The common neurotrophin receptor p75NTRhas been shown to initiate intracellular signaling that leads either to cell survival or to apoptosis depending on the cell type examined; however, the ...mechanism by which p75NTR initiates its intracellular transduction remains unclear. We show here that the tumor necrosis factor receptor-associated death domain protein (TRADD) interacts with p75NTR upon nerve growth factor (NGF) stimulation. TRADD could be immunodetected after p75NTRimmunoprecipitation from MCF-7 breast cancer cells stimulated by nerve growth factor. In addition, confocal microscopy indicated that NGF stimulation induced the plasma membrane localization of TRADD. Using a dominant negative form of TRADD, we also show that interactions between p75NTR and TRADD are dependent on the death domain of TRADD, thus demonstrating its requirement for binding. Furthermore, the p75NTR-mediated activation of NF-κB was inhibited by transfection with a dominant negative TRADD, resulting in an inhibition of NGF antiapoptotic activity. These results thus demonstrate that TRADD is involved in the p75NTR-mediated antiapoptotic activity of NGF in breast cancer cells.
(-)-Epigallocatechin (EGC), one of green tea polyphenols, has been shown to inhibit growth of cancer cells. However its mechanism of action is poorly known. We show here that EGC strongly inhibited ...the growth of breast cancer cell lines (MCF-7 and MDA-MB-231) but not that of normal breast epithelial cells. The inhibition of breast cancer cell growth was due to an induction of apoptosis, without any change in cell cycle progression. MCF-7 cells are known to express a wild-type p53 whereas MDA-MB-231 cells express a mutated p53. The fact that EGC induced apoptosis in both these cell lines suggests that the EGC-triggered apoptosis is independent of p53 status. Moreover, neutralizing antibodies against the death receptor Fas and inhibitors of caspases, such as caspase-8 and -10, efficiently inhibited the EGC-triggered apoptosis. In addition, immunoblotting revealed that EGC treatment was correlated with a decrease in Bcl-2 and an increase in Bax level. These results suggest that EGC-triggered apoptosis in breast cancer cells requires Fas signaling.
In breast cancer cells, the neurotrophin receptor p75(NTR) acts as a prosurvival factor able to stimulate resistance to apoptosis, but its mechanism of action remains incompletely defined. In this ...study, we investigated the global proteome modification induced by p75(NTR) overexpression in breast cancer cells treated by the pro-apoptotic agent tumor necrosis factor (TNF)-related-apoptosis-inducing-ligand (TRAIL). p75(NTR) was stably overexpressed in the MCF-7 breast cancer cells and the impact of a treatment by TRAIL was investigated in wild type vs. p75(NTR) overexpressing cells. Proteins were separated in two-dimensional electrophoresis, and regulated spots were detected by computer assisted analysis before identification by MALDI-TOF/TOF mass spectrometry. In the absence of TRAIL treatment, p75(NTR) did not induce any change in the proteome of breast cancer cells. In contrast, after treatment with TRAIL, fragments of cytokeratin-8, -18 and -19, as well as full length cytokeratin-18, were up-regulated by p75(NTR) overexpression. Of note, spectrin alpha-chain and the ribosomal protein RPLP0 were induced by TRAIL, independently of p75(NTR) level. Interestingly, the well known stress-induced protein HSP-27 was less abundant when p75(NTR) was overexpressed, indicating that p75(NTR) overexpression reduced TRAIL induced cell stress. These data indicate that overexpression of p75(NTR) induces proteome modifications in breast cancer cells and provide information on how this receptor contributes in tumor cell resistance to apoptosis.
We show here that nerve growth factor (NGF), the archetypal neurotrophic factor, is able to stimulate the proliferation of breast cancer cells (MCF-7 and MDA-MB-231 cell lines), although it is unable ...to stimulate growth of normal breast epithelial cells (NBEC). This stimulation induced cells in the G0 phase to reenter the cell cycle, as well as shortening cell cycle duration. Immunoblotting experiments revealed that both the two cancer cell lines and the NBEC express high affinity (p140(trk)) and low affinity (p75) NGF receptors. Inhibition of the NGF growth-promoting effect by the drugs K-252a and PD98059 indicated that activation of Trk-tyrosine kinase activity and the mitogen-activated protein kinase cascade are necessary to obtain the mitogenic effect. Activation of mitogen-activated protein kinase can be detected in breast cancer cells after 10 min of NGF stimulation, whereas no change was detected in NBEC. These results demonstrate that NGF is a mitogenic factor for human breast cancer cells and that it might constitute a new regulator of breast tumor growth.
Breast cancer is the most common form of cancer among women and the identification of markers to discriminate tumorigenic from normal cells, as well as the different stages of this pathology, is of ...critical importance. Two‐dimensional electrophoresis has been used before for studying breast cancer, but the progressive completion of human genomic sequencing and the introduction of mass spectrometry, combined with advanced bioinformatics for protein identification, have considerably increased the possibilities for characterizing new markers and therapeutic targets. Breast cancer proteomics has already identified markers of potential clinical interest (such as the molecular chaperone 14‐3‐3 sigma) and technological innovations such as large scale and high throughput analysis are now driving the field. Methods in functional proteomics have also been developed to study the intracellular signaling pathways that underlie the development of breast cancer. As illustrated with fibroblast growth factor‐2, a mitogen and motogen factor for breast cancer cells, proteomics is a powerful approach to identify signaling proteins and to decipher the complex signaling circuitry involved in tumor growth. Together with genomics, proteomics is well on the way to molecularly characterizing the different types of breast tumor, and thus defining new therapeutic targets for future treatment.
To explore how heparan sulfate (HS) controls the responsiveness of the breast cancer cell lines MCF-7 and MDA-MB-231 to fibroblast growth factors (FGFs), we have exposed them to HS preparations known ...to have specificity for FGF-1 (HS glycosaminoglycan (HSGAG A)) or FGF-2 (HSGAGB). Proliferation assays confirmed that MCF-7 cells were highly responsive to FGF-2 complexed with GAGB, whereas migration assays indicated that FGF-1/HSGAGA combinations were stimulatory for the highly invasive MDA-MB-231 cells. Quantitative polymerase chain reaction for the levels of FGF receptor (FGFR) isoforms revealed that MCF-7 cells have greater levels of FGFR1 and that MDA-MB-231 cells have greater relative levels of FGFR2. Cross-linking demonstrated that FGF-2/HSGAGB primarily activated FGFR1, which in turn up-regulated the activity of mitogen-activated protein kinase; in contrast, FGF-1/HSGAGA led to the phosphorylation of equal proportions of both FGFR1 and FGFR2, which in turn led to the up-regulation of Src and p125FAK. MDA-MB-231 cells were particularly responsive to vitronectin substrates in the presence of FGF-1/HSGAGA, and blocking antibodies established that they used the αvβ3 integrin to bind to it. These results suggest that the clustering of particular FGFR configurations on breast cancer cells induced by different HS chains leads to distinct phenotypic behaviors.
Nerve growth factor (NGF) and basic fibroblast growth factor (bFGF) stimulate neuronal differentiation, whereas epidermal growth factor (EGF) promotes only mitogenic responses in PC12 ...pheochromocytoma cells. The early changes in protein synthesis induced by bFGF, NGF, and EGF in these cells have been determined by two-dimensional PAGE of 35Smethionine-labeled proteins and computerized image analysis. The rate of synthesis of only 29 proteins (out of ≈1500 identified) was found to be modulated during the first several hours of growth factor stimulation. Individually, 12 were affected by EGF, 23 were affected by bFGF, and 20 were affected by NGF. Eight of these were regulated by all three growth factors, while 10 proteins were commonly induced by bFGF and NGF, in accordance with the essentially identical morphological responses induced by these two factors. In addition, the effects of bFGF and NGF were about equally divided between increases and decreases in the rate of synthesis of individual proteins, whereas EGF caused significantly more positive (increased) responses. All proteins modulated by NGF or FGF alone were negative in their response and those induced by only EGF were positive. Of particular interest, the rate of synthesis of two proteins of 55 kDa and pI 5.45 and 5.50 was dramatically and transiently induced during the first 2 hr of bFGF and NGF treatment and was not affected by EGF. This study indicates that all three factors elicit early increases and decreases in the synthesis of a quite limited number of proteins and provides molecular evidence for the specificity of a differentiative vs. a proliferative growth factor-induced signaling pathway in these cells.
Purpose: We have recently shown that breast tumors express high levels of TrkA compared with normal breast tissues, with TrkA overexpression enhancing breast cancer cell invasion in vitro and ...metastasis in animal models. In this study, we tried to identify molecules involved in TrkA overexpression‐mediated biological effects in breast cancer cells.
Experimental design: We used a proteomic‐based approach to identify proteins involved in TrkA overexpression‐stimulated invasion of MDA‐MB‐231 breast cancer cells. Proteins from control and TrkA overexpressing cells were separated using a cup‐loading two‐dimensional electrophoresis system before MALDI and LC‐MS/MS mass spectrometry analysis.
Results: Among several putative regulated proteins, Ku86 was found increased in TrkA overexpressing cells. Moreover, Ku86 was co‐immunoprecipitated with TrkA, suggesting the interaction of these two proteins in TrkA overexpressing cells. Interestingly, inhibition with small‐interfering RNA and neutralizing antibodies showed that Ku86 was required for TrkA‐stimulated cell invasion.
Conclusions and clinical relevance: These data allowed the identification of Ku86 as a new player involved in metastasis in breast cancer cells. Our findings suggest that TrkA and its down stream signaling pathways should be regarded as potential new targets for the development of future breast cancer therapy.
We performed a competitive binding study with 125I-labelled FGF (fibroblast growth factor)-2 and unlabelled FGF-2 in an unselected series of two hundred and thirty human primary breast cancers. One ...hundred and ninety-two breast cancer biopsies possessed FGF-2 low-affinity binding sites (FGF-2 LABS). The median dissociation constant was 2.4 nM (range, 1.03-18) and the median concentration of membrane protein was 6187.5 fmol/mg (range, 831-90,000). FGF-2 LABS concentrations were positively correlated to the progesterone receptor level. Cox univariate analyses showed that the FGF-2 LABS (> or = upper quartile) was associated to a longer overall survival (p = 0.05; RR = 0.042); node involvement, estrogen receptor progesterone receptor and histoprognostic grading were also prognostic. In Cox multivariate analyses, only the progesterone receptor, estrogen receptor, node involvement and FGF-2 LABS were prognostic factors; the FGF-2 LABS were associated with a longer overall survival (p = 0.033; RR = 0.068). The present study showed that FGF-2 LABS have only a limited role as a prognostic factor in breast cancer.
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