RASSF1C, an Isoform of the Tumor Suppressor RASSF1A, Promotes the Accumulation of B-Catenin by Interacting with Btrcp

Research Article

RASSF1C, an Isoform of the Tumor Suppressor RASSF1A, Promotes the Accumulation of B-Catenin by Interacting with BTrCP

Emilie Estrabaud,1 Irina Lassot,1 Guillaume Blot,1 Erwann Le Rouzic,1 Vale´rie Tanchou,3 Eric Quemeneur,3 Laurent Daviet,2 Florence Margottin-Goguet,1 and Richard Benarous1

1Institut Cochin, De´partementMaladies Infectieuses; Institut National de la Sante et de la Recherche Medicale, U567; Centre National de la Recherche Scientifique, UMR 8104; Universite´Paris Descartes, Faculte´deMe´decine Rene´Descartes, UMR-S 8104; 2Hybrigenics, Paris, France; and 3CEA Valrhoˆ, DSV, DIEP, SBTN, BP 17171, Bagnols sur Ce`zecedex, France

Abstract small-cell lung, breast, kidney, nasopharyngeal, prostate, and The Ras-association domain family 1 (RASSF1) gene has seven hepatocellular carcinoma, and retinoblastoma (reviewed in ref. 4). different isoforms; isoform A is a tumor-suppressor gene In contrast, the expression of another isoform of RASSF1, RASSF1C, (RASSF1A). The promoter of RASSF1A is inactivated in many has been shown not to be affected, and no epigenetic inactivation cancers, whereas the expression of another major isoform, of the RASSF1C promoter has been reported in these tumors (3, 4). RASSF1C, is not affected. Here, we show that RASSF1C, but not RASSF1A null mice that still express RASSF1C had a higher RASSF1A, interacts with BTrCP. Binding of RASSF1C to BTrCP incidence of spontaneous tumorigenesis and a lower survival rate than wild-type mice (5). The mechanism of action of RASSF1A at involves serine 18 and serine 19 of the SS18GYXS19 motif present in RASSF1C but not in RASSF1A. This motif is the molecular level remains poorly understood. RASSF1A and RASSF1C differ at their amino termini and share an identical reminiscent of the canonical phosphorylation motif recognized by BTrCP; however, surprisingly, the association sequence at their carboxyl termini. This common region includes a between RASSF1C and BTrCP does not occur via the BTrCP Ras association or RalGDS/AF-6 domain (RA domain; ref. 6) that substrate binding domain, the WD40 repeats. Overexpression may bind RasGTP similar to that of the Ras effector for apoptosis of RASSF1C, but not of RASSF1A, resulted in accumulation Nore1 (7–9). RASSF1A and RASSF1C both associate with micro- and transcriptional activation of the B-catenin oncogene, due tubules, but only RASSF1A stabilizes microtubules (10, 11). to inhibition of its BTrCP-mediated degradation. Silencing of RASSF1A has been implicated in the control of cell migration RASSF1A by small interfering RNA was sufficient for B-catenin (10) and in the control of cell cycle progression (4, 12, 13). In to accumulate, whereas silencing of both RASSF1A and proapoptotic pathways, RASSF1A is involved in activation of Bax RASSF1C had no effect. Thus, RASSF1A and RASSF1C have (14) and in a complex with the BH3 protein MAP1, linking death opposite effects on B-catenin degradation. Our results suggest receptor signaling to the apoptotic machinery through Bax that RASSF1C expression in the absence of RASSF1A could conformational change (15). We found that RASSF1A and RASSF1C had opposite effects on the accumulation of the h-catenin play a role in tumorigenesis. [Cancer Res 2007;67(3):1054–61] oncogene, a key effector of the Wnt signaling pathway (reviewed in ref. 16). Our results suggest that the inhibition of h-catenin Introduction accumulation could be one of the mechanisms by which RASSF1A Since the discovery of RASSF1A epigenetic inactivation in lung exerts its tumor suppressor function. tumors (1, 2), numerous reports have implicated the inactivation of the RASSF1A promoter by hypermethylation in tumorigenesis across a wide spectrum of human tumors (3, 4). The RASSF1 gene Materials and Methods has eight exons and is located within a 120-kb region of Plasmids. Vectors for HA-hTrCP, HA-hTrCPDF, HA-ATF4, and hTrCP- chromosome 3p21.3 that frequently undergoes allele loss in human GFP were described in Lassot et al. (17). The plasmid Top-tk-Luci tumors (3, 4). Differential promoter usage and alternative splicing expressing luciferase was kindly provided by H. Clevers (Department of generate seven different transcripts (RASSF1A–RASSF1G). Isoforms Pharmacology and Cancer Biology, Duke University Medical Center, A and C are ubiquitously expressed, whereas the expression of the Durham, NC). The RSV-Renilla luciferase vector is from Promega (Madison, other isoforms is tissue specific (4). A direct correlation between WI). Myc-RASSF1A, Myc-RASSF1C, and Myc-RA plasmids were kindly promoter methylation and loss of RASSF1A expression has been provided by M. White (Department of Cell Biology, UT Southwestern Medical Center, Dallas, TX). Vectors for Flag-RASSF1A and Flag-RASSF1C shown in many tumor cell lines, including small-cell lung, non– were obtained by transferring RASSF1A and RASSF1C into pSG (Sigma, St. Louis, MO). RASSF1DN (deletion of amino acids 1–49) was obtained by PCR from Myc-RASSF1C vector. Single point mutations in Myc-RASSF1C were obtained by PCR-directed Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). mutagenesis. Plamids for yeast two-hybrid have been described previously E. Estrabaud and I. Lassot contributed equally to this work. by Margottin et al. (18) and Hart et al. (19), except plex-hTrCP Nter 1 to 260, Requests for reprints: Florence Margottin-Goguet, Department of Infectious 192 to 569, and 260 to 569, which have been done by PCR using specific Diseases, Institut National de la Sante et de la Recherche Medicale, 27, rue du primers and plex-hTrCPDF, which was done by internal deletion of residues Faubourg Saint Jacques, Bat G Roussy, 5eme etage, 75014 Paris, France. Phone: 33-1-40- 51-66-16; Fax: 33-1-40-51-65-70; E-mail: [email protected] or Richard 148 to 192. Benarous, Department of Infectious Diseases, Institut National de la Sante et de la Yeast two-hybrid screening. Yeast two-hybrid screening was done Recherche Medicale, 27, rue du Faubourg Saint Jacques, Bat G Roussy, 5eme etage, according to Formstecher et al. (20), using the NH2-terminal domain 75014 Paris, France. Phone: 33-1-40-51-65-71; Fax: 33-1-40-51-65-70; E-mail: (1–291) of hTrCP as bait, against a random primed cDNA library cons- [email protected]. I2007 American Association for Cancer Research. tructed from human placental poly(A)+ mRNA. Two hybrid assays were doi:10.1158/0008-5472.CAN-06-2530 done as previously described (19).

Cancer Res 2007; 67: (3). February 1, 2007 1054 www.aacrjournals.org

Lysis, immunoprecipitation, and Western blot. Antibodies used were top). HA-hTrCP was not coimmunoprecipitated with Myc- as follows: mouse monoclonal anti-Flag (Sigma) and rabbit anti-Flag RASSF1A (Fig. 1A, lane 8). Similar results were found by using (Sigma), rat anti-hemagglutinin (anti-HA; High Affinity, Roche), mouse Flag-tagged RASSF1A and RASSF1C isoforms (Fig. 1B, lanes 1 and h monoclonal anti-Myc (Roche), rabbit anti- TrCP (gift from Klaus Strebel, 2, respectively). Deletion of a 49-amino-acid NH -terminal region Laboratory of Molecular Genetics, National Institute of Child Health and 2 specific to RASSF1C (and not present in RASSF1A) abolished its Human Development, Bethesda, MD) or goat anti-hTrCP (Santa Cruz Biotechnology, Santa Cruz, CA), mouse anti–a-tubulin (Sigma), mouse anti– h-catenin (Transduction Laboratories, Lexington, KY), goat anti-actin (Santa Cruz Biotechnology), and mouse anti-InBa (Santa Cruz Biotechnol- ogy). A rabbit polyclonal RASSF1C antiserum was produced. The immunogen was RASSF1C recombinant protein expressed as a (His)6- tagged protein. Antibody specificity was assessed by probing Western blots of 6-His–tagged RASSF1C recombinant protein (data not shown), lysates of Myc-RASSF1C–transfected HeLa cells (Fig. 1C, lane 4), and lysates of untransfected cells (Fig. 1C, lane 3). Anti-RASSF1C antibodies recognized both endogenous RASSF1C as well as transfected Myc-RASSF1C (Fig. 1C). Untransfected cells or cells at 24 h after transfection were harvested and lysed in 1% NP40, 150 mmol/L NaCl, 50 mmol/L Tris-HCl (pH 7.5), and 1 mmol/L EDTA. For immunoprecipitations, cell lysates were precleared with nonimmune antibodies and protein A or G agarose for 90 min, and supernatants were incubated overnight with the appropriate antibodies and then with protein A or G agarose beads (Sigma) for 1 h. Immunocomplexes were eluted with Laemmli buffer and were separated by SDS-PAGE. Immunostaining of cells. We used the protocol of Lassot et al. (17). HeLa cells were transfected, fixed, permeabilized, and incubated with mouse anti–h-catenin antibodies; washed in PBS; and incubated with anti- mouse Cy2 antibodies (Jackson Immunoresearch) or directly incubated with anti–Myc-TRITC (Santa Cruz Biotechnology). Confocal microscopy was carried out under fluorescent light. Pulse-chase experiments. HeLa cells were transiently transfected with 10 Ag of empty vector, Myc-RASSF1A, or Myc-RASSF1C expression vectors, and pulse-chase analysis of h-catenin was done as previously described for ATF4 by Lassot et al. (17), but using mouse anti–h-catenin antibodies. Ubiquitination assay. HeLa cells cotransfected with expression vectors and HA-ubiquitin plasmid were treated 4 h with MG132 (20 Amol/L). Cell pellets were resuspended in 1 volume of denaturing lysis buffer (50 mmol/L Tris, 10% SDS, 5 mmol/L NEM, 1 mmol/L NaOV, 10 mmol/L NaF, and 20 Amol/L MG132 with protease inhibitors), boiled for 10 min, and diluted in 9 volumes of 50 mmol/L Tris (pH 7.5), 150 mmol/L NaCl, 5 mmol/L EDTA, 1% NP40, 5 mmol/L NEM, 1 mmol/L NaOV, 10 mmol/L NaF, and 20 Amol/L MG132 with protease inhibitors; then, cells were immunoprecipitated with anti–h-catenin antibody. Ubiquitin species were revealed by Western blot using anti-HA antibodies. Small interfering RNAs. RNA oligonucleotides were purchased from Eurogentech (Seraing, Belgium). We used the oligonucleotide small interfering RNA A (siRNA A; ref. 12) to silence RASSF1A. The oligonucleotides siRNA A/C (forward, 5¶-gcugagauugagcagaagatt; reverse, 5¶-ucuucugcucaau- cucagctt) were designed to silence both RASSF1A and RASSF1C. We used the Figure 1. hTrCP coimmunoprecipitates with RASSF1C, but not with RASSF1A, siRNA luciferase as described in Bres et al. (21). After annealing, siRNAs were and not with the RA domain (Myc-RA) common to both isoforms. A, HeLa h transfected into HeLa cells or A549 cells using Oligofectamine. In Fig. 5A, cells were cotransfected with either HA- TrCP (lanes 2, 4, 6, and 8) or control plasmid (lanes 1, 3, 5, and 7) together with Myc-RA (lanes 3 and 4), 24 h later, cells were cotransfected with both siRNA and plasmid vectors using Myc-RASSF1C (lanes 5 and 6), or Myc-RASSF1A (lanes 7 and 8) expression LipofectAMINE (Invitrogen, Carlsbad, CA). Cells were harvested and lysed vectors. Immunoprecipitates (IP) using anti-Myc antibodies (top and middle) 24 h after transfection. and cell lysates were analyzed by Western blot using anti-HA-horseradish peroxidase (HRP; top and bottom) and anti-Myc-HRP antibodies (middle). B, the NH2-terminalregion of RASSF1C is required for hTrCP coimmunoprecipitation. 293T cells were cotransfected with HA-hTrCP Results (lanes 1–3) or controlplasmid( lanes 4–6) together with Flag-RASSF1A B h (lanes 1 and 5), Flag-RASSF1C (lanes 2 and 6), or the Flag-tagged TrCP binds RASSF1C but not RASSF1A. TrCP is the NH -terminal deletion fragment Flag-RASSF1DN(lanes 3 and 4) expression hTrCP 2 receptor subunit of the SCF ubiquitin ligase that recruits vectors. Anti-Flag immunoprecipitates (top and middle) and cell lysates (bottom) key signaling proteins, such as the oncogene h-catenin (19, 22), were analyzed by Western blot using anti-HA-HRP (top and bottom) and anti-Flag-HRP antibodies (middle). C, the hTrCP endogenous protein interacts InBa (23, 24), or ATF4 (17), and cell cycle regulators, such as Emi1 with endogenous RASSF1C. Lysates from nontransfected HeLa cells (lanes 1 (25, 26) for proteasomal degradation (reviewed in refs. 27–29). and 2) were immunoprecipitated with anti-hTrCP antibodies (lane 1) or, as a h control, with rabbit polyclonal anti-Flag antibodies (lane 2), and then analyzed by We did two-hybrid screening using a TrCP fragment from Western blot using anti-RASSF1C antibodies. A Western blot of cell lysates from residues 1 to 291 as bait, and we found that hTrCP interacts with nontransfected cells and Myc-RASSF1C–transfected cells is shown (lanes 3 RASSF1C (data not shown). This interaction was confirmed in and 4) to verify the specificity of the anti-RASSF1C antibodies and to identify h unambiguously the band corresponding to endogenous RASSF1C; the detection HeLa cells cotransfected with HA- TrCP and Myc-RASSF1C, and of both endogenous RASSF1C and Myc-RASSF1C bands by anti-RASSF1C coimmunoprecipitated with anti-Myc antibodies (Fig. 1A, lane 6, antibodies is indicated. www.aacrjournals.org 1055 Cancer Res 2007; 67: (3). February 1, 2007

Figure 2. RASSF1C but not RASSF1A promotes h-catenin accumulation. A, HeLa cells were either untransfected (lanes 1 and 6), transfected with increasing amounts of Myc-RASSF1C expression vector (lanes 2–5, top), or transfected with increasing amounts of Myc-RASSF1A expression vector (lanes 7–10, top), and were assessed for h-catenin accumulation by Western blot using anti-h-catenin monoclonal antibodies (middle). Anti–a-tubulin antibodies were used to controlfor equalamounts of protein. B, h-catenin immunofluorescence staining is greatly enhanced in the presence of RASSF1C. HeLa cells transfected with Myc-RASSF1A (right) or Myc-RASSF1C (left) were analyzed by indirect immunofluorescence using anti–Myc-TRITC (top) and anti-h-catenin antibodies (middle). The expression of h-catenin in transfected cells should be compared with the level of h-catenin barely detectable in neighboring untransfected cells. C, 35S h-catenin is stabilized in the presence of RASSF1C. HeLa cells were mock transfected (Mock, top), transfected with Myc-RASSF1A (middle), or with Myc-RASSF1C (bottom). Subsequently, we metabolically labeled cells for pulse-chase analysis, immunoprecipitated 35S h-catenin using anti-h-catenin antibodies, and did SDS-PAGE and autoradiography. The graph represents the radioactive bands corresponding to h-catenin detected at different times. These bands were quantified and the decay curves of h-catenin in untransfected (Mock) or in Myc-RASSF1A– or Myc-RASSF1C–transfected cells are shown. D, h-catenin ubiquitination is reduced in the presence of RASSF1C. HeLa cells were cotransfected with HA-ubiquitin and Myc-h-catenin in the absence (lane 1) or in the presence of Myc-RASSF1C (lane 2) or Myc-h-catenin was transfected alone (lane 3). Cells were treated with the proteasome inhibitor MG132. Ubiquitinated species of h-catenin were revealed using anti-HA antibodies after anti-h-catenin immunoprecipitation. h-Catenin and Myc-RASSF1C were detected in the lysates as indicated. WB, Western blot.

interaction with HA-hTrCP (Fig. 1B, lane 3), and the COOH- Overproduction of Myc-RASSF1A at similar levels had either no terminal RA domain alone was not able to associate with hTrCP such effect (Fig. 2A, compare lanes 7-10 with lane 6) or resulted in (Fig. 1A, lane 4). This indicated that the NH2-terminal fragment of less h-catenin expression (Fig. 2A, right). In Myc-RASSF1C– 49-amino-acid residues specific to RASSF1C was required for its transfected cells, there was substantial accumulation of h-catenin interaction with hTrCP. The association of RASSF1C with hTrCP as assessed by indirect immunofluorescence with anti–h-catenin was also detected for the endogenous proteins. RASSF1C coimmu- antibodies (Fig. 2B, left), whereas in neighboring untransfected cells noprecipitated with hTrCP when anti-hTrCP antibodies were used or in Myc-RASSF1A–transfected cells, h-catenin was barely for the immunoprecipitation (Fig. 1C, lane 1), but not when anti- detectable (Fig. 2B). Flag antibodies were used for the immunoprecipitation (negative We did pulse-chase experiments to investigate the mechanism control Fig. 1C, lane 2). of h-catenin accumulation. RASSF1C-mediated accumulation of RASSF1C promotes B-catenin accumulation. We overpro- h-catenin was due to stabilization of h-catenin, as detected in Myc- duced Myc-RASSF1C in HeLa cells and found that this led to an RASSF1C–transfected cells after 35S metabolic labeling (Fig. 2C, accumulation of h-catenin, as detected by immunoblot in lysates of top). After quantification of the labeled bands, we estimated transfected cells (Fig. 2A, compare lanes 2–5 with lane 1). that the stability of h-catenin was enhanced upon RASSF1C

Cancer Res 2007; 67: (3). February 1, 2007 1056 www.aacrjournals.org

Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 2007 American Association for Cancer Research. RASSF1C-Mediated b-Catenin Accumulation overexpression, with a half-life of 30 min in nontransfected cells We further considered the question of whether RASSF1A could and a half-life of 90 min in RASSF1C-transfected cells (Fig. 2C, play a role in RASSF1C-mediated h-catenin accumulation by first bottom). Transfection with RASSF1A had no effect on the degra- determining whether RASSF1A could interfere with the interac- dation rate of h-catenin (Fig. 2C). This accumulation of h-catenin tion between RASSF1C and hTrCP. We studied the interaction correlates with a strong inhibition of h-catenin ubiquitination between transfected HA-hTrCP and Myc-RASSF1C in the absence promoted by overexpression of RASSF1C in cells cotransfected with or in the presence of increasing concentrations of RASSF1A-Flag, HA-ubiquitin (Fig. 2D, compare lane 2 with lane 1). and we found that the level of expression of RASSF1A did not The RASSF1C-mediated accumulation of h-catenin led to an significantly affect the hTrCP/RASSF1C interaction analyzed by f10-fold increase in h-catenin transcriptional activity based on coimmunoprecipitation (Supplementary Fig. S3, compare lanes 3 experiments using a luciferase reporter gene under the control of and 4 with lane 2). the h-catenin–dependent TOP-flash synthetic promoter (ref. 19; RASSF1C-mediated B-catenin accumulation requires bind- Supplementary Fig. S1). This increase in h-catenin–dependent ing to RASSF1C via a SSGYXS motif. We isolated mutants of transcriptional activity was comparable with that obtained with RASSF1C deficient for interaction with hTrCP and examined their hTrCPDF, the hTrCP-negative transdominant mutant previously ability to promote h-catenin accumulation. We mutated two serine reported to inhibit the degradation rate of h-catenin (19). residues, S18 and S19, present in a SS18GYCS19 motif in the NH2- We studied the effect of RASSF1C overexpression on two other terminal region specific to RASSF1C. This SSGYCS motif is known substrates of hTrCP, InBa and ATF4, and we found that the reminiscent of the canonical phosphorylation motif DSGXXS, degradation rate of these hTrCP substrates was also inhibited by previously characterized as the binding site of hTrCP (18, 27, 28). overexpression of RASSF1C, whereas RASSF1A had no effect Constructs expressing wild-type Myc-RASSF1C or its mutants were (Supplementary Fig. S2 for InBa; data not shown for ATF4). Thus, cotransfected with HA-hTrCP to study the association between the effect of RASSF1C is not specific for h-catenin but is a more these proteins. The S18N, S19N, and S18-19N mutants did not general effect on the inhibition of hTrCP-mediated degradation of immunoprecipitate hTrCP (Fig. 3A, lanes 3-5), in contrast to wild- h several protein substrates of the SCF TrCP E3 ubiquitin ligase. type RASSF1C (Fig. 3A, lane 2), and the mutants did not promote

Figure 3. RASSF1C-mediated h-catenin accumulation requires binding to hTrCP. A, serine S18 and S19 of RASSF1C are required for binding to hTrCP. Lysates from transfected HeLa cells expressing HA-hTrCP alone (lane 1), or HA-hTrCP coexpressed with Myc-RASSF1C (WT), Myc-RASSF1C S18N, S19N, or S18-19N mutants (lanes 2–5) were immunoprecipitated with anti-Myc antibodies. Lysates and immunoprecipitates were subjected to Western blot using anti-Myc (top)or anti-HA antibodies (bottom). B, RASSF1C- mediated h-catenin accumulation requires binding to hTrCP. HeLa cells expressing wild-type Myc-RASSF1C (two top panels) or the mutants Myc-RASSF1C S18N (two middle panels), S19N (two bottom panels), or S18-19N (not shown) were analyzed by immunofluorescence using anti-Myc- TRITC (left panels) or anti–h-catenin antibodies (right panels). C, RASSF1C but not RASSF1A can inhibit h-catenin association to hTrCP. Lysates from HeLa cells coexpressing HA-hTrCP and increasing amounts of Myc-RASSF1C (top) or Myc-RASSF1A (bottom) were immunoprecipitated with anti-HA antibodies. Lysates and immunoprecipitates were analyzed by Western blot using anti–h-catenin, anti-HA, or anti-Myc antibodies as indicated. D, the hTrCP NH2-terminaldomain but not the COOH-terminalsubstrate binding domain interacts with RASSF1C. The yeast reporter strain L40 expressing the pairs of indicated hybrid proteins was analyzed for histidine auxotrophy and h-galactosidase expression. Transformants were plated on medium with histidine (+His, left), without histidine (ÀHis, middle), or replica-plated on Whatman filters and tested for h-galactosidase activity (b-gal, right). Growth in the absence of histidine and blue color in the h-galactosidase assay indicate interaction between hybrid proteins.

www.aacrjournals.org 1057 Cancer Res 2007; 67: (3). February 1, 2007

Figure 4. Exogenous RASSF1C can change the subcellular localization of hTrCP from nucleus to cytoplasm. HeLa cells expressing hTrCP-GFP alone (top), or together with Myc-RASSF1A (middle)or with Myc-RASSF1C (bottom), were analyzed by direct fluorescence and by indirect immunofluorescence using anti–Myc-TRITC antibodies. Cells were observed by confocalmicroscopy.

h-catenin accumulation, as shown by immunofluorescence using nor the F-box is required for RASSF1C binding. Further mapping anti–h-catenin antibodies (Fig. 3B). Interaction with hTrCP is showed that a fragment encompassing residues 192 to 260 present therefore required for RASSF1C to mediate h-catenin accumulation. between the F-box and the WD40 repeats conserved its ability to A hypothesis that could account for this ability of RASSF1C to interact with RASSF1C (data not shown). mediate h-catenin accumulation is that, by binding to hTrCP, RASSF1C delocalizes BTrCP from nucleus to cyctoplasm. RASSF1C inhibits the hTrCP-h-catenin interaction, thus impairing hTrCP is mostly localized to the nucleus in nonmitotic cells the proteasomal degradation of h-catenin. Consistent with this (17, 32), whereas RASSF1A and RASSF1C have been described as hypothesis, we found that increasing the amount of transfected cytoplasmic, microtubule-associated proteins (10, 12). Cells trans- RASSF1C inhibited the amount of endogenous h-catenin that could fected with hTrCP-GFP alone showed fluorescence in the nucleus be coimmunoprecipitated with transfected HA-hTrCP (Fig. 3C, top, (Fig. 4, top panels), in agreement with previous studies. In cells compare lanes 3-5 with lane 2), although the total expression level cotransfected with hTrCP-GFP and RASSF1C, there was substan- of h-catenin had increased as expected from RASSF1C transfection tial redistribution of hTrCP-GFP toward a mostly cytoplasmic (Fig. 3C, top). As a control, overexpression of RASSF1A had no staining (bottom panels), and this staining colocalized with that effect on coimmunoprecipitation of h-catenin with transfected HA- of RASSF1C on the cytoskeleton. As expected, Myc-RASSF1A, like hTrCP (Fig. 3C, bottom). From these data, we hypothesized that Myc-RASSF1C, was also associated with the cytoskeleton in the interaction with RASSF1C would require the substrate binding cytoplasm (Fig. 4, middle panels). However, when hTrCP-GFP was domain of hTrCP, comprising the WD40 repeats in the COOH- cotransfected with Myc-RASSF1A, the staining of hTrCP remained h terminal moiety of the protein, and not the NH2-terminal domain mostly nuclear, similar to that of TrCP-GFP transfected alone comprising the F-box, which connects to the proteasome through (middle panels), indicating that RASSF1A, unlike RASSF1C, did not Skp1 binding (30, 31). Surprisingly, RASSF1C still interacted with change the subcellular localization of hTrCP from the nucleus to the NH2-terminal moiety of hTrCP after removal of the all the the cytoplasm. B WD40 repeats (hTrCP NH2-terminal 1–260; Fig. 3D, lanes 1 and 2). Silencing of RASSF1A together with RASSF1C impairs - No interaction was found between RASSF1C and the COOH- catenin accumulation resulting from RASSF1A silencing. HeLa terminal substrate binding region, from residues 260 to 569 of cells express both RASSF1A and RASSF1C, as previously reported hTrCP, which, by contrast, interacted with the HIV-1 Vpu protein (refs. 1, 33; data not shown). We tested appropriate siRNAs to used as a control (Fig. 3D, lanes 5 and 11). hTrCP deleted from the silence specifically either RASSF1A or RASSF1C, or both, to mimic F-box motif and the NH2-terminal region upstream of the F-box the situation found in tumor cell lines in which RASSF1A (hTrCP 192-569) or hTrCP deleted only from the F-box (hTrCPDF) expression has been selectively lost by epigenetic methylation of still interacted with RASSF1C (Fig. 3D, lanes 4 and 3), whereas, as a the RASSF1A promoter. The efficiency of siRNA inhibition of the control, hTrCPDF does not interact with Skp1 (Fig. 3D, lane 8). expression of each isoform in HeLa cells transfected with Myc- Altogether, the binding data indicate that neither the WD40 repeats RASSF1A or Myc-RASSF1C was assessed by quantitative reverse

Cancer Res 2007; 67: (3). February 1, 2007 1058 www.aacrjournals.org

Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 2007 American Association for Cancer Research. RASSF1C-Mediated b-Catenin Accumulation transcription PCR and by Western blot using anti-Myc antibodies. effect on the expression of an unrelated protein, Myc (Fig. 5A, Using these assays, we could not find any siRNAs that specifically bottom). HeLa cells treated with siRNA A/C behaved similarly to silenced only isoform 1C and not isoform 1A. This is probably due those treated with siRNA Luc, with no detectable accumulation of to limited sequence differences between the mRNAs coding for the h-catenin (Fig. 5B, compare lanes 2 and 4). Therefore, we conclude RASSF1 isoforms. As previously reported (12, 33), siRNA A was that the silencing of RASSF1A was sufficient, in the absence of effective and could silence the expression of transfected Myc- overexpression of exogenous RASSF1C, to promote the accumula- RASSF1A by over 75%. However, the expression of transfected tion of h-catenin. This effect was observed despite the silencing of Myc-RASSF1C was also slightly inhibited (Fig. 5A, top, compare RASSF1A being incomplete, and despite partial silencing of lane 3 with lane 1, and lane 4 with lane 2). We found that in siRNA RASSF1C expression (Fig. 5A, lane 4). This effect of RASSF1A A–treated cells, the level of h-catenin expression was several times silencing is comparable with that observed upon siRNA silencing of higher than in control cells treated with siRNA luciferase (Luc; Fig. hTrCP1 and/or hTrCP2 (25). 5B, compare lane 3 with lane 2). Another siRNA, siRNA A/C, which Silencing of RASSF1C in a cell line that does not express targets a sequence common to RASSF1A and RASSF1C mRNAs, RASSF1A strongly decreases the expression of B-catenin. We almost completely silenced both isoforms (Fig. 5A, compare lane 5 further considered the question of whether RASSF1C could play a with lane 1, and lane 6 with lane 2, top), without any detectable role in controlling the expression level of h-catenin in a tumor cell line defective for RASSF1A expression, such as the A549 cell line. To address this question, we compared the effects of the siRNA A/C, which very efficiently suppresses the expression of RASSF1C (see Fig. 5A), with that of siRNA A, which should have no effect because RASSF1A is not expressed in these cells. As shown in Fig. 5C, treatment of A549 cells with siRNA A/C greatly reduced the expression level of h-catenin, whereas siRNA A had no effect, compared with siRNA Luc used as a control. Therefore, we conclude that the silencing of RASSF1C, in the absence of RASSF1A expression, was sufficient to strongly reduce h-catenin expression.

Discussion We found that RASSF1C interacts with hTrCP and as a result promotes the accumulation of h-catenin by inhibiting its hTrCP- mediated proteasomal degradation. This accumulation of h- catenin correlates with an increase of the half-life of h-catenin and a strong decrease of its ubiquitination. The stabilizing effect of RASSF1C on h-catenin does not seem to be restricted to this substrate of hTrCP, but seems also more generally to affect the degradation of other hTrCP substrates such as InBa and ATF4. Mutants of RASSF1C that did not associate with hTrCP had no effect on h-catenin. RASSF1C could inhibit the interaction of h- catenin with hTrCP and could change the subcellular localization of hTrCP from the nucleus to the cytoplasm, where both proteins colocalized. The isoform RASSF1A did not have these effects. The silencing of RASSF1A in HeLa cells, which express both RASSF1A and RASSF1C isoforms, was sufficient, without RASSF1C overexpression, to promote the accumulation of h-catenin. In contrast, the silencing of both RASSF1A and RASSF1C in these cells had no effect on h-catenin. In A549 lung cancer cells, which do not express RASSF1A, silencing of RASSF1A by siRNA A as expected had no effect, whereas silencing of RASSF1C by siRNA A/C h Figure 5. Accumulation of h-catenin resulting from RASSF1A silencing is decreased -catenin expression. Altogether, these observations impaired when RASSF1C is silenced together with RASSF1A. A, HeLa cells suggest that RASSF1A and RASSF1C isoforms have opposite effects transfected with either Myc-RASSF1A or Myc-RASSF1C expression vectors in controlling the degradation of h-catenin mediated by the were treated with siRNA Luc as a control( lanes 1 and 2), siRNA A (lanes 3 and hTrCP 4), or siRNA A/C (lanes 5 and 6). The effect of siRNAs on Myc-RASSF1A or SCF ubiquitin ligase. Myc-RASSF1C expression was analyzed by Western blot using anti-Myc Whether RASSF1C is a hTrCP substrate, and as such could antibodies. Endogenous Myc was used as a control. B, silencing of RASSF1A by compete for the binding of other substrates such as h-catenin or siRNA A promotes h-catenin accumulation, whereas silencing both RASSF1A and RASSF1C has no effect. HeLa cells treated with siRNA Luc (lane 2), siRNA InBa, remains an open question. However, several lines of evidence A(lane 3), or siRNA A/C (lane 4) were analyzed for the expression of h-catenin suggest that it is not the case: First, the association of RASSF1C by Western blot using anti–h-catenin antibodies (top). The blot was also probed h with antitubulin antibodies (bottom) as a control. HeLa cells transfected with with TrCP seems to be independent of the substrate binding RASSF1C (lane 1) were used as a positive controlfor RASSF1C-mediated domain of hTrCP; second, the association is very stable and does h-catenin accumulation. C, silencing of RASSF1C by siRNA A/C in A549 cells not require the addition of proteasome inhibitors to be unraveled prevents h-catenin accumulation. A549 cells treated with siRNA Luc (lane 1), h h siRNA A (lane 2), or siRNA A/C (lane 3) were analyzed for the expression of as for most TrCP substrates; last, knockdown of TrCP by siRNA h-catenin by Western blot (top). hardly affects RASSF1C levels whereas it dramatically increases www.aacrjournals.org 1059 Cancer Res 2007; 67: (3). February 1, 2007

Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 2007 American Association for Cancer Research. Cancer Research h-catenin levels (data not shown). Altogether, these observations hTrCP controls the stability of key signaling proteins and cell suggest that RASSF1C acts more likely as a hTrCP pseudosubstrate cycle regulators that are believed to be involved in carcinogenesis. h like hnRNP-U (32) and/or as a negative modulator of the SCF TrCP Expression defects or mutations in the bTrCP gene have been complex capable to inhibit the hTrCP-mediated degradation of found in some human tumors (18, 27–29, 36, 37). Inactivation of several substrates, although the association of RASSF1C to hTrCP hTrCP in transgenic mice, by overexpression of the negative depends on a SSGYCS motif. transdominant hTrCPDF, also resulted also in more frequent h-Catenin accumulation was promoted either by the over- tumors (38). expression of RASSF1C or by the silencing of RASSF1A, and this Additional investigations will be needed to better understand implies that the balance between the two isoforms is crucial for the the role of RASSF1C in tumorigenesis mediated by the absence of hTrCP-mediated degradation of h-catenin. When the equilibrium expression of RASSF1A in numerous tumors. Two previous reports between the RASSF1A and RASSF1C isoforms is disrupted, either indicated that RASSF1C could have, together with RASSF1A, by lack of RASSF1A expression or by RASSF1C overexpression, the tumor suppressor activity (39, 40). This hypothesis cannot be ruled h function of SCF TrCP is impaired and h-catenin accumulates. Thus, out today and RASSF1C could have multiple functions depending the ratio between RASSF1A and RASSF1C expression levels may be on the cell type. However, in many tumor cell lines, loss of crucial to tight regulation of h-catenin expression levels via RASSF1A expression has been observed (see ref. 4 for review), h constitutive degradation of h-catenin mediated by the SCF TrCP whereas changes in RASSF1C expression have not been reported ubiquitin ligase. In the presence of normal levels of RASSF1A in these tumors. Our present observations led us to postulate that expression, the inhibitory activity of RASSF1C on the degradation RASSF1C, through inhibition of h-catenin degradation, may be h of h-catenin mediated by the SCF TrCP seems to be prevented. In involved in the tumorigenesis events linked to the inactivation of the absence of RASSF1A expression, RASSF1C could inhibit h- RASSF1A, illustrating the opposite effects that two isoforms of the catenin degradation, thus promoting h-catenin accumulation. The RASSF1 gene may have in carcinogenesis. Consistent with this decrease in h-catenin expression we observed upon silencing of view, it was recently reported that RASSF1C could activate RASSF1C in RASSF1A-deficient cell line A549 strongly supports this osteoblast cell proliferation through interaction with IGFBP-5, interpretation. Furthermore, our observations agree with previous confirming that RASSF1C could play a role in carcinogenesis (41). reports which showed that the expression of cyclin D1, a target of h-catenin, was high in cells depleted of RASSF1A expression, and that cyclin D1 was then down-regulated upon reexpression of Acknowledgments RASSF1A (4, 12). Both hTrCP and RASSF1A act at the same phase Received 7/10/2006; revised 10/16/2006; accepted 11/28/2006. of the cell cycle: hTrCP controls the degradation of the APCCdc20 Grant support: Association pour la recherche sur le cancer, La Ligue Nationale Contre le Cancer (I. Lassot), Association nationale de recherche contre le sida, inhibitor Emi1 (26); and RASSF1A has been implicated in Sidaction (G. Blot), La Mairie de Paris, French Ministry of Education (I. Lassot, E. prometaphase arrest by also inhibiting the APCCdc20 complex and Estrabaud, and G. Blot), and Institut National de la Sante et de la Recherche Medicale (E.L. Le Rouzic). by subsequently stabilizing cyclin A (34). As discussed by Jackson The costs of publication of this article were defrayed in part by the payment of page (35), it is unclear why both RASSF1A and Emi1 would regulate the charges. This article must therefore be hereby marked advertisement in accordance levels of cyclin A. One possibility is that RASSF1A may control the with 18 U.S.C. Section 1734 solely to indicate this fact. We thank Michael White for the kind gift of plasmids and for fruitful discussions, activity or destruction of Emi1, perhaps involving an interaction and Klaus Strebel and Geoff Clark (Department of Cell and Cancer Biology, National between RASSF1C and hTrCP. Cancer Institute, NIH, Rockville, MD) for the kind gift of antibodies.

References 8. Vos MD, Martinez A, Ellis CA, Vallecorsa T, Clark GJ. signaling to Bax conformational change and cell death. The pro-apoptotic Ras effector Nore1 may serve as a Mol Cell 2005;18:637–50. 1. Dammann R, Li C, Yoon JH, Chin PL, Bates S, Pfeifer Ras-regulated tumor suppressor in the lung. J Biol Chem 16. Bienz M. h-Catenin: a pivot between cell adhesion GP. Epigenetic inactivation of a RAS association domain 2003;278:21938–43. and Wnt signalling. Curr Biol 2005;15:R64–7. family protein from the lung tumour suppressor locus 9. Ortiz-Vega S, Khokhlatchev A, Nedwidek M, et al. The 17. Lassot I, Segeral E, Berlioz-Torrent C, et al. ATF4 3p21.3. Nat Genet 2000;25:315–9. putative tumor suppressor RASSF1A homodimerizes degradation relies on a phosphorylation-dependent 2. Lerman MI, Minna JD. The 630-kb lung cancer and heterodimerizes with the Ras-GTP binding protein interaction with the SCF(hTrCP) ubiquitin ligase. Mol homozygous deletion region on human chromosome Nore1. Oncogene 2002;21:1381–90. Cell Biol 2001;21:2192–202. 3p21.3: identification and evaluation of the resident 10. Dallol A, Agathanggelou A, Fenton SL, et al. RASSF1A 18. Margottin F, Bour SP, Durand H, et al. A novel human candidate tumor suppressor genes. The International interacts with microtubule-associated proteins and WD protein, h-h TrCp, that interacts with HIV-1 Vpu Lung Cancer Chromosome 3p21.3 Tumor Suppressor modulates microtubule dynamics. Cancer Res 2004;64: connects CD4 to the ER degradation pathway through Gene Consortium. Cancer Res 2000;60:6116–33. 4112–6. an F-box motif. Mol Cell 1998;1:565–74. 3. Dammann R, Schagdarsurengin U, Seidel C, et al. The 11. Song MS, Chang JS, Song SJ, Yang TH, Lee H, Lim DS. 19. Hart M, Concordet JP, Lassot I, et al. The F-box tumor suppressor RASSF1A in human carcinogenesis: The centrosomal protein RAS association domain family protein h-TrCP associates with phosphorylated h- an update. Histol Histopathol 2005;20:645–63. protein 1A (RASSF1A)-binding protein 1 regulates catenin and regulates its activity in the cell. Curr Biol 4. Agathanggelou A, Cooper WN, Latif F. Role of the Ras- mitotic progression by recruiting RASSF1A to spindle 1999;9:207–10. association domain family 1 tumor suppressor gene in poles. J Biol Chem 2005;280:3920–7. 20. Formstecher E, Aresta S, Collura V, et al. Protein human cancers. Cancer Res 2005;65:3497–508. 12. Shivakumar L, Minna J, Sakamaki T, Pestell R, White interaction mapping: a Drosophila case study. Genome 5. van der Weyden L, Tachibana KK, Gonzalez MA, et al. MA. The RASSF1A tumor suppressor blocks cell cycle Res 2005;15:376–84. The RASSF1A isoform of RASSF1 promotes microtubule progression and inhibits cyclin D1 accumulation. Mol 21. Bres V, Kiernan RE, Linares LK, et al. A non- stability and suppresses tumorigenesis. Mol Cell Biol Cell Biol 2002;22:4309–18. proteolytic role for ubiquitin in Tat-mediated trans- 2005;25:8356–67. 13. Song MS, Song SJ, Ayad NG, et al. The tumour activation of the HIV-1 promoter. Nat Cell Biol 2003;5: 6. Wohlgemuth S, Kiel C, Kramer A, Serrano L, suppressor RASSF1A regulates mitosis by inhibiting the 754–61. Wittinghofer F, Herrmann C. Recognizing and defining APC-Cdc20 complex. Nat Cell Biol 2004;6:129–37. 22. Winston JT, Strack P, Beer-Romero P, Chu CY, true Ras binding domains I: biochemical analysis. J Mol 14. Vos MD, Dallol A, Eckfeld K, et al. The RASSF1A Elledge SJ, Harper JW. The SCFh-TRCP-ubiquitin ligase Biol 2005;348:741–58. tumor suppressor activates Bax via MOAP-1. J Biol complex associates specifically with phosphorylated 7. Khokhlatchev A, Rabizadeh S, Xavier R, et al. Chem 2005;281:4557–63. destruction motifs in InBa and h-catenin and stim- Identification of a novel Ras-regulated proapoptotic 15. Baksh S, Tommasi S, Fenton S, et al. The tumor ulates InBa ubiquitination in vitro. Genes Dev 1999;13: pathway. Curr Biol 2002;12:253–65. suppressor RASSF1A and MAP-1 link death receptor 270–83.

Cancer Res 2007; 67: (3). February 1, 2007 1060 www.aacrjournals.org

23. Kroll M, Margottin F, Kohl A, et al. Inducible 30. Wu G, Xu G, Schulman BA, Jeffrey PD, Harper JW, prostate cancers. Genes Chromosomes Cancer 2002; degradation of InBa by the proteasome requires Pavletich NP. Structure of a h-TrCP1-1-h-catenin 34:9–16. interaction with the F-box protein h-hTrCP. J Biol complex: destruction motif binding and lysine specific- 37. He N, Li C, Zhang X, et al. Regulation of lung cancer Chem 1999;274:7941–5. ity of the SCF(h-TrCP1) ubiquitin ligase. Mol Cell 2003; cell growth and invasiveness by h-TRCP. Mol Carcinog 24. Yaron A, Hatzubai A, Davis M, et al. Identification of 11:1445–56. 2005;42:18–28. the receptor component of the InBa-ubiquitin ligase. 31. Bai C, Sen P, Hofmann K, et al. SKP1 connects cell 38. Belaidouni N, Peuchmaur M, Perret C, Florentin A, Nature 1998;396:590–4. cycle regulators to the ubiquitin proteolysis machinery Benarous R, Besnard-Guerin C. Overexpression of 25. Guardavaccaro D, Kudo Y, Boulaire J, et al. Control of through a novel motif, the F-box. Cell 1996;86:263–74. human h TrCP1 deleted of its F box induces meiotic and mitotic progression by the F box protein h- 32. Davis M, Hatzubai A, Andersen JS, et al. Pseudosub- tumorigenesis in transgenic mice. Oncogene 2005;24: Trcp1 in vivo. Dev Cell 2003;4:799–812. strate regulation of the SCF(h-TrCP) ubiquitin ligase by 2271–6. 26. Margottin-Goguet F, Hsu JY, Loktev A, Hsieh HM, hnRNP-U. Genes Dev 2002;16:439–51. 39. Vos MD, Ellis CA, Elam C, Ulku AS, Taylor BJ, Clark Reimann JD, Jackson PK. Prophase destruction of Emi1 33. Ahmed-Choudhury J, Agathanggelou A, Fenton SL, GJ. RASSF2 is a novel K-Ras-specific effector and by the SCF(hTrCP/Slimb) ubiquitin ligase activates the et al. Transcriptional regulation of cyclin A2 by potential tumor suppressor. J Biol Chem 2003;278: anaphase promoting complex to allow progression RASSF1A through the enhanced binding of p120E4F to 28045–51. beyond prometaphase. Dev Cell 2003;4:813–26. the cyclin A2 promoter. Cancer Res 2005;65:2690–7. 40. Li J, Wang F, Protopopov A, et al. Inactivation of 27. Ang XL, Harper JW. SCF-mediated protein degrada- 34. Song MS, Lim DS. Control of APC-Cdc20 by the RASSF1C during in vivo tumor growth identifies it as a tion and cell cycle control. Oncogene 2005;17:2860–70. tumor suppressor RASSF1A. Cell Cycle 2004;3:574–6. tumor suppressor gene. Oncogene 2004;23:5941–9. 28. Fuchs SY, Spiegelman VS, Kumar KG. The many faces 35. Jackson PK. Linking tumor suppression, DNA dam- 41. Amaar YG, Baylink DJ, Mohan S. Ras-association of h-TrCP E3 ubiquitin ligases: reflections in the magic age and the anaphase-promoting complex. Trends Cell domain family 1 protein, RASSF1C, is an IGFBP-5 mirror of cancer. Oncogene 2004;23:2028–36. Biol 2004;14:331–4. binding partner and a potential regulator of osteo- 29. Nakayama KI, Nakayama K. Ubiquitin ligases: cell- 36. Gerstein AV, Almeida TA, Zhao G, et al. APC/ blast cell proliferation. J Bone Miner Res 2005;20: cycle control and cancer. Nat Rev Cancer 2006;5:369–81. CTNNB1 (h-catenin) pathway alterations in human 1430–9.

www.aacrjournals.org 1061 Cancer Res 2007; 67: (3). February 1, 2007

Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 2007 American Association for Cancer Research. RASSF1C, an Isoform of the Tumor Suppressor RASSF1A, Promotes the Accumulation of β-Catenin by Interacting with βTrCP

Emilie Estrabaud, Irina Lassot, Guillaume Blot, et al.

Cancer Res 2007;67:1054-1061.

Updated version Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/67/3/1054

Supplementary Access the most recent supplemental material at: Material http://cancerres.aacrjournals.org/content/suppl/2007/02/09/67.3.1054.DC1

Cited articles This article cites 39 articles, 14 of which you can access for free at: http://cancerres.aacrjournals.org/content/67/3/1054.full#ref-list-1

Citing articles This article has been cited by 8 HighWire-hosted articles. Access the articles at: http://cancerres.aacrjournals.org/content/67/3/1054.full#related-urls

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://cancerres.aacrjournals.org/content/67/3/1054. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.