Inflammation-Induced Abnormal Expression of Self-molecules on Epithelial Cells: Targets for Tumor Immunoprevention

Abstract

Tumor-associated antigens (TAA) are self-molecules abnormally expressed on tumor cells, which elicit humoral and cellular immunity and are targets of immunosurveillance. Immunity to TAAs is found in some healthy individuals with no history of cancer and correlates positively with a history of acute inflammatory and infectious events and cancer risk reduction. This suggests a potential role in cancer immunosurveillance for the immune memory elicited against disease-associated antigens (DAA) expressed on infected and inflamed tissues that are later recognized on tumors as TAAs. To understand probable sources for DAA generation, we investigated in vitro the role of inflammation that accompanies both infection and carcinogenesis. After exposure of normal primary breast epithelial cells to proinflammatory cytokines IL1β, IL6, and TNFα, or macrophages producing these cytokines, we saw transient overexpression of well-known TAAs, carcinoembryonic antigen and Her-2/neu, and overexpression and hypoglycosylation of MUC1. We documented inflammation-induced changes in the global cellular proteome by 2D difference gel electrophoresis combined with mass spectrometry and identified seven new DAAs. Through gene profiling, we showed that the cytokine treatment activated NF-κB and transcription of the identified DAAs. We tested three in vitro-identified DAAs, Serpin B1, S100A9, and SOD2, and found them overexpressed in premalignant and malignant breast tissues as well as in inflammatory conditions of the colon, stomach, and liver. This new category of TAAs, which are also DAAs, represent a potentially large number of predictable, shared, immunogenic, and safe antigens to use in preventative cancer vaccines and as targets for cancer therapies.

Figure 1:. Changes in expression of four known TAAs upon exposure of primary mammary epithelial cells (MEPiC) to pro-inflammatory cytokines.

(A) MUC1, Her-2/neu, hypoglycolysated MUC1 (hyp-MUC1), and CEA expression on untreated MEPiC (black) and following treatment for 72 hours with different cytokines or their combinations (indicated by different colors). MFI, mean fluorescence intensity. (B) Fold-increase in the expression of MUC1, hyp-MUC1, Her-2/neu, and CEA on MEPiC treated with different pro-inflammatory cytokines (indicated by different colors) for different durations of time, over untreated cells (dashed line). Results are presented as mean values±SEM of three experiments. Dunnett’s multiple comparisons test: ***p<0.001, **p<0.01, *p<0.05. The following cytokine concentrations were used: IL6 (50 ng/mL), IL1β (12.5 ng/mL), and TNFα (12.5 ng/mL).

Figure 2:. Changes in expression of four TAAs upon exposure of MCF10A cells to pro-inflammatory cytokines.

(A) MUC1, Her-2/neu, hypoglycolysated MUC1 (hyp-MUC1), and CEA expression on untreated MCF10A cells (black) and following treatment for 72 hours with different cytokines or their combinations (indicated by different colors). (B) Fold-increase of MUC1, hypoglycolysated MUC1 (Hyp-MUC1), Her-2/neu, and CEA expression by MCF10A cells treated with different pro-inflammatory cytokines (indicated by different colors) for different durations, over untreated cells (dashed line). Results are presented as mean values±SEM of three experiments. Dunnett’s multiple comparisons test: ***p<0.001, **p<0.01, *p<0.05. The following concentrations were used: IL6 (50 ng/mL), IL1β (12.5 ng/mL), and TNFα (12.5 ng/mL).

Figure 3:. Change in expression of TAAs in MCF10A cells exposed to supernatants of polarized primary macrophages.

(A) MUC1, hypoglycosylated MUC1, and Her-2/neu expression after 72 hours of incubation with supernatant from primary polarized macrophages compared to control MCF10A (gray). (B) Cytokine concentrations in macrophage supernatants used in (A). Supernatants were collected 24 hours after the end of polarization/activation and cytokine concentrations assessed by cytokine-based assay. M1-like (red), M2-like (green), or M0 (blue) macrophages.

Figure 4:. Changes in the overall proteome of MCF10A cells treated with pro-inflammatory cytokines and selection of differentially expressed DAAs.

(A) Proteins extracted from treated cells were labeled with Cy3-NHS (green); proteins from untreated cells were labeled with Cy5-NHS (red); labeled proteins were mixed and resolved on 2D-DIGE. Unchanged proteins in the two lysates migrate identically and appear as yellow spots. Proteins unique to untreated cells appear as red spots, and those unique to treated cells as green spots. Blue circles and numbers indicate spots that were picked for sequencing. (B) Total protein lysates from treated and untreated cells were resolved on SDS gels and immunoblotted with antibodies against SOD2, Serpin B1, S100A8, and S100A9. β-actin was used as a loading control. Results are representative of two independent experiments. (C) mRNA expression fold-change of 2D-DIGE-identified DAAs in cytokine-treated MCF10A cells relative to untreated cells (dashed line). Results are presented as mean values±SD of 5 biological replicates. Student T-test: ***p<0.001, **p<0.01, *p<0.05. Treated cells were exposed to IL6 (50 ng/mL), IL1β (12.5 ng/mL), and TNFα (12.5 ng/mL) for 72 hours.

Figure 5:. Biological response to the inflammatory cytokine treatment is associated with upregulation of genes involved in NF-κB signaling.

(A) Volcano plot displaying differentially expressed genes in treated mammary epithelial cells compared to untreated cells. Names of mRNA probes indicate genes that reached significance in difference of expression. The horizontal lines show the threshold of p<0.01 (continuous) or p<0.001(dashed). Vertical dashed lines represent the threshold for upregulation (Log2 FC>1) and downregulation (Log2 FC<−1). (B) Differential expression of the top 10 gene sets in treated cells compared to untreated cells. (C) Heatmap representation of expression of 12 genes involved in NF-κB signaling in MEPiC (orange) and MCF10A (purple) treated (yellow) or untreated (blue) cells. Names of genes significantly overexpressed in treated cells are framed in red. The color key was provided by the software and shows a gradient from low (log2 FC <–1) to high (log2 FC>1) expression compared to untreated cells. (D) Venn diagram of the highest differentially expressed genes in treated cells over untreated cells shared between MCF10A and MEPiC cells. Treated cells were exposed to IL6 (50 ng/mL), IL1β (12.5 ng/mL), and TNFα (12.5 ng/mL) for 72 hours.

Figure 6:. In vivo expression of DAAs/TAAs SOD2, S100A9, and Serpin B1.

Representative images of paraffin-embedeed tissue sections of breast hyperplasia, breast invasive ductal carcinoma, chronic esophagitis, chronic viral hepatitis, and chronic gastritis stained with relevant antibodies (see Materials and Methods). Corresponding normal tissues stained with the same antibodies are shown in smaller squares. All slides were scanned at 10X magnification in order to select for a high resolution images at 20X.