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

BACKGROUND: Rho 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. METHODS: This 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. RESULTS: Firstly, 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. CONCLUSIONS: This 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.

Kinetic signatures of microRNA modes of action.

MicroRNAs (miRNAs) are key regulators of all important biological processes, including development, differentiation, and cancer. Although remarkable progress has been made in deciphering the mechanisms used by miRNAs to regulate translation, many contradictory findings have been published that stimulate active debate in this field. Here we contribute to this discussion in three ways. First, based on a comprehensive analysis of the existing literature, we hypothesize a model in which all proposed mechanisms of microRNA action coexist, and where the apparent mechanism that is detected in a given experiment is determined by the relative values of the intrinsic characteristics of the target mRNAs and associated biological processes. Among several coexisting miRNA mechanisms, the one that will effectively be measurable is that which acts on or changes the sensitive parameters of the translation process. Second, we have created a mathematical model that combines nine known mechanisms of miRNA action and estimated the model parameters from the literature. Third, based on the mathematical modeling, we have developed a computational tool for discriminating among different possible individual mechanisms of miRNA action based on translation kinetics data that can be experimentally measured (kinetic signatures). To confirm the discriminatory power of these kinetic signatures and to test our hypothesis, we have performed several computational experiments with the model in which we simulated the coexistence of several miRNA action mechanisms in the context of variable parameter values of the translation.

Ovarian cancer: Stat3, RhoA and IGF-IR as therapeutic targets.

Seeking to improve ovarian cancer therapy, we compared biological characteristics of the moderately-aggressive OVCAR-3 cell line with two highly aggressive ovarian cancer cell populations: the SK-OV-3 cell line, and HASCJ primary cells isolated from the ascitic fluid of a patient with FIGO stage IV ovarian cancer. Secretion of angiogenic factors was not discriminative, whereas cell invasion through Matrigel and vasculogenic mimicry were much greater in the more aggressive cells. Among 10 agents tested for their ability to decrease cancer cell aggressivity using these two models, inhibitors of Stat3, IGF-IR and Rho GTPase were found to be the most promising.

Preferential association of irreversibly silenced E2F-target genes with pericentromeric heterochromatin in differentiated muscle cells.

The heterochromatin-associated H3K9 tri-methylase Suv39h1 is involved in the permanent silencing of E2F target genes in differentiating but not in quiescent cells. Here, we tested the hypothesis that permanent silencing of E2F target genes is associated with their subnuclear positioning close to the pericentromeric heterochromatin compartment, enriched in Suv39h1. Using fluorescence in situ hybridization, we analyzed the subnuclear localization of three E2F target genes relative to the pericentromeric heterochromatin, in cycling fibroblasts or differentiating myoblasts. We observed that all three E2F-target genes have a tendency to relocate closer to the pericentromeric heterochromatin, only when cells differentiate and undergo an irreversible cell cycle withdrawal. These data suggest that repression of E2F target genes in cycling or in differentiating cells is achieved through distinct mechanisms. In differentiating cells, permanent silencing is driven by a Suv39h1-dependent H3K9 tri-methylation and positioning close to the heterochromatin compartment, whereas repression in cycling cells seems independent from subnuclear positioning and requires distinct H3K9 methylation levels.

Stimulation of angiogenesis resulting from cooperation between macrophages and MDA-MB-231 breast cancer cells: proposed molecular mechanism and effect of tetrathiomolybdate.

BACKGROUND: Infiltration by macrophages (Mphi) indicates a poor prognosis in breast cancers, in particular by inducing angiogenesis. Our study aimed 1) to investigate the mechanism by which cooperation between Mphi and aggressive breast cancer cells (MDA-MB-231) induces angiogenesis; 2) to examine the effect of tetrathiomolybdate (TM) on this angiogenic activity. METHODS: Mphi coincubated with MDA-MB-231 were used as a model to mimic the inflammatory microenvironment. Angiogenesis induced by the culture media was tested in the chick chorioallantoic membrane (CAM). Mphi phenotype was evaluated by 1) expression of the M1 marker CD80, and secretion of interleukin 10 (IL-10), an M2 marker; 2) capacity to secrete Tumour Necrosis Factor alpha (TNFalpha) when stimulated by lipopolysaccharide/interferon gamma (LPS/IFNgamma); 3) ability to induce MDA-MB-231 apoptosis. To explore the molecular mechanisms involved, cytokine profiles of conditioned media from MDA-MB-231, Mphi and the coculture were characterised by an antibody cytokine array. All experiments were carried out both in presence and in absence of TM. RESULTS: Incubation of Mphi with MDA-MB-231 induced a pro-angiogenic effect in the CAM. It emerged that the angiogenic activity of the coculture is due to the capacity of Mphi to switch from M1 Mphi towards M2, probably due to an increase in Macrophage Colony Stimulating Factor. This M1-M2 switch was shown by a decreased expression of CD80 upon LPS/IFNgamma stimulation, an increased secretion of IL-10, a decreased secretion of TNFalpha in response to LPS/IFNgamma and an inability to potentiate apoptosis. At the molecular level, the angiogenic activity of the coculture medium can be explained by the secretion of CXC chemokines/ELR+ and CC chemokines. Although TM did not modify either the M2 phenotype in the coculture or the profile of the secreted chemokines, it did decrease the angiogenic activity of the coculture medium, suggesting that TM inhibited angiogenic activity by interfering with the endothelial cell signalling induced by these chemokines. CONCLUSIONS: Cooperation between Mphi and MDA-MB-231 transformed M1 Mphi to an angiogenic, M2 phenotype, attested by secretion of CXC chemokines/ELR+ and CC chemokines. TM inhibited this coculture-induced increase in angiogenic activity, without affecting either Mphi phenotype or cytokine secretion profiles.

Histone acetyltransferases and deacetylases in the control of cell proliferation and differentiation.

Histone acetylation and deacetylation are chromatin-modifying processes that have fundamental importance for transcriptional regulation. Transcriptionally active chromatin regions show a high degree of histone acetylation, whereas deacetylation events are generally linked to transcriptional silencing. Many of the acetylating and deacetylating enzymes were originally identified as transcriptional coactivators or repressors. Their histone-modifying enzymatic activity was discovered more recently, opening up a whole new area of research. Histone acetyltransferases such as CREB-binding protein (CBP) and PCAF are involved in processes as diverse as promoting cell cycle progression and regulating differentiation. A controlled balance between histone acetylation and deacetylation seems to be essential for normal cell growth. Both histone acetyltransferases and deacetylases are involved in the development of diseases, including neurodegenerative disorders and cancer. Treatments that target these enzymes are already under clinical investigation.