Nearly all deaths caused by solid cancers occur as a result of metastasis--the formation of secondary tumours in distant organs such as the lungs, liver, brain and bone. A major obstruction to the development of drugs with anti-metastatic efficacy is our fragmented understanding of how tumours 'evolve' and metastasize, at both the biological and genetic levels. Furthermore, although there is significant overlap in the metastatic process among different types of cancer, there are also marked differences in the propensity to metastasize, the extent of metastasis, the sites to which the tumour metastasizes, the kinetics of the process and the mechanisms involved. Here, we consider the case of breast cancer, which has some marked distinguishing features compared with other types of cancer. Considerable progress has been made in the development of preclinical models and in the identification of relevant signalling pathways and genetic regulators of metastatic breast cancer, and we discuss how these might facilitate the development of novel targeted anti-metastatic drugs.

The translation of basic research into improved therapies for breast cancer patients requires relevant preclinical models that incorporate spontaneous metastasis. We have completed a functional and molecular characterisation of a new isogenic C57BL/6 mouse model of breast cancer metastasis, comparing and contrasting it with the established BALB/c 4T1 model. Metastatic EO771.LMB tumours were derived from poorly metastatic parental EO771 mammary tumours. Functional differences were evaluated using both in vitro assays and spontaneous metastasis assays in mice. Results were compared to non-metastatic 67NR and metastatic 4T1.2 tumours of the 4T1 model. Protein and transcript levels of markers of human breast cancer molecular subtypes were measured in the four tumour lines, as well as p53 (Tp53) tumour-suppressor gene status and responses to tamoxifen in vivo and in vitro. Array-based expression profiling of whole tumours identified genes and pathways that were deregulated in metastatic tumours. EO771.LMB cells metastasised spontaneously to lung in C57BL/6 mice and displayed increased invasive capacity compared with parental EO771. By immunohistochemical assessment, EO771 and EO771.LMB were basal-like, as was the 4T1.2 tumour, whereas 67NR had a luminal phenotype. Primary tumours from all lines were negative for progesterone receptor, Erb-b2/Neu and cytokeratin 5/6, but positive for epidermal growth factor receptor (EGFR). Only 67NR displayed nuclear estrogen receptor alpha (ERα) positivity. EO771 and EO771.LMB expressed mutant p53, whereas 67NR and 4T1.2 were p53-null. Integrated molecular analysis of both the EO771/EO771.LMB and 67NR/4T1.2 pairs indicated that upregulation of matrix metalloproteinase-3 (MMP-3), parathyroid hormone-like hormone (Pthlh) and S100 calcium binding protein A8 (S100a8) and downregulation of the thrombospondin receptor (Cd36) might be causally involved in metastatic dissemination of breast cancer.

Targeted drug delivery offers an opportunity for the development of safer and more effective therapies for the treatment of cancer. In this study, we sought to identify short, cell-internalizing peptide ligands that could serve as directive agents for specific drug delivery in hematologic malignancies. By screening of human leukemia cells with a combinatorial phage display peptide library, we isolated a peptide motif, sequence Phe-Phe/TyrAny-Leu-Arg-Ser (F F / Y XLRS), which bound to different leukemia cell lines and to patient-derived bone marrow samples. The motif was internalized through a receptor-mediated pathway, and we next identified the corresponding receptor as the transmembrane glycoprotein neuropilin-1 (NRP-1). Moreover, we observed a potent anti-leukemia cell effect when the targeting motif was synthesized in tandem to the pro-apoptotic sequence D (KLAKLAK) 2 . Finally, our results confirmed increased expression of NRP-1 in representative human leukemia and lymphoma cell lines and in a panel of bone marrow specimens obtained from patients with acute lymphoblastic leukemia or acute myelogenous leukemia compared with normal bone marrow. These results indicate that NRP-1 could potentially be used as a target for liganddirected therapy in human leukemias and lymphomas and that the prototype CGFYWLRSC-GG-D (KLAKLAK) 2 is a promising drug candidate in this setting. (Blood. 2011;117(3):920-927) IntroductionThe development of targeted drug-delivery strategies for safer and more effective therapy in human hematologic malignancies has been a long-standing goal for clinical and basic investigators. We reasoned that profiling of human leukemia-and lymphoma-derived cell lines with combinatorial libraries might yield ligand peptide sequences that bind to specific internalizing receptors on cell surfaces and may potentially lead to the discovery of new or unrecognized therapeutic targets. Such targeting motifs could also serve as vehicles for the preferential delivery of cytotoxic agents to leukemia and lymphoma cells.Several cell surface-binding peptides recognizing receptors in the membranes of lymphoma and leukemia cell lines have been reported. [1][2][3][4][5] The selected peptide ligands are readily internalized by cells and may therefore be potentially useful in ligand-directed drug delivery. Recently, we described an arginine-rich motif that is internalized into leukemia and lymphoma cells through the macropinocytotic pathway; however, the precise cell surface receptor has yet to be identified. 6 In effect, there is currently a relative lack of well-defined ligand-receptor systems for targeting human leukemia or lymphoma cells. The identification and validation of ligandreceptor pairs for these hematologic cancer cells relative to normal leukocytes would potentially represent a differential strategy and perhaps even improve disease outcomes.In this study, we used a combinatorial phage display-based subtractive selection 7-9 to identify ligand motifs that bind to specific cell surface receptors on human leuk...

The TGFb 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 na€ ve 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-kB (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. Ó2014 AACR.

Significant research has been conducted to better understand the extensive, heterogeneous molecular features of triple-negative breast cancer (TNBC). We reviewed published TNBC molecular classifications to identify major groupings that have potential for clinical trial development. With the ultimate aim to streamline translational medicine, we linked these categories of TNBC according to their gene-expression signatures, biological function, and clinical outcome. To this end, we define five potential clinically actionable groupings of TNBC: 1) basal-like TNBC with DNA-repair deficiency or growth factor pathways; 2) mesenchymal-like TNBC with epithelial-to-mesenchymal transition and cancer stem cell features; 3) immune-associated TNBC; 4) luminal/apocrine TNBC with androgen-receptor overexpression; and 5) HER2-enriched TNBC. For each defined subtype, we highlight the major biological pathways and discuss potential targeted therapies in TNBC that might abrogate disease progression. However, many of these potential targets need clinical validation by clinical trials. We have yet to know how we can enrich the targets by molecular classifications.

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.

Most cancer patients with solid tumors who succumb to their illness die of metastatic disease. While early detection and improved treatment have led to reduced mortality, even for those with metastatic cancer, some patients still respond poorly to treatment. Understanding the mechanisms of metastasis is important to improve prognostication, to stratify patients for treatment, and to identify new targets for therapy. We have shown previously that expression of nephronectin (NPNT) is correlated with metastatic propensity in breast cancer cell lines. In the present study, we provide a comprehensive analysis of the expression pattern and distribution of NPNT in breast cancer tissue from 842 patients by immunohistochemical staining of tissue microarrays from a historic cohort. Several patterns of NPNT staining were observed. An association between granular cytoplasmic staining (in <10% of tumor cells) and poor prognosis was found. We suggest that granular cytoplasmic staining may represent NPNT-positive exosomes. We found that NPNT promotes adhesion and anchorage-independent growth via its integrin-binding and enhancer motifs and that enforced expression in breast tumor cells promotes their colonization of the lungs. We propose that NPNT may be a novel prognostic marker in a subgroup of breast cancer patients.

Purpose The major cause of morbidity in breast cancer is development of metastatic disease, for which few effective therapies exist. Since tumor cell dissemination is often an early event in breast cancer progression and can occur prior to diagnosis, new therapies need to focus on targeting established metastatic disease in secondary organs. We report an effective therapy based on targeting cell surface-localized glucose regulated protein 78 (GRP78). GRP78 is expressed normally in the endoplasmic reticulum, but many tumors and disseminated tumor cells are subjected to environmental stresses and exhibit elevated levels of GRP78, some of which is localized at the plasma membrane. Experimental Design and Results Here we show that matched primary tumors and metastases from patients who died from advanced breast cancer also express high levels of GRP78. We utilized a peptidomimetic targeting strategy that employs a known GRP78-binding peptide fused to a pro-apoptotic moiety (designated BMTP78) and show that it can selectively kill breast cancer cells that express surface-localized GRP78. Further, in preclinical metastasis models, we demonstrate that administration of BMTP78 can inhibit primary tumor growth as well as prolong overall survival by reducing the extent of outgrowth of established lung and bone micrometastases. Conclusions The data presented here provide strong evidence that it is possible to induce cell death in established micrometastases by peptide mediated targeting of cell surface localized GRP in advanced breast cancers. The significance to patients with advanced breast cancer of a therapy that can reduce established metastatic disease should not be underestimated.