Abstract 5073: Role of TIMP-1 glycosylation in lung carcinoma

Abstract

Abstract Tissue inhibitors of matrix metalloproteinases (TIMPs) were initially identified as endogenous physiological inhibitors of MMPs. However, over recent years novel functions have been attributed to TIMPs, especially TIMP-1. Amongst its complex and sometimes paradoxical functions,TIMP-1's role in inhibition of apoptosis and cell proliferation is well-documented. Our initial studies have shown that overexpressing TIMP-1 in a lung adenocarcinoma cells results in larger and more aggressive tumors when implanted in the mouse brain. This has also been corroborated in vitro, by demonstrating increased invasion and anchorage-independent growth as well as decreased apoptosis. In the present study, we have purified human TIMP-1 in order to examine its direct molecular activity in tumorigenesis. Non-small cell lung carcinoma cell line NCI-H460 was utilized because it normally produces high levels of TIMP-1 as confirmed by western blot analysis and ELISA. We found that TIMP-1 was secreted directly into the culture media and we could not identify presence of TIMP-1 in the exosome fraction. TIMP-1 was purified from serum-free conditioned media of NCI-H460 by ammonium precipitation, ion exchange chromatography followed by gel filtration, with resultant purity of greater than 90%. The addition of purified exogenous TIMP-1 to NCI-H2009, a low TIMP-1 producing NSCLC cell line, resulted in increased cell proliferation and reduced cytotoxicity from environmental stressors such as air exposure, H2O2, staurosporine and cycloheximide as determined by LDH assay. Flow cytometric analysis following addition of purified TIMP-1 to H2009 cells demonstrated reduced apoptosis. A549, a lung adenocarcinoma cell line that produces levels of TIMP-1, higher than H2009, was found to be relatively insensitive to exogenously added purified TIMP-1. This may be interpreted as indicating concentration dependent activity of TIMP-1. Deglycosylated TIMP-1 (by PNGase F treatment) as well as recombinant human TIMP-1 (from E.coli) did not exhibit any protective functions indicating that glycosylation is critical for the cell proliferative and anti-apoptotic functions of TIMP-1. To investigate whether the protective function of TIMP-1 is MMP-dependent, we constructed a T2G mutant of TIMP-1 where changing the second amino acid threonine to glycine resulted in impaired MMP-inhibition function. The T2G mutant was also as protective of cell survival as TIMP-1, supporting the concept that this is an MMP-independent function. Collectively our results indicate that TIMP-1 glycosylation is critical for exogenous TIMP-1 to induce cell proliferation and cell survival in an MMP-independent manner, most likely through receptors that recognize glycosylated TIMP-1. This study was supported in part by a Distinguished Cancer Scientist Award to AMR from the Georgia Research Alliance. Citation Format: Byung R. Lee, Ammar Kutiyanawalla, Sampa Ghosal-Gupta, Amyn M. Rojiani, Mumtaz V. Rojiani. Role of TIMP-1 glycosylation in lung carcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5073. doi:10.1158/1538-7445.AM2015-5073