Oncogene (2011) 30, 4765–4779 & 2011 Macmillan Publishers Limited All rights reserved 0950-9232/11 www.nature.com/onc ORIGINAL ARTICLE Conserved features of cancer cells define their sensitivity to HAMLET-induced death; c-Myc and glycolysis P Storm1,5, S Aits1,5, MK Puthia2,5, A Urbano2, T Northen3, S Powers4, B Bowen3, YChao2, W Reindl3, DY Lee3, NL Sullivan4, J Zhang4, M Trulsson1, H Yang2, JD Watson4 and C Svanborg1 1Division of Microbiology, Immunology and Glycobiology, Department of Laboratory Medicine, Lund University, Lund, Sweden; 2Singapore Immunology Network (SIgN), Biomedical Sciences Institutes, Agency for Science, Technology, and Research (A*STAR), Singapore; 3Lawrence Berkeley National Laboratory, Berkeley, CA, USA and 4Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, USA HAMLET is the first member of a new family of Introduction tumoricidal protein–lipid complexes that kill cancer cells broadly, while sparing healthy, differentiated cells. Many Despite the molecular complexity of oncogenic trans- and diverse tumor cell types are sensitive to the lethal formation, cancer cells frequently suffer from ‘oncogene effect, suggesting that HAMLET identifies and activates addiction’, and the disruption of a single oncogene can conserved death pathways in cancer cells. Here, we either reverse oncogenesis or be lethal (Weinstein and investigated the molecular basis for the difference in Joe, 2008). The c-Myc oncogene is a classic example sensitivity between cancer cells and healthy cells. Using a (Felsher and Bishop, 1999) and is deregulated in at least combination of small-hairpin RNA (shRNA) inhibition, 40% of all human cancers (Dang et al., 2009). The proteomic and metabolomic technology, we identified the c- broad transforming effect of c-Myc has been explained Myc oncogene as one essential determinant of HAMLET by its ability to bind to promoters of at least 30% of all sensitivity. Increased c-Myc expression levels promoted known genes (Dang et al., 2009), and in transgenic mice, sensitivity to HAMLET and shRNA knockdown of c-Myc c-Myc overexpression combined with inhibition of suppressed the lethal response, suggesting that oncogenic apoptosis is sufficient to drive pancreatic cancer forma- transformation with c-Myc creates a HAMLET-sensitive tion (Pelengaris et al., 2002). More recently, inhibition phenotype. Furthermore, HAMLET sensitivity was mod- of c-Myc was proposed to stop cancer growth and even ified by the glycolytic state of tumor cells. Glucose allow tissue repair and reversion to a functional deprivation sensitized tumor cells to HAMLET-induced phenotype (Soucek et al., 2008). Healthy cells, in cell death and in the shRNA screen, hexokinase 1 (HK1), contrast, retain a limited replicative potential, due to 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 1 and their susceptibility to programmed cell death, anti- hypoxia-inducible factor 1a modified HAMLET sensitiv- growth signals and other molecular interactions that ity. HK1 was shown to bind HAMLET in a protein array limit longevity and maintain normal tissue homeostasis containing B8000 targets, and HK activity decreased (Hanahan and Weinberg, 2000, 2011). within 15 min of HAMLET treatment, before morpholo- Oncogene activity may also cause a shift in the gical signs of tumor cell death. In parallel, HAMLET glycolytic machinery (Hsu and Sabatini, 2008), and triggered rapid metabolic paralysis in carcinoma cells. cancer cells are highly dependent on glycolysis (Mathu- Tumor cells were also shown to contain large amounts of pala et al., 1997). Glucose is first trapped inside cells by oleic acid and its derivatives already after 15 min. The hexokinases 1 and 2 (HK1 and HK2) (Wilson, 2003) and results identify HAMLET as a novel anti-cancer agent that then further converted to pyruvate. In contrast to killstumorcellsbyexploitingunifyingfeaturesofcancer healthy cells, cancer cells drive pyruvate to lactate cells such as oncogene addiction or the Warburg effect. conversion even in the presence of oxygen, a process Oncogene (2011) 30, 4765–4779; doi:10.1038/onc.2011.196; known as the Warburg effect (Warburg, 1956). Recent published online 6 June 2011 studies have shown that oxidative phosphorylation is functional in most cancer cells (Vander Heiden et al., Keywords: HAMLET; metabolism; c-Myc; glycolysis 2009) and have proposed that the Warburg effect may reflect the expression of the pyruvate kinase isoform M2 (PKM2) (Christofk et al., 2008), which catalyzes the dephosphorylation of phosphoenolpyruvate to pyruvate Correspondence: Professor C Svanborg, Department of Microbiology, and is responsible for net ATP production within the Immunology and Glycobiology (MIG), Institute of Laboratory glycolytic sequence. In contrast to healthy differentiated Medicine, Lund University, So¨lvegatan 23, S-223 62 Lund, Sweden. cells, embryonic cells and most cancer cells mainly E-mail: [email protected] express the M2 isoform (Mazurek et al., 2005), and in 5Shared first author. Received 22 December 2010; revised and accepted 19 April 2011; some tumors, replacement of M2 by M1 reverses the published online 6 June 2011 Warburg effect and reduces tumorigenicity (Christofk Metabolism and c-Myc defines HAMLET sensitivity P Storm et al 4766 et al., 2008). In addition to PKM2, c-Myc has a A small number of shRNA vectors were enriched or fundamental role in reprogramming metabolism in depleted in the population treated with HAMLET; 43 certain tumor cells and has been shown to enhance inhibitory and 44 sensitizing shRNAs showed an aerobic glycolysis by directly activating genes, such as absolute MaxMean score of 40.5 in both high- and HK2, PKM1 and LDHA (Shim et al., 1997; Dang et al., low-dose treatment groups or an absolute score of 42in 2009). The transcription factor hypoxia-inducible factor any group (Figure 1b and Supplementary Table S1). The 1 (HIF1) also regulates glycolysis by enhancing the identified genes included oncogenes like c-Myc and expression of glucose transporters, HKs and phospho- glycolysis-related proteins. shRNAs against HK1 and fructokinases (PFKs) (Denko, 2008). HIF1a sensitized the cells to HAMLET (Figures 1c and HAMLET is a complex of partially unfolded a- d), suggesting that increased HK1 and HIF1a expres- lactalbumin and oleic acid, which preferentially kills sion drives a phenotype more resistant to HAMLET. In cancer cells and immature cells (Svanborg et al., 2003). contrast, shRNA targeting 6-phosphofructo-2-kinase/ The broad tumoricidal activity includes leukemia cells, fructose-2,6-biphosphatase (PFKFB1) and c-Myc res- carcinoma cells and glioma cells, whereas healthy, cued the cells (Figures 1c and d). Biological pathways differentiated cells survive HAMLET challenge (Fischer enriched for by HAMLET were identified by gene set et al., 2004). HAMLET thus appears to target highly enrichment analysis (GSEA) using the canonical path- conserved survival mechanisms that are crucial for ways gene set collection. The GSEA algorithm tests for cancer cells. This selectivity has been confirmed in vivo the enrichment of a set of items within a larger ranked in a human glioblastoma xenograft model and in a list of such items. The identified pathways confirmed the murine bladder cancer model (Fischer et al., 2004; importance of glycolysis, as four glycolysis-related Mossberg et al., 2010). In clinical studies of skin pathways significantly modified HAMLET sensitivity papillomas and bladder cancers (Gustafsson et al., (Figures 1e and f). In addition, the Ras pathway, which 2004; Mossberg et al., 2007) HAMLET showed governs c-Myc expression, was identified as a determi- tumoricidal activity with limited side effects. nant of HAMLET sensitivity (Figure 1f). This study investigated the molecular basis of HAM- The shRNA screen suggested that oncogenic trans- LET sensitivity in cancer cells, using screening techni- formation by c-Myc sensitizes cells to HAMLET- ques to detect features of transcription, protein induced cell death and that the glycolytic machinery interaction and metabolism that alter survival after influences HAMLET sensitivity of cancer cells. HAMLET exposure. We show that sensitivity to HAMLET is dependent on c-Myc and glycolysis and identified the glycolytic enzyme HK1 as a potential The c-Myc oncogene determines the tumoricidal effect direct target of HAMLET. The results also indicated of HAMLET that HAMLET alters cancer cell metabolism and that it We subsequently examined whether HAMLET sensitiv- may disrupt glycolysis, which is altered in cancer cells ity reflects c-Myc expression levels. Human lung due to the Warburg effect. carcinoma cells (A549), kidney carcinoma cells (A498) and semi-differentiated healthy kidney cells (human renal proximal tubule cells) were exposed to HAMLET Results (21 mM), and ATP levels monitored (Figure 2a). A rapid reduction in ATP levels was observed in lung and kidney An shRNA screen identifies c-Myc and glycolytic-related carcinoma cells but healthy cells remained unaffected proteins as modifiers of HAMLET sensitivity after 3, 6 and 24 h. However, at higher HAMLET To get an unbiased overview of genes important for concentrations, a partial response was also seen in those HAMLET sensitivity, we used the RNA interference cells (Supplementary Figure S1A). The difference in c- barcode screening technology (Brummelkamp and Myc expression reflected the difference in HAMLET Bernards, 2003; Silva et al., 2005, 2008). Barcode sensitivity, as lung and kidney carcinoma cells show screens use DNA microarrays to follow the relative