Bruce Huang

Overexpression of the EMT driver brachyury in breast carcinomas: association with poor prognosis.

BACKGROUND The epithelial-mesenchymal transition (EMT) has been implicated as an important process in tumor cell invasion, metastasis, and drug resistance. The transcription factor brachyury has recently been described as a driver of EMT of human carcinoma cells. METHODS Brachyury mRNA and protein expression was analyzed in human breast carcinomas and benign tissues. The role of brachyury in breast tumor prognosis and drug resistance and the ability of brachyury-specific T cells to lyse human breast carcinoma cells were also evaluated. Kaplan-Meier analyses were used to evaluate the association between brachyury expression and survival. All statistical tests were two-sided. RESULTS The level of brachyury expression in breast cancer cells was positively associated with their ability to invade the extracellular matrix, efficiently form mammospheres in vitro, and resist the cytotoxic effect of docetaxel. A comparison of survival among breast cancer patients treated with tamoxifen in the adjuvant setting who had tumors with high vs low brachyury mRNA expression demonstrated that high expression of brachyury is associated as an independent variable with higher risk of recurrence (hazard ratio [HR] = 7.5; 95% confidence interval [CI] = 2.4 to 23.5; P = 5.14×10(-4)) and distant metastasis (HR = 15.2; 95% CI = 3.5 to 66.3; P = 3.01×10(-4)). We also demonstrated that brachyury-specific T cells can lyse human breast carcinoma cells. CONCLUSIONS The studies reported here provide the rationale for the use of a vaccine targeting brachyury for the therapy of human breast cancer, either as a monotherapy or in combination therapies.

Cancer Vaccines Targeting the Epithelial-Mesenchymal Transition: Tissue Distribution of Brachyury and Other Drivers of the Mesenchymal-Like Phenotype of Carcinomas

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.

WEE1 Inhibition Alleviates Resistance to Immune Attack of Tumor Cells Undergoing Epithelial–Mesenchymal Transition

Aberrant expression of the T-box transcription factor brachyury in human carcinomas drives the phenomenon of epithelial-mesenchymal transition (EMT), a phenotypic modulation that facilitates tumor dissemination and resistance to conventional therapies, including chemotherapy and radiotherapy. By generating isogenic cancer cell lines with various levels of brachyury expression, we demonstrate that high levels of brachyury also significantly reduce the susceptibility of cancer cells to lysis by both antigen-specific T cells and natural killer cells. Our results indicated that resistance of brachyury-high tumor cells to immune-mediated attack was due to inefficient caspase-dependent apoptosis, manifested as inefficient nuclear lamin degradation in the presence of activated effector caspases. We correlated this phenomenon with loss of cell-cycle-dependent kinase 1 (CDK1), which mediates lamin phosphorylation. In support of a causal connection, pretreatment of tumor cells with a specific inhibitor of WEE1, a negative regulator kinase of CDK1, could 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.

An autocrine loop between TGF-β1 and the transcription factor Brachyury controls the transition of human carcinoma cells into a mesenchymal phenotype

The epithelial–mesenchymal transition (EMT) is a process associated with the metastasis of solid tumors as well as with the acquisition of resistance to standard anticancer modalities. A major initiator of EMT in carcinoma cells is TGF-β, which has been shown to induce the expression of several transcription factors ultimately responsible for initiating and maintaining the EMT program. We have previously identified Brachyury, a T-box transcription factor, as an inducer of mesenchymal features in human carcinoma cells. In this study, a potential link between Brachyury and TGF-β signaling has been investigated. The results show for the first time that Brachyury expression is enhanced during TGF-β1–induced EMT in various human cancer cell lines, and that a positive feedback loop is established between Brachyury and TGF-β1 in mesenchymal-like tumor cells. In this context, Brachyury overexpression is shown to promote upregulation of TGF-β1 at the mRNA and protein levels, an effect mediated by activation of the TGF-β1 promoter in the presence of high levels of Brachyury. Furthermore, inhibition of TGF-β1 signaling by a small-molecule inhibitor of TGF-β receptor type I decreases Brachyury expression, induces a mesenchymal-to-epithelial transition, and renders cancer cells more susceptible to chemotherapy. This study thus has implications for the future development of clinical trials using TGF-β inhibitors in combination with other anticancer agents. Mol Cancer Ther; 12(9); 1805–15. ©2013 AACR.

Strategies to target molecules that control the acquisition of a mesenchymal-like phenotype by carcinoma cells.

The switch of carcinoma cells from an epithelial to a mesenchymal-like phenotype, via a process designated ‘epithelial-to-mesenchymal transition (EMT),’ has been recognized as a relevant step in the metastasis of solid tumors. Additionally, this phenotypic switch of carcinoma cells has been associated with the acquisition of tumor resistance mechanisms that reduce the antitumor effects of radiation, chemotherapy and some small-molecule-targeted therapies. As multiple signaling pathways and transcriptional regulators that play a role in this phenotypic switch are being identified, novel strategies can be designed to specifically target tumor cells with this metastatic and resistant phenotype. In particular, this review focuses on the potential use of cancer vaccine strategies to target tumor cells that exhibit a mesenchymal-like phenotype, with an emphasis on the characterization of a novel tumor antigen, Brachyury, which we have identified as a critical regulator of EMT in human cancer cells.

Brachyury, a driver of tumor invasiveness and resistance to multiple therapies, is a novel immunotherapy target

The epithelial-mesenchymal transition (EMT) is a normal developmental process that allows the conversion of epithelial, polarized and stationary cells into highly motile and invasive mesenchymal cells, a phenomenon required for the normal formation of the embryo. Recent investigations have demonstrated that a similar phenotypic switch can aberrantly take place during tumor progression, allowing epithelial cancer cells to lose cell polarity, epithelial markers and cell-to-cell contacts, while simultaneously acquiring mesenchymal-associated markers, cell motility and invasiveness. The T-box transcription factor brachyury has been recently characterized as a regulator of tumor EMT. As we demonstrate here, over-expression of brachyury in human carcinoma cells promotes tumor invasiveness in vitro and tumor dissemination in vivo, while endowing tumor cells with mechanisms of resistance to multiple therapies, including chemotherapy, radiation and some small targeted therapies. Targeting of brachyury, therefore, appears an interesting approach against tumor progression, however, due to its nuclear localization and the “undruggable” character of this kind of molecules, current options to target brachyury are limited. Our laboratory has investigated whether brachyury could be a target of an immunological approach. Our data indicate that brachyury is a tumor-associated antigen; by employing a murine monoclonal Ab against brachyury we have analyzed brachyury expression in a range of normal adult tissues and various tumor types. The brachyury protein was found expressed in non-small lung and breast carcinomas, both primary and metastases, while being absent in the majority of human adult normal tissues, with exception of low levels observed in testis and selected thyroid samples. In breast cancer, moreover, we have found an inverse association between the levels of brachyury expression and clinical outcome in patients receiving tamoxifen treatment. The immunogenicity of the brachyury protein has been characterized. A 9-mer epitope of brachyury has been identified and used to efficiently expand brachyury-specific T cells from the blood of cancer patients. We present data here on the use of these T cells to lyse brachyury-positive tumors in an antigen-specific, MHC-restricted fashion. Based on these studies, a brachyury-based yeast recombinant vaccine has been developed that is currently ongoing Phase I clinical testing in patients with advanced carcinomas.

Interleukin-8 drives epithelial-mesenchymal transition of human carcinomas

The phenomenon of epithelial-mesenchymal transition (EMT) has been proposed as a relevant event during carcinoma progression. Tumor EMT drives the phenotypic conversion of polarized, epithelial tumor cells into highly motile, mesenchymal-like cells, and promotes tumor dissemination and metastasis and the acquisition of tumor resistance to various therapies. In order to investigate the potential role of EMT in the modulation of the tumor microenvironment as well as to identify soluble factors that could initiate and/or maintain tumor EMT, isogenic tumor cell pairs with epithelial vs. mesenchymal-like features were generated via modulation of the expression of an EMT driver, the T-box transcription factor Brachyury. A characterization of the pattern of soluble factors secreted by each cell type showed that acquisition of a mesenchymal-like phenotype by epithelial tumor cells was associated with secretion of multiple cytokines and chemokines and, in particular, markedly enhanced the expression of the interleukin-8 (IL-8)/IL-8R axis. Interference with IL-8 signaling via IL-8 neutralization or blockade of IL-8 receptors was able to revert the invasive, mesenchymal phenotype of Brachyury-high tumor cells towards a non-invasive, epithelial one, therefore indicating that upregulation of the IL-8/IL-8R axis is critical for the maintenance of tumor EMT. The ability of IL-8 to induce EMT was also demonstrated on a panel of epithelial cancer cell lines that showed increased expression of mesenchmal markers, cell migration and invasion in response to recombinant IL-8. The correlation between Brachyury, tumor EMT and IL-8 expression was also investigated with various lung and colon cancer cell lines with acquired resistance to EGFR inhibition. These resistant cells exhibited changes characteristic of EMT, had high levels of brachyury and other EMT markers expression, and secreted significantly higher level of IL-8 compared to parental cells. Collectively, our results emphasize the role of the IL-8/IL-8R axis in the modulation of human tumor EMT and form the rationale for further investigations on the modulation of IL-8 signaling in the tumor microenvironment as an approach against carcinoma progression.

Abstract 1489: The T-box transcription factor Brachyury blocks cell cycle progression and mediates tumor resistance to chemotherapy and radiation.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC The T-box transcription factor Brachyury, a molecule frequently detected in human cancers but seldom found in normal adult tissue, has recently been proposed as a significant determinant of the epithelial-mesenchymal transition (EMT) in human carcinomas. In the current investigation, we present data demonstrating that in three different human lung carcinoma models, expression of Brachyury is associated with a mesenchymal phenotype. Additionally, elevated Brachyury expression is shown to strongly correlate with increased in vitro resistance to cytotoxic therapies, such as chemotherapy and radiation. Further investigation showed that chemotherapy treatment in vitro selected tumor cells that were high in Brachyury, and that the degree of resistance to therapy was comparable to the level of Brachyury expression. We also demonstrate that in vitro and in vivo, human lung carcinoma cells with greater levels of Brachyury divide at slower rates than those with lower levels of Brachyury, a phenomenon associated with marked downregulation of cyclin D1, phosphorylated Rb (pRb), and CDKN1A (p21). ChIP and luciferase resporter assays revealed that Brachyury represses p21 expression in carcinoma cells by directly binding to a half T-box consensus site located within the promoter region of the p21 gene, indicating a potential mechanism for the observed therapy resistance associated with Brachyury expression. Finally, we observed that in vivo treatment of tumor xenografts with chemotherapy resulted in the selective growth of resistant tumors that were high in Brachyury. Altogether, these results suggest that in addition to being a driver of EMT, Brachyury expression may attenuate cell cycle progression, and enable tumor cells to become less susceptible to chemotherapy and radiation in human carcinomas. Citation Format: Bruce K. Huang, Joseph Cohen, Romaine I. Fernando, Duane H. Hamilton, Mary T. Litzinger, James W. Hodge, Claudia M. Palena. The T-box transcription factor Brachyury blocks cell cycle progression and mediates tumor resistance to chemotherapy and radiation. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1489. doi:10.1158/1538-7445.AM2013-1489

Abstract A64: Brachyury-mediated epithelial-mesenchymal transition of human carcinoma cells is associated with an increased resistance to immune-mediated attack

The epithelial-mesenchymal transition (EMT) has recently been recognized as a relevant process during the progression of carcinomas. Epithelial tumor cells undergoing EMT have been shown to acquire a mesenchymal-like phenotype, cell motility, and invasiveness, in vivo metastatic propensity and stem cell-like properties, including the ability to resist cell death initiated by traditional cancer treatment modalities such as chemotherapy and radiation. The development of therapeutic interventions aimed at interfering with EMT is therefore emerging as a rational approach for the prevention of cancer metastasis and alleviation of therapeutic resistance. Promising results recently obtained in preclinical and clinical studies with cancer vaccines may lead to their use for the treatment of various types of cancer; the concept of utilizing a vaccine to specifically target essential regulators of EMT is yet to be exploited in the field of tumor immunology. Recently, we have demonstrated that Brachyury, a T-box transcription factor essential for embryonic development, plays a central role in the induction of EMT in human carcinoma cells. In contrast to other drivers of EMT, Brachyury is highly expressed in various types of human tumors, while its expression in most normal adult tissues is undetectable. Moreover, Brachyury-specific T cells capable of lysing human tumor cell lines in an antigen-specific, HLA-restricted manner can be expanded from the peripheral blood of cancer patients, making Brachyury an attractive target for a vaccination strategy designed to specifically target cells undergoing an EMT. In light of the documented resistance of mesenchymal-like tumor cells to chemotherapy and radiation, we have thoroughly investigated the susceptibility of tumor cells undergoing EMT to immune-mediated attack. Utilizing several epithelial human tumor cell lines stably transfected to express various levels of Brachyury, we have observed that tumor cells with low to moderate amounts of Brachyury can be efficiently lysed by various immune-mediated mechanisms, while very high levels of Brachyury expression correlate with increased resistance to lysis mediated by natural killer (NK) cells, lymphokine-activated killer (LAK) cells, and antigen-specific CD8+ T cells. It has been suggested that EMT is a reversible process, whereby cells can undergo a mesenchymal-epithelial transition (MET). By stably inhibiting the expression of Brachyury in various mesenchymal-like human tumor cell lines, we have also demonstrated that induction of MET restores susceptibility of tumor cells to immune-mediated attack. Currently, the mechanisms responsible for the acquisition of increased resistance to immune-mediated killing by cells undergoing an EMT are being investigated. The results from these studies will be fundamental in designing strategies aimed at rendering metastatic carcinoma cells more susceptible not only to chemotherapy and radiation, but also to immunotherapeutic interventions against the tumor. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr A64.

Abstract C53: Overexpression of Brachyury in human carcinoma cells drives the acquisition of resistance to anticancer therapeutics

The epithelial-mesenchymal transition (EMT) has been recognized as a process crucial to the progression of carcinomas, mediating the conversion of stationary epithelial tumor cells into mesenchymal-like, invasive tumor cells. Recent reports have also demonstrated a potential association between tumor EMT and the acquisition of resistance to cell death in carcinoma cells. We recently identified the T-box transcription factor Brachyury, a molecule prevalently expressed in human tumors but seldom found in normal adult tissues, as a novel driver of EMT in human carcinomas. Brachyury was demonstrated to induce the expression of molecules associated with the mesenchymal phenotype, tumor cell motility and invasiveness in vitro, as well as metastatic propensity in xenograft models. In the current study, we investigated whether deregulated expression of Brachyury in human lung tumor cells is also associated with acquisition of resistance to the conventional anti-cancer modalities, chemotherapy and radiation. Our results demonstrated that over-expression of Brachyury in epithelial lung tumor cells significantly improved their survival in response to treatment with various doses of Taxotere, Cisplatin, Vinorelbine, and combinations of Cisplatin plus Vinorelbine, as well as to various doses of gamma-radiation. Similarly, inhibition of Brachyury expression by using Brachyury-specific shRNA constructs in mesenchymal-like, human lung tumor cells resulted in enhanced susceptibility to the same cytotoxic agents. Analysis of clonal lines derived from single-cell isolates of human lung tumor cells expressing various amounts of Brachyury revealed an inverse relationship between Brachyury levels and tumor growth, and a positive correlation of Brachyury with the ability to resist treatment by Taxotere, Cisplatin, Vinorelbine, and Cisplatin plus Vinorelbine combinations. Detailed gene expression analysis demonstrated a positive association between Brachyury expression and markers of tumor stemness, such as the MDR1/ABCB1 transporter, and the self-renewal transcription factors Sox-2, Oct4, and Nanog. These results thus raise the possibility that in addition to being a mediator of EMT, Brachyury may also be a marker for human tumor cells bearing stem-like characteristics and resistance to conventional therapeutics. One approach that may overcome the therapeutic resistance of tumors undergoing Brachyury-mediated EMT is that of immune-mediated targeting – we have previously characterized the immunogenicity of the Brachyury protein and generated Brachyury-specific human T-cell lines that have the ability to lyse Brachyury-positive tumor cells. We hypothesize that the eradication of Brachyury-expressing tumor cells via Brachyury-based immunotherapeutic approaches could be efficient at eliminating tumor cells with metastatic propensity as well as at alleviating tumor resistance in response to conventional therapies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr C53.