Characterization of the Relationship between the IGF-1R Tyrosine Kinase Pathway and the Notch Signaling Pathway with Human Aspartyl (Asparaginyl) ß-Hydroxylase (HAAH) in Lung Cancer Cells

Abstract

Human Aspartyl-Asparaginyl β-hydroxylase (HAAH) is highly expressed in many cancer types including hepatocellular carcinoma, cholangiocarcinoma, breast, colon, intestinal, lung, pancreatic, and neuronal cancers. It plays an important role in the cellular transformation and invasive growth of malignant tumors. HAAH is specifically a cell surface antigen found on cancer cells, therefore it is easily accessible. This characteristic makes it a promising drug target for immunotherapy for various cancers because specific cells can be targeted unlike chemotherapy or radiation treatments where all cells are affected. It has been suggested that the IGF-1 pathway is an upstream mediator of HAAH and that HAAH is an upstream mediator of the Notch signaling pathway. In this study effects of the tyrosine kinase inhibitor PPP, or the y-secretase inhibitor DBZ on HAAH expression in the H460 lung cancer cell line were investigated. In addition, the effects of a novel drug candidate, anti-HAAH antibody PAN-622, were studied on the IGF-1R and Notch signaling pathways. Drug synergies between PPP and PAN-622 as well as DBZ and PAN-622 were also investigated. The H460 lung cancer cell line was treated with PPP, PAN-622, and DBZ, and tumor cell proliferation was evaluated. All three compounds were successful in blocking proliferation of H460 cells. IGF-1R, HAAH, and Notch protein expression were evaluated using western blot analysis. HAAH and Notch mRNA expression was measured by quantitative real-time PCR. These studies demonstrated that the IGF-1R pathway is likely inactive in untreated H460 cells, but can be up-regulated through inhibition of the Notch pathway. HAAH is an activator of the Notch pathway and its inhibition by the anti-HAAH antibody, PAN-622 results in an inhibition of the Notch pathway. Our findings have increased the understanding of the relationship between the IGF-1R and Notch pathways with HAAH and may be valuable in understanding the proliferation and survival of H460 cells in vitro. In summary, our results show that PPP, PAN-622, and DBZ are potent inhibitors of malignant cell growth in vitro. PAN-622 is currently in development as a novel therapeutic agent for cancer. These studies further the understanding of the mechanism of action of PAN-622 and the relationship of HAAH to the IGF-1R and Notch pathways.