Abstract B19: Redox factor 1 (Ref-1) signaling in the interaction between pancreatic tumor cells and cancer-associated fibroblasts

Pancreatic cancer (PDAC) is a deadly disease and is accompanied by a fibrotic phenotype that contributes to the resistance of the disease. Signaling between cancer-associated fibroblasts (CAFs) and tumor are important in the fibrotic phenotype and metastatic spread. Another feature of PDAC is the presence of metastatic disease at diagnosis and one mechanism contributing to this is perineural invasion (PNI), or invasion of cancer cells into the surrounding nerves. Thus, there is a critical need to understand the signaling within the cells of the tumor microenvironment (TME) and how it contributes to progression of the disease and resistance to treatment. The long-term goals of this work are understanding the critical signaling pathways within the tumor and the TME required for metastasis and resistance. A novel PDAC microenvironment target, AP endonuclease/Redox factor-1 (Ref-1), is a reduction-oxidation (redox) factor involved in the transcriptional regulation of gene expression. Transcriptional factors, HIF-1α, NF-κB, and STAT3, are regulated by Ref-1 and are activated in stromal cells as well as pancreatic tumors. The objective of this work is to determine the outcome of inhibiting Ref-1 in CAFs and the effects of that inhibition on proliferation and migration in PDAC. The central hypothesis of the proposed work is that Ref-1 redox activity plays a critical role in the signaling within the TME between tumor and CAFs. We are using several innovative methods to probe the tumor-CAF interaction including: 1) co-culture 2D and 3D models; 2) genetic approach via redox inactive-Ref-1 constructs and siRNA to Ref-1; 3) pharmacologic approach via a well-established small molecule inhibitor of Ref-1 redox activity, E3330; 4) quantitation of Ref-1 signaling via qPCR; and 5) in vivo mouse experiments with tumor-CAF co-injection. Our findings indicate that inhibition of Ref-1 is more effective in CAFs than tumor cells with nominal effect on normal fibroblasts. Furthermore, co-cultures of patient derived cells with normal fibroblasts do not show sensitivity to Ref-1 inhibition, in contrast to tumor-CAF co-cultures. Utilizing siRNA to reduce the levels of Ref-1 protein in the CAFs results in a decrease in the size and proliferation of 3D colonies that contain tumor cells plus CAFs that express ~80% reduced levels of Ref-1 protein. These data implicate the redox activity of Ref-1 and its regulation of critical transcription factors as significant in the signaling between the tumor and the CAFs. We are also investigating the effects of targeting STAT3 in the co-cultures based on our published data demonstrating that Ref-1 can activate STAT3 DNA binding and that dual targeting of Ref-1 and STAT3 is synthetic lethal to PDAC cells. Similar to Ref-1 inhibition, targeting of STAT3 is significant in the CAFs compared to normal fibroblasts. However, Gemcitabine treatment does not preferentially kill PDAC cells in co-culture with CAFs in contrast to what we observe with Ref-1 or STAT3 targeting. Due to PDAC’s fibrotic nature, targeting the interaction of tumor-stroma through Ref-1 inhibition is a promising avenue for combination treatment. The importance of not only targeting the tumor is clear, therefore, novel approaches that target the TME in addition to signaling pathways within the tumor may offer the most promise against this dreaded disease. Citation Format: Melissa L. Fishel, Huiwen Cheng, Murray Korc, Mark R. Kelley. Redox factor 1 (Ref-1) signaling in the interaction between pancreatic tumor cells and cancer-associated fibroblasts. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr B19.