Stromal genes discriminate preinvasive from invasive disease, predict outcome, and highlight inflammatory pathways in digestive cancers

Abstract

The stromal compartment is increasingly recognized to play a role in cancer. However, its role in the transition from preinvasive to invasive disease is unknown. Most gastrointestinal tumors have clearly defined premalignant stages, and Barrett's esophagus (BE) is an ideal research model. Supervised clustering of gene expression profiles from microdissected stroma identified a gene signature that could distinguish between BE metaplasia, dysplasia, and esophageal adenocarcinoma (EAC). EAC patients overexpressing any of the five genes (TMEPAI, JMY, TSP1, FAPalpha, and BCL6) identified from this stromal signature had a significantly poorer outcome. Gene ontology analysis identified a strong inflammatory component in BE disease progression, and key pathways included cytokine-cytokine receptor interactions and TGF-beta. Increased protein levels of inflammatory-related genes significantly up-regulated in EAC compared with preinvasive stages were confirmed in the stroma of independent samples, and in vitro assays confirmed functional relevance of these genes. Gene set enrichment analysis of external datasets demonstrated that the stromal signature was also relevant in the preinvasive to invasive transition of the stomach, colon, and pancreas. These data implicate inflammatory pathways in the genesis of gastrointestinal tract cancers, which can affect prognosis.

Fig. 1.

Hierarchical clustering showing discrimination between the different stages of progression (P < 10⁻⁴ for all comparisons).

Fig. 2.

Kaplan-Meier curve comparing survival in patients with (gray) and without (black) up-regulation of the five-gene signature (TMEPAI, JMY, TSP1, FAPα, or BCL6).

Fig. 3.

Functional role of BCL6, TSP1, and PSTN. (A) Expression of BCL6 and TP53 in FLO-1 after stimulation with 40 ng/mL IL-6. The data are expressed as -ΔCt (ΔCt = Ct GAPDH − Ct gene of interest), and the median ± SEM is represented. **P < 0.01; ***P < 0.001. (B) Intensity score of TSP1 in the index biopsy of a nested case–control study of patients with metaplasia who progressed to EAC over a mean of 6.5 years (range, 3–13 years) compared with patients with metaplasia who did not progress over a mean of 5.5 years (range, 3–9 years). **P < 0.01. (C) mRNA expression measured by quantitative PCR (qPCR) of POSTN in a panel of primary esophageal fibroblasts and a panel of epithelial cells of esophageal origin (FLO-1, TE7, OE33, GihTert, Chtert, and GohTert). The data are expressed as -ΔCt (ΔCt = Ct GAPDH − Ct POSTN), and the median is represented. ***P < 0.001. (D) Number of cells invading through matrigel per high-power field (×400). Data are represented as mean ± SEM. Cells were treated with 5% serum (FCS), or 5% serum with 10⁵ normal esophagus (NAF), dysplasia (DAF), or cancer-associated fibroblasts (CAF) were seeded in the lower chamber, or 100 ng/mL of POSTN was added to the top chamber. *P < 0.05.

Fig. 4.

Normalized enrichment score for the gene sets across progression to EAC. Normalized enrichment score for the set of up-regulated (red) and down-regulated (green) stromal genes and for the fibroblast gene set (blue) in four esophageal (A–D), one gastric (E), one colonic (F), two pancreatic (G: whole tissue; and H: epithelial only) and one breast (I).