Ectopic expression of pRb2/p130 suppresses the tumorigenicity of the ..... 8. aCells were plated at 16103 and 56103 cells/well in semi-solid medium containing ...
Oncogene (1999) 18, 651 ± 656 ã 1999 Stockton Press All rights reserved 0950 ± 9232/99 $12.00 http://www.stockton-press.co.uk/onc
Ectopic expression of pRb2/p130 suppresses the tumorigenicity of the c-erbB-2-overexpressing SKOV3 tumor cell line Serenella M Pupa1, Candace M Howard2, Anna M Invernizzi, Roberta De Vecchi1, Cristiana Giani1, Pier Paolo Claudio2, Maria I Colnaghi1, Antonio Giordano2 and Sylvie MeÂnard1 1
Division of Experimental Oncology E, Istituto Nazionale Tumori, 20133 Milan, Italy; 2Department of Pathology, Anatomy and Cell Biology, Sbarro Institute for Cancer Research and Molecular Medicine, Jeerson Medical College, Philadelphia, Pennsylvania 19107, USA
We investigated the in vitro and in vivo eects of the ectopic expression of the pRb2/p130 cell cycle regulator on c-erbB-2-associated tumorigenicity. SKOV3 ovarian cancer cells, which display c-erbB-2 gene ampli®cation and oncoprotein (p185HER2) overexpression, were stably transfected with a plasmid containing the coding sequence for human wild-type pRb2/p130 (wtRb2), or with pcDNA3 empty vector. Three wtRb2-transfected clones (cl. 24, cl. 49, cl. 100) and one empty vectortransfected clone (cl. mock) were randomly picked and further analysed. Western blot analysis revealed high levels of pRb2/p130 in the three clones compared to mock cells. Levels of p185HER2 and the extent of its tyrosine phosphorylation were similar in all transfectant clones, as were levels of pRb1 and p107. In anchorageindependent growth assays, the number of colonies from wtRb2 clone-transfectants was about 90% less than that arising from mock cells (P50.001). Tumor take rates of the three wtRb2-transfected clones xenografted in nu/nu mice were much lower than those of mock cells, and tumor volume was decreased by 80% (P50.001). A mutant version of pRb2/p130 deleted of the pocket region (mut-Rb2) was also transfected into SKOV3 cells and studied in parallel with the wtRb2-transfected and pcDNA empty vector-transfected bulk populations. mutRb2 transfected cells showed no inhibition of in vitro colony formation and were fully tumorigenic. Together, these ®ndings indicate that Rb2 acts as a tumor suppressor gene in vivo and in vitro in SKOV3 cells and that the intact pocket region is required for the suppressor activity. Keywords: Rb2; oncosuppressor; c-erbB-2; oncogene; ovary carcinoma; tumorigenicity
Introduction The human Rb2 gene, together with Rb1 and p107, form the retinoblastoma susceptibility gene family (Mayol et al., 1993). Products of these genes are characterized by a peculiar steric conformation, the `pocket region', responsible for most of the functional interactions that characterize the activity of these proteins in cell cycle homeostasis (Paggi et al., 1995). The Rb2 gene consists of 22 exons, spanning over
Correspondence: S MeÂnard Received 27 April 1998; revised 11 August 1998; accepted 11 August 1998
50 Kb of genomic DNA (Baldi et al., 1996a) and maps to human chromosome 16q12.2, an area in which deletions or loss of heterozygosity have been found in several human neoplasias (Yeung et al., 1993). pRb2/ p130 is a nuclear phosphoprotein whose phosphorylation is controlled by the cell cycle machinery (Claudio et al., 1996; Baldi et al., 1995). Like the other Rb family members, overexpression of pRb2/p130 inhibits in vitro cell growth of several tumor cell lines (Claudio et al., 1994), acting as a key regulator of the G1-S phase transition (Claudio et al., 1996). However, data on its in vivo suppressor activity have been lacking. The c-erbB-2 oncogene (HER2/neu) encodes a 185 kD transmembrane protein (p185HER2) with intrinsic tyrosine kinase activity that is related to the epidermal growth factor receptor (Coussens et al., 1985). c-erbB-2 is implicated in the pathogenesis of several human malignancies, and its overexpression correlates significantly with aggressive disease and poor prognosis (Kern et al., 1994). Overexpression of the rat neu proto-oncogene in the mammary glands of transgenic mice results in the development of focal mammary tumors that metastasize with high frequency (Guy et al., 1992). Moreover, in vitro studies using rodent cells indicate that p185HER2 overexpression per se is sucient to induce malignant transformation (Di Fiore et al., 1987). Together, these ®ndings identify HER2/neu as a potent oncogene. Cellular proliferation is triggered by the activation of growth factor receptors and is also controlled by cell cycle regulators (Zhu et al., 1993; DeCaprio et al., 1989). It has been reported that HER2/neu gene may be a target gene for the Rb protein and for Rb-binding proteins as E1A gene product (Yu et al., 1992, 1993). Indeed, pRb1 and E1A gene products can suppress cerbB-2-induced malignancy (Yu et al., 1992a), acting at the transcriptional level of both the human and rat HER2/neu oncogene (Yu et al., 1990). However, since a large number of cellular proteins can bind to Rb or E1A and many of them have not been well characterized, it is not yet clear which speci®c protein is involved in transcriptional regulation of HER2/neu by Rb and E1A (Yu et al., 1994). pRb exerts its control on cell cycle progression by formation of complexes with distinct members of the E2F family of transcription factors (Shirodkar et al., 1992). The pRbE2F complexes may, in turn, be modulated by the cell cycle machinery through phosphorylation of pRb by cyclin-dependent kinases, resulting in the release of active E2F species that stimulate transcription of genes involved in DNA synthesis and cell cycle progression (DeCaprio et al., 1992). The mechanism by which the
Rb2 as tumor suppressor gene SM Pupa et al
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other two members of the Rb family, p107 and pRb2/ p130, control cell cycle progression is not fully understood, although a pathway similar to that of pRb seems likely (Vairo et al., 1995; Hijmans et al., 1995; Shirodkar et al., 1992). In the present study, we asked whether the ectopic expression of pRb2/p130 aects p185HER2-mediated transforming activity. We ®nd that c-erbB-2-overexpressing SKOV3 human ovarian cancer cells transfected with wild-type (wt) Rb2 show impaired colony formation in soft agar and suppressed tumor growth in nu/nu mice, despite the expression of an active p185HER2 oncoprotein capable of autophosphorylation. By contrast SKOV3 cells transfected with a mutant Rb2 deleted in the `pocket region' (mut-Rb2) are fully tumorigenic both in vitro and in vivo. Our ®ndings demonstrate that wild-type pRb2/p130 acts as an oncosuppressor molecule in SKOV3 cells in vivo, and that pRb2/p130 requires an intact pocket region to exert this suppressive activity.
Results Transfection of Rb2 into SKOV3 ovarian carcinoma cells SKOV3 ovarian carcinoma cells were transfected with a plasmid containing the coding sequence for human wtRb2 or with the empty vector (mock transfection). Three pRb2/p130-transfected clones (cl. 24, cl. 49 and cl. 100) and one mock clone were randomly picked and further studied. Western blot analysis (Figure 1a) demonstrated an increase in pRb2/p130 protein level in the wtRb2-transfected clones as compared with protein levels in mock cells. The increase was also con®rmed at the mRNA level by Northern blot
Figure 1 (a) Western blot analyses with pRb2/p130 antiserum (1 : 500) on total lysates. Total protein extracts (100 mg) were electrophoresed in a 10% SDS ± polyacrylamide gel under nonreducing conditions, transferred onto Hybond-C paper and visualized by the ECL technique. pRb2/p130 protein levels were analysed and quantitated by densitometric scanning using Eagle EyeTM II (Stratagene). Values for mock, cl. 24, cl. 49 and cl. 100 were 54286, 85714, 85780 and 82336 for the upper bands, respectively, and 59093, 64494, 90197 and 92395 for the lower bands, respectively. (b) Western blot analyses with anti-c-erbB-2 Ab3 MAb (1.5 mg/ml) on total lysates. Total protein extracts (100 mg) were electrophoresed in a 7.5% SDS ± polyacrylamide gel under non-reducing conditions, transferred and visualized as above. Levels of p185HER2 quantitated by densitometric scanning showed a maximum variation of 10% between all lanes
analysis (data not shown). pRb2/p130 was detected as a double. In cl. 24, the intensity of the slower migrating band, corresponding to the putative inactive hyperphosphorylated form, was increased as compared to mock cells, whereas both bands, including the faster migrating one corresponding to the active hypophosphorylated form of pRb2/p130, were enhanced in cl. 49 and cl. 100 relative to mock cells. Expression levels of p185HER2 remained similar in mock-transfected cells and in the pRb2/p130-transfected clones (Figure 1b). Cyto¯uorimetric analysis of live mock cells and pRb2/p130 transfectants indicated similar levels of p185HER2 expression on the cell surface (data not shown). Analysis to estimate the p185HER2 intrinsic kinase activity using anti-c-erbB-2 immunoprecipitates incubated with [g-32P]ATP indicated that the receptor was functional in all cell populations tested, with higher activity in cl. 49 and cl. 100 (Figure 2a). The phosphorylation status of p185HER2, as determined by Western blot of anti-p185HER2 immunoprecipitates with anti-phosphotyrosine antibody, was high and similar in all cell populations (Figure 2b). Western blot analysis of the levels of the other two pocket proteins, pRb1 and p107, in lysates of the transfectant clones (Figure 3a,b) resolved both proteins as doublets that appeared to have the same functional status despite high or basal levels of pRb2/p130. In fact, the intensity of the hyperphosphorylated and hypophosphorylated bands of pRb1 was the same in all clones, whereas the hypophosphorylated band of p107 was more intense than the hyperphosphorylated band in all transfectants. In vitro and in vivo tumorigenicity of wtRb2-transfected clones Soft agar colony formation was signi®cantly inhibited (P50.001) in each of the pRb2/p130 transfectants
Figure 2 (a) p185HER2 intrinsic kinase activity estimated by precipitating 2 mg of total protein extracts from mock and pRb2/ p130 transfectants with anti-c-erbB-2 MGR2 MAb (+) or normal mouse serum (7) and incubating the immunocomplexes in the presence of 10 mCi of [g-32P]ATP. Immunocomplexes were electrophoresed in a 7.5% SDS ± polyacrylamide gel under nonreducing conditions and autoradiographed. The experiment was repeated two times with results varying by 5 ± 10%. (b) Tyrosinephosphorylated p185HER2 protein (P-Tyr) was detected by immunoprecipitating 2 mg of mock and clone cell extracts with anti-c-erbB-2 MGR2 MAb and blotting them with antiphosphotyrosine 4G10 MAb. Levels of tyrosine-phosphorylated p185HER2 as determined by densitometric scanning were 10% less and equal in mock cells and cl. 24, respectively, as compared to cl. 49 and cl. 100
Rb2 as tumor suppressor gene SM Pupa et al
compared with that of mock cells, with the number of foci originating from cl. 24, cl. 49 and cl. 100 decreased by 84, 98 and 94%, respectively (Table 1), and the foci size markedly reduced compared to that of mock cells (data not shown). In vivo injection of athymic (nu/nu) mice with the transfected cells gave rise to tumors in all 13 cl. 24injected mice, none of the 13 cl. 49-injected mice, and two of the 14 cl. 100-injected mice (Table 1). However, the tumor volume evaluated at 60 days after injection was signi®cantly lower in both the cl. 24- and cl. 100induced tumors (P50.001 and P=0.008, respectively) with a 79 and 85% reduction, as compared with tumors originating from mock-transfected cells (Table 1). In vitro and in vivo tumorigenicity of cells transfected with an Rb2 construct lacking the pocket region Cells transfected with mut-Rb2 gave rise to 333+9 colonies (Figure 4d), mock cells to 322+2 colonies (Figure 4b), and wtRb2-transfected cells to 103+2 colonies (Figure 4c), in colony formation assay. The decrease in colony growth of cells transfected with the functional Rb2 gene as compared with colony growth of mutated Rb2-transfectants was signi®cant (P50.0001). Soft agar colony formation was some-
Figure 3 Western blot analysis of transfectant clones for levels of pRb1 (a) and p107 (b). Total protein extracts (100 mg) were electrophoresed in a 10% SDS ± polyacrylamide gel under reducing conditions, transferred onto Hybond-C paper, blotted with anti-pRb1 xz77 MAb (1 : 1000) (a) or anti-p107 serum (1 : 500) (b) and visualized by the ECL technique. To normalize for protein content, blots were reprobed with anti-b actin serum (c)
Table 1
Discussion This study shows that ectopic expression of the cell cycle regulator pRb2/p130 in SKOV3 cells inhibits the tumorigenicity associated with overexpression of the c-erbB-2 oncoprotein in these cells both in vitro and in vivo. This is the ®rst demonstration that Rb2 can act as a tumor suppressor gene in vivo in human ovarian carcinoma cells. Unlike the Rb1 gene product (Matin and Hung, 1994; Yu et al., 1992), pRb2/p130 in our model did not repress c-erbB-2 expression, indicating that the Rb1
Figure 4 Colony formation assay of SKOV3 cells not transfected (a) or transfected with pcDNA3 (b), wtRb2 (c) or mutated Rb2 (d). Two independent experiments were carried out with similar results (less than 10% variation). Dierences were analysed by Student's t-test
In vitro and in vivo growth of transfected cells Clones
cl.mock
cl.24
Soft agar colony assay No. of colonies+s.da % decreasec
463+46 ±
74+11b 84
Tumor take Mice with tumor/totald Tumor volume (cm3)+s.d.e % decreasec
10/10 1.9+0.64 ±
13/13 0.4+0.16b 79
a
what decreased (41%) in the polyclonal bulk wtRb2transfected cells compared with that of mock and mutRb2 bulk transfectants, and three of seven mice injected with the wtRb2-transfected bulk population formed tumors, although tumor volume was significantly smaller than that of tumors grown in mice injected with bulk mock cells (P=0.001). mut-Rb2transfected cells were fully tumorigenic (Table 1).
Transfectants cl.49
cl.100
Mock
Bulk populations wtRb2
mut-Rb2
11+2b 98
26+7b 96
63+5 ±
37+10b 41
65+7 0
2/14b 0.3+0.05b 85
7/7 1.4+0.30 ±
3/7 0.4+0.03b 77
7/7 1.3+0.31 8
0/13b 0b ±
Cells were plated at 16103 and 56103 cells/well in semi-solid medium containing 0.35% Bacto agar over a 0.7% agarose layer for the bulk populations and the clones, respectively. Values represent the mean+s.d. of three replicates in two independent experiments. bP50.05 as compared with mock transfected cells. cAs compared with mock cells. dMice (6-week-old athymic mice) were injected s.c. in the right ¯ank under aseptic conditions with 46106 cells. eDetermined on day 45 for bulk populations and on day 60 for clones after tumor transplant
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Rb2 as tumor suppressor gene SM Pupa et al
654
and Rb2 genes are not redundant in this pathway despite their structural and functional homology (Claudio et al., 1994). Also, the levels of pRb1 and p107 remained unchanged in all transfectants clones and bulk populations independent of pRb2/p130 expression levels, indicating no cross-regulation between these proteins. pRb2/p130 activity is regulated by cyclin-dependent kinases through phosphorylation during the cell cycle (Claudio et al., 1996). As reported for pRb whose active hypophosphorylated form prevents progression into S phase, the presence of the hypophosphorylated form of pRb2/p130 in our model correlated with its ability to prevent progression into S phase (Paggi et al., 1996; Claudio et al., 1995). Consistent with this observation, the tumor take of the two clones, which display a high level of hypophosphorylated pRb2/p130, was completely abrogated or strongly inhibited. By contrast, the clone overexpressing mainly the hyperphosphorylated form of pRb2/p130, showed only a partial inhibition in vivo since only tumor volume and not tumor take decreased signi®cantly compared to control cells. These data support the notion it is mainly the hypophosphorylated form of pRb2/p130 that acts as a tumor suppressor molecule. The activity of the rat p185neu receptor, which shares up to 90% homology with the human p185HER2 oncoprotein in the catalytic domain, has been shown to be tightly regulated throughout the entire cell cycle, with the intrinsic kinase activity of p185neu gradually decreasing from the G0/G1 phase to the M phase in which it is least active (Kiyokawa et al., 1995). Since pRb2/p130 in its hypophosphorylated form acts as a key regulator of the G1-S phase transition mediating G1 growth arrest (Claudio et al., 1996), we speculate that cells deriving from cl. 49 and cl. 100, in which c-erbB-2-associated tumorigenicity is almost completely reversed, show a high p185HER2 intrinsic kinase activity because they are predominantly arrested in G1. The pocket region has been shown to be responsible for most of the speci®c and functionally relevant protein-protein interactions involving either some endogenous proteins or viral oncoproteins in¯uenced by the retinoblastoma cell cycle checkpoints (Paggi et al., 1996). The lack of tumor suppression obtained in SKOV3 cells with the plasmid containing the human Rb2 gene deleted in the pocket region sequence clearly indicates that this domain is the functional one in vivo. The cell cycle regulatory machinery is a focal point for the integration or convergence of the various signal transduction pathways that mediate mitogenic and anti-mitogenic signals between the environment and speci®c cell surface receptors. In this context and consistent with recent data obtained using the E1A model (Frisch and Dolter, 1995), pRb2/p130 behaves as a tumor suppressor gene that overcomes the c-erbB-2 receptor activity in SKOV3 cells. Immunohistochemical data also suggest an important role for pRb2/p130 in the pathogenesis and progression of lung and endometrial cancer, where its downregulation correlates with worse prognosis (Susini et al., 1998; Baldi et al., 1996b, 1997). Our data and those of others on the relationship between ectopic expression of cell cycle regulators and
the role of the c-erbB-2 oncoprotein in tumorigenesis (Yu et al., 1992, 1993, 1994) suggest that this gene acts as a dominant active oncogene only when cell cycle regulators and/or associated molecules, such as tumor viral oncoproteins, cyclins, cyclin-dependent kinases and inhibitors of cyclin-dependent kinases, are functionally inactive or not overexpressed. Our results on the role exerted by pRb2/p130 in a highly tumorigenic human ovarian carcinoma model strengthen the reported observations identifying Rb2 as a tumor suppressor gene and suggest the potential usefulness of therapeutic approaches that target this gene in cancer patients.
Materials and methods Cell culture, plasmids and transfection SKOV3 human ovarian carcinoma cells overexpressing p185HER2 (American Type Culture Collection, Rockville, MD, USA) were maintained at 378C in a humidi®ed atmosphere of 5% CO2 in air in RPMI-1640 medium (Sigma, St Louis, MO, USA) supplemented with 10% heatinactivated fetal calf serum (FCS) (Hyclone, Logan, UT, USA) and 2 mM L-glutamine. Plasmids pcDNA3-pRb2/ p130 (wtRb2) and the empty vector pcDNA3 have been described (Claudio et al., 1994). Plasmid pcDNA3:Rb2/321 encodes a truncated version of the wtRb2 deleted in aminoacids 322 ± 1082, which encompasses the pocket region of the gene, under the control of the CMV promoter (mut-Rb2). SKOV3 cells grown to 80% con¯uence in serum-free medium were transfected with 50 mg of Lipofectin (Gibco BRL, Paisley, UK) plus 20 mg of plasmid. Transfectants were kept overnight at 378C and maintained in culture for an additonal 48 h in complete medium. Cells were then trypsinized and plated in the presence of 500 mg/ml of G-418 (geneticin) (Gibco BRL). Individual colonies were randomly picked, expanded and maintained in the presence of 300 mg/ml of G-418. Soft agar colony formation assay Cells (16103 and 56103 cells/well) were plated in 6-well plates in semisolid medium containing 0.35% Bacto Agar (Difco, Detroit, MI, USA) supplemented with 30% FCS and 300 mg/ml G-418 over a 0.7% agarose layer. Colonies were scored after 27 days of incubation at 378C in 5% CO2 in air. Colony formation assay SKOV3 cells stably transfected with pcDNA3, pcDNA3Rb2/p130 or pcDNA3:Rb2/321 were plated (0.56106 cells in 10-cm dishes) in triplicate. After 3 weeks of selection in 500 mg/ml of G-418, cells were ®xed and stained with methylene blue in 50% ethanol (Difco) for 15 min and photographed. Mice and tumorigenicity assay Six-week-old athymic mice, purchased from Charles River (Calco, Italy) were used. Care and use of the animals was in accordance with institutional guidelines. Mice were injected subcutaneously in the right ¯ank under aseptic conditions with 46106 cells collected by trypsinization and washed twice in cold PBS. Tumors were measured twice weekly and tumor volume was calculated using the formula: 0.56d126d2, where d1 and d2 are the larger and smaller diameter, respectively.
Rb2 as tumor suppressor gene SM Pupa et al
Statistical analysis Data were evaluated using the Student's t-test. Dierences were considered signi®cant at P50.05. Western blot analysis Cells were trypsinized, washed twice with cold PBS, and solubilized for 50 min on ice in lysis buer (50 mM TrisHCl at pH 7.4, 5 mM EDTA, 250 mM NaCl, 50 mM NaF, 0.5% Triton X-100) containing protease plus phosphatase inhibitors, 10 mg/ml leupeptin, 1 mM phenylmethylsulfonyl¯uoride (PMSF), 1 mM sodium orthovanadate (Na3VO4) and 1 mM okadaic acid. Cell lysates were cleared by centrifugation at 48C for 10 min at 10 000 g. Protein concentration was determined by the BCA protein assay (Pierce Biochemical Company, St Louis, MO, USA). After heating to 958C for 5 min, 100 mg of total protein extract was electrophoresed in a 7.5% and 10% SDS-polyacrylamide gel. Separated proteins were then electrotransferred to Hybond-C paper (Amersham, Little Chalfont, UK) and incubated at room temperature for 1 ± 2 h with primary antibody followed by incubation with 125I-goat anti-mouse 7S immunoglobulin (Ig) (Amersham) and autoradiography, or by incubation with anti-rabbit Ig and/or anti-mouse Ig horseradish peroxidase-linked whole antibodies (1 : 10 000) (Amersham) and visualized using the ECL detection system (Amersham) according to the supplier's instructions. Monoclonal antibodies (MAbs) used were the following: anti-c-erbB-2 MGR2 (10 mg/ml) (Tagliabue et al., 1991), anti-c-erbB-2 Ab3 (1.5 mg/ml) (Oncogene Science, Cambridge, MA, USA), anti-phosphotyrosine 4G10 (2 mg/ml) (Upstate Biotechnology, Inc., Lake Placid, NY, USA) and anti-pRb1 xz77 (1 mg/ml) (Hu et al., 1991). Polyclonal antip107 serum (1 : 500) (Baldi et al., 1997), anti-pRb2/p130 serum (1 : 500) (Baldi et al., 1995) and anti-b actin serum
(1 : 2000) (Sigma Chemical Co, St Louis, MO, USA) were also used. Immunoprecipitation and in vitro kinase assay Cell lysates (2 mg protein/sample) obtained as described above were immunoprecipitated after preclearing for 30 min with GammaBind Plus Sepharose (Pharmacia Biotech, Uppsala, Sweden) by incubation on a rocker with speci®c antibodies or with rabbit or mouse normal sera as negative controls for 1 h at 48C. Sepharose was added (20 ml), and after 1 h incubation, immune complexes were separated by centrifugation and washed three times with lysis buer and once in kinase buer (20 mM HEPES, 10 mM magnesium acetate, 1 mM dithiothreitol, 20 mM ATP). In vitro kinase assay was performed as described (Giordano et al., 1991). Brie¯y, the pellet was incubated with 20 ml of kinase buer containing 10 mCi of [g-32P]ATP (Amersham) for 30 min at 308C. The reaction was terminated by addition of 20 ml of 2X Laemmli sample buer. Samples were boiled, electrophoresed in a 7.5 and 10% polyacrylamide gel, and autoradiographed.
Acknowledgements We sincerely thank Dr E Tagliabue and Dr A Baldi for critical review of the manuscript, L Mameli for secretarial support and M Azzini for photographic reproduction. This work was supported by grants from the AIRC/FIRC and the PF CNR-ACRO and from Sbarro Institute for Cancer Research and Molecular Medicine, NIH Grant RO1CA60999-01A1, and The Council for Tobacco Research and Sbarro Institute for Cancer Research and Molecular Medicine.
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