Françoise Fauvel-Lafève

Stromal cells from human benign prostate hyperplasia produce a growth‐inhibitory factor for LNCaP prostate cancer cells, identified as interleukin‐6

To understand specific interactions between stromal cells and epithelial cells in benign prostatic hyperplasia (BPH) and prostatic adenocarcinoma, we developed stromal‐cell cultures from normal human prostate (PNX) and BPH (BH101), composed of fibroblasts and myofibroblasts. Their role in epithelial‐cell growth was studied using the established cancer cell lines LNCaP, PC3 and DU 145 and an SV40 large T‐immortalized normal epithelial‐cell line, PNTIA, in double‐diffusion co‐culture chambers. PNTIA was stimulated by PNX (x 1.6) and more strongly by BH101 stromal cells (x 2.7). Conversely, LNCaP growth decreased by 50% in the presence of BH101 stromal cells (stromal/epithelial ratio: 10). A BH101‐conditioned medium (CM), obtained in serum‐free conditions, induced 90% inhibition of [3H]thymidine incorporation of the LNCaP androgen‐sensitive cell line. Two other androgen‐independent prostate cancer cell lines were either insensitive to BH101 CM (PC3) or slightly inhibited (40% for DU145). BH101 produced large amounts of IL‐Iβ, IL‐6 and IL‐8. HPLC gel filtration enabled separation of an inhibitory fraction which contained IL‐6. IL‐6 was demonstrated to be responsible for the strong inhibitory effect since an IL‐6‐neutralizing antibody abolished this inhibition, which was reproduced by human recombinant IL‐6. Recombinant IL‐6 growth inhibition was observed only on LNCaP prostate cancer androgen‐sensitive cells.

MICROFIBRILS FROM THE ARTERIAL SUBENDOTHELIUM

Microfibrillar structures of the subendothelium are represented by either type VI collagen or elastin-associated microfibrils which are also referred to as fibrillin-containing microfibrils. These structures are present throughout the subendothelium irrespective of the presence of elastin. The localization, structure, and protein composition of microfibrils are reviewed. The arterial subendothelium is thrombogenic despite its very low content in fibrillar collagens. This thrombogenicity is linked to the microfibrillar structures, essentially to type VI collagen and to thrombospondin-containing microfibrils. Their respective ability to bind the von Willebrand factor and to activate blood platelets is discussed.

Inhibition of platelets and tumor cell adhesion by the disintegrin domain of human ADAM9 to collagen I under dynamic flow conditions

This work aimed to investigate the role of the disintegrin domain of the human ADAM9 (ADAM9D) on the adhesion of breast tumor cells and platelets to collagen I, in a dynamic flow assay to simulate in vivo shear conditions. Recombinant ADAM9D was able to support tumor cell adhesion through binding to the beta1 integrin subunit and also to inhibit the invasion through matrigel in vitro. In a dynamic flow assay ADAM9D inhibited about 75% and 65% of MDA-MB-231 tumor cells and platelet adhesion to collagen I, respectively. In addition, it was demonstrated that alphaVbeta3 integrin is new interacting partner for ADAM9D. In conclusion, these results suggest a role for the disintegrin domain of ADAM9 in the metastatic process. Also, ADAM9D may be a tool for investigating the role of ADAMs in metastasis and cancer progression and for the design of selective inhibitors against the adhesion and extravasation of cancer cells.

Rac3 induces a molecular pathway triggering breast cancer cell aggressiveness: differences in MDA-MB-231 and MCF-7 breast cancer cell lines

BackgroundRho GTPases are involved in cellular functions relevant to cancer. The roles of RhoA and Rac1 have already been established. However, the role of Rac3 in cancer aggressiveness is less well understood.MethodsThis work was conducted to analyze the implication of Rac3 in the aggressiveness of two breast cancer cell lines, MDA-MB-231 and MCF-7: both express Rac3, but MDA-MB-231 expresses more activated RhoA. The effect of Rac3 in cancer cells was also compared with its effect on the non-tumorigenic mammary epithelial cells MCF-10A. We analyzed the consequences of Rac3 depletion by anti-Rac3 siRNA.ResultsFirstly, we analyzed the effects of Rac3 depletion on the breast cancer cells’ aggressiveness. In the invasive MDA-MB-231 cells, Rac3 inhibition caused a marked reduction of both invasion (40%) and cell adhesion to collagen (84%), accompanied by an increase in TNF-induced apoptosis (72%). This indicates that Rac3 is involved in the cancer cells’ aggressiveness. Secondly, we investigated the effects of Rac3 inhibition on the expression and activation of related signaling molecules, including NF-κB and ERK. Cytokine secretion profiles were also analyzed. In the non-invasive MCF-7 line; Rac3 did not influence any of the parameters of aggressiveness.ConclusionsThis discrepancy between the effects of Rac3 knockdown in the two cell lines could be explained as follows: in the MDA-MB-231 line, the Rac3-dependent aggressiveness of the cancer cells is due to the Rac3/ERK-2/NF-κB signaling pathway, which is responsible for MMP-9, interleukin-6, -8 and GRO secretion, as well as the resistance to TNF-induced apoptosis, whereas in the MCF-7 line, this pathway is not functional because of the low expression of NF-κB subunits in these cells. Rac3 may be a potent target for inhibiting aggressive breast cancer.

Role of thrombospondin in the adhesion of human endothelial cells in primary culture

SummaryThe role of thrombospondin on the adhesion of endothelial cells in primary culture was studied using a serum-free defined medium or thrombospondin-depleted fetal bovine serum. Under these conditions, only 6% of the cells adhered to gelatin-coated dishes, whereas cells adhering to gelatin in the presence of normal fetal bovine serum were considered as 100% adhesion. The percentage of cells attached to fibronectin or thrombospondin-coated dishes in thrombospondin-depleted serum was 66 and 32%, respectively. The addition of purified platelet thrombospondin to thrombospondin-depleted serum increased the adhesion of endothelial cells to gelatin and to thrombospondin, up to 32 and 59%, respectively, and restored the attachment to fibronectin to the same extent as that observed in the presence of normal serum. In contrast to the attachment, the spreading of the adhering cells was not further influenced by the addition of soluble thrombospondin. Subcultured cells did not require any protein for adhering to gelatin substrata. These observations indicate that thrombospondin plays a major role in the adhesion of endothelial cells in primary culture.

TRANSFORMING GROWTH FACTOR-BETA 1 INCREASES THE ADHESION OF MDA-MB-231 MAMMARY ADENOCARCINOMA CELLS TO THE MICROVASCULAR SUBENDOTHELIUM

The increase of tumor cell adhesion to the subendothelium in the presence of TGF-beta 1 is thought to be mediated by two major events: an enrichment of extracellular matrix proteins secreted by endothelial cells and an increase of the integrins on the surface of tumor cells. In this study, we analyzed the effect of TGF-beta 1 on the adhesion of a mammary adenocarcinoma cell line (MDA-MB-231) to the matrix of human microvascular endothelial cells (HMEC-1). The adhesion of TGF-beta 1-treated tumor cells to a non-treated matrix or to purified matrix proteins was enhanced, while no increase was observed when non-treated tumor cells were let to adhere to a matrix secreted by HMEC-1 in the presence of the cytokine. Thus, the increase of cell adhesion was due to the effect of TGF-beta 1 on tumor cells and not to the matrix enrichment induced by this cytokine. The hyper-adhesion was inhibited by the RGD peptide and EDTA indicating that integrins were involved. Integrin subunits concentrations (alpha 5, alpha v and beta 1) on the surface of TGF-beta 1-treated tumor cells were not modified, while confocal microscopy showed a reorganization of beta 1 integrin subunits on the cell surface and in the cytoplasm resulting in actin fibers reorganization in the cytoskeleton. This indicates that the enhanced adhesion of TGF-beta 1-treated MDA-MB-231 cells to the subendothelium is due to a qualitative change of integrins.