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

Fenil Shah, Emery Goossens, Nadia M. Atallah, Michelle Grimard, Mark Kelley, Melissa Fishel

Research output: Contribution to journalArticle

7 Citations (Scopus)

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.

Original languageEnglish (US)
Pages (from-to)1711-1732
Number of pages22
JournalMolecular Oncology
Volume11
Issue number12
DOIs
StatePublished - Dec 1 2017

Fingerprint

RNA Sequence Analysis
Adenocarcinoma
Gene Expression
Genes
Oxidation-Reduction
Pancreatic Neoplasms
Eukaryotic Initiation Factor-2
DNA-(Apurinic or Apyrimidinic Site) Lyase
Cell Line
Mitochondrial Genes
Sirolimus
Transcriptome
Small Interfering RNA
Neoplasms
Cell Survival
Proteins
Transcription Factors
Polymerase Chain Reaction
Therapeutics
Pharmaceutical Preparations

Keywords

  • APE1
  • pancreatic ductal adenocarcinoma
  • Ref-1
  • RNA sequencing
  • single cell

ASJC Scopus subject areas

  • Molecular Medicine
  • Genetics
  • Cancer Research

Cite this

APE1/Ref-1 knockdown in pancreatic ductal adenocarcinoma – characterizing gene expression changes and identifying novel pathways using single-cell RNA sequencing. / Shah, Fenil; Goossens, Emery; Atallah, Nadia M.; Grimard, Michelle; Kelley, Mark; Fishel, Melissa.

In: Molecular Oncology, Vol. 11, No. 12, 01.12.2017, p. 1711-1732.

Research output: Contribution to journalArticle

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