Catherine Buquet

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.

Survival of cord blood haematopoietic stem cells in a hyaluronan hydrogel for ex vivo biomimicry

Haematopoietic stem cells (HSCs) and haematopoietic progenitor cells (HPCs) grow in a specified niche in close association with the microenvironment, the so‐called ‘haematopoietic niche’. Scaffolds have been introduced to overcome the liquid culture limitations, mimicking the presence of the extracellular matrix (ECM). In the present study the hyaluronic acid scaffold, already developed in the laboratory, has been used for the first time to maintain long‐term cultures of CD34+ haematopoietic cells obtained from human cord blood. One parameter investigated was the impact on ex vivo survival of CD34+ cord blood cells (CBCs) on the hyaluronic acid surface, immobilized with peptides containing the RGD motif. This peptide was conjugated by coating the hyaluronan hydrogel and cultured in serum‐free liquid phase complemented with stem cell factor (SCF), a commonly indispensable cytokine for haematopoiesis. Our work demonstrated that these hyaluronan hydrogels were superior to traditional liquid cultures by maintaining and expanding the HPCs without the need for additional cytokines, and a colonization of 280‐fold increment in the hydrogel compared with liquid culture after 28 days of ex vivo expansion. Copyright

Rac-1 GTPase controls the capacity of human leukaemic lymphoblasts to migrate on fibronectin in response to SDF-1α (CXCL12).

Acute lymphoblastic leukaemia (ALL) is characterized by malignant cell infiltration of bone marrow, requiring chemotactic response to SDF-1α. Using time-lapse video, we measured the velocity of ALL cells on fibronectin, and found that SDF-1α increased their migration activity for 2 h, but had no effect after 4h, following internalization of its receptor CXCR4. Transfection of ALL cells with dominant-negative Rac1 mutant significantly prolonged their chemotactic response to SDF-1α, and this effect was associated with an alteration of CXCR4 internalization. These data suggest a regulatory role for Rac1 in the chemotactic response of ALL cells to SDF-1α via receptor processing.

Pyr3, a TRPC3 channel blocker, potentiates dexamethasone sensitivity and apoptosis in acute lymphoblastic leukemia cells by disturbing Ca2+ signaling, mitochondrial membrane potential changes and reactive oxygen species production

Dexamethasone (Dex) is used as a chemotherapeutic drug in the treatment of acute lymphoblastic leukemia (ALL) because of its capacity to induce apoptosis. However, some ALL patients acquire resistance to glucocorticoids (GC). Thus, it is important to explore new agents to overcome GC resistance. The aim of the present work was to assess the ability of Pyr3, a selective inhibitor of transient receptor potential canonical 3 (TRPC3), to sensitize human ALL cells to Dex. We show here, for the first time, that Pyr3 enhances Dex sensitivity through the distraction of Dex-mediated Ca(2+) signaling in ALL cells (in vitro) and primary blasts (ex vivo) associated with mitochondrial-mediated reactive oxygen species production in ALL cells. Pyr3 alone induced Ca(2+) signaling via only endoplasmic reticulum-released Ca(2+) and exerted inhibitory effect on store-operated Ca(2+) entry in dose-dependent manner in ALL cell lines. Pre-incubation of cells with Pyr3 significantly curtailed the thapsigargin- and Dex-evoked Ca(2+) signaling in ALL cell lines. Pyr3 synergistically potentiated Dex lethality, as shown by the induction of cell mortality, G2/M cell cycle arrest and apoptosis in ALL cell lines. Moreover, Pyr3 disrupted Dex-mediated Ca(2+) signaling and increased the sensitivity of Dex-induced cell death in primary blasts from ALL patients. Additional analysis showed that co-treatment with Dex and Pyr3 results in mitochondrial membrane potential depolarization and reactive oxygen species production in ALL cells. Together, Pyr3 exhibited potential therapeutic benefit in combination with Dex to inverse glucocorticoid resistance in human ALL and probably in other lymphoid malignancies.

Biomimetic Three Dimensional Cell Culturing: Colorectal Cancer Micro-Tissue Engineering

Background: Spheroid cultures are known to mimic closely the properties of tumor tissue than monolayer cultures with regard to growth kinetics and metabolic rates. The aim of this paper is to confirm that tumor micro-tissue in a 3D biocompatible microenvironment maintain the cells natural behavior when compared to 2D monolayer culturing. Method: In order to validate our 3D culture system, we compared the 3D culture within a cross-linked hydrogel of hyaluronic acid, one of the major components of the extracellular matrix and the conventional 2D culture system. Results: Interestingly within our culture system, cells could be analyzed either after retrieval from the scaffold or even without being extracted in the 3D form rendering the HA hydrogel an ideal tool for biological applications. We observed the difference in the cell cycle, cell proliferation and behavior in both culture systems. Additionally drug testing was carried out using a chemotherapeutic agent (cis-platinium) that is already in clinical use to unequivocally prove the clinical predictive significance of the test strategy as compared with less complex assay systems and more complex in vivo models. We observed the presence of cell cycle heterogeneity very similar to the situation in vivo human tumors. Moreover, we have confirmed that resistance to chemotherapeutic reagents within this 3D culture system is much higher than those used in 2D cultures, since the tight assembly of cells in 3D culture systems render them more resistant requiring chemotherapeutic doses that recapitulate the drug sensitivity of tumor cells in vivo. Additionally we have observed the difference of apoptotic protein expression between 2D and 3D cell culture.

Three Dimensional Tumor Engineering by Co-Culture of Breast Tumor and Endothelial Cells Using a Hyaluronic Acid Hydrogel Model

Besides tumor cells, the microenvironment harbors a variety of host-derived cells. To date, the most successful tissue engineering approaches have employed methods that recapitulate the composition, architecture and/or chemical presentation of the native microenvironment. Thus tumor engineering in biomimetic three dimensional conditions represents a dynamic cooperatively between different cell types in a spatially and functionally accurate fashion. Evidence has been provided that the cross-talk between tumor cells and stromal cells leads to enhanced tumor growth, metastasis and altered response to chemotherapeutic agents. It has been provided that endothelial cells play an important role in tumor in shaping the tumor microenvironment and controlling tumor development, in particular through neo-angiogenesis. We developed a 3D in vitro tumor model that encompasses a cross-linked hyaluronic acid hydrogel providing a physiologically relevant microenvironment for mammary tumor cell and endothelial cell co-culture. We investigated the morphological cross-talk between tumor and endothelial cells in a 3D configuration. Additionally, we observed the influence of co-culturing on the proliferation, angiogenic protein expression and secretion. We demonstrated that endothelial cells tend to acquire a spheroidal configuration with the mammary tumor cells surrounding the endothelial spheroid. We also observed that the levels of VEGF, MMP-2 and MMP-9 have tendencies to decrease within the first 6 days of co-culture, and tend to increase at day 12. This could be due to the restored polarity of the mammary tumor cells leading to a period of quiescence required to restore the malignant organization. These data confirm the importance of tissue architecture and polarity in malignant progression.

Integration of Ca 2+ signaling regulates the breast tumor cell response to simvastatin and doxorubicin

Recent studies have suggested that the lipid-lowering agent simvastatin holds great promise as a cancer therapeutic; it inhibits the growth of multiple tumors, including triple-negative breast cancer. Doxorubicin- and simvastatin-induced cytotoxicity has been associated with the modulation of Ca2+ signaling, but the underlying mechanisms remain incompletely understood. Here we identify how Ca2+ signaling regulates the breast tumor cell response to doxorubicin and simvastatin. These two drugs inhibit cell survival while increasing apoptosis in two human breast cancer cell lines and five primary breast tumor specimens through the modulation of Ca2+ signaling. Signal transduction and functional studies revealed that both simvastatin and doxorubicin trigger persistent cytosolic Ca2+ release, thereby stimulating the proapoptotic BIM pathway and mitochondrial Ca2+ overload, which are responsible for metabolic dysfunction and apoptosis induction. Simvastatin and doxorubicin suppress the prosurvival ERK1/2 pathway in a Ca2+-independent and Ca2+-dependent manner, respectively. In addition, reduction of the Ca2+ signal by chelation or pharmacological inhibition significantly prevents drug-mediated anticancer signaling. Unexpectedly, a scratch-wound assay indicated that these two drugs induce rapid cell migration, while inhibiting cell invasion and colony formation in a Ca2+-dependent manner. Further, the in vivo data for MDA-MB-231 xenografts demonstrate that upon chelation of Ca2+, the ability of both drugs to reduce the tumor burden was significantly reduced via caspase-3 deactivation. Our results establish a calcium-based mechanism as crucial for executing the cell death process triggered by simvastatin and doxorubicin, and suggest that combining simvastatin with doxorubicin may be an effective regimen for the treatment of breast cancer.

Importance of local hypoxia on endothelial phenotype for an in vitro approach to bone marrow angiogenesis

The vasculature of bone marrow differs from that in other organs, and its characteristics should be considered when exploring the medullar angiogenesis associated with hematological malignancies. We show here that the human bone marrow sinusoidal cell line HBME-1 has a specific expression pattern of angiogenic factors and receptors, characterized by a unique VEGFR3(+), Tie2(-) signature, that resembles the in vivo pattern. Moreover, the HBME-1 cultured for up to 3 days in hypoxic conditions, similar to those found in the bone marrow, specifically downregulated expression of VEGFR1, VEGFR2 and ETAR. Thus, a model using bone marrow sinusoidal cells cultured under reduced oxygen tension may be more relevant than classical in vitro endothelial cultures for understanding the interactions between endothelial and malignant cells in the medullar microenvironment.