Concomitant activation of pathways downstream of Grb2 and PI 3-kinase is required for MET-mediated metastasis.
The Met tyrosine kinase - the HGF receptor - induces cell transformation and metastasis when constitutively activated. Met signaling is mediated by phosphorylation of two carboxy-terminal tyrosines which act as docking sites for a number of SH2-containing molecules. These include Grb2 and p85 which couple the receptor, respectively, with Ras and PI 3-kinase. We previously showed that a Met mutant designed to obtain preferential coupling with Grb2 (Met2xGrb2) is permissive for motility, increases transformation, but - surprisingly - is impaired in causing invasion and metastasis. In this work we used Met mutants optimized for binding either p85 alone (Met2xPI3K) or p85 and Grb2 (MetPI3K/Grb2) to evaluate the relative importance of Ras and PI 3-kinase as downstream effectors of Met. Met2xPI3K was competent in eliciting motility, but not transformation, invasion, or metastasis. Conversely, MetP13K/Grb2 induced motility, transformation, invasion and metastasis as efficiently as wild type Met. Furthermore, the expression of constitutively active PI 3-kinase in cells transformed by the Met2xGrb2 mutant, fully rescued their ability to invade and metastasize. These data point to a central role for PI 3-kinase in Met-mediated invasiveness, and indicate that simultaneous activation of Ras and PI 3-kinase is required to unleash the Met metastatic potential. (+info)
Control of metastasis by Asn-linked, beta1-6 branched oligosaccharides in mouse mammary cancer cells.
Studies in cell lines and malignant human tissues have shown that increased cell-surface Asn-linked beta1-6(GlcNAcbeta1-6Man) branching is associated with increased tumorigenic and metastatic properties. In this study, three mouse mammary cancer cell lines were transfected with an expression vector containing the mouse cDNA for N-acetylglucosaminyltransferase V (GlcNAcT-V EC 22.214.171.124), the glycosyltransferase responsible for initiating beta1-6 branching on Asn-linked carbohydrates. The cell lines were screened for increased cytotoxicity to L-PHA, a lectin specific for beta1-6 branching structures. Cell lines exhibiting increased L-PHA cytotoxicity expressed increased levels of beta1-6 branching structures. Northern blots detected the presence of GlcNAcT-V transcribed from the expression vector in the L-PHA sensitive cell lines. After injection into the tail veins of mice, transfected cell lines with increased beta1-6 branching on the cell surface formed elevated levels of lung tumors relative to control transfected cell lines (P < 0.002). Western blots of membrane proteins from GlcNAcT-V transfected and control cells probed with the lectins DSA and WGA did not show an increase in polyN-acetyllactosamine and sialic acid content in the transfected cell lines. These results demonstrate that a specific increase in beta1-6 branching due to an elevation in GlcNAcT-V expression increases metastatic potential. (+info)
Role of thrombin receptor in breast cancer invasiveness.
Invasion, the ability of an epithelial cancer cell to detach from and move through a basement membrane, is a central process in tumour metastasis. Two components of invasion are proteolysis of extracellular matrix and cellular movement through it. A potential promoter of these two processes is thrombin, the serine proteinase derived from the ubiquitous plasma protein prothrombin. Thrombin promotes the invasion of MDA-MB231 breast tumour cells (a highly aggressive cell line) in an in vitro assay. Invasion by MDA-MB436 and MCF-7 cells, less aggressive cell lines, is not promoted by thrombin. Thrombin, added to the cells, is a stimulator of cellular movement; fibroblast-conditioned medium is the chemotaxin. Thrombin-promoted invasion is inhibited by hirudin. Stimulation of invasion is a receptor-mediated process that is mimicked by a thrombin receptor-activating peptide. Thrombin has no effect on chemotaxis in vitro. Thrombin receptor is detectable on the surface of MDA-MB231 cells, but not on the other two cell lines. Introduction of oestrogen receptors into MDA-MB231 cells by transfection with pHEO had no effect on thrombin receptor expression, in the presence or absence of oestradiol. This paper demonstrates that thrombin increases invasion by the aggressive breast cancer cell line MDA-MB231 by a thrombin receptor-dependent mechanism. (+info)
Development of a sensitive, specific reverse transcriptase polymerase chain reaction-based assay for epithelial tumour cells in effusions.
We developed a sensitive and specific method for the detection of epithelial cancer cells in effusions with a two-stage molecular-based assay which combined enrichment for cancer cells by immunomagnetic bead selection and reverse transcriptase polymerase chain reaction (RT-PCR) detection of epithelial glycoprotein 2 (EGP-2) RNA. Preliminary experiments indicated that immunobead selection was essential to avoid occasional false-positive RT-PCR results, and this method detected ten breast cancer cells electively added to 10(7) cytologically negative effusion cells. We studied 110 cases of pleural (n = 68) and peritoneal (n = 42) effusions (30 from patients with known carcinoma and 80 from those without known carcinoma), and the results were compared with cytological findings. Of 18 effusions that were cytologically positive or suspicious for malignant cells, 17 (94%) were positive for EGP-2 RNA (the one negative sample was from a patient who recently received combination chemotherapy). Of 92 cytologically negative samples, 11 (12%) were positive for EGP-2, including six patients with a history of previous or current carcinoma. Our method appears to be highly specific and increases the sensitivity of detection of malignant cells; it may be a useful adjunct to routine cytopathological examination. (+info)
Expression of tissue factor in non-small-cell lung cancers and its relationship to metastasis.
Tissue factor (TF) is an initiator of the extrinsic cascade of blood coagulation. Although recent studies have revealed a relationship between metastatic properties and TF expression in some neoplastic cells, the significance of TF in lung cancer, especially in non-small-cell lung cancer (NSCLC), is still unclear. In this study, TF was detected in NSCLC cell lines by functional study, Western blot analysis and immunocytochemical staining. TF levels in eight NSCLC cell lines were also quantitated by enzyme-linked immunosorbent assay (ELISA), and TF expression was evaluated in 55 specimens of surgically resected NSCLCs. NSCLC cell lines derived from metastatic lesions produced high levels of TF (48.3+/-23.5 ng 10(-6) cells, mean +/- s.e.m.), whereas those derived from primary lesions produced low levels of TF (0.2+/-0.1 ng 10(-6) cells). Immunohistochemical studies disclosed significantly stronger staining for TF in cells from NSCLC patients with metastasis than in those without metastasis. Among the 28 patients with metastasis, ten were strongly positive, 16 were moderately positive and two were negative for TF. In contrast, among the 27 patients without metastasis, only two were strongly positive, 18 were moderately positive and seven were negative for TF. Therefore, malignant cells from patients with lung cancer produce various levels of TF, and TF may play an important role in the metastatic process. (+info)
Peritoneal cytology in the surgical evaluation of gastric carcinoma.
Many patients undergoing surgery for gastric carcinoma will develop peritoneal metastases. A method to identify those patients at risk of peritoneal recurrence would help in the selection of patients for adjuvant therapy. Peritoneal cytology has received little attention in the West, but may prove a useful additional means of evaluating patients with gastric cancer. The aims of this study were to evaluate sampling techniques for peritoneal cytology in patients with gastric cancer, to assess the prognostic significance of free peritoneal malignant cells and to discover the effect of the operative procedure on dissemination of malignant cells. The study is based on 85 consecutive patients undergoing surgical treatment of gastric cancer and followed up for 2 years or until death. Peritoneal cytology samples were collected at laparoscopy, and at operation prior to resection by intraperitoneal lavage and serosal brushings. After resection, samples were taken by peritoneal lavage, imprint cytology of the resected specimen and post-operatively by peritoneal irrigation via a percutaneous catheter. Malignant cells were diagnosed by two independent microscopists. Preoperative peritoneal lavage yielded malignant cells in 16 out of 85 cases (19%). The yield of free malignant cells was increased by using serosal brushings (by four cases) and imprint cytology (by two cases); all of the cases had evidence of serosal penetration. One serosa-negative case exhibited positive cytology in the post-resection peritoneal specimen in which the preresection cytology specimen was negative. Survival was worse in the cytology-positive group (chi2 = 25.1; P< 0.0001). Among serosa-positive patients, survival was significantly reduced if cytology was positive, if cases yielded by brushings and imprint cytology were included (log-rank test = 8.44; 1 df, P = 0.004). In conclusion, free peritoneal malignant cells can be identified in patients with gastric cancer who have a poor prognosis; the yield can be increased with brushings and imprint cytology in addition to conventional peritoneal lavage. Evaluation of peritoneal cytology by these methods may have a role in the selection of patients with the poorest prognosis who may benefit most from adjuvant therapy. (+info)
Expression of pyrimidine nucleoside phosphorylase mRNA plays an important role in the prognosis of patients with oesophageal cancer.
To clarify the significance of the expression of pyrimidine nucleoside phosphorylase (PyNPase) mRNA as a predictive factor for the prognosis of patients with oesophageal carcinoma, the PyNPase mRNA in the tumours and normal tissues from 55 resected cases of oesophageal carcinoma was examined by a reverse transcription polymerase chain reaction (RT-PCR). As a result, a positive correlation was observed between the tumour/normal (T/N) ratio of the expression of PyNPase mRNA by RT-PCR and that of the enzyme activity of PyNPase based on the findings of an enzyme linked immunosolvent assay (r = 0.594, P = 0.009). The T/N ratio of the expression of PyNPase mRNA was significantly higher in the cases with lymph vessel invasion (P = 0.013), lymph node metastasis (P = 0.0016), and an advanced stage of the disease (P = 0.021) than those without these factors. The patients with a higher T/N ratio of PyNPase mRNA showed significantly worse prognosis than those with a lower T/N ratio (P = 0.023 with log-rank tests). A multivariate analysis for the cumulative survival rates revealed that a high T/N ratio of the expression of PyNPase mRNA was independently related to a poor prognosis. These findings suggested that the determination of PyNPase mRNA by RT-PCR thus appears to be a new useful parameter for identifying both a poor prognosis and a highly malignant potential of oesophageal carcinoma. (+info)
Phase I study of escalating doses of edatrexate in combination with paclitaxel in patients with metastatic breast cancer.
Motivated by the observation of preclinical synergy, a Phase I dose escalation study of edatrexate in combination with a 3-h paclitaxel infusion was performed in patients with advanced breast cancer to determine the maximum tolerated dose (MTD) of edatrexate and the toxicities associated with this combination and to report preliminary observations of efficacy with this novel combination. Thirty-six patients were enrolled in this Phase I trial. Thirty-five eligible patients were treated every 21 days in cohorts of at least three patients and were assessable for toxicity. One patient was ineligible due to hyperbilirubinemia. Stepwise dose escalations of edatrexate were administered until grade >3 nonhematological dose-limiting toxicities were reported. The initial dose level of edatrexate was 180 mg/m2; subsequent cohorts were treated with escalating doses of edatrexate (210, 240, 270, 300, 350, and 400 mg/m2). Edatrexate was administered by i.v. infusion over 1 h. Paclitaxel was administered 24 h later at a fixed dose of 175 mg/m2 as a 3-h infusion with standard dexamethasone, diphenhydramine, and cimetidine premedication. The MTD of edatrexate was reached at the 350 mg/m2 level in this study. Grade 3 diarrhea was seen in one patient at the 300 and 400 mg/m2 dose levels, requiring dose reductions. Two patients experienced grade 4 stomatitis at the 400 mg/m2 dose level and also required dose reduction, establishing the MTD as 350 mg/m2. Grade 3 nausea and vomiting were noted in two of three patients at the highest dose level. Of 35 patients, 4 patients reported grade 3 myalgias and 1 patient reported grade 3 neurosensory complaints, which were seen mostly at the 350 and 400 mg/m2 dose levels; however, 1 patient reported grade 3 myalgias at 180 mg/m2. No cumulative neurotoxicity was observed, and no patient experienced an allergic reaction to paclitaxel. In 23 patients with bidimensionally measurable disease, there were four complete (17%) and seven partial responses, with an overall response rate of 48% (95% confidence interval, 27-69%). All of the responses were seen in patients who had not received prior chemotherapy for stage IV disease. The median duration of response was not assessable because many responding patients went on to receive high-dose chemotherapy treatment with stem cell support. The combination of edatrexate and paclitaxel for treatment of metastatic breast cancer is a feasible and safe regimen. The MTD of edatrexate was 350 mg/m2 when combined with a 3-h infusion of paclitaxel (175 mg/m2) given 24 h later. Activity was noted even among patients who had relapsed shortly after receiving methotrexate- and/or doxorubicin-containing adjuvant regimens. Additional studies evaluating the sequences and dosing schema for this combination are warranted to improve the response proportion and define the duration of the response. (+info)