CCL2 Is a Vascular Permeability Factor Inducing CCR2-Dependent Endothelial Retraction during Lung Metastasis.

Increased levels of the chemokine CCL2 in cancer patients are associated with poor prognosis. Experimental evidence suggests that CCL2 correlates with inflammatory monocyte recruitment and induction of vascular activation, but the functionality remains open. Here, we show that endothelial Ccr2 facilitates pulmonary metastasis using an endothelial-specific Ccr2-deficient mouse model (Ccr2ecKO). Similar levels of circulating monocytes and equal leukocyte recruitment to metastatic lesions of Ccr2ecKO and Ccr2fl/fl littermates were observed. The absence of endothelial Ccr2 strongly reduced pulmonary metastasis, while the primary tumor growth was unaffected. Despite a comparable cytokine milieu in Ccr2ecKO and Ccr2fl/fl littermates the absence of vascular permeability induction was observed only in Ccr2ecKO mice. CCL2 stimulation of pulmonary endothelial cells resulted in increased phosphorylation of MLC2, endothelial cell retraction, and vascular leakiness that was blocked by an addition of a CCR2 inhibitor. These data demonstrate that endothelial CCR2 expression is required for tumor cell extravasation and pulmonary metastasis. IMPLICATIONS: The findings provide mechanistic insight into how CCL2-CCR2 signaling in endothelial cells promotes their activation through myosin light chain phosphorylation, resulting in endothelial retraction and enhanced tumor cell migration and metastasis.

Stromal Expression of Activated Leukocyte Cell Adhesion Molecule Promotes Lung Tumor Growth and Metastasis.

Activated leukocyte cell adhesion molecule (ALCAM) is expressed on various cell types, including leukocytes, endothelial cells, and certain tumor cells. Although ALCAM expression on tumor cells has been linked to tumor invasion and metastatic spread, the contribution of ALCAM expressed in cells forming the tumor stroma to cancer progression has not been investigated. In this study, ALCAM-deficient (ALCAM-/-) mice were used to evaluate the role of ALCAM in lung tumor growth and metastasis. ALCAM-/- mice displayed an altered blood vascular network in the lung and the diaphragm, indicative of an angiogenetic defect. The absence of ALCAM expression in cells forming the stromal tumor microenvironment profoundly affected lung tumor growth in three different i.v. metastasis models. In the case of Lewis lung carcinoma (LLC), an additional defect in tumor cell homing to the lungs and a resulting reduction in the number of lung tumor nodules were observed. Similarly, when LLC cells were implanted subcutaneously for the study of spontaneous tumor cell metastasis, the rate of LLC metastasis to the lungs was profoundly reduced in ALCAM-/- mice. Taken together, our work demonstrates for the first time the in vivo contribution of ALCAM to angiogenesis and reveals a novel role of stromally expressed ALCAM in supporting tumor growth and metastatic spread.

Monocyte Induction of E-Selectin-Mediated Endothelial Activation Releases VE-Cadherin Junctions to Promote Tumor Cell Extravasation in the Metastasis Cascade.

Tumor cells interact with blood constituents and these interactions promote metastasis. Selectins are vascular receptors facilitating interactions of tumor cells with platelets, leukocytes, and endothelium, but the role of endothelial E-selectin remains unclear. Here we show that E-selectin is a major receptor for monocyte recruitment to tumor cell-activated endothelium. Experimental and spontaneous lung metastasis using murine tumor cells, without E-selectin ligands, were attenuated in E-selectin-deficient mice. Tumor cell-derived CCL2 promoted endothelial activation, resulting in enhanced endothelial E-selectin expression. The recruitment of inflammatory monocytes to metastasizing tumor cells was dependent on the local endothelial activation and the presence of E-selectin. Monocytes promoted transendothelial migration of tumor cells through the induction of E-selectin-dependent endothelial retractions and a subsequent modulation of tight junctions through dephosphorylation of VE-cadherin. Thus, endothelial E-selectin shapes the tumor microenvironment through the recruitment, adhesion, and activation of monocytes that facilitate tumor cell extravasation and thereby metastasis. These findings provide evidence that endothelial E-selectin is a novel factor contributing to endothelial retraction required for efficient lung metastasis. Cancer Res; 76(18); 5302-12. ©2016 AACR.

Gene-gene interaction and RNA splicing profiles of MAP2K4 gene in rheumatoid arthritis.

We performed gene-gene interaction analysis, with HLA-DRB1 shared epitope (SE) alleles for 195 SNPs within immunologically important MAP2K, MAP3K and MAP4K gene families, in 2010 rheumatoid arthritis (RA) patients and 2280 healthy controls. We found a significant statistical interaction for rs10468473 with SE alleles in autoantibody-positive RA. Individuals heterozygous for rs10468473 demonstrated higher expression of total MAP2K4 mRNA in blood, compared to A-allele homozygous. We discovered a novel, putatively translated, "cassette exon" RNA splice form of MAP2K4, differentially expressed in peripheral blood mononuclear cells from 88 RA cases and controls. Within the group of RA patients, we observed a correlation of MAP2K4 isoform expression with carried SE alleles, autoantibody, and rheumatoid factor profiles. TNF-dependent modulation of isoform expression pattern was detected in the Jurkat cell line. Our data suggest a genetic interaction between MAP2K4 and HLA-DRB1, and the importance of rs10468473 and MAP2K4 splice variants in the development of autoantibody-positive RA.

Metastatic growth progression caused by PSGL-1-mediated recruitment of monocytes to metastatic sites.

Tumor cell-derived selectin ligands mediate contact to the endothelium, platelets, and leukocytes through binding to selectins that facilitates metastasis. Here, we describe the mechanism of how endogenous (non-tumor derived) selectin ligands contribute to metastasis using α(1,3)fucosyltransferase 7 (Fuc-TVII(-/-))-deficient mice. Experimental metastasis of MC-38GFP and Lewis lung (3LL) carcinoma cells was attenuated in Fuc-TVII(-/-) mice, which express minimal amount of selectin ligands. We show that metastasis is dependent on selectin ligands carried on hematopoietic cells. P-selectin glycoprotein ligand-1 (PSGL-1) was identified as the major ligand facilitating monocyte accumulation at metastatic sites. Reduced recruitment of monocytes to metastasizing tumor cells in Fuc-TVII(-/-) mice correlated with attenuated metastasis. Adoptive transfer of Fuc-T7(+) monocytes rescued metastasis in Fuc-TVII(-/-) mice, indicating that selectin ligand-dependent recruitment of monocytes is required for cancer progression. Cytokine analysis in metastatic lungs revealed high expression of CCL2 in C57BL/6 mice that was significantly lower in Fuc-TVII(-/-) mice. The absence of monocyte recruitment in Fuc-TVII(-/-) mice correlated with increased apoptosis of tumor cells. Thus, the recruitment of monocytes to metastasizing tumor cells is facilitated by endogenous selectin ligands on monocytes that enable efficient tumor cell survival, extravasation, and metastasis.

An activated JAK/STAT3 pathway and CD45 expression are associated with sensitivity to Hsp90 inhibitors in multiple myeloma.

The molecular chaperone Hsp90 is required to maintain the activity of many signaling proteins, including members of the JAK/STAT and the PI3K pathways. Inhibitors of Hsp90 (Hsp90-Is) demonstrated varying activity against multiple myeloma (MM) in clinical trials. We aimed to determine which signaling pathways that account for the differential sensitivity to the Hsp90-I 17DMAG on a panel of MM cell lines and freshly obtained MM cells. Three CD45(+) cell lines with an activated JAK/STAT3 pathway were sensitive to 17DMAG and underwent prominent apoptosis upon treatment, while the majority of CD45(-) cell lines, that were dependent on the activated PI3K pathway, were more resistant to the drug. Culturing the most resistant cell line, LP1, in the presence of IL-6 resulted in up-regulation of CD45 and pSTAT3, and sensitized to 17DMAG-induced apoptosis, primarily in the induced CD45(+) sub-population of cells. The high CD45 expressers among primary myeloma cells also expressed significantly higher levels of pSTAT3, as compared to the low CD45 expressers. Ex vivo treatment of primary myeloma cells with 17DMAG resulted in a stronger caspase3 activation in tumor samples with the prevalence of high CD45 expressers. STAT3 activity was efficiently inhibited by Hsp90-Is in both cell lines and primary cells suggesting an importance of STAT3 inactivation for the pro-apoptotic effects of HSP90-Is. Indeed, over-expression of STAT3C, a variant with an increased DNA binding activity, in U266 cells protected them from 17DMAG-induced cell death. The down-regulation of the STAT3 target gene Mcl-1 at both the mRNA and protein levels following 17DMAG treatment was significantly attenuated in STAT3C-expressing cells, and transient over-expression of Mcl-1 protected U266 cells from 17DMAG-induced cell death. The finding that CD45(+) MM cells with an IL-6-activated JAK/STAT3 pathway are particularly sensitive to Hsp90-Is as compared to the low CD45 expressers may provide a rational basis for selection of MM patients amenable to Hsp90-I treatment.