Xuefen Le Bourhis

Nerve growth factor stimulates proliferation and survival of human breast cancer cells through two distinct signaling pathways

We show here that the neurotrophin nerve growth factor (NGF), which has been shown to be a mitogen for breast cancer cells, also stimulates cell survival through a distinct signaling pathway. Breast cancer cell lines (MCF-7, T47-D, BT-20, and MDA-MB-231) were found to express both types of NGF receptors: p140 trkA and p75NTR. The two other tyrosine kinase receptors for neurotrophins, TrkB and TrkC, were not expressed. The mitogenic effect of NGF on breast cancer cells required the tyrosine kinase activity of p140 trkA as well as the mitogen-activated protein kinase (MAPK) cascade, but was independent of p75NTR. In contrast, the anti-apoptotic effect of NGF (studied using the ceramide analogue C2) required p75NTR as well as the activation of the transcription factor NF-kB, but neither p140 trkA nor MAPK was necessary. Other neurotrophins (BDNF, NT-3, NT-4/5) also induced cell survival, although not proliferation, emphasizing the importance of p75NTR in NGF-mediated survival. Both the pharmacological NF-κB inhibitor SN50, and cell transfection with IkBm, resulted in a diminution of NGF anti-apoptotic effect. These data show that two distinct signaling pathways are required for NGF activity and confirm the roles played by p75NTR and NF-κB in the activation of the survival pathway in breast cancer cells.

Store depletion and store-operated Ca2+ current in human prostate cancer LNCaP cells: involvement in apoptosis

1 In the present study, we investigated the mechanisms involved in the induction of apoptosis by the Ca2+‐ATPase inhibitor thapsigargin (TG), in androgen‐sensitive human prostate cancer LNCaP cells. 2 Exposure of fura‐2‐loaded LNCaP cells to TG in the presence of extracellular calcium produced an increase in intracellular Ca2+, the first phase of which was associated with depletion of intracellular stores and the second one with consecutive extracellular Ca2+ entry through plasma membrane, store‐operated Ca2+ channels (SOCs). 3 For the first time we have identified and characterized the SOC‐mediated membrane current (Istore) in prostate cells using whole‐cell, cell‐attached, and perforated patch‐clamp techniques, combined with fura‐2 microspectrofluorimetric and Ca2+‐imaging measurements. 4 I store in LNCaP cells lacked voltage‐dependent gating and displayed an inwardly rectifying current‐voltage relationship. The unitary conductance of SOCs with 80 mM Ca2+ as a charge carrier was estimated at 3.2 ± 0.4 pS. The channel has a high selectivity for Ca2+ over monovalent cations and is inhibited by Ni2+ (0.5–3 mM) and La3+ (1 μM). 5 Treatment of LNCaP cells with TG (0.1 μM) induced apoptosis as judged from morphological changes. Decreasing extracellular free Ca2+ to 200 nM or adding 0.5 mM Ni2+ enhanced TG‐induced apoptosis. 6 The ability of TG to induce apoptosis was not reduced by loading the cells with intracellular Ca2+ chelator (BAPTA‐AM). 7 These results indicate that in androgen‐sensitive prostate cancer cells the depletion of intracellular Ca2+ stores may trigger apoptosis but that there is no requirement for the activation of store‐activated Ca2+ current and sustained Ca2+ entry in induction and development of programmed cell death.

Nerve growth factor is a potential therapeutic target in breast cancer.

We show here that nerve growth factor (NGF), the prototypic neurotrophin, can be targeted in breast cancer to inhibit tumor cell proliferation, survival, and metastasis. Analysis of a series of biopsies revealed widespread expression of NGF in the majority of human breast tumors, with anti-NGF immunoreactivity concentrated in the epithelial cancer cells. Moreover, immunodeficient mice xenografted with human breast cancer cells and treated with either anti-NGF antibodies or small interfering RNA against NGF displayed inhibited tumor growth and metastasis. Such treatments directed against NGF induced a decrease in cell proliferation with a concomitant increase in apoptosis of breast cancer cells and an inhibition of tumor angiogenesis. Together, these data indicate that targeting NGF in breast cancer may have therapeutic ramifications.

Epigallocatechin (EGC) of green tea induces apoptosis of human breast cancer cells but not of their normal counterparts.

Abstract(−)-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.

P21 WAF1/CIP1 is dispensable for G1 arrest, but indispensable for apoptosis induced by sodium butyrate in MCF-7 breast cancer cells

Sodium butyrate (NaB) has been proposed as a potential anticancer agent. However, its mechanism of action is not totally elucidated. Here, we showed that NaB-induced cell cycle arrest and apoptosis were associated with an increase of P21waf1/cip1 in MCF-7 breast cancer cells. This increase was more important in the nuclei, as revealed by immunofluorescence analysis. Transient transfections of MCF-7 cells with p21 deficient for interaction with CDK, but not with p21 deficient for interaction with PCNA (p21PCNA−), abrogated NaB-induced cell cycle arrest. This indicated that cell cycle blockage involved the interaction of P21waf1/cip1 with CDK. However, P21waf1/cip1 was dispensable, since p21 antisense did not modify cell cycle arrest. On the other hand, NaB-induced apoptosis was abolished by p21 antisense or p21PCNA−. In addition, NaB decreased PCNA levels, but increased the association of PCNA with P21waf1/cip1. These results suggested that NaB-induced apoptosis required P21waf1/cip1 and its interaction with PCNA.

Sodium butyrate induces P53-independent, Fas-mediated apoptosis in MCF-7 human breast cancer cells

This study was performed to determine the effect and action mechanisms of sodium butyrate (NaB) on the growth of breast cancer cells. Butyrate inhibited the growth of all breast cancer cell lines analysed. It induced cell cycle arrest in G1 and apoptosis in MCF‐7, MCF‐7ras, T47‐D, and BT‐20 cells, as well as arrest in G2/M in MDA‐MB‐231 cells. Transient transfection of MCF‐7 and T47‐D cells with wild‐type and antisense p53 did not modify butyrate‐induced apoptosis. Pifithrin‐α, which inhibits the transcriptional activity of P53, did not modify cell growth or apoptosis of MCF‐7 and T47‐D cells treated with butyrate. These results indicate that P53 was not involved in butyrate‐induced growth inhibition of breast cancer cells. Treatment of MCF‐7 cells with anti‐Fas agonist antibody induced cell death, indicating that Fas was functional in these cells. Moreover, butyrate potentiated Fas‐induced apoptosis, as massive apoptosis was observed rapidly when MCF‐7 cells were treated with butyrate and anti‐Fas agonist antibody. In addition, butyrate‐induced apoptosis in MCF‐7 cells was considerably reduced by anti‐Fas antagonist antibody. Western blot analysis showed that butyrate increased Fas and Fas ligand levels (Fas L), indicating that butyrate‐induced apoptosis may be mediated by Fas signalling. These results demonstrate that butyrate inhibited the growth of breast cancer cells in a P53‐independent manner. Moreover, it induced apoptosis via the Fas/Fas L system and potentiated Fas‐triggered apoptosis in MCF‐7 cells. These findings may open interesting perspectives in human breast cancer treatment strategy.

Nerve Growth Factor Receptors and Signaling in Breast Cancer

Nerve growth factor (NGF) has long been known for its effects on neuronal cell survival and differentiation. This prototypical neurotrophic factor stimulates neurons through two distinct classes of membrane receptors: the TrkA tyrosine kinase receptor, and the tumor necrosis factor receptor family member p75NTR, also known as the common neurotrophin receptor. Somewhat surprisingly, there is a growing body of evidence indicating that NGF is also a major stimulator of breast cancer cell growth. Both the survival and proliferation of breast cancer cells are strongly stimulated by NGF, mediated by TrkA and p75NTR respectively, utilising signaling pathways similar to those described for neurons. In addition, although NGF is produced by breast cancer cells, it is not in normal breast epithelial cells, giving rise to an autocrine stimulation of tumor growth. Therefore, NGF receptors and signaling are thus looking increasingly promising as potential drug targets for breast cancer.

H19 non coding RNA-derived miR-675 enhances tumorigenesis and metastasis of breast cancer cells by downregulating c-Cbl and Cbl-b

H19 is a long non-coding RNA precursor of miR-675microRNA. H19 is increasingly described to play key roles in the progression and metastasis of cancers from different tissue origins. We have previously shown that the H19 gene is activated by growth factors and increases breast cancer cell invasion. In this study, we established H19/miR-675 ectopic expression models of MDA-MB-231 breast cancer cells to further investigate the underlying mechanisms of H19 oncogenic action. We showed that overexpression of H19/miR-675 enhanced the aggressive phenotype of breast cancer cells including increased cell proliferation and migration in vitro, and increased tumor growth and metastasis in vivo. Moreover, we identified ubiquitin ligase E3 family (c-Cbl and Cbl-b) as direct targets of miR-675 in breast cancer cells. Using a luciferase assay, we demonstrated that H19, through its microRNA, decreased both c-Cbl and Cbl-b expression in all breast cancer cell lines tested. Thus, by directly binding c-Cbl and Cbl-b mRNA, miR-675 increased the stability and the activation of EGFR and c-Met, leading to sustained activation of Akt and Erk as well as enhanced cell proliferation and migration. Our data describe a novel mechanism of protumoral action of H19 in breast cancer.

Brain-Derived Neurotrophic Factor and Neurotrophin-4/5 Are Expressed in Breast Cancer and Can Be Targeted to Inhibit Tumor Cell Survival

Purpose: Given that nerve growth factor has previously been shown to be involved in breast cancer progression, we have tested here the hypothesis that the other neurotrophins (NT) are expressed and have an influence in breast tumor growth. Experimental Design: The expression of brain-derived neurotrophic factor (BDNF), NT-3 and NT-4/5, as well as the neurotrophin receptor p75NTR, TrkB, and TrkC, was studied by RT-PCR, Western blotting, and immunohistochemistry in cell lines and tumor biopsies. The biological impacts of neurotrophins, and associated mechanisms, were analyzed in cell cultures and xenografted mice. Results: BDNF and NT-4/5 were expressed and secreted by breast cancer cells, and the use of blocking antibodies suggested an autocrine loop mediating cell resistance to apoptosis. The corresponding tyrosine kinase receptor TrkB was only rarely observed at full length, whereas the expression of TrkB-T1, lacking the kinase domain, as well as p75NTR, were detected in all tested breast cancer cell lines and tumor biopsies. In contrast, NT-3 and TrkC were not detected. SiRNA against p75NTR and TrkB-T1 abolished the antiapoptotic effect of BDNF and NT-4/5, whereas the pharmacological inhibitors K252a and PD98059 had no effect, suggesting the involvement of p75NTR and TrkB-T1, but not kinase activities from Trks and MAPK. In xenografted mice, anti-BDNF, anti-NT-4/5, anti-p75NTR, or anti-TrkB-T1 treatments resulted in tumor growth inhibition, characterized by an increase in cell apoptosis, but with no change in proliferation. Conclusion: BDNF and NT-4/5 contribute to breast cancer cell survival and can serve as prospective targets in attempts to inhibit tumor growth. Clin Cancer Res; 17(7); 1741–52. ©2011 AACR.

Stable expression of sialyl-Tn antigen in T47-D cells induces a decrease of cell adhesion and an increase of cell migration

SummarySialyl-Tn is a carbohydrate antigen overexpressed in several epithelial cancers including breast cancer, and usually associated with poor prognosis. Sialyl-Tn is synthesized by a CMP-Neu5Ac: GalNAc α2,6-sialyltransferase: ST6GalNAc I, which catalyzes the transfer of a sialic acid residue in α2,6-linkage to the GalNAcα1-O-Ser/Thr structure. The resulting disaccharide (Neu5Acα2-6GalNAcα1-O-Ser/Thr) cannot be further elongated and sialyl-Tn expression results therefore in a shortening of the O-glycan chains. However, usual breast cancer cell lines express neither ST6GalNAc I nor sialyl-Tn antigen. We have previously shown that stable transfection of MDA-MB-231 cells with the hST6GalNAc I cDNA induces the sialyl-Tn antigen expression at the cell surface and leads to a decreased cell growth and an increased cell migration. We describe herein the generation of new T47-D clones expressing sialyl-Tn antigen after hST6GalNAc I cDNA stable transfection. sialyl-Tn antigen is carried by several high molecular weight membrane bound O-glycoproteins, including MUC1. We show that sialyl-Tn expression induces a decrease of cell growth and adhesion, and an increase of cell migration in sialyl-Tn positive clones compared to mock transfected cells. These observations show that the alteration of the O-glycans pattern is sufficient to modify the biological features of cancer cells. These T47-D sialyl-Tn expressing clones might allow further in vivo investigation to determine precisely the impact of such O-glycosylation modifications on breast cancer development.