Tsipi Meshel

A Possible Role for CXCR4 and Its Ligand, the CXC Chemokine Stromal Cell-Derived Factor-1, in the Development of Bone Marrow Metastases in Neuroblastoma

The homing of hemopoietic stem cells to the bone marrow is mediated by specific interactions occurring between CXCR4, which is expressed on hemopoietic stem cells, and its ligand, stromal cell-derived factor-1 (SDF-1), a CXC chemokine secreted by bone marrow stromal cells. In the present study we evaluated the possibility that neuroblastoma cells use a mechanism similar to that used by hemopoietic stem cells to home to the bone marrow and adhere to bone marrow stromal cells. Our study suggests that CXCR4 expression may be a general characteristic of neuroblastoma cells. SH-SY5Y neuroblastoma cells express not only CXCR4, but also its ligand, SDF-1. CXCR4 expression on SH-SY5Y neuroblastoma cells is tightly regulated by tumor cell-derived SDF-1, as demonstrated by the ability of neutralizing Abs against human SDF-1α to up-regulate CXCR4 expression on the tumor cells. The reduction in CXCR4 expression following short term exposure to recombinant human SDF-1α can be recovered as a result of de novo receptor synthesis. Recombinant human SDF-1α induces the migration of CXCR4-expressing SH-SY5Y neuroblastoma cells in CXCR4- and heterotrimeric G protein-dependent manners. Furthermore, SH-SY5Y cells interact at multiple levels with bone marrow components, as evidenced by the fact that bone marrow-derived constituents promote SH-SY5Y cell migration, adhesion to bone marrow stromal cells, and proliferation. These results suggest that SH-SY5Y neuroblastoma cells are equipped with adequate machinery to support their homing to the bone marrow. Therefore, the ability of neuroblastoma tumors to preferentially form metastases in the bone marrow may be influenced by a set of complex CXCR4-SDF-1 interactions.

Inflammatory mediators in breast cancer: Coordinated expression of TNFα & IL-1β with CCL2 & CCL5 and effects on epithelial-to-mesenchymal transition

BackgroundThe inflammatory chemokines CCL2 (MCP-1) & CCL5 (RANTES) and the inflammatory cytokines TNFα & IL-1β were shown to contribute to breast cancer development and metastasis. In this study, we wished to determine whether there are associations between these factors along stages of breast cancer progression, and to identify the possible implications of these factors to disease course.MethodsThe expression of CCL2, CCL5, TNFα and IL-1β was determined by immunohistochemistry in patients diagnosed with: (1) Benign breast disorders (=healthy individuals); (2) Ductal Carcinoma In Situ (DCIS); (3) Invasive Ducal Carcinoma without relapse (IDC-no-relapse); (4) IDC-with-relapse. Based on the results obtained, breast tumor cells were stimulated by the inflammatory cytokines, and epithelial-to-mesenchymal transition (EMT) was determined by flow cytometry, confocal analyses and adhesion, migration and invasion experiments.ResultsCCL2, CCL5, TNFα and IL-1β were expressed at very low incidence in normal breast epithelial cells, but their incidence was significantly elevated in tumor cells of the three groups of cancer patients. Significant associations were found between CCL2 & CCL5 and TNFα & IL-1β in the tumor cells in DCIS and IDC-no-relapse patients. In the IDC-with-relapse group, the expression of CCL2 & CCL5 was accompanied by further elevated incidence of TNFα & IL-1β expression. These results suggest progression-related roles for TNFα and IL-1β in breast cancer, as indeed indicated by the following: (1) Tumors of the IDC-with-relapse group had significantly higher persistence of TNFα and IL-1β compared to tumors of DCIS or IDC-no-relapse; (2) Continuous stimulation of the tumor cells by TNFα (and to some extent IL-1β) has led to EMT in the tumor cells; (3) Combined analyses with relevant clinical parameters suggested that IL-1β acts jointly with other pro-malignancy factors to promote disease relapse.ConclusionsOur findings suggest that the coordinated expression of CCL2 & CCL5 and TNFα & IL-1β may be important for disease course, and that TNFα & IL-1β may promote disease relapse. Further in vitro and in vivo studies are needed for determination of the joint powers of the four factors in breast cancer, as well as analyses of their combined targeting in breast cancer.

CXCL10 Promotes Invasion-Related Properties in Human Colorectal Carcinoma Cells

CXCL10 was recently shown to exert antimalignancy functions by influencing the tumor microenvironment. Here, we have taken a different approach, investigating the effects of CXCL10 directly on tumor-promoting functions in colorectal carcinoma (CRC) cells. CXCL10 expression was detected in preferred metastatic sites of CRC (liver, lungs, and lymph nodes), and its CXCR3 receptor was expressed by eight CRC cell lines (detected: reverse transcription-PCR and/or flow cytometry). Detailed analysis was done on two cell lines derived from primary CRC tumors (SW480, KM12C) and their metastatic descendents (SW620 and KM12SM). The three known variants of CXCR3 (CXCR3-A, CXCR3-B, and CXCR3-alt) were detected in all four cell lines. CXCR3 expression was also observed on colorectal tumor cells in biopsies of CRC patients (immunohistochemistry). CXCL10 and CXCR3 expression were potently induced in CRC cells by Interferon gamma and all four CRC cell lines responded to CXCL10 by extracellular signal-regulated kinase 1/2 dephosphorylation. The chemokine did not affect tumor cell growth or angiogenesis-related functions in the tumor cells, such as CXCL8 and vascular endothelial growth factor secretion. Importantly, CXCL10 significantly up-regulated invasion-related properties in CRC cells: It promoted matrix metalloproteinase 9 expression and induced CRC cell migration. Of note, CXCL10-induced migration was detected only in the two metastatic cells and not in their primary counterparts. Also, CXCL10 promoted the adhesion of metastatic cells to laminin. These results suggest that CXCL10 can be exploited by CRC cells toward their progression, thus possibly antagonizing the antimalignancy effects of the chemokine on the tumor microenvironment. Therefore, care should be taken when considering CXCL10 as a therapeutic antitumor modality for CRC treatment.

Transendothelial migration of lymphocytes mediated by intraendothelial vesicle stores rather than by extracellular chemokine depots

Chemokines presented by the endothelium are critical for integrin-dependent adhesion and transendothelial migration of naive and memory lymphocytes. Here we found that effector lymphocytes of the type 1 helper T cell (TH1 cell) and type 1 cytotoxic T cell (TC1 cell) subtypes expressed adhesive integrins that bypassed chemokine signals and established firm arrests on variably inflamed endothelial barriers. Nevertheless, the transendothelial migration of these lymphocytes strictly depended on signals from guanine nucleotide–binding proteins of the Gi type and was promoted by multiple endothelium-derived inflammatory chemokines, even without outer endothelial surface exposure. Instead, transendothelial migration–promoting endothelial chemokines were stored in vesicles docked on actin fibers beneath the plasma membranes and were locally released within tight lymphocyte-endothelial synapses. Thus, effector T lymphocytes can cross inflamed barriers through contact-guided consumption of intraendothelial chemokines without surface-deposited chemokines or extraendothelial chemokine gradients.

Tumor-Microenvironment Interactions The Fucose-Generating FX Enzyme Controls Adhesive Properties of Colorectal Cancer Cells

Extravasation of tumor cells is a pivotal step in metastasis formation. This step is initiated by an interaction of extravasating tumor cells with endothelial cells. Among the molecules mediating tumor-endothelium interactions are selectins and their fucosylated ligands. In a previous study, we demonstrated that the fucose-generating FX enzyme regulates the expression of selectin ligands by B and T lymphocytes and by head and neck squamous cell carcinoma cells. It was also shown that the FX enzyme regulated important interaction parameters between these cancer cells and endothelial cells. The present study was aimed to determine whether the FX enzyme controls adhesive interactions between colorectal cancer cells and endothelial cells. The results clearly indicate that this is indeed the case. Overexpressing the FX enzyme by the transfer of FX cDNA to low FX-expressing colorectal cancer cells resulted in an increased adhesive capacity of the transfectants to activated endothelial cells and to recombinant E-selectin. Down-regulating FX levels in colorectal cancer cells expressing high levels of endogenous FX by transfection with small-interfering RNA resulted in a down-regulated expression of the selectin ligand sialyl Lewis-a and a decrease in the adhesive capacity of the transfectants to activated endothelial cells and to recombinant E-selectin. These transfection experiments also indicated that manipulating the levels of the FX enzyme affected global cellular fucosylation and altered the interaction of colorectal cancer cells with some extracellular matrix components such as fibronectin. We also found that highly metastatic colorectal cancer variants express higher levels of FX and of sialyl Lewis-a than low metastatic variants originating in the same tumors. These results lead us to hypothesize that the FX enzyme controls the capacity of colorectal cancer to extravasate and form metastasis. If this hypothesis will be confirmed the FX enzyme could become a target molecule for metastasis prevention.

The metastatic microenvironment: brain-residing melanoma metastasis and dormant micrometastasis.

Brain metastasis occurs frequently in melanoma patients with advanced disease whereby the prognosis is dismal. The underlying mechanisms of melanoma brain metastasis development are not well understood. We generated a reproducible melanoma brain metastasis model, consisting of brain‐metastasizing variants and local, subdermal variants that originate from the same melanomas thus sharing a common genetic background. The brain‐metastasizing variants were obtained by intracardiac inoculation. Brain metastasis variants when inoculated subdermally yielded spontaneous brain dormant micrometastasis. Cultured cells from the spontaneous brain micrometastasis grew very well in vitro and generated subdermal tumors after an orthotopic inoculation. Expression analysis assays indicated that the brain metastasis and micrometastasis cells expressed higher levels of angiopoietin‐like 4, prostaglandin‐synthesizing enzyme cyclooxygenase‐2, matrix metalloproteinase‐1 and preferentially expressed antigen in melanoma and lower levels of claudin‐1 and cysteine‐rich protein 61 than the corresponding cutaneous variants. The reproducible models of human melanoma metastasizing experimentally and spontaneously to the brain will facilitate the identification of novel biomarkers and targets for therapy and contribute to the deciphering of mechanisms underlying melanoma metastasis.

Chemokine–chemokine receptor axes in melanoma brain metastasis

Brain metastasis confers an extremely unfavorable prognosis upon melanoma patients. The mechanisms underlying the homing of metastatic melanoma to the brain and survival of metastatic melanoma cells in the brain are unknown. Tumor cells, including melanoma, use chemokine receptor-ligand axes to home to specific organ sites. To identify chemokine receptors that might be involved in brain-targeted melanoma metastasis, we first established a chemokine receptor profile of cultured melanoma cells (3 cell lines of cutaneous melanoma and 5 cell lines of melanoma brain metastasis). The expression of the membrane-bound chemokine CX3CL1 by these lines was also determined. We show that out of 19 receptors tested, cultured melanoma cells express CCR3, CCR4, CXCR3, CXCR7, CX3CR1 and membrane CX3CL1. Utilizing cells from newly created variants of human melanoma xenografts, we found that the expression of CCR4 was significantly higher in one brain metastatic variant compared to its expression in the corresponding local variant. Local and metastatic variants stimulated with the CCR4 ligand, CCL22, showed a differential AKT phosphorylation pattern. These findings may suggest the involvement of CCR4 in the process of brain metastasis in human melanoma, and that CCR4 may be a novel molecular biomarker for the identification of melanoma cells likely to metastasize to the brain.

The involvement of the fractalkine receptor in the transmigration of neuroblastoma cells through bone-marrow endothelial cells

Transendothelial migration (TEM) of tumor cells is a crucial step in metastasis formation. The prevailing paradigm is that the mechanism underlying TEM of tumor cells is similar to that of leukocytes involving adhesion molecules and chemokines. Fractalkine (CX3CL1) is a unique membrane-bound chemokine that functions also as an adhesion molecule. CX3CL1 can be cleaved to a soluble fragment, capable of attracting fractalkine receptor (CX3CR1)-expressing cells. In the present study, we asked if CX3CR1 is involved in the TEM of neuroblastoma cells. We demonstrated that biologically functional CX3CR1 is expressed by several neuroblastoma cell lines. Most importantly, CX3CR1-expressing neuroblastoma cells were stimulated by CX3CL1 to transmigrate through human bone-marrow endothelial cells. A dose dependent phosphorylation of ERK1/2 and AKT was induced in CX3CR1-expressing neuroblastoma cells by soluble CX3CL1. In addition to CX3CR1, neuroblastoma cells also express the CX3CL1 ligand. Membrane CX3CL1 expression was downregulated and the shedding of soluble CX3CL1 was upregulated by PKC activation. Taken together, the results of this study indicate that CX3CR1 plays a functional role in transmigration of neuroblastoma cells through bone-marrow endothelium. These results led us to hypothesize that the CX3CR1-CX3CL1 axis takes part in bone-marrow metastasis of neuroblastoma.

Astrocytes facilitate melanoma brain metastasis via secretion of IL-23.

Melanoma is the leading cause of skin cancer mortality. The major cause of melanoma mortality is metastasis to distant organs, frequently to the brain. The microenvironment plays a critical role in tumourigenesis and metastasis. In order to treat or prevent metastasis, the interactions of disseminated tumour cells with the microenvironment at the metastatic organ have to be elucidated. However, the role of brain stromal cells in facilitating metastatic growth is poorly understood. Astrocytes are glial cells that function in repair and scarring of the brain following injury, in part via mediating neuroinflammation, but the role of astrocytes in melanoma brain metastasis is largely unresolved. Here we show that astrocytes can be reprogrammed by human brain‐metastasizing melanoma cells to express pro‐inflammatory factors, including the cytokine IL‐23, which was highly expressed by metastases‐associated astrocytes in vivo. Moreover, we show that the interactions between astrocytes and melanoma cells are reciprocal: paracrine signalling from astrocytes up‐regulates the secretion of the matrix metalloproteinase MMP2 and enhances the invasiveness of brain‐metastasizing melanoma cells. IL‐23 was sufficient to increase melanoma cell invasion, and neutralizing antibodies to IL‐23 could block this enhanced migration, implying a functional role for astrocyte‐derived IL‐23 in facilitating the progression of melanoma brain metastasis. Knocking down the expression of MMP2 in melanoma cells resulted in inhibition of IL‐23‐induced invasiveness. Thus, our study demonstrates that bidirectional signalling between melanoma cells and astrocytes results in the formation of a pro‐inflammatory milieu in the brain, and in functional enhancement of the metastatic potential of disseminated melanoma cells. Copyright

Regulation of the inflammatory profile of stromal cells in human breast cancer: prominent roles for TNF-α and the NF-κB pathway

IntroductionBreast cancer progression is promoted by stromal cells that populate the tumors, including cancer-associated fibroblasts (CAFs) and mesenchymal stem/stromal cells (MSCs). The activities of CAFs and MSCs in breast cancer are integrated within an intimate inflammatory tumor microenvironment (TME) that includes high levels of tumor necrosis factor α (TNF-α) and interleukin 1β (IL-1β). Here, we identified the impact of TNF-α and IL-1β on the inflammatory phenotype of CAFs and MSCs by determining the expression of inflammatory chemokines that are well-characterized as pro-tumorigenic in breast cancer: CCL2 (MCP-1), CXCL8 (IL-8) and CCL5 (RANTES).MethodsChemokine expression was determined in breast cancer patient-derived CAFs by ELISA and in patient biopsies by immunohistochemistry. Chemokine levels were determined by ELISA in (1) human bone marrow-derived MSCs stimulated by tumor conditioned media (Tumor CM) of breast tumor cells (MDA-MB-231 and MCF-7) at the end of MSC-to-CAF-conversion process; (2) Tumor CM-derived CAFs, patient CAFs and MSCs stimulated by TNF-α (and IL-1β). The roles of AP-1 and NF-κB in chemokine secretion were analyzed by Western blotting and by siRNAs to c-Jun and p65, respectively. Migration of monocytic cells was determined in modified Boyden chambers.ResultsTNF-α (and IL-1β) induced the release of CCL2, CXCL8 and CCL5 by MSCs and CAFs generated by prolonged stimulation of MSCs with Tumor CM of MDA-MB-231 and MCF-7 cells. Patient-derived CAFs expressed CCL2 and CXCL8, and secreted CCL5 following TNF-α (and IL-1β) stimulation. CCL2 was expressed in CAFs residing in proximity to breast tumor cells in biopsies of patients diagnosed with invasive ductal carcinoma. CCL2 release by TNF-α-stimulated MSCs was mediated by TNF-RI and TNF-RII, through the NF-κB but not via the AP-1 pathway. Exposure of MSCs to TNF-α led to potent CCL2-induced migration of monocytic cells, a process that may yield pro-cancerous myeloid infiltrates in breast tumors.ConclusionsOur novel results emphasize the important roles of inflammation-stroma interactions in breast cancer, and suggest that NF-κB may be a potential target for inhibition in tumor-adjacent stromal cells, enabling improved tumor control in inflammation-driven malignancies.