Abstract 4725: Elevated co-expression of CCL2 and CX3CL1 is associated with apoptosis and good prognosis in soft tissue sarcoma patients
Authors: Nolte Elke , Kehlen Astrid , Greither Thomas , Wach Sven , Kappler Matthias , Bache Matthias , Holzhausen Hans-Jürgen , Lautenschläger Christine , Göbel Steffen , Würl Peter , Immel Uta-Dorothee , Agaimy Abbas , Wullich Bernd , Taubert Helge 
Published: May 2015

Abstract
Background: Chemokines represent a group of chemotactic cytokines, which are involved in both negative and positive regulation of inflammatory processes, angiogenesis, cancer cell proliferation and chemoattraciton of tumor cells to metastatic sites.
Material and methods: mRNA expression of CCL2 and CX3CL1 was determined in 126 patients with soft-tissue sarcoma using quantitative real-time PCR. Patient groups displaying low or high expression in their tumors were separated by the median expression of the markers. Kaplan-Meier analysis and multivariate Cox's regression analysis were performed to assess the correlation between chemokine expression levels and disease-specific survival in STS. To gain insight into the cell physiological consequences of inhibition of CCL2 and CX3CL1, siRNA-mediated knockdown was used. The effect on proliferation was analyzed by a BrdU Elisa. The effect on inhibition of apoptosis was analyzed by adding etoposide after transfection with the respective siRNA, followed by FACS analysis.
Results: The mRNA levels of CCL2, CCL7, and CX3CL1 were analyzed in tumor tissues from 126 STS patients using qPCR. Low mRNA expression of CCL2 and CX3CL1 was significantly correlated with a worse prognosis (RR = 1.98; p = 0.019 and RR = 2.10; p = 0.014; multivariate Cox's regression analysis). A combined low expression of CCL2 and CX3CL1 was associated with a significantly increased risk of tumor-related death compared to patients with high expression levels of both chemokines (RR = 3.08; p = 0.003). A gender-specific multivariate analysis revealed that female STS patients with low CX3CL1 mRNA expression had a 3.46-fold increased risk of death (p = 0.004). Low expression of both CCL2 and CX3CL1 mRNAs resulted in an additive 5.37-fold increased risk of tumor-related death (p = 0.003) compared to those with high expression of both parameters in female patients.
Treatment with siRNA against CX3CL1 revealed a significantly increased cell proliferation after 24h but this effect disappeared after 48h and 72 h, respectively. To test the effect of siRNA treatment against CCL2 or CX3CL1 on induction of apoptosis, after transfection with the respective siRNA, etoposide was added to induce apoptosis. A significant decrease in apoptosis induction could be detected after combined treatment with etoposide and siRNA against CCL2 (p = 0.0047) or against CX3CL1 (p = 0.0043) compared with the etoposide and non-targeting siRNAs treated control cells.
Conclusion: Our findings demonstrate that mRNA expression of CCL2 and CX3CL1 was significantly correlated with prognosis. Interestingly, there was a gender-specific impact of CCL2 on disease-specific survival. A significant decrease in apoptosis induction could be detected when cells were transfected with siRNA targeting CCL2 or CX3CL1 compared to the cells transfected with non-targeting siRNA.
Citation Format: Elke Nolte, Astrid Kehlen, Thomas Greither, Sven Wach, Matthias Kappler, Matthias Bache, Hans-Jürgen Holzhausen, Christine Lautenschläger, Steffen Göbel, Peter Würl, Uta-Dorothee Immel, Abbas Agaimy, Bernd Wullich, Helge Taubert. Elevated co-expression of CCL2 and CX3CL1 is associated with apoptosis and good prognosis in soft tissue sarcoma patients. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4725. doi:10.1158/1538-7445.AM2014-4725