ABSTRACT

No abstract

Small-cell lung cancer (SCLC) is an aggressive, rapidly metastasizing neoplasm. The chemokine stromal cellderived factor-1 (SDF-1/CXCL12) is constitutively secreted by marrow stromal cells and plays a key role for homing of hematopoietic cells to the marrow. Here, we report that tumor cells from patients with SCLC express high levels of functional CXCR4 receptors for the chemokine CXCL12. Reverse transcriptase-polymerase chain reaction and flow cytometry demonstrated CXCR4 mRNA and CXCR4 surface expression in SCLC cell lines. Immunohistochemistry of primary tumor samples from SCLC patients revealed high expression of CXCR4. CXCL12 elicited CXCR4 receptor endocytosis, actin polymerization, and a robust activation of phospho-p44/ 42 mitogen-activated protein kinase in SCLC cells. Furthermore, CXCL12 induced SCLC cell invasion into extracellular matrix and firm adhesion to marrow stromal cells. Stromal cell adhesion of SCLC cells was significantly inhibited by the specific CXCR4 antagonist T140, pertussis toxin, antivascular cell adhesion molecule-1(VCAM-1) antibodies, and CS-1 peptide, demonstrating the importance of CXCR4 chemokine receptor activation and a4b1 integrin binding, respectively. In addition, CXCL12 enhanced the adhesion of SCLC cells to immobilized VCAM-1, demonstrating that CXCR4 chemokine receptors can induce integrin activation on SCLC cells. As SCLC has a high propensity for bone marrow involvement, our findings suggest that CXCR4 chemokine receptors and a4b1 integrins play a critical role in the interaction of SCLC cells with stromal cells in the tumor microenvironment.

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 a2, a4, a5, and b1 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.

It is largely unknown how hematopoietic progenitors are positioned within specialized niches of the bone marrow microenvironment during development. Chemokines such as CXCL12, previously called stromal cell–derived factor 1, are known to activate cell integrins of circulating leukocytes resulting in transient adhesion before extravasation into tissues. However, this short-term effect does not explain the mechanism by which progenitor cells are retained for prolonged periods in the bone marrow. Here we show that in human bone marrow CXCL12 triggers a sustained adhesion response specifically in progenitor (pro- and pre-) B cells. This sustained adhesion diminishes during B cell maturation in the bone marrow and, strikingly, is absent in circulating mature B cells, which exhibit only transient CXCL12-induced adhesion. The duration of adhesion is tightly correlated with CXCL12-induced activation of focal adhesion kinase (FAK), a known molecule involved in integrin-mediated signaling. Sustained adhesion of progenitor B cells is associated with prolonged FAK activation, whereas transient adhesion in circulating B cells is associated with short-lived FAK activation. Moreover, sustained and transient adhesion responses are differentially affected by pharmacological inhibitors of protein kinase C and phosphatidylinositol 3-kinase. These results provide a developmental cell stage–specific mechanism by which chemokines orchestrate hematopoiesis through sustained rather than transient activation of adhesion and cell survival pathways.

Hematopoietic stem/progenitor cells (HSC/P) reside in the bone marrow in distinct anatomic locations (niches) to receive growth, survival and differentiation signals. HSC/P localization and migration between niches depend on cell-cell and cell-matrix interactions, which result from the cooperation of cytokines, chemokines and adhesion molecules. The CXCL12-CXCR4 pathway, in particular, is essential for myelopoiesis and B lymphopoiesis but the molecular mechanisms of CXCL12 action remain unclear. We previously noted a strong correlation between prolonged CXCL12-mediated focal adhesion kinase (FAK) phosphorylation and sustained pro-adhesive responses in progenitor B cells, but not in mature B cells. Although FAK has been well studied in adherent fibroblasts, its function in hematopoietic cells is not defined. We used two independent approaches to reduce FAK expression in (human and mouse) progenitor cells. RNA interference (RNAi)-mediated FAK silencing abolished CXCL12-induced responses in human pro-B leukemia, REH cells. FAK-deficient REH cells also demonstrated reduced CXCL12-induced activation of the GTPase Rap1, suggesting the importance of FAK in CXCL12-mediated integrin activation. Moreover, in FAK flox/flox hematopoietic precursor cells, Cre-mediated FAK deletion resulted in impaired CXCL12-induced chemotaxis. These studies suggest that FAK may function as a key intermediary in signaling pathways controlling hematopoietic cell lodgment and lineage development.

IntroductionChemokines were initially defined as molecules abundantly produced at sites of inflammation and responsible for the recruitment of cells to participate in local immune responses. 1 Indeed, chemokines have been shown to direct the movement of cells between intravascular and extravascular compartments, within specific zones of secondary lymphoid organs, and to drive cells to recirculate between tissues and lymphoid organs. 2,3 It has recently become clear that this family of molecules also controls multiple cell functions involving angiogenesis, embryogenesis, lymphocyte development, and hematopoiesis. 4,5 The growth and differentiation of progenitor B cells are dependent on factors such as chemokines and cytokines, which are either bound or secreted by bone marrow stromal cells. 6 Within the extravascular compartment of bone marrow, there are several well-known movements of progenitor B cells in which chemokines might be involved. The earliest progenitor B cells (eg, pro-B cells), are located within or near the endosteum. 7,8 In subsequent stages of development, the differentiating B cells move toward sinusoids along stromal cell processes. 8,9 After beginning to express surface IgM (sIgM), the immature and mature B cells migrate into the sinusoid segments and are released into the circulation.The CXCL12/CXCR4 axis appears essential for B-cell development during early ontogeny. Mice in which the gene for CXCR4 or CXCL12 is disrupted show impaired B lymphopoiesis in the bone marrow. 10,11 In other experiments, the transfer of CXCR4-deficient stem cells into wild-type mice results in high circulating levels of progenitor B cells, suggesting that the CXCL12/CXCR4 axis is important for the retention/positioning of early-lineage B cells in the bone marrow cavity. 12,13 Furthermore, in support of this hypothesis is the observation that in both mice and humans CXCL12-mediated responsiveness decreases with B-cell maturation, despite comparable high levels of CXCR4 expression at all developmental B-cell stages. [14][15][16] In contrast, little data are available as to the role of CC chemokine receptors during B-cell development. We reasoned that CC chemokines and corresponding receptors might also participate in the positioning of progenitor B cells in the bone marrow.In the current study we therefore characterized CCR5 expression and function on primary progenitor B-cell populations (pro-, pre-, immature, and mature B cells) For personal use only. on May 10, 2018. by guest www.bloodjournal.org From primary bone marrow B cells to subsequent CXCR4/CXCL12-mediated calcium mobilization, extracellular signal-regulated kinase 1 (ERK1) and 2 (ERK2) and p38 phosphorylation as well as chemotaxis responses. We also established that this inhibitory effect was dependent on the specific interaction of these chemokines with CCR5. To our knowledge, the present data represent the first example of heterologous desensitization of CXCR4 by signaling through CCR5.Thus, CCR5-binding chemokines can trigger biologically signifi...

CXCL12-induced chemotaxis and adhesion to VCAM-1 decrease as B cells differentiate in the bone marrow. However, the mechanisms that regulate CXCL12/CXCR4-mediated signaling are poorly understood. We report that after CXCL12 stimulation of progenitor B cells, focal adhesion kinase (FAK) and PI3K are inducibly recruited to raft-associated membrane domains. After CXCL12 stimulation, phosphorylated FAK is also localized in membrane domains. The CXCL12/CXCR4-FAK pathway is membrane cholesterol dependent and impaired by metabolic inhibitors of Gi, Src family, and the GTPase-activating protein, regulator of G protein signaling 1 (RGS1). In the bone marrow, RGS1 mRNA expression is low in progenitor B cells and high in mature B cells, implying developmental regulation of CXCL12/CXCR4 signaling by RGS1. CXCL12-induced chemotaxis and adhesion are impaired when FAK recruitment and phosphorylation are inhibited by either membrane cholesterol depletion or overexpression of RGS1 in progenitor B cells. We conclude that the recruitment of signaling molecules to specific membrane domains plays an important role in CXCL12/CXCR4-induced cellular responses.