Inhibition of Cytokine Receptor gp130 Signaling in Breast Cancer Cells Markedly Suppresses Secretion of Tissue Factor-Expressing Tumor Microparticles.
Authors: de Idiaquez Diego A. , Branam Dannielle E. , Lamb Larry , Chen Dongquan , Teng Lihong , Selander Katri S. , Harris Kevin W. 
Published: October 2019

Abstract
gp130 is the common signaling receptor subunit for the IL-6 family of cytokines. We have previously shown that expression of a dominant negative (DN) gp130 inhibitory protein in MDA-MB-231 breast cancer cells decreased tumor invasion and angiogenesis in an orthotopic animal model (Cancer Research64:6924, 2004). In order to better understand the molecular mechanisms of this decreased malignancy, we determined global MDA-MB-231 gene expression in the presence of DN gp130. RNA was obtained from multiple independent single cell clones of MDA-MB-231 cells expressing either DN gp130 (n=5) or vector-only (n=5). Each clone was studied using Affymetrix U133A chips containing >22,000 genes. We identified 237 genes that were up- or down-regulated in the presence of DN gp130 at least 2-fold with p<0.05. Genes of interest were further studied by real-time PCR, western blot, and flow cytometry. We focused on extracellular proteins involved in proteolytic pathways. Six coagulation regulatory proteins were found to be differentially regulated by DN gp130: tissue factor (decreased), PAI-1 (decreased), alpha-2 antiplasmin (decreased), TFPI (increased), thrombomodulin (increased), and MMP-14 (increased). Thus, inhibition of gp130 signaling results in an anticoagulant phenotype in these cells. Tissue factor regulation was further studied. Cell surface tissue factor protein was down-regulated 10-fold in MDA-MB-231 DN gp130 cells compared to MDA-MB-231 control cells. Tumor microparticles (MP) were isolated from conditioned media by differential centrifugation. MDA-MB-231 MP contained uPA and MMP-9, as previously reported for MP from other tumors. Tissue factor was strongly expressed in control MP and nearly absent in MP from DN gp130 cells. Flow cytometry of tumor MP was used to quantitate tissue factor-expressing MP. There was a 50-fold decrease in double-labeled annexin V positive/tissue factor positive MP obtained from DN gp130 cells compared to control cells. In addition, the absolute number of tumor MP was decreased by 50% in the presence of DN gp130. In order to determine the mechanism of this regulation of breast cancer MP by gp130, the above list of 237 differentially expressed genes was re-examined. Proteins known to be enriched in MP- CD55, CD59, and three members of the tetraspanin family (including CD9) were significantly decreased in the presence of DN gp130. The expression of three proteins that are involved in the cellular production of MP were also significantly affected by DN gp130: Ca2+-dependent activator of protein for secretion 2, Rab11 interacting protein, and syntaxin 3. Our data supports a hypothesis in which signaling via IL-6 family cytokines induces a procoagulant gene expression program in breast cancer cells. As part of this program, the production of tumor MP are increased via gene regulation of cellular proteins that control MP processing. Additionally, the procoagulant properties of the tumor MP are increased due to an increase in tissue factor content. It has been reported that both activation of coagulation and production of tumor MP are involved in solid tumor invasion and metastasis. Inflammatory signaling by IL-6 family members in the tumor milieu could be important in this process. This hypothesis has clinical significance since potential pharmacologic approaches to specific gp130 inhibition are available.