3' RNA modification enzymes regulate tumor angiogenesis in non-small lung cancer

Lung cancer is the leading cause of cancer-related mortality in the United States, and non-small cell lung cancer (NSCLC) comprises over 80 percent of all types of lung cancer. The tumor microenvironment plays an important role in cancer progression and tumor endothelial cells (TECs) are critical components of this

microenvironment. TECs harbor molecular abnormalities that keeps them in a persistent “activated” state and are also chronically stimulated by cytokines in the tumor microenvironment. In this proposal, we will study the significance of a molecular abnormality in TECs, that is induced by vascular endothelial growth factor (VEGF)

and regulates the levels of microRNAs (miRNAs). MiRNAs are small (21-22 nucleotides) noncoding RNAs that regulate gene expression by recruiting messenger RNAs to the RNA-induced silencing complex (RISC). MiR-1 is a flagship example of a tumor suppressor miRNA. We have previously shown that miR-1 levels in TECs isolated from NSCLC patients are

significantly lower than endothelial cells isolated from the non-cancerous lung tissues. Moreover, overexpression of miR-1 specifically in endothelium decreased tumor burden and vascularity in KRAS-mutant, P53 knock-out (KP) mice, showing that the downregulation of miR-1 is critical for NSCLC tumor angiogenesis and progression.

A mechanistic investigation on miR-1 downregulation in endothelial cells showed that VEGF downregulates mature miR-1 without altering the transcription, processing, or loading of its precursors on RISC. i.e it degrades mature miR-1. One of the main mechanisms of selective mature miRNA degradation is the

modification and trimming of its 3' end. This process produces 3’ miRNA variants (isomiRs). A small RNA sequencing analysis on VEGF-stimulated endothelial cells revealed the emergence of unique 3’-adenylated and -trimmed isomiRs in these cells, suggesting that 3’-adenylation triggers miR-1 degradation. In accord with this

finding, VEGF stimulation selectively increased the expression of terminal nucleotidyltransferase 2 (TENT2), the main enzyme known to adenylate miRNA 3’ end. TENT2 expression was also increased after VEGF stimulation in TECs isolated from NSCLC patients. We preliminarily assessed the role of TENT2 in miR-1 regulation and

angiogenesis by testing the effects of its knockdown. These experiments showed that TENT2 knockdown increases miR-1 levels, inhibits endothelial cell proliferation, and decreases the tumor burden in KP mice. Based on these findings we have hypothesized that: TENT2 triggers miR-1 degradation in TECs via the non-templated

addition of adenines to its 3' end and through this mechanism regulates tumor angiogenesis and progression in NSCLC. To test this hypothesis, we propose following aims: Aim 1: To determine the role of TENT2 in miRNA adenylation and degradation in the tumor endothelium. Aim 2: To determine the role of endothelial TENT2 in NSCLC tumor angiogenesis and progression