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P032. 5-ASA inhibits phospholipase D-dependent mammalian target of rapamycin signaling in colorectal cancer

B. Baan1, A. Dihal2, E. Hoff2, C. Bos3, P. Voorneveld2, P. Koelink2, H.W. Verspaget2, D. Richel4, J. Hardwick2, D.W. Hommes2, M. Peppelenbosch5, G.R. van den Brink1

1Tytgat Institute for Liver and Intestinal Research, Amsterdam, The Netherlands; 2Department of Gastroenterology & Hepatology, Leiden University Medical Center, Leiden, The Netherlands; 3Department of Pathology, VU University Medical Center, Amsterdam, The Netherlands; 4Department of Medical Oncology, Academic Medical Center, Amsterdam, The Netherlands; 5Department of Gastroenterology and Hepatology, Erasmus MC, University Medical Center Rotterdam, Rotterdam, The Netherlands

Aim: Mesalazine or 5-aminosalicylic acid (5-ASA) is a cornerstone in the induction and maintenance of remission of patients with ulcerative colitis. In addition to its anti-inflammatory activity it may also protect against the development of inflammation-associated colorectal cancer. The molecular mechanism of the anti-inflammatory and anti-cancer actions of 5-ASA remain to be fully determined. Here we focus on mammalian Target of Rapamycin (mTOR), an important regulator of cell cycle progression, and examine the anti-proliferative effects of 5-ASA on colorectal cancer in vitro and in vivo and aim to dissect the signal transduction events that lead to 5-ASA mediated inhibition of proliferation in colorectal cancer cells.

Materials and Methods: We examined the effect of 5-ASA on mTOR signaling in a panel of cancer cell lines originating from different tissues. Effects of 5-ASA on the pathways that control mTOR activity were studied in detail in two different colorectal cancer cell lines, using western blot, siRNA, a phospholipase D (PLD) activity assay and proliferation assays. The phosphorylation status of mTOR and its downstream target, ribosomal protein S6, was studied in colorectal cancer biopsies taken from patients before and after topical 5-ASA treatment.

Results: Treatment of colorectal cancer with 5-ASA inhibited mTOR signaling in vitro and in vivo. This effect seemed to be generic, as 5-ASA treatment also reduced mTOR signaling in several cancer cell lines originating from tissues other then colon. In colorectal cancer cells, 5-ASA did not affect any of the pathways known to control the tuberous sclerosis complex (TSC), a major regulator of mTOR activity, including the AMPK, PKB/Akt and MAPK pathways. Indeed the 5-ASA effect was independent of TSC-integrity as assessed by knockdown of TSC-component TSC2. Using inhibitors and knockdown experiments, we were able to show that in colorectal cancer cells both proliferation and mTOR activity depended on PLD, an enzyme that generates phosphatidic acid (PA). 5-ASA treatment inhibited both PLD-activity and proliferation, and these effects could be rescued with exogenous PA.

Conclusion: 5-ASA interferes with proliferation of colorectal cancer cells via inhibition of PLD-dependent generation of PA and consequent loss of mTOR signaling.