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The plasminogen activation system regulates the activity of the serine protease, plasmin. The role of plasminogen receptors in cancer progression is being increasingly appreciated as key players in modulation of the tumor microenvironment. The interaction of plasminogen with cells to promote plasminogen activation requires the presence of proteins exposing C-terminal lysines on the cell surface. Plg-R KT is a structurally unique plasminogen receptor because it is an integral membrane protein that is synthesized with and binds plasminogen via a C-terminal lysine exposed on the cell surface. Here, we have investigated the expression of Plg-R KT in human breast tumors and human breast cancer cell lines. Breast cancer progression tissue microarrays were probed with anti-Plg-R KT mAB and we found that Plg-R KT is widely expressed in human breast tumors, that its expression is increased in tumors that have spread to draining lymph nodes and distant organs, and that Plg-R KT expression is most pronounced in hormone receptor (HR)-positive tumors. Plg-R KT was detected by Western blotting in human breast cancer cell lines. By flow cytometry, Plg-R KT cell surface expression was highest on the most aggressive tumor cell line. Future studies are warranted to address the functions of Plg-R KT in breast cancer.

Purpose: Individual or combined strategies of cellular therapy with alloreactive CTLs (alloCTL) and gene therapy using retroviral replicating vectors (RRV) encoding a suicide prodrug activating gene were explored for the treatment of breast tumors metastatic to the brain., Experimental Design: AlloCTL, sensitized to the HLA of MDA-MB-231 breast cancer cells, were examined in vitro for antitumor functionality toward breast cancer targets. RRV encoding the yeast cytosine deaminase (CD) gene was tested in vivo for virus spread, ability to infect, and kill breast cancer targets when exposed to 5-fluorocytosine (5-FC). Individual and combination treatments were tested in subcutaneous and intracranial xenograft models with 231BR, a brain tropic variant., Results: AlloCTL preparations were cytotoxic, proliferated, and produced IFN-γ when coincubated with target cells displaying relevant HLA. In vivo, intratumorally placed alloCTL trafficked through one established intracranial 231BR focus to another in contralateral brain and induced tumor cell apoptosis. RRV-CD efficiently spread in vivo, infected 231BR and induced their apoptosis upon 5-FC exposure. Subcutaneous tumor volumes were significantly reduced in alloCTL and/or gene therapy-treated groups compared to control groups. Mice with established intracranial 231BR tumors treated with combined alloCTL and RRV-CD had a median survival of 97.5 days compared with single modalities (50-83 days); all experimental treatment groups survived significantly longer than sham-treated groups (median survivals 31.5 or 40 days) and exhibited good safety/toxicity profiles., Conclusion: The results indicate combining cellular and suicide gene therapies is a viable strategy for the treatment of established breast tumors in the brain., (©2013 AACR.)

Several markers identify cancer stem cell-like populations, but little is known about the functional roles of stem cell surface receptors in tumor progression. Here, we show that the endothelial protein C receptor (EPCR), a stem cell marker in hematopoietic, neuronal and epithelial cells, is crucial for breast cancer growth in the orthotopic microenvironment of the mammary gland. Mice with a hypomorphic allele of EPCR show reduced tumor growth in the PyMT-model of spontaneous breast cancer development and deletion of EPCR in established PyMT tumor cells significantly attenuates transplanted tumor take and growth. We find expansion of EPCR(+) cancer stem cell-like populations in aggressive, mammary fat pad-enhanced human triple negative breast cancer cells. In this model, EPCR-expressing cells have markedly increased mammosphere- and tumor-cell initiating activity compared to another stable progenitor-like subpopulation present at comparable frequency. We show that receptor blocking antibodies to EPCR specifically attenuate in vivo tumor growth initiated by either EPCR(+) cells or the heterogenous mixture of EPCR(+) and EPCR(-) cells. Furthermore, we have identified tumor associated macrophages as a major source for recognized ligands of EPCR, suggesting a novel mechanism by which cancer stem cell-like populations are regulated by innate immune cells in the tumor microenvironment.

The chemokine receptor CXCR4 is functionally expressed on the cell surface of various cancer cells, and plays a role in cell proliferation and migration of these cells. Specifically, in breast cancer cells the CXCR4/CXCL12 axis has been implicated in cell migration in vitro and in metastasis in vivo, but the underlying signaling mechanisms are incompletely understood. The xenograft-derived MDA-MB-231 breast cancer cell line (231mfp), which was shown previously to grow more aggressively than the parent cells, showed increased CXCR4 expression at the mRNA, total protein and cell surface expression level. This correlated with an enhanced response to CXCL12, specifically in augmented and prolonged Akt activation in a G(i), Src family kinase and PI-3 kinase dependent fashion. 231mfp cells migrated towards CXCL12--in contrast to the parent cell line--and this chemotaxis was blocked by inhibition of G(i), Src family kinases, PI-3 kinase and interestingly, Akt itself, as could be shown with two pharmacological inhibitors, a dominant negative Akt construct and with Akt shRNA. Collectively, we have demonstrated that prolonged Akt activation is an important signaling pathway for breast cancer cells expressing CXCR4 and is necessary for CXCL12-dependent cell migration.

Coagulation activation by tissue factor (TF) is implicated in cancer progression, cancer-associated thrombosis and metastasis. The role of direct TF signaling pathways in cancer, however, remains incompletely understood. Here we address how TF contributes to primary tumor growth by using a unique pair of isotype-matched antibodies that inhibit either coagulation (monoclonal antibody [Mab]-5G9) or direct signaling (Mab-10H10). We demonstrate that the inhibitory antibody of direct TF-VIIa signaling not only blocks TF-VIIa mediated activation of PAR2, but also disrupts the interaction of TF with integrins. In epithelial and TF-expressing endothelial cells, association of TF with beta1 integrins is regulated by TF extracellular ligand binding and independent of PAR2 signaling or proteolytic activity of VIIa. In contrast, alpha3beta1 integrin association of TF is constitutive in breast cancer cells and blocked by Mab-10H10 but not by Mab-5G9. Mab-5G9 has antitumor activity in vivo, but we show here that Mab-10H10 is at least as effective in suppressing human xenograft tumors in 2 different models. Breast tumor growth was also attenuated by blocking PAR2 signaling. These results show that tumor cell TF-PAR2 signaling is crucial for tumor growth and suggest that anti-TF strategies can be applied in cancer therapy with minor impairment of TF-dependent hemostatic pathways.

Protease-activated receptor 2 (PAR2) is a G-protein coupled receptor that is cleaved and activated by serine proteases including the coagulation protease factor VIIa (FVIIa). There is evidence that PAR2 function contributes to angiogenesis, but the mechanisms involved are poorly defined. Here we show that PAR2 activation in human breast cancer cells leads to the upregulation of vascular endothelial growth factor (VEGF). Activation of PAR2 with agonist peptide (AP), trypsin or FVIIa results in a robust increase of VEGF message and protein. Incubation of cells with PAR1-AP, PAR3-AP, PAR4-AP, or thrombin has only a modest effect on VEGF production. Cleavage blocking antibodies show that FVIIa-mediated VEGF production is PAR2 mediated. Mitogen-activated protein kinase (MAPK) pathway inhibitors U0126 and SB203580 inhibit PAR2-mediated VEGF production. Incubation of cells with PAR2-AP leads to significant extracellular regulated kinase1/2 (ERK1/2) and p38 MAPK phosphorylation and activation. Collectively, these data suggest that PAR2 signaling through MAPK pathways leads to the production of proangiogenic VEGF in breast cancer cells.

Direct proliferative effects of estrogen (E(2)) on estrogen receptor-positive tumors are well documented; however, the potential for E(2) to mediate effects selective for the host (i.e., angiogenesis, vascular permeability, or stromal effects), which influence tumor growth and/or metastasis, has received less attention. In this study, we examine the capacity for E(2) to promote tumor growth and/or metastasis independent of direct effects on tumor cells. In these studies, we distinguish host versus tumor compartment components of E(2) action in tumor growth and metastasis by analysis of E(2)-nonresponsive tumor cells implanted in ovariectomized (OVX) mice that contain s.c. implants of placebo (OVX) or E(2)-containing slow-release pellets (OVX + E(2)). We show that the D121 lung carcinoma cell line is E(2)-nonresponsive, and following s.c. implantation in OVX versus OVX + E(2) mice, E(2) action on the host compartment leads to an increase in spontaneous metastasis but not primary tumor growth or neovascularization. Similarly, experimental lung metastasis of E(2)-nonresponsive 4T1 mammary carcinoma cells also leads to increased tumor burden in the lungs of OVX + E(2) mice. These results suggest that the E(2) status of the host compartment influences late steps in tumor cell metastasis that can provide important insights into the role of E(2) in the tumor versus host compartments.

Human cell lines constitute powerful model systems for the in vitro and in vivo analysis of cancer. Cancer lines that are invasive in culture often form tumors and metastases in immune deficient mice. It is generally assumed that, in such cases, the principal population of cancer cells in culture corresponds to the tumor-forming cells in vivo. Here, we high-light a recent functional proteomics investigation that suggests the contrary. In this study, cells derived from orthotopic xenograft tumors formed by the invasive breast cancer line MDA-MB-231 were found to exhibit profound differences in their enzyme activity profiles and increased tumor growth rates and metastasis when compared to the parental line. These findings suggest that the in vivo microenvironment of the mouse mammary fat pad cultivates the growth of human breast cancer cells with elevated tumorigenic properties. Characterization of the unique molecular properties of these tumor-forming cells may reveal new strategies for the diagnosis and treatment of breast cancer.

Cancer research depends on the use of human cell lines for both the in vitro (culture) and in vivo (xenograft) analysis of tumor progression and treatment. However, the extent to which cultured preparations of human cancer lines display similar properties in vivo, where important host factors may influence tumor biology, remains unclear. Here, we address this question by conducting a functional proteomic analysis of the human breast cancer line MDA-MB-231 grown in culture and as orthotopic xenograft tumors in the mammary fad pad of immunodeficient mice. Using a suite of activity-based chemical probes, we identified carcinoma (human) enzyme activities that were expressed selectively in culture or in xenograft tumors. Likewise, distinct groups of stromal (mouse) enzyme activities were found that either infiltrated or were excluded from xenograft tumors, indicating a contribution by specific host components to breast cancer development. MDA-MB-231 cells isolated from tumors exhibited profound differences in their enzyme activity profiles compared with the parent cell line, including the dramatic posttranscriptional up-regulation of the serine proteases urokinase plasminogen activator and tissue plasminogen activator and down-regulation of the glycolytic enzyme phosphofructokinase. These altered enzyme activity profiles correlated with significantly greater tumor growth rates and metastases for xenograft-derived MDA-MB-231 cells upon reintroduction into mice. Collectively, these data indicate that the in vivo environment of the mouse mammary fat pad cultivates the growth of human breast cancer cells with elevated tumorigenic properties and highlight the value of activity-based protein profiling for identifying proteomic signatures that depict such changes in cancer cell biology.