Belinda S. Parker

Silencing of Irf7 pathways in breast cancer cells promotes bone metastasis through immune escape

Breast cancer metastasis is a key determinant of long-term patient survival. By comparing the transcriptomes of primary and metastatic tumor cells in a mouse model of spontaneous bone metastasis, we found that a substantial number of genes suppressed in bone metastases are targets of the interferon regulatory factor Irf7. Restoration of Irf7 in tumor cells or administration of interferon led to reduced bone metastases and prolonged survival time. In mice deficient in the interferon (IFN) receptor or in natural killer (NK) and CD8+ T cell responses, metastasis was accelerated, indicating that Irf7-driven suppression of metastasis was reliant on IFN signaling to host immune cells. We confirmed the clinical relevance of these findings in over 800 patients in which high expression of Irf7-regulated genes in primary tumors was associated with prolonged bone metastasis–free survival. This gene signature may identify patients that could benefit from IFN-based therapies. Thus, we have identified an innate immune pathway intrinsic to breast cancer cells, the suppression of which restricts immunosurveillance to enable metastasis.

Alterations in vascular gene expression in invasive breast carcinoma.

The molecular signature that defines tumor microvasculature will likely provide clues as to how vascular-dependent tumor proliferation is regulated. Using purified endothelial cells, we generated a database of gene expression changes accompanying vascular proliferation in invasive breast cancer. In contrast to normal mammary vasculature, invasive breast cancer vasculature expresses extracellular matrix and surface proteins characteristic of proliferating and migrating endothelial cells. We define and validate the up-regulated expression of VE-cadherin and osteonectin in breast tumor vasculature. In contrast to other tumor types, invasive breast cancer vasculature induced a high expression level of specific transcription factors, including SNAIL1 and HEYL, that may drive gene expression changes necessary for breast tumor neovascularization. We demonstrate the expression of HEYL in tumor endothelial cells and additionally establish the ability of HEYL to both induce proliferation and attenuate programmed cell death of primary endothelial cells in vitro. We also establish that an additional intracellular protein and previously defined metastasis-associated gene, PRL3, appears to be expressed predominately in the vasculature of invasive breast cancers and is able to enhance the migration of endothelial cells in vitro. Together, our results provide unique insights into vascular regulation in breast tumors and suggest specific roles for genes in driving tumor angiogenesis.

Primary Tumor Hypoxia Recruits CD11b+/Ly6Cmed/Ly6G+ Immune Suppressor Cells and Compromises NK Cell Cytotoxicity in the Premetastatic Niche

Hypoxia within a tumor acts as a strong selective pressure that promotes angiogenesis, invasion, and metastatic spread. In this study, we used immune competent bone marrow chimeric mice and syngeneic orthotopic mammary cancer models to show that hypoxia in the primary tumor promotes premetastatic niche formation in secondary organs. Injection of mice with cell-free conditioned medium derived from hypoxic mammary tumor cells resulted in increased bone marrow-derived cell infiltration into the lung in the absence of a primary tumor and led to increased metastatic burden in mammary and melanoma experimental metastasis models. By characterizing the composition of infiltrating bone marrow-derived cells, we identified CD11b+/Ly6Cmed/Ly6G+ myeloid and CD3-/NK1.1+ immune cell lineages as key constituents of the premetastatic niche. Furthermore, the cytotoxicity of natural killer (NK) cells was significantly decreased, resulting in a reduced antitumor response that allowed metastasis formation in secondary organs to a similar extent as ablation of NK cells. In contrast, metastatic burden was decreased when active NK cells were present in premetastatic lungs. Together, our findings suggest that primary tumor hypoxia provides cytokines and growth factors capable of creating a premetastatic niche through recruitment of CD11b+/Ly6Cmed/Ly6G+ myeloid cells and a reduction in the cytotoxic effector functions of NK cell populations.

HOXB7, a Homeodomain Protein, Is Overexpressed in Breast Cancer and Confers Epithelial-Mesenchymal Transition

Epithelial-mesenchymal transition (EMT) is increasingly recognized as a mechanism whereby cells in primary noninvasive tumors acquire properties essential for migration and invasion. Microarray analyses of microdissected epithelial cells from bone metastasis revealed a HOXB7 overexpression that was 3-fold higher than in primary breast carcinomas and 18-fold higher compared with normal breast. This led us to investigate the role of HOXB7 in neoplastic transformation of breast cells. Expression of HOXB7 in both MCF10A and Madin-Darby canine kidney (MDCK) epithelial cells resulted in the acquisition of both phenotypic and molecular attributes typical of EMT. Loss of epithelial proteins, claudin 1 and claudin 7, mislocalization of claudin 4 and E-cadherin, and the expression of mesenchymal proteins, vimentin and alpha-smooth muscle actin, were observed. MDCK cells expressing HOXB7 exhibited properties of migration and invasion. Unlike MDCK vector-transfected cells, MDCK-HOXB7 cells formed highly vascularized tumors in mice. MDCK-HOXB7 cells overexpressed basic fibroblast growth factor (bFGF), had more active forms of both Ras and RhoA proteins, and displayed higher levels of phosphorylation of p44 and p42 mitogen-activated protein kinase (MAPK; extracellular signal-regulated kinases 1 and 2). Effects initiated by HOXB7 were reversed by specific inhibitors of FGF receptor and the Ras-MAPK pathways. These data provide support for a function for HOXB7 in promoting tumor invasion through activation of Ras/Rho pathway by up-regulating bFGF, a known transcriptional target of HOXB7. Reversal of these effects by HOXB7-specific siRNA further suggested that these effects were mediated by HOXB7. Thus, HOXB7 overexpression caused EMT in epithelial cells, accompanied by acquisition of aggressive properties of tumorigenicity, migration, and invasion.

Cathepsin B Inhibition Limits Bone Metastasis in Breast Cancer

Metastasis to bone is a major cause of morbidity in breast cancer patients, emphasizing the importance of identifying molecular drivers of bone metastasis for new therapeutic targets. The endogenous cysteine cathepsin inhibitor stefin A is a suppressor of breast cancer metastasis to bone that is coexpressed with cathepsin B in bone metastases. In this study, we used the immunocompetent 4T1.2 model of breast cancer which exhibits spontaneous bone metastasis to evaluate the function and therapeutic targeting potential of cathepsin B in this setting of advanced disease. Cathepsin B abundancy in the model mimicked human disease, both at the level of primary tumors and matched spinal metastases. RNA interference-mediated knockdown of cathepsin B in tumor cells reduced collagen I degradation in vitro and bone metastasis in vivo. Similarly, intraperitoneal administration of the highly selective cathepsin B inhibitor CA-074 reduced metastasis in tumor-bearing animals, a reduction that was not reproduced by the broad spectrum cysteine cathepsin inhibitor JPM-OEt. Notably, metastasis suppression by CA-074 was maintained in a late treatment setting, pointing to a role in metastatic outgrowth. Together, our findings established a prometastatic role for cathepsin B in distant metastasis and illustrated the therapeutic benefits of its selective inhibition in vivo.

Interferon-ε Protects the Female Reproductive Tract from Viral and Bacterial Infection

A Role for IFN-ɛ Type I interferons (IFNs) are critical cytokines involved in host defense against pathogens, particularly viruses. IFN-ɛ is an IFN-like gene encoded within the type I IFN locus in mice and humans whose function has not been characterized. Fung et al. (p. 1088) created mice with a genetic deletion in Ifn-ɛ and found that, like other type I IFNs, IFN-ɛ signals through the IFN-α receptors 1 and 2. However, unlike these other cytokines, which are primarily expressed by immune cells and are induced upon immune cell triggering, IFN-ɛ was expressed exclusively by epithelial cells of the female reproductive tract in both mice and humans and its expression was hormonally regulated. IFN-ɛ–deficient mice were more susceptible to infection with herpes simplex virus 2 and Chlamydia muridarum, two common sexually transmitted pathogens. The cytokine interferon-ε is expressed in the female reproductive tract and protects against sexually transmitted diseases. The innate immune system senses pathogens through pattern-recognition receptors (PRRs) that signal to induce effector cytokines, such as type I interferons (IFNs). We characterized IFN-ε as a type I IFN because it signaled via the Ifnar1 and Ifnar2 receptors to induce IFN-regulated genes. In contrast to other type I IFNs, IFN-ε was not induced by known PRR pathways; instead, IFN-ε was constitutively expressed by epithelial cells of the female reproductive tract (FRT) and was hormonally regulated. Ifn-ε–deficient mice had increased susceptibility to infection of the FRT by the common sexually transmitted infections (STIs) herpes simplex virus 2 and Chlamydia muridarum. Thus, IFN-ε is a potent antipathogen and immunoregulatory cytokine that may be important in combating STIs that represent a major global health and socioeconomic burden.

Primary tumour expression of the cysteine cathepsin inhibitor Stefin A inhibits distant metastasis in breast cancer

Using the clinically relevant 4T1‐derived syngeneic murine model of spontaneous mammary metastasis to bone, we have identified the cysteine cathepsin inhibitor Stefin A as a gene differentially expressed in primary and metastatic mammary tumours. In primary tumours, Stefin A expression correlated inversely with metastatic potential in 4T1‐derived lines and was not detected in tumour cells in culture, indicating induction only within the tumour microenvironment. Enforced expression of Stefin A in the highly metastatic 4T1.2 cell line significantly reduced spontaneous bone metastasis following orthotopic injection into the mammary gland. Consistent with the mouse data, Stefin A expression correlated with disease‐free survival (absence of distant metastasis) in a cohort of 142 primary tumours from breast cancer patients. This was most significant for patients with invasive ductal carcinoma expressing Stefin A, who were less likely to develop distant metastases (log rank test, p = 0.0075). In a multivariate disease‐free survival analysis (Cox proportional hazards model), Stefin A expression remained a significant independent prognostic factor in patients with invasive ductal carcinoma (p = 0.0014), along with grade and progesterone receptor (PR) status. In human lung and bone metastases, we detected irregular Stefin A staining patterns, with expression often localizing to micrometastases (<0.2 mm) in direct contact with the stroma. We propose that Stefin A, as a cysteine cathepsin inhibitor, may be a marker of increased cathepsin activity in metastases. Using immunohistology, the cathepsin inhibitor was detected co‐expressed with cathepsin B in lung and bone metastases in both the murine model and human tissues. We conclude that Stefin A expression reduces distant metastasis in breast cancer and propose that this may be due to the inhibition of cysteine cathepsins, such as cathepsin B. Copyright

Integrin-dependent response to laminin-511 regulates breast tumor cell invasion and metastasis

The basement membrane protein, laminin (LM)‐511, is a potent adhesive and migratory substrate for metastatic breast tumor cells in vitro. Its expression correlates with tumor grade and metastatic potential in vivo. These observations suggest that responsiveness to autocrine or paracrine‐derived LM‐511 may be an important property regulating breast cancer metastasis in vivo. To address this, we compared the metastatic potential of 4T1 mammary carcinoma cells to that of 4T1 variants isolated by repeated chemotactic migration toward LM‐511 in vitro (4T1LMF4) followed by serial injection into the mammary gland and recovery of spontaneous metastases from bone (4T1BM2). Variant subpopulations exhibited a distinct morphology on LM‐511 and increased expression of β1 and β4 integrins compared to parental 4T1 cells. Importantly, mice inoculated with 4T1LMF4 and 4T1BM2 variants showed a 2.5‐ to 4‐fold increase in the incidence of spontaneous metastasis to bone compared to 4T1 tumor‐bearing mice. Functionally, 4T1BM2 variants were more adherent and more invasive toward LM‐511 than parental 4T1 cells. Treatment of 4T1BM2 cells with lebein‐1, a disintegrin with selectivity toward LM‐type integrin receptors, potently inhibited their migration and invasion toward LM‐511. Similarly, α3β1 integrin‐dependent migration and invasion of human MDA‐MB‐231 breast carcinoma cells toward LM‐511 were significantly inhibited by lebein‐1. Taken together, these results provide strong evidence that LM‐511 contributes to the metastasis of breast tumors and suggest that targeting integrin‐LM‐511 interactions with lebein‐1 or other inhibitors of LM‐511 receptors may have therapeutic potential for patients with advanced breast cancer.

Vascular Normalization by Loss of Siah2 Results in Increased Chemotherapeutic Efficacy

Tumor hypoxia is associated with resistance to antiangiogenic therapy and poor prognosis. The Siah E3 ubiquitin ligases regulate the hypoxic response pathway by modulating the turnover of the master proangiogenic transcription factor hypoxia-inducible factor-1α (Hif-1α). In this study, we show that genetic deficiency in the Siah family member Siah2 results in vascular normalization and delayed tumor growth in an established transgenic model of aggressive breast cancer. Tumors arising in a Siah2(-/-) genetic background showed increased perfusion and pericyte-associated vasculature, similar to that occurring with antiangiogenic therapy. In support of the role of Siah2 in regulating levels of Hif-1α, expression of angiogenic factors was decreased in Siah2(-/-) tumors. Blood vessel normalization in Siah2(-/-) tumors resulted in an increased response to chemotherapy and prolonged survival. Together, our findings offer a preclinical proof of concept that targeting Siah2 is sufficient to attenuate Hif-1α-mediated angiogenesis and hypoxia signaling, thereby improving responses to chemotherapy.

BMP4 Inhibits Breast Cancer Metastasis by Blocking Myeloid-Derived Suppressor Cell Activity

The TGFβ growth factor family member BMP4 is a potent suppressor of breast cancer metastasis. In the mouse, the development of highly metastatic mammary tumors is associated with an accumulation of myeloid-derived suppressor cells (MDSC), the numbers of which are reduced by exogenous BMP4 expression. MDSCs are undetectable in naïve mice but can be induced by treatment with granulocyte colony-stimulating factor (G-CSF/Csf3) or by secretion of G-CSF from the tumor. Both tumor-induced and G-CSF-induced MDSCs effectively suppress T-cell activation and proliferation, leading to metastatic enhancement. BMP4 reduces the expression and secretion of G-CSF by inhibiting NF-κB (Nfkb1) activity in human and mouse tumor lines. Because MDSCs correlate with poor prognosis in patients with breast cancer, therapies based on activation of BMP4 signaling may offer a novel treatment strategy for breast cancer. Cancer Res; 74(18); 5091-102. ©2014 AACR.