Introduction: Pancreatic cancer (PC) has the worst prognosis of all the major cancers. The role of inflammation in tumor progression and metastasis, particularly in PC, is widely accepted. Our lab has recently shown that levels of circulating cell-free DNA (cfDNA) and associated proteins (histones, HMGB-1, etc.) are elevated in PC patients at baseline and increase in response to standard therapies. It is also known that components of the innate immune system respond to cfDNA and associated proteins. Specifically, the toll-like receptors (TLRs) have been shown to be activated by cfDNA and associated proteins. These TLRs, specifically TLR-4 and TLR-9, have been shown to promote tumor progression and metastasis in other cancers such as breast, colorectal, and skin cancers. We wanted to investigate the role of TLRs in PC by characterizing receptor expression and downstream function. We also attempted to inhibit the downstream effects of TLR signaling using nucleic acid binding polymers (NABPs) that our lab has previously utilized in other disease models such as systemic lupus erythematosus.
Methods: Three human (MiaPaCa-2, BxPC-3, PANC-1) and one murine (KPC-4580P) PC cell line were characterized for TLR-4 and TLR-9 expression by western blot. Briefly, cells were plated and lysed using RIPA buffer. Total protein concentration was quantified using bicinchoninic acid (BCA) assay and normalized prior to running the cell lysates on SDS-PAGE. The protein bands were then transferred to immunoblotting membrane and probed with specific primary antibodies, followed by fluorescent secondary antibodies. Blots were exposed and quantified via LI-COR. Downstream function of TLR expression was tested using Transwell-matrigel invasion assays wherein cells were plated in the Transwell system in the presence of TLR-4 and TLR-9 specific agonists (LPS and CpG or mtDNA, respectively). These cells were also treated with NABPs to inhibit TLR activation.
Results: All PC cell lines expressed TLR-4 and TLR-9. The invasion assays indicated that in the presence of TLR-4 and TLR-9 specific agonists, PC cells exhibited increased invasion. Moreover, PC cells that invaded in response to TLR-9 agonists were inhibited in the presence of NABPs. Our lab has also prepared conditioned media (CM) from cells treated with clinically relevant doses of radiation. Our PC cell lines displayed increased invasiveness in response to CM and decreased with NABP treatment.
Conclusion: We have previously shown that there are elevated levels of cfDNA and associated proteins in PC patient serum and have now shown that these factors can have direct effects on PC cells via TLRs that are expressed by the cancer cells. Treating cells with NABPs in the presence of TLR agonists can abrogate TLR mediated invasion in vitro. Our goal is to further characterize the effects of TLR signaling on cancer cells including RNA transcription and protein translation profiling.
Citation Format: Ibtehaj A. Naqvi, Jessica McDade, Jaewoo Lee, Rebekah White. Toll-like receptor expression and ligation promotes in vitro invasion in pancreatic cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4554.