mRNA expression levels of the biological factors uPAR, uPAR-del4/5, and rab31, displaying prognostic value in breast cancer, are not clinically relevant in advanced ovarian cancer.

High tumor tissue mRNA expression of the tumor biological factors uPAR, uPAR-del4/5, or rab31 is associated with shorter distant metastasis-free and overall survival in breast cancer patients. To evaluate whether these factors are also clinically relevant in ovarian cancer, we quantified the respective mRNA levels in primary tumor tissue of advanced ovarian cancer patients (n=103) and evaluated their association with clinicopathological parameters and patients' prognosis. mRNA expression levels of all three markers did not show any significant association with overall or progression-free survival, demonstrating that these factors have no prognostic value in advanced ovarian cancer.

Primary metastatic leiomyosarcoma of the fallopian tube: a rare case report.

BACKGROUND: Leiomyosarcoma of the fallopian tube is an extremely unusual gynecologic neoplasm. Since 1886, only 19 of about 35 sarcomas of the fallopian tube have been identified as leiomyosarcomas. As such, clinical diagnosis and therapy management are difficult. CASE REPORT: We report on the case of a 59-year-old woman with leiomyosarcoma of the fallopian tube and liver metastases at the time of diagnosis. After initial tumor debulking, she received palliative chemotherapy with gemcitabine 900 mg/m(2) (d1+8) and docetaxel 100 mg/m(2) (d8) (q21). For additional bone metastases, she started local radiation plus bisphosphonates (q28). After 2 cycles of chemotherapy, the disease progressed, and the patient died within 8 months of diagnosis. A review of the literature is given. CONCLUSIONS: Primary metastatic leiomyosarcoma of the fallopian tube is a progressive disease with limited therapy options. For better prognostic evaluation and disease management in such rare cases, it is important to report and compare more cases regarding course of disease and outcome.

FoxM1 is a downstream target and marker of HER2 overexpression in breast cancer.

The tyrosine kinase receptor, HER2 is a crucial prognostic marker and therapeutic target for breast cancer; however, the downstream targets and biological effectors of HER2 remain unclear. We investigated the relationship between HER2 and the transcription factor FoxM1 in breast cancer. HER2 and FoxM1 expression levels were compared in breast carcinoma cell lines, paraffin-embedded breast cancer patient samples and at the mRNA level in purified breast epithelial cells. To further examine the relationship between HER2 and FoxM1 expression, we either overexpressed or siRNA-mediated depleted endogenous HER2 in breast cancer cell lines. Additionally, a mammary epithelium-targeted HER2 (neu) transgenic mouse model was also used to assess the effect of HER2 on FoxM1 levels. Furthermore, the effect of the HER2-tyrosine kinase inhibitor lapatinib on FoxM1 in HER2 positive breast cancer cells was investigated. HER2 protein levels directly correlated with FoxM1 expression in both breast carcinoma cell lines and paraffin-embedded breast cancer patient samples. Moreover, in purified breast epithelial cells, overexpression of HER2 was associated with high levels of FoxM1 mRNA, suggesting that the upregulation of FoxM1 expression is at least partially mediated transcriptionally. Furthermore, overexpression or ablation of endogenous HER2 resulted in parallel changes in FoxM1 expression. Critically, mammary epithelium-targeted HER2 mouse tumours also resulted in increased FoxM1 expression, suggesting that HER2 directed FoxM1 expression occurs in vivo and may be a critical downstream effector of HER2-targeting therapies. Indeed, treatment of breast cancer cells with lapatinib reduced FoxM1 expression at protein, mRNA and gene promoter levels. Moreover, analysis of normal and breast cancer patient samples revealed that elevated FoxM1 expression at protein and mRNA levels correlated with breast cancer development, but not significantly with cancer progression and survival. Our results indicate that the HER2 receptor regulates the expression of the FoxM1 transcription factor, which has a role in breast cancer development.

Gefitinib (Iressa) represses FOXM1 expression via FOXO3a in breast cancer.

Gefitinib (Iressa) is a specific and effective epidermal growth factor receptor inhibitor. An understanding of the downstream cellular targets of gefitinib will allow the discovery of biomarkers for predicting outcomes and monitoring anti-epidermal growth factor receptor therapies and provide information for overcoming gefitinib resistance. In this study, we investigated the role and regulation of FOXM1 in response to gefitinib treatment in breast cancer. Using the gefitinib-sensitive breast carcinoma cell lines BT474 and SKBR3 as well as the resistant lines MCF-7, MDA-MB-231, and MDA-MB-453, we showed that gefitinib represses the expression of the transcription factor FOXM1 in sensitive, but not resistant, cells. FOXM1 repression by gefitinib is associated with FOXO3a activation and is mediated at the transcriptional level and gene promoter level. These results were verified by immunohistochemical staining of biopsy samples from primary breast cancer patients obtained from a gefitinib neoadjuvant study. We also showed that ectopic expression of an active FOXO3a represses FOXM1 expression, whereas knockdown of FOXO3a expression using small interfering RNA can up-regulate FOXM1 and its downstream targets polo-like kinase, cyclin B1, and CDC25B and rescue sensitive BT474 cells from gefitinib-induced cell proliferative arrest. These results suggest that gefitinib represses FOXM1 expression via FOXO3a in breast cancer. We further showed that overexpression of a wild-type FOXM1 or a constitutively active FOXM1, DeltaN-FOXM1, abrogates the cell death induced by gefitinib, indicating that FOXM1 has a functional role in mediating the gefitinib-induced proliferative arrest and in determining sensitivity to gefitinib. In summary, our study defined FOXM1 as a cellular target and marker of gefitinib activity in breast cancer.

The transcription factor FOXO3a is a crucial cellular target of gefitinib (Iressa) in breast cancer cells.

Gefitinib is a specific inhibitor of the epidermal growth factor receptor (EGFR) that causes growth delay in cancer cell lines and human tumor xenografts expressing high levels of EGFR. An understanding of the downstream cellular targets of gefitinib will allow the discovery of biomarkers for predicting outcomes and monitoring anti-EGFR therapies and provide information for key targets for therapeutic intervention. In this study, we investigated the role of FOXO3a in gefitinib action and resistance. Using two gefitinib-sensitive (i.e., BT474 and SKBR3) as well as three other resistant breast carcinoma cell lines (i.e., MCF-7, MDA-MB-231, and MDA-MB-453), we showed that gefitinib targets the transcription factor FOXO3a to mediate cell cycle arrest and cell death in sensitive breast cancer cells. In the sensitive cells, gefitinib treatment causes cell cycle arrest predominantly at the G(0)-G(1) phase and apoptosis, which is associated with FOXO3a dephosphorylation at Akt sites and nuclear translocation, whereas in the resistant cells, FOXO3a stays phosphorylated and remains in the cytoplasm. The nuclear accumulation of FOXO3a in response to gefitinib was confirmed in tumor tissue sections from breast cancer patients presurgically treated with gefitinib as monotherapy. We also showed that knockdown of FOXO3a expression using small interfering RNA (siRNA) can rescue sensitive BT474 cells from gefitinib-induced cell-proliferative arrest, whereas reintroduction of active FOXO3a in resistant MDA-MB-231 cells can at least partially restore cell-proliferative arrest and sensitivity to gefitinib. These results suggest that the FOXO3a dephosphorylation and nuclear localization have a direct role in mediating the gefitinib-induced proliferative arrest and in determining sensitivity to gefitinib.

G-CSF in the prevention of febrile neutropenia in chemotherapy in breast cancer patients.

The most common chemotherapeutic agents in the treatment of breast cancer are anthracyclines and taxanes. The major dose-limiting toxicities associated with these agents are myelosuppression and associated febrile neutropenia (FN). FN can significantly impact the ability to deliver full-dose chemotherapy on schedule and as a result may increase the risk of disease recurrence and eventual disease-related mortality. The use of granulocyte colony stimulating factors (G-CSFs) significantly improves the management of FN, both in a therapeutic and in a prophylactic approach. Nevertheless, the high cost of these agents limits their widespread prophylactic use. Therefore, the identification of patients who are at a higher risk of developing FN and who will benefit from the prophylactic use of G-CSFs has become the subject of several clinical and cost-effectiveness studies. Recently, new data have been accumulated concerning the risk of FN in different chemotherapy regimens, and different risk models have been developed to assess the neutropenic risk with all its complications. This article reviews and summarizes cutting-edge, disease-specific data as well as national and international guidelines regarding the use of G-CSFs to prevent chemotherapy-induced FN, with focus on the treatment of breast cancer.

Ex vivo detection of apoptotic trophoblast cells applying flow cytofluorometry and immunocytochemistry using M30 antibody directed to the cytokeratin 18 neo-epitope.

Apoptosis of placental trophoblast cells has become the subject of intensive research. Recently, a monoclonal antibody (M30) directed against a neo-epitope of cytokeratin 18, that is formed after cleavage of this cytoskeletal protein by caspases, was shown to be of advantage over other tests for the detection of trophoblast cell apoptosis. In the present study, we describe a method for the enrichment of highly pure villous trophoblast cells based on the proteolytic digestion of placental tissue, density gradient separation of dissected cells, and immunoelimination of contaminating, non-trophoblast cells employing an antibody to the HLA class I antigen. The high purity (94-99%) of the trophoblast cell preparation was shown by antibody staining for cytokeratin 7 and absence of vimentin. Furthermore, we demonstrate that after a simple permeabilization and fixation step with 90% methanol and using the M30 CytoDeath, FITC-conjugated antibody, apoptotic trophoblast cells could be distinguished from non-apoptotic cells by flow cytofluorometry in a highly quantitative and sensitive fashion. Our protocol is an improvement over previously used methods such as immunocytochemistry as it allows to differentiate rapidly between competent and apoptotic trophoblast cells by the quantitative method of flow cytofluorometry.

Novel bi- and trifunctional inhibitors of tumor-associated proteolytic systems.

Serine proteases, cysteine proteases, and matrix metalloproteinases (MMPs) are involved in cancer cell invasion and metastasis. Recently, a recombinant bifunctional inhibitor (chCys-uPA19-31) directed against cysteine proteases and the urokinase-type plasminogen activator (uPA)/plasmin serine protease system was generated by introducing the uPA receptor (uPAR)-binding site of uPA into chicken cystatin (chCysWT). In the present study, we designed and recombinantly produced multifunctional inhibitors also targeting MMPs. The inhibitors comprise the N-terminal inhibitory domain of human TIMP-1 (tissue inhibitor of matrix metalloproteinase-1) or TIMP-3, fused to chCys-uPA19-31 or chCysWT. As demonstrated by various techniques, these fusion proteins effectively interfere with all three targeted protease systems. In in vitro Matrigel invasion assays, the addition of recombinant inhibitors strongly reduced invasion of ovarian cancer cells (OV-MZ-6#8). Additionally, OV-MZ-6#8 cells were stably transfected with expression plasmids encoding the various inhibitors. Synthesis and secretion of the inhibitors was verified by a newly developed ELISA, which selectively detects the recombinant proteins. Invasive capacity of inhibitor-producing cells was significantly reduced compared to vector-transfected control cells. Thus, these novel, compact, and small-size inhibitors directed against up to three different tumor-associated proteolytic systems may represent promising agents for prevention of tumor cell migration and metastasis.

Inhibition of intraperitoneal tumor growth of human ovarian cancer cells by bi- and trifunctional inhibitors of tumor-associated proteolytic systems.

Several proteolytic systems are involved in (anti)adhesive, migratory, and proteolytic processes, necessary for tumor progression and metastasis. We analyzed whether multifunctional inhibitors of different tumor-associated proteolytic systems reduce tumor growth and spread of human ovarian cancer cells in vivo. Bifunctional inhibitors are composed of the N-terminal domain of either the human matrix metalloproteinase inhibitors TIMP-1 or TIMP-3 and the cysteine protease inhibitor chicken cystatin (chCysWT); trifunctional inhibitors are composed of N-TIMP-1 or -3 and a chicken cystatin variant harboring the uPAR binding site of uPA, chCys-uPA19-31, which in addition to its inhibitory activity toward cysteine proteases interferes with the interaction of the serine protease uPA with its receptor. OV-MZ-6#8 cancer cells, stably transfected with plasmids expressing the multifunctional inhibitors, displayed similar proliferative and adhesive features as the vector-transfected control, but showed significant reduction in their invasive behavior in vitro. The cell lines expressing the multifunctional inhibitors were inoculated into the peritoneum of nude mice. Expression of three of the four inhibitor variants (N-hTIMP-1-chCysWT, N-hTIMP-1-chCys-uPA19-31, and N-hTIMP-3-chCysWT) resulted in a significant reduction of tumor burden compared to the vector-control cell line. These compact and small inhibitors may represent promising agents for gene therapy of solid malignant tumors.

Loss of microglial ramification in microglia-astrocyte cocultures: involvement of adenylate cyclase, calcium, phosphatase, and Gi-protein systems.

Reduction in microglial branching is a common feature in brain pathology and culminates in the transformation into small, rounded, microglia-derived phagocytes in the presence of neural debris. The molecular factors responsible for this transformation are unknown. Here we explored the effect of different classes of intra- and extracellular stimuli in vitro on the morphology of ramified microglia cultured on a confluent astrocyte substrate. These studies showed a strong dose-dependent effect for the Ca(2+) ionophore calcimycine/A21837 (50 microM) and for dibutyryl-cAMP (1 mM), with a loss of microglial ramification. Direct activation of the adenylate cyclase with forskolin (0.1 mM) also led to the disappearance of microglial branching. Okadaic acid (70 nM), the inhibitor of protein phosphatases 1 and 2A (PP1/PP2A), and pertussis toxin (12.5 microg/ml), a G(i)-protein inhibitor, also showed similar effects. No effect was observed for dibutyryl-cGMP or for UTP; addition of ATP had a moderate effect, but only at very high, probably nonphysiological concentrations (100 mM). Extracellular matrix components such as keratatan-sulfate, integrin receptor blockers, the disintegrins kistrin, echistatin, and flavoridin, or the serine protease thrombin all had no effect. Addition of prostaglandin D(2) (PGD(2)), a molecule produced by activated microglial cells, had a transforming effect, but at concentrations two orders of magnitude higher than that of established PGD(2) receptors. In summary, addition of agents causing intracellular elevation of Ca(2+) and cAMP or inhibition of G(i)-proteins and phosphatases to ramified microglia cultured on top of confluent astrocytes leads to a rapid loss of microglial branching. Signaling cascades controlled by these molecules may play an important role in the regulation of this common physiological process in the injured brain.