APE1/Ref‐1 knockdown in pancreatic ductal adenocarcinoma – characterizing gene expression changes and identifying novel pathways using single‐cell RNA sequencing

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.

Architecture of The Human Ape1 Interactome Defines Novel Cancers Signatures

“…These 11 gene sets were then subjected to survival analysis for a better understanding of their role according to a clinical perspective. LIHC, LUAD and PAAD networks, which were composed of bad prognostic genes in high percentages, confirmed previous studies on the important role of APE1 in these cancers [52][53][54][55][57][58][59][60][61][62][63][64][65] . Kaplan-Meier estimation was used to calculate survival probability associated with these genes; the ones having significantly lower survival prognosis (p < 0.05) were used to create cancer-specific PPI subnetworks ( Fig.…”

“…In order to focus our attention on the most relevant cancer types, we based our bioinformatic analysis on the eleven datasets having the highest number of bad prognostic genes. Among those, we were interested in particular in liver (LIHC) [52][53][54][55][56] , lung (LUAD) [57][58][59][60] and pancreatic (PAAD) [61][62][63][64][65] cancer datasets, as the essential role of APE1 in the tumorigenic processes of these cancer types is already well established. Notably, the LUAD dataset was the third having the highest number of bad prognostic genes (n = 153).…”

LTMG: A novel statistical modeling of transcriptional expression states in single-cell RNA-Seq data

“…APEX1 is a multifunctional protein that interact with multiple transcriptional factors (TFs) to regulate the genes involvement in response to DNA damage, hypoxia and oxidative stress [49]. Our previous study identified significant roles of APEX1 in the regulation of Pa03c cell's response to microenvironmental stresses [50]. Functional enrichment of the differentially expressed genes identified by the methods were further examined.…”

“…Comparing to MAST, SCDE, SC2P, EdgeR and DESeq, the downregulated genes in APE1/Ref-1-KD vs. control under hypoxia conditions identified by LTMG-DGE are more significantly enriched to the pathways such as glycolysis, TCA cycle and respiration chain, apoptosis, and lipid metabolism pathways, as well as genes regulated by HIF1A and STAT3 ( Figure 5B and Supplementary Table 7). Note that APE1/Ref-1 directly interacts with HIF1A and STAT3 [50,51], and regulates oxidative stress response, glucose and lipid metabolism, and relevant mitochondrial functions. These results suggest LTMG-DGE method can detect more functionally relevant genes than other tested methods.…”

Cytoplasmic APE1 promotes resistance response in osteosarcoma patients with cisplatin treatment

“…APE1 is the key regulator in the cellular oxidative stress pathway, which maintains the activity of numerous cellular transcription factors, such as AP‐1, NF‐κB, Myb, HIF‐1α, HLF, PAX, and p53, through keeping specific cysteine residues in the DNA binding domain reduced state. Moreover, some of these transcription factors are closely related to chemotherapy resistance …”