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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 twodimensional, cultures. The three-dimensional model may reflect a more clinically relevant in vitro setting in which mTOR exhibits anti-apoptotic properties.Resistance to apoptosis is a distinguishing characteristic of all tumors (1) and underlies their resistance to therapy (2). Understanding the molecular foundations of apoptotic resistance could lead to improvements in the utility of current therapies or provide new therapies altogether. In vitro, apoptotic resistance of tumor cells is usually studied using two-dimensional monolayers. However, cells can attain increased resistance when grown in three-dimensional structures, a phenomenon referred as the acquisition of multicellular resistance (3). This acquired property of tumor cells may help explain why promising findings from in vitro studies have not been easily translated into therapy (4). Consequently, in the last decade, multicellular spheroids have become a valuable tool in the study of solid tumors by representing a threedimensional system of intermediate cellular complexity between monolayer cell cultures and tumors in vivo (5, 6).Human malignant pleural mesothelioma, an aggressive thoracic cancer that arises from the pleural mesothelium, is characterized by a profound resistance to standard anti-neoplastic therapies. At present, no curative therapy is available (7). To investigate the apoptotic resistance of mesothelioma, we are increasingly utilizing in vitro three-dimension...

Approximately 45% of metastatic colorectal cancer (mCRC) patients with wild-type KRAS exon 2 are resistant to cetuximab treatment. We set out to identify additional genetic markers that might predict the response to cetuximab treatment. Fifty-three wild-type KRAS exon 2 mCRC patients were treated with cetuximab/irinotecan-based chemotherapy as a first- or third-line therapy. The mutational statuses of 10 EGFR pathway genes were analyzed in primary tumors using next-generation sequencing. BRAF, PIK3CA, KRAS (exons 3 and 4), NRAS, PTEN, and AKT1 mutations were detected in 6, 6, 5, 4, 1, and 1 patient, respectively. Four of the BRAF mutations were non-V600 variants. Four tumors harbored multiple co-existing (complex) mutations. All patients with BRAF mutations or complex mutation patterns were cetuximab non-responders. All patients but one harboring KRAS, NRAS, or BRAF mutations were non-responders. Mutations in any one of these three genes were associated with a poor response rate (7.1%) and reduced survival (PFS = 8.0 months) compared to wild-type patients (74.4% and 11.6 months). Our data suggest that KRAS, NRAS, and BRAF mutations predict response to cetuximab treatment in mCRC patients.