Angiogenic profile associated with KRAS dependency upon modulation of TIMP1

Non-small cell lung carcinoma (NSCLC) remains a leading cause of cancer-related deaths, with KRAS mutations conferring oncogene addiction and chemoresistance to therapy. Our previous work demonstrated that Tissue Inhibitor of Metalloproteinase-1 (TIMP-1) inversely correlates with KRAS dependency and promotes epithelial-mesenchymal transition (EMT). Given the role of TIMP-1 in tumor progression, we investigated how TIMP-1 modulation impacts the expression of angiogenic mediators in NSCLC cells.KRAS-dependent (H441) and KRAS-independent (H460) NSCLC cell lines were modulated to overexpress (OE) or knock down (KD) TIMP-1, respectively. Serum free conditioned media from these cells was analyzed using angiogenesis proteome profiler array (R&D systems). We found that the levels of pro-angiogenic factors VEGF1 and CXCL8 increased upon TIMP1 overexpression and decreased upon knocking down TIMP1. On the other hand, Thrombospondin-1, an angiogenic inhibitor was downregulated when TIMP1 was over-expressed and upregulated when TIMP1 was knocked down. Additionally, TIMP-1 overexpression in H441 cells increased Angiogenin and decreased Endostatin, Angiostatin, Serpin E1, and Platelet Factor 4 (PF4). Conversely, TIMP-1 knockdown in H460 cells showed the opposite trend, indicating that TIMP1 is pro-angiogenic. RNA-seq data analysis across five NSCLC lines revealed a reciprocal TIMP1:THBS1 expression pattern with strong CXCL8 induction under TIMP1 overexpression, most pronounced in H441. The TIMP1-THBS1 inverse relationship supports a CXCL8-dominant angiogenic axis.As studies have shown that KRAS dependency is associated with upregulation of c-Myc and ERK, leading to a decrease in TSP-1 levels, we assessed c-Myc levels in the lysates using Western blot. We found that the levels of c-MYC were high in KRAS-dependent parental cells and further increased upon TIMP-1 overexpression. In KRAS-independent cells however, TIMP-1 knockdown did not significantly alter c-MYC levels. Ongoing studies continue to evaluate the effects of TIMP-1 modulation on angiogenic responses under normoxic and hypoxic conditions.Our findings highlight TIMP-1 as a potential regulator of tumor angiogenesis in NSCLC in a KRAS dependency specific manner, incorporating a CXCL8-dominant axis.