PI3K inhibitors mesalamine (43) or LY294002 (4) protect against colitis. PI3K has been described as mediating proliferation and activation of AKT and β-catenin in epithelial stem cells resulting in crypt architectural changes that predispose to colitis (4). However, we show with unbiased in situ staining of human colonic tissue sections that the relative frequency of detectable PI3K active cells in the stroma and submucosa is more than 10 fold above that in of epithelial cells and increases with colitis and cancer. In contrast, in the IL-10^−/− mouse colon, this ratio is reversed. This difference between human and mouse tissue may have masked the significance of PI3K activity of tissue infiltrating cells in predisposition to colitis and progression to cancer.

PI3K activity is essential for the differentiation, homing, and functions of a number of key pro-inflammatory cell types that drive colitis and progression to cancer. PI3K is essential for mast cell differentiation and function, to the extent that inhibition of PI3K by over-expression of the dominant negative inhibitor −p85 leads to a significant decline in MC degranulation via antigen-induced Ca2⁺ signals (44, 45), and mice deficient for PI3K activity are devoid of mast cells (46, 47). Furthermore, earlier mouse studies have shown that pro-inflammatory macrophages with tumor promoting properties have high levels of PI3K activity(13). We have shown in the past that MCs are an inherent component of intestinal carcinogenesis in mouse models of hereditary polyposis (11, 18). MCs increase in ulcerative colitis (UC) and in colon cancer (11). High densities of MCs correlate with active angiogenesis and poor clinical outcome for colorectal cancer patients (39, 48, 49). We showed that treatment of mice with the PI3K inhibitor LY294002 blocks degranulation of MCs.

Macrophages are among the major and most extensively pro-inflammatory bone marrow derived cells that accumulate in tumors and contribute to CRC progression and invasion (22, 25, 50). We showed that pAKT levels steadily increased in the submucosa and co-localized with CD68⁺ TAMs as colitis progressed to invasive cancer. Furthermore, we showed that inhibition of PI3K activity hinders the ability of these cells to promote tumor cell proliferation and invasion. We found TAMs from UC patients promoted HT-29 invasion, either through their secreted soluble factors in the conditioned medium (experimental setup-1) or direct cell-cell contact (experimental setup-2). Indeed, when these cells were co-cultured, invasion was even more pronounced (experimental setup-2); LY294002 treatment of TILs reduced their ability to promote HT-29 invasion in both setups.

Tissue-infiltrating MCs and macrophages are sensitive to inhibition of PI3K and are abundantly present in increasing numbers during progression to colitis and cancer. Therapies that target the PI3K pathway need to take into account that tumor cells may not be the primary target cells. Our findings demonstrate the role of PI3K in tumor-infiltrating cells and their communication with tumor cells, drawing attention to the role of PI3K signaling in the tissue and tumor environment in predisposition to cancer.

Statement of Translational Relevance.

Stromal interactions that sustain chronic inflammation and predispose to cancer are poorly understood. Experimental models of colitis implicate the phosphatidylinositide 3-kinase (PI3K) pathway in its activation of gut enterocytes and tissue remodeling. While mouse models are important for gaining mechanistic insights into diseases that affect us, validation of the findings in humans remains the only way to evaluate their clinical relevance. Here we show that in contrast to mouse models of colitis, by far the greatest fraction of PI3K-active cells are tissue infiltrating pro-inflammatory cells. We use a potent inhibitor of PI3K that is currently in clinical use in combination with ex vivo assays and animal modeling to elucidate the contribution of PI3K activity to the recruitment of inflammatory cells and predisposition to cancer. Our findings point to stromal interactions as the prime site of action of PI3K inhibitors in prevention and therapy of inflammation-induced colon cancer.