Abstract 3368: TIMP-1 and angiogenesis: A role for CDH5 and eNOS

Abstract

Abstract Tissue inhibitors of metalloproteinases (TIMP) have emerged as diverse molecules with novel roles in apoptosis, angiogenesis and metastasis. TIMP-1, a well-documented inhibitor of apoptosis, has been shown to inhibit or promote angiogenesis. In recent years, a strong association has also been demonstrated between high levels of TIMP-1 and poor prognosis in many cancers. It has been recognized as a multifunctional protein affecting cell growth and angiogenesis. Conflicting studies have shown it to function either as a negative or a positive regulator of angiogenesis. In earlier studies, we have documented that lung adenocarcinoma cell line H2009, when transfected to overexpress TIMP-1 and injected into nude mice resulted in larger, more aggressive tumors with increased microvessel density, which was supported by in vitro angiogenesis assays whereby enhanced capillary network formation was seen. We have also shown that TIMP-1 expression levels can be correlated with KRAS independency in non-small-cell lung carcinoma (NSCLC) cell lines harboring KRAS mutations. The present study was undertaken to address the role of TIMP-1 in angiogenesis in the context of KRAS in NSCLC cell lines. KRAS dependent cell line H2009 and its TIMP-1 overexpressing clone HB1 and a KRAS independent cell line A549 and its TIMP-1 knockdown (KD) clone SH3 were examined by angiogenesis specific PCR array. Comparison of the angiogenesis associated profiles of these cell lines identified a marked increase in vascular endothelial cadherin CDH5 in TIMP-1 over-expressing clone HB1. KRAS independent A549 cells expressed high level of CDH5, however its TIMP-1 KD clone exhibited a marked decrease in CDH5 level. A similar profile was identified for endothelial nitric oxide synthase (eNOS/NOS3). CDH5 is an indispensable element of normal vascular development and maintenance, and any perturbations of normal levels will impact vessel sprouting and growth. eNOS is primarily active through production of nitric oxide, maintaining vascular tone as well as expressing anti-proliferative and antithrombotic properties. We confirmed our earlier observation that serum free conditioned media (SFCM) from TIMP-1 overexpressing HBI cells caused increased and more complex network formation. SFCM from A549 TIMP-1 KD SH3 clone showed a marked inhibition of endothelial network formation compared to SFCM from A549 cells. To confirm that TIMP-1 was responsible for the changes observed we purified secretory TIMP-1 by combined fast protein liquid chromatography and gel filtration from another high TIMP-1 producing NSCLC cell line (H460) and also observed more capillary network formation in human umbilical vein endothelial cells. Our studies suggest that TIMP-1 modulates angiogenesis by impacting CDH5 and eNOS positively. These results define an important function of TIMP-1 in angiogenesis and provide additional therapeutic targets for managing NSCLC. Citation Format: Sampa Ghoshal-Gupta, Ian A. Coe, Ammar Kutiyanawalla, Byung R. Lee, Amyn M. Rojiani, Mumtaz V. Rojiani. TIMP-1 and angiogenesis: A role for CDH5 and eNOS. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3368.