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J. Biol. Chem., Vol. 283, Issue 19, 13021-13030, May 9, 2008

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Mammalian Target of Rapamycin Contributes to the Acquired Apoptotic Resistance of Human Mesothelioma Multicellular Spheroids^*

Dario Barbone, Tsung-Ming Yang, Jeffrey R. Morgan^¶, Giovanni Gaudino, and V. Courtney Broaddus¹

From the Lung Biology Center, San Francisco General Hospital, University of California, San Francisco, California 94110, the Dipartimento di Scienze Chimiche, Alimentari, Farmaceutiche e Farmacologiche, Universita' degli Studi del Piemonte Orientale "Amedeo Avogadro", Novara 28100, Italy, and the ^¶Department of Molecular Pharmacology, Physiology and Biotechnology, Brown University, Providence, Rhode Island 02912

When grown as three-dimensional structures, tumor cells can acquire an additional multicellular resistance to apoptosis that may mimic the chemoresistance found in solid tumors. We developed a multicellular spheroid model of malignant mesothelioma to investigate molecular mechanisms of acquired apoptotic resistance. We found that mesothelioma cell lines, when grown as multicellular spheroids, acquired resistance to a variety of apoptotic stimuli, including combinations of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), ribotoxic stressors, histone deacetylase, and proteasome inhibitors, that were highly effective against mesothelioma cells when grown as monolayers. Inhibitors of the phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin (mTOR) pathway, particularly rapamycin, blocked much of the acquired resistance of the spheroids, suggesting a key role for mTOR. Knockdown by small interference RNA of S6K, a major downstream target of mTOR, reproduced the effect of rapamycin, thereby confirming the role of mTOR and of S6K in the acquired resistance of threedimensional spheroids. Rapamycin or S6K knockdown increased TRAIL-induced caspase-8 cleavage in spheroids, suggesting initially that mTOR inhibited apoptosis by actions at the death receptor pathway; however, isolation of the apoptotic pathways by means of Bid knockdown ablated this effect showing that mTOR actually controls a step distal to Bid, probably at the level of the mitochondria. In sum, mTOR and S6K contribute to the apoptotic resistance of mesothelioma cells in three-dimensional, not in two-dimensional, cultures. The three-dimensional model may reflect a more clinically relevant in vitro setting in which mTOR exhibits anti-apoptotic properties.

Received for publication, November 27, 2007 , and in revised form, February 13, 2008.

^* This work was supported, in whole or in part, by National Institutes of Health Grant R01 CA95671 (to V. C. B.). This work was also supported by a Buzzi-Unicem Foundation grant (to G. G.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1–S5.

¹ To whom correspondence should be addressed: San Francisco General Hospital, University of California, 1001 Potrero Ave., Bldg. 1, Rm. 150, San Francisco, CA 94110. Tel.: 415-206-6656; Fax: 415-206-4123; E-mail: cbroaddus{at}medsfgh.ucsf.edu.

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