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Critical Role for Transcriptional Repressor Snail2 in Transformation by Oncogenic RAS in Colorectal Carcinoma Cells

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Critical Role for Transcriptional Repressor Snail2 in Transformation by Oncogenic RAS in Colorectal Carcinoma Cells Oncogene (2010) 29, 4658–4670 & 2010 Macmillan Publishers Limited All rights reserved 0950-9232/10 www.nature.com/onc ORIGINAL ARTICLE Critical role for transcriptional repressor Snail2 in transformation by oncogenic RAS in colorectal carcinoma cells Y Wang1,4, VN Ngo2, M Marani1, Y Yang2, G Wright3, LM Staudt2 and J Downward1 1Signal Transduction Laboratory, Cancer Research UK London Research Institute, London, UK; 2Metabolism Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA and 3Biometric Research Branch, DCTD, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA Activating mutations in the KRAS gene are among RAS-activating mutations, especially KRAS muta- the most prevalent genetic changes in human cancers. tions, are one of the most prevalent genetic changes To identify synthetic lethal interactions in cancer cells found in cancer, occurring in about 20% of human harbouring mutant KRAS, we performed a large-scale tumours (Downward, 2003; Karnoub and Weinberg, screen in isogenic paired colon cancer cell lines that differ 2008). In these tumours, the activated RAS protein by a single allele of mutant KRAS using an inducible short contributes significantly to several aspects of the hairpin RNA interference library. Snail2, a zinc finger malignant phenotype, including the deregulation of transcriptional repressor encoded by the SNAI2 gene, was tumour cell growth, invasiveness and the ability to found to be selectively required for the long-term survival induce new blood-vessel formation, and the suppression of cancer cells with mutant KRAS that have undergone of programmed cell death. Thus, identifying synthetic epithelial–mesenchymal transition (EMT), a transdiffer- lethal genetic interactions in the context of mutant entiation event that is frequently seen in advanced tumours KRAS would provide additional drug targets for and is promoted by RAS activation. Snail2 expression therapeutic exploration and also shed new light on is regulated by the RAS pathway and is required for RAS signalling pathways (Cully and Downward, 2008). EMT. Our findings support Snail2 as a possible target With the advent of RNA interference technology, it for the treatment of the broad spectrum of human cancers has become possible to systematically determine the of epithelial origin with mutant RAS that have undergone functional consequence of gene suppression in cancer EMT and are characterized by a high degree of cell lines (Downward, 2004; Bernards et al., 2006; Iorns chemoresistance and radioresistance. et al., 2007). Isogenic paired cancer cell lines that differ Oncogene (2010) 29, 4658–4670; doi:10.1038/onc.2010.218; by a single oncogenic lesion can be used to identify published online 21 June 2010 potential targets for selectively killing tumour cells. In this study, we screened a small hairpin RNA Keywords: KRAS; synthetic lethal; oncogene addiction; (shRNA) library for those genes the inhibition of which epithelial–mesenchymal transition shows synthetic lethality with the KRAS oncogene. Using paired colon cancer cell lines that differ in the expression of mutant KRAS, we identified a zinc finger transcriptional repressor, Snail2, which is selectively Introduction required for the survival of cancer cells with mutant KRAS. We further showed that Snail2 is regulated by the The concept of synthetic lethality was originally defined RAS pathway and is very important for the epithelial– in fruit fly genetics (Dobzhansky, 1946) and was mesenchymal transition (EMT) initiated in part by RAS elaborated in a series of yeast genetic studies by pathway activation. Our findings also support Snail2 as a Hartwell et al., (1997). Synthetic lethality occurs when target for treatment of a broad spectrum of human alteration of a gene or treatment with a drug results in cancers that have undergone EMT, associated at least in cell death only in the presence of another nonlethal part with mutational activation of RAS. genetic alteration, such as a cancer-associated mutation (Kaelin, 2005). Targeting a gene that is synthetic lethal to a cancer-specific mutation should kill only cancer cells and spare normal cells without such a mutation. Results Correspondence: Dr J Downward, Signal Transduction Laboratory, Identification of genes required for survival in cells Cancer Research UK London Research Institute, 44 Lincoln’s Inn with mutant KRAS Fields, London WC2A 3PX, UK. To identify those genes the targeting of which selectively E-mail: [email protected] kills cancer cells with an activating KRAS mutation, we 4Current address: Ludwig Institute for Cancer Research, University of Oxford Branch, Oxford OX3 7DQ, UK. performed large-scale loss-of-function RNA interference Received 16 October 2009; revised 29 April 2010; accepted 6 May 2010; screens using a pair of human isogenic colon cancer cell published online 21 June 2010 lines containing a mutant KRAS allele (HCT-116 Snail2 in RAS induced EMT YWanget al 4659 parental) or only wild-type (wt) KRAS (HKe-3 isogenic HCT-116 cells. To expand our repertoire of shRNAs counterpart). HCT-116 cells carry an endogenous for functional validation studies, we also constructed a activating KRAS G13D point mutation required for fourth Snail2 shRNA that effectively decreased the maintaining their oncogenic state. Their isogenic coun- expression of its cognate mRNA (Gupta et al., 2005; terpart, HKe-3, was created by genetic disruption of Supplementary Figure S1a). the activated KRAS allele and is impaired in both To confirm and extend the results from the bar-code anchorage-independent growth and the ability to form screen, we inducibly expressed shRNAs targeting Snail2 tumours in mice (Shirasawa et al., 1993). We screened in HCT-116, HKe-3 and HKh-2 cells. HKh-2 cells each cell line with a doxycycline-inducible retroviral are another isogenic counterpart of HCT-116 cells, in shRNA library targeting 2500 human genes, including which the activated KRAS allele was also disrupted the majority of known protein kinases and cancer- using targeted homologous recombination (Shirasawa related genes. The library was screened in six pools using et al., 1993). In agreement with the primary screen, the a protocol described previously (Ngo et al., 2006; knockdown of Snail2 killed HCT-116 cells much more Shaffer et al., 2008). We analysed the change in the than HKe-3 cells (Po0.05), and HKh-2 cells were also bar code abundance of each shRNA by microarray to relatively resistance to Snail2 knockdown compared identify those that are essential for cell survival and with HCT-116 cells (Figure 1b; Po0.05). In addition, are thus depleted from the surviving cell population. We we found that shRNA-mediated knockdown of Snail2 compared the lethality signature of HCT-116 and HKe- expression in HCT-116 cells by two different shRNAs 3 cells to identify those shRNAs showing selective also severely impaired colony formation in soft agar depletion in the KRAS mutant, but not in KRAS wt (Figure 1c), thus confirming the inhibition of Snail2 cells. The strongest hit that we found to be selectively to be sufficient for suppressing the malignant phenotype lost from HCT-116 cells relative to Hke-3 cells was of HCT-116 cells. Snail2, shRNAs targeting which were depleted from To further determine whether our finding of a HCT-116 cultures by twofold (Figure 1a), but were correlation between mutant KRAS dependency and not lost from HKe-3 cell cultures (P40.05). Two out sensitivity to Snail2 knockdown is true in a different of three Snail2 shRNAs were selectively lost from system, we investigated the effects of Snail2 suppression Snail2 Snail2 shRNA-3 1.5 HCT 116 2 2 HKe 3 HKh 2 1 1.0 0 * shRNA-1 shRNA-2 shRNA-3 0.5 -1 * Live cell number -2 HCT 116 (induced/uninduced) HKe 3 0.0 -3 Barcode abundance log 0246810 [shRNA-uninduced/induced] Days after shRNA induction HCT 116 1.5 1.5 ** ** 1.0 1.0 SW48 wt SW48 KRAS G13D 0.5 0.5 Colony formation Normalized viability (induced/uninduced) 0.0 0.0 Snail2 Snail2 1 2 shRNA-3 shRNA-4 Snail2 siRNA Lipid only RISC free control-shRNA Figure 1 Snail2 is required for the survival of cells with mutant KRAS. (a) HCT-116 and HKe-3 cells were screened using a retrovirally delivered, doxycycline-inducible, shRNA library to identify genes required for cell survival. Depletion of cells bearing three Snail2-targeted shRNAs in shRNA-uninduced versus induced cells is plotted; error bars represent the standard deviation of triplicate measurements. (b) A Snail2 shRNA is more toxic to HCT-116 cells compared with HKe-3 and HKh-2 cells. A vector for inducible expression of Snail2 shRNA was introduced into cell lines and cell numbers were monitored at indicated days after doxycycline addition. Data were the ratio of live cell number in shRNA-induced versus uninduced cells. *Po0.05; error bars indicate s.d. (c) Snail2 knockdown causes strong inhibition effects on soft agar colony formation of HCT-116 cells. Data were the ratio of soft agar colonies in shRNA-induced versus uninduced cells. Error bars represent the s.d. of triplicates. (d) Snail2 knockdown is more toxic to SW48 KRAS G13D cells compared with SW48 wt cells. siRNAs against Snail2 were transfected into both cell lines and cell viability was measured. **Po0.01; error bars indicate s.d. Oncogene Snail2 in RAS induced EMT Y Wang et al 4660 ** 2.0 ** HCT 116 * 3 * HKe 3 SW48 wt * 1.5 HKh 2 SW48 KRAS G13D 2 1.0 1 against HKe 3 0.5 against SW48 wt Relative expression Relative expression (normalized by GAPDH) 0.0 (normalized
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