The effect of blocking the prosurvival AKT/P13K/mTOR and mutant KRAS–signaling pathways on chemotherapy resistance of pancreatic cancer.

Abstract

e13514 Background: Pancreatic adenocarcinoma continues to be a highly lethal malignancy (5 yr survival 5%) is poorly responsive to all chemotherapy and has only modest sensitivity to the current best. C6 ceramide demonstrates chemo enhancement of gemcitabine, cetuximab and other agents in vitro. Exploration of the C6 mechanism suggests reversal of chemo resistance pathways – pro-survival and mutant KRAS-signaling in pancreatic cancer cells. Methods: In vivo: 2-3 month SCID male mice with pancreatic (pan) tumor implants (2 x 106 L3.6 cells) received intraperitoneal (I.P.) injections (3x/wk) of paclitaxel (Pax) (3.0mg/gm), gemcitabine (Gem, 10mg/gm) with/without C6 ceramide (10mg/mL) and were observed 6 weeks and tumor response/survival recorded. In vitro: drug dose Pax 3ug/mL, Gem 3 ug/mL, C6 ceramide 10 ug/mL. The MTT anti tumor response was measured in 3 pancreatic lines L3.6, PANC-1, and MIA (Kras mutated). Signaling activation by Western blot utilized antibodies: LY294002, and Wortmannin to block PI3K/AKT pathway, U0126 and PD 98059 to block ERK pathway and rapamycin to block mTORC1 pathway. Results: Administration of Gem or Pax with C6 ceramide induced a significant increase in cell death and apoptosis in pancreatic cancer cells (L3.6, PANC-1 and MIA PaCa-2) which were associated with inhibiting perturbations of cell signaling pathways including pro-survival PI3K/Akt/mTOR, and ERK/MAPK, Ras signaling pathways. Pharmacological inhibition with specific signaling pathway inhibitors enhanced cytotoxicity by Gem or Pax, suggesting that survival pathway inhibition might be the key mechanism contributing to C6 ceramide inhibition synergism of Pax or Gem antitumor effects. Conclusions: C6 Ceramide an apoptosis signal potentiates the anti tumor effects of chemo agents Gem and Pac and the biologic EGFR inhibitor cetuximab against 3 aggressive pancreatic cancer cell lines by apparent inhibition of the pro-survival PI3K/AKT/mTOR pathways and the Kras mutated ERK/MAPK pathway.