Dependence of fibroblast infiltration in tumor stroma on type IV collagen-initiated integrin signal through induction of platelet-derived growth factor.

Cancer-associated fibroblasts play a crucial role in accelerating tumor progression, but there is a knowledge gap regarding the chemotactic signal activated in a tumor microenvironment. In this study, the expression of type IV collagen was knocked down using a lentiviral-mediated short hairpin RNA strategy. Although there was no obvious effect on cell growth in vitro, silencing the Col4-α1 gene decreased the tumorigenicity of B16F10 in C57BL/6 mice, which was accompanied by a reduction in the infiltration of alpha-smooth muscle actin-positive (α-SMA+) fibroblasts. Silencing the Col4-α1 gene or disrupting integrin engagement by blocking the antibody reduced the expression of platelet-derived growth factor A (PDGF-A), a potent chemotactic factor for fibroblasts. Furthermore, ectopic expression of the autoclustering integrin mutant significantly stimulated PDGF-A expression in murine B16F10 and human U118MG and Huh7 cells. PDGF-A-specific sh-RNA and neutralizing anti-PDGF-A antibody effectively inhibited the transwell migration of fibroblasts. Adding recombinant PDGF-A back to shCol cell-conditioned media restored the fibroblast-attraction ability indicating that PDGF-A is a major chemotactic factor for fibroblasts in the current study model. The integrin-associated PDGF-A production correlated with the activation of Src and ERK. High type IV collagen staining intensity colocalized with elevated PDGF-A expression was observed in tumor tissues obtained from hepatoma and glioma patients. The integrin signal pathway was activated by collagen engagement through Src and ERK, leading to enhanced PDGF-A production, which serves as a key regulator of fibroblast recruitment.

Expression of Th17-related genes in PHA/IL-2-activated human T cells by Fas signaling via caspase-1- and Stat3-dependent pathway.

T helper 17 (Th17) cells, which produce interleukin 17 (IL-17), are involved in the pathogenesis of autoimmune diseases and inflammatory conditions. Th17 cells have been detected in many Fas ligand-positive tumors. This study investigates the expression of Th17-related genes in PHA/IL-2-activated human T cells upon Fas ligation. Activated T cells transiently express RORγt, IL-17A, and IL-17F. A subsequent Fas receptor stimulation or contact with FasL-expressing glioma cells significantly prolongs the induction of RORγt and Th17-related cytokines. Treatments with inhibitors of caspase-1 and Stat3 reduce the Fas-signal-associated induction of RORγt, IL-17A, and IL-17F, as well as the phosphorylation of Stat3. Although the ligation of Fas results in caspase-8 cleavage and ERK1/2 phosphorylation, inhibitors for caspase-8 and MEK have no effect on the expressions of RORγt, IL-17A, and IL-17F. The results suggest that the Fas signal favors the Th17-phenotypic features of human T cells through the caspase-1/Stat3 signaling pathway.

Fas ligand enhances malignant behavior of tumor cells through interaction with Met, hepatocyte growth factor receptor, in lipid rafts.

Many late-stage cancer cells express Fas ligand (FasL) and show high malignancy with metastatic potential. We report here a novel signaling mechanism for FasL that hijacks the Met signal pathway to promote tumor metastasis. FasL-expressing human tumor cells express a significant amount of phosphorylated Met. The down-regulation of FasL in these cells led to decreased Met activity and reduced cell motility. Ectopic expression of human FasL in NIH3T3 cells significantly stimulated their migration and invasion. The inhibition of Met and Stat3 activities reverted the FasL-associated phenotype. Notably, FasL variants activated the Met pathway, even though most of their intracellular domain or Fas binding sites were deleted. FasL interacted with Met through the FasL(105-130) extracellular region in lipid rafts, which consequently led to Met activation. Knocking down Met gene expression by RNAi technology reverted the FasL-associated motility to basal levels. Furthermore, treatment with synthetic peptides corresponding to FasL(117-126) significantly reduced the FasL/Met interaction, Met phosphorylation, and cell motility of FasL(+) transfectants and tumor cells. Finally, the transfectants of truncated FasL showed strong anchorage-independent growth and lung metastasis potential in null mice. Collectively, our results establish the FasL-Met-Stat3 signaling pathway and explains the metastatic phenotype of FasL-expressing tumors.

Extracellular matrix of glioblastoma inhibits polarization and transmigration of T cells: the role of tenascin-C in immune suppression.

Dense accumulations of T cells are often found in peritumoral areas, which reduce the efficiency of contact-dependent lysis of tumor cells. We demonstrate in this study that the extracellular matrix (ECM) produced by tumors can directly regulate T cell migration. The transmigration rate of several T cells including peripheral blood primary T cell, Jurkat, and Molt-4 measured for glioma cells or glioma ECM was consistently low. Jurkat cells showed reduced amoeba-like shape formation and delayed ERK activation when they were in contact with monolayers or ECM of glioma cells as compared with those in contact with HepG2 and MCF-7 cells. Phospho-ERK was located at the leading edge of migrating Jurkat cells. Glioma cells, but not MCF-7 and HepG2 cells, expressed tenascin-C. Knocking down the tenascin-C gene using the short hairpin RNA strategy converted glioma cells to a transmigration-permissive phenotype for Jurkat cells regarding ERK activation, transmigration, and amoeba-like shape formation. In addition, exogenous tenascin-C protein reduced the amoeba-like shape formation and transmigration of Jurkat cells through MCF-7 and HepG2 cell monolayers. A high level of tenascin-C was visualized immunohistochemically in glioma tumor tissues. CD3(+) T cells were detected in the boundary tumor area and stained strongly positive for tenascin-C. In summary, glioma cells can actively paralyze T cell migration by the expression of tenascin-C, representing a novel immune suppressive mechanism achieved through tumor ECM.

Zap70 controls the interaction of talin with integrin to regulate the chemotactic directionality of T-cell migration.

Aberrant lymphocyte infiltration is crucial for many disorders such as tumor immune escape and autoimmunity. In this study, we have investigated T-cell migration in a three-dimensional collagen matrix containing tumor spheroids and by using micro-Slide chemotaxis and found that Zap70 regulates directionality during cell chemotaxis. Jurkat cells actively migrated toward SDF-1, nutrition, and spheroids of MCF-7 breast carcinoma cells embedded in collagen matrix. Inhibition of Zap70 activity impaired transmigration and mu-Slide chemotaxis but not the random movement of T cells in the collagen/fibronectin matrix. P116 cells, a Zap70 deficient variant of Jurkat, showed active random movement but failed to migrate against chemoattractants. P116 cells exhibited a reduced polarization of cell morphology, showing less lamellipodia formation accompanied with a fast pseudopod turnover rate. Instead of direct interacting with F-actin, Zap70 formed a complex with talin which is an integrin scaffold for F-actin. SDF-1 enhanced Zap70 phosphorylation and also stimulated binding of talin and beta1 integrin activation. P116 cells showed reduced complex of talin and beta1 integrin in parallel with impaired integrin activation. Collectively, Zap70 modulates integrin activation by interacting with talin, which contributes to directionality of T-cell migration, severing as a potential target for anti-inflammation therapy.