Abstract 3838: KRAS dependency and thrombospondin-1 levels are inversely associated with TIMP1

Abstract Beyond its role as an inhibitor of matrix metalloproteinases, tissue inhibitor of metalloproteinase 1 (TIMP1) has been associated with poor prognosis in many cancer types including non-small cell lung cancer (NSCLC), and substantial evidence suggests it plays a role in tumor progression. The most frequent mutation identified in NSCLC is KRAS. Recently, we described an inverse relationship between expression of KRAS and TIMP1 in NSCLC cell lines. These cells fall into two distinct categories, KRAS dependent and KRAS independent, depending upon their need for sustained expression of KRAS or if the cells have become independent of KRAS expression. We have shown that KRAS-dependent cells with high levels of KRAS, exhibit lower levels of TIMP1. Inversely, KRAS-independent cells with low levels of KRAS show high levels of TIMP1. We have demonstrated that modulation of TIMP1 using knockdown clones or TIMP1 overexpressing cells, alters KRAS dependency features. In the present study, based on preliminary observations on TIMP1 and angiogenesis, we postulate a similar inverse relationship between KRAS-dependent cells expressing high levels of Thrombospondin-1 (TSP1), whereas the independent cells expressing low levels of TSP1. Overexpression of TIMP1 led to a decrease in thrombospondin (TSP1) levels, while TIMP1 knockdown resulted in an upregulation of TSP1. Furthermore, serum-free conditioned media (SFCM) from TIMP1 overexpressing (OE) cells significantly enhanced capillary networking tube formation of HUVEC cells in Matrigel, indicating increased angiogenic potential. By contrast, SFCM from TIMP1 knockdown (KD) clones reduced capillary networking and tube formation compared to parental cells. Additionally, CDH5 (VE-cadherin), a marker of endothelial cells, was upregulated in TIMP1-OE cells and downregulated in the TIMP1-KD clone relative to parental cells. These findings suggest that TIMP1 modulates endothelial cell behavior, likely through regulation of TSP1 and CDH5. In KRAS dependent cells, we also observed the upregulation of VEGFα secreted in the SFCM of TIMP1-OE cells. Furthermore, we investigated the clinical significance of TSP1 and TIMP1 in the TCGA LUAD dataset. Immune deconvolution analysis revealed that TIMP1 expression is associated with a higher infiltration of immune cells, along with a significant enrichment of exhausted immune phenotypes compared to TSP1. These findings highlight the dichotomy between TIMP1 and TSP1, demonstrating both quantitative and qualitative impacts on immune infiltration, with crucial therapeutic implications. Future studies will be directed at investigating the underlying mechanisms linking KRAS dependency and TIMP1 in angiogenesis along with the complex interplay of TSP1, which could provide novel insights into therapeutic strategies targeting TIMP1 to impair angiogenesis and tumor progression in NSCLC. Citation Format: Ilamathi M. Thirusenthilarasan, Bilal Siddiqui, Pankaj Ahluwalia, Ravindra Kolhe, Amyn M. Rojiani, Mumtaz V. Rojiani. KRAS dependency and thrombospondin-1 levels are inversely associated with TIMP1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3838.