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1.
Clin Cancer Res ; 23(11): 2806-2816, 2017 Jun 01.
Article in English | MEDLINE | ID: mdl-27923843

ABSTRACT

Purpose: AXL receptor tyrosine kinase has been described as a relevant molecular marker and a key player in invasiveness, especially in triple-negative breast cancer (TNBC).Experimental Design: We evaluate the antitumor efficacy of the anti-AXL monoclonal antibody 20G7-D9 in several TNBC cell xenografts or patient-derived xenograft (PDX) models and decipher the underlying mechanisms. In a dataset of 254 basal-like breast cancer samples, genes correlated with AXL expression are enriched in EMT, migration, and invasion signaling pathways.Results: Treatment with 20G7-D9 inhibited tumor growth and bone metastasis formation in AXL-positive TNBC cell xenografts or PDX, but not in AXL-negative PDX, highlighting AXL role in cancer growth and invasion. In vitro stimulation of AXL-positive cancer cells by its ligand GAS6 induced the expression of several EMT-associated genes (SNAIL, SLUG, and VIM) through an intracellular signaling implicating the transcription factor FRA-1, important in cell invasion and plasticity, and increased their migration/invasion capacity. 20G7-D9 induced AXL degradation and inhibited all AXL/GAS6-dependent cell signaling implicated in EMT and in cell migration/invasion.Conclusions: The anti-AXL antibody 20G7-D9 represents a promising therapeutic strategy in TNBC with mesenchymal features by inhibiting AXL-dependent EMT, tumor growth, and metastasis formation. Clin Cancer Res; 23(11); 2806-16. ©2016 AACR.


Subject(s)
Antibodies, Anti-Idiotypic/administration & dosage , Cell Proliferation/drug effects , Proto-Oncogene Proteins/immunology , Receptor Protein-Tyrosine Kinases/immunology , Triple Negative Breast Neoplasms/therapy , Animals , Antibodies, Anti-Idiotypic/immunology , Cell Movement/drug effects , Cell Movement/immunology , Epithelial-Mesenchymal Transition/drug effects , Epithelial-Mesenchymal Transition/immunology , Female , Gene Expression Regulation, Neoplastic/drug effects , Heterografts , Humans , Mice , Neoplasm Metastasis , Proto-Oncogene Proteins/antagonists & inhibitors , Receptor Protein-Tyrosine Kinases/antagonists & inhibitors , Signal Transduction/drug effects , Triple Negative Breast Neoplasms/genetics , Triple Negative Breast Neoplasms/immunology , Triple Negative Breast Neoplasms/pathology , Xenograft Model Antitumor Assays , Axl Receptor Tyrosine Kinase
2.
Cancer Lett ; 385: 97-107, 2017 01 28.
Article in English | MEDLINE | ID: mdl-27816489

ABSTRACT

The AP-1 transcription factor Fra-1 is aberrantly expressed in a large number of cancers and plays crucial roles in cancer development and progression by stimulating the expression of genes involved in these processes. However, the control of Fra-1 transactivation ability is still unclear and here we hypothesized that PKCθ-induced phosphorylation could be necessary to obtain a fully active Fra-1 protein. Using MCF7 stable cells overexpressing equivalent levels of unphosphorylated Fra-1 or PKCθ-phosphorylated Fra-1, we showed that PKCθ-induced phosphorylation of Fra-1 was crucial for the stimulation of MMP1 and IL6 expression. Consistently, we found a significant positive correlation between PRKCQ (coding for PKCθ) and MMP1 mRNA expression levels in human breast cancer samples. PKCθ-induced phosphorylations, in part at T217 and T227 residues, strongly and specifically increased Fra-1 transcriptional activity through the stimulation of Fra-1 transactivation domain, without affecting JUN factors. More importantly, these phosphorylations were required for Fra-1-induced migration of breast cancer cells and phosphorylated Fra-1 expression was enriched at the invasion front of human breast tumors. Taken together, our findings indicate that PKCθ-induced phosphorylation could be important for the function of Fra-1 in cancer progression.


Subject(s)
Biomarkers, Tumor/metabolism , Breast Neoplasms/enzymology , Cell Movement , Gene Expression Regulation, Neoplastic , Isoenzymes/metabolism , Protein Kinase C/metabolism , Proto-Oncogene Proteins c-fos/metabolism , Biomarkers, Tumor/genetics , Breast Neoplasms/genetics , Breast Neoplasms/pathology , Extracellular Signal-Regulated MAP Kinases/metabolism , Female , Humans , Interleukin-6/genetics , Interleukin-6/metabolism , Isoenzymes/genetics , MCF-7 Cells , Matrix Metalloproteinase 1/genetics , Matrix Metalloproteinase 1/metabolism , Neoplasm Invasiveness , Phosphorylation , Protein Kinase C/genetics , Protein Kinase C-theta , Protein Stability , Proto-Oncogene Proteins c-fos/genetics , RNA Interference , Signal Transduction , Transcription, Genetic , Transfection
3.
Cancer Res ; 72(23): 6268-78, 2012 Dec 01.
Article in English | MEDLINE | ID: mdl-23054396

ABSTRACT

Induction of epithelial-to-mesenchymal transition (EMT) by TGF-ß1 requires Ras signaling. We recently identified the transcriptional repressor Blimp-1 (PRDM1) as a downstream effector of the NF-κB, RelB/Bcl-2/Ras-driven pathway that promotes breast cancer cell migration. As the RelB/Blimp-1 pathway similarly required Ras signaling activation, we tested whether Blimp-1 plays a role in TGF-ß1-mediated EMT. Here, TGF-ß1 treatment of untransformed NMuMG mammary epithelial and MDA-MB-231 breast cancer cells was shown to induce Blimp-1 expression, which promoted an EMT signature and cell migration. TGFB1 and BLIMP1 RNA levels were correlated in patient breast tumors. BLIMP1 gene transcription was activated by TGF-ß1 via a c-Raf (RAF1) to AP-1 pathway. Blimp-1 induced expression of the EMT master regulator Snail (SNAI1) via repressing BMP-5, which inhibited Snail expression upon TGF-ß1 treatment. Interestingly, a similar cascade was observed during postnatal mouse mammary gland development. RelB expression was detected early in pregnancy followed progressively by Blimp-1 and then Snail; whereas, BMP-5 levels were high in nulliparous and regressing glands. Finally, lower BMP5 RNA levels were detected in patient breast tumors versus normal tissues, and correlated with cancer recurrence. Thus, the Ras effector Blimp-1 plays an essential role in TGF-ß1-induced EMT via repression of BMP-5 in breast cancer.


Subject(s)
Bone Morphogenetic Protein 5/metabolism , Breast Neoplasms/pathology , Epithelial-Mesenchymal Transition/drug effects , Repressor Proteins/metabolism , Transforming Growth Factor beta1/pharmacology , Animals , Bone Morphogenetic Protein 5/genetics , Breast Neoplasms/metabolism , Cell Line, Tumor , Female , Gene Knockout Techniques , Humans , Immunoblotting , MCF-7 Cells , Mice , Peptides/metabolism , Positive Regulatory Domain I-Binding Factor 1 , Pregnancy , Proto-Oncogene Proteins c-raf/metabolism , Repressor Proteins/biosynthesis , Repressor Proteins/deficiency , Repressor Proteins/genetics , Transfection , Transforming Growth Factor beta1/genetics , Transforming Growth Factor beta1/metabolism
4.
Anticancer Agents Med Chem ; 11(1): 78-88, 2011 Jan.
Article in English | MEDLINE | ID: mdl-21235435

ABSTRACT

Protein tyrosine phosphorylation plays a major role in many cellular functions implicated in cancer development and progression, but only a few of the known protein tyrosine phosphatases have yet been clearly classified as oncogenes or tumor suppressors. PTPL1 interacts with tumor-associated proteins, suggesting a link between PTPL1, the PTPN13 gene product, and tumorigenesis or cancer progression. However, the impact of PTPL1 on cancer is divided between its capacity to counteract the activity of oncogenic tyrosine kinases and its inhibitory interaction with the death receptor, Fas. In this manuscript, we review the PTPL1-interacting proteins implicated in cancer. In addition, we examine the phenotypic arguments concerning both the PTPL1/Fas interaction and the ability of PTPL1 to inhibit signaling from growth factor receptors or oncogenes with tyrosine kinase activity. Finally, we compare the alterations in expression and the genetic and epigenetic arguments supporting an oncogenic or an anti-oncogenic impact of PTPL1.


Subject(s)
Neoplasms/metabolism , Neoplasms/pathology , Protein Tyrosine Phosphatase, Non-Receptor Type 13/metabolism , Animals , Humans , Protein Tyrosine Phosphatase, Non-Receptor Type 13/genetics
5.
Cancer Res ; 70(12): 5116-26, 2010 Jun 15.
Article in English | MEDLINE | ID: mdl-20501847

ABSTRACT

The protein tyrosine phosphatase PTPL1/PTPN13, the activity of which is decreased through allelic loss, promoter methylation, or somatic mutations in some tumors, has been proposed as a tumor suppressor gene. Moreover, our recent clinical study identified PTPL1 expression level as an independent prognostic indicator of a favorable outcome for patients with breast cancer. However, how PTPL1 can affect tumor aggressiveness has not been characterized. Here, we first show that PTPL1 expression, assessed by immunohistochemistry, is decreased in breast cancer and metastasis specimens compared with nonmalignant tissues. Second, to evaluate whether PTPL1 plays a critical role in breast cancer progression, RNA interference experiments were performed in poorly tumorigenic MCF-7 breast cancer cells. PTPL1 inhibition drastically increased tumor growth in athymic mice and also enhanced several parameters associated with tumor progression, including cell proliferation on extracellular matrix components and cell invasion. Furthermore, the inhibition of Src kinase expression drastically blocked the effects of PTPL1 silencing on cell growth. In PTPL1 knockdown cells, the phosphorylation of Src on tyrosine 419 is increased, leading to the activation of its downstream substrates Fak and p130cas. Finally, substrate-trapping experiments revealed that Src tyrosine 419 is a direct target of the phosphatase. Thus, by identification of PTPL1 as the first phosphatase able to inhibit Src through direct dephosphorylation in intact cells, we presently describe a new mechanism by which PTPL1 inhibits breast tumor aggressiveness.


Subject(s)
Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Protein Tyrosine Phosphatase, Non-Receptor Type 13/metabolism , src-Family Kinases/metabolism , Animals , Blotting, Western , Breast Neoplasms/genetics , Cell Adhesion , Cell Line, Tumor , Cell Movement , Cell Proliferation , Female , Fluorescent Antibody Technique , Humans , Immunoenzyme Techniques , Lymphatic Metastasis , Mice , Mice, Nude , Neoplasm Invasiveness , Phosphorylation , Protein Tyrosine Phosphatase, Non-Receptor Type 13/antagonists & inhibitors , Protein Tyrosine Phosphatase, Non-Receptor Type 13/genetics , RNA, Small Interfering/pharmacology , Signal Transduction , Tissue Array Analysis , src-Family Kinases/antagonists & inhibitors
6.
Horm Cancer ; 1(2): 63-70, 2010 Apr.
Article in English | MEDLINE | ID: mdl-21761351

ABSTRACT

Cancer cells require both nutrients and mitogens to multiply and survive in the unfavorable microenvironment of solid tumors or metastases before angiogenesis. Most cancer cells do not use fatty acids (FA) from the circulation but synthesize them in situ especially to make membranes and lipid signals required for continuously dividing cells. Three lipogenic enzymes are overexpressed, induced by sex steroid hormones and responsible for the in situ increased lipogenesis in cancer cells. We propose that in the early stages of human breast and prostate carcinogenesis, an increased activity of sex steroid receptors is partly responsible for the overexpression of FA synthase (FASN) whose regulation in cancer cells has been particularly studied. Increased activity of androgen receptor (AR), via different mechanisms, was extensively reported in prostate cancer. An increased level/and or activity of progesterone receptors, correlated with an increased expression of FASN, was found both in early stages of breast carcinogenesis and during the hormone replacement therapy of menopausal women. While the majority of recent targeted therapies are based on inhibition of mitogenic pathways, the inhibition of cancer cell nutrition by interfering with lipid synthesis and hormone action should open the way to new therapeutic and preventive approaches of hormone-dependent cancers.


Subject(s)
Breast Neoplasms/metabolism , Fatty Acid Synthases/metabolism , Lipogenesis , Prostatic Neoplasms/metabolism , Receptors, Progesterone/metabolism , Receptors, Steroid/metabolism , Female , Humans , Male , Models, Biological , Receptors, Androgen/metabolism
7.
Mol Cell Biol ; 29(20): 5488-504, 2009 Oct.
Article in English | MEDLINE | ID: mdl-19687301

ABSTRACT

Interleukin-6 (IL-6), involved in cancer-related inflammation, acts as an autocrine and paracrine growth factor, which promotes angiogenesis, metastasis, and subversion of immunity, and changes the response to hormones and to chemotherapeutics. We explored transcription mechanisms involved in differential IL-6 gene expression in breast cancer cells with different metastatic properties. In weakly metastatic MCF7 cells, histone H3 K9 methylation, HP1 binding, and weak recruitment of AP-1 Fra-1/c-Jun, NF-kappaB p65 transcription factors, and coactivators is indicative of low chromatin accessibility and gene transcription at the IL-6 gene promoter. In highly metastatic MDA-MB231 cells, strong DNase, MNase, and restriction enzyme accessibility, as well potent constitutive transcription of the IL-6 gene promoter, coincide with increased H3 S10 K14 phosphoacetylation and promoter enrichment of AP-1 Fra-1/c-Jun and NF-kappaB p65 transcription factors and MSK1, CBP/p300, Brg1, and Ezh2 cofactors. Complementation, silencing, and kinase inhibitor experiments further demonstrate involvement of AP-1 Fra-1/c-Jun and NF-kappaB p65/RelB members, but not of the alpha estrogen receptor in promoting chromatin accessibility and transcription across the IL-6 gene promoter in metastatic breast cancer cells. Finally, the natural withanolide Withaferin A was found to repress IL-6 gene transcription in metastatic breast cancer cells upon dual inhibition of NF-kappaB and AP-1 Fra-1 transcription factors and silencing of IL-6 promoter chromatin accessibility.


Subject(s)
Breast Neoplasms/genetics , Chromatin/metabolism , Gene Expression Regulation, Neoplastic , Interleukin-6/genetics , Transcription Factor AP-1/metabolism , Transcription Factor RelA/metabolism , Breast Neoplasms/pathology , Cell Line, Tumor , Ergosterol/analogs & derivatives , Ergosterol/pharmacology , Gene Expression Regulation, Neoplastic/drug effects , Histones/metabolism , Humans , Neoplasm Metastasis , Promoter Regions, Genetic/physiology , Proto-Oncogene Proteins c-fos/antagonists & inhibitors , Proto-Oncogene Proteins c-fos/metabolism , Proto-Oncogene Proteins c-jun/metabolism , RNA, Small Interfering/metabolism , Transcription Factor AP-1/antagonists & inhibitors , Transcription Factor RelA/antagonists & inhibitors , Withanolides
8.
Mol Cell Biol ; 29(14): 3832-44, 2009 Jul.
Article in English | MEDLINE | ID: mdl-19433448

ABSTRACT

Aberrant constitutive expression of NF-kappaB subunits, reported in more than 90% of breast cancers and multiple other malignancies, plays pivotal roles in tumorigenesis. Higher RelB subunit expression was demonstrated in estrogen receptor alpha (ERalpha)-negative breast cancers versus ERalpha-positive ones, due in part to repression of RelB synthesis by ERalpha signaling. Notably, RelB promoted a more invasive phenotype in ERalpha-negative cancers via induction of the BCL2 gene. We report here that RelB reciprocally inhibits ERalpha synthesis in breast cancer cells, which contributes to a more migratory phenotype. Specifically, RelB is shown for the first time to induce expression of the zinc finger repressor protein Blimp1 (B-lymphocyte-induced maturation protein), the critical mediator of B- and T-cell development, which is transcribed from the PRDM1 gene. Blimp1 protein repressed ERalpha (ESR1) gene transcription. Commensurately higher Blimp1/PRDM1 expression was detected in ERalpha-negative breast cancer cells and primary breast tumors. Induction of PRDM1 gene expression was mediated by interaction of Bcl-2, localized in the mitochondria, with Ras. Thus, the induction of Blimp1 represents a novel mechanism whereby the RelB NF-kappaB subunit mediates repression, specifically of ERalpha, thereby promoting a more migratory phenotype.


Subject(s)
Estrogen Receptor alpha/metabolism , Repressor Proteins/biosynthesis , Transcription Factor RelB/metabolism , Base Sequence , Breast Neoplasms/genetics , Breast Neoplasms/metabolism , Cell Line, Tumor , Cell Movement , DNA Primers/genetics , Estrogen Receptor alpha/genetics , Female , Gene Expression , Genes, bcl-2 , Humans , Neoplasms, Hormone-Dependent/genetics , Neoplasms, Hormone-Dependent/metabolism , Phenotype , Positive Regulatory Domain I-Binding Factor 1 , Proto-Oncogene Proteins c-bcl-2/metabolism , Repressor Proteins/genetics , Repressor Proteins/metabolism , ras Proteins/metabolism
9.
Int J Cancer ; 124(3): 638-43, 2009 Feb 01.
Article in English | MEDLINE | ID: mdl-19004008

ABSTRACT

Although it is well established that some protein tyrosine kinases have a prognostic value in breast cancer, the involvement of protein tyrosine phosphatases (PTPs) is poorly substantiated for breast tumors. Three of these enzymes (PTP-gamma, LAR, and PTPL1) are already known to be regulated by estrogens or their antagonists in human breast cancer cells. We used a real-time reverse transcriptase polymerase chain reaction method to test the expression levels of PTP-gamma, LAR and its neuronal isoform, and PTPL1 in a training set of RNA from 59 breast tumors. We sought correlations between levels of these molecular markers, current tumor markers, and survival. We then quantified the expression level of the selected phosphatase in 232 additional samples, resulting in a testing set of 291 breast tumor RNAs from patients with a median follow-up of 6.4 years. The Spearman nonparametric test revealed correlations between PTPL1 expression and differentiation markers. Cox univariate analysis of the overall survival studies demonstrated that PTPL1 is a prognostic factor [risk ratio (RR)=0.45], together with the progesterone receptor (PR) (RR=0.52) and node involvement (RR=1.58). In multivariate analyses, PTPL1 and PR retained their prognostic value (RRs of 0.48 and 0.55, respectively). This study demonstrates for the first time that PTPL1 expression level is an independent prognostic indicator of favorable outcome for patients with breast cancer. In conjunction with our mechanistic studies, this finding identifies PTPL1 as an important regulatory element of human breast tumor aggressiveness and sensitivity to treatments such as antiestrogens and antiaromatase.


Subject(s)
Biomarkers, Tumor/genetics , Breast Neoplasms/genetics , Genes, Tumor Suppressor , Protein Tyrosine Phosphatase, Non-Receptor Type 13/biosynthesis , Adult , Aged , Aged, 80 and over , Breast Neoplasms/mortality , Breast Neoplasms/pathology , Female , Gene Expression , Humans , Kaplan-Meier Estimate , Middle Aged , Pilot Projects , Prognosis , RNA, Messenger/analysis , Receptors, Progesterone/metabolism , Reverse Transcriptase Polymerase Chain Reaction
10.
Cancer Res ; 67(14): 6806-13, 2007 Jul 15.
Article in English | MEDLINE | ID: mdl-17638892

ABSTRACT

The protein tyrosine phosphatase (PTP) PTPL1/PTPN13 is a candidate tumor suppressor gene. Indeed, PTPL1 activity has been reported recently to be decreased through somatic mutations, allelic loss, or promoter methylation in some tumors. We showed previously that its expression was necessary for inhibition of Akt activation and induction of apoptosis by antiestrogens in breast cancer cells. Implications of the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway in cancer progression are now well established, and our study was therefore designed to define whether PTPL1 is sufficient to inhibit this pathway and, if so, to identify a direct substrate of this PTP, which may trigger a proapoptotic effect. We first show by complementary approaches that PTPL1 specifically dephosphorylates insulin receptor substrate-1 (IRS-1) in vitro and in cellulo. Next, our experiments using a dominant-negative mutant and RNA interference confirm the crucial role of PTPL1 in IRS-1 dephosphorylation. Finally, we report that PTPL1 expression is sufficient to block the IRS-1/PI3K/Akt signaling pathway, to inhibit the insulin-like growth factor-I effect on cell survival, and to induce apoptosis. Altogether, these data provide the first evidence for a direct positive role of the putative tumor suppressor gene PTPL1/PTPN13 on apoptosis and identify its target in the IRS-1/PI3K/Akt signaling pathway.


Subject(s)
Apoptosis , Phosphoproteins/metabolism , Protein Tyrosine Phosphatases/physiology , Catalytic Domain , Cell Line, Tumor , DNA Fragmentation , Disease Progression , HeLa Cells , Humans , Insulin Receptor Substrate Proteins , Microscopy, Fluorescence , Phosphatidylinositol 3-Kinases/metabolism , Phosphorylation , Plasmids/metabolism , Protein Tyrosine Phosphatase, Non-Receptor Type 13 , Protein Tyrosine Phosphatases/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Signal Transduction
11.
J Biol Chem ; 282(39): 28328-28334, 2007 Sep 28.
Article in English | MEDLINE | ID: mdl-17631492

ABSTRACT

ICI182,780 (Fulvestrant) is a pure anti-estrogen used in adjuvant therapies of breast cancer. This compound not only inhibits the transcriptional activities of the estrogen receptor-alpha (ER alpha) but also induces its proteasome-dependent degradation. The latter activity is believed to be required for the antiproliferative effects of ICI182,780. Estrogen receptor-related receptor-alpha (ERR alpha) is an orphan member of the nuclear receptor superfamily that is expressed in a wide range of tissues including breast tumors, in which its high expression correlates with poor prognosis. Although not regulated by any natural ligand, ERR alpha can be deactivated by the synthetic molecule XCT790. Here we demonstrate that this compound also induces a proteasome degradation of ERR alpha. We also show that although it does not act directly on the steady-state level of ER alpha, XCT790 potentiates the ICI182,780-induced ER alpha degradation. We suggest that treatment with XCT790 could thus enhance the efficacy of ICI182,780 in ER alpha-dependent pathologies such as breast cancer.


Subject(s)
Antineoplastic Agents, Hormonal/pharmacology , Breast Neoplasms/metabolism , Estradiol/analogs & derivatives , Nitriles/pharmacology , Proteasome Endopeptidase Complex/metabolism , Thiazoles/pharmacology , Antineoplastic Agents, Hormonal/agonists , Antineoplastic Agents, Hormonal/therapeutic use , Breast Neoplasms/drug therapy , Breast Neoplasms/pathology , Cell Line, Tumor , Drug Synergism , Estradiol/agonists , Estradiol/pharmacology , Estradiol/therapeutic use , Estrogen Receptor alpha/agonists , Female , Fulvestrant , Gene Expression Regulation, Neoplastic/drug effects , Humans , Nitriles/agonists , Nitriles/therapeutic use , Prognosis , Receptors, Estrogen/agonists , Thiazoles/agonists , Thiazoles/therapeutic use , ERRalpha Estrogen-Related Receptor
12.
Mol Cell Biol ; 27(11): 3936-50, 2007 Jun.
Article in English | MEDLINE | ID: mdl-17371847

ABSTRACT

Fra-1, a transcription factor that is phylogenetically and functionally related to the proto-oncoprotein c-Fos, controls many essential cell functions. It is expressed in many cell types, albeit with differing kinetics and abundances. In cells reentering the cell cycle, Fra-1 expression is transiently stimulated albeit later than that of c-Fos and for a longer time. Moreover, Fra-1 overexpression is found in cancer cells displaying high Erk1/2 activity and has been linked to tumorigenesis. One crucial point of regulation of Fra-1 levels is controlled protein degradation, the mechanism of which remains poorly characterized. Here, we have combined genetic, pharmacological, and signaling studies to investigate this process in nontransformed cells and to elucidate how it is altered in cancer cells. We report that the intrinsic instability of Fra-1 depends on a single destabilizer contained within the C-terminal 30 to 40 amino acids. Two serines therein, S252 and S265, are phosphorylated by kinases of the Erk1/2 pathway, which compromises protein destruction upon both normal physiological induction and tumorigenic constitutive activation of this cascade. Our data also indicate that Fra-1, like c-Fos, belongs to a small group of proteins that may, under certain circumstances, undergo ubiquitin-independent degradation by the proteasome. Our work reveals both similitudes and differences between Fra-1 and c-Fos degradation mechanisms. In particular, the presence of a single destabilizer within Fra-1, instead of two that are differentially regulated in c-Fos, explains the much faster turnover of the latter when cells traverse the G(0)/G(1)-to-S-phase transition. Finally, our study offers further insights into the signaling-regulated expression of the other Fos family proteins.


Subject(s)
MAP Kinase Signaling System/physiology , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3/metabolism , Neoplasms/metabolism , Proteasome Endopeptidase Complex/metabolism , Proto-Oncogene Proteins c-fos/metabolism , Ubiquitin/metabolism , Amino Acid Sequence , Animals , Cell Cycle/physiology , Cell Line , Humans , Mitogen-Activated Protein Kinase 1/genetics , Mitogen-Activated Protein Kinase 3/genetics , Molecular Sequence Data , Phosphorylation , Protein Structure, Tertiary , Proto-Oncogene Proteins c-fos/genetics , Sequence Alignment , Serine/metabolism , Threonine/metabolism
13.
Nat Cell Biol ; 9(4): 470-8, 2007 Apr.
Article in English | MEDLINE | ID: mdl-17369819

ABSTRACT

Aberrant constitutive expression of c-Rel, p65 and p50 NF-kappaB subunits has been reported in over 90% of breast cancers. Recently, we characterized a de novo RelB NF-kappaB subunit synthesis pathway, induced by the cytomegalovirus (CMV) IE1 protein, in which binding of p50-p65 NF-kappaB and c-Jun-Fra-2 AP-1 complexes to the RELB promoter work in synergy to potently activate transcription. Although RelB complexes were observed in mouse mammary tumours induced by either ectopic c-Rel expression or carcinogen exposure, little is known about RelB in human breast disease. Here, we demonstrate constitutive de novo RelB synthesis is selectively active in invasive oestrogen receptor alpha (ERalpha)-negative breast cancer cells. ERalpha signalling reduced levels of functional NF-kappaB and Fra-2 AP-1 and inhibited de novo RelB synthesis, leading to an inverse correlation between RELB and ERalpha gene expression in human breast cancer tissues and cell lines. Induction of Bcl-2 by RelB promoted the more invasive phenotype of ERalpha-negative cancer cells. Thus, inhibition of de novo RelB synthesis represents a new mechanism whereby ERalpha controls epithelial to mesenchymal transition (EMT).


Subject(s)
Estrogens/physiology , Proto-Oncogene Proteins c-bcl-2/genetics , Signal Transduction/physiology , Transcription Factor RelB/genetics , Animals , Blotting, Northern , Breast Neoplasms/genetics , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Cell Line, Tumor , Cell Movement/genetics , Cell Movement/physiology , Cell Transformation, Neoplastic/genetics , Electrophoretic Mobility Shift Assay , Estrogen Receptor alpha/genetics , Estrogen Receptor alpha/metabolism , Estrogen Receptor alpha/physiology , Female , Fos-Related Antigen-2/genetics , Fos-Related Antigen-2/metabolism , Gene Expression Regulation, Neoplastic , Humans , Immunoblotting , Mice , Microscopy, Fluorescence , NF-kappa B/genetics , NF-kappa B/metabolism , Phenotype , Protein Binding , Proto-Oncogene Proteins c-bcl-2/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Transcription Factor RelB/metabolism
14.
Int J Cancer ; 120(2): 224-9, 2007 Jan 15.
Article in English | MEDLINE | ID: mdl-17044016

ABSTRACT

Progestins increase the risk of breast cancer in the hormone therapy of menopause, and progesterone receptor-induced fatty acid synthase (FAS) is a potential therapeutical target of breast cancer. In a first attempt to specify in which lesions at risk of breast cancer progestins might be acting, we have compared the progesterone receptor (PR) and FAS expression in preinvasive breast lesions and in adjacent "normal" mammary glands. We used archive paraffin-embedded tissues from 116 patients, with 164 lesions of increasing histological risk from nonproliferative "benign" breast disease (BBD) to in situ breast carcinomas. Immunostaining using our FAS antibody and a PR antibody from Dako was quantified as continuous variables by computer-assisted image analysis. FAS level increased (p < 10(-3) by the Kruskall-Wallis test) in all lesions, starting from nonproliferative BBD, and was maximal in in situ carcinoma. The % of PR-positive cells increased from nonproliferative BBD and was higher in proliferative atypia (p < 10(-3)). It was very low in high-grade DCIS corresponding to a likely different carcinogenesis pathway. There was a trend for a positive correlation between FAS and PR in normal glands. However, the 2 markers increased independently in BBD and were negatively correlated in in situ carcinomas. FAS and PR were positively correlated with Ki67 in BBD. The increased PR level in premalignant steps of mammary carcinogenesis suggests an early increased responsiveness to progestins. The increased FAS expression, in lesions parallel to their increased breast cancer risk, suggests further studies to develop new markers of high-risk lesions and to prevent breast cancer.


Subject(s)
Biomarkers, Tumor/analysis , Breast Neoplasms/diagnosis , Cell Transformation, Neoplastic/metabolism , Fatty Acid Synthases/analysis , Receptors, Progesterone/analysis , Adult , Aged , Breast Neoplasms/pathology , Cell Transformation, Neoplastic/pathology , Early Diagnosis , Female , Humans , Immunohistochemistry , Ki-67 Antigen/analysis , Middle Aged
15.
Oncogene ; 24(8): 1434-44, 2005 Feb 17.
Article in English | MEDLINE | ID: mdl-15608675

ABSTRACT

Breast cancer progression is likely a multistep process involving the activation and inactivation of a number of genes. Previously, we showed that the mRNA coding for Fra-1, a FOS family member and an AP-1 transcription factor component, was highly expressed in the more invasive estrogen receptor negative (ER-) breast cancer cell lines. We used a tet-off system to stably overexpress Fra-1 in MCF7 ER+ cells and evaluate the impact of Fra-1 on this aggressive phenotype. Conversely, Fra-1 was silenced in highly invasive ER-MDA-MB231 cells using RNA interference. We report that in both systems the Fra-1 expression level was positively associated with cell proliferation, cell motility and invasiveness assessed in vitro. In addition, Fra-1 inhibition in fibroblastoid ER- cells, which formed colonies with large stellate projections in Matrigel, resulted in morphological changes. Cells acquired an epithelioid shape and had a spherical appearance in Matrigel. Fra-1 regulated several genes, implicated in invasion, angiogenesis and cell proliferation independently of beta1-integrin activation, and directly induced MMP-1 and MMP-9 promoter activity. These overall results show that high Fra-1 expression is associated with a more malignant cell phenotype and suggest that Fra-1 could have a pivotal role in breast cancer progression.


Subject(s)
Breast Neoplasms/metabolism , Neoplasm Invasiveness , Proto-Oncogene Proteins c-fos/metabolism , Breast Neoplasms/genetics , Breast Neoplasms/pathology , Cell Differentiation , Cell Line, Tumor , Cell Proliferation/drug effects , Collagen/metabolism , Doxycycline/pharmacology , Drug Combinations , Female , Gene Expression Regulation, Neoplastic , Humans , Integrin beta1/physiology , Laminin/metabolism , Matrix Metalloproteinase 1/biosynthesis , Matrix Metalloproteinase 9/biosynthesis , Neoplasm Invasiveness/genetics , Proteoglycans/metabolism , Proto-Oncogene Proteins c-fos/genetics , Proto-Oncogene Proteins c-fos/physiology , RNA, Small Interfering/genetics , Tissue Inhibitor of Metalloproteinase-1/biosynthesis , Transcription Factor AP-1/physiology , Vascular Endothelial Growth Factor A/biosynthesis
16.
Oncogene ; 23(36): 6105-14, 2004 Aug 12.
Article in English | MEDLINE | ID: mdl-15208657

ABSTRACT

We have recently reported that interleukin-8 (IL-8) expression was inversely correlated to estrogen receptor (ER) status and was overexpressed in invasive breast cancer cells. In the present study, we show that IL-8 overexpression in breast cancer cells involves a higher transcriptional activity of IL-8 gene promoter. Cloning of IL-8 promoter from MDA-MB-231 and MCF-7 cells expressing high and low levels of IL-8, respectively, shows the integrity of the promoter in both cell lines. Deletion and site-directed mutagenesis of the promoter demonstrate that NF-kappaB and AP-1 and to a lesser extent C/EBP binding sites play a crucial role in the control of IL-8 promoter activity in MDA-MB-231 cells. Knockdown of NF-kappaB and AP-1 activities by adenovirus-mediated expression of an NF-kappaB super-repressor and RNA interference, respectively, decreased IL-8 expression in MDA-MB-231 cells. On the contrary, restoration of Fra-1, Fra-2, c-Jun, p50, p65, C/EBPalpha and C/EBPbeta expression levels in MCF-7 cells led to a promoter activity comparable to that observed in MDA-MB-231 cells. Our data constitute the first extensive study of IL-8 gene overexpression in breast cancer cells and suggest that the high expression of IL-8 in invasive cancer cells requires a complex cooperation between NF-kappaB, AP-1 and C/EBP transcription factors.


Subject(s)
Breast Neoplasms/genetics , Gene Expression Regulation, Neoplastic , Interleukin-8/genetics , Binding Sites , Breast Neoplasms/metabolism , CCAAT-Enhancer-Binding Protein-beta/metabolism , Cell Line, Tumor , Female , Humans , Interleukin-8/biosynthesis , NF-kappa B/metabolism , Promoter Regions, Genetic , Transcription Factor AP-1/metabolism , Transcription, Genetic , Transcriptional Activation , Tumor Cells, Cultured
17.
Eur J Biochem ; 270(20): 4216-25, 2003 Oct.
Article in English | MEDLINE | ID: mdl-14519134

ABSTRACT

The matrix metalloproteinase (MMP) family degrades the extracellular matrix. One member of this family, MMP-1, initiates the breakdown of interstitial collagens. The expression of MMP-1 is controlled by the mitogen activated protein kinase (MAPK) pathway(s) via the activity of activator protein-1 (AP-1) and polyoma enhancing activity-3/E26 virus (PEA3/ETS) transcription factors through consensus binding sites present in the promoter. Another ETS site in the MMP-1 promoter is created at -1607 bp by a single nucleotide polymorphism (SNP), which contains two guanines (5'-GGAT-3'; '2G SNP'), rather one guanine (5'-GAT-3'; '1G SNP'), adjacent to an AP-1 binding site at -1602 bp. The 2G SNP displays greater transcriptional activity than the 1G SNP, and AP-1 and Ets families of transcription factors cooperate to increase transcription. The 2G SNP has been linked to the incidence and the progression of several cancers and is also associated with non-neoplastic diseases; although the underlying mechanism(s) has yet to be elucidated. In this study we demonstrate that the expression of Fos-like region antigen (Fra-1), an AP-1 transcription factor component that also correlates strongly with neoplastic disease, is necessary for MMP-1 transcription in A2058 melanoma cells. The inhibition of Fra-1 expression preferentially downregulates transcription from the MMP-1 promoter DNA containing the 2G SNP, compared to DNA containing the 1G SNP. This study provides evidence that, in cooperation with the 2G DNA polymorphism, the AP-1 family member, Fra-1, contributes to the high constitutive expression of MMP-1 in melanoma cells.


Subject(s)
Matrix Metalloproteinase 1/genetics , Polymorphism, Single Nucleotide , Promoter Regions, Genetic , Proto-Oncogene Proteins c-fos/metabolism , DNA Mutational Analysis , Enzyme Inhibitors/pharmacology , Flavonoids/pharmacology , Humans , Mitogen-Activated Protein Kinases/antagonists & inhibitors , Proto-Oncogene Proteins c-fos/drug effects
18.
Mol Endocrinol ; 17(2): 287-99, 2003 Feb.
Article in English | MEDLINE | ID: mdl-12554755

ABSTRACT

In the presence of estradiol, estrogen receptor-alpha (ERalpha) increases transcription triggered by activator protein-1 (AP-1). We have previously shown that induction is mediated by the direct interaction between c-Jun and ERalpha, which stabilizes a multiprotein complex containing the coactivator GRIP1 (glucocorticoid receptor interacting protein 1). The effect of receptor-interacting protein 140 (RIP140) in this regulation was assessed in the present study. We report that overexpression of RIP140 inhibits estradiol-induced AP-1-dependent transcription in a dose-dependent manner. Inhibition is not affected by trichostatin A, suggesting that histone deacetylase recruitment is not implicated. RIP140, which binds Jun proteins in pull-down assays and in intact cells, as shown by coimmunoprecipitation analysis and a mammalian one-hybrid system, participates in a multiprotein complex containing c-Jun and ERalpha. Moreover, the negative effect of RIP140 on AP-1-mediated transcription is relieved by GRIP1 overexpression and, conversely, RIP140 inhibits the stimulatory effect of GRIP1. The two cofactors compete for binding to c-Jun and ERalpha both in vitro and in intact cells, and GRIP1 interaction with both ERalpha and c-Jun is required for an efficient competition. These overall results suggest that the ratio between RIP140 and GRIP1 could determine, as proposed for hormone response element-mediated responses, the efficacy of estradiol in stimulating transcription of genes under AP-1 control.


Subject(s)
Estradiol/pharmacology , Nuclear Proteins/metabolism , Proto-Oncogene Proteins c-jun/metabolism , Transcription Factor AP-1/metabolism , Transcription Factors/metabolism , Adaptor Proteins, Signal Transducing , Binding Sites , Binding, Competitive , Cells, Cultured , Dose-Response Relationship, Drug , Enzyme Inhibitors/pharmacology , Estradiol/metabolism , Estrogen Receptor alpha , Histone Deacetylase Inhibitors , Humans , Hydroxamic Acids/pharmacology , Macromolecular Substances , Multiprotein Complexes , Nuclear Proteins/genetics , Nuclear Receptor Coactivator 2 , Nuclear Receptor Interacting Protein 1 , Proto-Oncogene Proteins c-jun/genetics , Receptors, Estrogen/drug effects , Receptors, Estrogen/genetics , Receptors, Estrogen/metabolism , Repressor Proteins/genetics , Repressor Proteins/metabolism , Transcription Factor AP-1/drug effects , Transcription Factor AP-1/genetics , Transcription Factors/genetics
19.
Oncogene ; 21(7): 1097-107, 2002 Feb 07.
Article in English | MEDLINE | ID: mdl-11850827

ABSTRACT

Fibulin-1 is an extracellular matrix protein induced by estradiol in estrogen receptor (ER) positive ovarian cancer cell lines. Alternative splicing of fibulin-1 mRNA results in four different variants named A, B, C and D that may have distinct biological functions. We studied the relative expression of fibulin-1 mRNA variants and their estrogen regulation in human ovarian cancer cells. In ovarian tissues and cancer cell lines, fibulin-1C and -1D are the predominant forms, whereas fibulin-1A and -1B are weakly expressed. We developed a competitive PCR assay based on coamplification of fibulin-1C and -1D to study the relative expression of these fibulin-1 variants in human ovarian samples. In ovarian cancer cell lines and ovarian cancer samples, there was a marked increase in the fibulin-1C:1D and fibulin-1C:HPRT mRNA ratios as compared to normal ovaries. In the BG1 estrogen receptor positive ovarian cancer cell line, fibulin-1C mRNA was induced by estradiol in a dose- and time-dependent manner. Since others and we have previously shown an increased expression of ERalpha as compared to ERbeta in ovarian cancer cells, we investigated whether ERalpha or ERbeta is involved in this induction. For this aim, MDA-MB-231 breast cancer cell line, which expresses both low basal levels of ERs and fibulin-1, was infected with recombinant ERalpha or ERbeta encoding adenovirus and treated with estradiol. Fibulin-1C was induced by estradiol in ERalpha- but not ERbeta-infected cells, suggesting that fibulin-1C induction is mediated through ERalpha. In ovarian tumors, a trend towards a correlation between fibulin-1C and ERalpha expression levels was noted. In conclusion, this study showed an increased fibulin-1C:-1D mRNA ratio in ovarian cancer cells as compared to normal ovaries. This finding suggests that the C variant may be involved in ovarian carcinogenesis. Fibulin-1C overexpression may thus be a clue for the understanding of a putative role of estrogens in ERalpha promoted ovarian tumor progression.


Subject(s)
Calcium-Binding Proteins/biosynthesis , Estradiol/pharmacology , Ovarian Neoplasms/metabolism , Transcriptional Activation , Aged , Calcium-Binding Proteins/genetics , Calibration , Female , Humans , Kinetics , Middle Aged , Ovarian Neoplasms/genetics , Ovary/metabolism , Plasmids , Polymerase Chain Reaction/methods , Protein Isoforms/biosynthesis , Protein Isoforms/genetics , RNA, Messenger/biosynthesis , RNA, Neoplasm/biosynthesis , Receptors, Estrogen/genetics , Receptors, Estrogen/physiology , Tumor Cells, Cultured
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