Identification of Novel Pathways Regulated by APE1/Ref-1 in Human Retinal Endothelial Cells

Abstract

APE1/Ref-1 (apurinic/apyrimidinic endonuclease 1, APE1 or APEX1; redox factor-1, Ref-1) is a dual-functional enzyme with crucial roles in DNA repair, reduction/oxidation (redox) signaling, and RNA processing and metabolism. The redox function of Ref-1 regulates several transcription factors, such as NF-κB, STAT3, HIF-1α, and others, which have been implicated in multiple human diseases, including ocular angiogenesis, inflammation, and multiple cancers. To better understand how APE1 influences these disease processes, we investigated the effects of APEX1 knockdown (KD) on gene expression in human retinal endothelial cells. This abolishes both DNA repair and redox signaling functions, as well as RNA interactions. Using RNA-seq analysis, we identified the crucial signaling pathways affected following APEX1 KD, with subsequent validation by qRT-PCR. Gene expression data revealed that multiple genes involved in DNA base excision repair, other DNA repair pathways, purine or pyrimidine metabolism signaling, and histidine/one carbon metabolism pathways were downregulated by APEX1 KD. This is in contrast with the alteration of pathways by APEX1 KD in human cancer lines, such as pancreatic ductal adenocarcinoma, lung, HeLa, and malignant peripheral nerve sheath tumors. These results highlight the unique role of APE1/Ref-1 and the clinical therapeutic potential of targeting APE1 and pathways regulated by APE1 in the eye. These findings provide novel avenues for ocular neovascularization treatment.

Activation of the integrated stress response (ISR) pathways in response to Ref-1 inhibition in human pancreatic cancer and its tumor microenvironment

“…qRT-PCR cycling conditions were 1 min at 95°C, 10 min at 95°C, 15 s at 95°C and 1 min at 60°C for 40 cycles. Relative changes in mRNA expression levels were assessed by the 2 –ΔΔ CT method, and changes in mRNA expression of the target gene were normalized to that of β-actin gene ( 1 , 24 ).…”

In vitro and In vivo evidence demonstrating chronic absence of Ref-1 Cysteine 65 impacts Ref-1 folding configuration, redox signaling, proliferation and metastasis in pancreatic cancer

“…Cells were lysed in 1% SDS extraction buffer supplemented with protease inhibitors (Santa Cruz Biotechnology, TX, USA) [ 26 , 33 ]. Briefly, cell extract was heated at 95 °C for 5 min, then sonicated (4 pulses, 4 cycles) to shear the DNA in the samples as previously described [ 26 ].…”

New Ref-1/APE1 targeted inhibitors demonstrating improved potency for clinical applications in multiple cancer types

“…AP endonuclease-1 / Redox factor-1 (APE1/Ref-1 or Ref-1) is one such regulatory protein that is upstream of numerous TFs such as NFκB, HIF1α, STAT3, AP-1 (Fos/Jun) and stimulates their DNA binding activity [ 22 , 23 ]. Ref-1, a multifunctional protein with redox signaling and DNA repair as its major functions, and additional activities including RNA processing, DNA maintenance, RNA quality control, miRNA metabolism, regulation of stress-response gene expression and mitochondrial DNA (mtDNA) repair is dysregulated in cancer cells [ 23 ]. Its endonuclease function is the rate-limiting step in the Base Excision Repair (BER) pathway and is considered indispensable for cell survival [ 24 ].…”