Aberrant expression of the embryonic transcription factor brachyury in human tumors detected with a novel rabbit monoclonal antibody

Abstract

The embryonic transcription factor brachyury is overexpressed in a variety of human tumors, including lung, breast, colon and prostate carcinomas, chordomas and hemangioblastomas. In human carcinoma cells, overexpression of brachyury associates with the occurrence of the phenomenon of epithelial-mesenchymal transition (EMT), acquisition of metastatic propensity and resistance to a variety of anti-cancer therapeutics. Brachyury is preferentially expressed in human tumors vs. normal adult tissues, and high levels of this molecule associate with poor prognosis in patients with lung, colon and prostate carcinomas, and in breast cancer patients treated with adjuvant tamoxifen. Brachyury is immunogenic in humans and vaccines against this novel oncotarget are currently undergoing clinical investigation. While our group and others have employed various anti-brachyury antibodies to interrogate the above findings, we report here on the development and thorough characterization of a novel rabbit monoclonal antibody (MAb 54-1) that reacts with distinct high affinity and specificity with human brachyury. MAb 54-1 was successfully used in ELISA, western blot, immunofluorescence and immunohistochemistry assays to evaluate expression of brachyury in various human tumor cell lines and tissues. We propose the use of this antibody to assist in research studies of EMT and in prognostic studies for a range of human tumors.

Keywords: EMT; brachyury; monoclonal antibody; prognosis marker; tumor antigen.

Figure 1. Characterization of a novel anti-brachyury rabbit MAb

(A) Western blot of protein lysates from lung H460 cells using culture supernatants from nine hybridomas generated from a rabbit vaccinated with the full-length brachyury protein. (B) Immunofluorescent analysis of the H460 and ES2 cell lines grown in glass coverslips utilizing MAb 54-1 (green signal). Western blot of protein lysates from H460 Con shRNA vs. Br shRNA and chordoma U-CH1 cells (C), and PANC-1 pBr clones (D) with MAb 54-1. (E) Comparison between the levels of actin-normalized brachyury protein detected with MAb 54-1 (black bars) and brachyury mRNA (blue bars) assessed in the same clones by quantitative PCR. Indicated is the Pearson's correlation coefficient. (F) Immunofluorescent analysis of three PANC-1 pBr clones using MAb 54-1 or a control rabbit IgG Ab (green signal).

Figure 2. Specificity of the anti-brachyury MAb 54-1

(A) The ability of MAb 54-1 to discriminate between brachyury and the highly homologous TBX19 protein was assessed utilizing a TBX19-specific ELISA assay using MAb 54-1, anti-TBX19, or a control rabbit IgG Ab. (B) MAb 54-1 selectively captures soluble recombinant brachyury protein, but not recombinant PSA protein in an ELISA assay. (C) Schematic representation of each of the peptide pools as compared to the full-length protein, and resulting absorbance obtained when using each peptide pool as the capture protein in an ELISA assay with MAb 54-1.

Figure 3. MAb 54-1 binds brachyury with a high affinity

(A) Comparison between MAb 54-1 and commercial anti-brachyury antibodies (H-210 and ab57480) by ELISA. (B) Western blot analysis of endogenously expressed brachyury in three chordoma cell lines using the indicated antibodies. Bottom panels: Level of actin-normalized protein for each cell line as detected with the indicated antibodies, corresponding to the full length (49 KDa) and the shorter (45 KDa) protein band. (C) Western blot of protein lysates from H460 Con shRNA vs. Br shRNA utilizing the H-210 polyclonal Ab. (D) Western blot of PANC-1 cells expressing a control plasmid (pCMV), a shorter isoform (pBr-Short), or the full-length brachyury protein (pBr) using MAb 54-1.

Figure 4. Immunohistochemical detection of brachyury protein in human lung cancers using MAb 54-1

Transmitted light photomicrographs of a primary bronchioloalveolar carcinoma stained with (A) MAb 54-1 versus (B) control isotype IgG. Also shown is a representative staining of normal lung (C) with MAb 54-1. Expression of brachyury was analyzed by immunohistochemistry with MAb 54-1 in 30 cases of primary lung cancer and 10 lung cancer metastases. Shown (D) is the number of brachyury positive and brachyury negative cases for each tumor type. (E-J) Transmitted light photomicrographs of representative primary bronchioloalveolar (E) and large cell (F) primary lung carcinomas. Also shown are matched pairs of primary adenocarcinoma (G) and its corresponding bone metastasis (H) and a primary adenosquamous carcinoma (I) and corresponding matched bone metastasis (J). The brown signal corresponds to brachyury. Magnification 20X, scale bars = 100 μm.