Mary T. Litzinger

IL-8 Signaling Plays a Critical Role in the Epithelial–Mesenchymal Transition of Human Carcinoma Cells

The switch of tumor cells from an epithelial to a mesenchymal-like phenotype [designated as epithelial-to-mesenchymal transition (EMT)] is known to induce tumor cell motility and invasiveness, therefore promoting metastasis of solid carcinomas. Although multiple studies have focused on elucidating the signaling events that initiate this phenotypic switch, there has been so far no characterization of the pattern of soluble mediators released by tumor cells undergoing EMT, and the potential impact that this phenotypic switch could have on the remodeling of the tumor microenvironment. Here we show that induction of EMT in human carcinoma cells via overexpression of the transcription factor Brachyury is associated with enhanced secretion of multiple cytokines, chemokines, and angiogenic factors and, in particular, with the induction of the IL-8/IL-8R axis. Our results also indicate the essential role of interleukin 8 (IL-8) signaling for the acquisition and/or maintenance of the mesenchymal and invasive features of Brachyury-overexpressing tumor cells and show that IL-8 secreted by tumor cells undergoing EMT could potentiate tumor progression by inducing adjacent epithelial tumor cells into EMT. Altogether, our results emphasize the potential role of EMT in the modulation of the tumor microenvironment via secretion of multiple soluble mediators and suggest that IL-8 signaling blockade may provide a means of targeting mesenchymal-like, invasive tumor cells.

The T-box transcription factor Brachyury promotes epithelial-mesenchymal transition in human tumor cells

Metastatic disease is responsible for the majority of human cancer deaths. Understanding the molecular mechanisms of metastasis is a major step in designing effective cancer therapeutics. Here we show that the T-box transcription factor Brachyury induces in tumor cells epithelial-mesenchymal transition (EMT), an important step in the progression of primary tumors toward metastasis. Overexpression of Brachyury in human carcinoma cells induced changes characteristic of EMT, including upregulation of mesenchymal markers, downregulation of epithelial markers, and an increase in cell migration and invasion. Brachyury overexpression also repressed E-cadherin transcription, an effect partially mediated by Slug. Conversely, inhibition of Brachyury resulted in downregulation of mesenchymal markers and loss of cell migration and invasion and diminished the ability of human tumor cells to form lung metastases in a xenograft model. Furthermore, we found Brachyury to be overexpressed in various human tumor tissues and tumor cell lines compared with normal tissues. We also determined that the percentage of human lung tumor tissues positive for Brachyury expression increased with the stage of the tumor, indicating a potential association between Brachyury and tumor progression. The selective expression of Brachyury in tumor cells and its role in EMT and cancer progression suggest that Brachyury may be an attractive target for antitumor therapies.

The Human T-Box Mesodermal Transcription Factor Brachyury Is a Candidate Target for T-Cell–Mediated Cancer Immunotherapy

Purpose: Identification of tumor antigens is essential in advancing immune-based therapeutic interventions in cancer. Particularly attractive targets are those molecules that are selectively expressed by malignant cells and that are also essential for tumor progression. Experimental Design and Results: We have used a computer-based differential display analysis tool for mining of expressed sequence tag clusters in the human Unigene database and identified Brachyury as a novel tumor antigen. Brachyury, a member of the T-box transcription factor family, is a key player in mesoderm specification during embryonic development. Moreover, transcription factors that control mesoderm have been implicated in the epithelial-mesenchymal transition (EMT), which has been postulated to be a key step during tumor progression to metastasis. Reverse transcription-PCR analysis validated the in silico predictions and showed Brachyury expression in tumors of the small intestine, stomach, kidney, bladder, uterus, ovary, and testis, as well as in cell lines derived from lung, colon, and prostate carcinomas, but not in the vast majority of the normal tissues tested. An HLA-A0201 epitope of human Brachyury was identified that was able to expand T lymphocytes from blood of cancer patients and normal donors with the ability to lyse Brachyury-expressing tumor cells. Conclusions: To our knowledge, this is the first demonstration that (a) a T-box transcription factor and (b) a molecule implicated in mesodermal development, i.e., EMT, can be a potential target for human T-cell–mediated cancer immunotherapy.

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.

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.

Comparative analysis of MVA-CD40L and MVA-TRICOM vectors for enhancing the immunogenicity of chronic lymphocytic leukemia (CLL) cells

Adenoviral transduction with CD40L and poxviral transduction with B7-1, ICAM-1, and LFA-3 (TRICOM) have been used to enhance the antigen-presenting capacity of chronic lymphocytic leukemia (CLL) cells. This study compares the same vector (modified vaccinia virus strain Ankara (MVA)) encoding CD40L or TRICOM for its ability to enhance the immunogenicity of CLL cells. CLL cells from some patients showed differential responses to each vector in terms of induction of autologous T-cell responses. This study supports the rationale for the use of CLL cells modified ex vivo with pre-specified recombinant MVA vectors as a whole tumor-cell vaccine for immunotherapy in CLL patients.

Chronic lymphocytic leukemia (CLL) cells genetically modified to express B7-1, ICAM-1, and LFA-3 confer APC capacity to T cells from CLL patients

In chronic lymphocytic leukemia (CLL), malignant B cells and nonmalignant T cells exhibit dysfunction. We previously demonstrated that infection of CLL cells with modified vaccinia Ankara (MVA) expressing the costimulatory molecules B7-1, ICAM-1, and LFA-3 (designated TRICOM) increased expression of these costimulatory molecules on the surface of CLL cells and thus augmented their antigen-presenting capability. Here, we evaluate the effect of MVA-TRICOM-modified CLL cells on T cells. Following incubation with irradiated MVA-TRICOM-modified CLL cells, allogeneic and autologous CD4+ and CD8+ T cells expressed significantly higher levels of B7-1, ICAM-1, and LFA-3. We show that this increase was the result of physical acquisition from the antigen-presenting cells (APCs), and that purified T cells that acquired costimulatory molecules from MVA-TRICOM-modified CLL cells were able to stimulate the proliferation of untreated T cells. These results demonstrate for the first time that T cells from CLL patients can acquire multiple costimulatory molecules from autologous CLL cells and can then act as APCs themselves. Given the immunodeficiencies characteristic of CLL, enhancing the antigen-presenting function of CLL cells and T cells simultaneously could be a distinct advantage in the effort to elicit antitumor immune responses.

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 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.