Our laboratory has identified the T-box transcription factor brachyury as a driver of the epithelial-mesenchymal switch (EMT) in human carcinomas. We have demonstrated that high levels of expression of brachyury drive carcinoma cells into a mesenchymal-like phenotype inducing up-regulation and down-regulation of mesenchymal and epithelial proteins, respectively. Brachyury was also shown to increase tumor cell motility and invasiveness in vitro and to enhance the tumor's ability to disseminate in vivo in animal models of cancer. In addition to promoting tumor dissemination, we have now shown that brachyury expression induces tumor resistance to a variety of cancer therapies, including chemotherapy, radiation, and small molecule targeted therapies. With the aim of investigating the expression of brachyury in various types of cancer, our laboratory has now developed and thoroughly characterized a novel rabbit monoclonal antibody (MAb 54-1) that reacts with high affinity and specificity with at least two isoforms of human brachyury. We demonstrated that brachyury protein is expressed both in primary and metastatic lesions of lung and breast cancer. This pattern of expression in tumors contrasted with the expression of brachyury in normal tissues, which was limited to normal testis and some thyroid tissues. Utilizing the MAb 54-1 antibody we have now characterized the pattern of brachyury nuclear vs. cytosolic expression in a range of cancer types, including in chordoma and other types of rare carcinomas. In breast carcinomas, a comprehensive study of brachyury demonstrated that (a) this EMT driver is over-expressed in primary as well as metastatic breast cancer tissues; (b) high levels of brachyury mRNA in primary breast tumors correlate with poor clinical outcome in patients treated with tamoxifen therapy for 5 years post-surgery; (c) brachyury induces resistance of breast carcinoma cells to the chemotherapy docetaxel and correlates with markers of tumor stemness; (d) brachyury-specific T cells can effectively lyse brachyury-positive breast tumor cells in an MHC-restricted manner. These studies provided a rationale for the use of vaccine approaches targeting brachyury for the therapy of human breast cancer, either as monotherapy or in combination therapies. Currently, two vaccine platforms, a recombinant yeast-brachyury vaccine and a pox viral vector brachyury vaccine are undergoing Phase I and II clinical trials. As brachyury (and the phenomenon of EMT in general) are able to induce tumor resistance to cell death, we also evaluated in detail whether brachyury-induced EMT could induce resistance to immune-mediated lysis of tumor cells. By generating isogenic cancer cell lines with various levels of brachyury, we demonstrated that high levels of this transcription factor can also significantly reduce the susceptibility of cancer cells to lysis by both antigen-specific T cells and natural killer (NK) cells. Our results indicated that resistance of brachyury-high tumor cells to immune-mediated lysis was due to inefficient caspase dependent apoptosis, manifested as inefficient nuclear lamin degradation in the presence of activated effector caspases. The resistance of brachyury-high tumor cells was attributed to the loss of cell-cycle-dependent kinase 1 (CDK1), which mediates lamin phosphorylation. Pretreatment of tumor cells with a specific inhibitor of WEE1, a negative regulator kinase of CDK1, was shown to counter the defective apoptosis of tumor cells expressing high levels of brachyury. Thus, our findings suggested that reconstituting CDK1 activity to threshold levels may be sufficient to restore immunosurveillance of mesenchymal-like cancer cells that have escaped previous immune detection or eradication.

National Institute of Health (NIH)
National Cancer Institute (NCI)
Scientific Cores Intramural Research (ZIC)
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Hamilton, Duane H; McCampbell, Kristen K; Palena, Claudia (2018) Loss of the Cyclin-Dependent Kinase Inhibitor 1 in the Context of Brachyury-Mediated Phenotypic Plasticity Drives Tumor Resistance to Immune Attack. Front Oncol 8:143
Hamilton, Duane H; David, Justin M; Dominguez, Charli et al. (2017) Development of Cancer Vaccines Targeting Brachyury, a Transcription Factor Associated with Tumor Epithelial-Mesenchymal Transition. Cells Tissues Organs 203:128-138
David, Justin M; Dominguez, Charli; McCampbell, Kristen K et al. (2017) A novel bifunctional anti-PD-L1/TGF-? Trap fusion protein (M7824) efficiently reverts mesenchymalization of human lung cancer cells. Oncoimmunology 6:e1349589
Dominguez, Charli; McCampbell, Kristen K; David, Justin M et al. (2017) Neutralization of IL-8 decreases tumor PMN-MDSCs and reduces mesenchymalization of claudin-low triple-negative breast cancer. JCI Insight 2:
Dominguez, Charli; David, Justin M; Palena, Claudia (2017) Epithelial-mesenchymal transition and inflammation at the site of the primary tumor. Semin Cancer Biol 47:177-184
Shah, Sagar R; David, Justin M; Tippens, Nathaniel D et al. (2017) Brachyury-YAP Regulatory Axis Drives Stemness and Growth in Cancer. Cell Rep 21:495-507
Heery, Christopher R; Palena, Claudia; McMahon, Sheri et al. (2017) Phase I Study of a Poxviral TRICOM-Based Vaccine Directed Against the Transcription Factor Brachyury. Clin Cancer Res 23:6833-6845
David, Justin M; Dominguez, Charli; Palena, Claudia (2017) Pharmacological and immunological targeting of tumor mesenchymalization. Pharmacol Ther 170:212-225
Tsang, Kwong Y; Fantini, Massimo; Fernando, Romaine I et al. (2017) Identification and characterization of enhancer agonist human cytotoxic T-cell epitopes of the human papillomavirus type 16 (HPV16) E6/E7. Vaccine 35:2605-2611
Fernando, Romaine I; Hamilton, Duane H; Dominguez, Charli et al. (2016) IL-8 signaling is involved in resistance of lung carcinoma cells to erlotinib. Oncotarget :

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