RB Family Tumor Suppressor Activity May Not Relate to Active Silencing of E2F Target Genes

Author Manuscript Published OnlineFirst on July 23, 2014; DOI: 10.1158/0008-5472.CAN-13-3706 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

RB family tumor suppressor activity may not relate to active silencing of E2F target genes

Running title: Tumor suppressor activity of pRB-N750F

Tinke L. Vormer1, Kamila Wojciechowicz1, Marleen Dekker1, Sandra de Vries1, Anja van der Wal1, Elly Delzenne-Goette1, Sjalin H. Naik2, Ji-Ying Song3, Jan-Hermen Dannenberg1, Jacob B. Hansen1,4,* and Hein te Riele1,*

1 The Netherlands Cancer Institute, Division of Biological Stress Response, Amsterdam, The Netherlands 2 The Netherlands Cancer Institute, Division of Immunology, Amsterdam, The Netherlands 3 The Netherlands Cancer Institute, Department of Animal Pathology, Amsterdam, The Netherlands 4 Department of Biology, University of Copenhagen, Copenhagen, Denmark

* Corresponding authors Jacob B. Hansen Hein te Riele University of Copenhagen The Netherlands Cancer Institute Department of Biology Division of Biological Stress Response Universitetsparken 13 Plesmanlaan 121 2100 Copenhagen Ø 1066 CX Amsterdam Denmark The Netherlands Phone +45 4075 9544 Phone: 31-20-512-2084 [email protected] Email: [email protected]

Financial support: This work was supported by the Dutch Cancer Society, grant numbers NKI 2000-2232 and NKI 2002-2634, and a fellowship from the Federation of European Biochemical Societies to JBH.

Disclosure of Conflicts of Interest: The authors declare no conflicts of interest

Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 2014 American Association for Cancer Research. Author Manuscript Published OnlineFirst on July 23, 2014; DOI: 10.1158/0008-5472.CAN-13-3706 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Abstract The retinoblastoma protein pRB and its two homologs p130 and p107 form the family of pocket proteins and play a major role in cell cycle regulation and suppression of human and mouse tumorigenesis. Pocket proteins regulate the activity of E2F transcription factors during

G1/S transition. Two mechanisms have been described: (i) pocket-protein-binding blocks the transactivation domain of activator E2Fs, inhibiting E2F-dependent transcription; (ii) E2F- bound pocket proteins can recruit chromatin-remodeling proteins containing an LxCxE motif (x encoding any amino acid), resulting in active repression of E2F target genes. To investigate the importance of pRB’s LxCxE-interacting motif in cell cycle control and tumor suppression, we generated MEFs and mice expressing a mutant pRB protein carrying an asparagine for phenylalanine substitution at position 750, abrogating LxCxE binding. Since p130 may compensate for loss of pRB, we studied pRBN750F activity in the presence and absence of p130. The pRB-LxCxE interaction was not required for cell cycle arrest upon mitogen deprivation and cell-cell contact, but did contribute to RASV12- and radiation-induced cell cycle arrest. Remarkably, the pRB-LxCxE interaction was not required for suppression of in vitro and in vivo transformation, even in the absence of p130. These results indicate that pRB’s tumor suppressor activity is not effectuated by active silencing of E2F target genes, but rather by regulation of activator E2Fs or another unidentified mechanism. Furthermore, the in vitro response of pocket-protein-perturbed cells to mitogen deprivation and cell-cell contact seems a better predictor of tumor development than the response to ectopic RASV12 expression.

Keywords: pRB, p130, LxCxE binding, E2F regulation, transformation

Introduction Loss of the retinoblastoma suppressor gene, RB1, is a frequent event in human cancer. pRB and its homologs p130 and p107 form the family of pocket proteins and play a key role in cell cycle regulation. They collectively regulate the family of E2F transcription factors,

whose activity is essential for the G1-S transition. Eleven E2F transcription factors have been identified, classified as ‘activator E2Fs’ (E2F1, E2F2 and E2F3a) and ‘repressor E2Fs’ (E2F3b, E2F4, E2F5, E2F6a, E2F6b, E2F7a, E2F7b and E2F8). Whereas pRB can interact with E2F1-4, p130 and p107 interact with E2F4 and E2F5. E2F6-8 do not bind pocket proteins.(1-3) Pocket protein-E2F complex formation inhibits transcription of E2F target genes via several mechanisms. First, pocket-protein binding blocks E2F’s transactivation domain, preventing E2F-mediated transcription. The balance between pocket protein-bound and free E2Fs is regulated by cyclin-dependent kinases that phosphorylate pocket proteins, causing release of E2Fs. This enables activating E2Fs to promote transcription and cell cycle progression.(3) Second, pocket protein ‘repressor’ complexes pRB-E2F3b, pRB-E2F4, p130/p107-E2F4 and p130/p107-E2F5 can recruit chromatin-remodeling proteins to E2F target genes by the capacity of pocket proteins to bind proteins containing an LxCxE-like motif (x encoding any amino acid).(4) The LxCxE motif, originally identified in viral oncoproteins, is present in a variety of chromatin remodeling proteins that favor a chromatin state incompatible with transcription.(4) These include histone deacetylase 1 (HDAC1), HDAC2, heterochromatin protein 1 (HP1), the histone methyltransferase Suv39h1, and the CtBP-interacting protein (CtIP).(3-7) Consistent with their role in cell cycle control, loss of pocket proteins intitiates tumorigenesis. In mice, Rb loss induces pituitary and thyroid tumors, and the tumor spectrum is extended by the additional loss of p130 or p107.(8-10) However, it is unclear which of the E2F-regulating activities of pocket proteins is critical for tumor suppression. One view is that regulation of ‘activator’, rather than ‘repressor’ E2Fs, mediates pRB’s tumor suppressor activity.(11) However, pocket protein-chromatin remodeling complexes have been implicated in (irreversible) cell cycle arrest and senescence, which are considered critical for tumor

suppression. The recruitment of p130-E2F4 to silenced promoters in G0/G1-arrested mouse and human cells suggests active repression.(12,13) Furthermore, pRB and LxCxE-containing proteins have been implicated in repression of E2F target genes upon serum starvation (14,15), RASV12 expression (16) and ectopic expression of p16INK4A (17).

Although LxCxE-containing proteins have thus been implicated in processes counteracting transformation, the requirement for these proteins to suppress tumorigenesis in vivo remains elusive. To address this issue, we generated MEFs and mice expressing a pRB variant in which asparagine at position 750 was replaced for phenylalanine. pRBN750F protein still inhibited E2F transactivation activity, but was impaired in binding an LxCxE-containing protein and therefore likely unable to recruit chromatin remodelers to E2F-regulated promoters. Because of functional compensation within the pocket protein family (8) (this

communication), the essential role of p130-repressor complexes in G0/G1 (12,13) and the ability of p130 to recruit chromatin remodeling proteins via its LxCxE binding site (18,19), we anticipated the phenotypic consequences of pRBN750F to be masked by p130 activity. Therefore, we also generated RbN750F/N750Fp130-/- MEFs and RbN750F/p130 mutant mice. Our results show that the pRB-LxCxE interaction contributes to cell cycle arrest upon RASV12 expression or γ-irradiation. Remarkably, attenuation of LxCxE-dependent interactions did not promote tumor formation, even in the absence of p130, suggesting that pRB’s tumor suppressor activity mainly operates via inhibition of activator E2Fs or an LxCxE-independent repressor mechanism.

Materials and Methods Constructs The 6xE2F-luciferase reporter, CMV-HA-E2F1, CMV-HA-E2F4, CMV-Renilla and pJ3-mRb were provided by Dr. R. Bernards, GAL4-TK-luciferase by Dr. R. Medema and pECE-pRbΔcdk-HA by Dr. J. Lukas. Addition of an N-terminal HA-tag to pRB, mutation of the Rb cDNA and cloning into pcDNA3.1(-) is described in Supplementary Methods. GAL4- pRB vectors were generated by cloning PCR-amplified Rb fragments (wild type or mutant) into pM (Clontech) (Supplementary Methods). pSG5-Tag was provided by Dr. J. A. DeCaprio, pBABE-RASV12-puro by Dr. T. Brummelkamp and pCL-Eco by Dr. D. Peeper. The pEYK-TBX2 vector was previously isolated from the pEYK-MCF7 library (20) obtained from Dr. G. Q. Daley.

Reporter assays Rb-/- 3T9 cells were seeded onto a 12-well plate and transfected the next day with 0.4 μg DNA per well using FuGENE6 Transfection Reagent (Roche) with a DNA-FuGENE ratio of 1 μg to 3 μl. All transfections included either 6xE2F-luciferase or GAL4-TK-luciferase

reporter plasmid, Renilla luciferase expression plasmid and pcDNA3.1(-)-HA-mRb or pM- GAL4-pRB plasmids to a total of 0.4 μg DNA/well. For detailed concentrations: see Supplemental Methods. Twenty-four hours post transfection, cells were washed in PBS, lysed and measured for Luciferase and Renilla luminescence activity using the Dual-Luciferase Reporter Assay System (Promega E1910). Luciferase luminescence was divided by Renilla luminescence.

MEF isolation Crossings of RbN750F/wt animals (21) and Rbwt/- animals (22) with p130wt/- mice (22) and intercrossings of the resulting offspring, enabled us to isolate RbN750F/N750Fp130-/-, RbN750F/N750Fp130wt/wt, Rb-/-p130wt/wt, Rbwt/wtp130-/- and Rbwt/wtp130wt/wt MEFs at embryonic day (E) 14.5 and Rb-/-p130-/- at E13.5. Rb-/-p130-/-p107-/- MEFs were previously generated (22).

Cell culture MEFs were cultured with 100 units/ml penicillin (Invitrogen/Gibco) and 100 μg/ml streptomycin (Invitrogen/Gibco).(22) Cells were plated and treated as follows: Mitogen deprivation: 8 x 105 cells/10-cm dish; three hours after plating, cells were washed in PBS and medium without fetal calf serum was added. Contact inhibition: 8 x 105 cells/10-cm dish; cells were analyzed 10 days after plating. γ-irradiation: 5 x 105 cells/10-cm dish; cells were irradiated the next day with 5.5, 10 or 15 Gy and analyzed 16 h post irradiation. All assays: cells were harvested for FACS at the indicated time points and, if applicable, 10 μM BrdU was added 1 h before harvesting. BrdU labeling and FACS: See Supplementary methods. Production of retroviruses, retroviral infections and soft agar plating was performed as described (20). For serial infections, MEFs were cultured in non-virus containing media for at least 36 h between infections and reseeded before infection to obtain optimal cell density.

Proliferation curves Cells were seeded in triplicate at a density of 2.5 x 104 cells/well of a 12-well plate (Corning Incorporated, 3512). At the indicated time points (time point 0: 3 h after seeding), cells were fixed in 4% formaldehyde/PBS, washed in demineralized water, stained with 0.1% crystal violet solution, washed and dryed (dark area). The dye was extracted in 1 ml 10% acetic acid/well and the optical density at 590 nm was measured using a microplate reader (M200 Tecan).

Protein isolation, immunoprecipitation and immunoblot Protein isolation, separation on 3-8% Tris-Acetate and 4-12% Bis-Tris NuPage gradient gels (Invitrogen) and immunoblotting were performed according to standard protocols. Cells were lysed in 150 mM NaCl, 50 mM Hepes pH 7.5, 5 mM EDTA, 0.1% NP- 40 and 1 tablet complete protease inhibitor cocktail (Roche) per 50 ml. Protein concentrations were determined using Bio-Rad protein assay (Bio-Rad). For immunoprecipitation experiments, C33A cells co-transfected with pSG5-TAg plus pcDNA3.1(-)-HA-mRb (wild-type, mutant or empty) were used. Protein isolation was performed in the presence of 5 mM NaF, 0.5 mM vanadate, 20 mM β-glycerolphosphate and 1mM PMSF. Lysates were incubated with anti-HA antibody and 50% protein A/G beads (Santa Cruz) in lysis buffer, separated on 12% polyacrylamide gels and immunoblotted. For antibodies, see Supplemenary methods.

Mice RbN750F/N750Fp130wt/-, RbN750F/wtp130-/- and RbN750F/N750Fp107wt/- mice plus appropriate control genotypes, were inspected twice a week and sacrificed when ill. Mice were examined histologically; blood was collected into heparinized tubes and diluted 2x in PBS. Red- and white blood cell counts were determined using a Coulter-counter.

Cellular preparations and FACS analysis Cell populations from spleen, bone marrow, blood (leukocytes) and thymus were stained in antibody cocktails and analyzed by FACS as described in the Supplementary methods.

Results Mutating the LxCxE binding site of pRB impairs transcriptional repression Binding of pRB to LxCxE-containing proteins involves four conserved residues in the B domain: Asn757, Tyr756, Lys713 and Tyr709. (23) Substituting asparagine at position 757 for phenylalanine disrupted LxCxE binding whereas binding to E2F1 remained intact.(24,25) We introduced the corresponding N750F substitution in murine pRB and found this to fully abrogate interaction with the LxCxE containing protein SV40 large T antigen (TAg) (Figure 1A). As controls, we used pRBC699F (human pRBC706F) and pRBR654W (human pRBR661W). pRBC706F is incapable of binding E2Fs (26) and LxCxE proteins (27), impaired in

transcriptional repression (28) and unable to inhibit proliferation of SAOS2 cells.(26) pRBR661W is a low-penetrance pRB mutant defective in binding E2Fs and LxCxE-containing proteins. (29,30) To test the ability of pRBN750F to reduce transcription, we introduced a luciferase gene preceded by six E2F binding sites in mouse Rb-/- 3T9 cells. Luciferase activity induced by ectopic expression of E2F1 (Figure 1B, lanes 1 and 2), was efficiently suppressed by co- expression of wild-type pRB (pRBwt) or pRBN750F (Figure 1B, compare lanes 3 to 5 with lane 6 to 8), but not by pRBC699F (Figure 1B, lanes 9 to 11). Also without ectopically-expressed E2F1, pRBwt and pRBN750F repressed the luciferase reporter, whereas pRBC699F did not (Figure 1C). These experiments demonstrate pRBN750F to effectively block E2F transactivation activity. E2F4 generally acts as a transcriptional repressor (12,13), likely by forming E2F4- pocket protein-chromatin remodeling complexes. However, E2F4 can also function as a transcriptional activator (31) (Figure 1D, compare lanes 1 and 2). pRBN750F only partly repressed the E2F-luciferase reporter induced by E2F4 (Figure 1D, compare lanes 2, 3 and 4). We propose that pRBwt suppressed E2F4-mediated transcription by simultaneously inhibiting transactivation and forming E2F4-pRB-chromatin remodeling complexes and that pRBN750F was impaired in the latter. Consistently, when pRB was fused to a GAL4-DNA binding motif, targeting pRB to a co-transfected GAL4-TK-luciferase reporter, pRBN750F was severely, but perhaps not completely, impaired in repressing transcription (Figure 1E). We conclude that pRBN750F is able to bind E2F transcription factors but unable to establish complete transcriptional repression, likely because it is impaired in recruiting chromatin remodeling complexes.

E2F target gene expression in RbN750F/N750F and RbN750F/N750Fp130-/- MEFs To study E2F target gene expression in a physiological setting, we generated RbN750F/N750F primary embryonic fibroblasts (MEFs) carrying the N750F substitution at the endogenous Rb gene. (21) (Figure S1A). As the consequences of abrogating pRB’s LxCxE- binding capacity may be masked by p130 and/or p107 (8,22) and p130 is the main pocket N750F/N750F -/- protein recruited to repressed promoters in G0/G1, we also generated Rb p130 MEFs and appropriate controls (Rb-/-, p130-/- and Rb-/-p130-/-). Unfortunately, we have not obtained RbN750F/N750Fp107-/- MEFs as in our mouse background p107 deficiency alone already affected embryonic development.

pRB-N750F protein level was similar to wild-type pRB (Figure S1B and C). RbN750F/N750F MEFs displayed elevated expression of the E2F target genes Cyclin E and p107 during asynchronous proliferation (Figure S1B, lanes 1 and 3), upon expression of RASV12 (Figure S1B, lanes 4 and 6) and after serum starvation (Figure S1B, lanes 7 and 9), indicative for similar derepression as in Rb-/- cells. The effect on Cyclin A appeared less prominent. Strikingly, derepression was not enhanced by the absence of p130 (Fig. S1C). Thus, the pRB- LxCxE interaction contributes to repression of E2F target genes in a physiological setting.

Cell cycle regulation in RbN750F/N750F and RbN750F/N750Fp130-/- MEFs We tested the ability of RbN750F/N750F and RbN750F/N750Fp130-/- MEFs to arrest in response to anti-proliferative signals.

Mitogen deprivation and confluency Bypass of cell cycle arrest in response to mitogen deprivation or cell-cell contact only occurred upon the combined loss of pRB and p130 (Table 1). RbN750F/N750Fp130-/- MEFs arrested as efficiently as wild-type and p130-/- MEFs (Table 1), showing that the LxCxE interaction of pRB did not contribute to cell cycle arrest.

Ionizing radiation -/- G1 arrest in response to γ-irradiation relies on pRB.(32) Indeed, Rb MEFs were severely impaired in arresting the cell cycle in response to γ-irradiation: at 5.5 Gy, wild-type and p130-/- MEFs displayed a 45-50% reduction in S phase cells, opposed to only 25% in Rb-/- MEFs (Figure 2A, lanes 1-6). This effect was not aggravated upon concomitant ablation of

p130 (Figure 2A, lanes 7-8). Also the fold increase in (G1+G2)/S ratio of irradiated versus untreated cells revealed an impaired arrest in Rb-/- MEFs, irrespective of p130 (Figure 2B, lanes 1-4). Similarly irradiated RbN750F/N750F MEFs displayed a reduction in S phase cells of - 35% (Figure 2A, lanes 9 and 10) and the fold increase in (G1+G2)/S ratio was similar as in Rb /- MEFs (Figure 2B, lanes 2 and 5). Again, this effect was not modulated by p130 (Figure 2A, lanes 9-12; Figure 2B, lanes 5 and 6). At higher doses of irradiation, RbN750F/N750F MEFs displayed an intermediate phenotype: at 10 or 15 Gy, RbN750F/N750F MEFs arrested less efficiently than wild-type MEFs, but more efficiently than Rb-/- MEFs (Figure S2A, lanes 4-6 and 7-9). Moreover, irradiation N750F/N750F -/- with 15 Gy induced a shift from G1 towards G2 arrest in both Rb MEFs and Rb MEFs (Figure S3).

To measure the stringency of radiation-induced cell cycle arrest, serum-starved RbN750F/N750F, Rb-/- and Rbwt/wt MEFs were re-stimulated with serum and irradiated 7 h later. Rb-/- MEFs entered S phase earlier than Rbwt/wt MEFs, with RbN750F/N750F MEFs showing an intermediate phenotype (Figure S2B). Consistent with earlier findings (33), these results

indicate that the pRB-LxCxE interaction contributes to DNA-damage-induced G1 arrest.

Expression of RASV12 Whereas pRB alone is the central player in the cellular response to ionizing radiation, bypass of RASV12-induced growth inhibition required concomitant loss of pRB and p130, pRB and p107, or all three proteins.(8,34) We therefore studied the effect of RASV12 expression in a p130-defective backgound. Similar to Rb-/-p130-/- MEFs, RbN750F/N750Fp130-/- MEFs maintained a high proliferation rate upon RASV12 expression, whereas Rbwt/wtp130-/- MEFs decelerated proliferation (Figure 3). This result indicates the pRB-LxCxE interaction is critical for RASV12-induced inhibition of proliferation.

RASV12-induced transformation We previously showed that Rb-/-p130-/- MEFs, but not Rb-/- or p130-/- MEFs, could proliferate anchorage-independently upon expression of RASV12 and TBX2.(20) We therefore studied the transforming capacity of RASV12 in RbN750F/N750Fp130-/- MEFs rather than in RbN750F/N750F MEFs. Surprisingly, RbN750F/N750Fp130-/- MEFs did not grow anchorage- independently upon expression of RASV12 and TBX2 (Figure 4). Thus, ablation of the pRB- LxCxE interaction alleviated RASV12-induced growth inhibition (in the absence of p130) but was not sufficient to support RASV12/TBX2-induced transformation.

p130 compensates for loss of pRB-LxCxE interactions in embryonic development To investigate whether the pRB-LxCxE interaction is critical for pRB’s role in embryonic development, we intercrossed RbN750F/wt mice. While full ablation of Rb was embryonic lethal (35), RbN750F/N750F animals were born according to mendelian ratio and survived into adulthood. In contrast, in crosses with p130+/- animals, life-born RbN750F/N750Fp130-/- animals were not obtained (Table S1). RbN750F/N750Fp130-/- embryos were detected at embryonic day 18 but died prenatally. We could not test the effect of p107 disruption on the viability of RbN750F/N750F mice as p107-/- mice died in utero. However, combination of three mutant alleles,

RbN750F/N750Fp130+/-, RbN750F/wtp130-/- and RbN750F/N750Fp107+/-, allowed normal embryonic development and survival into adulthood.

Concomitant loss of pRB-LxCxE interactions and p130/p107 is not sufficient to drive tumorigenesis Ablation of the pRB LxCxE-interacting domain did not affect lifespan or tumorigenesis (Figure 5A). The absence of tumor development in RbN750F/N750F mice sharply contrasted with the high incidence of pituitary and thyroid tumors in Rb+/- and chimeric Rb-/- mice.(9,10) We previously showed that the tumor spectrum induced by Rb loss was broadened by concomitant loss of p130 or p107, and included retinoblastoma, osteocarcoma, lymphosarcoma, pheochromocytoma and coecal adenocarcinoma.(8,36) We therefore wondered whether tumor development in RbN750F/N750F mice was suppressed by compensatory activities of p130 or p107. Since RbN750F/N750F animals were not viable in the absence of p130 or p107 (Table S1), we studied tumor incidence in mice with three mutant alleles, i.e., RbN750F/N750Fp130+/-, RbN750F/wtp130-/- or RbN750F/N750Fp107+/-, anticipating that similar to Rb+/- p130-/- or Rb+/-p107-/- chimeric mice, tumor development may ensue from loss of the respective wild-type alleles.(10) Although hemizygosity for p107, and perhaps also for p130, reduced the survival of RbN750F/N750F mice (Figure 5D and B, respectively), RbN750F/N750Fp130+/- and RbN750F/N750Fp107+/- animals were not predisposed to tumorigenesis (Table S2). Also RbN750F/wtp130-/- mice were not tumor prone although they showed a severe reduction in life span (Figure 5C), their median survival being reduced from >70 weeks in control littermates to 32 weeks. RbN750F/wtp130-/- animals were smaller and skinnier than control littermates and had an irregular fur. Of 37 animals, 24 had to be sacrificed due to sudden illness and 8 suddenly died before 60 weeks of age. Surprisingly, microscopical examination of pituitary (n=10), thyroid (n=16), brain (n=24), eyes (n=23), digestive system (stomach, intestines, liver, pancreas; n=22), testes (n=7), ovary (n=15), heart (n=15), lungs (n=22), adrenals (n=16), spleen (n=22), thymus (n=19), and bone marrow (sternum, hind leg, head; n=16) did not reveal abnormalities, except in the spleen (see below). Only 3 animals, older than 50 weeks, were diagnosed with tumor(s): 1 alveolar-bronchiolar adenoma, 2 adenomas in the pituitary gland and 1 lymphoma (Table S2). For comparison, Rb+/- animals developed pituitary tumors between 32 and 36 weeks (10), and in Rb+/-p130-/- chimeric mice, 5 out of 15 animals developed a tumor.(8) Taken together, we conclude that RbN750F/wtp130-/- animals had a reduced life span but were not predisposed to tumorigenesis.

Hematopoiesis in RbN750F/wtp130-/- animals Twenty-four percent (n=21) of RbN750F/wtp130-/- animals showed a reduced number of hematopoietic cells in the red pulp of the spleen (‘reduced cellularity’), a phenomenon not seen in RbN750F/wtp130+/- and Rbwt/wtp130-/- animals (both n=6). Of the five cases only two showed reduced hematopoiesis in the bone marrow (not shown). Furthermore, red and white blood cell counts were sometimes reduced in ill RbN750F/wtp130-/- animals (data not shown), but this did not correlate with the reduced cellularity in the spleen. Therefore, the cause and significance of this observation remain elusive. FACS analysis revealed normal percentages of LSK, LT-HSC, ST-HSC, myeloid progenitors, lymphoid progenitors, erythroid progenitors, monocytes and granulocytes in the bone marrow of RbN750F/wtp130-/- mice (not shown). However, 2/4 RbN750F/wtp130-/- animals had a decreased population of cells with intermediate expression of CD19 and B220, possibly reflecting a defective pre-B cell population (mouse a and c in Table S3; 0.75 and 1.7% respectively, versus on average 6.6% in RbN750F/wtp130+/- and Rbwt/wtp130-/- control animals). However, mature B cells, characterized by high CD19/B220 expression, where only mildly reduced (1.7 and 2.1%, versus on average 3.3% in control animals, Table S3) and B and T cell numbers were normal in spleen, peripheral blood and thymus (not shown), arguing against a B cell defect in RbN750F/wtp130-/- mice. In conclusion, although pocket proteins have been implicated in proliferation and mobilization of hematopoietic stem cells in the bone marrow (37,38), both microscopic and FACS analyses argue against a major bone marrow defect in RbN750F/wtp130-/- animals.

Decreased fat deposition in RbN750/wtp130-/- animals A defect that could relate to early death of RbN750/wtp130-/- mice was suggested by their skinny appearance. Indeed, at the time they became ill, RbN750/wtp130-/- mice showed strongly reduced or even absent subcutaneous adipose depots (Figure S4A) that lacked unilocular lipid droplet-containing adipocytes (Figure 6A). Fat deposits, if present, looked like brown rather than white fat. The back skin of RbN750/wtp130-/- animals was thinner and more transparent than in control mice (Figure S4A): in particular, the adipose tissue layer in the lower dermis was thin and in some RbN750/wtp130-/- animals totally absent (Figure 6B). Four to seven weeks old RbN750/wtp130-/- animals already showed reduced thickness of the dermal adipose layer containing fewer adipocytes and smaller lipid droplets (Figure S4B). Anterior and posterior subcutaneous fat deposits were present, but again the size of the lipid droplets was smaller

than in control littermates (Figure S4C). These observations indicate a shift of white to brown fat in RbN750/wtp130-/- animals, which may impact on thermoregulation and viability of the animals.

Discussion Given the proposed role of the pRB-LxCxE interaction in silencing of E2F target genes (4,14), we asked whether this interaction is essential for pRB’s role in cell cycle regulation, development and suppression of tumorigenesis. To this aim, we abrogated the LxCxE binding site of mouse pRB by substituting asparagine at position 750 for phenylalanine. pRBN750F retained the ability to inhibit E2F1 transcriptional activity, but was unable to bind an LxCxE-containing protein and to effectively mediate E2F4-dependent transcriptional repression. We introduced the N750F substitution at the endogenous Rb gene and studied its consequences in the absence of p130.

The role of pRB-LxCxE interactions in cell cycle regulation We found the pRB-LxCxE interaction to contribute to growth inhibition in response to γ-irradiation and expression of RASV12, but not after growth factor deprivation or contact inhibition.

Growth factor deprivation and ionizing radiation Although serum starvation was found to induce pRB- and HDAC1-dependent deacetylation and silencing of the Cyclin E promoter (15) and a mutation in the LxCxE binding site of pRB increased Cyclin E expression during serum starvation (14) (our experiments), RbN750F/N750Fp130-/- MEFs arrested normally in response to serum starvation, whereas Rb-/- p130-/- MEFs did not. Therefore, cell cycle arrest after serum withdrawal does not rely on pRB’s LxCxE binding site but is effectuated by other pocket protein functions, possibly- inhibition of E2F transactivation or another activity of pRB. In contrast, pRB’s critical role in cell cycle arrest after γ-irradiation partially relied on its interaction with LxCxE proteins. Similar observations were made in response to UV irradiation and cisplatin exposure.(33,39) Cycle arrest upon γ-irradiation coincided with reduced CDK2 kinase activity, indicative for pRB-HDAC-mediated repression of the CDK2 regulator Cyclin E.(32) However, other LxCxE-proteins may be involved as well, such as the LxCxE containing protein RF-Cp145, which enhanced survival after DNA damage in the

presence of pRB (39); LxCxE members of the p200 family, which may stabilize the hypo- phosphorylated form of pRB upon DNA damage (40); the APC-Cdh1 complex that binds pRB in an LxCxE-dependent manner causing downregulation of Skp2 and stabilization of p27KIP1.(41)

RASV12-induced senescence and transformation Also RASV12-induced growth inhibition required pRB’s LxCxE interacting domain. Consistently, expression of the DNA binding domain of E2F1 (E2F-DB) could bypass RASV12-induced senescence.(42) Since E2F-DB cannot activate transcription but only displace endogenous E2Fs from promoter regions, this results implied that relieve of E2F- mediated transcriptional repression can overcome RASV12-induced senescence. Additionally, Narita and colleagues detected recruitment of HP1γ and the presence of methylated H3K9 to promoter regions in RASV12-induced senescent cells, but not in confluent quiescent cells.(16) Surprisingly, while loss of pRB’s LxCxE-binding activity in RbN750F/N750Fp130-/- MEFs allowed proliferation in the presence of RASV12, it did not allow transformation by RASV12 and TBX2. Possibly, derepression of Cyclin E in RbN750F/N750Fp130-/- MEFs induced CDK2 activity to a level sufficient to bypass RASV12-induced senescence, but insufficient to counteract the dramatic drop in kinase activities that occurs upon loss of anchorage.(20) It is also possible that anchorless growth is prohibited by inhibiton of E2F transactivation activity or another function of pRB that was not affected by the N750F mutation. Of note, others reported that ablation of the pRB-LxCxE interaction promoted anchorless growth upon expression of dominant-negative p53 and RASV12 (43) or RASV12 alone.(44) However, in the first case, the effect was modest (twofold) and no comparison was made with Rb-/- cells; in the second, cells were plated at low density prior to removal of anchorage, which may have caused selection for (rare) cells that had escaped cell cycle arrest. Importantly, our in vitro and in vivo results are consistent: ablation of pRB-LxCxE interactions did not promote anchorless growth in vitro or tumorigenesis in vivo (see below).

In vivo significance of pRB-LxCxE interaction Our in vitro experiments show that ablation of the pRB-LxCxE interaction attenuated cell cycle arrest upon γ-irradiation and expression of RASV12, but did not alleviate cell cycle arrest upon serum starvation or cell-cell contact and was not sufficient to support anchorage- independent growth. It was therefore of interest to determine whether recruitment of LxCxE-

containing proteins by pRB is critical for mouse development and survival and for tumor suppression, in particular in the absence of p130 or p107.

Development The perinatal death of RbN750F/N750Fp130-/- animals as opposed RbN750F/N750F mice suggests that recruitment of chromatin remodeling complexes via LxCxE-mediated interactions is essential for completion of embryonic development. This is consistent with embryonic failure of p130-/- p107-/- and E2F4-/-E2F5-/- mice.(45,46) Triple RbN750F/N750Fp130+/- and RbN750F/wtp130-/- mutant mice developed to term and survived into adulthood, although the lifespan of the latter was severely reduced. This may be related to strongly reduced dermal and subcutaneous fat deposits, with white adipose tissue having acquired a morphology resembling brown fat. Previous reports have shown the involvement of pocket proteins in the control of white versus brown fat cell differentiation, the latter predominating in the absence of Rb or p107.(47,48) Perhaps RbN750F/wtp130-/- mice suffered from excessive heat loss due to the reduced presence of dermal white adipocytes and thin skin. Heat loss may be compensated for by ‘browning’ of residual subcutaneous white fat depots through recruitment of thermogenic adipocytes in normally white adipose depots. This may result in excessive energy consumption in adipose tissue, possibly depleting energy-rich substrates required for maintaining normal thermoregulation. However, further experiments are required to support this scenario.

Tumorigenesis Our previous studies demonstrated that Rb+/-p107-/- and Rb+/-p130-/- chimeric mice developed a wide spectrum of tumors following loss of the wild-type Rb allele.(8) Surprisingly, not only RbN750F/N750F mice, but also RbN750F/N750Fp130+/-, RbN750F/wtp130-/- and RbN750F/N750Fp107+/- mice were not tumor prone. While one may argue that the wasting syndrome in RbN750F/wtp130-/- mice may have precluded tumor development, this cannot explain the absence of tumor development in RbN750F/N750Fp130+/- and RbN750F/N750Fp107+/- mice that did not show this syndrome. Therefore, the absence of tumor susceptibility in these triple allele mutant mice implies that RbN750F/N750Fp130-/- and RbN750F/N750Fp107-/- cells, which are expected to frequently arise by spontaneous loss of heterozygosity, do not readily undergo oncogenic transformation. Our results therefore argue against a major role for LxCxE- containing proteins in tumor suppression.

It remains possible that loss of the pRB-LxCxE interaction stimulates tumor formation only under specific conditions. E.g., mice with an ablated pRB-LxCxE interaction were prone to genotoxin-induced liver tumorigenesis, consistent with the role of pRB-LxCxE interactions in DNA damage response.(33) Also, abrogation of the pRB-LxCxE interaction enhanced tumorigenesis in combination with mutant or inactive p53 (43,49), an effect that may be related to the involvement of pRB’s LxCxE binding activity in chromosome condensation and cohesion.(49,50) The absence of tumor development in RbN750F/p130 and RbN750F/p107 mutant mice indicates that disturbance of transcriptional regulation by loss of pRB-LxCxE interactions is insufficient to promote spontaneous tumor development. Thus, pRB’s tumor suppressor activity may be effectuated through regulation of activator E2Fs rather than by active silencing of E2F target genes. However, in some tissues, activator E2F activity was dispensable for proliferation. (51-53) It may therefore be possible that pRB-LxCxE and pRB- activator E2F interactions are each dispensable for tumor suppression but not both together. Alternatively, a different function of pRB is critical for tumor suppression, such as LxCxE- independent transcriptional repression or a function of pRB in S-phase during DNA replication. Irrespective of the mechanism, our results suggest that the in vitro cellular response to mitogen deprivation or cell-cell contact is a better predictor of tumorigenesis than RASV12-induced growth inhibition.

Acknowledgments We thank our colleagues Marieke Aarts, Sietske Bakker, Rob Dekker, Tanja van Harn, Camiel Wielders and Eva Wielders for helpful discussions and comments on the manuscript, and members of our animal and animal pathology facilities for technical assistance. We acknowledge financial support from the Dutch Cancer Society (NKI 2002-2634) and the Federation of European Biochemical Societies (JBH).

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Figure and Table legends

Figure 1. Characterization of murine pRBN750F. (A) pRBN750F does not interact with SV40 TAg. C33A cells were transfected with pSG5-TAg plus empty pcDNA3.1(-) (vector) or pcDNA3.1(-) encoding HA-tagged wild-type or mutant pRB. Upper panel: Westernblotting of immunoprecipitated HA-tagged pRB proteins and co-immunoprecipitated SV40 TAg using anti-TAg and anti-pRB antibodies. Lower panel: total protein extracts (input). (B-E) Transcriptional repression by wild-type and mutant pRB in luciferase reporter assays. Rb-/- 3T9 cells were transfected with the indicated constructs confrom the concentrations described in Supplemental Methods. Luciferase/Renilla luminescence is plotted in B-D and set to 100% in lane 2 (B and D) or lane 1 (C). Fold repression, defined as (Luciferase/Renilla

luminescence)GAL4 / (Luciferase/Renilla luminescence)GAL4-pRB wildtype or mutant, is plotted in E. For each analysis, two independent experiments were performed and carried out in triplo (Fig. 1B, C and E) or in duplo (Fig. 1D); a representative experiment is shown.

Figure 2. The pRB-LxCxE interaction is involved in cell cycle arrest after γ-irradiation. (A) MEFs of the indicated genotypes were either untreated or irradiated with 5.5 Gy and harvested 16 h post irradiation. BrdU was added 1 h before harvesting. Harvested cells were stained for BrdU and propidium iodide incorporation and analyzed by FACS. (B) Fold

increase in (G1+G2)/S in 5.5 Gy-irradiated versus untreated MEFs. Depicted is the average fold increase, calculated from 2 independent experiments. One of these experiments is shown in (A).

Figure 3. The pRB-LxCxE interaction is involved in RASV12-induced senescence. p130-/-, RbN750F/N750Fp130-/- and Rb-/-p130-/- MEFs were infected with pBABE-RASV12-puro (+ RASV12) or pBABE-puro (- RASV12) and plated for proliferation assays 4 days after infection. Cells were fixed and stained with crystal violet at the indicated time points. The relative cell

number was determined by dividing OD590 at time point x by OD590 at time point 0. Two independent experiments (upper and lower panels), performed in triplicate, are shown.

Figure 4. Ablation of the pRB-LxCxE interaction in a p130 null background is not sufficient for anchorage-independent growth upon expression of TBX2 and RASV12. RbN750F/N750Fp130-/- and Rb-/-p130-/- MEFs were infected with pEYK-TBX2 and subsequently with pBABE-

RASV12-puro and then plated in soft agar. Pictures were taken after 2 weeks using a non- phase-contrast lens (2.5x magnification) in (A) and a phase-contrast lens (5x magnification) in (B). RASV12 and TBX2 were expressed at comparable levels in the two cell lines (not shown).

Figure 5. Kaplan-Meier survival curves showing the life span of Rb, Rb/p130 and Rb/p107 mutant mice. (A) RbN750F/N750F mice have a normal life span. (B) Decreased life span of RbN750F/N750Fp130+/- mice. (C) Strongly decreased life span of RbN750F/wtp130-/- mice. (D) Decreased life span of RbN750F/N750Fp107+/- mice. Animals of the indicated genotypes were followed for 111 (A) or 70 (B, C, D) weeks and sacrificed when ill.

Figure 6. Hematoxilin and eosin staining of skin samples from RbN750F/wtp130-/- (left panels) and RbN750F/wtp130+/- (right panels). (A) Staining of subcutaneous adipose depots shows the absence of lipid droplets in RbN750F/wtp130-/- mice and their abundant presence in RbN750F/wtp130+/- mice. (B) Back skin samples showing absence of dermal adipose tissue in RbN750F/wtp130-/- mice.

Table 1. RbN750F/N750Fp130-/- MEFs arrest efficiently in response to growth factor deprivation or cell-cell contact. MEFs were cultured in the presence or absence of serum, stained for BrdU and propidium iodide incorporation and analyzed by FACS. The S-phase population was defined as the BrdU-positive population of cells with a DNA content ranging from 2N

(G1) to 4N (G2). Subconfluent and contact-inhibited MEFs were stained with propidium iodide and analyzed by FACS. Under both conditions, attenuated cell cycle arrest was seen in Rb-/-p130-/- MEFs but not in RbN750F/N750F p130-/- MEFs.

Table 1. Response of MEFs to serum starvation and contact inhibition Author manuscriptshavebeenpeerreviewedandacceptedforpublicationbutnotyetedited. Author ManuscriptPublishedOnlineFirstonJuly23,2014;DOI:10.1158/0008-5472.CAN-13-3706

Genotype Condition (G1+G2)/S Fold increase Condition (G1+G2)/S Fold increase

+ serum 2.4 subconfluent 6.2 cancerres.aacrjournals.org wild-type 11.6 5.5 7 days starved 27.6 contact inhibited 34.1

+ serum 1.6 subconfluent 6.8 Rb-/- 9.6 4.0 7 days starved 15.5 contact inhibited 27.0

+ serum 1.8 subconfluent 6.4 p130-/- 10.0 4.1 7 days starved 18.2 contact inhibited 26.1

on September 28, 2021. © 2014American Association for Cancer + serum 1.6 subconfluent 8.2 Rb-/-p130-/- 6.3 2.8 7 days starved 10.2 contact inhibited 23.1 Research. + serum 1.7 subconfluent 4.7 RbN750F/N750F 11.8 7.6 7 days starved 20.0 contact inhibited 35.6

+ serum 1.7 subconfluent 5.3 RbN750F/N750F p130-/- 10.9 6.3 7 days starved 18.7 contact inhibited 33.0

subconfluent 6.2 Rb-/-p130-/-p107-/- 1.2 contact inhibited 7.8 RbN750F/N750Fp130-/- MEFs arrest efficiently in response to growth factor deprivation or cell-cell contact. MEFs were cultured in the presence or absence of serum, stained for BrdU and propidium iodide incorporation and analyzed by FACS. G1-, and G2-phase populations were defined as the populations of cells with a 2N or 4N DNA content as apparent in the propidium iodide profile. The S- phase population was defined as the BrdU-positive population of cells with a DNA content ranging from 2N to 4N. Subconfluent and contact-inhibited MEFs were stained with propidium iodide and analyzed by FACs. Under both conditions, attenuated cell cycle arrest was seen in Rb-/-p130-/- MEFs but not in RbN750F/N750F p130-/- MEFs.

Author Manuscript Published OnlineFirst on July 23, 2014; DOI: 10.1158/0008-5472.CAN-13-3706 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Fig. 1 A B + SV40 TAg 1 2 3 4 5 6 7 8 9 10 11 160 140 wt N750F R654W C699F

RB 120 100 vector HA-pRB HA-p HA-pRB HA-pRB IP: anti-HA 80

pRB 60 % Luciferase Activity % Luciferase TAg 40 20 Input 0 pRB pRBwt pRBN750F pRBC699F TAg + E2F1 + E2F-luciferase reporter C D E N750F C699F wt 4-pRB wt N750F C699F wt N750F C699F vector vector pRB pRB pRB GAL4-pRB vector pRB pRB pRB GAL4 GAL4-pRB GAL 120 120 9 8 100 100 7 80 80 6 5 60 60

uciferase Activity uciferase 4 40 40 repression fold 3 % L % Luciferase Activity % Luciferase 2 20 20 1 0 0 0 1 2 3 4 1 2 3 4 5 1 2 3 4 + E2F-luciferase reporter + E2F4 + GAL4-TK- Downloaded from cancerres.aacrjournals.org on SeptemberLuciferase 28, reporter 2021. © 2014 American Association for Cancer + E2F-luciferase reporter Research. Author Manuscript Published OnlineFirst on July 23, 2014; DOI: 10.1158/0008-5472.CAN-13-3706 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Fig. 2 -/- A B -/- p130 p130 -/- -/- -/- -/- p130 p130 -/- -/- N750F/N750F N750F/N750F -/- N750F/N750F -/- N750F/N750F p130 p130 Rb Rb Rb Rb wild-type Rb wild-type Rb Rb Rb - + - + - + - + - + - + irradiation ++ + + + + irradiation 100 % (5.5 Gy) 2.5 (5.5 Gy ) 90 % 80 % 2.0 70 % /S)

8N 2 60 % 1.5 +G

G2 1 50 % S (BrdU+) 40 % 1.0 30 % G1 20 % 0.5 10 % Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 2014 American Association for Cancer Fold increase ( G increase Fold 0 % untreated versus irradiated 0.0 1 2 3 4 5 6 7 8 9 10 11 12 1 2 3 Research. 4 5 6 Author Manuscript Published OnlineFirst on July 23, 2014; DOI: 10.1158/0008-5472.CAN-13-3706 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Fig. 3 + RASV12 - RASV12 7 12 -/- -/- -/- -/- Rb p130 Rb p130 6 RbN750F/N750Fp130-/- 10 RbN750F/N750Fp130-/- -/- -/- 5 p130 p130 8 4 6 3 4 lative cell numer 2 e R 1 R elative cell numer 2

0 0 0 2 4 6 8 10 12 0 2 4 6 8 10 12 Time (days) Time (days)

16 14 Rb-/-p130-/- Rb-/-p130-/- 14 RbN750F/N750Fp130 -/- 12 RbN750F/N750Fp130 -/- -/- -/- 12 p130 10 p130 10 8 8 6 6

lative cell numer 4

e 4 R R elative cell numer 2 2 0 0 0 2 4 6 8 10 12 0 2 4 6 8 10 12 Time (days) Time (days)

Fig. 4

A TBX2 + RASV12 Rb-/-p130-/- RbN750F/N750Fp130-/-

B TBX2 + RASV12 Rb-/-p130-/- RbN750F/N750Fp130-/-

Fig. 5 A 100

40 Survival (%)

20 Rbwt/wt (n=62) RbN750F/wt (n=25) RbN750F/N750F (n=10) 0 0 20 40 60 80 100 120 Time (weeks)

B 100

40 Survival (%)

20 RbN750F/wtp130+/- (n=22) RbN750F/N750Fp130+/- (n=10) 0 0 20 40 60 80 Time (weeks)

C 100

40 Survival (%)

20 RbN750F/wtp130+/- (n=22) Rbwt/wtp130-/- (n=11) RbN750F/wtp130-/- (n=37) 0 0 20 40 60 80 Time (weeks)

D 100

40 Survival (%)

20 RbN750F/wtp107+/- (n=14) N750F/N750F +/- 0 Rb p107 (n=31) 0 20 40 60 80 Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 2014 American Association for Cancer Time (weeks) Research. Author Manuscript Published OnlineFirst on July 23, 2014; DOI: 10.1158/0008-5472.CAN-13-3706 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Fig. 6

Anterior Subcutaneous Adipose Tissue

RbN750F/wt RbN750F/wt p130-/- p130+/-

Posterior Subcutaneous Adipose Tissue

RbN750F/wt RbN750F/wt p130-/- p130+/-

dermal adipose tissue

RbN750F/wt RbN750F/wt p130Ex -/- Cp130on+/ - Tabl e 1: RbN7 50F/N 750Fp 130- /- Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 2014 American Association for Cancer MEF Research. Author Manuscript Published OnlineFirst on July 23, 2014; DOI: 10.1158/0008-5472.CAN-13-3706 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

RB family tumor suppressor activity may not relate to active silencing of E2F target genes

Tinke L Vormer, Kamila Wojciechowicz, Marleen Dekker, et al.

Cancer Res Published OnlineFirst July 23, 2014.

Updated version Access the most recent version of this article at: doi:10.1158/0008-5472.CAN-13-3706

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