semaxinib and Breast-Neoplasms

We used inflammatory breast cancer (IBC) as a model disease to investigate biological changes associated with an antiangiogenesis agent, SU5416, combined with doxorubicin.. Patients with stage IIIB or IV IBC were treated neoadjuvantly with the combination of SU5416 and doxorubicin for induction therapy. The dose of SU5416 (administered on days 1 and 4, every 3 weeks) and doxorubicin (administered on day 1 every 3 weeks) were escalated in cohorts of three patients starting at 110 and 60 mg/m2, respectively, for a total of five cycles leading up to mastectomy. Patients underwent serial assessment (pharmacokinetic sampling, biopsy of breast, tumor blood flow dynamic contrast-enhanced magnetic resonance imaging, plasma angiogenesis, and endothelial cell damage markers) prior to treatment, at the end of cycles no. 2 and no. 5, and after mastectomy.. Eighteen patients were enrolled; neutropenia was dose-limiting, and overall median survival was not reached (50 months of study follow-up). Four patients (22%) experienced congestive heart failure, which resolved and were likely attributable to a smaller volume of distribution and higher Cmax of doxorubicin in combination with SU5416. We did observe a significant decline in tumor blood flow using Kep calculated by Brix (pretreatment versus post-cycle no. 5; P = 0.033), trend for a decline in tumor microvessel density after treatment, and low baseline levels of soluble intracellular adhesion molecule were associated with improved event-free survival.. This study showed evidence of an unfavorable cardiac interaction between SU5416 and doxorubicin, which prohibits further investigation of this combination. However, this study supports the importance of using IBC as a model for investigating angiogenesis inhibitors.

Topics: Adult; Aged; Angiogenesis Inhibitors; Antineoplastic Combined Chemotherapy Protocols; Breast Neoplasms; Disease-Free Survival; Doxorubicin; Female; Humans; Indoles; Inflammation; Male; Maximum Tolerated Dose; Middle Aged; Neovascularization, Pathologic; Pyrroles; Treatment Outcome

Endoglin, a transforming growth factor-β co-receptor, is highly expressed on angiogenic endothelial cells in solid tumors. Therefore, targeting endoglin is currently being explored in clinical trials for anti-angiogenic therapy. In this project, the redundancy between endoglin and vascular endothelial growth factor (VEGF) signaling in angiogenesis and the effects of targeting both pathways on breast cancer metastasis were explored. In patient samples, increased endoglin signaling after VEGF inhibition was observed. In vitro TRC105, an endoglin-neutralizing antibody, increased VEGF signaling in endothelial cells. Moreover, combined targeting of the endoglin and VEGF pathway, with the VEGF receptor kinase inhibitor SU5416, increased antiangiogenic effects in vitro and in a zebrafish angiogenesis model. Next, in a mouse model for invasive lobular breast cancer, the effects of TRC105 and SU5416 on tumor growth and metastasis were explored. Although TRC105 and SU5416 decreased tumor vascular density, tumor volume was unaffected. Strikingly, in mice treated with TRC105, or TRC105 and SU5416 combined, a strong inhibition in the number of metastases was seen. Moreover, upon resection of the primary tumor, strong inhibition of metastatic spread by TRC105 was observed in an adjuvant setting. To confirm these data, we assessed the effects of endoglin-Fc (an endoglin ligand trap) on metastasis formation. Similar to treatment with TRC105 in the resection model, endoglin-Fc-expressing tumors showed strong inhibition of distant metastases. These results show, for the first time, that targeting endoglin, either with neutralizing antibodies or a ligand trap, strongly inhibits metastatic spread of breast cancer in vivo.

Topics: Animals; Antibodies, Monoclonal; Breast Neoplasms; Endoglin; Female; Humans; Indoles; Neoplasm Metastasis; Neovascularization, Pathologic; Pyrroles; Smad1 Protein; Vascular Endothelial Growth Factor A; Zebrafish

The WASF3 (WAVE3) gene is an important mediator of cell motility, invasion and metastasis and is expressed at high levels in some advanced stage tumors. In our survey of breast cancer cells, we now demonstrate that exposure to hypoxic conditions increases WASF3 expression levels in MDA231, SKBR3 and MCF7 cells. The WASF3 promoter region contains HIF1A response elements (HRE). ChIP assays demonstrate that HIF1A binds to these HRE elements in the promoter region, and luciferase reporter assays using the WASF3 gene minimal promoter shows that hypoxia results in its upregulation. Phosphorylation of WASF3 is required for its ability to affect invasion and increased phosphoactivation of WASF3 is also seen in cells challenged with hypoxia. These cells also show increased motility in the scratch wound assay. Cells in which WASF3 has been knocked down show no response to hypoxia as expected, implicating the specificity of the hypoxic response to WASF3. Overall, these experiments demonstrate WASF3 is a HIF1A-regulated gene and suggests a mechanism to explain the observation of elevated expression of WASF3 in advanced stage tumors.

Topics: Breast Neoplasms; Cell Hypoxia; Female; Gene Expression Regulation, Neoplastic; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Indoles; Neoplasm Metastasis; Pyrroles; Transcription, Genetic; Tumor Microenvironment; Vascular Endothelial Growth Factor A; Wiskott-Aldrich Syndrome Protein Family

The progression of breast cancer is associated with oxidative stress.  However, the effects of pyrrolidine dithiocarbamate (PDTC), a known antioxidant, on the development of breast cancer are poorly understood.  The present study evaluates the effects of PDTC on tumor growth, the expression of vascular endothelial growth factor (VEGF), and angiogenesis of breast cancer in female mice.  Eight week old female mice (C57BL/6J) were given PDTC at 100 to 200 mg/kg/day for 3 weeks (n=10).  The control mice received regular drinking water only.  In the 2nd wk, 5x10^5 E0771 (mouse breast cancer) cells were injected in the pad of the fourth mammary gland of the mice.  Tumor size was monitored using dial calipers.  At the end of the experiment, the tumors were isolated and measured for tumor size, intratumoral microvessel (IM) density using CD31 immunohistochemistry staining, NFκB activation using EMSA, and VEGF protein levels using ELISA.  PDTC treatment caused a significant decrease in tumor weight compared to the control (0.64±0.22 vs. 1.43±0.31 g; n=8; P< 0.01), and a significant decrease in IM density (66.1±5.3 vs. 84.2±9.4 IM# /mm^2; P< 0.01).  There was a significant decrease in tissue protein levels of VEGF (22.6±2.1 vs. 32.4±2.6 pg/mg) and a 43% reduction NFκB activation in the breast tumors of mice treated with PDTC compared to the control group (P< 0.01).  Western blot indicated that estrogen receptor-α (ERα), VEGF receptor-1 (Flt-1), and VEGF receptor-2 (Flk-1) were expressed in E0771 cells.  VEGF receptor inhibitor SU5416 and PDTC synergistically suppressed the proliferation of E0771 cells.  PDTC also significantly inhibited the migration of cultured E0771 cells.  These results support the hypothesis that PDTC suppresses tumor angiogenesis, growth, and migration of breast cancer via inhibiting autocrine and paracrine effects of VEGF through the reduction of NFκB activation and VEGF expression.

Topics: Angiogenesis Inhibitors; Animals; Antineoplastic Agents; Breast Neoplasms; Cell Line, Tumor; Cell Movement; Cell Proliferation; Drug Synergism; Estrogen Receptor alpha; Female; Gene Expression Regulation, Neoplastic; Humans; Indoles; Mice; Mice, Inbred C57BL; Neovascularization, Pathologic; NF-kappa B; Pyrroles; Pyrrolidines; Thiocarbamates; Tumor Burden; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factor Receptor-1; Vascular Endothelial Growth Factor Receptor-2

Doxorubicin is a useful antineoplastic drug with multiple mechanisms of cytotoxicity. One such mechanism involves the reductive bioactivation of the quinone ring to a semiquinone radical, which can exert direct toxic effects and/or undergo redox cycling. We hypothesized that human NADPH-cytochrome p450 reductase (CYPRED) catalyzes doxorubicin reduction and that overexpression of this enzyme sensitizes human breast cancer cell lines to the aerobic cytotoxicity of doxorubicin. cDNA-expressed human CYPRED catalyzed doxorubicin reduction, measured as the rate of doxorubicin-stimulated NADPH consumption. Using a bank of 17 human liver microsomal samples, the rate of doxorubicin reduction correlated with CYPRED catalytic activity and CYPRED protein immunoreactivity. Diphenyliodonium chloride, a mechanism-based inactivator of CYPRED, inhibited CYPRED activity and doxorubicin reduction in human liver microsomes with similar concentration dependence. Stably transfected clones of MDA231 human breast cancer cells overexpressing human CYPRED immunoreactive protein and catalytic activity showed enhanced sensitivity to the aerobic cytotoxicity of tirapazamine, a bioreductive drug known to be activated by CYPRED; however, no sensitization to the cytotoxic effects of doxorubicin was observed. Although human CYPRED is an important catalyst of doxorubicin reduction, overexpression of this enzyme does not confer enhanced sensitivity of human breast cancer cells to the aerobic cytotoxicity of doxorubicin.

Topics: Aerobiosis; Antibiotics, Antineoplastic; Breast Neoplasms; Cell Line, Tumor; Doxorubicin; Humans; Indoles; NADPH-Ferrihemoprotein Reductase; Pyrroles; Transfection