cathepsin-g and Breast-Neoplasms

Breast cancer is primarily classified into ductal and lobular types, as well as into noninvasive and invasive cancer. Invasive cancer involves lymphatic and hematogenous metastasis. In breast cancer patients with distant metastases, a neutrophil-derived serine protease; cathepsin G (Cat G), is highly expressed in breast cancer cells. Cat G induces cell migration and multicellular aggregation of MCF-7 human breast cancer cells; however, the mechanism is not clear. Recently, platelet-activating factor (PAF)-acetylhydrolase (PAF-AH), the enzyme responsible for PAF degradation, was reported to be overexpressed in some tumor types, including pancreatic and breast cancers. In this study, we investigated whether PAF-AH is involved in Cat G-induced aggregation and migration of MCF-7 cells. We first showed that Cat G increased PAF-AH activity and elevated PAFAH1B2 expression in MCF-7 cells. The elevated expression of PAFAH1B2 was also observed in human breast cancer tissue specimens by immunohistochemical analysis. Furthermore, knockdown of PAFAH1B2 in MCF-7 cells suppressed the cell migration and aggregation induced by low concentrations, but not high concentrations, of Cat G. Carbamoyl PAF (cPAF), a nonhydrolyzable PAF analog, completely suppressed Cat G-induced migration of MCF-7 cells. In addition, PAF receptor (PAFR) inhibition induced cell migration of MCF-7 cells even in the absence of Cat G, suggesting that Cat G suppresses the activation of PAFR through enhanced PAF degradation due to elevated expression of PAFAH1B2 and thereby induces malignant phenotypes in MCF-7 cells. Our findings may lead to a novel therapeutic modality for treating breast cancer by modulating the activity of Cat G/PAF signaling.

Topics: 1-Alkyl-2-acetylglycerophosphocholine Esterase; Breast Neoplasms; Cathepsin G; Cell Movement; Female; Humans; MCF-7 Cells; Microtubule-Associated Proteins; Neutrophils; Platelet Activating Factor

Cathepsin G (CG), a neutrophil serine protease, induces cell migration and multicellular aggregation of human breast cancer MCF-7 cells in a process that is dependent on E-cadherin and CG enzymatic activity. While these tumor cell aggregates can cause tumor emboli that could represent intravascular growth and extravasation into the surrounding tissues, resulting in metastasis, the molecular mechanism underlying this process remains poorly characterized. In this study, we aimed to identify the signaling pathway that is triggered during CG-mediated stimulation of cell aggregation. Screening of a library of compounds containing approximately 90 molecular-targeting drugs revealed that this process was suppressed by the insulin-like growth factor-1 (IGF-1) receptor (IGF-1R)-specific kinase inhibitor OSI-906, as well as the multikinase inhibitors axitinib and sunitinib. Antibody array analysis, which is capable of detecting tyrosine phosphorylation of 49 distinct receptor tyrosine kinases, and the results of immunoprecipitation studies indicated that IGF-1R is phosphorylated in response to CG treatment. Notably, IGF-1R neutralization via treatment with a specific antibody or silencing of IGF-1R expression through siRNA transfection suppressed cell aggregation. Furthermore, CG treatment of MCF-7 cells resulted in increased release of IGF-1 into the medium for 24 h, while antibody-mediated IGF-1 neutralization partially prevented CG-induced cell aggregation. These results demonstrate that autocrine IGF-1 signaling is partly responsible for the cell aggregation induced by CG.

Topics: Autocrine Communication; Axitinib; Breast Neoplasms; Cathepsin G; Cell Aggregation; Female; Humans; Imidazoles; Indazoles; Indoles; Insulin-Like Growth Factor I; MCF-7 Cells; Phosphorylation; Pyrazines; Pyrroles; Receptor, IGF Type 1; Receptors, Somatomedin; Signal Transduction; Small Molecule Libraries; Sunitinib

IL-20 is a proinflammatory cytokine involved in rheumatoid arthritis, atherosclerosis, and stroke. However, little is known about its role in breast cancer. We explored the function of IL-20 in tumor growth and metastasis, as well as in clinical outcome. Tumor expression of IL-20 was assessed by immunohistochemical staining among 198 patients with invasive ductal carcinoma of the breast, using available clinical and survival data. IL-20 expression was associated with advanced tumor stage, greater tumor metastasis, and worse survival. Reverse transcription quantitative polymerase chain reaction showed that clinical breast tumor tissue expressed higher levels of IL-20 and its receptors than did nontumorous breast tissue. IL-20 was also highly expressed in breast cancer bone-metastasis tissue. In vitro, IL-20 upregulated matrix metalloproteinase-9, matrix metalloproteinase-12, cathepsin K, and cathepsin G, and enhanced proliferation and migration of breast cancer cells, which were inhibited by anti-IL-20 mAb 7E. In vivo, we generated murine models to evaluate the therapeutic potential of 7E, using luminescence intensity, radiological scans, and micro-computed tomography. 7E reduced tumor growth, suppressed bone colonization, diminished tumor-mediated osteolysis, and lessened bone density decrement in mice injected with breast cancer cells. In conclusion, our results suggest that IL-20 plays pivotal roles in the tumor progression of breast cancer. IL-20 expression in breast cancer tissue is associated with a poor clinical outcome. Anti-IL-20 mAb 7E suppressed bone colonization and decreased osteolytic bone lesions. Therefore, IL-20 may be a novel target in treating breast tumor-induced osteolysis.

Topics: Animals; Antibodies, Monoclonal; Bone and Bones; Bone Neoplasms; Breast; Breast Neoplasms; Carcinoma, Ductal, Breast; Cathepsin G; Cathepsin K; Cell Line, Tumor; Cell Movement; Cell Proliferation; Disease Progression; Female; Humans; Interleukins; Matrix Metalloproteinase 12; Matrix Metalloproteinase 9; Mice; Mice, Inbred BALB C; Neoplasm Metastasis; Osteolysis

Transforming growth factor (TGF)-beta signaling makes a significant contribution to the pathogenesis of breast cancer bone metastasis. In other tumor types, TGF-beta has been shown to promote tumor vascularity. Here, we report that inhibition of TGF-beta significantly reduces microvessel density in mammary tumor-induced bone lesions, mediated by decreased expression of both vascular endothelial growth factor (VEGF) and monocyte chemotactic protein (MCP)-1, both known angiogenic factors. Cathepsin G upregulation at the tumor-bone interface has been linked to increased TGF-beta signaling, and we also report that inhibition of Cathepsin G reduced tumor vascularity, as well as VEGF and MCP-1 expression.

Topics: Animals; Antibodies; Bone Neoplasms; Breast Neoplasms; Cathepsin G; Cell Line, Tumor; Chemokine CCL2; Female; Gene Expression Regulation, Neoplastic; Mice; Mice, Inbred BALB C; Neovascularization, Pathologic; RNA, Messenger; Serine Proteinase Inhibitors; Signal Transduction; Tosylphenylalanyl Chloromethyl Ketone; Transforming Growth Factor beta; Up-Regulation; Vascular Endothelial Growth Factor A

In tumor metastasis, multicellular aggregates of tumor cells form and disseminate into the blood or lymph vessels from the tumor mass, following the formation of tumor cell emboli in distant vessels. However, the mechanism by which aggregates form in the tumor mass is unknown. Neutrophils often exist in tumors and are considered to affect tumor development. We observed that neutrophils had the capacity to induce the aggregation of MCF-7 human breast carcinoma cells adhering to culture substrates. When MCF-7 cells were cultured with rat inflammatory neutrophils, the soluble fraction of their lysate, and the conditioned medium of neutrophils stimulated with N-formyl-Met-Leu-Phe plus cytochalasin B, multicellular aggregates formed within 16 h, and tightly aggregated 3-D spheroids formed when the cultures were prolonged. The spheroid-inducing reaction was reversible and energy-dependent. The MCF-7 cells induced to aggregate by the neutrophil extract showed growth potential, although the growth rate of the cells was slightly reduced. The aggregation was dependent on E-cadherin, because the spheroids dispersed into isolated cells on incubation with EGTA or anti-E-cadherin antibody following pipetting. The aggregation-inducing activity in neutrophils was completely inhibited by soybean trypsin-chymotrypsin inhibitor. Moreover, the commercially available human neutrophil elastase and cathepsin G induced the aggregation of MCF-7 cells and formation of spheroids. The proteases secreted by infiltrated neutrophils in tumors are implicated in the dissemination of tumor aggregates from primary tumor sites.

Topics: Adenocarcinoma; Animals; Breast Neoplasms; Cadherins; Cathepsin G; Cathepsins; Cell Adhesion; Female; Humans; Neoplasm Metastasis; Neutrophils; Pancreatic Elastase; Rats; Serine Endopeptidases; Spheroids, Cellular; Tumor Cells, Cultured

Despite years of research, it is still unclear which women with node-negative (N-) breast cancer will need adjuvant chemotherapy and which women are being treated unnecessarily. Our goal was to determine which factors best predicted disease free survival (DFS) or cancer-specific overall survival (OS) and, therefore, select the correct patients for treatment. A total of 11 parameters were measured: estrogen receptor (ER), progesterone receptor (PR), age, race, ploidy status, %G0/G1 (% non-DNA synthesis), %S (% S-phase), cathepsin D status, size, stage, and histologic grade.. In this prospective study, we followed 556 N- patients diagnosed between 1991 and 1996. The tumors were 56% ER+, 51% PR+, 30% diploid, with a mean %S of 8.9%. The level of cathepsin D ranged from 0.50 to 155 pmol/mg of protein with a mean of 42.9 pmol/mg of protein. There were 87 recurrences (16%) and 72 cancer deaths (13%), with a median follow-up of 7.8 years. Ploidy status (p = 0.01), S-phase activity (p = 0.003), G1 phase activity (p = 0.02) and age (p = 0.01) were able to significantly predict DFS in a univariate manner. All of the measurable factors were significant or borderline significant in predicting OS in a univariate manner except for age, race, and ER status. In multivariate analysis with S-phase included, it was the only remaining factor in DFS and OS; with S-phase excluded, age and ploidy status remained as factors for DFS in stepwise regression, while PR, size, and cathepsin D were the remaining factors that predicted cancer-specific OS. The effect of adjuvant treatment on prognosis was also analyzed.. Both biochemical and clinical parameters have the potential to predict prognosis for N- breast cancer. In this large prospective clinical trial, with a median follow-up of 7.8 years, no individual marker adequately predicted the prognosis for an individual patient. %S activity was the best independent marker, but only 77% of the tumors provided this value. Subset analysis provided improved prognostication, but there were limits to its utility. These data represents a definitive study starting in 1991 and ending in 2002.

Topics: Adult; Age Factors; Aged; Aged, 80 and over; Biomarkers, Tumor; Breast Neoplasms; Cathepsin G; Cathepsins; Cell Cycle; Disease-Free Survival; Female; Humans; Middle Aged; Multivariate Analysis; Neoplasm Recurrence, Local; Ploidies; Predictive Value of Tests; Prognosis; Prospective Studies; Serine Endopeptidases

In MCF7 cells, transcription of the lysosomal protease cathepsin D is stimulated by estrogens via a non-consensus estrogen responsive element (ERE). By contrast, in estrogen responsive Ishikawa endometrial cancer cells, the cathepsin D gene is unresponsive to estrogens. We now show that the transfected cathepsin D promoter, which can be induced by estrogens in several cell types, is insensitive in Ishikawa cells. The block is not due to a mutation in the cathepsin D promoter or estrogen receptor, but involves the cathepsin D ERE, and implies a C at position 3 of the ERE sequence. Our results suggest that in Ishikawa cells, cathepsin D insensitivity to estrogen most likely occurs through a specific interaction with the ER, or with an endometrial factor which may compete with the ER for binding to the cathepsin D ERE.

Topics: 3T3 Cells; Animals; Base Sequence; Binding Sites; Breast Neoplasms; Cathepsin G; Cathepsins; Cell Line; Chloramphenicol O-Acetyltransferase; Chlorocebus aethiops; DNA; Endometrium; Estradiol; Female; HeLa Cells; Humans; Mice; Molecular Sequence Data; Mutagenesis, Insertional; Oligodeoxyribonucleotides; Promoter Regions, Genetic; Restriction Mapping; Serine Endopeptidases; Transfection; Tumor Cells, Cultured