Lynn M. Herbert

Up-Regulation of Matrix Metalloproteinase-9 in T Lymphocytes of Mammary Tumor Bearers: Role of Vascular Endothelial Growth Factor

Matrix metalloproteinase-9 (MMP-9), a matrix-degrading enzyme, is crucial in tumor invasion and metastasis and is implicated in leukocyte extravasation. In this report, we demonstrate that during growth of the D1–7,12-dimethylbenzanthracene-3 mammary tumor in BALB/c mice, there is progressive up-regulation of MMP-9 in splenic T cells at both the transcriptional and translational levels. Our previous work has identified several factors produced by this tumor, including PGE2, GM-CSF, and phosphatidyl serine; however, none of these agents induces increased production of MMP-9 by normal splenic T cells. Although not produced by the tumor, TNF-α and IL-6 are up-regulated in both macrophages and B cells in tumor-bearing mice. Exposure of normal T cells to these two cytokines, however, also fails to up-regulate MMP-9 production. Vascular endothelial growth factor (VEGF) is produced by many tumors, and we determined that the mammary tumor used in our studies expresses high levels of this angiogenic growth factor. Importantly, splenic T cells from tumor bearers constitutively produce increased amounts of VEGF, and treatment of normal T cells with VEGF results in up-regulated MMP-9 production. Of crucial importance is the finding that tumor-infiltrating T cells also produce high levels of VEGF and MMP-9. Our studies indicate that VEGF can act directly on T lymphocytes and that elevated VEGF levels may contribute to the aberrant MMP-9 secretion by mammary tumor bearers’ T cells.

MUC1/sec-Expressing Tumors Are Rejected In Vivo by a T Cell-Dependent Mechanism and Secrete High Levels of CCL2

MUC1/sec is a secreted form of the glycoprotein mucin 1 (MUC1). To characterize the role that MUC1 and MUC1/sec have in tumor progression, these genes were expressed in DA-3 mammary tumor cells. DA-3 cells and DA-3 cells expressing the transmembrane MUC1 gene (DA-3/TM) grow with similar kinetics in BALB/c mice. Surprisingly, DA-3 cells expressing and secreting MUC1/sec (DA-3/sec) fail to form tumors in vivo. The mechanism of rejection was evaluated using mice deficient in constituents of the immune system. All mice lacking IFN-γ, NK, NKT, or macrophages formed DA-3/sec tumors that regressed shortly after implantation. However, progressively growing DA-3/sec tumors developed in mice devoid of T lymphocytes. The importance of T lymphocytes in the rejection of DA-3/sec tumors was further supported by detection of DA-3-specific CTL in mice challenged with the DA-3/sec tumor. Recruitment of appropriate APC and effector cells is an important first step in the tumor clearance. Indeed, DA-3/sec cells or cell supernatants recruited 3–4 times as many macrophages as DA-3/TM cells in vivo, suggesting that a secreted chemotactic product is produced from DA-3/sec cells. RNA and protein analysis of DA-3/sec cells revealed that several genes are up-regulated by MUC1/sec expression, including MCP-1 (CCL-2). These results suggest DA-3/sec cells are capable of recruiting immune cells, and that rejection of DA-3/sec tumors, although aided by cells of the innate immune response, is ultimately due to T cell-mediated events.

A Unique Mucin Immunoenhancing Peptide with Antitumor Properties

Implantation of DA-3 mammary tumor cells into BALB/c mice results in tumor growth, metastatic lesions, and death. These cells were transfected with genes encoding for either the transmembrane (DA-3/TM) or secreted (DA-3/sec) form of human mucin 1 (MUC1). Although the gene for the secreted form lacks the transmembrane and cytoplasmic domains, the 5′ sequences of these mucins are identical; however, the gene for the secreted mucin isoform ends with a sequence encoding for a unique 11 amino acid peptide. The DA-3/TM or DA-3 cells transfected with the neomycin vector only (DA-3/neo) have the same in vivo growth characteristics as the parent cell line. In contrast, DA-3/sec cells fail to grow when implanted in immunocompetent BALB/c animals. DA-3/sec cells implanted in nude mice resulted in tumor development verifying the tumorigenic potential of these cells. Pre-exposure of BALB/c mice to DA-3/sec cells afforded protection against challenge with DA-3/TM or DA-3/neo mammary tumors and the unrelated tumors K7, an osteosarcoma, and RENCA, a renal cell carcinoma. Partial protection against subsequent tumor challenges was also achieved by substituting the 11 amino acid peptide found only in the secreted MUC1 isoform, for the live DA-3/sec cells. Notably, the efficacy of this peptide is not strain restricted because it also retarded the growth of Lewis lung carcinoma cells in C57 BL/6 mice. These findings reveal that a unique peptide present in the secreted MUC1 has immunoenhancing properties and may be a potential agent for use in immunotherapy.

Antitumor Effects of Mucin 1/sec Involves the Modulation of Urokinase-Type Plasminogen Activator and Signal Transducer and Activator of Transcription 1 Expression in Tumor Cells

Expression of the transmembrane isoform of Mucin 1 (MUC1/TM) in an aggressive murine mammary tumor line, DA-3, does not alter tumor development and metastasis, leading to death of the host. However, tumor cells expressing a secreted isoform of MUC1 (MUC1/sec) fail to develop tumors in immunocompetent mice. The rejection of MUC1/sec-expressing tumor cells is immunologically mediated, as, initially, innate cells and, ultimately, T cells are required. After gene array analysis, and confirmation at the protein level, it was discovered that MUC1/sec-expressing tumor cells (DA-3/sec) have a significant reduction in expression of urokinase-type plasminogen activator (uPA) relative to the parental tumor line and tumor cells expressing MUC1/TM. The serine protease uPA has been found to be involved in growth-promoting signaling, angiogenesis, and induction of matrix remodeling leading to metastasis. Although the tumor-promoting Stat3 transcription factor was unaltered in these tumor cells, the tumor-suppressive and IFN-responsive signal transducer and activator of transcription 1 (Stat1) is dramatically up-regulated in DA-3/sec cells. In addition, treatment of various murine and human cell lines with conditioned medium containing MUC1/sec results in up-regulation of Stat1. DA-3/sec tumor cells are also sensitized to the antiproliferative effects of IFN-gamma. Furthermore, transfection of the Stat1 gene into DA-3 tumor cells leads to a down-regulation of uPA and delays tumor progression. Thus, Stat1 up-regulation in DA-3/sec cells seems to play a significant role in the mechanism(s) by which rejection of tumor cells expressing MUC1/sec may be occurring.

Cytotoxic Effector Mechanisms Involved in the Immunity Against Mammary Tumors

There is considerable evidence that the initiation and growth of tumor masses can be modulated by the immune system. This influence not only applies to the primary tumor burdens, but is also apparent in the establishment and development of metastases. Several immunologic components are involved in these processes, however, the relevance of the cell-mediated compartment has been amply documented. The interactions among regulatory, helper, and effector cells are undeniably important, however, in many instances, the direct actions of several types of immune cells with lytic potential represent an important aspect of tumor immunology. Using animal models of breast cancer and, on a smaller scale, clinical experimentation, much information has been generated in several laboratories, providing insight into the relative contributions of various types of cytotoxic effectors operative against mammary tumors. Our understanding of the function of such cells in the context of in vivo tumor development is a first step in devising logical protocols for immu-notherapeutic approaches.

Abstract 3916: Inhibition of GREB1 mRNA and protein limits ER+ breast tumor cell proliferation

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Discovering the mechanism by which estrogen regulates breast cancer growth will aid in the identification of patients who will benefit from endocrine therapy. Estrogen (E2) binding to the estrogen receptor (ER) stimulates the proliferation of normal mammary cells and some breast cancers. Preventing ER expression or receptor-ligand interaction in hormone-responsive breast cancers inhibits cell division and promotes tumor cell death. However, the mechanism of E2-regulated breast cancer cell growth remains unclear. Although E2-regulated genes have been identified, those critically involved in growth regulation remain elusive. Identification of critical E2-regulated genes involved in mammary cell proliferation would elucidate the key pathway(s) supporting hormone-mediated tumor growth as well as provide insight into mechanisms of resistance to endocrine therapies and potential prognostic and therapeutic targets for ER+ breast cancer treatment. Gene regulated in breast cancer 1 (GREB1) is an estrogen-regulated gene that has been implicated in hormone-stimulated cell proliferation and is a candidate clinical marker for response to endocrine therapy. GREB1 mRNA and protein expression correlate with ER expression in breast tumors and the addition of estrogen to ER+ breast cancer cell lines leads to an increase in GREB1 expression and cell proliferation. The function of GREB1 and its role in ER+ breast tumors remains undefined. Experiments were designed to elucidate the role of GREB1 in ER+ breast cancer cell lines. ER+ breast cancer cell lines were transfected with GREB1-specific siRNA and control species. These tumor cell populations were evaluated for GREB1 mRNA and protein expression, cell growth and proliferation as well as invasion and migration in vitro. Sequence-specific siRNA effectively inhibited GREB1 mRNA and protein expression for more than 96 hours in MCF7 and T47D ER+ breast tumor cell lines. Utilizing the real-time Xcelligence system to monitor cell growth in culture suggested tumor cells treated with siRNA targeting GREB1 had significantly retarded cell proliferation. These observations suggest that GREB1 is a critical E2-regulated gene involved in breast tumor cell proliferation and is an exploitable target for novel breast cancer therapies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3916. doi:1538-7445.AM2012-3916

P5-01-15: The Functional Role of the Estrogen-Regulated Gene GREB1: Characterization of a Novel GREB1 Knockout Mouse Model.

Background: Gene regulated in breast cancer 1 (GREB1) was initially discovered in breast cancers as an estrogen-regulated gene that mediates estrogen-stimulated cell proliferation and is a candidate clinical marker for response to endocrine therapy. However, little is known of the functional role of GREB1 protein in normal breast tissue or breast cancers. Methods: To address this unknown role, our laboratory designed and created a novel Greb1 Knockout Mouse model (C57/bl MEL Greb1 KO). This constitutive model results in the loss of Greb1 mRNA and protein expression in cells where expression of Cre recombinase promotes the cleavage of exon 1 and intron 1 of the gene encoding Greb1. ROSA26 Cre C57/b1 MEL Greb1 KO mice heterozygous for the floxed Greb1 allele were crossed to generate experimental litters. Initial experiments were designed to evaluate if the complete loss of Greb1 expression in offspring homozygous for the floxed Greb1 allele was lethal during gestation. Experimental litters were tail clipped and genotyped using gDNA and genotype-specific PCR. Results: Offspring homozygous for the floxed Greb1 allele were identified in expected Mendelian ratios with wild type and heterozygous siblings. Loss of Greb1 expression was confirmed using RT-PCR, in situ hybridization and immunoblotting. Loss of both Greb1 alleles was not observed to be lethal during gestation for either male or female pups. Preliminary gross observation of these homozygous KO mice revealed no overt anatomical differences, however, they were 25–30% smaller than their heterozygous and wild-type siblings. Breeding experiments are underway to determine the fertility of crossbred Greb1 homozygous KO mice. Imaging experiments and necropsy with histochemical analysis of tissues will reveal any alteration in architecture and function. These findings will be summarized in this presentation. Discussion: As GREB1 has been identified as an estrogen-regulated gene involved in breast cancer cell proliferation and a potential target for new therapeutic strategies, it is important to understand the contribution of GREB1 to the differentiation, development and function of normal tissues as well as in breast cancers. Characterization of this novel Greb1 KO mouse model will provide answers to these functional questions surrounding GREB1. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P5-01-15.

Abstract P2-06-18: The Role of GREB1 in Breast Cancer: Identifying GREB1 Binding Proteins and Functional Pathways

Gene regulated in breast cancer 1 (GREB1) was initially discovered in breast cancers as an estrogen-regulated gene that mediates estrogen-stimulated cell proliferation and is a candidate clinical marker for response to endocrine therapy. However, little is known of the functional role of GREB1 protein in breast cancers, its interactive protein partners or the critical cellular pathways where GREB1 is involved. Our team employed a unique two-hybrid system to begin identifying GREB1 binding proteins from normal and cancerous breast cells as well as elucidating the molecular pathways with GREB1 involvement. The Matchmaker Gold Yeast Two-Hybrid System (Clontech Laboratories Inc., Mountain View, CA) was used to identify GREB1 binding proteins from immortalized human mammary epithelial cells (HMECs) and breast cancer cell lines. GREB1 served as a “bait” protein (pGBKT7-GREB1) to screen “prey” proteins generated from cDNA libraries created from immortalized HMECs, GREB1+ (MCF7, T47D) and GREB1- (SKBR3, MDA-464) breast cancer cells lines in yeast (Y187 MATα strain). Protein interactions were stringently screened using GAL4 activation and binding domainmediated reporters including aureobasidin A antibiotic resistance, two nutritional reporters (Histidine and Adenine) and a colorimetric reporter. Prey plasmid DNA, containing cDNA for GREB1 interactive protein candidates, was isolated from selected yeast colonies and sequenced to identify potential GREB1 binding proteins. These individual prey plasmids were subsequently re-introduced to the Y2H system to verify GREB1 interaction and measure the strength of this interaction. Confirmed GREB1 binding protein candidates were further screened using the Matchmaker Mammalian Two-Hybrid System, where interactions between GREB1 and a candidate GREB1-binding protein are verified in mammalian cell background (HEK 293 cells) using a quantitative secreted alkaline phosphatase (SEAP) reporter. This second screen in the mammalian background, unique to this Y2H system, ensures the conformation of GREB1 and the prey protein is similar to their natural state in breast cells and reflects interactions between proteins with greater authenticity than can be achieved in yeast alone. Finally, commercial monoclonal antibodies for GREB1 binding proteins identified using these two-hybrid systems were used to perform confirmatory co-immunoprecipitation experiments with the novel GREB1 monoclonal antibody developed in our laboratory and breast cell line lysates. Several GREB1 binding proteins, including PTMS, CRABP2, DHTKD1 and SEC23B, were confirmed by yeast and mammalian two-hybrid systems using cDNAs from GREB1+ breast cancer cell lines as well as co-immunoprecipitation assays. Subsequent pathway analysis using public databases (ex: GeneGo, Ingenuity) with GREB1 and these interactive partners suggest that GREB1 may be involved in regulation of gene transcription and cell cycle progression. These preliminary findings substantiate a critical role for GREB1 in breast cancer cell proliferation. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P2-06-18.