Our laboratory has previously identified the T-box transcription factor brachyury as a driver of human carcinoma EMT. We have shown that high levels of expression of brachyury drive carcinoma cells into a mesenchymal-like phenotype, increase tumor cell motility and invasiveness in vitro, and enhance tumor cell dissemination in vivo in animal models. High levels of brachyury have been reported in various types of carcinomas, including lung, colon, prostate and breast, and our laboratory has recently demonstrated the predominant overexpression of this embryonic transcription factor in triple negative breast cancer (TNBC). Analysis of breast cancer datasets demonstrated a predominant expression of brachyury mRNA in TNBC and in basal vs luminal or HER2 molecular breast cancer subtypes. At the protein level, variable levels of brachyury expression were detected both in primary and metastatic TNBC lesions. A strong association was observed between nuclear brachyury protein expression and the stage of disease, with nuclear brachyury being more predominant in metastatic vs primary tumors. Survival analysis also demonstrated an association between high levels of brachyury in the primary tumor and poor prognosis. Two brachyury-targeting cancer vaccines are currently undergoing clinical evaluation; the data presented here provide rationale for using brachyury-targeting immunotherapy approaches for the treatment of TNBC. 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, small-molecule targeted therapies and immune-mediated cytotoxicity. In search for mechanisms of immunotherapy resistance of tumor cells undergoing EMT, we have also reported a positive association between brachyury and MUC1, a tumor antigen overexpressed in most types of carcinomas, which mediates oncogenic signaling and confers resistance to genotoxic agents. We found that MUC1 is upregulated in tumor cell lines that highly express brachyury, a positive association that was also observed with patient-derived tumor tissues. Inhibition of MUC1 by siRNA-based gene silencing markedly enhanced the susceptibility of brachyury-high tumor cells to killing by immune cytotoxic cells, thus suggesting that inhibition of MUC1 can restore the susceptibility of mesenchymal-like cancer cells to immune attack. In collaboration with a team of scientists at the National Center for Advancing Translational Sciences (NCATS), we recently performed a high-throughput screening of compound libraries to identify drugs that may be used to enhance the sensitivity of mesenchymalized, highly invasive carcinoma cells to immune-mediated lysis. This led us to the identification of fulvestrant, an estrogen receptor antagonist approved and widely used in humans, for sensitization of lung cancer cells to immune-mediated lysis. The results of these studies also uncovered a novel association between the acquisition of mesenchymal features by lung carcinoma cells and the expression of estrogen receptor 1 (ESR1). We showed that acquisition of resistance to chemotherapy results in enhanced expression of ESR1, and while chemo-resistant lung cancer cells are also resistant to immune effector cells, blockade of estrogen signaling via fulvestrant was shown to improve lysis. Our data supports further investigations on the use of fulvestrant in combination with chemotherapy or immunotherapy for the treatment of lung cancer. A signaling pathway that is frequently deregulated in human carcinomas and has been explored as a therapeutic target involves the activation of the epidermal growth factor receptor (EGFR). Studies conducted in our laboratory exploring EGFR inhibition via erlotinib in EGFR-mutated NSCLC cells showed that short-term pre-treatment with low dose erlotinib leads to the enhancement of tumor cell lysis mediated by innate NK cells or antigen-specific T cells, in the context of phenotypic modulation. This effect, however, was lost when erlotinib was utilized for long periods of time, which resulted in tumor mesenchymalization in vitro or in vivo, with decreased (rather than increased) tumor lysis in response to immune effector mechanisms. Interestingly, inhibition of EGFR via the small-molecule inhibitors erlotinib and gefitinib commonly results in tumor resistance, even in patients with EGFR-mutant tumors that initially show substantial clinical responses. We have now demonstrated that generation of erlotinib-resistant lung cancer cells in vitro results in phenotypic alterations reminiscent of an EMT, concomitant with a robust upregulation of the IL-8/IL-8R axis. Blockade of IL-8 signaling was able to effectively reduce mesenchymal features of the resistant cells and also to markedly enhance their susceptibility to erlotinib. These results provided the rationale for the development of new therapeutic approaches involving blockade of IL-8 signaling for the management of acquired resistance to EGFR inhibition in patients with lung cancer. In recent years, encouraging clinical results have been achieved in various tumor types using monoclonal antibodies that target immune checkpoints such as cytotoxic T lymphocyte associated protein-4 (CTLA-4), programmed cell death-1 (PD-1), and programmed cell death ligand-1 (PD-L1). Recent advances have revealed that EMT may also suppress antitumor immunity through upregulation of PD-L1. Our laboratory has now demonstrated the effect of TGF-B1, a cytokine known to induce EMT and to suppress antitumor immunity, on tumor PD-L1 expression in several epithelial NSCLC cell lines. We showed that upregulation of PD-L1 takes place in the context of TGF-B1-mediated mesenchymalization, a phenomenon that occurs at the transcriptional level via phosphorylation of Smad2, a key downstream effector of TGF-B signaling. Utilizing a novel bifunctional fusion protein designated M7824, which consists of an anti-PD-L1 antibody moiety linked to the extracellular domain of two TGFBRII molecules, we demonstrated that TGF-B1-dependent mesenchymalization can be prevented/reduced by M7824 treatment in NSCLC cells. Moreover, TGF-B1-mediated upregulation of PD-L1 was found to enhance the susceptibility of NSCLC cells to ADCC mediated by M7824. These findings identify upregulation of PD-L1 as an additional mechanism of TGF-B1-induced suppression of antitumor immunity, and provide further rationale for using this novel agent, M7824, to treat patients with NSCLC and potentially other malignancies.

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National Cancer Institute (NCI)
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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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