Abstract B55: Molecular characterization of epithelial and stromal crosstalk associated with TRIM28 expression levels in colorectal cancer

Abstract

Abstract Background: TRIM28 is a universal transcriptional co-repressor with pleotropic effects in both normal and tumor cells. We have previously shown that varying TRIM28 levels in epithelial cells and stromal fibroblasts have a prognostic value in colorectal cancer patients. The pathophysiological role of TRIM28 in carcinogenesis may therefore be associated with TRIM28 expression levels and cell type of expression within the tumor microenvironment. In this study we aim to dissect the molecular pathways associated with TRIM28 expression ratios within the tumor microenvironment by isolating individual populations of neoplastic epithelial and stromal cells and investigating their protein signaling networks. Methods: TRIM28 expression was analyzed by immunohistochemistry on FFPE tissue from 19 colorectal cancer patients. TRIM28 staining was evaluated in both epithelial and stromal compartments. IHC scoring of at least 2 units of difference in staining intensity between stromal fibroblasts and epithelial cells was defined as a high TRIM28 expression ratio and a low TRIM28 expression ratio was defined as 1 or 0 units of difference in staining intensity. Reverse-phase protein microarrays (RPMA) were constructed from laser capture micro-dissection (LCM) enriched tumor epithelium and stroma isolated from fresh-frozen tissue of the same patient cohort. The protein signaling networks were measured for 32 downstream signaling endpoints. Spearman rank analysis was used to assess the correlations between individual protein pairs. Correlation coefficient ρ ≥ 0.75 with P ≤ 0.01 was considered significant. Results: Immunohistochemical analysis of the FFPE tissue sections identified 10 TRIM28 high ratio cases and 9 TRIM28 low ratio cases. Proteomic networks were assessed in TRIM28 high and low ratio cases in fresh-frozen tissue using RPMA. Spearman ρ rank correlation analyses for the 32 signaling proteins revealed that 184 highly correlated protein pairs exclusive to the TRIM28 high ratio group, whereas 157 protein pairs were found exclusively in the TRIM28 low ratio group. In addition 191 protein pairs were shared across both TRIM28 high and low ratio groups. The caspases 3 and 7, as well as the cell surface receptor RAGE, were prominent in the high TRIM28 ratio group proteomic network, whereas the Metalloprotease MMP9 and the GTPase Ras-GRF1 were exclusive to the low TRIM28 ratio group. Furthermore we found Survivin and JNK to be prominent in the stromal compartment of the high TRIM28 cases. Conclusions: We characterized molecular pathways associated with TRIM28 expression ratios within the tumor microenvironment. Proteomic analysis revealed distinct protein signaling networks associated with varying TRIM28 expression levels in epithelial and stromal compartments. The study presents a novel way of deciphering molecular crosstalk between the epithelial and stromal compartments within the microenvironment. Citation Format: Seán Fitzgerald, Virginia Espina, Katherine M. Sheehan, Robert Cummins, Anthony O'Grady, Dermot Kenny, Richard O'Kennedy, Lance Liotta, Elaine W. Kay, Gregor Kijanka. Molecular characterization of epithelial and stromal crosstalk associated with TRIM28 expression levels in colorectal cancer. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr B55. doi:10.1158/1538-7445.CHTME14-B55