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Introduction: The effect of TNF-α on pancreatic tumorigenesis is controversial due to the differential signaling pathways initiated after binding its receptors TNFR1 and TNFR2. TNFR1 activation by TNF-α leads to cell apoptosis, whereas TNFR2 signaling is believed to be involved in cell survival through the activation of NF kappa B. TNF-α gene delivery has been suggested as a potentially useful therapeutic approach to improve gemcitabine treatment of pancreatic ductal adenocarcinoma (PDA), but its exact mechanism of action is not clearly understood. Although TNF-α has been shown to increase the expression of the lipogenesis promoting enzyme, fatty acid synthase (FAS) in liver steatosis, its impact on de novo lipogenesis in tumor cells has not been determined. In this study, we investigated effect of TNF-α on fatty acid synthase (FAS) in PDA cells and in fibroblasts as part of the tumor micro-environment.
Methods: PDA cells (MIAPACA-2 and AsPC-1) and the fibroblast cell line, hTERT-BJ were transfected with TNF-α gene by lentivirus-vector transduction. Control cells were transfected with the empty vector. FAS mRNA and protein were analyzed by real time PCR and Western blotting, respectively. Total- and phospho-AMPK, total- and phospho-Acetyl CoA carboxylase (ACC), FAS, and LKB/STK11 were analyzed by Western immunoblotting. The effects of TNF-α on sterol regulatory element binding protein-1, SREBP-1, the transcription factor responsible for FAS transcription, LKB1/STK11 (a tumor suppressor and the established upstream regulator of AMPK) and ACC (the downstream target of AMPK and the rate-limiting enzyme of fatty acid synthesis) were evaluated by real time PCR. MTT and Wound healing assays were used to determine cell survival and migration, respectively.
Results: TNF-α significantly (P<0.05) reduced PDA and fibroblast cell survival and migration. This was associated with significant reduction of FAS mRNA and protein expression levels in PDA cells (P= 0.02) but not the fibroblasts. Cells overexpressing TNF-α also showed significantly (p<0.05) reduced SREBP-1 and ACC. Reduction of FAS by TNF-α was inhibited when either SREBP-1 or ACC was knocked down by siRNA. No significant differences were seen in AMPK phosphorylation in cells that overexpress TNF-α.
Conclusion: Our data demonstrate a previously unknown involvement of TNF-α in PDA and microenvironment lipogenesis and suggest that targeted introduction of intratumor TNF-α can have the potential as a novel therapeutic anti-lipogenic agent in human PDA.