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Published on: May 26, 2023
APE1/Ref-1 knockdown in pancreatic ductal adenocarcinoma - characterizing gene expression changes and identifying
Fenil Shah1, Emery Goossens2, Nadia M Atallah3
1Department of Pediatrics, Herman B Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN, USA.
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.
Insights
Apurinic/apyrimidinic endonuclease 1 (APE1) regulates cancer pathways. Researchers used single-cell RNA sequencing to identify 2837 genes affected by APE1 levels, revealing new therapeutic targets and pathways for cancer treatment.
Area of Science:
- Molecular Biology
- Genetics
- Cancer Research
Background:
- Apurinic/apyrimidinic endonuclease 1/redox factor-1 (APE1/Ref-1 or APE1) is a key regulator of transcription factors in cancer pathways.
- APE1's essential role in cell viability has hindered previous studies using knockout models.
- Understanding APE1's regulatory network is crucial for developing targeted cancer therapies.
Purpose of the Study:
- To identify genes and pathways regulated by APE1 using a novel single-cell RNA sequencing approach.
- To investigate the impact of APE1 levels on gene expression without complete knockout.
- To explore potential therapeutic strategies targeting APE1 in cancer.
Main Methods:
- Utilized single-cell RNA sequencing to analyze gene expression changes in relation to APE1 protein levels.
- Employed a novel statistical design to identify differentially expressed genes (DEGs) upon APE1 knockdown.
- Validated findings using siRNA knockdown, qRT-PCR, and treatment with the APE1 redox inhibitor APX3330.
Main Results:
- Identified 2837 genes significantly affected by APE1 knockdown.
- Discovered novel APE1-regulated pathways, including EIF2 signaling, mTOR, and mitochondrial pathways.
- Demonstrated that APE1's effect on gene expression is dose-dependent, not requiring complete knockout.
- Observed consistent responses in patient-derived pancreatic cancer cells for specific genes (ITGA1, TNFAIP2, COMMD7, RAB3D).
- Confirmed the role of APE1's redox function in regulating mitochondrial and proliferation genes.
Conclusions:
- APE1 significantly influences a broad spectrum of genes and pathways, including novel ones not previously associated with its function.
- The study provides a comprehensive gene expression profile regulated by APE1, offering new targets for cancer therapy.
- Findings support the use of APE1 inhibitors, like APX3330, in combination therapies for pancreatic and other cancers, highlighting tumor subtype specificity.
