The ETS transcription factor ETV4 is involved in the main steps of organogenesis and is also a significant mediator of tumorigenesis and metastasis, such as in breast cancer. Indeed, ETV4 is ...overexpressed in breast tumors and is associated with distant metastasis and poor prognosis. However, the cellular and molecular events regulated by this factor are still misunderstood. In mammary epithelial cells, ETV4 controls the expression of many genes, MMP13 among them. The aim of this study was to understand the function of MMP13 during ETV4-driven tumorigenesis.
Different constructs of the MMP13 gene promoter were used to study the direct regulation of MMP13 by ETV4. Moreover, cell proliferation, migration, invasion, anchorage-independent growth, and in vivo tumorigenicity were assayed using models of mammary epithelial and cancer cells in which the expression of MMP13 and/or ETV4 is modulated. Importantly, the expression of MMP13 and ETV4 messenger RNA was characterized in 456 breast cancer samples.
Our results revealed that ETV4 promotes proliferation, migration, invasion, and anchorage-independent growth of the MMT mouse mammary tumorigenic cell line. By investigating molecular events downstream of ETV4, we found that MMP13, an extracellular metalloprotease, was an ETV4 target gene. By overexpressing or repressing MMP13, we showed that this metalloprotease contributes to proliferation, migration, and anchorage-independent clonogenicity. Furthermore, we demonstrated that MMP13 inhibition disturbs proliferation, migration, and invasion induced by ETV4 and participates to ETV4-induced tumor formation in immunodeficient mice. Finally, ETV4 and MMP13 co-overexpression is associated with poor prognosis in breast cancer.
MMP13 potentiates the effects of the ETV4 oncogene during breast cancer genesis and progression.
Ets‐1 overexpression in human breast cancers is associated with invasiveness and poor prognosis. By overexpressing Ets‐1 or a dominant negative mutant in MMT breast cancer cells, we previously ...highlighted the key role of Ets‐1 in coordinating multiple invasive features of these cells. Interestingly, we also noticed that Ets‐1 decreased the density of breast cancer cells cultured in three‐dimensional extracellular matrix gels. The 3D context was instrumental to this phenomenon, as such downregulation was not observed in cells grown on two‐dimensional plastic or matrix‐coated dishes. Ets‐1 overexpression was deleterious to anchorage‐independent growth of MMT cells in soft agar, a standard model for in vitro tumorigenicity. The relevance of this mechanism was confirmed in vivo, during primary tumor growth and in a metastatic assay of lung colonization. In these models, Ets‐1 was associated with epithelial‐to‐mesenchymal transition features and modulated the ratio of Ki67‐positive cells, while hardly affecting in vivo apoptotic cell death. Finally, siRNA‐mediated knockdown of Ets‐1 in human breast cancer cell lines also decreased colony growth, both in anchorage‐independent assays and 3D extracellular matrix cultures. These in vitro and in vivo observations shed light on an unsuspected facet of Ets‐1 in breast tumorigenesis. They show that while promoting malignancy through the acquisition of invasive features, Ets‐1 also attenuates breast tumor cell growth and could therefore repress the growth of primary tumors and metastases. This work also demonstrates that 3D models may reveal mechanisms of tumor biology that are cryptic in standard 2D models.
What's New?
Overexpression of the Ets‐1 transcription factor in human breast cancers is associated with invasiveness and poor prognosis. Here the authors report the unexpected finding that Ets‐1 expression decreases cellular density when culturing breast cancer cells in three‐dimensional extracellular matrix gels. The 3D context was essential for this finding as it was not observed under 2D culturing conditions but was supported by studies in mice. This data uncovers a new dual role of Ets‐1 in tumor biology: while promoting malignant invasiveness, Ets‐1 attenuates cell growth in breast tumors as well as metastases, a new property of the proto‐oncogene with potential therapeutic implications.
ERM, ER81 and PEA3 are three highly related transcription factors belonging to the ETS family. Together they form the PEA3 group within this family. Little data is yet available regarding the roles ...of these three genes during embryonic development. A prerequisite to investigations in this field is to obtain an accurate spatio-temporal expression map for the erm, er81 and pea3 genes. To this end, we have used in situ hybridization to compare their expression patterns during critical stages of murine embryogenesis. We report that all three genes are expressed in numerous developing organs coming from different embryonic tissues. The three genes appeared co-expressed in different organs but presented specific sites of expression, so that the resultant expression pattern could in fact reveal several distinct functions depending upon isolated and/or various combinations of the PEA3 member expression. These results suggest that erm, er81 and pea3 genes are differentially regulated, probably to serve important functions as cell proliferation control, tissue interaction mediator or cell differentiation, all over successive steps of the mouse organogenesis.
We measured the expression of ERM gene, a nuclear transcription factor belonging to the ets family, in a series of 364 unselected primary breast cancers from patients who underwent locoregional ...surgery in the Centre Oscar Lambret between May 1989 and December 1991. The expression of ERM was quantified with a real-time one-step reverse transcription-PCR assay based on the 5'-nuclease activity of the TaqDNA polymerase and with an Abi Prism 7700 Sequence Detector System (Applied Biosystems, Courtaboeuf, France). ERM was positively correlated (Spearman test) to epidermal growth factor receptor (EGFR; P < 0.001, r = 0.296) and to histoprognostic grading (P = 0.044, r = 0.112), whereas it was negatively correlated to estradiol receptors (P = 0.019, r = -0.124), HER3 (c-erbB-3; P = 0.01, r = -0.135), and HER4 (c-erbB-4; P = 0.003, r = -0.154). Using the chi2 test, a positive relationship was found between the expression of ERM and EGFR (chi2 = 7.795, P = 0.007). In overall survival studies, Cox univariate analyses demonstrated a prognostic value of ERM (P = 0.006; risk ratio, 2.95) besides the classical prognostic factors histoprognostic grading, node involvement, tumor size, estradiol receptors, progesterone receptors, EGFR, HER3, and HER4. In multivariate analyses, ERM preserved its prognostic value (P = 0.004; risk ratio, 3.779) together with histoprognostic grading, tumor size, estradiol receptors, and progesterone receptors. In relapse-free survival studies, univariate analyses demonstrated that histoprognostic grading, node involvement, tumor size, and HER4 were prognostic factors. These parameters, except histoprognostic grading, retained their prognostic value in multivariate analyses. This study demonstrates for the first time that ERM gene expression is an independent adverse prognostic factor for overall survival in breast cancer patients.
The receptor tyrosine kinase Met and its ligand, the hepatocyte growth factor/scatter factor, are essential for embryonic development, whereas deregulation of Met signaling pathways is associated ...with tumorigenesis and metastasis. The presenilin‐regulated intramembrane proteolysis (PS‐RIP) is involved in ligand‐independent downregulation of Met. This proteolytic process involves shedding of the Met extracellular domain followed by γ‐secretase cleavage, generating labile intracellular fragments degraded by the proteasome. We demonstrate here that upon shedding both generated Met N‐ and C‐terminal fragments are degraded directly in the lysosome, with C‐terminal fragments escaping γ‐secretase cleavage. PS‐RIP and lysosomal degradation are complementary, because their simultaneous inhibition induces synergistic accumulation of fragments. Met N‐terminal fragments associate with the high‐affinity domain of HGF/SF, confirming its decoy activity which could be reduced through their routing to the lysosome at the expense of extracellular release. Finally, the DN30 monoclonal antibody inducing Met shedding promotes receptor degradation through induction of both PS‐RIP and the lysosomal pathway. Thus, we demonstrate that Met shedding initiates a novel lysosomal degradation which participates to ligand‐independent downregulation of the receptor.
The term in situ hybridization (ISH) refers to all methods allowing the detection of specific DNA (gene loci) or RNA (gene expression products) sequences, using molecular hybridization (base pairing) ...of labeled nucleic acid probes to target molecules within "intact" cell populations in tissue sections or whole organisms, cultured cells, or chromosomal spreads. For more than two decades, ISH has been one of the main approaches used to characterize gene expression patterns in all laboratory animal models, especially in the context of embryonic development, as well as in human tissue or cell samples for both research and diagnostic purposes. Here, we describe several ISH protocols applied to the analysis of mouse embryos and tissues; this organism has become a reference for mammalian experimental genetics. These protocols use in vitro transcribed RNAs as probes for detection. Radiolabeled probes (using 35S as a radioisotope) allow sensitive ISH on sections of paraffin-embedded material, whereas nonradioactively (digoxigenin) labeled probes can be used both for hybridization of whole embryos (whole-mount ISH) and frozen tissue sections.
The Ets family transcription factor Pea3 (ETV4) is involved in tumorigenesis especially during the metastatic process. Pea3 is known to induce migration and invasion in mammary epithelial cell model ...systems. However, the molecular pathways regulated by Pea3 are still misunderstood. In the current study, using in vivo and in vitro assays, Pea3 increased the morphogenetic and tumorigenic capacity of mammary epithelial cells by modulating their cell morphology, proliferation, and migration potential. In addition, Pea3 overexpression favored an epithelial-mesenchymal transition (EMT) triggered by TGF-β1. During investigation for molecular events downstream of Pea3, Cyclin D2 (CCND2) was identified as a new Pea3 target gene involved in the control of cellular proliferation and migration, a finding that highlights a new negative regulatory loop between Pea3 and Cyclin D2. Furthermore, Cyclin D2 expression was lost during TGF-β1-induced EMT and Pea3-induced tumorigenesis. Finally, restored Cyclin D2 expression in Pea3-dependent mammary tumorigenic cells decreased cell migration in an opposite manner to Pea3. As such, these data demonstrate that loss of the negative feedback loop between Cyclin D2 and Pea3 contributes to Pea3-induced tumorigenesis.
This study reveals molecular insight into how the Ets family transcription factor Pea3 favors EMT and contributes to tumorigenesis via a negative regulatory loop with Cyclin D2, a new Pea3 target gene.
Pea3 and Erm are transcription factors expressed in normal developing branching organs such as the mammary gland. Deregulation of their expression is generally associated with tumorigenesis and ...particularly breast cancer. By using RNA interference (RNAi) to downregulate the expression of Pea3 and/or Erm in a mammary cancer cell line, we present evidence for a role of these factors in proliferation, migration and invasion capacity of cancer cells. We have used different small interfering RNAs (siRNAs) targeting pea3 and erm transcripts in transiently or stably transfected cells, and assessed the physiological behavior of these cells in in vitro assays. We also identified an in vivo alteration of tumor progression after injection of cells that overexpress pea3 and/or erm short hairpin RNAs (shRNAs) in immunodeficient mice. Using transcriptome profiling in Pea3- or Erm-targeted cells, two largely independent gene expression programs were identified on the basis of their shared phenotypic modifications. A statistically highly significant part of both sets of target genes had previously been already associated with the cellular signaling pathways of the `proliferation, migration, invasion' class. These data provide the first evidence, by using endogenous knockdown, for pivotal and complementary roles of Pea3 and Erm transcription factors in events crucial to mammary tumorigenesis, and identify sets of downstream target genes whose expression during tumorigenesis is regulated by these transcription factors.
The Pea3 transcription factor (which belongs to the PEA3 group) from the Ets family has been shown to be involved in mammary embryogenesis and oncogenesis. However, except for proteinases, only few ...of its target genes have been reported. In the present report, we identified bax as a Pea3 up-regulated gene. We provide evidence of this regulation by using Pea3 overexpression and Pea3 silencing in a mammary cell line. Both Pea3 and Erm, another member of the PEA3 group, are able to transactivate bax promoter fragments. Although the minimal Pea3-regulated bax promoter does not contain an Ets-binding site, two functional upstream stimulatory factor-regulated E boxes are present. We further demonstrate the ability of Pea3 and USF-1 to cooperate for the transactivation of the bax promoter, mutation of the E boxes dramatically reducing the Pea3 transactivation potential. Although Pea3 did not directly bind to the minimal bax promoter, we provide evidence that USF-1 could form a ternary complex with Pea3 and DNA. Taken together, our results suggest that Pea3 may regulate bax transcription via the interaction with USF-1 but without binding to DNA.
Aeromonas spp., considered as emerging opportunistic pathogens, belong to the family Vibrionaceae. Among the criteria currently used for their classification is the presence of a single FeSOD ...(iron-containing superoxide dismutase), which distinguishes them from Enterobacteriacea. In this paper the cloning of the sodA and sodB genes encoding two different SODs in Aeromonas hydrophila ATCC 7966 is reported. The sodB gene encoded an FeSOD (196 amino acids, 21.5 kDa), was constitutively expressed and showed 75% homology with the E. coli FeSOD. The sodA gene encoded a protein of 206 amino acids (22.5 kDa) with MnSOD (manganese-containing SOD) activity and showed 55% homology with the Escherichia coli MnSOD. The MnSOD of A. hydrophila was detected only during the stationary phase of growth under high aeration or when induced by lack of iron. Nevertheless, paraquat had no detectable effect on its production. The amino-terminal part of the Mn-containing protein contained a putative signal sequence which could permit a periplasmic localization.
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