S1885 TGFβ Utilizes NF-κB to Mediate PTEN Suppression in a Smad-Independent Manner in Pancreatic Cancer Cells

Abstract

Background: Transforming growth factor-β (TGFβ) is a key tumor suppressor that is involved in cell differentiation and normal pancreatic cell homeostasis. In pancreatic cancer, the suppressor function is lost in part by events that affect the TGFβ-SMAD signaling pathway, such as bi-allelic loss of SMAD4 and oncogenic K-RAS activation that slows nuclear translocation of receptor-activated SMADs. With loss of TGFβ-SMAD signaling, TGFβ promotes cellular proliferation via SMAD-independent signaling pathways. We have shown that TGFβ downregulates the tumor suppressor PTEN in a SMAD-independent manner via activation of PKCα in pancreatic cancer cells, but the mechanisms leading to PKCα activation by TGFβ are unexplored. We examined if TGFβ could activate a Ca2+-dependent pathway, particularly Ca2+ entry through the plasmamembrane store-operated Ca2+ channel (SOC).We hypothesized that TGFβ increases cytoplasmic free Ca2+ ([Ca]cyt) via SOC after activation of the IP3 receptor (IP3R) on the endoplasmic reticulum membrane. Methods: BxPc-3 human pancreatic cancer cells were treated with 10 ng/ml TGFβ, 100 mM 2-APB (inhibitor for both IP3R and SOC), 30 mM La3+ (a selective SOC inhibitor), and the effect on [Ca]cyt were measured by a digital MetaFluor Imaging System using the fluorescent Ca2+-sensitive dye, fura 2-AM. Separated membrane and cytosolic fraction proteins were probed for PKCα by Western blotting. Results: TGFβ activates PKCα by 15 minutes after treatment, as evidenced by translocation from the cytosolic to the membrane fraction. TGFβ increased [Ca]cyt within 100 seconds after treatment (∆F 0.25 ± 0.02, n=38, P<0.01), while pretreatment with 2-APB inhibited increase in [Ca]cyt (∆F 0.01 ± 0.01, 98% inhibition, n=46, P<0.001). Pre-treatment of the cells with 2-APB prevented TGFβ-induced PKCα activation. Pretreatment with La3+ totally abolished the TGFβ-induced increase in [Ca]cyt (n=34, P<0.01). Conclusion: TGFβ may activate IP3R that triggers release of intracellular Ca2+ and Ca2+ entry via SOC. These intracellular Ca2+ stores are important for TGFβ-induced PKCα activation and subsequent tumor cell proliferation. Our data is the first to demonstrate that TGFβ can mobilize intracellular Ca2+ stores to activate proliferative PKCα signaling in pancreatic cells. Future treatments of pancreatic cancer and other cancers may involve manipulation of intracellular Ca2+ levels by interventions towards Ca2+-dependent pathways.