VentX trans-Activates p53 and p16ink4a to Regulate Cellular Senescence

Cell senescence is a process of irreversible arrest of cell proliferation and plays an important role in tumor suppression. Recent studies showed that Wnt inhibition is a trigger of cellular senescence. Using methods of reverse genetics, we recently identified VentX, a human homolog of the vertebrate Xenopus Vent family of homeobox genes, as a novel Wnt repressor and a putative tumor suppressor in lymphocytic leukemia. Here, we show that VentX is a direct transcriptional activator of p53-p21 and p16ink4a-Rb tumor suppression pathways. Ectopic expression of VentX in cancer cells caused an irreversible cell cycle arrest with a typical senescence-like phenotype. Conversely, inhibition of VentX expression by RNA interference ameliorated chemotherapeutic agent-induced senescence in lymphocytic leukemia cells. The results of our study further reveal the mechanisms underlying tumor suppression function of VentX and suggest a role of VentX as a potential target in cancer prevention and treatment.

In parallel with its prominent role in development, recent studies suggest a critical role for Wnt signaling in cellular senescence, an irreversible process of cell proliferation arrest (14). Initially described as a cellular mechanism underlying physiological aging of fibroblasts, cellular senescence is being recognized as playing critical roles in tumor suppression (15)(16)(17). Similar to primary fibroblasts, tumor cells also retain the ability to undergo senescence in response to genetic manipulation or treatment with chemotherapeutic drugs (18 -21). Senescent cells display positive staining for senescence-associated (SA) ␤-galactosidase and form senescence-associated heterochromatic foci (22,23). It has been shown that down-regulation of Wnt signaling triggers the formation of the SA heterochromatic foci and onset of cellular senescence (14).
The p53-p21 and Rb-p16 ink4a are two critical tumor suppression pathways implicated in cellular senescence (16,17). p53 is a well established tumor suppressor gene and exerts its function in part by transcriptional activation of p21, an inhibitor of cyclin-dependent kinases (CDKs) (24). Rb exerts its function by binding to the E2F family of transcriptional factors and inhibiting the downstream transcriptional cascades required for cell cycle entry (25). The inhibitory effects of Rb on E2F are abolished through phosphorylation of Rb by cyclin/CDK complexes which, in turn, are inhibited by p16 ink4a and p15 ink4b (26). Clinical genetics studies have showed that silencing of the p53-p21 pathway occurs in ϳ50% of cases of acute lymphocytic leukemia. Likewise, deletion or epigenetic silencing of p16 ink4a and p15 ink4b occurs frequently in acute lymphocytic leukemia (27)(28)(29).
VentX is a novel Wnt repressor implicated in the pathogenesis of lymphocytic leukemia (9). To explore the mechanisms underlying VentX tumor suppression function, we screened for VentX effects on the expression of a panel of key regulators of cell proliferation. Here, we report that VentX is a direct transcriptional activator of the p53-p21 and Rb-p16 ink4a tumor suppressor pathways. We found that VentX expression induces a senescence phenotype in several tumor cell lines and that down-regulation of VentX expression by RNA interference is associated with reduced senescence and increased resistance of leukemia cells to chemotherapeutic agents. Our data suggest a potential role for VentX as a novel therapeutic target in cancer treatment.

EXPERIMENTAL PROCEDURES
Cell Culture-Human embryonic kidney cell line 293 (HEK293), human cervical cancer cell line HeLa, human osteosarcoma U2OS cell line, and human primary fibroblasts IMR90 were cultured in DMEM supplemented with 10% FBS and 1% antibiotics. Human acute lymphoblastic leukemia cell line Nalm16 was maintained in RPMI 1640 medium. Primary CD19 ϩ B lymphocytes were purified from peripheral blood mononuclear cells by positive selection using a magnetic cell separator (MACS; Miltenyi Biotec, Auburn, CA). The lymphocytes were seeded at a density of 10 6 /ml in RPMI 1640 medium supplemented with 10% FBS.
Western Blotting-Cells were lysed in solution A (50 mM Tris-HCl, pH 7.8, 420 mM NaCl, 1 mM EDTA, 0.5% Nonidet P-40, 0.34 M sucrose, 10% glycerol, 1 mM Na 3 VO 4 , 10 mM NaF, ␤-glycerophosphate, 1 mM PMSF, and protease inhibitor mixture). Lysates were cleared by centrifugation, and protein concentration was determined by the Bradford assay (Bio-Rad). Proteins resolved by SDS-PAGE were transferred onto PVDF membrane, which were detected with optimal dilutions of primary antibodies, followed by horseradish peroxidase-linked secondary antibodies. The blots were developed by enhanced chemiluminescence (Amersham Biosciences). Primary antibodies used were from Cell Signaling (Danvers, MA) except anti-VentX sera, which were generated in our laboratory.
Luciferase Reporter Assay-The luciferase reporters of p53 (Ϫ2.4 kb) and p16 ink4a (Ϫ869 bp) promoters have been described previously (30,31). The p21 luciferase reporter was a gift from Dr. W Gu as described (32). Cells seeded in 24-well plates were transfected with 500 ng of reporter plasmid and increasing amounts of pcDNA-VentX plasmid. Total plasmid transfected was brought to the same amount by the addition of empty pcDNA vector. 10 ng of Renilla luciferase plasmid was included for each transfection to normalize reporter activity. Cells were harvested at 48 h after transfection and analyzed with Dual Luciferase Reporter Assay System (Promega, Madison, WI).
Generation of Stable Cell lines Expressing VentX under Control of Tetracycline (Tet)-inducible Promoter-The U2OS cell line expressing Tet-inducible VentX was generated through transfection of a TetR-expressing U2OS cell line (a gift from Dr. Yao, Department of Surgery, Brigham and Women's Hospital, Boston) with pcDNA TM 4/TO-VentX plasmid. Positively transfected cells were selected with 2 g/ml Zeocin (Invitrogen). Single colonies were selected out by limiting dilution. IMR90 cell lines expressing GFP-VentX (or GFP as control) were generated through co-transduction of pRetroX-GFP-VentX (or pRetroX-GFP) and pRetroX-Tet-On advanced retroviruses (Retro-X TM Tet-On Advanced Expression System, Clontech). GFP-positive cells were sorted by FACS after a brief incubation with Tet (24 h) and maintained in the absence of Tet.
RNA Interference-Small hairpin RNA (shRNA) retroviral vectors targeting VentX (pRS vectors) with a puromycin selection marker were purchased from Origene (Rockville, MD) and described in our previous publication (9). Stable knockdown of VentX expression in Nalm16 cells was described as previously (9). Transient knockdown of VentX in primary CD19 ϩ B lymphocytes was achieved through electroporation of duplex RNA oligonucleotides (siRNA) with a kit from Lonza (Allendale, NJ) following the manufacturer's instructions. Retroviral shRNA against human p16 ink4a was a gift from Dr. Lowe (Cold Spring Harbor Laboratory). pMKO.1-shp53 and pBabe-SV40 large T (SV40LT) antigen retroviral vectors were obtained from Addgene.
␤-Galactosidase Staining-Senescence of U2OS, Nalm16, and IMR90 cells was evaluated by a SA ␤-galactosidase staining kit from Cell Signaling. Cell counts were made on five random fields of at least 200 cells.
Cell Viability Assay-Nalm16 cells were subjected for the following treatments: 800 nM all-trans-retinoic acid (ATRA) for 10 days, 100 ng/ml doxorubicin (Doxo) for 4 days, 5 M 5fluorouracil for 3 days, or 1 M camptothecin for 2 days. Stable knockdown of VentX in Nalm16 cells and the tetrazolium compound (MTS) assay of cells viability were performed as described previously (9).
FACS Analysis of Ki67-Nalm16 cells were treated with 800 nM ATRA for 5 days, 100 ng/ml Doxo for 2 days, or mock treated. Cells were then harvested for intracellular staining of phycoerythrin-conjugated Ki67 antibody with protocol provided by the manufacturer (eBioscience, San Diego, CA) and analyzed with a FACScan flow cytometer (BD Bioscience) using FlowJo software.

RESULTS
Identification of p53, p21, and p16 ink4a as Downstream Targets of VentX-Our recent study showed that VentX is a negative regulator of cell proliferation. Mechanistically, we found that VentX exerts its function on cell proliferation at least in part through inhibition of Wnt signaling and its downstream targets, such as cyclin D1 (9). Given the broad implications of Wnt signaling in cell proliferation, we sought to determine further the molecular pathways underlying VentX-induced cell growth arrest through a targeted screen of cell cycle regulators, using a transient transfection assay. HEK293 cells were transfected with expression constructs encoding VentX or vector control. At 48 h after transfection, total cell lysates were harvested, and the expression levels of a panel of cell cycle regulatory proteins were determined by Western blot analysis. As shown in Fig. 1, we found that VentX expression led to a significant increase in the protein levels of p53 and two CDK inhibitors: p21 and p16 ink4a (Fig.  1). Consistent with our previous finding, the cyclin D1 protein level was down-regulated upon the expression of VentX (9). No obvious expression change was detected for other tested proteins (Fig. 1).
VentX trans-Activates p53-p21 Pathway-p53 and p21 are key regulators of cell proliferation and are broadly implicated in tumorigenesis. To corroborate our screening findings, we used U2OS cells to explore further the effects of VentX on the expression of p53 and p21. Plasmid encoding VentX or vector control was transiently transfected into U2OS cells. At 48 h after transfection, total proteins were extracted, and the expression levels of p53 and p21 were determined by Western blot analysis. Again, we observed an increase in the protein levels of p53 and p21 in U2OS cells upon VentX expression ( Fig. 2A, left  panel). The effect of VentX on the protein level of p53 was also observed in HeLa cells (data not shown). Collectively, these data suggest that the p53-p21 pathway is a downstream target of VentX. Either increased transcription or reduced proteolysis may account for the effects of VentX on p53 protein level. To elaborate the mechanism of VentX action, we tested whether VentX affects the stability of p53. U2OS cells were treated with cycloheximide after transfection of VentX or control plasmid. As shown in Fig. 2A (right panel), we did not observe significant alteration on the kinetics of p53 protein degradation upon transfection of VentX, suggesting that VentX does not exert significant effects on p53 stability. Next, we explored the effects of VentX on transcriptional activation of p53-p21 pathway. As shown in Fig. 2B (left panel), in comparison with the vector control, VentX expression led to increased mRNA levels of p53 and p21 in U2OS cells. In addition to its effects on p53 in U2OS cells, VentX also up-regulated p53 mRNA level in Sw480 cells, which harbor a mutant p53 protein (Fig. 2B, right panel) (33). These data further suggest that p53 is a transcriptional target of VentX. Consistently, we showed that ectopic expression of VentX trans-activated the human p53 and p21 promoter-luciferase reporters in a dose-dependent manner (Fig. 3). Human p53 promoter region contains several putative homeodomain core binding sequences (ATTA) (Fig. 2G and Ref. 31). A ChIP assay therefore was performed to examine a potential direct interaction between VentX and the p53 promoter (Fig. 2G). As shown in Fig. 2F, the ChIP assay revealed a specific binding of VentX to the p53 promoter. As negative controls, VentX was found not to bind to the region 3.7 kb upstream of the VentX binding site on p53 promoter. Further, VentX did not bind to the constant region of immunoglobulin M heavy chain gene (C) (9) or the GAPDH promoter.
VentX trans-Activates p16 ink4a -Rb Pathway-The p16 ink4a -Rb pathway has been implicated in the pathogenesis of a variety of cancers, including lymphocytic leukemia (29). Previous studies showed that p16 ink4a inhibits phosphorylation of Rb by CDKs (26). Our recent study indicated that VentX is a putative tumor suppressor in lymphocytic leukemia (9). The finding that VentX up-regulates the expression of p16 ink4a (Fig. 1) prompted us to test the hypothesis that VentX also exerts its function by transactivation of the p16 ink4a -Rb tumor suppressor pathway. HeLa cells, in which the p16 ink4a -Rb pathway is functional (34), were employed to test this hypothesis. As shown in Fig. 2 D (left panel), with increased expression of VentX, there was a corresponding increase of p16 ink4a protein expression. In comparison to the elevated expression of p16 ink4a , no substantial change of Rb protein level was observed. However, when the level of phosphorylated Rb was examined with specific antibody, we found that VentX expression is associated with significant reduction of the hyperphosphorylated form of Rb (Fig. 2D,  left panel). Our findings support the hypothesis that VentX promotes the expression of p16 ink4a , which, in turn, inhibits the phosphorylation of Rb by CDKs (35). The potential transcriptional activation of p16 ink4a by VentX was suggested by the increased p16 ink4a mRNA levels upon ectopic expression of VentX (Fig. 2 D, right panel). Using p16 ink4a promoterluciferase reporter assay, we further demonstrated that VentX promoted p16 ink4a transactivation in a dose depend- ent manner (Fig. 2 E). Human p16 promoter also contains putative homeodomain binding sites (Fig. 2 G). Consistently, the ChIP assay demonstrated a specific binding of VentX to the p16 ink4a promoter (Fig. 2 F).

Knockdown of VentX Is Associated with Reduced p53-p21 and p16 ink4a Expression in Primary Lymphocytes-VentX is expressed predominately in hematopoietic cells. Expression analysis of VentX in peripheral blood leukocytes shows that
VentX is expressed in both myeloid and lymphoid lineages (9). To determine whether VentX regulates p53, p21 and p16 ink4a expression under physiological condition in primary hematopoietic cells, we examined the effects of VentX knockdown in primary B cells. As shown in Fig. 3 A, VentX expression in isolated primary B cells was efficiently down-regulated by electroporation of VentX siRNA. Correspondingly, with the downregulation of VentX, there was significant decreases of p53, p21 and p16 ink4a expression at both mRNA and protein levels (Fig. 3  B and C). These data suggest that VentX plays a critical physiological role in regulating the expression of p53, p21 and p16 ink4a in primary lymphocytes.
VentX Induces Irreversible Cell Growth Arrest and a Senescence Phenotype-Originally described during normal aging of fibroblasts, senescence pathways have been shown to play critical roles in preventing tumorigenesis (17,36). Previous investigations showed that the p53-p21 and p16 ink4a -Rb tumor suppressor pathways are critical mediators of cellular senescence following various stimuli (16,17). Therefore, the findings that VentX activates both pathways in transient transfection studies prompted us to investigate whether VentX is able to exert a tumor suppression function by inducing cellular senescence in tumor cells. To address this question, we generated a stable U2OS cell line, U2OS/VentX Tet , in which the expression of VentX is under control of a tetracycline (Tet)-inducible promoter. As shown in Fig. 4A, U2OS/VentX Tet cells express VentX in a Tet-dependent manner. Upon addition of Tet, there was a strong induction of p53 and p21 expression in U2OS/ VentX Tet cells (Fig. 4A). Consistent with its role as an inhibitor of cell proliferation, induction of VentX expression is associated with significant inhibition of U2OS/VentX Tet cell growth (Fig. 4B). Interestingly, VentX expression caused a striking morphological change with U2OS/VentX Tet cells appeared to be enlarged and flattened. These cells displayed positive staining for SA ␤-galactosidase, a characteristic marker of cellular senescence (Fig. 4C). Because irreversible growth arrest is a key feature of cellular senescence, we examined whether the growth inhibitory effect of VentX was irreversible. As shown in Fig. 4D, when Tet was removed from the culture medium after 3 days of exposure, the expression of VentX and p53 gradually p53 and p16 ink4a promoter regions but not with the region 3.7 kb upstream of the VentX binding site on p53 promoter, immunoglobulin C region, and GAPDH promoter region. Increasing amounts of input or precipitated DNA were used as templates for PCR analysis. G, schematic diagrams depict the putative homeodomain binding sites and the position of primers used for ChIP assay on the promoter regions of p53 an p16 ink4a . The primers amplify the regions that encompass the most proximal potential homeodomain binding sequences to the transcription start sites.

FIGURE 3. Knockdown of VentX in primary B cells is associated with decreased p53-p21 and p16 ink4a expression.
Human primary B cells isolated from three different donors were electroporated with VentX siRNA or control siRNA as described under "Experimental Procedures." Cells were then cultured in the presence of CD40 ligand and IL4 for 3 days. A, mRNA level of VentX was determined by reverse transcription and quantitative PCR. B, mRNA level of p53, p21, and p16 ink4a was determined by quantitative PCR. C, whole cell lysates were prepared from B cells and used to probe protein levels of p53, p21, and p16 ink4a with corresponding antibodies. decreased (Fig. 4D, left panel). However, the U2OS/VentX Tet cells remained positive for SA ␤-galactosidase activity (Fig. 4D,  middle panel). Moreover, the growth arrest of the U2OS/ VentX Tet cells continued (Fig. 4D, right panel). In addition to U2OS cells, stable VentX expression also provoked a senescent phenotype in HeLa cells. 3 We next explored the potential dependence of VentX-induced cellular senescence on the p53 and p16 ink4a pathways. For this purpose, we employed the IMR90 primary human fibroblasts, which possess intact p53-p21 and p16 ink4a -Rb pathways and have been widely used as a model to study cellular senescence (37,38). As shown in Fig. 4E, inducible expression of VentX caused a typical senescence phenotype in early passage IMR90 cells as indicated by enlarged and flattened cell morphology, positive staining for SA ␤-galactosidase activity (Fig. 4E, left panel, Control). First, p53 and p16 ink4a shRNAs were used to down-regulate the expression p53 and p16 ink4a , and the efficacy of the p53 and p16 ink4a shRNA was confirmed by Western blot analysis (data not shown). We found that, although suppression of p53 or p16 ink4a individually exerted few effects on VentX-induced senescence, the combined suppression of both p53 and p16 significantly ameliorated the senescence phenotype induced by VentX (Fig. 4E). Previous studies have showed that SV40 large T antigen (SV40LT) inhibits the function of both p53 and p16 ink4a pathways through direct binding to p53 and Rb (39). Consistently, we found that ectopic expression of SV40LT substantially ameliorated the  Nalm16 cells were treated with 1 M camptothecin (CPT) for 2 days or mock treated. The mRNA and protein levels of p53 were determined by RT-PCR and Western blotting, respectively. C, effect of VentX knockdown on the growth curve of Nalm16 cells after chemotherapeutic treatments. 0.5 million Nalm16 cells were treated with 800 nM ATRA for 6 days, 100 ng/ml Doxo for 4 days, or mock treated. Viable cells were counted every day and plotted. (D) 1.0 M Nalm16 cells were subjected for the following treatments: 800 nM ATRA for 10 days, 100 ng/ml Doxo for 4 days, and 5 M 5-fluorouracil for 3 days. The viability of Nalm16 cells (control and VentX knockdown) after drug treatments was determined as described under "Experimental Procedures." E, representative image of SA ␤-galactosidase staining in control versus VentX-suppressed Nalm16 cells after treatments with ATRA for 10 days or Doxo for 4 days is shown. F, SA ␤-galactosidase positivity in control versus VentX-suppressed Nalm16 cells at different time points after drug treatments was analyzed quantitatively. G, Ki67 staining in control versus VentX-suppressed Nalm16 cells after treatments with ATRA for 5 days or Doxo for 2 days is shown. cellular senescence induced by VentX (Fig. 4E). Collectively, our data suggest that VentX induces cellular senescence, at least in part, through p53/p16 ink4a -mediated mechanisms.

Knockdown of VentX Results in Amelioration of Senescence and Enhanced Chemotherapeutic Resistance in Leukemic Cells-
Chemotherapy remains a main therapeutic modality for leukemia patients. Chemotherapeutic drugs such as Doxo and ATRA have been show to induce premature senescence in tumor cells (19,40,41). Our previous study showed that lymphoblastic leukemia cells Nalm16 express low levels of VentX (9). Using the Nalm16 model, we found that VentX expression can be induced in Nalm16 cells by various chemotherapeutic agents (Fig. 5A).
To determine whether VentX plays a role in mediating senescence of Nalm16 cells induced by chemotherapeutic agents, we down-regulated VentX expression in Nalm16 cells with specific shRNA (Fig. 5B, left panel). We found that Nalm16 cells transfected with shVentX became more resistant to chemotherapeutic agent-induced growth inhibition (Fig. 5C) and showed enhanced cellular viability (Fig. 5D). Supporting a role of VentX in mediating chemotherapeutic agent-induced cellular senescence, Nalm16 cells transfected with shVentX displayed significantly lower SA ␤-galactosidase staining upon exposure to chemotherapeutic agents compared with the control cells (Fig.  5, E and F). The shVentX-transfected Nalm16 cells also showed a stronger Ki67 staining, a cell proliferation marker (42) (Fig.  5G). In addition, down-regulation of VentX diminished the DNA-damaging agent camptothecin-induced up-regulation of p53 (Fig. 5B, right panel), suggesting that VentX may also be involved in the DNA damage response.

DISCUSSION
VentX is a human homeobox gene that shares strong homology to the Xenopus Vent gene family (9,43). Besides its ventralizing activity in the zebrafish model, little is known about the function of VentX (43). Our recent investigation found that VentX is a negative regulator of cellular proliferation and a LEF/TCF-associated Wnt repressor (9). The present study was undertaken to dissect further the mechanisms of the VentX tumor suppression function. We screened a variety of proteins involved in cell cycle regulation and identified novel downstream targets of VentX in the cell cycle regulatory machinery. We demonstrated that VentX was able to activate two cyclindependent kinase inhibitors: p21 and p16 ink4a . When ectopically expressed, VentX up-regulated p53-p21 and p16 ink4a protein level in multiple cell lines. In contrast, knockdown of VentX in primary B cells led to reduced expression of p53-p21 and p16 ink4a (Fig. 3). Combined with other biochemical analyses (luciferase reporter and ChIP, as shown in Fig. 2), we conclude that VentX is a direct transcriptional activator of p53-p21 and p16 ink4a .
Our recent finding that VentX is an inhibitor of oncogenic Wnt/␤-catenin signaling and is down-regulated in blood samples of chronic lymphoblastic leukemia patients led us to propose that VentX is a putative tumor suppressor (9). Here, we found that VentX is a potent activator of p53-p21 and p16 ink4a -Rb tumor suppressor pathways and is down-regulated in acute lymphoblastic leukemia samples. 3 The current study further showed that VentX expression can be induced upon chemotherapeutic treatments. Consistent with a role as a tumor suppressor, we demonstrated that knockdown of VentX expression in lymphoblastic leukemic Nalm16 cells led to enhanced resistance of these cells to cytotoxic effects of chemotherapeutic agents. Thus, data from this and our previous studies strongly support that VentX is a novel tumor suppressor and a cancer therapeutic target. Recently, Rawat et al. reported that knockdown of VentX in human acute myeloid leukemia cell lines inhibited their proliferative potential. It was suggested that functions of VentX may depend on cellular context (44).
VentX expression induced pronounced senescence phenotypes in several tumor cell lines and primary fibroblasts, and down-regulation of VentX was associated with a reduction in chemotherapeutic drug-induced senescence in lymphoblastic leukemia cells. Our study indicated that p53-p21 and p16 ink4a -Rb, previously defined as key players of cellular senescence (16,17), may be important mediators of VentX-induced senescence (Fig. 4E). Our findings are consistent with the previous findings that Wnt inhibition activates cellular senescence pathway (14). Therefore, VentX appears to provide a link between Wnt inhibition and activation of p53 and p16 ink senescence pathways. It should be noted, however, in contrast to oncogene-induced senescence, which is usually accompanied by mitogenic effects and activation of the DNA damage response (37,45,46), VentX-induced senescence triggered immediate growth inhibition without evidence of the DNA damage response (lack of phospho-Chk1 Ser 317 /Ser 345 and Chk2 Thr 68 ). 3