Defective function of dendritic cells (DC) in cancer has been recently described and may represent one of the mechanisms of tumor evasion from immune system control. We have previously shown in vitro that vascular endothelial growth factor (VEGF), produced by almost all tumors, is one of the tumor-derived factors responsible for the defective function of these cells. In this study, we investigated whether in vivo infusion of recombinant VEGF could reproduce the observed DC dysfunction. Continuous VEGF infusion, at rates as low as 50 ng/h (resulting in serum VEGF concentrations of 120 to 160 pg/mL), resulted in a dramatic inhibition of dendritic cell development, associated with an increase in the production of B cells and immature Gr-1+ myeloid cells. Infusion of VEGF was associated with inhibition of the activity of the transcription factor NF-κB in bone marrow progenitor cells. Experiments in vitro showed that VEGF itself, and not factors released by VEGF-activated endothelial cells, affected polypotent stem cells resulting in the observed abnormal hematopoiesis. These data suggest that VEGF, at pathologically relevant concentrations in vivo, may exert effects on pluripotent stem cells that result in blocked DC development as well as affect many other hematopoietic lineages.

Matrix metalloproteinases (MMPs) have been implicated in diverse roles in breast cancer development and progression. While many of the different MMPs expressed in breast cancer are produced by stromal cells MMP-9 is produced mainly by the tumor cells themselves. To date, the functional role of tumor cell-produced MMP-9 has remained unclear. Here, we show that human breast cancer cell-produced MMP-9 is specifically required for invasion in cell culture and for pulmonary metastasis in a mouse orthotopic model of basal-like breast cancer. We also find that tumor cell-produced MMP-9 promotes tumor vascularization with only modest impact on primary tumor growth, and that silencing of MMP-9 expression in tumor cells leads to an altered transcriptional program consistent with reversion to a less malignant phenotype. MMP-9 is most highly expressed in human basal-like and triple negative tumors, where our data suggest that it contributes to metastatic progression. Our results suggest that MMP9 may offer a target for anti-metastatic therapies for basal-like triple negative breast cancers, a poor prognosis subtype with few available molecularly targeted therapeutic options.

Lymphatic metastasis is the main prognostic factor for survival of patients with breast cancer and other epithelial malignancies. Mounting clinical and experimental data suggest that migration of tumor cells into the lymph nodes is greatly facilitated by lymphangiogenesis, a process that generates new lymphatic vessels from pre-existing lymphatics with the aid of circulating lymphatic endothelial progenitor cells. The key protein that induces lymphangiogenesis is vascular endothelial growth factor receptor-3 (VEGFR-3), which is activated by vascular endothelial growth factor-C and -D (VEGF-C and VEGF-D). These lymphangiogenic factors are commonly expressed in malignant, tumor-infiltrating and stromal cells, creating a favorable environment for generation of new lymphatic vessels. Clinical evidence demonstrates that increased lymphatic vessel density in and around tumors is associated with lymphatic metastasis and reduced patient survival. Recent evidence shows that breast cancers induce remodeling of the local lymphatic vessels and the regional lymphatic network in the sentinel and distal lymph nodes. These changes include an increase in number and diameter of tumor-draining lymphatic vessels. Consequently, lymph flow away from the tumor is increased, which significantly increases tumor cell metastasis to draining lymph nodes and may contribute to systemic spread. Collectively, recent advances in the biology of tumor-induced lymphangiogenesis suggest that chemical inhibitors of this process may be an attractive target for inhibiting tumor metastasis and cancer-related death. Nevertheless, this is a relatively new field of study and much remains to be established before the concept of tumor-induced lymphangiogenesis is accepted as a viable anti-metastatic target. This review summarizes the current concepts related to breast cancer lymphangiogenesis and lymphatic metastasis while highlighting controversies and unanswered questions.

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The concept of inflammation-induced lymphangiogenesis (ie, formation of new lymphatic vessels) has long been recognized, but the molecular mechanisms remained largely unknown. The 2 primary mediators of lymphangiogenesis are vascular endothelial growth factor receptor-3 (VEGFR-3) and Prox1. The key factors that regulate inflammation-induced transcription are members of the nuclear factor-kappaB (NF-B) family; however, the role of NF-B in regulation of lymphatic-specific genes has not been defined. Here, we identified VEGFR-3 and

Emerging evidence suggests that cytotoxic therapy may actually promote drug resistance and metastasis while inhibiting the growth of primary tumors. Work in preclinical models of breast cancer have shown that acquired chemoresistance to the widely used drug paclitaxel (PXL) can be mediated by activation of the Toll-like receptor TLR4 in cancer cells. In this study, we determined the pro-metastatic effects of tumor-expressed TLR4 and PXL therapy and we investigated the mechanisms mediating these effects. While PXL treatment was largely efficacious in inhibiting TLR4-negative tumors, it significantly increased the incidence and burden of pulmonary and lymphatic metastasis by TLR4-positive tumors. TLR4 activation by PXL strongly increased the expression of inflammatory mediators, not only locally in the primary tumor microenvironment but also systemically in the blood, lymph nodes, spleen, bone marrow and lungs. These pro-inflammatory changes promoted the outgrowth of Ly6C+ and Ly6G+ myeloid progenitor cells and their mobilization to tumors, where they increased blood vessel formation but not invasion of these vessels. In contrast, PXL-mediated activation of TLR4-positive tumors induced de novo generation of deep intratumoral lymphatic vessels that were highly permissive to invasion by malignant cells. These results suggest that PXL therapy of patients with TLR4-expressing tumors may activate systemic inflammatory circuits that promote angiogenesis, lymphangiogenesis and metastasis, both at local sites and premetastatic niches where invasion occurs in distal organs. Taken together, our findings suggest that efforts to target TLR4 on tumor cells may simultaneously quell local and systemic inflammatory pathways that promote malignant progression, with implications for how to prevent tumor recurrence and the establishment of metastatic lesions, either during chemotherapy or after it is completed.

The differentiation-related gene-1 (Drg-1) was first identified as a gene strongly upregulated by induction of differentiation in colon carcinoma cells in vitro, and later the same gene was shown to suppress tumorigenicity of human bladder cancer cells in vivo. On the other hand, we and others have demonstrated that the Drg-1 gene suppresses prostate and colon cancer metastases in mouse models. In the context of such potential organ-specific differential function of the Drg-1 gene, the present study was designed to clarify the expression status, regulation and function of Drg-1 in the case of human breast cancer. We found that the expression of the Drg-1 protein was significantly reduced in breast tumor cells, particularly in patients with lymph node or bone metastasis as compared to those with localized breast cancer. Drg-1 expression also exhibited significant inverse correlation with the disease-free survival rate of patients and emerged as an independent prognostic factor. The downregulation of the Drg-1 gene appeared to be largely at the RNA level, and the DNA methylation inhibitor, 5-Azacytidine, significantly elevated the Drg-1 gene expression in various breast tumor cell lines. Furthermore, we found that overexpression of the Drg-1 gene suppresses the invasiveness of breast cancer cells in vitro, and this suppression was also achieved by treatment of cells with 5-Azacytidine. Together, our results strongly suggest functional involvement of the Drg-1 gene in suppressing the metastatic advancement of human breast cancer.

Semaphorin 3B (SEMA3B) is a secreted member of the semaphorin family, important in axonal guidance. We and others have shown that SEMA3B can act as a tumor suppressor by inducing apoptosis either by reexpression in tumor cells or applied as a soluble ligand. The common method of inactivation of SEMA3B is by allele loss and tumor-acquired promoter methylation. We studied the mechanism of SEMA3B-induced tumor cell apoptosis and found that vascular endothelial growth factor (VEGF) 165 significantly decreased the proapoptotic and antimitotic effect of transfected or secreted SEMA3B on lung and breast cancer cells. VEGF 165 binds to neuropilin, receptors for SEMA3B, and we found that SEMA3B competed for binding of 125 I-VEGF165 to lung and breast cancer cells. We also found that small interfering RNA knockdown of tumor-produced VEGF-A or the use of an anti-VEGF neutralizing antibody (Ab) significantly inhibited tumor cell growth in vitro. By contrast, VEGF 121, a VEGF variant that lacks binding to neuropilin (NP)-1 or NP-2 receptors, was not expressed in tumor cells and had no effect on SEMA3B growth-suppressing activities. In conclusion, we hypothesize that VEGF 165, produced by tumor cells, acts as an autocrine survival factor and that SEMA3B mediates its tumor-suppressing effects, at least in part, by blocking this VEGF autocrine activity.