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provided by Elsevier - Publisher Connector Current Biology 22, 2063–2068, November 6, 2012 ª2012 Elsevier Ltd All rights reserved http://dx.doi.org/10.1016/j.cub.2012.09.013 Report Genetic Deletion of RALA and RALB Small GTPases Reveals Redundant Functions in Development and Tumorigenesis

Pascal Peschard,1 Afshan McCarthy,1 (Figures 1A and 1B) and loss of RALB proteins in tissues of Vale´rie Leblanc-Dominguez,1 Maggie Yeo,1 adult mice (Figure 1C). The expression of RALB proteins was Sabrina Guichard,1 Gordon Stamp,2 unchanged in Rala null cells and vice versa. and Christopher J. Marshall1,* To investigate the role of RALA in embryonic development, 1Oncogene Team, Division of Cancer Biology, Institute of we examined embryos from Rala+/2 intercrosses. Between Cancer Research, London SW3 6JB, UK embryonic day 10.5 (E10.5) and E19.5, we observed that 10 2Department of Histopathology, Royal Marsden Hospital, out of 33 of the Rala null embryos displayed exencephaly London SW3 6JJ, UK (Figures 1E and 1F), a condition caused by a failure of closure of the neural tube along the hindbrain region [15]. We also observed exencephaly in Rala null embryos generated from Summary a gene-trap embryonic stem (ES) clone (Figures 1G and S1E– S1I). To examine whether RALA and RALB have overlapping RAL small GTPases, encoded by the Rala and Ralb genes, functions in neural tube closure, we generated compound are members of the RAS superfamily of small GTPases and mutants: 14 of 14 Rala2/2;Ralb+/2 embryos displayed a severe can act as downstream effectors of RAS [1]. Although highly neural tube defect (NTD) at E10.5–E13.5, with open neural tube similar, distinct functions have been identified for RALA and at the forebrain/midbrain boundary and hindbrain/cervical RALB: RALA has been implicated in epithelial cell polarity boundary, as well as caudally (Figures 1H–1J). These embryos [2], insulin secretion [3], GLUT4 translocation [4, 5], neurite also exhibited developmental retardation. We did not observe branching, and neuronal polarity [6, 7], and RALB in tumor NTDs in Rala+/2;Ralb2/2 embryos (0 of 8), although some of cell survival [8], migration/invasion [9–12], TBK1 activation them displayed developmental retardation. These observa- [13], and autophagy [14]. To investigate RAL GTPases tions demonstrate a role for RALA during neural tube closure in vivo, we generated null and conditional knockout mice. that can only be partially compensated by RALB. Importantly, Ralb null mice are viable with no overt phenotype; the Rala we failed to identify Rala2/2;Ralb2/2 embryos between E10.5 null leads to exencephaly and embryonic lethality. The exen- and E13.5, establishing that RALA and RALB share functions cephaly phenotype is exacerbated in Rala2/2;Ralb+/2 that are essential during early embryogenesis (Figure 1J). In embryos; embryos null for Rala and Ralb do not live past further support of the conclusion that RALA and RALB have gastrulation. Using a Kras-driven non-small cell lung carci- overlapping functions, we found that when the Ral-floxed noma mouse model, we found that either RALA or RALB is alleles were crossed to an adipocyte-specific Cre [16], sufficient for tumor growth. However, deletion of both Ral absence of either RALA or RALB in adipocytes had no effect genes blocks tumor formation. Either RALA or RALB is on viability, but absence of both RAL proteins led to perinatal sufficient for cell proliferation, but cells lacking both fail to lethality (data not shown). proliferate. These studies demonstrate functions of RAL proteins in development, tumorigenesis, and cell prolifera- RALA and RALB Act Redundantly in RAS-Driven tion and show that RALA and RALB act in a redundant Tumorigenesis fashion. To investigate the functions of RALA and RALB in RAS-medi- ated tumorigenesis, we crossed the Ral-targeted mice to the LSL-KrasG12D Results and Discussion non-small cell lung carcinoma (NSCLC) model, where the expression of an oncogenic allele of Kras requires RAL Small GTPases Are Required for Neural Tube Closure the excision of a LoxP-stop-LoxP cassette by Cre recombi- in the Mouse nase [17]. This mouse model develops tumors that are histo- To investigate the functions of RAL GTPases in mouse devel- logically similar to human adenocarcinoma, 30% of which opment and tumorigenesis, we generated null and conditional harbor KRAS mutations [18]. Rala Ralb Because Rala null mice exhibit embryonic lethality, we used alleles of the and loci using the Cre-lox system. The lox Rala and Ralb genes have similar intron-exon structures, with mice with two copies of the Rala conditional allele so that five exons and the translation start site located in exon 2. We excision of Rala only occurred in lung epithelial cells express- Rala Ralb ing Cre recombinase. Because Ralb null mice are viable, we flanked exons 2 and 3 in and exon 2 in with LoxP 2/2 lox/2 sites (see Figures S1A–S1D available online). To obtain null used null (Ralb ) and conditional (Ralb ) alleles. Cohorts alleles, mice carrying the targeted alleles were crossed to the were treated with adenovirus expressing Cre (AdCre) and PGK-Cre transgenic strain. Intercrossing Ralb+/2 mice gener- sacrificed 6 months later [17, 19]. Histological analysis re- Ralb vealed a similar number of tumors and tumor grade distribu- ated null pups at the expected Mendelian ratio that were lox/lox lox/2 2/2 viable, displayed no obvious abnormalities (Figure 1D), and tion among WT, Rala , Ralb , and Ralb animals developed to fertile adults. Rala+/2 intercrosses did not (Figures 2A and 2D). We quantified tumor burden by measuring generate Rala null mice, implying that Rala is required for the volume of lesions via microcomputed tomography (mi- croCT) imaging, which revealed no significant difference embryonic development. We confirmed the loss of RALA and lox RALB proteins in null mouse embryonic fibroblasts (MEFs) between the genotypes (Figure 2G). To show whether Rala alleles were efficiently recombined in tumors, we performed immunohistochemistry (IHC) on lung cross-sections with an *Correspondence: [email protected] anti-RALA antibody (Figures 2E and 2F); 80% of tumors in Current Biology Vol 22 No 21 2064

Figure 1. RAL GTPases Are Required for Mouse Embryonic Development (A–C) Western blot analysis of RALA and RALB protein expression in Rala2/2 (A) and Ralb2/2 (B) MEFs and in tissues from Ralb2/2 mice (C). (D) Offspring from Ralb+/2 intercrosses were genotyped 3 weeks after birth. (E) Embryos from Rala+/2 intercrosses were collected between E9.5 and E19.5 and examined for the presence of neural tube defects (NTDs). (F and G) Images of Rala null embryos generated either by gene targeting (F) or gene trap (GT) (G) displaying exencephaly beside a control littermate. (H and I) Images of Rala2/2;Ralb+/2 embryos. Arrows indicate where the neural tube remained open. (J) Analysis of embryos dissected between E10.5 and E13.5 from either Rala+/2;Ralb2/2 3 Rala+/2;Ralb+/2 or Rala+/2;Ralb+/2 3 Rala+/2;Ralb+/2 crosses. Embryos were scored for NTDs and somite count and then genotyped. See also Figure S1.

Ralalox/lox mice had completely lost RALA expression (Fig- examined their ability to proliferate. Rala and Ralb null MEFs ure 2H). Hence, the absence of RALA or RALB does not affect proliferated as well as their WT counterparts (Figure 4A). To the formation of NSCLC. We obtained similar results using rule out selection for a compensatory mechanism during the 7,12-dimethylbenzanthracene-12-O-tetradecanoylphorbol- embryogenesis, we infected Ralalox/lox and Ralblox/lox MEF 13-acetate (DMBA-TPA)-induced, Hras-driven skin carcino- populations with adenovirus expressing Cre and green fluo- genesis tumor model [20] using Ralb null mice or K14-Cre rescent protein (AdCreGFP) and observed that they pro- [21] and Ralalox/lox alleles to specifically excise Rala from the liferated as well as their counterparts infected with adenovirus epidermis; in both cases, tumor formation was not impaired expressing GFP alone (data not shown). However, three inde- (Figure S3). pendent populations of Ralalox/lox;Ralb2/2 MEFs infected with Because the deletion of either Rala or Ralb has no apparent AdCreGFP viruses failed to proliferate (Figure 4B). The effi- effect on the formation of NSCLC, we tested whether deleting ciency of excision was confirmed by western blotting (Fig- both RAL GTPases would impair NSCLC formation. Mice with ure 4C). Hence, deletion of both RAL GTPases seriously a KrasG12D;Ralalox/lox;Ralb2/2 genotype had fewer tumors than impairs the ability of MEFs to proliferate. We assume that their control KrasG12D;Ralalox/+;Ralb2/2 littermates (Figures 3A the proliferating cells in the AdCreGFP-infected Ralalox/lox; and 3B). Histological analysis showed a 60% decrease in Ralb2/2 MEF population retained RALA expression. Consis- tumor area (Figure 3E). Similarly, quantification of tumor tent with this, although we were able to select WT MEF popu- burden by microCT showed a 60% decrease in KrasG12D; lations stably expressing Cre recombinase using a retrovirus Ralalox/lox;Ralb2/2 mice (Figure 3F). In marked contrast to encoding Cre, we were unable to do this with Ralalox/lox; tumors in KrasG12D;Ralalox/lox animals (Figure 2H), we observed Ralb2/2 MEFs. that RALA was expressed in all tumors arising in KrasG12D; We have shown that RAL function is essential for cell prolif- Ralalox/lox;Ralb2/2 mice (Figures 3D and 3G). These data eration, but the fact that either RALA or RALB is sufficient show that RAL function is essential for tumorigenesis in this implies that they act redundantly. This redundancy raises the NSCLC model, the expression of either RALA or RALB being issue of whether there is compensatory activation of a RAL sufficient. isoform in the absence of the other isoform; therefore, we measured the amount of activated GTP-loaded RALB in Rala Redundant Activity of RALA and RALB in Cell Proliferation null MEFs and vice versa. We observed that RALB-GTP was To investigate whether the requirement for RAL GTPases significantly increased in Rala null MEFs (Figures 4D and 4E) in tumor formation reflects a role in cell proliferation, we gener- and that RALA-GTP was increased in Ralb null MEFs (Figures ated Rala2/2, Ralb2/2, and Ralalox/lox;Ralb2/2 MEFs and 4F and 4G). This increase in activation occurred without any RAL GTPase Overlapping Functions In Vivo 2065

Figure 2. RALA and RALB Are Not Singly Required for KrasG12D-Induced NSCLCs Cohorts of mice carrying one LSL-KrasG12D allele and the indicated Ral alleles were treated once with adenoCre viruses by intranasal instillation. Six months later, the mice were sacrificed and the lungs processed for histological and microCT analysis. (A–D) Hematoxylin and eosin (H&E) staining of lung sections from mice of the indicated genotype. (E and F) Immunohistochemistry (IHC) performed using an antibody against RALA on lung sections (43 magnification). (G) Quantification of the tumor burden in the lungs of the mice of the indicated genotypes. Left lobes were processed for microCT scanning, and the ratio of total tumor volume to total lung volume was measured for each animal; mean 6 SD. (H) Tumors in Ralalox/lox mice were scored for RALA protein expression using images as shown in (F); 97 tumors from 8 animals were scored. See also Figure S2. apparent increase in the level of RAL proteins (Figures 4D different functions to the isoforms. However, no previous and 4F). We obtained similar results using MEFs carrying work has used rigorous germline approaches; therefore, to Ral-floxed alleles infected with AdCreGFP (data not shown). investigate the role of RALA and RALB in mouse development Hence, the loss of one RAL protein is partially compensated and tumorigenesis, we generated null and conditional alleles. by an increase in the activation of the other isoform, suggest- We found that RALA but not RALB is required for embryonic ing that it is the overall level of RAL activity that is important development, implying that RALA has a unique role. Ralb null rather than a specific isoform. mice develop apparently normally and are viable and fertile. The different functions ascribed to RALA and RALB have Nearly 30% of Rala null embryos displayed exencephaly; been rationalized in part by different subcellular localizations 100% of Rala2/2;Ralb+/2 embryos displayed a more severe [2, 22]. To investigate subcellular localization, we generated NTD. This indicates that RALA has a specific function in neural HeLa and MEF cell populations stably coexpressing tube closure but that RALB is also involved. Possibly the mCherry-RALA and GFP-RALB at levels similar to the endog- unique requirement for RALA may reflect differences in enous proteins. In both cell types, most RALA and RALB expression rather than protein function, although it has been proteins colocalize at interphase (HeLa, thresholded Pear- reported that both Ral genes have very similar expression son’s correlation coefficient [PCC] > 0.600), the majority of patterns between E9.5 and E16.5 [26]. RAL proteins being present at the plasma membrane while Strikingly, we observed that either RALA or RALB is suffi- a fraction are on intracellular puncta, as previously reported cient for proliferation of MEFs in culture but that deletion of (HeLa, Figure S4; MEFs, data not shown) [2, 23, 24]. However, both Rala and Ralb blocks proliferation. Together with the every cell examined displayed intracellular structures that more severe NTD in Rala2/2; Ralb+/2 embryos, these data were positive for RALA but not RALB. The identity of the struc- argue for overlapping, redundant functions of RAL proteins. tures remains to be determined. Interestingly, an even higher The requirement for RAL GTPases for cell proliferation may fraction of RALA and RALB proteins colocalized during the explain the early lethality of the Rala2/2;Ralb2/2 embryos different stages of mitosis (HeLa, thresholded PCC > 0.750) because no embryos were present at E10.5. The redundancy (Figure S4). of RAL GTPases in cell proliferation that we observed Vertebrate Rala and Ralb genes derive from a common contrasts with a reported nonredundant function during ancestor in metazoa and fungi, Rala being most similar to its cytokinesis, where HeLa cells depleted of RALA and RALB metazoan orthologs [25]. Despite the high similarity between have a binucleated phenotype similar to cells depleted of RALA and RALB proteins, many studies have attributed RALA only [27]. The fact that a cell proliferation defect was Current Biology Vol 22 No 21 2066

Figure 3. Ablation of Both RALA and RALB Impairs the Formation of NSCLCs Comparison of tumor formation in KrasG12D;Ralalox/+;Ralb2/2 and KrasG12D;Ralalox/lox;Ralb2/2 mice. The experiment was conducted as in Figure 2. (A and B) H&E staining of two representative lung sections of the indicated genotype. (C and D) IHC against RALA on sections from KrasG12D;Ralalox/+;Ralb2/2 (C) and KrasG12D;Ralalox/lox;Ralb2/2 (D) animals (43 magnification). (E) Quantification of tumor burden by measuring the area of tumors and total area of lungs on serial cross-sections of the right lobes; mean 6 SD. (F) Quantification of tumor burden by microCT as described in Figure 2G; mean 6 SD, representative of three independent experiments (n R 7 for each genotype in each experiment). (G) Tumors in KrasG12D;Ralalox/lox;Ralb2/2 mice were scored for RALA protein expression; 40 tumors from 8 animals. See also Figure S3.

not observed under RNAi conditions may be because argument that RALA and RALB have redundant functions in complete depletion of RAL proteins is required to reveal this tumorigenesis. It will be necessary to induce the genetic phenotype. MEFs lacking expression of all three RAS genes deletion of Rals in established tumors to gain further insight do not proliferate but can be rescued by expression of consti- into their role in tumor maintenance and to study the require- tutive activation of RAF, but not RAL [28]. This suggests that ment for RAL GTPases in the formation of tumors that are RAF activation is the key signaling event downstream of RAS not driven by oncogenic RAS. for proliferation of MEFs. Because our results show that RAL Overall, this work reveals new roles for RAL GTPases during is also required for proliferation of MEFs, they argue that embryonic development and cell proliferation. Our data RAL activation is not downstream of RAS signaling in cell strongly support the conclusion that much of RAL signaling proliferation. can be performed by either RALA or RALB. A number of studies suggest that RALA and RALB have distinct roles in cancer cells, RALA being required for tumor Experimental Procedures cell proliferation and RALB for tumor cell survival and migra- tion [1]. These studies used RNAi in human cancer cell culture Plasmids and Reagents models of pancreatic [9], bladder, and prostate origin [10, 12]. Adenoviruses were obtained from the Gene Transfer Vector Core at the University of Iowa. We used the following primary antibodies: Rala (BD More recently, a requirement for RALA in cell proliferation and Biosciences, 610222), Ralb (LifeSpan BioSciences, LS-C38719; SCBT, survival was shown in human NSCLC cells [29]. Hence, we SC-1531; Millipore, 04-037), and GAPDH (Novus Biologicals, NB300-221). were surprised to discover that the deletion of either Rala or Human RALA (GC-Z1257) and RALB (GC-B0022) cDNAs were obtained Ralb does not impair the formation of KRASG12D-driven tumors from GeneCopoeia and transferred using Gateway Technology into pLVX- in a mouse model of NSCLC. Similarly, we observed that tumor puro lentiviral vectors (Clontech) containing either enhanced GFP or formation was not compromised in the absence of either mCherry (kind gift of Jasmine Abella) and the RfC.1 Gateway cassette (Invitrogen). Lentiviruses were produced in 293T cells cotransfected RALA or RALB in DMBA-TPA-induced skin carcinogenesis or G12D with pMD2.G and psPAX2 packaging vectors (http://tronolab.epfl.ch/ a mouse model of KRAS -driven pancreatic adenocarci- lentivectors). noma (J. Morton, O. Sansom, P.P., and C.J.M., unpublished data). The discrepancy between these results and the RNAi- Mice based experiments may be due to the fact that in RNAi studies, The targeting vectors to generate the Rala and Ralb gene-targeted mice RAL is depleted in fully transformed cells, whereas in our were made by inserting the arms of homology into the pGKNeoF2L2FDTA genetic system, the Ral gene is deleted at tumor initiation, vector (Phillipe Soriano, Addgene plasmid 13445 [30]). The homologous possibly allowing time for adaptation and compensation recombination was performed in Bruce4 ES cells of C57BL/6J origin (Ozgene). The positive clones were identified by PCR and confirmed by during the process of cell transformation. However, if such Southern blotting before being injected into albino C57BL/6J blastocysts adaptation is the explanation for the difference between our to generate chimeras in a pure C57BL/6J background. The Rala- and genetic experiments and RNAi studies, it reinforces the Ralb-targeted mice were crossed to the PGK-Neo or PGK-Flp transgenic RAL GTPase Overlapping Functions In Vivo 2067

Figure 4. MEFs Null for Rala and Ralb Fail to Proliferate (A and B) Proliferation curves. Cells (5.5 3 103/cm2) were seeded in triplicate and counted daily for 3 days. Each point represents the average 6 SEM of three independent experiments. In (A), two independent MEF populations were used in each experiment. In (B), three independent Ralalox/lox;Ralb2/2 MEF pop- ulations Pop1 (population 1), Pop2 (population 2), and Pop3 (population 3) were infected with either adenoCre or adenoCreGFP and seeded 3 days later for the proliferation assay. (C) Western blot analysis performed with lysates from adeno-infected (Ad) Ralalox/lox;Ralb2/2 MEF populations collected at day 1 of the proliferation assay. The membrane was probed with anti-RALA and anti-GAPDH antibodies. (D) Western blot analysis showing the levels of RALB-GTP and total RALB in two independent populations of WT and Rala2/2 MEFs cultured in 10% serum. (E) Quantification of western blots of three independent experiments; mean 6 SD (**p % 0.01). (F) Levels of RALA-GTP and total RALA in two independent populations of WT and Ralb2/2 MEFs. (G) Quantification of data from (F), as in (E); mean 6 SD. See also Figure S4. mice (provided by the Cancer Research UK transgenic facility) to generate and Rala null MEFs that were formalin fixed and paraffin embedded the knockout and conditional knockout mice, respectively. The DD1113 (Figure S1I). Rala gene-trap ES clone was obtained from the Sanger Institute, and the mice were derived at the Cancer Research UK transgenic facility. Images MicroCT Analysis of embryos were captured using a M2FLIII microscope and DC500 camera After being fixed in formalin, lungs were dehydrated in ethanol (35%, 2 hr; (Leica Microsystems). The LSL-KrasG12D mice were a generous gift of Dave 70%, 2 hr; 80%, 2 hr; 90%, 2 hr; 100%, 12 hr), transferred to hexamethyldisi- Tuveson [31]. The lung tumor experiments were performed on mice with lazane (Sigma-Aldrich, #52619) for 2 hr, and dried for 12 hr. The lungs were a pure C57BL/6J genetic background. Briefly, the mice were anesthetized scanned in a SkyScan 1076 microCT scanner (Bruker microCT) with the with ketamine and xylazine and treated once by intranasal instillation using following settings: Styrofoam bed, no filter, 18 mm resolution, 40 kV, 120 ms either 0.5 or 1 3 108 pfu/mouse of adenoCre viruses as described in [19]. For exposure, and 0.4 rotation step. The scans were reconstructed and analyzed the DMBA-TPA-induced skin carcinogenesis experiment, the K14-Cre mice using NRecon and CTAn software, respectively (Bruker microCT). (FVB-Tg(K14-Cre)8Brn) [32] were acquired from the National Cancer Institute Mouse Models of Human Cancers Consortium, and the Ral mice Cell Culture and Pull-Down Assays were backcrossed in a FVB/N genetic background for at least six genera- MEFs were isolated from E13.5 embryos and cultured in a low-oxygen (3%) tions. The animals were shaved and treated topically with a single dose of incubator [34] in Dulbecco’s modified Eagle’s medium containing 10% fetal 7,12-dimethylbenzanthracene (DMBA, Sigma-Aldrich) followed by 12-O- bovine serum. For analysis of RAL-GTP levels, 1 3 106 cells were lysed in tetradecanoylphorbol-13-acetate (TPA, Sigma-Aldrich) twice weekly for 650 ml of ice-cold Ral lysis buffer (50 mM Tris [pH 7.4], 10% glycerol, 1%

20 weeks as described in [33]. The FABP4-Cre (AP2-Cre) mice [16] used NP40, 5 mM MgCl2, 100 mM NaCl, 1 mM dithiothreitol [DTT], and cOmplete to delete floxed Ral alleles in adipocytes were obtained from the Institute EDTA-free Protease Inhibitor; Roche). The lysates were spun at 13,000 rpm of Metabolic Science at the University of Cambridge. Animals were handled at 4C for 15 min. Next, 400 ml of the supernatant was incubated with 25 ml in strict accordance with UK Home Office regulations and institutional ethics of glutathione Sepharose beads bound to approximately 50 mg glutathione approval. S-transferase-RalBP1 RBD proteins at 4C for 45 min. The beads were spun at 4C for 5 min at 1,000 3 g and washed twice with 1 ml of Ral wash Histopathology and Immunohistochemistry buffer (Tris-buffered saline, 10 mM MgCl2, 1 mM DTT, and cOmplete EDTA- Lungs were perfused and fixed in 10% buffered formalin for 24 hr. The right free Protease Inhibitor) before being resuspended in Laemmli loading buffer lobes were embedded in paraffin, and the left lobe was processed for mi- containing 100 mM DTT. Immunoblotting analyses were performed using croCT imaging. For IHC against RALA, paraffin sections were pretreated an Odyssey Imaging System (LI-COR Biosciences) according to the manufac- for 2 min in a pressure cooker in citrate buffer (pH 6), washed, blocked turer’s instructions. Confocal images were acquired with a Zeiss LSM 7 micro- 1 hr in mouse IgG block (VECTASTAIN) and then 5 min in protein block scope, and images were processed and analyzed with Volocity (PerkinElmer). (Dako X0909), incubated 1 hr with anti-RALA antibody (1:250; BD Biosci- ences, 610222), washed, incubated 30 min in Mouse EnVision (Dako), Supplemental Information washed, incubated 5 min in diaminobenzene, washed, and counterstained with Gill’s hematoxylin. The washes were all performed using Dako FLEX Supplemental Information includes four figures and can be found with this Wash Buffer (K8000). The RALA antibody was validated for IHC with WT article online at http://dx.doi.org/10.1016/j.cub.2012.09.013. Current Biology Vol 22 No 21 2068

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