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

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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

against HKe 3 0.5 against SW48 wt Relative expression Relative expression (normalized by GAPDH) 0.0 (normalized by GAPDH) 0 Snail2 Snail1 Snail2 Snail1

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Snail1 Snail1 HCT-116 KRAS shRNA 4 4 1.5 Day 0 Day 3 3 3 Day 6 1.0 2 2 0.5 1 1 against mock against mock against uninduced Relative expression Relative expression 0 Relative expression 0 0.0 (normalized by GAPDH) (normalized by GAPDH) (normalized by GAPDH) 6 hours Day 1 Day 2 Day 3 0 12.5 25 50 Snail2 Snail1 OHT 50nM OHT (nM) Figure 2 Snail2 and Snail expression levels are regulated by the RAS pathway. (a) mRNA expression of Snail2 or Snail1 is higher in HCT-116 than in HKe-3 and HKh-2 cells. *Po0.05; **Po0.01; error bars indicate s.d. (b) mRNA expression of Snail2, but not of Snail1, is higher in SW48 KRAS G13D cells, compared with SW48 wt cells. *Po0.05; error bars indicate s.d. (c) Protein expression of Snail2 and Snail1 in HCT-116/HKe-3/HKh-2 cells, SW48 wt KRAS G13D cells and HKe-3 ER:HRAS V12 cells treated with or without OHT. (d) Snail2 mRNA is regulated by RAS pathway in a dose-dependent manner. HKe-3 ER:HRAS V12 cells were treated with different concentrations of OHT and Snail2 expression was measured by quantitative reverse transcriptase PCR after 3 days of OHT treatment, normalized to ethanol (EtOH)-treated cells. Error bars represent the s.d. of triplicates. (e) Snail2 mRNA is regulated by the RAS pathway in a time-dependent manner. HKe-3 ER:HRAS V12 cells were treated with 50 nM OHT, and Snail2 expression was measured by quantitative reverse transcriptase PCR (RT–PCR) at indicated time points after OHT treatment, normalized to EtOH-treated cells. Error bars represent the s.d. of triplicates. (f, g) Snail2 expression induced by RAS pathway activation is inhibited by LY294002 or UO126. HKe-3 ER:HRAS V12 cells were pretreated with LY294002 (16 mM) or UO126 (10 mM) for 30 min, then 50 nM OHT was introduced together with these inhibitors. After 6 h, Snail2 expression was measured by quantitative RT–PCR (f)orby western blots (g). Snail2 mRNA level was normalized to EtOH-treated cells. Error bars represent the s.d. of triplicates. LY, LY294002; UO, UO126. (h) Snail1 mRNA is regulated by RAS pathway in a dose-dependent manner. HKe-3 ER:HRAS V12 cells were treated with different concentrations of OHT, and Snail1 expression was measured by quantitative RT–PCR after 3 days of OHT treatment, normalized to EtOH-treated cells. Error bars represent the s.d. of triplicates. (i) Snail1 mRNA is regulated by RAS pathway in a time- dependent manner. HKe-3 ER:HRAS V12 cells were treated with 50 nM OHT and Snail1 expression was measured by quantitative RT–PCR at indicated time points after OHT treatment, normalized to EtOH-treated cells. Error bars represent the s.d. of triplicates. (j) Snail2 expression was reduced following KRAS knockdown. HCT-116 KRAS shRNA cells were treated with doxycycline. Snail2 or Snail1 expression was measured by quantitative RT–PCR at indicated time points and normalized to uninduced cells. Error bars represent the s.d.

Oncogene Snail2 in RAS induced EMT YWanget al 4661 in another colon cancer cell pair. SW48 cells are wt for et al., 2000; Katoh, 2005; Cobaleda et al., 2007; Peinado KRAS, whereas SW48 KRAS G13D cells were created et al., 2007). Snail1 mRNA expression is also regulated by knock-in of an activating mutation into one KRAS by RAS activation, but to a much lesser extent than allele (Di Nicolantonio et al., 2008). We found that Snail2. Snail1 expression has previously been reported Snail2 knockdown using two small interfering RNA to be induced by RAS and transforming growth factor-b (siRNA) oligos differentially killed SW48 KRAS G13D (TGF-b) signalling through pathways involving both cells, but had a minimal effect on SW48 wt cells MAP kinase and PI3-kinase activities (Peinado et al., (Figure 1d, Po0.01), even though Snail2 knockdown 2003; Barbera et al., 2004). With the ER:HRAS V12 effects were stronger in SW48 wt cells (Supplementary system in HKe-3 cells, we showed that the Snail1 Figure S1b). mRNA level was increased around 2.5-fold with 50 nM These experiments showed that Snail2 is preferentially OHT after 3 days of induction (Figures 2h and i). Snail1 required by cells that rely on mutant KRAS for their expression was also regulated by RAS both in a dose- survival, but not otherwise by isogenic cells harbouring dependent and time-dependent manner. Snail1 expres- wt KRAS. sion level was relatively higher in HCT-116 cells than in both HKe-3 and HKh-2 cells (Figures 2a and c), but expression of Snail1 was not increased in SW48 KRAS Snail2 and Snail1 expression levels are both regulated G13D cells (Figures 2b and c). by RAS The regulation of Snail2 by RAS was further Our observations suggested a causal relationship be- confirmed by knocking down of KRAS in HCT-116 tween the presence of a transforming RAS mutation and cells. Following doxycycline treatments in HCT-116 the requirement for Snail2. To confirm whether Snail2 KRAS shRNA cells, the expression of KRAS was expression is regulated by RAS activity, we first checked decreased (Figure 4a). Meanwhile, Snail2 mRNA Snail2 mRNA and protein level in HCT-116/HKe-3/ expression was markedly reduced on day 6 after HKh2 cells and SW48 wt/SW48 KRAS G13D cells. We induction of KRAS shRNA, whereas expression of found that, in HCT-116 cells, Snail2 mRNA expression Snail1 was not significantly altered (Figure 2j). was around 1.5-fold that seen in HKe-3 cells (Figure 2a; Overall, these data suggest that the RAS pathway Po0.05), whereas Snail2 expression in HKh-2 cells was regulates Snail2 expression and also the expression of very low (Figure 2a). Expression of Snail2 was also Snail1, although to a much lesser extent. Induction of higher in SW48 KRAS G13D cells, around 2.5-fold Snail2 expression by RAS pathway activation can be higher than that in SW48 wt cells (Figure 2b). Similar blocked by inhibition of either the PI3K or mitogen- proteinexpressionpatternwasalsoobservedinthese activated protein kinase pathway. cells (Figure 2c). These data suggest that cells with mutant KRAS tend to have a higher expression of Snail2 compared with their isogenic counterparts with RAS pathway activation leads to EMT wt KRAS. As expression of activated mutant RAS in the HCT-116 We then introduced into HKe-3 cells a regulatable cell system leads to elevated expression of Snail2 and RAS construct made up of mutant HRAS fused to the Snail1, both very important mediators of the transdif- oestrogen receptor (ER) ligand-binding domain that is ferentiation process known as EMT (Hemavathy et al., conditionally responsive to 4-hydroxytamoxifen (OHT; 2000; Nieto, 2002), we next sought to determine Dajee et al., 2002). Addition of OHT acutely activates whether these cells expressing mutant RAS have under- the RAS pathway in HKe-3 cells expressing ER:HRAS gone EMT. V12. As shown in Figures 2c–e, Snail2 protein and To test this hypothesis, we performed microarray mRNA level were massively increased after activating analysis in HCT-116/HKe-3/HKh-2 cells. We found the RAS pathway, with more than a 60-fold change in HCT-116 cells have increased levels of mesenchymal mRNA after 3 days of treatment with 50 nM OHT. markers such as VCAN, EFNB2, and lower levels of Snail2 expression was upregulated as early as 6 h epithelial markers such as CDH1, OCLN and CLDN3, (Figure 2e). To determine whether the mitogen-activated compared with HKe-3 and HKh-2 cells (Figure 3a), protein kinase or phosphatidylinositol 3-kinase (PI3K) suggesting that HCT-116 cells have undergone at least a pathway is involved in RAS-mediated activation of partial EMT relative to their mutant KRAS-deleted Snail2 expression, HKe-3 ER:HRAS V12 cells were derivatives. This point was also confirmed by E-cadherin treated with OHT and inhibitors for these pathways. staining in HCT-116/HKe-3/HKh-2 cells. The alterna- Treatment with the MEK inhibitor UO126 partially tive EMT inducers, ZEB2, Twist and E47, do not show abolished Snail2 mRNA and protein upregulation significant differences in expression under these different because of activation of the RAS pathway, and the conditions (data not shown). As shown in Figure 3b, PI3K inhibitor LY294002 could completely abolish there was much less E-cadherin membrane staining in these effects (Figures 2f and g). Together, these results HCT-116 cells compared with HKe-3 and HKh-2 cells. show that Snail2 mRNA expression is regulated by This was also the case in SW48 KRAS G13D, relative to RAS, with requirement for both the PI3K and mitogen- wt SW48 (Figure 3c). In HKe-3 ER:HRAS V12 cells, activated protein kinase pathways. RAS pathway activation by adding OHT resulted in a Snail2 belongs to the Snail superfamily of zinc finger marked reduction in E-cadherin and a scattered cellular transcription repressors, along with Snail1 (Hemavathy phenotype (Figures 3c and d). These results were

Oncogene Snail2 in RAS induced EMT Y Wang et al 4662 consistent with a recent microarray analysis in a human This point was further strengthened using the colon cancer cell system (Joyce et al., 2009), showing ER:HRAS V12 system in HKe-3 cells. E-cadherin is that human colon cancer cells with mutant RAS have a an important component of the epithelial phenotype molecular signature for EMT that is reﬂected in many and downregulation of E-cadherin is of direct relevance markers such as E-cadherin and Snail2. to EMT (Thiery and Sleeman, 2006). Throughout the

HCT 116 HKe 3 HKh 2 Microarray data 4 HCT 116 3 HKe 3 HKh 2 2

0 Relative abundance to HCT 116 KRAS ZEB1 CDH1 VCANEFNB2CLDN3 OCLN Mesenchymal Epithelial markers markers

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Oncogene Snail2 in RAS induced EMT YWanget al 4663 course of activated RAS induction in these cells at least a partial EMT, and knockdown of Snail2 (confirmed by increased phosphorylation of ERK and expression reverses the mesenchymal status. AKT, Figures 3f and h), E-cadherin mRNA expression and protein level were both strongly decreased in a dose- dependent and time-dependent manner (Figures 3e–h). Cells that have undergone EMT require continued Snail2 After 3 days of 50 nM OHT treatment, the mRNA level and Snail1 expression of E-cadherin was reduced to only 20% compared with Using the Oncomine research online tool (www. mock treatment, whereas the protein level was reduced oncomine.com; Supplementary Figure S2), we found to less than half. an inverse correlation between Snail2 expression and This was also confirmed by knocking down KRAS in E-cadherin expression in many types of cancer. In breast HCT-116 cells. After doxycycline treatment of HCT-116 cancer cell lines, BT-549 and Hs-578-T cells have KRAS shRNA cells to inhibit KRAS expression, undergone EMT as shown by reduced expression of microarray data showed that epithelial markers E-cadherin and higher abundance of Snail2 expression (such as CDH1, OCLN and CLDN3) were upregulated, compared with MCF7 and T47D cells (Figures 5a–c), whereas some mesenchymal markers (such as VCAN consistent with previous reports (Hajra et al., 2002; and EFNB2, although not ZEB1) were reduced Come et al., 2006). Of these lines, only Hs-578-T cells (Figure 4a), suggesting that HCT-116 cells underwent carry an activating mutation in a RAS oncogene mesenchymal–epithelial transition after KRAS knock- (HRAS). Expression of Snail2, rather than that of down. This was further supported by E-cadherin Snail1, was strongly correlated with the reduced staining in HCT-116 KRAS shRNA cells treated with expression of E-cadherin (Figures 5a–c), implying that or without doxycycline (Figure 4b). Snail2 is more likely to be a significant in vivo repressor As RAS pathway activation leads to massive upregu- of E-cadherin transcription in breast cancer. lation of Snail2 expression with simultaneous reduction To investigate whether cells that have undergone of E-cadherin levels (Figures 2 and 3), we attempted EMT are more sensitive to Snail2 or Snail1 knockdown, to knock down Snail2 expression in HKe-3 ER:RAS we introduced siRNAs against Snail2 or Snail1 into cells treated with OHT. We found that even at very low these four breast cancer cells. SiRNA-induced knock- concentrations of OHT, Snail2 mRNA level was still down of Snail1 or Snail2 (Supplementary Figures S1g strongly elevated, whereas E-cadherin mRNA expres- and h) significantly killed BT-549 and Hs-578-T cells. sion was reduced to half (Figures 4c and d). These data Cell viability was reduced to around 50% in both cell further confirmed that RAS pathway activation in- lines, with two independent siRNA oligos against Snail1 creases Snail2 expression and leads to EMT. The Snail2 or Snail2 (Figure 5d). We also observed more apoptosis siRNA oligo caused a significant decrease in Snail2 after Snail1 or Snail2 knockdown in both cell lines, as mRNA level (Figure 4c), and Snail2 knockdown shown by caspase-3 assays (Figure 5e). In contrast, there allowed upregulation of E-cadherin mRNA, despite were no significant effects of Snail1 or Snail2 knock- activated mutant RAS expression (Figure 4d). Both down on cell viability or apoptosis in MCF7 or T47D HCT-116 and SW48 KRAS G13D cells express mutant cells. These data confirmed that breast cancer cells that RAS and have less E-cadherin compared with their have undergone some degree of EMT are more sensitive isogenic counterparts with wt RAS. Knockdown of to Snail2 or Snail1 knockdown than primarily epithelial Snail2 expression caused a return of E-cadherin protein breast cancer cells. expression in both HCT116 and SW48 KRAS G13D This conclusion was further strengthened by the cells (Figures 4e–h), whereas knockdown of Snail1 study of a pair of KRAS mutant colon cancer cell expression in HCT-116 cells did not have much effect lines, SW480 and SW620, derived from the same on E-cadherin expression (Figure 4f). Snail2 knockdown patient; SW480 was from the primary tumour site and had no effect on KRAS expression or activation, or the SW620 was from a metastatic site. The metastatic phosphorylation state of ERK or Akt in HCT-116 or tumour line SW620 has a more mesenchymal phenotype SW48 cell systems (data not shown). as shown by a lower E-cadherin expression and a higher Our above data confirm the hypothesis that some level of Snail2 and Snail1 than the primary tumour line carcinoma cell lines with mutant RAS have undergone SW480 (Figure 5f). SW620 cells were more sensitive to

Figure 3 RAS pathway activation leads to EMT process. (a) Microarray data show higher levels of mesenchymal markers (ZEB1, VCAN, EFNB2), lower levels of epithelial markers (CDH1, OCLN, CLDN3) and KRAS expression in HCT-116 cells compared with HKe-3 and HKh-2 cells. (b) Immunoﬂuorescence staining of E-cadherin in HCT-116/HKe-3/HKh-2 cells (upper panel). Lower panel: phase contrast pictures. (c) Western blot analysis of lysates from SW48 wt and SW48 KRAS G13D cells or from HKe-3 ER:HRAS V12 cells after OHT treatment, showing effects on E-cadherin, occludin, ZEB1 and activation status of ERK. p, phospho. (d) Immunoﬂuorescence staining of HRAS and E-cadherin in HKe-3 ER:HRAS V12 cells treated with or without OHT. (e, f) E-cadherin is regulated by the RAS pathway in a dose-dependent manner. HKe-3 ER:HRAS V12 cells were treated with different concentrations of OHT, and E-cadherin expression was measured by quantitative RT–PCR (e) or western blot analysis (f) after 3 days of OHT treatment. Error bars represent the s.d. of triplicates. (g, h) E-cadherin is regulated by the RAS pathway in a time-dependent manner. HKe-3 ER:HRAS V12 cells were treated with OHT, and E-cadherin expression was measured by quantitative RT–PCR (g) or by western blot analysis (h) at indicated time points after OHT treatment. Error bars represent the s.d. of triplicates. In f and h, for western blot analysis, the data are representative of three independent experiments.

Oncogene Snail2 in RAS induced EMT Y Wang et al 4664 Snail1 or Snail2 knockdown than SW480 cells 2 weeks resulted in a marked reduction of E-cadherin (Figure 5g). (at both mRNA and protein level), coupled with an To show a causal relationship between Snail2 or increased expression of mesenchymal markers ZEB1 Snail1 dependency and the mesenchymal phenotype, we and vimentin (Figures 5h and i), and a scattered cellular tested the possibility that induction of EMT could phenotype (data not shown). These cells are typical in induce Snail1 or Snail2 dependency. Exposure of KRAS showing that RAS mutation alone is not sufﬁcient to mutant H358 cells to TGF-b1, a promoter of EMT, over induce EMT, but that it requires coordinate activation

HCT-116 KRAS shRNA HCT-116 KRAS shRNA-Microarray data EtOH Doxycycline 2.0 Day 0 Day 3 1.5 Day 6

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SW48 HCT-116 HKe-3 HKh-2 Snail2 shRNA wt KRAS G13D Doxycycline ---+++ Lipid Snail2 Lipid Snail2 only siRNA only siRNA E-Cadherin E-Cadherin GAPDH GAPDH

Oncogene Snail2 in RAS induced EMT YWanget al 4665 of other pathways, such as TGF-b signalling. We down of Snail2 or Snail1 significantly sensitizes them to observed a marked increase in Snail2 expression after treatment with this drug, to a level comparable with that TGF-b1 treatment in H358 cells and also a moderate seen for breast cancer cell lines with a more epithelial increase in Snail1 expression (Figure 5h), suggesting phenotype. potential roles of Snail2 and/or Snail1 in TGF-b1- induced EMT in H358 cells. Whereas parental H358 cells seemed resistant to Snail2 or Snail1 knockdown, mesenchymal H358 human TGF-b cells responded to Discussion the siRNAs against Snail2 or Snail1 with reduction in viability (Figure 5j; Po 0.05). Functional RNA interference cell-based screens for Our above data indicate that cells that have under- genes that are differentially required, dependent on a gone EMT in part because of RAS mutation or other particular genotype or phenotype, have been performed pathways have relatively high Snail2 or Snail1 expres- recently by several groups (Ngo et al., 2006; Shaffer sion and are sensitive to their knockdown. et al., 2008; Luo et al., 2009; Scholl et al., 2009). The primary screen in this study was similar in design to that Synergistic loss of viability due to Snail2 or Snail1 of Luo et al., using a pooled shRNA library and knockdown and DNA-damaging drug treatment an isogenic colon cancer cell line pair differing only in We next tried to investigate whether there are synergistic the presence or absence of an activated mutant KRAS effects between Snail2 or Snail1 knockdown and allele. shRNAs that were selectively toxic to KRAS treatment with the DNA-damaging anthracycline drug, mutant cells were identified by comparative hybridiza- doxorubicin. tion on custom microarrays. However, for reasons that As shown in Figures 6a and b, breast cancer cell are not immediately obvious, the hits identified in our lines BT-549 and Hs-578-T, which have a mesenchymal screen and that of Luo et al. did not overlap. phenotype, were relatively resistant to doxorubicin The strongest hit that we identified in this screen, as treatment, whereas two other breast cancer cell lines selectively required for the survival of mutant KRAS, with an epithelial phenotype, MCF-7 and T47D, were but not KRAS wt, colon cancer cells, was the zinc finger sensitive. Cell viability of MCF-7 and T47D was transcriptional repressor Snail2, which is encoded by reduced to a minimum of around 50% at the indicated the SNAI2 gene. Snail2 belongs to the Snail superfamily concentrations of doxorubicin (Figure 6a), and apopto- of zinc finger transcriptional repressors. It is an sis was induced in these two lines (Figure 6b). There important regulator of EMT and has a significant were no significant effects after doxorubicin treatment in role in the malignant progression of cancer (Cobaleda BT-549 and Hs-578-T cells. et al., 2007; Peinado et al., 2007; Alves et al., 2009). To determine the effects of Snail2 or Snail1 knock- Snail2-overexpressing mice develop mesenchymal tu- down in combination with doxorubicin treatment, mours, whereas Snail2-deficient mice are resistant to siRNAs against Snail2 or Snail1 were transfected into BCR-ABL-induced leukaemogenesis (Perez-Mancera BT-549 or Hs-578-T cells and, 24 h later, cells were et al., 2005). Snail2 overexpression has been described treated with doxorubicin. Without Snail2 or Snail1 in a wide spectrum of human cancers including breast knockdown, no significant killing effects were observed cancer, colorectal cancer, gastric carcinoma, oesopha- in BT-549 cells after doxorubicin treatment at 90 or geal carcinoma and other tumour types and seems to be 180 nM (Figures 6c and d). We found that transfections important for tumour progression toward invasion of two independent siRNA oligos against Snail2 and metastasis, in most cases involving E-cadherin or Snail1, in combination with doxorubicin treatment, downregulation (Cobaleda et al., 2007; Peinado et al., led to further reduced cell viability and more induction 2007; Alves et al., 2009). Snail2 expression has been of apoptosis (Figures 6c and d). Similar results were reported to be important for the metastatic spread of observed in Hs-578-T cells (Figures 6e and f). RAS mutant melanoma cells in a mouse model Thus, breast tumour cell lines that have undergone (Gupta et al., 2005) and to be an independent prognostic EMT and are of mesenchymal phenotype are relatively marker for poor survival in colorectal cancer patients resistance to doxorubicin treatment; however, knock- (Shioiri et al., 2006).

Figure 4 Knockdown of KRAS or Snail2 promotes reversal of EMT. (a) Microarray data show upregulation of epithelial markers (CDH1, OCLN, CLDN3) and downregulation of mesenchymal markers (VCAN, EFNB2) following KRAS knockdown in HCT-116 cells. (b) Immunofluorescence staining of E-cadherin in HCT-116 KRAS shRNA cells treated without or with doxycycline (25 ng/ml) for 1 week (upper panel). Lower panel: phase contrast pictures. (c, d) RAS pathway activation leads to increased Snail2 expression and reduced E-cadherin expression, and Snail2 knockdown promotes increased expression of E-cadherin. HKe-3 ER:HRAS V12 cells were transfected with siRNA against Snail2. After 24 h, cells were treated with 5 or 10 nM OHT for 48 h before being collected and measured by quantitative reverse transcriptase PCR, normalized to EtOH-treated cells. Error bars represent the s.d. of triplicates. (e–h) Snail2 knockdown enhances E-cadherin expression. (e) Immunofluorescence staining of E-cadherin in HCT-116 Snail2 shRNA cells treated without or with doxycycline (25 ng/ml) for 1 week (upper panel). Lower panel: phase contrast pictures. (f) Immunofluorescence staining of E-cadherin in HCT-116 cells transfected with siRNA against Snail2 or Snail1 for 72 h (upper panel). Lower panel: phase contrast pictures. (g) Western blot analysis of lysates from HCT-116/HKe-3/HKh-2 cells expressing an inducible Snail2 shRNA after doxycycline addition showing effects on E-cadherin. (h) Western blot analysis of lysates from SW48 wt/KRAS G13D cells transfected with RISC free or Snail2 siRNA, showing effects on E-cadherin.

Oncogene Snail2 in RAS induced EMT Y Wang et al 4666 Our observations indicate a relationship between ERK (Conacci-Sorrell et al., 2003; Wang et al.,2007) RAS signalling and Snail2 expression, with a down- and also by AKT (Saegusa et al., 2009). Snail2 knock- stream effect on the EMT state of the cell. Previous down does have a relatively modest effect on studies have shown that activated RAS can increase E-cadherin mRNA, even in cells without RAS activation, Snail2 expression in rat intestinal epithelial cells suggesting that there is at least some activity of Snail2 (Schmidt et al., 2005) and that Snail2 expression can be independent of elevated RAS signalling. It is possible that induced by multiple RAS effector pathways, by Raf and this could be due to basal, growth factor-stimulated RAS

BT 549 BT549Hs578T MCF7 T47D 1000 Hs-578-T 100 MCF 7 Snail2 10 T47D 1 Snail1 0.1 0.01 against MCF 7] 0.001 GAPDH lg[Relative expression 0.0001 E-Cadherin Snail1 Snail2

BT 549 Hs-578-T MCF-7 T47D

BT 549 Hs-578-T BT 549 1.5 8 Hs-578-T MCF 7 T47D MCF 7 6 T47D 1.0

0.5 2 Normalized viability Normalized apoptosis 0.0 0

1 2 1 2 1 2 1 2 Snail1 Snail2 Snail1 Snail2 Lipid OnlyRISC free siRNA siRNA Lipid OnlyRISC free siRNA siRNA Figure 5 Cells that have undergone EMT have high Snail2 and Snail1 expression and are sensitive to their knockdown. (a) mRNA expression of E-cadherin, Snail1 and Snail2 in four breast cancer cell lines. Error bars indicate s.d. (b) Protein expression of Snail1 and Snail2 in four breast cancer cell lines. (c) Immunoﬂuorescence staining of E-cadherin in four breast cancer cell lines (upper panel). Lower panel: phase contrast pictures. (d, e) Snail2 or Snail1 knockdown is more toxic to BT-549 and Hs-578-T cells than to MCF-7 and T47D cells. siRNAs against Snail2 or Snail1 were transfected into these cell lines and cell viability (d) or apoptosis (e) was measured 4 days after transfection. Error bars indicate s.d. (f) Expression of E-cadherin, Snail1 and Snail2 in SW480 and SW620 cells. Error bars indicate s.d. (g) Snail2 or Snail1 knockdown is more toxic to SW620 cells. siRNAs against Snail2 or Snail1 were transfected into both lines and cell viability was measured. *Po0.05; **Po0.01; error bars indicate s.d. (h, i) TGF-b induces EMT in H358 cells and increases the expression of Snail2 and Snail1. Expression of E-cadherin, Snail1 and Snail2 was measured after TGF-b treatment at indicated time points (h). Error bars indicate s.d. Western blot analysis with the indicated antibodies of lysates collected from H358 cells treated with or without TGF-b at indicated time points. (j) Snail2 or Snail1 knockdown is more toxic to H358 cells treated with TGF-b. H358 cells were treated with hTGF-b for 10 days, then transfected with siRNAs against Snail2 or Snail1 for 3 days. Cell viability was measured thereafter. *Po0.05; **Po0.01; error bars indicate s.d.

Oncogene Snail2 in RAS induced EMT YWanget al 4667 1.5 SW480 4 SW480 SW620 ** ** * ** SW620 3 1.0

2 0.5 1 Normalized viability (normalized by GAPDH) 0 0.0 E-Cadherin Snail1 Snail2 1 2 1 2 Relative expression against SW480 Snail1 Snail2 Lipid OnlyRISC free siRNA siRNA

H358 1 week 2 weeks 10 9 H358 control TGFβ -- ++-- 8 H358 TGFβ 1 week E-Cadherin 7 H358 TGFβ 2 weeks 6 5 ZEB1 4 3 2 Vimentin 1

Relative expression against 0

mock (normalized by GAPDH) GAPDH E-Cadherin Snail1 Snail2

1.5

* ** ** * 1.0 H358 control H358 TGFβ

0.5 Normalized viability 0.0

1 2 1 2 Snail1 Snail2 Lipid OnlyRISC free siRNA siRNA Figure 5 Continued. signalling, or signalling from other pathways. We find that factor in the ability of RAS pathway activation in expression of the related transcriptional regulator Snail1 is promoting EMT, with knockdown of Snail2 expression also induced by RAS, but to a much lesser extent at least partially reversing the mesenchymal phenotype. compared with Snail2. The appearance of Snail2 as the strongest hit in our Although we find that RAS activation can clearly KRAS synthetic lethal screen and its critical role in induce Snail2 expression in a given cell system, there is EMT, in which RAS has a part, suggests that the no evidence of a general correlation between RAS lethality associated with inhibition of Snail2 in HCT-116 mutational status and Snail2 expression levels in cells, but not in their mutant KRAS-deleted derivatives, tumours and cancer cell lines. This is likely due to the may be linked to forcing a reversal of EMT on cells that complexity of the regulation of Snail2 expression, with are at least part of the way toward transdifferentiation several pathways contributing to its control, including from epithelial to mesenchymal phenotypes. Targeting TGF-b, Wnt and p53 (Wu et al., 2005; Cobaleda et al., Snail2 is selectively damaging to RAS mutant cells with 2007; Peinado et al., 2007; Wang et al., 2009). It is well a more mesenchymal, rather than epithelial, phenotype. established that activation of RAS in epithelial cells can It has recently been found that the requirement of RAS cooperate with other pathways, such as TGF-b, to lead mutant cancer cell lines for continued expression of to EMT (Oft et al., 1996; Lehmann et al., 2000; Huber the RAS oncogene, so-called oncogene addiction, is et al., 2005; Larue and Bellacosa, 2005; Thiery and restricted to cells with an epithelial phenotype (Singh Sleeman, 2006). We find here that Snail2 is an important et al., 2009). By contrast, mesenchymal cells with

Oncogene Snail2 in RAS induced EMT Y Wang et al 4668 BT 549 1.5 Hs-578-T 3 BT 549 MCF 7 Hs-578-T T47D MCF 7 1.0 2 T47D

0.5 1 Normalized viability Normalized apoptosis 0.0 0 16 32 64 128 256 512 16 32 64 128 256 512

log2[Doxorubicin (nM)] log2[Doxorubicin (nM)]

BT 549 BT-549 Doxorubicin 0 nM 4 1.5 Doxorubicin 90 nM Doxorubicin 0 nM Doxorubicin 180 nM Doxorubicin 90 nM 3 1.0 Doxorubicin 180 nM 2

0.5 1 Normalized viability Normalized apoptosis 0.0 0 1 2 1 2 1 2 1 2 Snail1 Snail2 Snail1 siRNA Snail2 siRNA Lipid OnlyRISC free siRNA siRNA Lipid OnlyRISC free

Hs-578-T Hs-578-T Doxorubicin 0 nM 3 Doxorubicin 90 nM 1.5 Doxorubicin 0 nM Doxorubicin 180 nM Doxorubicin 90 nM 2 1.0 Doxorubicin 180 nM

0.5 1 Normalized viability Normalized apoptosis 0.0 0 1 2 1 2 1 2 1 2 Snail1 Snail2 Snail1 siRNA Snail2 siRNA Lipid OnlyRISC free Lipid OnlyRISC free siRNA siRNA Figure 6 Synergistic effects between Snail2 or Snail1 knockdown and doxorubicin treatment. (a, b) BT-549 and Hs-578-T cells are relatively resistant to doxorubicin treatments. Cell lines were treated with doxorubicin at indicated concentrations for 72 h and subjected to cell viability (a) and apoptosis assays (b). Error bars represent the s.d. of triplicate measurements. (c, d) Knockdown of Snail2 or Snail1 sensitizes BT-549 cells to doxorubicin treatments. BT-549 cells were treated 24 h after being transfected with siRNAs against Snail2 or Snail1 with the indicated concentration of doxorubicin for 48 h and subjected to cell viability (c) and apoptosis assays (d). Error bars represent the s.d. of triplicate measurements. (e, f) Knockdown of Snail2 or Snail1 sensitizes Hs-578-T cells to doxorubicin treatments. Hs-578-T cells were treated for 24 h after being transfected with siRNAs against Snail2 or Snail1 with the indicated concentration of doxorubicin for 48 h and subjected to cell viability (e) and apoptosis assays (f). Error bars represent the s.d. of triplicate measurements.

mutant RAS do not show RAS oncogene addiction, Snail1 knockdown by siRNA in breast carcinoma although this may reﬂect a broader resistance to cells has a somewhat greater effect on the induction induction of cell death associated with the mesenchymal of cell death than does the knockdown of Snail2. state; for example, the sensitivity of non-small-cell lung In contrast, in the colon cancer line SW620 and in the cancer cell lines that are wt for EGF receptor to EGF lung cancer line H358, Snail2 knockdown has the same receptor inhibitors correlates with their differentiation effect as Snail1 knockdown on induction of cell death, state, with mesenchymal cell lines being resistant possibly reﬂecting a tissue-type difference. It is certainly (Thomson et al., 2005). established that Snail1 provides an important part of the

Oncogene Snail2 in RAS induced EMT YWanget al 4669 signal leading to the establishment of the mesenchymal media containing 0.35% low-melting point agarose and phenotype. However, expression of Snail2 is markedly colonies were counted 2 weeks later with Giemsa stain. more responsive to input from RAS signalling pathways, suggesting that it may be more important than Snail1 in Quantitative reverse transcription–PCR the context of RAS mutation, as in colon and lung Real-time reverse transcriptase–PCR was performed using cancers with a mesenchymal phenotype, than in tumour gene-specific primers (QuantiTect Primer Assays) for Snail2, types in which RAS mutation is rare, such as breast. Snail1 or GAPDH with the FastLane Cell SYBR Green Kit The significance of finding that more mesenchymal (Qiagen, Hilden, Germany). Relative transcript levels of target RAS mutant tumour cells are dependent on continued genes were normalized to GAPDH mRNA levels. Snail2 expression lies in the fact that it is precisely these cells that are most resistant to existing therapies. Their Immunoblot analysis lack of RAS oncogene addiction suggests that they For western blot analysis, cells were lysed in NuPage LDS sample buffer (Invitrogen, Carlsbad, CA, USA). Proteins were resolved would not respond to the RAS pathway inhibitors by electrophoresis on NuPage 4–12% Bis–Tris gels (Invitrogen) currently in clinical trials, such as RAF, MEK, PI3- and immunoblots were developed using the ECL Western kinase and Akt inhibitors. In addition to causing loss of Blotting Detection Reagent (Amersham, Munich, Germany). viability in RAS mutant mesenchymal cells, Snail2 The following antibodies were used: anti-E-cadherin, anti-ZEB1 knockdown increases the sensitivity of these cells to and anti-KRAS, anti-lamin B (Santa Cruz Biotechnology, cytotoxic drugs. Snail2 inhibition has previously been Santa Cruz, CA, USA), anti-occludin (Invitrogen), anti-GAPDH shown to sensitize cells to DNA-damaging agents, in (Abcam, Cambridge, UK), anti-Snail1, anti-Snail2, anti-phospho part by directing the p53 response away from apop- (Ser473)-AKT, anti-phospho (Thr202/Tyr204)-ERK and ERK tosis (Inoue et al., 2002; Perez-Losada et al., 2003; (Cell Signaling Technology, Danvers, MA, USA). Kajita et al., 2004; Wu et al., 2005; Vannini et al., 2007; Vitali et al., 2008; Kurrey et al., 2009). Although the Immunofluorescence function of Snail2 as a transcriptional regulator does not Cells were fixed in 4% phosphate-buffered saline–paraformal- make it an attractive target for pharmacological dehyde for 15 min, incubated in 0.2% Triton-X-100 for 5 min, then in 0.2% fish skin gelatine in phosphate-buffered saline for targeting, the data presented here suggest that its 10 min and stained for 1 h with antibodies against E-cadherin inhibition could be a good way of targeting some of (1:100; Santa Cruz Biotechnology) or HRAS (1:50; Calbio- the most intractable tumour types, those with a mutant chem, San Diego, CA, USA). Cells were then stained with the RAS oncogene and a mesenchymal phenotype. The secondary antibody, followed by visualization under a relatively mild phenotype of Snail2 knockout mice fluorescence microscope. (Jiang et al., 1998; Inoue et al., 2002) suggests that on- Further materials and methods are described in the target inhibition of this factor may also be relatively free Supplementary Material. from unwanted side effects.

Conﬂict of interest Materials and methods The authors declare no conﬂict of interest. Molecular biology For cellular growth curves, cells were seeded in six-well plates and cell number was measured using a Vi-CELL Cell Viability Analyzer (Beckman Coulter, Brea, CA, USA). For cell Acknowledgements viability assays, cells were seeded in 96-well plates and cell number was measured using CellTiter-Blue (Promega, Madi- We thank Alberto Bardelli for providing SW48 and SW48 son, WI, USA). Apoptosis was measured using the Apo-ONE KRAS G13D cells and Senji Shirasawa for providing HCT- Homogeneous Caspase-3/7 Assay (Promega). Caspase-3/7 116, HKe-3 and HKh-2 cells. This work was supported by activity was normalized to relative cell number using the funding from Cancer Research UK and from the Intramural CellTiter-Blue assay. For anchorage-independent colony Research Program of the NIH, National Cancer Institute, assays, cells were seeded in each well of a six-well plate in Center for Cancer Research.

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Supplementary Information accompanies the paper on the Oncogene website (http://www.nature.com/onc)

Oncogene