Abstract 1894: Gremlin-1 is an important regulator of invasiveness in malignant mesothelioma
Authors: Yin Miao , Tissari Mira , Joensuu Emmi I. , Tamminen Jenni , Ylivinkka Irene , Rönty Mikko , Lehti Kaisa , Hyytiäinen Marko , Myllärniemi Marjukka , Koli Katri 
Published: July 2017

Abstract
The purpose of this study was to clarify the role of BMP inhibitor gremlin 1 in invasion and migration in mesothelioma.
Primary mesothelioma cells isolated from patient pleural fluid as well as mesothelioma cell lines were used for in vitro studies. Cells were transfected with siRNAs or transduced with lentiviral expression vectors. Invasive growth was analyzed in 3D Matrigel or collagen I matrices. mRNA expressions were analyzed using a commercial PCR array and quantitative RT-PCR. Migration assays were performed using scratch wound assay or transwell migration assay with fibronectin or collagen coating. TGF-β and BMP signaling activity was measured with luciferase reporter assays. For in vivo mouse xenograft experiment cells were additionally transduced to express a luciferase marker. Subcutaneous cell injection with Matrigel matrix was performed in the flank of nude mice.
Malignant mesothelioma is an aggressive cancer that develops from mesothelial cells, most often in the pleural lining of the lung. We have previously shown that the BMP inhibitor protein gremlin 1 is highly expressed in mesothelioma and induces a mesenchymal and chemoresistant phenotype in mesothelioma cells. Since mesothelioma tumors are locally very invasive and has poor prognosis, we analyzed the role of gremlin 1 in mesothelioma cell migration and invasive growth.
We found that mesothelioma cells expressing gremlin 1 showed invasive sprouting when tumor cell spheroids were imbedded into 3D collagen matrix. Silencing of gremlin 1 expression significantly reduced invasive growth. In addition, cells overexpressing gremlin 1 gained more invasive phenotype. This was associated with increased mRNA expression levels of Slug and matrix metalloproteinases (MMP) as well as reduced expression of E-cadherin. The cells were more migratory and exhibited increased expression of certain integrins, especially the αv subunit. Gremlin 1 induced invasive growth was dependent on MMP activity and associated with increased TGF-β activity. Intrapleural injection of mesothelioma cells overexpressing gremlin 1 isolated from a patient with epithelioid mesothelioma, produced tumors in 2/4 mice over 5 months after injection. Mice with cells transduced with vector only did not develop tumors (0/4). When cells were injected subcutaneously together with Matrigel gremlin 1 overexpressing tumors appeared more slowly, but exhibited comparable luciferase signal 2.5 months after injection. However, gremlin 1 tumors showed more vascularization and in contrast to control tumors some also developed metastases (2/6 mice).
As conclusion, we identified gremlin 1 as an important regulator of mesothelioma invasive growth and chemoresistance. Blocking gremlin 1 function may overcome drug resistance and reduce invasive behavior of mesothelioma.
Citation Format: Miao Yin, Mira Tissari, Emmi I. Joensuu, Jenni Tamminen, Irene Ylivinkka, Mikko Rönty, Kaisa Lehti, Marko Hyytiäinen, Marjukka Myllärniemi, Katri Koli. Gremlin-1 is an important regulator of invasiveness in malignant mesothelioma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1894. doi:10.1158/1538-7445.AM2017-1894