Eric Adriaenssens

H19 mRNA-like Noncoding RNA Promotes Breast Cancer Cell Proliferation through Positive Control by E2F1

The imprinted H19 gene has riboregulatory functions. We show here that H19 transcription is up-regulated during the S-phase of growth-stimulated cells and that the H19 promoter is activated by E2F1 in breast cancer cells. H19 repression by pRb and E2F6 confirms the E2F1-dependent control of the H19 promoter. Consistently, we demonstrate by chromatin immunoprecipitation assays that endogenous E2F1 is recruited to the H19 promoter in vivo. The functionality of E2F promoter sites was further confirmed by gel shift and mutagenesis experiments, revealing that these sites are required for binding and promoter response to E2F1 exogenous expression and serum stimulation. Furthermore, we show that H19 overexpression confers a growth advantage on breast cancer cells released from growth arrest as well as in asynchronously growing cells. The H19 knockdown by small interfering RNA duplexes impedes S-phase entry in both wild-type and stably H19-transfected cells. Based on these findings, we conclude that the H19 RNA is actively linked to E2F1 to promote cell cycle progression of breast cancer cells. This clearly supports the H19 oncogenic function in breast tumor genesis.

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.

TrkA overexpression enhances growth and metastasis of breast cancer cells

The Trk family of neurotrophin tyrosine kinase receptors is emerging as an important player in carcinogenic progression in non-neuronal tissues. Here, we show that breast tumors present high levels of TrkA and phospho-TrkA compared to normal breast tissues. To further evaluate the precise functions of TrkA overexpression in breast cancer development, we have performed a series of biological tests using breast cancer cells that stably overexpress TrkA. We show that (1) TrkA overexpression promoted cell growth, migration and invasion in vitro; (2) overexpression of TrkA per se conferred constitutive activation of its tyrosine kinase activity; (3) signal pathways including PI3K-Akt and ERK/p38 MAP kinases were activated by TrkA overexpression and were required for the maintenance of a more aggressive cellular phenotype; and (4) TrkA overexpression enhanced tumor growth, angiogenesis and metastasis of xenografted breast cancer cells in immunodeficient mice. Moreover, recovered metastatic cells from the lungs exhibited enhanced anoikis resistance that was abolished by the pharmacological inhibitor K252a, suggesting that TrkA-promoted breast tumor metastasis could be mediated at least in part by enhancing anoikis resistance. Together, these results provide the first direct evidence that TrkA overexpression enhances the tumorigenic properties of breast cancer cells and point to TrkA as a potential target in breast cancer therapy.

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.

A Novel H19 Antisense RNA Overexpressed in Breast Cancer Contributes to Paternal IGF2 Expression

ABSTRACT The H19/IGFf2 locus belongs to a large imprinted domain located on human chromosome 11p15.5 (homologue to mouse distal chromosome 7). The H19 gene is expressed from the maternal allele, while IGF2 is paternally expressed. Natural antisense transcripts and intergenic transcription have been involved in many aspects of eukaryotic gene expression, including genomic imprinting and RNA interference. However, apart from the identification of some IGF2 antisense transcripts, few data are available on that topic at the H19/IGF2 locus. We identify here a novel transcriptional activity at both the human and the mouse H19/IGF2 imprinted loci. This activity occurs antisense to the H19 gene and has the potential to produce a single 120-kb transcript that we called the 91H RNA. This nuclear and short-lived RNA is not imprinted in mouse but is expressed predominantly from the maternal allele in both mice and humans within the H19 gene region. Moreover, the transcript is stabilized in breast cancer cells and overexpressed in human breast tumors. Finally, knockdown experiments showed that, in humans, 91H, rather than affecting H19 expression, regulates IGF2 expression in trans.

Nerve growth factor overexpression and autocrine loop in breast cancer cells

We show here that nerve growth factor (NGF), the canonical neurotrophic factor, is synthesized and released by breast cancer cells. High levels of NGF transcript and protein were detected in breast cancer cells by reverse transcription-PCR, Western blotting, ELISA assay and immunohistochemistry. Conversely, NGF production could not be detected in normal breast epithelial cells at either the transcriptional or protein level. Confocal analysis indicated the presence of NGF within classical secretion vesicles. Breast cancer cell-produced NGF was biologically active, as demonstrated by its ability to induce the neuronal differentiation of embryonic neural precursor cells. Importantly, the constitutive growth of breast cancer cells was strongly inhibited by either NGF-neutralizing antibodies or K-252a, a pharmacological inhibitor of NGF receptor TrkA, indicating the existence of an NGF autocrine loop. Together, our data demonstrate the physiological relevance of NGF in breast cancer and its potential interest as a marker and therapeutic target.

H19 overexpression in breast adenocarcinoma stromal cells is associated with tumor values and steroid receptor status but independent of p53 and Ki-67 expression

In a previous study we described the expression of the H19 gene by in situ hybridization (ISH) in normal breast and in benign or malignant breast tumors (Dugimont T, Curgy JJ, Wernert N, Delobelle A, Raes MB, Joubel A, Stehelin D, Coll J: Biol Cell 1995, 85:117-124). In the present work, 1) we extend the previous one to a statistically useful number of adenocarcinomas, including 10 subclasses, 2) we provide information on the precise ISH localization of the H19 RNA by using, on serial tissue sections, antibodies delineating specifically the stromal or the epithelial component of the breast, and 3) we consider relationships between the H19 gene expression and various clinicopathological information as tumor values (T0 to T4), grades, steroid receptors, lymph node status, and molecular features as the p53 gene product and the Ki-67/MIB1 protein, which is specific to proliferating cells. Data indicate that 1) in 72.5% of studied breast adenocarcinomas an overall H19 gene expression is increased when compared with healthy tissues, 2) the H19 gene is generally overexpressed in stromal cells (92.2%) and rarely in epithelial cells (2.9% only), 3) an up-regulation of the H19 gene is significantly correlated with the tumor values and the presence of both estrogen and progesterone receptors, and 4) at the cellular level, the H19 gene demonstrates an independent expression versus accumulation of both the p53 protein and the Ki-67/MIB-1 cell-cycle marker.

The H19 TATA-less promoter is efficiently repressed by wild-type tumor suppressor gene product p53

The developmentally regulated H19 gene displays several remarkable properties: expression of an apparently non-translated mRNA, genomic imprinting (maternal allele only expressed), relaxation of the imprinting and/or epigenetic lesions demonstrated in some tumors. Despite several observations after relaxation of imprinting status of the gene, data on trans and cis-acting factors required for the human H19 gene expression are still missing. As a first approach to address identification of factors involved in the regulation of the gene, we found that cells from a p53 antisense-transfected HeLa clone displayed increased amounts of H19 transcripts when compared to the non-transfected cells. Moreover, a HeLa clone stably transfected with a temperature sensitive (ts) 143 Ala p53 mutant exhibited temperature-dependent regulation of H19 expression. This preliminary indication of the repressing effect of the p53 protein on H19 expression has been confirmed by transient cotransfection experiments in HeLa cells, using luciferase surrogate constructs under the control of the 823 bp sequence immediately upstream of the transcription start point of the H19 gene, and different constructs containing sense, antisense or a ts 143 Ala mutant p53 cDNA. We observed an increase of H19 promoter-driven activity in transient cotransfections with the antisense p53 cDNA and the temperature sensitive mutant p53 at the non-permissive temperature, but a decrease with sense wild-type p53 cDNA. Furthermore, the cotransfection experiments were repeated in a cell line lacking endogenous p53. (Calu 6 cells) and the results provided additional evidence for a down regulation of the expression of the H19 gene by the p53 protein.

The antiapoptotic effect of fibroblast growth factor-2 is mediated through nuclear factor-κB activation induced via interaction between Akt and IκB kinase-β in breast cancer cells

Fibroblast growth factor-2 (FGF-2) is known for its mitogenic and motogenic effects on breast cancer cells. Here, we demonstrate that FGF-2 is also a potent stimulator of breast cancer cell survival, as it counteracts the apoptotic activity of the C2 ceramide analogue and various chemotherapeutic agents (5-fluorouracil, camptothecin, etoposide) in MCF-7, T47-D and BT-20 cells. The use of pharmacological inhibitors (PD98059, wortmannin, LY294002, SN50) and transfection with negative dominants (IκBm, p110(PI3K (phosphoinositide 3-kinase))*ΔK, AktND) or small interfering RNA targeted against Akt indicated that PI3K/Akt and nuclear factor-κB (NF-κB), but not p42/p44 MAP-kinases, were required to stimulate FGF-2 antiapoptotic activity. The activation of NF-κB was dependent on PI3K/Akt, and using a combination of approaches based on immunoprecipitation, Western blotting and proteomics (two-dimensional electrophoresis and mass spectrometry), we identified the beta form of IκB kinase (IKKβ) as a target of Akt signaling. The selective disruption of IKKβ using small interfering RNA induced a potent inhibition of Akt-mediated activation of NF-κB and cell survival, indicating the functional involvement of IKKβ in FGF-2 antiapoptotic signaling. Together, these results demonstrate Akt/IKKβ interaction in NF-κB pathways, thereby emphasizing the potential of these proteins as therapeutic targets in breast cancer.

The valosin-containing protein (VCP) is a target of AKT signaling required for cell survival

The serine/threonine kinase Akt is a key mediator of cell survival and growth, but its precise mechanism of action, and more specifically, the nature of its signaling partners largely remain to be elucidated. We show, using a proteomics-based approach, that the valosin-containing protein (VCP), a member of the AAA (ATPases associated with a variety of cellular activities) family, is a target of Akt signaling. SDS-PAGE of Akt co-immunoprecipitated proteins obtained from MCF-7 breast cancer cells revealed the increase of a 97-kDa band under Akt activation. Mass spectrometry analysis allowed the identification of VCP, and we have shown a serine/threonine phosphorylation on an Akt consensus site upon activation by growth factors. Site-directed mutagenesis identified Ser-351, Ser-745, and Ser-747 as Akt phosphorylation sites on VCP. Confocal microscopy indicated a co-localization between Akt and VCP upon Akt stimulation. Interestingly, small interfering RNA against VCP induced an inhibition of the growth factor-induced activation of NF-κB and a potent pro-apoptotic effect. Together, these data identify VCP as an essential target of Akt signaling.