(LIMD1), a Tumor Suppressor Encoded at Chromosome 3P21.3, Binds Prb and Represses E2F-Driven Transcription

LIM domains-containing protein 1 (LIMD1), a tumor suppressor encoded at chromosome 3p21.3, binds pRB and represses E2F-driven transcription

Tyson V. Sharp*†, Fernando Munoz*, Dimitra Bourboulia*, Nadege Presneau*, Eva Darai‡, Hsei-Wei Wang*, Mark Cannon*, David N. Butcher§, Andrew G. Nicholson§, George Klein‡, Stephan Imreh‡, and Chris Boshoff*†

*Cancer Research UK, Viral Oncology Group, Wolfson Institute for Biomedical Research, Cruciform Building, University College London, London WC1E 6BT, United Kingdom; ‡Microbiology and Tumor Biology Center, Karolinska Institute, S-171 77 Stockholm, Sweden; and §Department of Histopathology, Royal Brompton Hospital, London SW3 6NP, United Kingdom

Contributed by George Klein, September 29, 2004 LIM domains-containing protein 1 (LIMD1) is encoded at chromo- FuGene (Roche Diagnostics) per the manufacturer’s instructions. some 3p21.3, a region commonly deleted in many solid malignan- The reporter plasmid pGL2–3xE2F-luciferase contains three cop- cies. However, the function of LIMD1 is unknown. Here we show ies of the E2F DNA-binding site derived from the adenovirus E2A that LIMD1 speciﬁcally interacts with retinoblastoma protein (pRB), gene promoter and has been described (11). Luciferase activity in inhibits E2F-mediated transcription, and suppresses the expression cell lysates was normalized to cotransfected ␤-galactosidase values of the majority of genes with E2F1-responsive elements. LIMD1 [relative E2F-luciferase (E2F-luc) activity]. blocks tumor growth in vitro and in vivo and is down-regulated in the majority of human lung cancer samples tested. Our data Plasmids. To create N-terminal hemagglutinin (HA)-tagged indicate that LIMD1 is a tumor-suppressor gene, the protein prod- LIMD1, we performed PCR amplification on human LIMD1 uct of which functionally interacts with pRB and the loss of which cDNA (kindly provided by S.I.) by using the 5Ј-HA-START promotes lung carcinogenesis. primer (5Ј-GCAGCATGTACCCATACGATGTTCCAGA- TTACGCTATGGATAAGTATGACGACCTGGGCCTGGA- lung cancer ͉ retinoblastoma GGCC-3Ј) and the 3Ј-STOP primer (5Ј-GAAGTTGTGCGT- GTGAAGGGCTGTAGATGAGGGTCT-3Ј). The PCR prod- he archetypal tumor-suppressor gene (TSG) is represented by uct was TA cloned into pcDNA3.1 Topo (Invitrogen) and Tthe retinoblastoma 1 (RB1) gene (1, 2). Loss of its function sequence-verified. results in susceptibility to retinoblastoma, a sporadic or hereditary pHR-CMV-HA-LIMD1-IRES-GFP was produced as follows: pediatric neoplasm arising from retinal cells harboring either pcDNA3.1-HA-LIMD1 was cut with HindIII, treated with the deleted or mutated RB alleles (1, 3, 4). To identify binding partners Klenow fragment of DNA polymerase to produce blunt ends, of retinoblastoma protein (pRB), we performed a yeast two-hybrid purified and cut with XhoI, and then ligated directionally into the screen using full-length pRB fused to the GAL4 DNA-binding SmaI͞XhoI-cut pHR-CMV-IRES-GFP plasmid downstream of the domain as bait. We identified the LIM domains-containing protein cytomegalovirus (CMV) promoter and upstream of the internal 1 (LIMD1) sequence, which is localized within the common elim- ribosome entry site (IRES)-GFP. inated region 1 (C3CER1; also called CER1) on chromosome Xpress (Invitrogen)-tagged LIMD1 and deletion mutant plas- 3p21.3 (5, 6). C3CER1 is one of the putative tumor-suppressor mids were created as follows. LIMD1 cDNA was used as a template regions identified by the ‘‘elimination test,’’ a functional test system for PCR to create consecutive deletions in a C- to N-terminal that identifies regularly lost (eliminated) chromosome segments in direction. PCR amplification was performed by using the 5Ј- microcell hybrid-derived severe combined immunodeficiency LIMD1-START primer (5Ј-GAGATCGAATTCGCAATG- (SCID) tumors (7). Within chromosome 3p are several TSG GATAAGTATGACGACCTGGGCCTG-3Ј) in combination with regions. Nine homozygous deletion regions have been described, the 3Ј primer, which introduced the indicated C-terminal deletion four of which were found in lung tumors. C3CER1 (megabases followed by a stop codon. Consecutive LIMD1 deletions in an N- to 43.32–45.74) is located between two such regions, AP20 and LUCA C-terminal direction were achieved by using the 3Ј-LIMD1-STOP (8, 9). Deletions in this chromosome 3p region are a common event primer (5Ј-CTCTGCAGGTCGACCTAGAAGTGGTGCT- in solid malignancies including breast, gastric, colorectal, ovarian, GGTGAAGGGCTGTA-3Ј) in combination with the indicated 5Ј and renal (6). Furthermore, C3CER1 loss of heterozygosity (LOH) primer, which annealed at the indicated amino acid and introduced exceeds 90% in lung tumors compared to the putative TSG FHIT an ATG start codon before this residue. All PCR products pro- (65%) and the TSG VHL (72%) (6), which map at 3p14.2 and duced by this PCR deletion mutagenesis method were gel-purified 3p25.3, respectively. Therefore, because deletion in this region is a and TOPO cloned into pcDNA4͞HisMax (Invitrogen), which common event in lung tumors, chromosome 3p is under intense resulted in an N-terminal Xpress-tagged LIMD1 (when using just study for the identification of putative TSGs (10). the 5Ј-START and 3Ј-STOP primers) or the indicated deletion Here, we confirm the specific binding of LIMD1 to pRB and mutants. All PCR full-length and deletion mutants of LIMD1 were its ability to repress E2F-driven transcription and cell prolifer- sequence-verified. ation. Furthermore, we demonstrate that LIMD1 inhibits tumor pGEX6P-1-LIMD1 plasmid was constructed as follows. Hu- growth in both in vitro and in vivo models. Finally, we show that man LIMD1 cDNA was PCR-amplified by using primers de- LIMD1 expression is down-regulated in lung cancer.

Materials and Methods Abbreviations: LIMD1, LIM domains-containing protein 1; pRB, retinoblastoma protein; TSG, tumor suppressor gene; C3CER1, common eliminated region 1; LOH, loss of heterozy- Cell Culture, Transfection, and Reporter Assays. All cell lines were gosity; HA, hemagglutinin; VO, vector only; qRT-PCR, quantitative RT-PCR; LMB, leptomy- cultured in DMEM (GIBCO͞BRL) supplemented with 10% FCS, cin B; NES, nuclear export signal. 2 mM glutamine, 50 units͞ml penicillin, and 50 ␮g͞ml streptomy- †To whom correspondence may be addressed. E-mail: [email protected] or

cin. For transient transfections, 8 ϫ 105 adherent cells were plated [email protected]. CELL BIOLOGY in a 25-cm2 flask and the following day were transfected by using © 2004 by The National Academy of Sciences of the USA

www.pnas.org͞cgi͞doi͞10.1073͞pnas.0407123101 PNAS ͉ November 23, 2004 ͉ vol. 101 ͉ no. 47 ͉ 16531–16536 Downloaded by guest on October 1, 2021 scribed above to incorporate an EcoRI and SalI site. The was used for transfections (16). According to standard protocols, resulting PCR product was restriction enzyme-digested with 50–70% confluent monolayer A9 cells were transfected with a EcoRI and SalI and ligated into similarly cut pGEX6P-1 vector mixture of 1–2 ␮g of PAC DNA and 10 ␮l of Lipofectamine (Life (Amersham Pharmacia Biotech). The plasmid with the correct Technologies, Rockville, MD) per well, in six-well plates. The insert was sequence-verified on both DNA strands. transfectants were selected on blasticidin (1–2 ␮g͞ml). Two positive pHR-CMV-E7-IRES-GFP plasmid was constructed by BamHI clones were chosen and expanded in vitro for inoculation into SCID digestion of pLXSN-E7 (a gift from David Beach, Queen Mary’s mice. All transfectants were analyzed by fluorescent in situ hybrid- School of Medicine and Dentistry, London) to excise the E7 cDNA, ization (FISH) and PCR. which was then ligated into similarly cut pHR-CMV-IRES-GFP. One million A9 and A9-LIMD1-PAC transfectants were inoculated s.c. into 6-week-old SCID mice in three sites, with a fourth site Construction, Production, and Infection of Lentivirus Expressing used for A9 controls. The mice were observed for tumor formation LIMD1. To obtain pseudotyped lentivirus [recombinant HIV-1 with once a week up to 6 weeks. The two positive LIMD1-PAC clones vesicular stomatitis virus G (VSV-G) envelope protein], which were inoculated in two series into four mice per clone and three expresses HA-tagged LIMD1 or E7 protein, we used the gene inoculation sites per mouse. A9 cells were inoculated as controls at delivery and production system developed by Naldini et al. (12). one site per mouse. Tumor growth was monitored once a week for 6 weeks. Yeast Two-Hybrid Screen. The cloning of full-length pRB into the pAS2.1 bait vector has been described (13). The resulting plasmid BrdUrd Cell Proliferation Assay. The Cell Proliferation ELISA, BrdU (pAS2.1-pRB) was pretransformed into the Saccharomyces cerevi- (Colorimetric) (Roche Applied Science) immunoassay for the siae PJ69-4a (MATa trp1-90 leu2-3,112 ura3-52 his3-200 quantification of cell proliferation, based on the measurement of gal4⌬gal80⌬LYS2ϻGAL1-HIS3 GAL2-ADE2 met2ϻGAL7-lacZ) BrdUrd incorporation during DNA synthesis, was used per the (14) reporter strain by using a modified lithium acetate protocol manufacturer’s instructions. (see Yeast Protocols Handbook, BD Biosciences Clontech). Subse- quently, this strain was cotransformed with the BC3 cDNA library Microarray Processing and Analysis. RNA preparation, microarray (produced by using the Stratagene HybriZAP cDNA library kit) in processing, and data analysis of the MDA-MB435 cell line (here- the GAL4 DNA activation domain (GAL4AD) fusion ‘‘prey’’ after referred to as MB435) were performed as described by Wang vector. Selection for positive colonies and cDNA clone isolation et al. (17). Affymetrix (Santa Clara, CA) Hg-U133 Plus 2.0 chips were as previously described (15). were used in this analysis. The human orthologs for the E2F1- responsive genes were selected by using lists previously obtained by Generation of Recombinant GST–LIMD1 Fusion Protein. Full-length Ma et al. (18). The false discovery rate q of 0.05 assures that Ͻ5% LIMD1 was expressed as a recombinant GST fusion protein from of genes with q values Ͻ0.05 can be expected to be false positives. the pGEX6P-1-LIMD1 plasmid. Protein was produced and purified as previously described for other GST fusion proteins (15). Quantitative RT-PCR (qRT-PCR). Total RNA from normal and tumor samples was treated with DNase I (RNase-Free, Ambion, Oxon, Anti-LIMD1 mAb Production and Purification. Recombinant LIMD1 U.K.) before cDNA synthesis according to the manufacturer’s was used in the production of the 3F2͞G6 anti-LIMD1 mAb. The instructions. cDNA was generated from 1 ␮g of total RNA by using method for this production has been described previously (15). The the SuperScript III First-Strand Synthesis System for RT-PCR epitope on LIMD1 for the 3F2͞G6 mAb was determined to be (Invitrogen). between amino acids 144 and 214 (data not shown). Real-time qRT-PCR was performed on an ABI PRISM 7700 sequence detector (Applied Biosystems) by using the SYBR Green Immunoprecipitations, Immunoblots, and Immunofluorescence. Im- PCR Master Mix (Applied Biosystems) in duplicate, with triplicate munoprecipitations and immunoblots were as previously described nontemplate controls (NTCs) in a 25-␮l PCR reaction. One mi- (15). Briefly, transfected cells were washed three times with ice-cold croliter of cDNA was used in a 25-␮l PCR mixture containing 1ϫ PBS, fixed with 4% paraformaldehyde for 15 min at room temper- SYBR Green PCR Master Mix and 0.9 ␮M LIMD1 forward primer ature, washed as above, and then permeabilized with 0.5% Triton (LIMD1 5Ј-TACCACAAGGTGCTGGCCCCCAAG) and 0.3 X-100 for 13 min and immunostained as described (15). The ␮M reverse primer (LIMD1 3Ј-TGGCCATCTTCATCATT- primary Abs used were anti-Xpress mAb (R910-25, Invitrogen) and GAGCTCCAGAC) (which produced a product of 163 base pairs anti-HA mAb (12CA5, Roche Applied Science). and spans nucleotides 1765–1928 of LIMD1 cDNA) or 0.3 ␮M GAPDH forward and reverse primers (GAPDH 5Ј-GGA GTC In Vivo Studies. The human non-small cell lung cancer (NSCLC) AAC GGA TTT GGT CGT A and GAPDH 3Ј-GGC AAC AAT tumor cell line A549 was used for determining the inhibitory effects ATC CAC TTT ACC AGA GT, respectively). The comparative Ct of lentivirus-LIMD1-HA on the establishment of metastatic tu- method was used as described previously (19). mors. Briefly, A549 tumor cells were infected with either lentivirus [empty, vector only (VO)] or lentivirus-LIMD1-HA at a multiplic- Results and Discussion ity of infection (moi) of 100 in cell culture. Three days later, cells LIMD1 Binds the pRB Tumor Suppressor. To identify binding partners were harvested, washed with PBS, and resuspended in sterile PBS of pRB, we performed a yeast two-hybrid screen using full-length at a density of 1 ϫ 106 viable cells per 200 ␮l. Female nude mice pRB as bait fused to the GAL4 DNA-binding domain. From this were injected in the tail vein with lentivirus-infected (empty) or screen, LIMD1 was independently identified three times as a lentivirus-LIMD1-HA-infected A549 tumor cells. Eight animals specific pRB-interacting protein (Fig. 1A). LIMD1 is a member of were used in each group. Two weeks after injection, animals were the LIM domain family of proteins (5, 20). LIM domains consist of killed, injected intratracheally with 15% India ink, and fixed in a cysteine-rich consensus sequence containing two distinct zinc- Fekete’s solution (60% ethanol, 8% formaldehyde, and 4% glacial binding subdomains that mediate protein–protein interactions (20, acetic acid). Lung tumor formation was observed under a dissecting 21). LIM-domain-containing proteins play a role in intracellular stereomicroscope, and the number of lung tumors was counted. For signaling, transcriptional regulation, and cellular differentiation the A9 fibrosarcoma tumorigenicity assay in severe combined during development (18, 19). The predicted protein structure of the immunodeficient (SCID) mice, a P1-derived artificial chromosome human LIMD1 gene reveals three LIM domains located at the C (PAC) fragment, RP6-3307, that contained full-length LIMD1 gene terminus (Fig. 1A), indicating that it belongs to group 3 of this in a blasticidin-selectable pPAC4 vector and empty pPAC4 vector family (Fig. 1B) (5).

16532 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0407123101 Sharp et al. Downloaded by guest on October 1, 2021 326–608) interaction depends on the pRB C-terminal 136 amino acids. We examined the ability of these two proteins to interact in vivo. LIMD1 (73 kDa) containing an Xpress tag was transfected into U2OS (pRBϩ/ϩ) cells, and endogenous pRB specifically coimmu- noprecipitated with the anti-Xpress mAb only (Fig. 1E). Next, a series of Xpress-tagged LIMD1 deletion mutants was constructed and transfected into U2OS cells. This localized the pRB-binding site to amino acids 404–442 (see Fig. 1 F and G for summarization). LIMD1 did not bind the retinoblastoma family member p130 in vivo (Fig. 1F), indicating the specificity of the pRB interaction.

Subcellular Localization of LIMD1. Nuclear localization is a prereq- uisite for proteins that bind and regulate active pRB; therefore, we examined the subcellular localization of LIMD1. Indirect immunofluorescence assay (IFA) on U2OS cells transfected with Xpress-tagged LIMD1 show that LIMD1 is localized predominantly to the cytoplasm with a small but consistently specific fraction detected in the nucleus but not nucleoli (Fig. 2A Left). The staining of LIMD1 in the cytoplasm consisted of a diffuse, punctuated perinuclear, as well as cytoskeletal, pattern (Fig. 2A Left). In addition, many cells show vesicle-like staining distrib- uted throughout the cytoplasm (Fig. 2B Right). On average, 14% of LIMD1-expressing cells showed predominantly nuclear localized LIMD1 (Fig. 2B, arrowheads). The group 3 LIM domain family of proteins shares the ability to shuttle between the cytoplasm and the nucleus (20, 22–24). To determine whether LIMD1 shares this function, we treated LIMD1-transfected cells with leptomycin B (LMB), a drug that blocks nuclear export by preventing the formation of the nuclear export signal (NES)͞chromosomal region maintenance 1 (CRM1)͞ Ran–GTP nuclear export complex (25). This resulted in nuclear accumulation of LIMD1 (Fig. 2C), suggesting that LIMD1 is Fig. 1. pRB binds to LIMD1, a member of the zyxin LIM domain family of actively exported and may contain a NES that facilitates cytosolic͞ proteins. (A) A yeast two-hybrid screen of a HeLa cDNA library with GAL4 nuclear shuttling. DNA-binding domain pRB obtained a cDNA encoding amino acids 326–608 of Using a series of deletion mutants in IFAs in the absence of LMB, LIMD1. The predicted protein structure of the human LIMD1 gene reveals we localized the NES to a region spanning amino acids 54–134 (Fig. three LIM domains located at the C terminus and a unique N-terminal region (amino acids 1–68) that encodes a LEM domain (amino acids 18–68) as 2 D and E and data not shown). This NES is leucine-rich, a previously reported (bioinformatics analyses of both the LIMD1 gene and characteristic shared with other group 3 family NESs. However, no product can be found at www.dsi.univ-paris5.fr͞genatlas and http:͞͞ motifs within this NES matched those reported for other family bioinfo.weizmann.ac.il͞cards͞index.shtml). (B) LIMD1 belongs to group 3 of members (data not shown). Regions important for import of the gene family encoding LIM motifs. Shown are phylogenetic relationships LIMD1 to the nucleus were localized to the LIM domains, because between human LIMD1 and other family members containing three LIM LIMD1⌬472–676 in the presence of LMB is predominantly cyto- domains, Ajuba (24), LPP (lipoma preferred partner) (40), Trip6 (thyroid re- plasmic and ⌬472–676 (LIM domains only) is totally nuclear (Fig. ceptor interacting protein 6) (41), and zyxin (42). Numbers indicate relative 2E). Similarly, N-terminally truncated forms of Ajuba, Trip6, distances of branches. (C). The specificity of the pRB–LIMD1 interaction in the paxillin, LPP, and Hic-5 containing essentially only the LIM yeast two-hybrid assay was confirmed by combining these two proteins with positive and negative controls and then assayed by prototrophy for histidine domains localize to the nucleus (23, 26–29). (His) and adenine (Ade). The indicated GAL4 binding domain (BD) and GAL4 activation domain (AD) fusion or VO plasmids were cotransformed into yeast LIMD1 Regulates E2F1-Driven Transcription. pRB represses the tran- strain PJ69-4a. Growth on medium lacking histidine and adenine (ϪHis ϪAde) scriptional activity of E2F transcription factors. Because LIMD1 is indicative of specific interactions. LAMINC, lamin C. (D) Amino acids 326– binds specifically to pRB in vitro and in vivo and shuttles to the 608 of LIMD1 interact directly with the C terminus of pRB (amino acids nucleus, we tested the functional relevance of this interaction by 763–928). [35S]Methionine-labeled LIMD1 (amino acids 326–608) was pro- performing E2F-luc reporter assays. HEK293 cells were transfected duced by using the Promega in vitro TNT kit and incubated with the indicated with VO or increasing concentrations of LIMD1 in the presence of GST-pRB fusion proteins. GST pull-down assays were performed as described a 3xE2F-luc (E2F1-luc) transcriptional reporter plasmid. LIMD1 (15). (E) Endogenous pRB and transfected Xpress-tagged LIMD1 interact in vivo. IP, immunoprecipitation. (F) pRB binds to amino acids 404–442 of LIMD1 represses E2F1-driven transcription in a concentration-dependent in vivo. U2OS cells were transfected with VO, full-length LIMD1 (WT), or the manner (Fig. 3A). indicated deletion mutant (⌬). Immunoprecipitations were performed as Different deletion mutants of LIMD1 were examined for their above. Anti-human pRB mAb, clone G99-549, was from BD Biosciences Pharm- ability to repress E2F-driven transcription (Fig. 3B). Deletion ingen, and anti-Xpress mAb was from Invitrogen. (G) Graphical summary of mutants without the pRB-binding region have a Ͼ50% reduction the pRB-binding domain as determined by in vivo coimmunoprecipitations. in their ability to repress E2F-mediated transcription (Fig. 3B, compare WT with ⌬224–676 and ⌬404–676). Whereas addition of the pRB-binding region significantly increases repression of E2F- The yeast two-hybrid interaction between LIMD1 and pRB was transcription (Fig. 3B, compare ⌬404–676 with ⌬443–676). Dele- specific (Fig. 1C) and confirmed the use of the indicated GST–pRB tion of the NES caused activation of E2F-transcription (Fig. 3B). fusion proteins in an in vitro pull-down assay with [S35]methionine- The activation domain localized to the three LIM domains at the

labeled LIMD1 (Fig. 1D). Both the yeast two-hybrid and GST C terminus (Fig. 3B, ⌬1–467). LIMD1 deletion mutants (⌬1–134 CELL BIOLOGY pull-down interaction assays indicated that LIMD1 (amino acids and ⌬1–467) can therefore act in a dominant negative fashion by

Sharp et al. PNAS ͉ November 23, 2004 ͉ vol. 101 ͉ no. 47 ͉ 16533 Downloaded by guest on October 1, 2021 Fig. 2. LIMD1 is a cytosolic protein that shuttles between the nucleus and the cytoplasm via its nuclear localizing LIM domains and its NES, respectively. (A) U2OS cells transfected with Xpress-tagged LIMD1 were grown on coverslips Fig. 3. LIMD1 is a transcriptional regulator that represses E2F-driven tran- and 24 h posttransfection were ﬁxed and subjected to IFA with anti-Xpress tag scription. (A Upper) HEK293 cells were transiently transfected with VO or mAb. Images of localized proteins were obtained by using confocal laser LIMD1 expression vectors. Luciferase activity was determined 48 h later. scanning microscopy. (B) LIMD1 localizes to the nucleus. A typical example of (Lower) Cell extracts were probed for LIMD1 expression with anti-Xpress mAb. such localization is shown (arrowheads). LIMD1 was visualized with anti- (B) The ability of LIMD1 to repress E2F-luc-driven transcription depends on the Xpress mAb, and DNA was visualized with Hoechst 33342. (C) LMB treatment presence of the pRB-binding domain. HEK293 cells were transfected with the of cells causes LIMD1 accumulation in the nucleus. U2OS cells previously indicated LIMD1 deletion mutants and the E2F-luc reporter. E2F-luc transcrip- transfected with Xpress-tagged LIMD1 were analyzed by IFA using anti-Xpress tion is shown relative to VO, which is normalized to zero. Blue bars indicate primary Ab. (Upper) Untreated U2OS cells (ϪLMB). (Lower) U2OS cells treated LIMD1 and its mutants that localized predominantly to the cytosol; red bars with LMB (10 ng͞ml)for4h(ϩLMB). (D) LIMD1 contains a NES. A series of indicate LIMD1 deletion mutants that localized to the nucleus. (C) The ability Xpress-tagged LIMD1 deletion mutants was constructed and transfected into of LIMD1 to repress E2F-driven transcription depends partly on the presence U2OS cells, and the subcellular localization of these mutants was visualized of pRB. U2OS (pRBϩ/ϩ) and SAOS2 (pRBϪ/Ϫ) were transiently transfected with with anti-Xpress mAb by IFA. Two such deletion mutants, LIMD1⌬1–54 and VO or LIMD1, and the E2F-luc levels were determined. (D) Inhibition of pRb LIMD1⌬1–134, are shown because they show the switch between cytoplasmic͞ function by E7 reduces the transcriptional repressive ability of LIMD1. U2OS perinuclear and nuclear localization, respectively, upon deletion of amino cell lines previously transduced with lentivirus VO or lentivirus E7 were trans- acids 54–134. (E) Summary of localization data with the indicated LIMD1 fected with the indicated plasmid in the presence of the E2F-luc reporter. deletion mutants. Localization in U2OS cells was determined as described Relative E2F-luc activity was determined 48 h posttransfection. (E) LIMD1 is not above in the absence (ϪLMB) or presence (ϩLMB and data not shown) of LMB. expressed in MB435 cells. HA-LIMD1 was transduced into these cells by using The three LIM domain modules of LIMD1 have inherent nuclear localizing lentivirus HA-LIMD1 or lentivirus only (VO). Expression of endogenous LIMD1 properties (NLS). C, predominant cytoplasmic localization; N, predominant and HA-LIMD1 was detected by using the anti-LIMD1 mAb (3F2͞G6). (F) nuclear localization. E2F1-responsive genes are down-regulated by LIMD1. cDNA microarray analysis of MB435 cells transduced with LIMD1-expressing lentivirus (L1–3) or lentivirus only (V1–3). Heat map of 64 E2F1-responsive genes with down- activating E2F-mediated transcription (Fig. 3B). It remains to be regulated genes shown in blue and up-regulated genes shown in red. The determined whether this ability is due to the loss of a transcriptional green bar indicates down-regulated genes upon LIMD1 expression; the purple repressive domain that overlaps with the NES or to the disruption bar indicates up-regulated genes upon LIMD1 expression. Heat map color of endogenous LIMD1͞pRB͞E2F repressive complexes upon nu- scale (at right) displays units of standard deviation from the mean of each gene. (G) LIMD1 inhibits cell proliferation. A BrdUrd ELISA was performed on clear retention of these mutants. These findings are similar to those the LIMD1-negative cell line MB435 transduced with lentivirus, with or with- for the group 3 family member Trip6, which has transcriptional out LIMD1 cDNA. Cells were plated 48 h posttransduction into a 96-well plate activation and repressive functions that overlap with its NES (23). at 3 ϫ 103 cells per well. Cells were incubated with media containing BrdUrd for 1 h before the anti-BrdUrd ELISA was performed. LIMD1 Has pRB-Dependent and -Independent Transcriptional Repres- sor Activities. The deletion of the pRB-binding site significantly reduced, but did not completely abolish, LIMD1-mediated LIMD1 to repress E2F-driven transcription in SAOS2 cells is repression (Fig. 3B). To address whether LIMD1 repression of reduced by 48% compared with LIMD1 repression in U2OS E2F-driven transcription depends on pRB, we examined E2F- cells (100%) (Fig. 3C), suggesting that Ϸ50% of the repressive luc reporter activity in pRB-positive (U2OS and pRBϩ/ϩ) and ability of LIMD1 depends partly on the presence of pRB. We -negative (SAOS2 and pRBϪ/Ϫ) cell lines (30). The ability of also tested E2F repression by LIMD1 in the presence or

16534 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0407123101 Sharp et al. Downloaded by guest on October 1, 2021 absence of human papillomavirus (HPV) E7 (which abrogates the function of pRB family members). In the presence of E7, the ability of LIMD1 to repress E2F-driven transcription was reduced by 52% (Fig. 3D). This is consistent with the Ϸ50% loss of repression seen between pRB-positive and -negative cell lines (Fig. 3C). The pRB-independent mechanisms of LIMD1 repression remain to be elucidated. However, within amino acids 18–68 of LIMD1 is a LEM domain (Fig. 1A). This is an Ϸ40 amino acid residue sequence that can specifically bind barrier-to-autointegration (BAF), a conserved chromatin protein that is a component of the SWI͞SNF chromatin- remodeling complex (31–33). Therefore, LIMD1 may also induce repression by way of recruiting chromatin-remodeling proteins.

LIMD1 Expression Reduces Transcription of E2F1-Responsive Genes. The LIMD1-negative cell line MB435 [as determined by RT-PCR (data not shown) and Western blot analysis (Fig. 3E)] was transduced with lentivirus expressing HA-LIMD1 (or no cDNA insert as control). Cells were harvested and processed 48 h postinfection for gene expression microarray (GEM) analysis (Fig. 3F); 3,102 genes were significantly (q Ͻ0.11) (17) differentially expressed between LIMD1-transduced cells and control cells. Of these significantly expressed genes, we analyzed 64 E2F1-responsive genes previously reported to be activated by E2F1 (18). We found that 55 of 64 (85.9%) E2F1-responsive genes were down-regulated by LIMD1 expression (Fig. 3F). We also examined 28 genes that were not activated by E2F1 (18). Of these genes, 19 of 28 (67%) were down-regulated, indicating that LIMD1 does not have a global transcription-repressive effect, but that the effect on E2F1- responsive genes is specific. (For the gene list, see Tables 1 and 2, which are published as supporting information on the PNAS web site.)

LIMD1 Inhibits Cell Proliferation. Because the pRB͞E2F1 complex can regulate cell cycle progression (transition from G1 to S phase) by the regulation of E2F-responsive genes necessary for Fig. 4. LIMD1 causes growth suppression in vitro and in vivo.(A Top) LIMD1 S phase progression and thus proliferation, we examined the inhibits colony formation and growth when transiently expressed in A549 cells. (Middle) Expression of HA-LIMD1 is shown 24 h posttransfection. This effects of LIMD1 expression on cell proliferation in the experiment was repeated at least three times in triplicate with A549 (Bottom) LIMD1-negative cancer cell line MB435 (Fig. 3E). DNA and HEK293 (data not shown) cells. (B) LIMD1 reduces growth rate in the synthesis was determined by measuring BrdUrd incorporation. mouse A9 fibrosarcoma model. Shown are cumulative results of tumor size of A significant inhibition of BrdUrd incorporation in MB435 mouse fibrosarcoma A9 and A9-LIMD1 P1-derived artificial chromosome (PAC) cells was observed in the presence of WT LIMD1 relative to transfectants (LIMD1) versus weeks postinjection. Circles represent outliers. vector control (Fig. 3G), indicating that LIMD1 expression (C) LIMD1 significantly (P Ͻ 0.05) reduces development of A549 experimental inhibits cell proliferation. lung metastases in nu͞nu mice. Lentivirus expressing either LIMD1 or VO control was transduced into A549 cells and injected via tail veins into mice. LIMD1 Represses Colony Formation. To test whether the transcrip- (Middle) Metastatic colonies (tumor nodules) on the surface of the lung were counted without knowledge of the treatment group. (D) The expression of tional and proliferate suppressive effects of LIMD1 translate endogenous LIMD1 is reduced in lung cancer cell lines. Using an anti-LIMD1 to a tumor-suppressive phenotype, we tested the growth- mAb (Upper), we examined the expression of endogenous LIMD1 in six lung suppressive effects of ectopically expressed LIMD1 on trans- cancer cell lines equalized for protein concentration (Lower, anti-actin) and formed cell lines. Colony formation assays were performed by compared with a positive control cell line (U2OS). (E) mRNA levels for LIMD1 selecting for the Zeocin-resistance gene carried by our LIMD1 are reduced in all lung tumors tested compared with adjacent normal lung. expression plasmids. Plasmids containing the full ORF of Total RNA was extracted from paired lung tumor and adjacent normal tissue LIMD1 and the VO control were transfected into non-small and subjected to qRT-PCR. All LIMD1 mRNA levels in tumor samples (hatched cell lung cancer (NSCLC) A549 and HEK293 cells. The bars) are shown relative to the patient-paired normal tissue (filled bars), number of Zeocin-resistant colonies after LIMD1 transfection normalized to one. The tumors analyzed included five adenocarcinomas Ͼ (ADCs), six squamous cell carcinomas (SQCCs), two large cell undifferentiated was reduced by 80% in both cell types (Fig. 4A and data not carcinomas (LCUCs), and one small cell lung carcinoma (SCLC). (F) Protein levels shown). LIMD1 mutants unable to interact with pRB but of LIMD1 are reduced in lung tumors. Two adenocarcinomas (ADCs) and one expressed at comparable levels did not inhibit colony forma- squamous cell carcinoma (SQCC), together with their matched normal tissue tion (data not shown). controls, were picked from those in E, and protein extracts were normalized and blotted for the expression of LIMD1. Anti-actin blotting confirmed com- LIMD1 A9 Fibrosarcoma Mouse Model. To further evaluate the parable loading of protein. growth-inhibitory effects of LIMD1 expression in vivo, we used the A9 mouse fibrosarcoma cells that form xenografts and were used to obtain the initial functional evidence, demonstrating that chro- human chromosome 3 encompassing 3p21–22. Five weeks postin- mosome 3p contains tumor-suppressive activity (34). In these jection (p.i.), LIMD1 expression caused a significant (P Ͻ 0.05)

previous experiments (34), the in vivo growth of A9 cells in nude decrease in tumor size (Fig. 4B). This same effect on tumor growth CELL BIOLOGY mice was inhibited by transfection with a 2-megabase piece of was observed 6 weeks p.i., although this was not statistically

Sharp et al. PNAS ͉ November 23, 2004 ͉ vol. 101 ͉ no. 47 ͉ 16535 Downloaded by guest on October 1, 2021 significant (Fig. 4B). These data indicate that LIMD1 suppresses or the matched samples and confirmed to be decreased in the tumors delays the growth of tumor cells in an experimental model. compared with normal tissue (Fig. 4F). Chromosome 3p is known to possess tumor-suppressive activity LIMD1 Inhibits Development of Experimental Lung Metastases in Vivo. (34). Of the 3p regions analyzed, the highest deletion frequency in We evaluated the efficacy of LIMD1 in suppressing in vivo tumor solid tumors occurs at the C3CER1 region within 3p21.3 (6). growth and metastases. A549 cells were transduced with a lentivirus Furthermore, in the majority of tumors analyzed, including lung expressing HA-LIMD1 or lentivirus only. More than 95% of the cancer, LOH at the C3CER1 region (containing LIMD1)ismore cells consistently expressed LIMD1, as determined by IFA for the frequent than LOH at either the FHIT or the VHL TSG regions (6). HA tag and by fluorescent activated cell sorting (FACS) for GFP The functional role in tumor formation of the majority of the 19 (see Materials and Methods). Once expression was confirmed, cells active genes encoded within C3CER1 is unknown (37). The lacto- were grown for a further 24 h, and an equal number of viable cells transferrin gene has been shown to suppress growth in fibrosarcoma was injected into the tail veins of athymic nude mice. LIMD1 cells (38) and, like LIMD1, inhibits v-ras transformation of NIH 3T3 expression led to a significantly reduced incidence of lung metas- cells (unpublished work). LIMD1’s effect on cell proliferation and tases (Fig. 4C Middle and Bottom). tumor growth (Figs. 3 and 4), linked to its ability to bind pRB and Overexpression of E2F1 is associated with increased tumor repress E2F-driven transcription (Figs. 1 and 3), suggests that cell growth and metastatic progression (35, 36). Therefore, our LIMD1 is a principal TSG candidate within C3CER1. In addition, data showing that LIMD1 induced down-regulation of E2F1- reduced expression of LIMD1 in all lung cancers tested (Fig. 4 E and responsive genes (Fig. 3F) concur with the tumor growth and F) supports the finding that LOH at the C3CER1 region represents metastatic inhibitory phenotype induced by LIMD1 in vitro and an early and recurrent event in the development of lung cancer (6). in vivo (Fig. 4 A, B, and C, respectively). Because pRB mutation, or loss, does not occur early in lung cancer development (39), early LIMD1 LOH (through 3p͞ LIMD1 Expression Is Reduced in Lung Tumors. Finally, we tested C3CER1 deletion) may indirectly represent a loss of pRB regula- whether LIMD1 expression is reduced in human lung cancer cell tion and be a critical early step in the development of lung cancer. lines and lung cancers. Five of the six (83%) cell lines had reduced expression of LIMD1 protein compared with U2OS cells when We thank D. Lagos, H. Laman, H. Ye, and H. Kiss for helpful equalized for protein concentration and actin expression (Fig. 4D). discussions; the Department of Thoracic Surgery, Royal Brompton We next tested LIMD1 transcript levels in 14 paired malignant Hospital, led by Professor P. Goldstraw, for help in collection of samples versus adjacent normal lung tissue samples using real-time qRT- of lung tumors; and J. Timms (University College London, London) for PCR (Fig. 4E). All of the tumor samples had significantly decreased providing the MB435 cell line. This work was supported by funds from LIMD1 mRNA levels compared with their adjacent normal lung Cancer Research UK (to T.V.S., D.B., and C.B.) and The Wellcome tissue (Fig. 4F). LIMD1 protein levels were determined in three of Trust (to H.-W.W.).

1. Knudson, A. G., Jr. (1971) Proc. Natl. Acad. Sci. USA 68, 820–823. 25. Fornerod, M., Ohno, M., Yoshida, M. & Mattaj, I. W. (1997) Cell 90, 2. Knudson, A. G., Jr. (1978) Semin. Oncol. 5, 57–60. 1051–1060. 3. Knudson, A. G. (2001) Nat. Rev. Cancer 1, 157–162. 26. Yang, L., Guerrero, J., Hong, H., DeFranco, D. B. & Stallcup, M. R. (2000) 4. Hinds, P. W. & Weinberg, R. A. (1994) Curr. Opin. Genet. Dev. 4, 135–141. Mol. Biol. Cell 11, 2007–2018. 5. Kiss, H., Kedra, D., Yang, Y., Kost-Alimova, M., Kiss, C., O’Brien, K. P., 27. Kanungo, J., Pratt, S. J., Marie, H. & Longmore, G. D. (2000) Mol. Biol. Cell Fransson, I., Klein, G., Imreh, S. & Dumanski, J. P. (1999) Hum. Genet. 105, 11, 3299–3313. 552–559. 28. Fujita, H., Kamiguchi, K., Cho, D., Shibanuma, M., Morimoto, C. & Tachibana, 6. Petursdottir, T. E., Thorsteinsdottir, U., Jonasson, J. G., Moller, P. H., Huiping, K. (1998) J. Biol. Chem. 273, 26516–26521. C., Bjornsson, J., Egilsson, V., Imreh, S. & Ingvarsson, S. (2004) Genes 29. Thomas, S. M., Hagel, M. & Turner, C. E. (1999) J. Cell Sci. 112, 181–190. Chromosomes Cancer 41, 232–242. 30. Ewen, M. E., Sluss, H. K., Sherr, C. J., Matsushime, H., Kato, J. & Livingston, 7. Imreh, S., Kholodnyuk, I., Allikmetts, R., Stanbridge, E. J., Zabarovsky, E. R. D. M. (1993) Cell 73, 487–497. & Klein, G. (1994) Genes Chromosomes Cancer 11, 237–245. 8. Zabarovsky, E. R., Lerman, M. I. & Minna, J. D. (2002) Oncogene 21, 31. Wang, W., Cote, J., Xue, Y., Zhou, S., Khavari, P. A., Biggar, S. R., 6915–6935. Muchardt, C., Kalpana, G. V., Goff, S. P., Yaniv, M., et al. (1996) EMBO J. 9. Imreh, S., Klein, G. & Zabarovsky, E. R. (2003) Genes Chromosomes Cancer 15, 5370–5382. 38, 307–321. 32. Bruder, C. E., Dumanski, J. P. & Kedra, D. (1999) Biochem. Biophys. Res. 10. Kaye, F. J. (2001) Lung Cancer 34, Suppl. 2, S35–S41. Commun. 257, 886–890. 11. Zamanian, M. & La Thangue, N. B. (1992) EMBO J. 11, 2603–2610. 33. Guidi, C. J., Sands, A. T., Zambrowicz, B. P., Turner, T. K., Demers, D. A., 12. Naldini, L., Blomer, U., Gallay, P., Ory, D., Mulligan, R., Gage, F. H., Verma, Webster, W., Smith, T. W., Imbalzano, A. N. & Jones, S. N. (2001) Mol. Cell. I. M. & Trono, D. (1996) Science 272, 263–267. Biol. 21, 3598–3603. 13. Radkov, S. A., Kellam, P. & Boshoff, C. (2000) Nat. Med. 6, 1121–1127. 34. Killary, A. M., Wolf, M. E., Giambernardi, T. A. & Naylor, S. L. (1992) Proc. 14. James, P., Halladay, J. & Craig, E. A. (1996) Genetics 144, 1425–1436. Natl. Acad. Sci. USA 89, 10877–10881. 15. Sharp, T. V., Wang, H. W., Koumi, A., Hollyman, D., Endo, Y., Ye, H., Du, 35. Zhang, S. Y., Liu, S. C., Johnson, D. G. & Klein-Szanto, A. J. (2000) Cancer M. Q. & Boshoff, C. (2002) J. Virol. 76, 802–816. Res. 60, 5972–5976. 16. Ioannou, P. A., Amemiya, C. T., Garnes, J., Kroisel, P. M., Shizuya, H., Chen, 36. Banerjee, D., Gorlick, R., Liefshitz, A., Danenberg, K., Danenberg, P. C., C., Batzer, M. A. & de Jong, P. J. (1994) Nat. Genet. 6, 84–89. Danenberg, P. V., Klimstra, D., Jhanwar, S., Cordon-Cardo, C., Fong, Y., et al. 17. Wang, H. W., Trotter, M. W., Lagos, D., Bourboulia, D., Henderson, S., (2000) Cancer Res. 60, 2365–2367. Makinen, T., Elliman, S., Flanagan, A. M., Alitalo, K. & Boshoff, C. (2004) Nat. 37. Kiss, H., Yang, Y., Kiss, C., Andersson, K., Klein, G., Imreh, S. & Dumanski, Genet. 36, 687–693. J. P. (2002) Eur. J. Hum. Genet. 10, 52–61. 18. Ma, Y., Croxton, R., Moorer, R. L., Jr., & Cress, W. D. (2002) Arch. Biochem. 38. Bezault, J., Bhimani, R., Wiprovnick, J. & Furmanski, P. (1994) Cancer Res. 54, Biophys. 399, 212–224. 2310–2312. 19. Heid, C. A., Stevens, J., Livak, K. J. & Williams, P. M. (1996) Genome Res. 6, 986–994. 39. Wistuba, I. I., Berry, J., Behrens, C., Maitra, A., Shivapurkar, N., Milchgrub, 20. Bach, I. (2000) Mech. Dev. 91, 5–17. S., Mackay, B., Minna, J. D. & Gazdar, A. F. (2000) Clin. Cancer Res. 6, 21. Dawid, I. B., Breen, J. J. & Toyama, R. (1998) Trends Genet. 14, 156–162. 2604–2610. 22. Wang, Y. & Gilmore, T. D. (2003) Biochim. Biophys. Acta 1593, 115–120. 40. Petit, M. M., Mols, R., Schoenmakers, E. F., Mandahl, N. & Van de Ven, W. J. 23. Wang, Y. & Gilmore, T. D. (2001) Biochim. Biophys. Acta 1538, 260–272. (1996) Genomics 36, 118–129. 24. Goyal, R. K., Lin, P., Kanungo, J., Payne, A. S., Muslin, A. J. & Longmore, 41. Yi, J. & Beckerle, M. C. (1998) Genomics. 49, 314–316. G. D. (1999) Mol. Cell. Biol. 19, 4379–4389. 42. Beckerle, M. C. (1997) Bioessays 19, 949–957.

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