Notch signalling drives bone marrow stromal cell-mediated chemoresistance in acute myeloid leukemia

Both preclinical and clinical investigations suggest that Notch signalling is critical for the development of many cancers and for their response to chemotherapy. We previously showed that Notch inhibition abrogates stromal-induced chemoresistance in lymphoid neoplasms. However, the role of Notch in acute myeloid leukemia (AML) and its contribution to the crosstalk between leukemia cells and bone marrow stromal cells remain controversial. Thus, we evaluated the role of the Notch pathway in the proliferation, survival and chemoresistance of AML cells in co-culture with bone marrow mesenchymal stromal cells expanded from both healthy donors (hBM-MSCs) and AML patients (hBM-MSCs*). As compared to hBM-MSCs, hBM-MSCs* showed higher level of Notch1, Jagged1 as well as the main Notch target gene HES1. Notably, hBM-MSCs* induced expression and activation of Notch signalling in AML cells, supporting AML proliferation and being more efficientin inducing AML chemoresistance than hBM-MSCs*. Pharmacological inhibition of Notch using combinations of Notch receptor-blocking antibodies or gamma-secretase inhibitors (GSIs), in presence of chemotherapeutic agents, significant lowered the supportive effect of hBM-MSCs and hBM-MSCs* towards AML cells, by activating apoptotic cascade and reducing protein level of STAT3, AKT and NF-κB.These results suggest that Notch signalling inhibition, by overcoming the stromal-mediated promotion of chemoresistance,may represent a potential therapeutic targetnot only for lymphoid neoplasms, but also for AML

Inhibition of GSK-3 Signalling Enhances Sensitivity of Non-Promyelocitic Acute Myeloid Leukemia (AML) Cell to Chemotherapy

Background: GSK-3 is a serine-threonine kinase involved in metabolic regulation as well as in the control of many pathways associated to cancer development, including Notch Wnt/\u3b2-catenin, Hedgehog, and AKT. It has been demonstrated that association of GSK-3 inhibitors with All-trans-retinoic acid (ATRA) significantly improves ATRA-mediated differentiation and cell death of acute promyelocytic (APL) leukaemia cells. However, little is currently known about the contribution of GSK-3 role to non-promyelocytic AML cell response to treatment with chemotherapeutic agents. Aims: In this study, we aim to validate GSK-3 signalling as potent successful therapeutic target in non-promyelocytic AML. For this purpose we tested different GSK-3 for their ability to influence AML cells proliferation and response to Cytarabine (Ara-C) or Idarubicin treatments. Methods: GSK-3 expression was analyzed by Western blot or flow cytometry inAML cell lines (HL-60, THP1, U937) or primary non-promyelocyticAML blast cells (30 samples). AML cellscultured alone or in presence ofhuman bone marrow mesenchymal stromal cells (hBM-MSCs) were treated with GSK-3 inhibitors, including LiCL, AR-A014418, SB 216763, in association or not with Cytarabine (Ara-C) or Idarubicin. Cell proliferation and cell death were measured by CFSE dilution and TOPRO-3/Annexin-V staining, respectively. Results: Flow cytometry and Western blot analysis in AML samples revealed high expression levels of all GSK-3forms, including total GSK-3\u3b1, (Ser21) GSK-3\u3b1, total GSK-3\u3b2, and (Ser 21) GSK-3\u3b2; theseforms were all down-modulated when AML cells were cultured in presence of hBM-MSCs, thus suggesting that GSK-3 plays an important role in transducting micro-environmental signals in AML cells interacting with bone marrow stroma. The treatment of AML cells with increasing concentrations of each GSK-3 inhibitors decreased AML cell viability in a dose-dependent manner; interestingly, hBM-MSCs or peripheral blood mononuclear cells were less sensitive to GSK-3inhibitors. The addition of each inhibitor increased dramatically the AML cell apoptotic rate induced by the addition of Ara-C or Idarubicin in vitro. Notably, LiCl and AR-A014418 were capable of abrogating hBM-MSC-mediated AML cell resistance to apoptosis induced by Ara-C or Idarubicin. Conclusion: Overall our data clearly demonstrated that inhibition of GSK-3 reduced proliferation and chemoresistance of non promyelocytic AML cells. Thus GSK-3 inhibition represents a therapeutic strategy not only for APL but also for other AML subtypes

Role of Wnt/\u3b2-Catenin Signalling in Acute Myeloid Leukemia (AML) Cell Response to Chemotherapy

Background: Growing evidences from both preclinical and clinical investigations reveal the critical role of Wnt signalling for the development of many cancers and for their response to chemotherapy. Although recent studies suggest that aberrant Wnt signalling can be involved in the neoplastic myeloid cell growth, the contribution of the Wnt/\u3b2-catenin pathway to AML survival and chemoresistance is still unclear. Aims: In this study, we investigated the contribution of WNT/\u3b2-CATENIN signalling to AML survival and chemoresistance. For this purpose we tested different modulators of Wnt/\u3b2-Catenin pathway for their ability to influence AML cells proliferation and response to Cytarabine (Ara-C) or Idarubicin treatment. Methods: AML primary blast cells(30 samples) or AML cell lines cultured alone or in presence of human bone marrow mesenchymal stromal cells (hBM-MSCs), were treated with with Cytarabine (Ara-C) or Idarubicin, in presence or absence of Wnt modulators, including ligands (Wnt3a, Wnt5a/5b), Porcupine inhibitors (IWP-2), LRP6 inhibitors (Niclosamide), or antagonists of TCF/\u3b2-catenin (PKF118-310, PNU-74654). Results: In silico analysis showed the enrichment of Wnt signalling components in AML samples. Western Blot and flow cytometry showed the presence of total \u3b2-catenin only in about 2/3 of primary samples analyzed, while . \u3b2-catenin positive samples had different degree of activation of the pathway, as revealed by the expression of active forms of \u3b2-catenin, including (Ser675)\u3b2-catenin and non-phospho-(Ser33/37/Thr41) \u3b2-catenin. Notably, we found that active forms of \u3b2-catenin increased in AML samples in co-culture with hBM-MSCs, thus suggesting that Wnt signalling could be involved in the crosstalk between bone marrow stroma and AML cells. The addition of Wnt or pharmacological inhibitors, such as IWP-2, PNU-74654 and Niclosamide, to the culture medium of \u3b2-catenin-positive AML samples, either cultured alone or in co-culture with hBM-MSCs, reduced AML cell proliferation with slight effect on cell death. When associated to Idarubicin, all Wnt inhibitors except IWP-2 synergycally induced a dramatic cell death in AML cells in both culture conditions. However, when Idarubicin was replaced by Ara-C the synergism was observed only with Niclosamide and PKF. Cell death was mainly due to apoptosis, as shown by Annexin-V staining. Conclusion: Overall our data show that Wnt inhibitors reduce proliferation and chemoresistance of AML cells in culture or co-culture with bone marrow stroma cells. Wnt/\u3b2-catenin signalling may represent a potential therapeutic strategy to improve AML treatment, overcoming bone marrow stromal-mediated anti-apoptotic and chemoresistance effects

Notch Signaling Molecules as Prognostic Biomarkers for Acute Myeloid Leukemia

International audienceThe role of Notch signaling in acute myeloid leukemia (AML) is still under investigation. We have previously shown that high levels of Notch receptors and ligands could interfere with drug response. In this study, the protein expression of 79 AML blast samples collected from newly diagnosed patients was examined through flow cytometry. Gamma-secretase inhibitors were used in AML mouse xenograft models to evaluate the contribution of Notch pharmacological inhibition to mouse survival. We used univariate analysis for testing the correlation and/or association between protein expression and well-known prognostics markers. All the four receptors (Notch1–4) and some ligands (Jagged2, DLL-3) were highly expressed in less mature subtypes (M0–M1). Notch3, Notch4, and Jagged2 were overexpressed in an adverse cytogenetic risk group compared to good cytogenetic risk patients. Chi-square analysis revealed a positive association between the complete remission rate after induction therapy and weak expression of Notch2 and Notch3. We also found an association between low levels of Notch4 and Jagged2 and three-year remission following allogeneic stem cell transplantation (HSCT). Accordingly, Kaplan–Meier analysis showed improved OS for patients lacking significant expression of Notch4, Jagged2, and DLL3. In vivo experiments in an AML mouse model highlighted both improved survival and a significant reduction of leukemia cell burden in the bone marrow of mice treated with the combination of Notch pan-inhibitors (GSIs) plus chemotherapy (Ara-C). Our results suggest that Notch can be useful as a prognostic marker and therapeutic target in AML

Docosahexaenoic acid induces proteasome-dependent degradation of beta-catenin, downregulation of survivin and apoptosis in human colorectal cancer cells not expressing COX-2.

n-3 Polyunsaturated fatty acids have been shown to powerfully inhibit the growth of colon cancer cells, mainly acting as pro-apoptotic agents through inhibition of COX-2 expression. Since dysregulation of beta-catenin expression is frequently found at early stage of colorectal carcinogenesis, we analyzed whether docosahexaenoic acid (DHA) may modify the expression of beta-catenin in colon cancer cells (SW480 and HCT116) overexpressing this protein, but lacking COX-2. Futhermore, we investigated if alterations in beta-catenin expression may be associated with apoptosis induction. Treatment of cells with increasing concentrations of DHA induced a dose- and time-dependent inhibition of beta-catenin protein expression which, however, was not accompanied by modifications in beta-catenin transcription. Conversely, the proteasomal inhibitors MG132 and lactacystin prevented DHA-induced beta-catenin decrease, suggesting that DHA may regulate the proteasomal degradation of beta-catenin. The reduced levels of beta-catenin were accompanied by decreased translocation of beta-catenin into the nucleus, where it acts as a transcription factor in concert with TCF. DHA, at the same range of concentrations, was also able to induce apoptosis by a caspase-3 dependent mechanism and to cause a dose- and time-dependent decrease of survivin, an apoptosis inhibitor undetectable in normal tissues and expressed in colorectal cancer through TCF/beta-catenin stimulation. Several other proteins regulated by the TCF/beta-catenin pathway and involved in regulation of tumor growth were down-regulated by DHA, including PPAR-delta, MT1-MMP, MMP-7, and VEGF. The present study, thus, raises the possibility that DHA may exert pro-apoptotic and antitumoral effects through proteasomal regulation of beta-catenin levels and alterations in the expression of TCF/beta-catenin-target genes

Growth, viability, adhesion potential and fibronectin expression in fibroblasts cultured on zirconia or feldspatic ceramics in vitro

Zirconia, a biomaterial widely used in dentistry, has recently attracted much attention for its mechanical strength and toughness. Previously, its lack of mutagenic and carcinogenic power was reported. We describe here other essential aspects to be taken into account to define in vitro the biocompatibility of a material: the growth rate, viability, and adhesion capacity of normal stabilized cells growing on it. To this aim, immortalized RAT-1 fibroblasts, growing either on zirconia and on feldspatic (FE) ceramics were compared. In particular, the level of expression and the intra- and extra-cellular organization of fibronectin, a glycoprotein involved in cellular adhesion and migration during tissue repair, was analyzed. Fibroblasts cultured on zirconia showed a higher growth rate, and underwent necrosis at lower levels than cells on FE ceramic, whereas either materials did not stimulate apoptosis. Adhesion capacity of fibroblasts was evaluated measuring adherent cell nucleic acids with the fluorimetric CyQuant(R) assay, and it was found significantly higher in cells cultured on zirconia than on FE ceramic. This finding may be explained by the higher and more precocious expression of the adhesion protein fibronectin observed by indirect immunofluorescence in fibroblasts on zirconia. Overall, the results suggest that zirconia, exerting low cytotoxicity and strongly inducing adhesion capacity, increases cellular growth rate of fibroblasts. All these features suggest that zirconia could represent a more suitable biomaterial than FE ceramic for prosthesis in dentistry