Identification of the Axis ß-Catenin-BTK in the Dynamic Adhesion of Chronic Lymphocytic Leukemia Cells to Their Microenvironment.

In the microenvironment, cell interactions are established between different cell types to regulate their migration, survival and activation. ß-Catenin is a multifunctional protein that stabilizes cell-cell interactions and regulates cell survival through its transcriptional activity. We used chronic lymphocytic leukemia (CLL) cells as a cellular model to study the role of ß-catenin in regulating the adhesion of tumor cells to their microenvironment, which is necessary for tumor cell survival and accumulation. When co-cultured with a stromal cell line (HS-5), a fraction of the CLL cells adhere to stromal cells in a dynamic fashion regulated by the different levels of ß-catenin expression. In non-adherent cells, ß-catenin is stabilized in the cytosol and translocates into the nucleus, increasing the expression of cyclin D1. In adherent cells, the level of cytosolic ß-catenin is low but membrane ß-catenin helps to stabilize the adhesion of CLL to stromal cells. Indeed, the overexpression of ß-catenin enhances the interaction of CLL with HS-5 cells, suggesting that this protein behaves as a regulator of cell adhesion to the stromal component and of the transcriptional regulation of cell survival. Inhibitors that block the stabilization of ß-catenin alter this equilibrium and effectively disrupt the support that CLL cells receive from the cross-talk with the stroma.

Stabilization of ß-catenin upon B-cell receptor signaling promotes NF-kB target genes transcription in mantle cell lymphoma.

B-cell receptor (BCR) signaling pathways and interactions with the tumor microenvironment account for mantle cell lymphoma (MCL) cells survival in lymphoid organs. In several MCL cases, the WNT/ß-catenin canonical pathway is activated and ß-catenin accumulates into the nucleus. As both BCR and ß-catenin are important mediators of cell survival and interaction with the microenvironment, we investigated the crosstalk between BCR and WNT/ß-catenin signaling and analyzed their impact on cellular homeostasis as well as their targeting by specific inhibitors. ß-catenin was detected in all leukemic MCL samples and its level of expression rapidly increased upon BCR stimulation. This stabilization was hampered by the BCR-pathway inhibitor Ibrutinib, supporting ß-catenin as an effector of the BCR signaling. In parallel, MCL cells as compared with normal B cells expressed elevated levels of WNT16, a NF-κB target gene. Its expression increased further upon BCR stimulation to participate to the stabilization of ß-catenin. Upon BCR stimulation, ß-catenin translocated into the nucleus but did not induce a Wnt-like transcriptional response, i.e., TCF/LEF dependent. ß-catenin rather participated to the regulation of NF-κB transcriptional targets, such as IL6, IL8, and IL1. Oligo pull down and chromatin immunoprecipitation experiments demonstrated that ß-catenin is part of a protein complex that binds the NF-κB DNA consensus sequence, strengthening the idea of an association between the two proteins. An inhibitor targeting ß-catenin transcriptional interactions hindered both NF-κB DNA recruitment and induced primary MCL cells apoptosis. Thus, ß-catenin likely represents another player through which BCR signaling impacts on MCL cell survival.

Protein kinase D-dependent CXCR4 down-regulation upon BCR triggering is linked to lymphadenopathy in chronic lymphocytic leukaemia.

In Chronic Lymphocytic Leukemia (CLL), infiltration of lymph nodes by leukemic cells is observed in patients with progressive disease and adverse outcome. We have previously demonstrated that B-cell receptor (BCR) engagement resulted in CXCR4 down-regulation in CLL cells, correlating with a shorter progression-free survival in patients. In this study, we show a simultaneous down-regulation of CXCR4, CXCR5 and CD62L upon BCR triggering. While concomitant CXCR4 and CXCR5 down-regulation involves PKDs, CD62L release relies on PKC activation. BCR engagement induces PI3K-δ-dependent phosphorylation of PKD2 and 3, which in turn phosphorylate CXCR4 Ser324/325. Moreover, upon BCR triggering, PKD phosphorylation levels correlate with the extent of membrane CXCR4 decrease. Inhibition of PKD activity restores membrane expression of CXCR4 and migration towards CXCL12 in BCR-responsive cells in vitro. In terms of pathophysiology, BCR-dependent CXCR4 down-regulation is observed in leukemic cells from patients with enlarged lymph nodes, irrespective of their IGHV mutational status. Taken together, our results demonstrate that PKD-mediated CXCR4 internalization induced by BCR engagement in B-CLL is associated with lymph node enlargement and suggest PKD as a potential druggable target for CLL therapeutics.

Inhibitors of BCR signalling interrupt the survival signal mediated by the micro-environment in mantle cell lymphoma.

Several studies provide evidences for mantle cell lymphoma (MCL) cell survival relying on B-cell receptor (BCR)-mediated signalling pathways, whereas the nature of this activation is unknown. Significant progress in MCL treatment is achieved through therapies targeting BCR-associated kinases, i.e., Ibrutinib and Fostamatinib, inhibitors of BTK and SYK, respectively. Our study addresses survival signals emanating from the BCR or the tumour environment and how inhibiting BCR signalling effectors might impact these survival signals. We found that BTK was constitutively activated and that SYK phosphorylation was highly increased and sustained upon BCR activation of primary MCL cells. Moreover, MCL cells from leukaemic patients secreted high amount of IL-1ß, IL-6, IL-8 and CCL5. Activation of the BCR induced (i) cell survival, (ii) STAT3 activation and (iii) increased autocrine secretion of IL-1ß, IL-6, IL-8, CCL5, IL-10, TNFα and VEGF. Specific inhibition of BTK by Ibrutinib or SYK by Fostamatinib (R406) reversed these protective effects and decreased both basal and BCR-induced autocrine cytokine secretions associated with STAT3 phosphorylation. Interestingly, targeting BTK and SYK prevented and inhibited BCR-induced MCL cell adhesion to human bone marrow stromal cells (HMSCs) in short- and long-term co-culture. We demonstrated that BCR-induced survival relies on autocrine secretion of IL-1ß, TNFα and CCL5 that might facilitate adhesion of MCL cells to HMSC. Treatment with Ibrutinib or Fostamatinib blocked the chemotactic signal thus increasing apoptosis.

Targeting early B-cell receptor signaling induces apoptosis in leukemic mantle cell lymphoma.

BACKGROUND: We previously showed that B-cell receptor (BCR) signaling pathways are important for in vitro survival of mantle cell lymphoma (MCL) cells. To further identify early BCR-activated signaling pathways involved in MCL cell survival, we focused our study on BCR-proximal kinases such as LYN whose dysregulations could contribute to the aggressive course of MCL. METHODS: Primary MCL cells were isolated from 14 leukemic patients. Early BCR-induced genes were identified by qRT-PCR array. The basal and BCR-induced phosphorylation of LYN and JNK were evaluated by immunoblottting. Cell survival signals were evaluated by apoptosis using flow cytometry. RESULTS: We showed that LYN was constitutively phosphorylated in MCL cell lines and in 9/10 leukemic MCL cases. Treatment with dasatinib or with a specific inhibitor of Src kinases such as PP2 suppressed constitutive LYN activation and increased in vitro spontaneous apoptosis of primary MCL cells. BCR engagement resulted in an increase of LYN phosphorylation leading to activation of c-JUN NH2-terminal kinase (JNK) and over-expression of the early growth response gene-1 (EGR-1). Inhibition of JNK with SP600125 induced apoptosis and reduced level of basal and BCR-induced expression of EGR-1. Furthermore, decreasing EGR1 expression by siRNA reduced BCR-induced cell survival. Treatment with PP2 or with dasatinib suppressed BCR-induced LYN and JNK phosphorylation as well as EGR-1 upregulation and is associated with a decrease of cell survival in all cases analysed. CONCLUSIONS: This study highlights the importance of BCR signaling in MCL cell survival and points out to the efficiency of kinase inhibitors in suppressing proximal BCR signaling events and in inducing apoptosis.

Extracellular matrix metalloproteinase inducer (EMMPRIN/CD147) as a novel regulator of myogenic cell differentiation.

Matrix metalloproteinases (MMPs) are thought to play an important role in skeletal muscle cell growth and differentiation. In view of the MMP inducing function of EMMPRIN/CD147, its role in myogenic cell differentiation was investigated. EMMPRIN level increased during differentiation of both rat primary myoblasts derived from satellite cells and mouse C2.7 myogenic cells and was associated with an alteration in its molecular forms. In parallel, expression of pro-MMP-9 gradually decreased and that of pro-MMP-2 and active MMP-2 increased. While small interfering RNA (siRNA) inhibition of EMMPRIN expression accelerated cell differentiation, exogenously added recombinant EMMPRIN inhibited differentiation by an MMP-mediated mechanism, as the MMP inhibitor marimastat abrogated EMMPRIN's effect. Our results further suggest that EMMPRIN regulates differentiation through an MMP activation of transforming growth factor beta (TGFß), a known inhibitor of myoblast's differentiation, as the increased activation and signaling of TGFß by EMMPRIN was attenuated in the presence of marimastat. EMMPRIN inhibition may thus represent a novel strategy in the treatment of muscular degenerative disorders.

Constitutive and B-cell receptor-induced activation of STAT3 are important signaling pathways targeted by bortezomib in leukemic mantle cell lymphoma.

BACKGROUND: The deregulation of several transcription factors contribute to the aggressive course of mantle cell lymphoma. This study focuses on survival signals emanating from the tumor environment and involving the signal transducer and activator of transcription (STAT) 3 through cytokines or antigen recognition. DESIGN AND METHODS: Primary mantle cell lymphoma cells were isolated from 20 leukemic patients. The phosphorylation status of STAT3 was evaluated by immunoblottting and immunofluorescence, the levels of cytokine secretion by enzyme-linked immunosorbent assay and the cell survival signals by apoptosis and cell viability assays. RESULTS: STAT3 was constitutively phosphorylated in the Jeko-1 mantle cell lymphoma cell line and in 14 out of 20 (70%) cases of leukemic mantle cell lymphoma as the result of an autocrine secretion of interleukin-6 and/or interleukin-10. In addition, B-cell receptor engagement resulted in an increase of both in vitro cell survival and STAT3 phosphorylation in primary mantle cell lymphoma cells. Inhibition of the Janus-activated kinase/STAT3 pathway increased spontaneous apoptosis and suppressed B-cell receptor-induced cell survival in all cases analyzed. The impact of in vitro exposure to the proteasome inhibitor bortezomib was next evaluated in primary mantle cell lymphoma cells. Bortezomib induced apoptosis and a decrease of both interleukin-6/interleukin-10 secretion and STAT3 phosphorylation. In addition, bortezomib inhibited B-cell receptor-triggered STAT3 phosphorylation and cell survival. CONCLUSIONS: We demonstrated that STAT3 was activated in primary mantle cell lymphoma cells either constitutively through a cytokine autocrine loop or in response to B-cell receptor engagement, both processes leading to a survival signal inhibited by bortezomib. STAT3 appears, therefore, to play a pivotal role in mantle cell lymphoma and represents a promising therapeutic target.

Down-regulation of CXCR4 and CD62L in chronic lymphocytic leukemia cells is triggered by B-cell receptor ligation and associated with progressive disease.

Progressive cases of B-cell chronic lymphocytic leukemia (CLL) are frequently associated with lymphadenopathy, highlighting a critical role for signals emanating from the tumor environment in the accumulation of malignant B cells. We investigated on CLL cells from 30 untreated patients the consequence of B-cell receptor (BCR) triggering on the membrane expression of CXCR4 and CD62L, two surface molecules involved in trafficking and exit of B-lymphocytes from lymph nodes. BCR stimulation promoted a strictly simultaneous down-regulation of CXCR4 and CD62L membrane expression to a variable extent. The variable BCR-dependent decrease of the two proteins was strikingly representative of the heterogeneous capacity of the CLL cells to respond to BCR engagement in a given patient. Functionally, cells down-regulating CXCR4 and CD62L in response to BCR engagement displayed a reduction in both migration toward CXCL12 and adhesion to lymphatic endothelial cells. Remarkably, the ability of CLL cells to respond to BCR ligation was correlated with unfavorable prognostic markers and short progression-free survival. In conclusion, BCR signaling promotes decrease of CXCR4 and CD62L membrane expression in progressive cases only. These results are consistent with the hypothesis that BCR-mediated signaling pathways favor accumulation of a proliferative pool within the lymph nodes of progressive CLL cases.

The shedding of syndecan-4 and syndecan-1 from HeLa cells and human primary macrophages is accelerated by SDF-1/CXCL12 and mediated by the matrix metalloproteinase-9.

We recently demonstrated that stromal cell-derived factor-1 (SDF-1/CXCL12) forms complexes with CXCR4, but also with syndecan-4 expressed by human primary lymphocytes and macrophages, and HeLa cells. We also suggested that syndecan-4 behaves as a SDF-1-signaling molecule. Here, we demonstrate that SDF-1 strongly accelerates the shedding of syndecan-4 ectodomains and to a lesser extent that of syndecan-1 from HeLa cells. The fact that this acceleration was not inhibited by the CXCR4 antagonist AMD3100, anti-CXCR4 mAb 12G5, and CXCR4 gene silencing suggests its CXCR4-independence. Pre-treating the cells with heparitinases I, III, or with the protein kinase C (PKC) inhibitor, bisindolylmaleimide, significantly inhibited this accelerated shedding, which suggests the involvement of both cell-surface heparan sulfate and PKC transduction pathway. In contrast, Map Kinase or NF-kappaB pathway inhibitors had no effect. Moreover, SDF-1 increases the matrix metalloproteinase-9 (MMP-9) mRNA level as well as MMP-9 activity in HeLa cells, and MMP-9 silencing by RNA interference strongly decreases the syndecan-1 and -4 ectodomain shedding accelerated by SDF-1. Finally, SDF-1 also accelerates in a CXCR4-independent manner, the shedding of syndecan-1 and -4 from human primary macrophages, which is significantly inhibited by anti-MMP-9 antibodies. This strongly indicates the role of MMP-9 in these events occurring in both a tumoral cell line and in human primary macrophages. Because MMP-9 plays a crucial role in extracellular matrix degradation during cancer cell metastasis and invasion, and shed ectodomains of syndecans may likely be involved in tumor cell proliferation, these data further indicate the multiplicity of the roles played by SDF-1 on tumor cell biology.

A new dimethyl ester bisphosphonate inhibits angiogenesis and growth of human epidermoid carcinoma xenograft in nude mice.

Bisphosphonates are extensively used in the treatment of patients with metastasis-induced osteolysis. The major drawback in the efficacy of all bisphosphonates lies in their high hydrophilic nature, which results in poor membrane permeability and low availability for soft tissues. A reasonable approach to overcome these problems consists in masking one or more ionizable groups of bisphosphonates, notably by esterification of the hydroxyl functions. We have previously shown that the novel non-nitrogen-containing bisphosphonate BP7033 inhibited angiogenesis and growth of primary tumors in nude mice. The present study focuses on the dimethyl-esterified analog of this compound (Me-BP7033). In-vitro, Me-BP7033 inhibited proliferation of human carcinoma A431 cells as well as their invasive activity based on a transwell invasion assay. in-vivo, administration of Me-BP7033 (0.3 mg/kg) twice a week for 5 weeks inhibited the tumor growth of A431 cells xenografted in nude mice by 65%. Immunostaining of endothelial cells (ECs) in tumor sections revealed that Me-BP7033 inhibited the intratumor ECs density by 60%. The in-vivo anti-angiogenic properties of Me-BP7033 were also demonstrated in an in-vivo angiogenesis assay showing that Me-BP7033 reduced the vascular endothelial growth factor-stimulated infiltration of ECs in a Matrigel plug by 70%. In summary, we demonstrated for the first time that a diesterified bisphosphonate exhibited in vivo both anti-tumoral and anti-angiogenic activities with no apparent sign of toxic effects. These new diesterified compounds, which could display enhanced bioavailability and pharmacokinetics, thus represent interesting candidates for therapeutic applications such as cancer treatment.

Structure-activity relationships of a new class of aromatic bisphosphonates that inhibit tumor cell proliferation in vitro.

We previously reported a simple and efficient one-pot procedure for synthesis of 1-hydroxymethylene-1,1-bisphosphonic acids (HMBP). According to this method, we synthesized a series of new aromatic HMBP and investigated structure-activity relationships by evaluating their anti-proliferative activity against A431 human tumor cell line. Our results showed that the introduction of an extra methylene group in a pyridyl-containing R2 side chain increased 100-fold the anti-proliferative activity of the HMBP. In contrast, this chemical modification did not modify the anti-proliferative activity of compounds substituted with a phenyl-containing R2 side chain. Para-substitution of the phenyl ring with various groups markedly influenced the HMBP activity, the order of potency (bromine > chlorine > fluorine = none) closely matching the atomic volume of the substituted group. Moreover, changes in the substitution position of the bromine group also affected the anti-proliferative activity, the more potent activity being obtained with para-substitution of the phenyl ring. In conclusion, this structure-activity study led us to identify the new aromatic HMBP [(4-Bromo-phenyl)-hydroxy-phosphono-methyl]-phosphonic acid as a potent in vitro anti-proliferative molecule against tumor cell lines (IC50 value of 9.5 x 10(-5) M). Interestingly, this compound can be further easily esterified on its phosphonic acid functions according to our chemical method and, thus, represents a potential candidate for the development of new esterified HMBP with enhanced pharmacokinetics.

An engineered biopolymer prevents mucositis induced by 5-fluorouracil in hamsters.

Oral mucositis is a common, treatment-limiting, and costly side effect of cancer treatments whose biological underpinnings remain poorly understood. In this study, mucositis induced in hamsters by 5-fluorouracil (5-FU) was observed after cheek-pouch scarifications, with and without administration of RGTA (RG1503), a polymer engineered to mimic the protective effects of heparan sulfate. RG1503 had no effects on 5-FU-induced decreases in body weight, blood cell counts, or cheek-pouch and jejunum epithelium proliferation rates, suggesting absence of interference with the cytotoxic effects of 5-FU. Extensive mucositis occurred in all of the untreated animals, and consisted of severe damage to cheek pouch tissues (epithelium, underlying connective tissue, and muscle bundles). Only half of the RG1503-treated animals had mucositis, over a mean area 70% smaller than in the untreated animals. Basement membranes were almost completely destroyed in the untreated group but was preserved in the RG1503 group. RG1503 blunted or abolished the following 5-FU-induced effects: increases in matrix metalloproteinase (MMP)-2, MMP-9, and plasmin, and decreases in tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2. These data indicate that mucositis lesions are related to massive release of proteolytic enzymes and are improved by RG1503 treatment, this effect being ascribable in part to restoration of the MMP-TIMP balance. RG1503 given with cancer treatment might protect patients from mucositis.

A novel non-containing-nitrogen bisphosphonate inhibits both in vitro and in vivo angiogenesis.

Bisphosphonates (BP) are powerful inhibitors of bone resorption and are widely used in the treatment of patients with metastasis-induced osteolysis. In the present study, we show that a novel non-nitrogen-containing BP (BP7033) that exhibits antitumor activity is a potent inhibitor of both in vivo and in vitro angiogenesis. When administered to mice, BP7033 inhibited tumoral angiogenesis (65% at 0.06mg/injection) as well as tumor growth (65% at 0.006mg/injection) in a tumor model of A431 cells xenografted in nude mice, with no sign of toxicity. Additionally, in vivo angiogenesis induced by vascular endothelial growth factor-containing Matrigel implants was reduced by 90% in the presence of BP7033 (0.6mg/plug). In vitro, BP7033 inhibited proliferation of human umbilical vein endothelial cells (HUVEC) (IC(50) value 3x10(-4) M) and completely prevented the formation of capillary-like tubules by HUVEC in Matrigel. Moreover, treatment of A431 cells by BP7033 induced an inhibition of Ras processing and a decrease in the secretion of both vascular endothelial growth factor and matrix metalloproteinase-2, two well-known stimulators of the proliferation and migration of endothelial cells. These findings indicate that this new BP compound has marked antiangiogenic properties and thus represents a promising candidate for treatment of malignant diseases with an angiogenic component.

Heparin-like dextran derivatives as well as glycosaminoglycans inhibit the enzymatic activity of human cathepsin G.

Some synthetic dextran derivatives that mimic the action of heparin/heparan sulfate were previously shown to inhibit neutrophil elastase and plasmin. Here we report that these derivatized dextrans also inhibit cathepsin G (CatG). Dextran containing carboxymethyl and benzylamide groups (RG1150) as well as those containing carboxymethyl, sulfate and benzylamide groups (RG1192), were the most efficient inhibitors of CatG activity. RG1192 and RG1150 bind CatG with a K(i) of 0.11 and 0.17 nM, respectively, while carboxymethylated sulfated dextran (RG1503) as well as heparin, heparan sulfate and dermatan sulfate bind CatG with a 7- to 30-fold lower affinity. Variation of K(i) with ionic strength indicates that ionic interactions account for 26% of the RG1503-CatG binding energy, while binding of RG1192 or RG1150 to CatG is mainly governed by non-electrostatic interactions. This, together with the fact that these compounds both protect fibronectin and laminin against CatG-mediated degradation, suggest that specific dextran derivatives can contribute to the regulation of CatG activity.

FGF-2 and TPA induce matrix metalloproteinase-9 secretion in MCF-7 cells through PKC activation of the Ras/ERK pathway.

Matrix metalloproteinases (MMPs) play an important role in cancer metastasis. Here, we investigated the effect of fibroblast growth factor-2 (FGF-2) and 12-O-tetradecanoylphorbol-13-acetate (TPA) on the secretion of type IV collagenases (MMP-2, MMP-9) in breast cancer MCF-7 cells. As shown by gelatin zymography, both FGF-2 and TPA stimulated the secretion of MMP-9 in MCF-7 cells while they did not change the level of MMP-2 secretion. Signaling cascade studies indicated that both FGF-2 and TPA induced Ras activation, c-Raf phosphorylation, mitogen-activated protein kinase/ERK kinase (MEK(1/2)) phosphorylation, and extracellular signal-regulated kinase (ERK(1/2)) phosphorylation. The FGF-2- and TPA-induced MMP-9 secretion was significantly inhibited by transient transfection of MCF-7 cells with dominant negative Ras (Ras-N17) and by treatment with MEK(1/2) inhibitor PD98059. A pan-protein kinase C (PKC) inhibitor, GF109203X, was found to totally abolish the FGF-2- and TPA-induced MMP-9 secretion and ERK(1/2) phosphorylation. Use of isoform-specific PKC inhibitors such as Rotllerin and Gö6976 suggested, moreover, that the PKC-delta isoform is a likely component of FGF-2 and TPA trophic signaling. These results demonstrated that FGF-2 and TPA induce MMP-9 secretion in MCF-7 cells mainly through PKC-dependent activation of the Ras/ERK(1/2) signaling pathway.