Priyanka Swami

The receptor for advanced glycation end products (RAGE) contributes to many cellular aspects of pancreatic cancer including cell proliferation, migration, and survival. Studies have shown that RAGE activation by its ligands promotes pancreatic tumor growth by stimulating both cell proliferation and migration. In this study, we investigated the effect of RAGE up-regulation on the proliferation and migration of the human pancreatic cancer Panc-1 cell-line. We show that moderate overexpression of RAGE in Panc-1 cells results in increased cell proliferation, but decreased cell migration. The observed cellular changes were confirmed to be RAGE-specific and reversible by using RAGE-specific siRNAs and the small molecule RAGE inhibitor FPS-ZM1. At the molecular level, we show that RAGE up-regulation was associated with decreased activity of FAK, Akt, Erk1/2, and NF-κB signaling pathways and greatly reduced levels of α2 and β1 integrin expression, which is in agreement with the observed decreases in cell migration. We also demonstrate that RAGE up-regulation changes the expression of key molecular markers of epithelial-to-mesenchymal transition (EMT). Our results suggest that in the absence of stimulation by external ligands, RAGE up-regulation can differently modulate cell proliferation and migration in pancreatic cancer cells and regulates partly EMT.

Pancreatic ductal adenocarcinoma (PDAC) remains a very difficult cancer to treat. Recent in vitro and in vivo studies suggest that the activation of the receptor for advanced glycation end products (RAGE) by its ligands stimulates pancreatic cancer cell proliferation and tumor growth. Additional studies show that, in the RAGE ligand, the high mobility group box 1 (HMGB1) protein plays an important role in chemoresistance against the cytotoxic agent gemcitabine by promoting cell survival through increased autophagy. We hypothesized that blocking the RAGE/HMGB1 interaction would enhance the cytotoxic effect of gemcitabine by reducing cell survival and autophagy. Using a preclinical mouse model of PDAC and a monoclonal antibody (IgG 2A11) as a RAGE inhibitor, we demonstrate that RAGE inhibition concurrent with gemcitabine treatment enhanced the cytotoxic effect of gemcitabine. The combination of IgG 2A11 and gemcitabine resulted in decreased autophagy compared to treatment with gemcitabine combined with control antibodies. Notably, we also observed that RAGE inhibition protected against excessive weight loss during treatment with gemcitabine. Our data suggest that the combination of gemcitabine with a RAGE inhibitor could be a promising therapeutic approach for the treatment of pancreatic cancer and needs to be further investigated.

Pancreatic cancer (PC) is a lethal disease with the current 5-year survival of only about 8%. Despite decades of research, gemcitabine remains the standard of therapy for PC. However, after an initial good response, eventual development of chemo-resistance is a major challenge faced by gemcitabine therapy. One principal cause of chemo-resistance is the release of high mobility group box 1 (HMGB1) protein from dying tumor cells, upon treatment with gemcitabine. When released, HMGB1 interacts with the receptor for advanced glycation end-products (RAGE), stimulating distinct signaling pathways leading to increased autophagy and tumor cell proliferation along with a reduction in apoptosis. RAGE activation by HMGB1 thereby supports tumor cell survival, making the tumor less sensitive towards gemcitabine. We hypothesized that inhibiting RAGE-HMGB1 interaction, in combination with gemcitabine therapy could reduce autophagy and tumor cell proliferation as well as facilitate apoptosis. This would ultimately reduce chemo-resistance and make pancreatic tumor more sensitive towards gemcitabine therapy. In our study, we intended to determine the efficacy of RAGE inhibitors (anti-RAGE antibody IgG2A11 and small molecule RAGE inhibitor FPS-ZM1) in combination with gemcitabine in PC. We employed an orthotopic mouse model of PC to test our hypothesis. In this model, the murine #5508 KPC pancreatic cancer cell line was implanted in the pancreas of C57BL/6 mice. The implanted mice received different treatments (saline, RAGE inhibitor, gemcitabine or gemcitabine with RAGE inhibitor). At the end of the study, tumors obtained from different treatment groups were assessed for their weight. Additionally, these tumors were analyzed for the expression of markers of autophagy, cell proliferation and apoptosis by western blot. We observed smaller tumors in mice treated with the combination of anti-RAGE antibody IgG2A11 & gemcitabine as well as lower levels of phosphorylated ERK in the combination treated group. This data was presented at “EB 2018” conference. We now show that the smaller tumors in the combination group are also associated with an increase in p62 levels, supporting a reduction in autophagy. In addition, we observed higher levels of cleaved PARP in this combination group suggesting higher levels of apoptosis in tumor cells. In our second study where we used the small molecule RAGE inhibitor FPS-ZM1 in combination with gemcitabine, we also observed significantly smaller tumors in the combination group as compared to the gemcitabine alone treatment group. In conclusion, our data strongly suggests that inhibiting RAGE in combination with gemcitabine could be a valid therapeutic approach for the treatment of pancreatic adenocarcinoma. Support or Funding Information This work was in part supported by the College of Health Professions at NDSU and by the NIH Grant Number P20GM109024 from the National Institute of General Medicine (NIGMS). This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Pancreatic cancer is currently the fourth leading cause of cancer deaths in the US with a dismal 5‐year survival rate of 7%. Despite several advancements in therapy, gemcitabine remains the standard treatment for pancreatic cancer. However, the modest survival advantage presented by gemcitabine evokes the need for a targeted therapeutic approach in addition to conventional treatment. The Receptor for advanced glycation end‐products (RAGE) is a cell surface receptor which has been demonstrated to be involved in pancreatic cancer progression. RAGE is also reported to facilitate pancreatic tumor cell survival by supporting autophagy and restricting apoptosis. Recent reports have illustrated RAGE as a potential mediator of chemo‐resistance in pancreatic cancer. HMGB1, one of the important ligands of RAGE, is released from necrotic cells upon treatment with gemcitabine. This HMGB1 triggers upregulation of RAGE in the tumor, making it resistant to chemotherapy. We intend to target pancreatic cancer with RAGE inhibitors in combination with gemcitabine to improve survival. We hypothesize that RAGE inhibitors can prevent the interaction between RAGE and its ligands, consequently sensitizing pancreatic cancer for gemcitabine. To accomplish the aim of our study we employed orthotopic mouse model of pancreatic cancer. KPC cells, derived from mice tumors, were implanted in the pancreas of C57BL/6 mice. The mice were divided into four treatment groups: (i) Saline control (ii) Gemcitabine (iii) RAGE inhibitor (anti‐RAGE antibody, IgG2A11) (iv) RAGE inhibitor + gemcitabine. At the end of the study, the tumors obtained from the different treatment groups were assessed for their size, weight and volume. Additionally, the tumors from the four treatment groups were analyzed for the expression of markers of cell proliferation (Ki67), autophagy (LC3), apoptosis (cleaved caspase 3) and cell survival (BCl2) by western blot and immunohistochemistry. We also compared the phosphorylation of ERK1/2 in the tumors from the four treatment groups by western blot. The data showed a statistically significant reduction in tumor weight in mice treated with the combination of anti‐RAGE antibody, IgG2A11 and gemcitabine as compared to the control group. We could also observe a significant decrease in the expression of LC3 and an increase in cleaved caspase 3 in the tumors treated with the combination as compared to the control group. There was also a significant reduction in the phosphorylation of ERK1/2 in tumors treated with anti‐RAGE antibody IgG2A11 and gemcitabine. These results imply that the combination of RAGE inhibitors and gemcitabine can be used as an approach to mitigate chemo‐resistance in pancreatic cancer. Support or Funding Information College of Health Professions at NDSU NIH Grant Number P20 GM109024 from the National Institute of General Medicine (NIGMS) This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

Pancreatic cancer (PC) is currently the fourth leading cause of cancer deaths in the US. For decades, gemcitabine has been used as the standard treatment for PC. However, it offers a modest survival advantage which can be attributed to dense stroma and restricted vasculature in pancreatic tumors, as well as development of chemo-resistance. Receptor for Advanced Glycation End products (RAGE), a cell surface receptor, has been demonstrated to contribute in PC progression. RAGE interacts with several ligands and promotes pancreatic tumor cell survival by supporting autophagy and limiting apoptosis. Recent reports have illustrated that gemcitabine treatment results in the release of HMGB1 from necrotic cells. HMGB1, an important ligand for RAGE, leads to RAGE upregulation in tumor, making it chemo-resistant. We intend to utilize a combination of RAGE inhibitors and gemcitabine to target pancreatic cancer. We propose that RAGE inhibitors can prevent the RAGE-ligand interaction, thereby sensitizing PC for gemcitabine.