Mariko Muta

Significance of vascular endothelial growth factor (VEGF)/soluble VEGF receptor-1 relationship in breast cancer

Angiogenesis, the formation of new blood vessels, is controlled by a balance between positive and negative endothelial regulatory factors. Soluble vascular endothelial growth factor receptor‐1 (sVEGFR1), a naturally occurring soluble form of VEGFR1, is a negative counterpart of the vascular endothelial growth factor (VEGF) signaling pathway, which has been characterized as one of the most important endothelial regulators in human tumor angiogenesis. In our study, we examined the expression of sVEGFR1 in 110 primary breast carcinomas, and assessed its clinical significance. Ninety‐four of 110 tumors showed ≥0.1 ng/mg protein of sVEGFR1 (range:0. 1–6.9 ng/mg protein; median: 1.03 ng/mg protein) as determined by a specific enzyme‐linked immunosorbent assay (ELISA). Immunoblot analysis confirmed the presence of sVEGFR1 in breast tumor tissues. The levels of sVEGFR1 were correlated significantly with the levels of VEGF. There was no significant correlation between the levels of sVEGFR1 and any clinico‐pathological factors including age, menopause, nodal involvement and hormone receptor status. A univariate prognosis analysis showed that the intratumoral VEGF status, as determined by ELISA, was a significant prognostic indicator, but sVEGFR1 status was not. In the combined analysis, however, the ratio of sVEGFR1 and VEGF levels provided more statistically significant prognostic value than VEGF status alone. Tumors in which the sVEGFR1 levels exceeded VEGF levels 10‐fold had a markedly favorable prognosis. Multivariate analysis also demonstrated that the ratio of sVEGFR1 and VEGF was an independent prognostic indicator after nodal status. In conclusion, sVEGFR1, an intrinsic inhibitor of VEGF, frequently co‐expressed with VEGF in primary breast cancer tissues. The intratumoral balance between sVEGFR1 and VEGF levels might be crucial for the progression of breast cancer.

Expression of Macrophage Migration Inhibitory Factor in Human Breast Cancer: Association with Nodal Spread

Macrophage migration inhibitory factor (MIF) is known to exert pleiotropic functions including inhibition of macrophage migration, anchoring, and counteraction of the anti‐inflammatory and immunosuppressive activity of glucocorticoids. Ninety‐three primary breast cancer tissues and 64 sera of primary breast cancer patients were analyzed for the expression of MIF. The clinico‐pathological significance of MIF expression was evaluated. It was found that MIF was frequently over‐expressed in primary breast cancer tissues. RT‐PCR and western blotting analysis confirmed that wild‐type MIF is expressed, and immunohistochemical analysis showed that MIF expression was localized at tumor cells as well as stromal cells, including tumor‐associated macrophages. Intra‐tumoral MIF protein concentrations detected by enzyme‐linked immunosorbent assay (ELISA) varied with a median value of 1821 ng/mg protein (range: 8–8126 ng/mg protein), and correlated inversely with nodal involvement (P=0.039). No significant correlation was observed with other clinico‐pathological factors including tumor size, menopausal status and hormone receptors. The circulating level of MIF protein ranged up to 105.7 ng/ml (median: 17.3 ng/ml), and it was also found to correlate inversely with the number of involved nodes (P=0.02). A comparative study with other soluble inflammatory mediators showed that intratumoral levels of MIF were significantly associated with those of interleukin‐1β, suggesting that interactions between tumor cells and tumor‐associated macrophages play an important role in the up‐regulation of MIF. The multifunctional inflammatory/immune mediator MIF was frequently expressed in primary breast cancer, and its expression level was inversely associated with nodal spread. Thus, MIF seems to play a role in tumor‐stroma interactions of primary breast cancers, particularly those with a phenotype of node‐negative or minimal nodal spread.

A Nonfucosylated Anti-HER2 Antibody Augments Antibody-Dependent Cellular Cytotoxicity in Breast Cancer Patients

Purpose: Removal of fucose residues from the oligosaccharides of human antibody is a powerful approach to enhance antibody-dependent cellular cytotoxicity (ADCC), a potential important antitumor mechanism of therapeutic antibodies. To provide clinically relevant evidence of this mechanism, we investigated ADCC of a fucose-negative version of trastuzumab [anti–human epidermal growth factor receptor 2 (HER2) humanized antibody] using peripheral blood mononuclear cells (PBMC) from breast cancer patients as effector cells. Experimental Design: Thirty volunteers, including 20 breast cancer patients and 10 normal healthy control donors, were recruited randomly, and aliquots of peripheral blood were collected. ADCC of commercial trastuzumab (fucosylated) and its fucose-negative version were measured using PBMCs drawn from the volunteers as effector cells and two breast cancer cell lines with different HER2 expression levels as target cells. Relationships between cytotoxicity and characteristics of the patients, such as content of natural killer cells in PBMCs, type of therapy, FCGR3A genotypes, etc. were also analyzed. Results: ADCC was significantly enhanced with the fucose-negative antibody compared with the fucose-positive antibody using PBMCs from either normal donors or breast cancer patients. Enhancement of ADCC was observed irrespective of the various clinical backgrounds of the patients, even in the chemotherapy cohort that presented with a reduced number of natural killer cells and weaker ADCC. Conclusions: This preliminary study suggests that the use of fucose-negative antibodies may improve the therapeutic effects of anti-HER2 therapy for patients independent of clinical backgrounds.

Significance of Membrane Type 1 Matrix Metalloproteinase Expression in Breast Cancer

Expression of matrix metalloproteinases (MMPs) plays an essential role in tumor metastasis and invasion through the degradation of extracellular matrix (ECM). MT1‐MMP (membrane type 1 matrix metalloproteinase), a membrane‐type MMP, is responsible for the activation of MMP2. In this study the significance of MT1‐MMP expression in human breast tumors was investigated by immunocytochemical assay, and its correlation with clinicobiological features was analyzed. MT1‐MMP expression was detected in tumor cells and/or stromal cells, and there was a strong correlation between the expressions of MT1‐MMP in the two cell types. Out of 183 primary tumors, 103 (56.2%) showed positive staining of MT1‐MMP in tumor cells. MT1‐MMP expression showed no significant correlation with any of the clinicobiological parameters examined, including hormone receptor status and angiogenesis. In postoperative survival analysis, MT1‐MMP expression itself was not a significant prognostic factor. However, in the particular subgroup with the accumulation of thymidine phosphorylase (TP)‐positive stromal cells, which have been activated by various stimuli, such as cytokines and hypoxia, MT1‐MMP expression had a significant prognostic value. These data suggested that MT1‐MMP might function cooperatively with tumor‐associated stromal cells for the progression of breast cancer.

Role of MDM2 Overexpression in Doxorubicin Resistance of Breast Carcinoma

Several oncoproteins or tumor suppressor gene products have been indicated to be of value as predictors of the de novo resistance to cytotoxic agents. In this study, we have investigated the role of MDM2 (murine double minutes) overexpression in doxorubicin resistance of breast cancer. Immunocytochemical analysis demonstrated that MDM2‐positive tumors, even with p53‐negative pheno‐type, were significantly more resistant to doxorubicin treatment compared to MDM2‐negative tumors. An in vitro experimental model using stable mdm2‐transfected MCF‐7 cells carrying wild‐type p53 confirmed that the cells become approximately 3‐fold more resistant to doxorubicin as a result of MDM2 overexpression, and the wild‐type p53 function, such as the induction of p21Waf1 following DNA damage, was significantly suppressed. MDM2 overexpression is suggested to be a novel marker for predicting lack of response to doxorubicin treatment in breast cancer patients.

Establishment of bone marrow-derived endothelial cell lines from ts-SV40 T-antigen gene transgenic rats

Purpose. Postneonatal neovascularization is thought to result exclusively from the proliferation, migration, and remodeling of fully differentiated endothelial cells (ECs). Recently, it has been reported that bone marrow contains cells which can differentiate into ECs and contribute to neoangiogenesis in adult species. In this study, we tried to establish conditionally immortalized endothelial cell lines (TR-BME) derived from rat bone marrow.Methods. Mononuclear cells were isolated and differentiated into ECs at 37°C from the bone marrow of a transgenic rat harboring temperature-sensitive SV40 large T-antigen (ts T-Ag) gene. Then, the cells were transferred and incubated at 33°C, a permissive temperature for ts T-Ag. Expression of vascular endothelial growth factor (VEGF) receptor (VEGFR)-1, 2, Tie-1, 2 and von Willebrand factor (VWF) were assayed by reverse transcriptase-mediated polymerase chain reaction (RT-PCR).Results. We have established three cell lines incorporating 1,1′-dioctadecyl-3,3,3′,3-tetramethylindo-carbocyanine perchlorate (DiI-Ac-LDL) with a spindle shape. One of these, clone 2, strongly expressed VEGFR-2, and weakly expressed VEGFR-1 and VWF. In contrast, clone 8 showed strong expression of Tie-1, 2, and VWF, and weak expression of VEGFR-1,2. All markers were expressed strongly in clone 3.Conclusions. These data confirm that the above three TR-BME cells are novel ECs derived from bone marrow progenitors.

Genome-wide copy number analysis in primary breast cancer

Introduction: Carcinogenesis is considered to be a multistep process that may involve cumulative genomic alterations. Loss of chromosomal material would inactivate tumor suppressor genes and gain of chromosomal material has the potential to activate tumor-promoting genes. Areas covered: Recent intensive studies by array comparative genomic hybridization (aCGH) have demonstrated frequent alterations in multiple regions of the genome. This suggests that these regions contain a variety of oncogenes and tumor suppressor genes associated with breast cancer development. The patterns of copy number variations (CNVs) have been suggested to be associated with breast cancer subtypes, indicating the importance of genomic instability in the development of breast cancer. Expert opinion: To further clarify the complexity of gene alterations, one approach is to employ a CNV-targeted platform that harbors a large number of direct CNV markers located in the repeat-rich unstable regions of the human genome. Next generation sequencing is another approach to overcome the limitations of aCGH such as the repeat-rich regions. Genomic analysis should be combined with expression analysis to elucidate individual genes relevant to breast cancer development and progression. The elucidation of the functions of the affected genes would lead to identification of new molecular targets for breast cancer eradication.

Effect of Low-Dose Paclitaxel and Docetaxel on Endothelial Progenitor Cells

Objective: Bone marrow (BM)-derived endothelial progenitor cells (EPC) play an important role in neovascularization and tumor growth. It has been reported that docetaxel and paclitaxel inhibit angiogenesis, but the effect of docetaxel and paclitaxel on EPC-induced neovascularization has not been examined. We aimed to clarify the cytotoxic and inhibitory effects of these drugs on EPC. Methods: The effects of drugs on growth, tube formation, and migration of EPC were analyzed in vitro using a rat BM-derived EPC cell line (TR-BME). Fluorescence-labeled TR-BME cells were injected into tumor-bearing rats and accumulation at the tumor site was analyzed by fluorescence-activated cell sorting (FACS). Results: In in vitro cytotoxicity assays of these drugs in TR-BME, rat endothelial cell line TR-BBB and rat tumor cell line Walker 256, the IC50 values for TR-BME were higher than those for TR-BBB or Walker 256. Both drugs inhibited tube formation and migration of TR-BME at lower concentrations than the cytotoxic IC50. In vivo studies showed that a low dose of both drugs inhibited EPC accumulation at the tumor site in tumor-bearing rats, as determined by FACS, and caused a decrease in microvessel density. Conclusion: Docetaxel and paclitaxel directly inhibited EPC-initiated vasculogenesis at low (non-cytotoxic) concentrations, causing suppression of tumor growth.

Tumor size significantly correlates with postoperative liver metastases and COX-2 expression in patients with resectable pancreatic cancer.

Background/Aim: Local treatment often fails in patients with resectable pancreatic cancer due to the postoperative development of distant metastases, especially liver metastases. We determined the prognostic factors for postoperative liver metastases in pancreatic cancer patients following surgical resection with combined radiotherapy. Methods: Sixty-four patients with nonmetastatic, resectable pancreatic cancer were entered into this study. All of these patients had pancreatic resection surgery combined with radiotherapy. The development of postoperative liver metastases was carefully followed, and the survival ratio was evaluated using the Kaplan-Meier method. The prognostic importance of clinicopathological factors and molecular characteristics was analyzed by the Cox proportional hazards model. The correlation study was performed using Fisher’s exact test. Results: Tumor size, curability, and histological type of differentiation were statistically significant independent prognostic factors. On multivariate analysis, curability and histological type of differentiation were statistically significant. Only tumor size (≧3 cm) was significantly correlated with postoperative liver metastases, as well as cyclooxygenase-2 expression. Conclusions: There were three significant prognostic factors in patients with resectable pancreatic cancer who had local therapy. Patients who have a large tumor require particularly careful follow-up for postoperative liver metastases.

Mechanical analysis of tumor growth regression by the cyclooxygenase-2 inhibitor, DFU, in a Walker256 rat tumor model: Importance of monocyte chemoattractant protein-1 modulation

Cyclooxygenase (COX)-2 inhibition results in tumor regression; however, little is known about the mechanism. In the present study, using a Walker256 tumor model and a rat bone marrow–derived endothelial cell line TR-BME-2, we analyzed the effects of a new selective COX-2 inhibitor, 5,5-dimethyl-3-(3-fluorophenyl)-4-(4-methylsulphonyl)phenyl-2-(5H)-furanone (DFU), on the production of chemokines and growth factors and on the neovascularization. The oral administration of DFU (5 mg/kg/d) significantly suppressed the tumor growth with decreasing microvessel density in vivo, although it showed no direct inhibition of Walker256 cell proliferation in vitro. It was newly found that the recruitment of systemically injected TR-BME-2 cells into the tumor site was significantly inhibited by DFU treatment. In addition, we found that DFU significantly reduced the production of monocyte chemoattractant protein-1 (MCP-1) both in tumor tissues and in the systemic circulation (P < 0.001 and P < 0.001, respectively). Such reduction was not observed in other chemotactic factors, vascular endothelial growth factor and stromal cell–derived factor-1. The induced chemotaxis of TR-BME-2 by serum of tumor-bearing rats was significantly reduced in DFU-treated rat serum, although DFU showed no direct inhibition for TR-BME-2 cells, either cell growth or chemotaxis. Treatment with neutralizing antibodies to soluble mediators, including MCP-1, significantly suppressed the chemotaxis. Regarding the down-regulation machinery of MCP-1 production in vivo, tumor-associated macrophages seem to play crucial roles, because DFU eliminated MCP-1 production in the activated macrophages remarkably but not in Walker256 tumor cells in vitro. In conclusion, COX-2 inhibitor DFU exerts tumor regression activity in a Walker256 tumor model by suppressing MCP-1 production in tumor tissues and in the circulation.