The microenvironment provides essential growth and survival signals to chronic lymphocytic leukemia (CLL) cells and contributes to their resistance to cytotoxic agents. Pharmacologic inhibition of ...spleen tyrosine kinase (SYK), a key mediator of B-cell receptor (BCR) signaling, induces apoptosis in primary CLL cells and prevents stroma contact-mediated cell survival. This report demonstrates a role of SYK in molecularly defined pathways that mediate the CLL-microenvironmental crosstalk independent from the BCR. Chemokine and integrin stimulation induced SYK phosphorylation, SYK-dependent Akt phosphorylation, and F-actin formation in primary CLL cells. Inhibition of SYK by 2 pharmacologic inhibitors and siRNA-knockdown abrogated downstream SYK signaling and morphologic changes induced by these stimuli. CLL cell migration toward CXCL12, the major homing attractor, and CLL cell adhesion to VCAM-1, a major integrin ligand expressed on stromal cells, were markedly reduced by SYK inhibition. In combination with fludarabine, the SYK inhibitor R406 abrogated stroma-mediated drug resistance by preventing up-regulation of the antiapoptotic factor Mcl-1 in CLL cells. SYK blockade in CLL is a promising therapeutic principle not only for its inhibition of the BCR signaling pathway, but also by inhibiting protective stroma signals in a manner entirely independent of BCR signaling.
Growth and survival of chronic lymphocytic leukemia (CLL) B cells are favored by interactions between CLL and nontumoral accessory cells. CLL cells express CXCR4 chemokine receptors that direct ...leukemia cell chemotaxis. Marrow stromal cells or nurselike cells constitutively secrete CXCL12, the ligand for CXCR4, thereby attracting and rescuing CLL B cells from apoptosis in a contact-dependent fashion. Therefore, the CXCR4-CXCL12 axis represents a potential therapeutic target in CLL. We evaluated the most active CXCR4-specific antagonists (T140, TC14012, TN14003) for their capacity to inhibit CXCL12 responses in CLL cells. T140, or its analogs, inhibited actin polymerization, chemotaxis, and migration of CLL cells beneath stromal cells. CXCL12-induced phosphorylation of p44/42 mitogen-activated protein kinase (MAPK) and signal transducer and activator of transcription 3 (STAT3) was abolished by CXCR4 antagonists. TC14012 and TN14003 antagonized the antiapoptotic effect of synthetic CXCL12 and stromal cell-mediated protection of CLL cells from spontaneous apoptosis. Furthermore, we found that stromal cells protected CLL cells from chemotherapy-induced apoptosis. Treatment with CXCR4 antagonists resensitized CLL cells cultured with stromal cells to fludarabine-induced apoptosis. These findings demonstrate that CXCR4 blocking agents effectively antagonize CXCL12-induced migratory and signaling responses and stromal protection of CLL cells from spontaneous or fludarabine-induced apoptosis. As such, small molecular CXCR4 antagonists may have activity in the treatment of patients with this disease. (Blood. 2005;106:1824-1830)
Small cell lung cancer (SCLC) is an aggressive, rapidly metastazising neoplasm with a high propensity for marrow involvement. SCLC cells express high levels of functional CXCR4 receptors for the ...chemokine stromal-cell-derived factor-1 (SDF-1/CXCL12). Adhesion of SCLC cells to extracellular matrix or accessory cells within the tumor microenvironment confers resistance to chemotherapy via integrin signaling and thus may be responsible for residual disease and relapses commonly seen in SCLC. We examined the signaling mechanisms that regulate CXCL12-induced adhesion of SCLC cells to fibronectin, collagen, and stromal cells and the effects on SCLC cell chemoresistance. We found that CXCL12-induced integrin activation which resulted in an increased adhesion of SCLC cells to fibronectin and collagen. This was mediated by alpha2, alpha4, alpha5, and beta1 integrins along with CXCR4 activation, which could be inhibited by CXCR4 antagonists. Stromal cells protected SCLC cells from chemotherapy-induced apoptosis, and this protection could also be antagonized by CXCR4 inhibitors. We conclude that activation of integrins and CXCR4 chemokine receptors co-operate in mediating adhesion and survival signals from the tumor microenvironment to SCLC cells. Therefore, CXCR4 antagonists in combination with cytotoxic drugs should be explored in SCLC to overcome CXCL12-mediated adhesion and survival signals in the tumor microenvironment.
Lung cancer is one of the leading causes of cancer related death worldwide with more than a million deaths per year. The poor prognosis is due to its high aggressiveness and its early metastasis. ...Although the exact mechanisms are still unknown, the process of epithelial to mesenchymal transition (EMT) seems to be involved in these neoplastic processes. We already demonstrated that serum levels of CCL18, a primate specific chemokine, are highly elevated in patients with lung cancer and correlate with their survival time of patients with adenocarcinoma of the lung. Therefore, we hypothesized that CCL18 may be directly involved in pathological processes of lung cancer, e.g. EMT. We investigated the effect of CCL18 on A549, an adenocarcinoma cell line of the lung, on EMT and other cell functions like proliferation, chemotaxis, invasion, chemoresistance and proliferation. Exposure of A549 lung cancer cells to CCL18 in various concentrations decreases the epithelial marker E-cadherin, whereas FSP-1, a marker of the mesenchymal phenotype increases. Accordingly, CCL18 induced the transcriptional EMT regulator SNAIL1 in a dose dependent fashion. In contrast, an increasing CCL18 concentration was associated with a decline of cell proliferation rate. In addition, CCL18 induced chemotaxis of these cells and increased their chemoresistance. Therefore, CCL18 may be an interesting therapeutic target for NSCLC.
A subset of blood cells from patients with B-cell chronic lymphocytic leukemia (CLL) spontaneously differentiates in vitro into large, round, or fibroblast-like adherent cells that display stromal ...cell markers, namely vimentin and STRO-1. These cells also express stromal cell–derived factor-1 (SDF-1), a CXC chemokine that ordinarily is secreted by marrow stromal cells. Leukemia B cells attach to these blood-derived adherent cells, down-modulate their receptors for SDF-1 (CXCR4), and are protected from undergoing spontaneous apoptosis in vitro. Neutralizing antibodies to SDF-1 inhibit this effect. Moreover, the rapid deterioration in the survival of CLL B cells, when separated from such cells, is mitigated by exogenous SDF-1. This chemokine also results in the rapid down-modulation of CXCR4 and activation of p44/42 mitogen-activated protein-kinase (ERK 1/2) by CLL B cells in vitro. It is concluded that the blood of patients with CLL contains cells that can differentiate into adherent nurse-like cells that protect leukemia cells from undergoing spontaneous apoptosis through an SDF-1–dependent mechanism. In addition to its recently recognized role in CLL B-cell migration, SDF-1–mediated CLL B-cell activation has to be considered a new mechanism involved in the microenvironmental regulation of CLL B-cell survival.
Nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) is known to play an important role in the pathogenesis of chronic lymphocytic leukemia (CLL). Several NF-κB inhibitors were shown to ...successfully induce apoptosis of CLL cells
Since the microenvironment is known to be crucial for the survival of CLL cells, herein, we tested whether NF-κB inhibition may still induce apoptosis in these leukemic cells in the presence of protective stromal interaction. We used the specific NF-κB inhibitor dehydroxymethylepoxyquinomicin (DHMEQ). Microenvironmental support was mimicked by co-culturing CLL cells with bone marrow-derived stromal cell lines (HS-5 and M2-10B4). NF-κB inhibition by DHMEQ in CLL cells could be confirmed in both the monoculture and co-culture setting. In line with previous reports, NF-κB inhibition induced apoptosis in the monoculture setting by activating the intrinsic apoptotic pathway resulting in poly (ADP-ribose) polymerase (PARP)-cleavage; however, it was unable to induce apoptosis in leukemic cells co-cultured with stromal cells. Similarly, small interfering ribonucleic acid (siRNA)-mediated
downregulation induced apoptosis of CLL cells cultured alone, but not in the presence of supportive stromal cells. B-cell activating factor (BAFF) was identified as a microenvironmental messenger potentially protecting the leukemic cells from NF-κB inhibition-induced apoptosis. Finally, we show improved sensitivity of stroma-supported CLL cells to NF-κB inhibition when combining the NF-κB inhibitor with the SYK inhibitor R406 or the Bruton's tyrosine kinase (BTK) inhibitor ibrutinib, agents known to inhibit the stroma-leukemia crosstalk. We conclude that NF-κB inhibitors are not promising as monotherapies in CLL, but may represent attractive therapeutic partners for ibrutinib and R406.
Background Five different G protein–coupled sphingosine-1-phosphate (S1P) receptors (S1P1-S1P5) regulate a variety of physiologic and pathophysiologic processes, including lymphocyte circulation, ...multiple sclerosis (MS), and cancer. Although B-lymphocyte circulation plays an important role in these processes and is essential for normal immune responses, little is known about S1P receptors in human B cells. Objective To explore their function and signaling, we studied B-cell lines and primary B cells from control subjects, patients with leukemia, patients with S1P receptor inhibitor–treated MS, and patients with primary immunodeficiencies. Methods S1P receptor expression was analyzed by using multicolor immunofluorescence microscopy and quantitative PCR. Transwell assays were used to study cell migration. S1P receptor internalization was visualized by means of time-lapse imaging with fluorescent S1P receptor fusion proteins expressed by using lentiviral gene transfer. B-lymphocyte subsets were characterized by means of flow cytometry and immunofluorescence microscopy. Results Showing that different B-cell populations express different combinations of S1P receptors, we found that S1P1 promotes migration, whereas S1P4 modulates and S1P2 inhibits S1P1 signals. Expression of CD69 in activated B lymphocytes and B cells from patients with chronic lymphocytic leukemia inhibited S1P-induced migration. Studying B-cell lines, normal B lymphocytes, and B cells from patients with primary immunodeficiencies, we identified Bruton tyrosine kinase, β-arrestin 2, LPS-responsive beige-like anchor protein, dedicator of cytokinesis 8, and Wiskott-Aldrich syndrome protein as critical signaling components downstream of S1P1. Conclusion Thus S1P receptor signaling regulates human B-cell circulation and might be a factor contributing to the pathology of MS, chronic lymphocytic leukemia, and primary immunodeficiencies.
Small cell lung cancer (SCLC) is a highly aggressive subtype of lung cancer with very poor prognosis due to early metastatic spread and development of chemoresistance. In the last 30 years the study ...of SCLC has been constrained by a lack of primary human tumor specimen thus highlighting the need of a suitable mouse model. In this article we present the establishment of an orthotopic xenograft mouse model which accurately reproduced the clinical course of SCLC. Orthotopic implantation enabled engraftment of primary lung tumors in all injected mice. Furthermore, immunodeficiency of mice allowed formation of spontaneous metastases in characteristic organs. Bioluminescence Imaging, Magnetic Resonance Imaging and Positron emission tomography were applied to monitor engraftment, metabolism and the exact growth of tumors over time. In order to mimic the extensive disease stage, mice were injected with aggressive human chemoresistant cells leading to development of chemoresistant tumors and early metastatic spread. As a proof of concept treatment of tumor-bearing mice with conventional chemotherapeutics reduced tumor volumes, but a complete regression of tumors was not achieved. By mimicking the extensive disease stage our mouse model can facilitate the study of mechanisms contributing to chemoresistance and metastasis formation, as well as drug screening and evaluation of new treatment strategies for SCLC patients.
CC-chemokine ligand 18 (CCL18) is mainly expressed by alternatively activated macrophages and DCs and plays an important role in lung fibrosis, arthritis and other diseases. Here CCL18 was measured ...in sera of 31 healthy volunteers and 170 patients with lung cancer and correlated these data with histology, tumor stage and clinical parameters. Mean CCL18 serum level of the patients with non-small-cell lung cancer was 150(857) ng/ml vs. 32(61) ng/ml in the healthy control group. Patient groups differ significantly according their histology (adenocarcinoma 143(528) ng/ml vs squamous cell carcinoma 187(857) ng/ml, p<0.02). In addition, we found a significant difference between patients with lower versus higher T-stage (p<0.003). Receiver operating characteristic (ROC) analyses revealed a cutoff point of 83 ng/ml (area under the curve (AUC): 0.968; p<0.0001) to discriminate between healthy controls and non-small-cell lung cancer patients. ROC analyses to discriminate between patients, who died because of cancer related death and those who died for other reasons did not lead to a valid AUC. To stratify the tumor patients, a criterion value plot was performed leading to a point of equal sensitivity and specificity (54%) of 162 ng/ml. Patients with a CCL18 serum level higher than 160 ng/ml had a mean survival time of 623 days. In contrast, those in patients with a baseline level between 83 ng/ml and 160 ng/ml the mean survival time was 984 days (p<0.005). Survival-analysis revealed in adenocarcinoma a mean survival of 1152 days in the group below 83 ng/ml. In the median group the mean survival time was 788 days and in the group with the highest levels the mean survival time was 388 days (p<0.001). In contrast, we found no correlation between the FEV1 and the CCL18 baseline level. In conclusion, in patients suffering from adenocarcinoma increased serum CCL18 levels predict a diminished survival time.
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