Abstract B55: Chemokine biased agonists regulate pancreatic cancer migration and metastasis through bioenergetic signaling
Authors: Roy Ishan , McAllister Donna , Gorse Egal , Dixon Kate , Cheng Gang , Getschman Anthony , Engle Dannielle , Tsai Susan , Tuveson David , Evans Douglas , Volkman Brian , Kalyanaraman Balaraman , Dwinell Michael 
Published: October 2015

Abstract
Across all stages of initial diagnosis, the majority of patients with pancreatic ductal adenocarcinoma (PDAC) eventually succumb to metastatic disease. Yet, there are no drugs approved for disruption of the specific mechanisms behind PDAC metastasis. As soluble/secreted proteins, chemokines provide a unique opportunity to manipulate cancer cell movement and metastasis. Our prior reports show that chemokines such as CXCL12 regulate movement in a biphasic concentration dependent fashion, wherein low doses drive migration and higher doses are cytostatic. This biphasic migration response reflects the dose dependent oligomerization of the ligand, with monomeric CXCL12 as pro-migratory and dimeric CXCL12 as anti-migratory. Previously, we demonstrated that the CXCL12 gene is silenced in human PDAC tumor cells and, when re-expressed, is a tumor suppressor decreasing growth and metastasis. Herein, we examine how CXCL12 inhibits PDAC malignancy and explore CXCL12 use as a biologic therapy for PDAC metastasis. Established and patient-derived PDAC cells along with tumor cells derived from the B6-KRasLSL.G12D/+-p53R172H/+-PdxCretg/+ (KPC) model were used to test the hypothesis that CXCL12 biphasic chemotactic signaling reflects biased agonist regulation of bioenergetic signaling. In transwell assays, human PDAC cells responded to recombinant wild type CXCL12 (L12WT) in a biphasic manner, with 10 nM L12WT as pro-migratory and concentrations ≥ 100 nM as cytostatic. As predicted by the biased agonist model, migratory and cytostatic doses of L12WT stimulated equivalent intracellular calcium mobilization. Though calcium signaling has previously been associated with activity of AMP-Kinase (AMPK), a known regulator of metabolic stress, no prior studies have established a link between bioenergetics and chemotactic migration. Given CXCL12 dual regulation of growth and migration, we next tested the hypothesis that CXCL12 alters bioenergetic metabolism using a Seahorse XF analyzer. In MiaPaCa2 cells, while cell bioenergetics were unchanged by migratory doses, cytostatic doses of L12WT significantly decreased both ATP-linked oxidative phosphorylation and reserve glycolysis capacity. In parallel, cytostatic doses of L12WT significantly increased levels of phosphorylated AMPK in human and murine PDAC cells. L12WT-induced AMPK activity was calcium/calmodulin-kinase II dependent. Anti-motile effects of CXCL12 were dependent on AMPK signaling as the specific inhibitor Compound C completely prevented the cytostasis induced by L12WT. Subsequent experiments established that cytostatic doses of L12WT drove inhibitory phosphorylation of MYPT1, preventing dephosphorylation of myosin light chain (MLC). Thus, use of the AMPK inhibitor reversed the CXCL12 biphasic phenomenon by restoring balance to MLC dephosphorylation-phosphorylation cycling, allowing PDAC cells to once again migrate. CXCL12 biphasic migration was mimicked with locked mutants of CXCL12, with monomer (L121) as pro-migratory but equal levels of dimer (L122) as anti-migratory. Stimulation of PDAC cells with L122 lead to more sustained AMPK activity when compared with L121. Finally, in mouse orthotopic xenografts of two different PDAC cell lines, intraperitoneal administration of 5 μM L12WT significantly decreased PDAC metastasis to the liver and lung. Together, these data demonstrate for the first time that the cytostatic component of CXCL12 function is AMPK dependent. Given the ability of CXCL12 to regulate bioenergetic migratory signaling, our data show the promise of recombinant CXCL12 and biased agonist variants, as a biologic therapy for abrogating PDAC metastasis.
Citation Format: Ishan Roy, Donna McAllister, Egal Gorse, Kate Dixon, Gang Cheng, Anthony Getschman, Dannielle Engle, Susan Tsai, David Tuveson, Douglas Evans, Brian Volkman, Balaraman Kalyanaraman, Michael Dwinell. Chemokine biased agonists regulate pancreatic cancer migration and metastasis through bioenergetic signaling. [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 B55.