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

by Selleckchem | Sep 09 2023

Pancreatic cancer or pancreatic ductal adenocarcinoma (PDAC) is characterized by a profound inflammatory tumor microenvironment (TME) with high heterogeneity, metastatic propensity, and extreme hypoxia. The integrated stress response (ISR) pathway features a family of protein kinases that phosphorylate eukaryotic initiation factor 2 (eIF2) and regulate translation in response to diverse stress conditions, including hypoxia. We previously demonstrated that eIF2 signaling pathways were profoundly affected in response to Redox factor-1 (Ref-1) knockdown in human PDAC cells. Ref-1 is a dual function enzyme with activities of DNA repair and redox signaling, responds to cellular stress, and regulates survival pathways. The redox function of Ref-1 directly regulates multiple transcription factors including HIF-1α, STAT3, and NF-κB, which are highly active in the PDAC TME. However, the mechanistic details of the crosstalk between Ref-1 redox signaling and activation of ISR pathways are unclear. Following Ref-1 knockdown, induction of ISR was observed under normoxic conditions, while hypoxic conditions were sufficient to activate ISR irrespective of Ref-1 levels. Inhibition of Ref-1 redox activity increased expression of p-eIF2 and ATF4 transcriptional activity in a concentration-dependent manner in multiple human PDAC cell lines, and the effect on eIF2 phosphorylation was PERK-dependent. Treatment with PERK inhibitor, AMG-44 at high concentrations resulted in activation of the alternative ISR kinase, GCN2 and induced levels of p-eIF2 and ATF4 in both tumor cells and cancer-associated fibroblasts (CAFs). Combination treatment with inhibitors of Ref-1 and PERK enhanced cell killing effects in both human pancreatic cancer lines and CAFs in 3D co-culture, but only at high doses of PERK inhibitors. This effect was completely abrogated when Ref-1 inhibitors were used in combination with GCN2 inhibitor, GCN2iB. We demonstrate that targeting of Ref-1 redox signaling activates the ISR in multiple PDAC lines and that this activation of ISR is critical for inhibition of the growth of co-culture spheroids. Combination effects were only observed in physiologically relevant 3D co-cultures, suggesting that the model system utilized can greatly affect the outcome of these targeted agents. Inhibition of Ref-1 signaling induces cell death through ISR signaling pathways, and combination of Ref-1 redox signaling blockade with ISR activation could be a novel therapeutic strategy for PDAC treatment.

Comments:

The passage you provided describes a study that investigated the role of Redox factor-1 (Ref-1) and the integrated stress response (ISR) pathway in pancreatic ductal adenocarcinoma (PDAC), a type of pancreatic cancer. The study found that Ref-1, an enzyme involved in DNA repair and redox signaling, plays a role in regulating survival pathways in PDAC cells.

The researchers observed that knocking down Ref-1 affected the eIF2 signaling pathways, which are involved in regulating protein translation under stress conditions such as hypoxia (low oxygen levels). They found that under normal oxygen conditions (normoxia), Ref-1 knockdown induced the ISR. However, under hypoxic conditions, ISR activation occurred regardless of Ref-1 levels.

Inhibition of Ref-1's redox activity increased the expression of phosphorylated eIF2 (p-eIF2) and ATF4, a transcription factor involved in the ISR, in multiple PDAC cell lines. The effect on eIF2 phosphorylation was found to be dependent on the PERK kinase, which is one of the kinases involved in the ISR pathway. Treatment with a PERK inhibitor resulted in the activation of another ISR kinase called GCN2 and increased levels of p-eIF2 and ATF4 in both tumor cells and cancer-associated fibroblasts (CAFs), which are cells found in the tumor microenvironment.

The researchers also investigated the combination effects of Ref-1 inhibitors and PERK inhibitors on cell viability in 3D co-culture models of PDAC. They found that high doses of PERK inhibitors, in combination with Ref-1 inhibitors, enhanced cell killing effects in both human pancreatic cancer cell lines and CAFs. However, when a GCN2 inhibitor was added to the combination treatment, the effect was completely abolished.

Based on these findings, the study suggests that targeting Ref-1's redox signaling can activate the ISR in PDAC cells and inhibit tumor growth. Combining Ref-1 inhibition with ISR activation could be a potential therapeutic strategy for treating PDAC. The study also emphasizes the importance of using physiologically relevant 3D co-culture models to evaluate the effects of these targeted agents accurately.