Cancer vaccines targeting the epithelial-mesenchymal transition: tissue distribution of brachyury and other drivers of the mesenchymal-like phenotype of carcinomas

Abstract

The epithelial-mesenchymal transition (EMT) is thought to be a critical step along the metastasis of carcinomas. In addition to gaining motility and invasiveness, tumor cells that undergo EMT also acquire increased resistance to many traditional cancer treatment modalities, including chemotherapy and radiation. As such, EMT has become an attractive, potentially targetable process for therapeutic interventions against tumor metastasis. The process of EMT is driven by a group of transcription factors designated as EMT transcription factors, such as Snail, Slug, Twist, and the recently identified T-box family member, Brachyury. In an attempt to determine which of these drivers of EMT is more amenable to targeted therapies and, in particular, T-cell-mediated immunotherapeutic approaches, we have examined their relative expression levels in a range of human and murine normal tissues, cancer cell lines, and human tumor biopsies. Our results demonstrated that Brachyury is a molecule with a highly restricted human tumor expression pattern. We also demonstrated that Brachyury is immunogenic and that Brachyury-specific CD8(+) T cells expanded in vitro are able to lyse Brachyury-positive tumor cells. We thus propose Brachyury as an attractive target for vaccination strategies designed to specifically target tumor cells undergoing EMT.

Figure 1. Relative mRNA expression of four EMT mediators in human normal tissues and carcinoma cell lines

Real-time PCR was performed for Brachyury (A), Snail (B), Slug (C), and Twist (D) in a multi-tissue, commercially available cDNA panel library (Clontech), and several human lung and colon carcinoma cell lines as previously described. All values are expressed as a ratio to the endogenous control GAPDH.

Figure 2. Relative mRNA expression of three EMT mediators in murine normal tissues and tumor cell lines

Real-time PCR was performed for Brachyury (A), Snail (B), and Twist (C) in a panel of normal murine tissue cDNAs (Clontech) and several murine tumor cell lines. The following TaqMan probes (AB Biosystems) were used: Brachyury (Mm01318249_m1), Snail (Mm00441533_g1), Twist (Mm00442036_m1). All values are expressed as a ratio to the endogenous control GAPDH as previously reported.

Figure 3. Relative mRNA expression of Brachyury in lung tumor biopsies, normal adjacent tissues and several human lung and colon carcinoma cell lines

Relative Brachyury mRNA expression in a commercial panel of cDNAs (Origene TissueScan qPCR Arrays) generated from 80 lung tumors at various stages of disease (I–IV), along with 16 histologically normal lung tissues adjacent to the tumor. All values and the median for each group are expressed as a ratio to the endogenous control GAPDH.

Figure 4. Lysis of Brachyury positive tumor cells by Brachyury-specific T cells

Brachyury-specific human T cells were expanded from the blood of normal donors and utilized in vitro for lysis of H441 (HLA-A2+/Brachyury+), SW1463 (HLA-A2+/Brachyury−), NCI-H460 (HLA-A2− Brachyury+) or AsPC-1 (HLA-A2−/Brachyury−) carcinoma cell lines. (Reprinted with permission from the American Association for Cancer Research; see reference )