Abstract
Apurinic/apyrimidinic endonuclease 1/redox factor‐1 (APE1/Ref‐1 or APE1) is a multifunctional protein that regulates numerous transcription factors associated with cancer‐related pathways. Because APE1 is essential for cell viability, generation of APE1‐knockout cell lines and determining a comprehensive list of genes regulated by APE1 has not been possible. To circumvent this challenge, we utilized single‐cell RNA sequencing to identify differentially expressed genes (DEGs) in relation to APE1 protein levels within the cell. Using a straightforward yet novel statistical design, we identified 2837 genes whose expression is significantly changed following APE1 knockdown. Using this gene expression profile, we identified multiple new pathways not previously linked to APE1, including the EIF2 signaling and mechanistic target of Rapamycin pathways and a number of mitochondrial‐related pathways. We demonstrate that APE1 has an effect on modifying gene expression up to a threshold of APE1 expression, demonstrating that it is not necessary to completely knockout APE1 in cells to accurately study APE1 function. We validated the findings using a selection of the DEGs along with siRNA knockdown and qRT‐PCR. Testing additional patient‐derived pancreatic cancer cells reveals particular genes (ITGA1,TNFAIP2,COMMD7,RAB3D) that respond to APE1 knockdown similarly across all the cell lines. Furthermore, we verified that the redox function of APE1 was responsible for driving gene expression of mitochondrial genes such as PRDX5 and genes that are important for proliferation such as SIPA1 and RAB3D by treating with APE1 redox‐specific inhibitor, APX3330. Our study identifies several novel genes and pathways affected by APE1, as well as tumor subtype specificity. These findings will allow for hypothesis‐driven approaches to generate combination therapies using, for example, APE1 inhibitor APX3330 with other approved FDA drugs in an innovative manner for pancreatic and other cancer treatments.
Highlights
This SCR/siAPE1 analysis resulted in 1950 differentially expressed genes (DEGs) (Fig. 2B) and allowed us to identify several new genes and pathways impacted by APE1, including the eukaryotic initiation factor 2 (EIF2) signaling and mechanistic target of Rapamycin (mTOR) pathways and a significant number of mitochondrialrelated genes and pathways
This study takes an unbiased statistical approach to determine the effects of APE1 knockdown in pancreatic ductal adenocarcinoma (PDAC) cells
While it has been long known that APE1 regulates various essential transcription factors (Cardoso et al, 2012; Fishel et al, 2015; Gaiddon et al, 1999; Jiang et al, 2010; Kelley et al, 2012; Lando et al, 2000; Logsdon et al, 2016), the amplified effect of APE1 knockdown on downstream targets of those transcription factors has not been previously elucidated
Summary
Apurinic/apyrimidinic endonuclease 1/redox factor-1 (APE1/Ref-1; referred to as APE1) is a multifunctional protein that is involved in repairing DNA damage via its endonuclease activity in base excision repair (Fung and Demple, 2005; Izumi et al, 2005; Jiang et al, 2009; Kelley et al, 2014), and using its redox protein–protein signaling function to control the activity of numerous transcription factors such as STAT3, NFjB, AP-1, p53, and hypoxia-inducible factor 1a (HIF1a), among others (Cardoso et al, 2012; Fishel et al, 2015; Gaiddon et al, 1999; Jiang et al, 2010; Kelley et al, 2012; Lando et al, 2000; Logsdon et al, 2016). It is not possible to generate stable APE1-knockout cell lines (Tell et al, 2009) Approaches to circumvent this dilemma have utilized conditional knockouts and siRNA knockdowns (Fung and Demple, 2005; Izumi et al, 2005; Jiang et al, 2010). Using siRNA knockdowns, our laboratory has previously identified APE1 directly regulating STAT3 transcriptional activity (Cardoso et al, 2012), suppressing Nrf2-induced gene expression (Fishel et al, 2015) and, most recently, regulating carbonic anhydrase 9 (CA9) via HIF-1a under hypoxic conditions (Logsdon et al, 2016)
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