p16 Modulates VEGF Expression via Its Interaction With HIF-1α in Breast Cancer Cells

Keywords: p16, VEGF, HIF-1α, Breast cancer

Cell culture and medium

Dulbecco’s modified Eagle medium (D-MEM) and RPM1-1640 was purchased from Gibco BRL (Gaithersburg, MD), and fetal bovine serum (FBS) from Hyclone Laboratories (Logan, UT). Human embryonic kidney 293 cells (American Type Culture Collection, Rockville, MD) were grown in D-MEM with 10% heat-inactivated FBS. Human breast cancer cell lines MDA-MB-231 (ATCC) were grown in RPM1-1640 medium with 10% FBS. All cell lines were grown in medium containing 100 units/ml penicillin, and 100 μg/ml streptomycin at 37°C in 5% CO₂.

Adenoviral vector construction

The construction of AdRSVp16, a replication-defective, recombinant adenoviral vector expressing a human wild-type p16 cDNA gene under the control of a Rous sarcoma virus (RSV) promoter, was described previously (25). The construction of the control virus, AdRSVlacZ, in which a bacterial β-galactosidase gene was under the control of the RSV promoter, was also described previously (26). The construction of recombinant adenovirus expressing the HIF-1α, Ad-HIF-1α, was done as follows: The HaloTag pHT2 vector (Promega, Madison, WI) was cut with EcoR V and Not I to release an 890-bp HT2 cDNA, which encodes a HaloTag reporter gene (Promega). The HT2 cDNA was subcloned into the pacAd5CMVNpA (University of Iowa, Iowa City, IA) to generate the adenoviral shuttle vector pacAd5-HT2. A 2.5-kb PCR product of human HIF-1α cDNA was generated from HIF-1α expression vector (pHIF-1α, a generous gift from Dr. B. Jiang of West Virginia University; see (13)) using a set of primers specific to the human HIF-1α cDNA gene, the primers contained an introduced Bam HI site at both ends of the PCR products. After digestion with Bam HI on both the 2.5-kb PCR product of HIF-1α and pacAd5-HT2, the HIF-1α cDNA was inserted into at the 5′-terminal of the HT2 gene to create an adenoviral shuttle vector pacAd5-HIF-1α/HT2. The pacAd5-HIF-1α/HT2 was cotransfected with adenoviral genome vector into the 293 cells (27) to generate the recombinant adenovirus Ad-HIF-1α.

Adenoviral vector preparation, titration, and transduction

Individual clones of adenoviral vector were propagated in 293 cells. The culture medium of the 293 cells showing the complete cytopathic effect was collected, and adenoviral vectors were purified by BD Adeno-X Virus Purification Kits (BD Biosciences, Palo Alto, CA) according to the manufacturer’s instructions. The viral titration and transduction were performed by standard procedures as has been described previously (28).

Construction of p16 doxycycline

An EcoR I and Bam H I-digested 960-bp human p16 cDNA (25) was subcloned into Bam HI and Xho I sites of pLenti-Tet-pgk-puro (University of Tennessee Viral Vector Core Laboratory, Memphis, TN) to generate a doxycycline (Dox)-inducible lentiviral vector pLenti-Tet-p16-pgk-puro. In this vector, p16 gene was driven by tetracycline-regulated Tet-on promoter (Clontech, Mountain View, CA). In order to activate the p16 expression, we constructed another lentiviral vector, pcFUW-rtTA3-IERS-puro, in which the third-generation reverse Tet-transactivator (rtTA3) cDNA was placed under the control of a constitutively active ubiqutin C promoter. In brief, rtTA3 cDNA was amplified by PCR from pTRIPZ Tet-on vector (Openbiosystems, Huntsville, AL) and subcloned into XbaI and BamHI sites of pcFUW (a gift from Dr. Carlos, MIT) to generate pcFUW-rtTA3-IERS-puro. Both lentiviral vectors pLenti-Tet-p16-pgk-puro and pcFUW-rtTA3-IRES-puro, respectively, were produced in 293FT cells (Invitrogen, Carlsbad, CA) with cotransfection of ViraPower Packaging Mix (Invitrogen) that contains different expression plasmids including pLP/VSVG, pLP1/gag-pol, and pLP2-Rev to express all the essential elements for generating lentivirus. The supernatant was collected 60 hr post-transfection and centrifuged for 15 min at 3000 g at 4°C to remove cell debris, then passed through 0.45-μm filter and collected for ultra-centrifugation at 17,000 g for 3 hr at 4°C. The pellet was dissolved in phosphate buffer solution (PBS) and kept at −80°C.

Generation of stably transfected, Dox-inducible (Tet-on), p16-expressing breast cancer cell lines

MDA-MB-231 cells were cotransduced by both viruses pLenti-Tet-p16-pgk-puro and pcFUW-rtTA3-IRES-puro at multiplicity of infection (moi) of 10 for each virus, together with 6-μg/ml polybrene (Millipore, Bedford, MA). The MDA-MB-231 cells stably transfected with lentivirus were enriched by incubated and transfected cells in medium containing 1-μg/ml puromycin. The stably transfected cells are named as MDA-MB-231/Tet-on p16.

Immunohistochemistry

For in vitro immunohistochemistry, the culture cells were grown on SlideFlasks with detachable bottom slides (Nalge Nunc, Naperville, IL) that could be directly used for immunohistochemistry staining later. The samples (slides) were incubated with first antibody against human p16 (mouse anti-human p16 antibody, Santa Cruz Biotechnology, Inc., Santa Cruz, CA) for 16 hr at 4°C, then by a corresponding second antibody (goat anti-mouse antibody) and the Universal Elite ABC Kit (Vector Laboratories, Inc., Burlingame, CA) according to the manufacturer’s protocol. The reaction was visualized with DAB solution (75-mg 3,3′-Diaminobenzidine and 30-μl 50% H₂O₂ in 150-ml PBS) for 3–10 min.

Enzyme-linked immunosorbent assay (ELISA) for detecting VEGF

Cells were grown in 10-cm culture dishes and were either untreated or transduced with AdRSVlacZ or AdRSVp16 at moi of 200. After 90-min viral infection, the viral medium was replaced with exactly 10-ml fresh medium in each sample dish. The cell medium (supernatant) was collected 72 hr after viral transduction, and the cells attached on the culture dish were counted. The supernatant was processed to determine the secreted amount of VEGF₁₆₅ protein by a VEGF immunoassay kit (Quantikine VEGF ELISA Kit, R&D Systems, Minneapolis, MN). The procedures followed the methods according to the manufacturers’ manual, and results were normalized based on the same amount of analyzed cells.

Transactivation assay of VEGF promoter

A chimeric construct containing a 2.4-kb VEGF promoter and a luciferase reporter gene (pVEGF/Luc) (a generous gift from Dr. B.H. Jiang of West Virginia University; see (13)) was cotransfected with or without p16 expression vector, pAvsp16 (25), or HIF-1α expression vector, pHIF-1α (13), or pHIF-1α plus pAvsp16, into MDA-MB-231 cells by Fugene 6 (Roche, Indianapolis, IN) according to the manufacturer’s instruction. After 48 transfections, the cells were lysed using 1x Reporter Lysis Buffer (Promega) and processed to luciferase activity assay using a Luciferase Assay Kit (Promega). The detection of luciferase activity was performed using a luminometer (Turner Designs Instrument, Model TD2020, Sunnyvale, CA), and the activity of luciferase was normalized with protein concentration. In the instance of MDA-MB-231/Tet-on p16 cells, the cells were incubated in the medium containing 1-μg/ml Dox for 2 days prior to cotransfection with (10:1 ratio, w:w) pVEGF/Luc and phRLuc-TK (Promega), a plasmid expressing Renilla luciferase as an internal control for the normalization of transfection efficiency. After 48 hr of transfection, the cell extracts were harvested and analyzed for luciferase activity using Dual-Luciferase Assay Kit (Promega) as instructed by the manufacturer’s instruction. The normalized luciferase activity was represented as the ratio of Firefly luciferase activity (pVEGF/Luc) over Renilla luciferase activity (phRLuc-TK).

In vivo angiogenic assay using a dorsal air sac model

Cells were either untreated or transduced with control virus AdRSVlacZ or AdRSVp16 at moi of 200. After 48 hr the cells were harvested and suspended in PBS at a concentration of 1 × 10⁸ cells/ml. This suspension (0.1 ml in PBS) was injected into a chamber (Millipore) consisting of a ring with a filter (pore size, 0.22 μm) on both sides. The semi-permeable membrane chamber allowed for diffusion of growth factor, such as VEGF, but not cells. The chamber was implanted into a dorsal air sac produced by the injection of 10 ml of air in the dorsum of a 6-week-old nude female mouse (Harlan Sprague-Dawley, Indianapolis, IN). The mouse was sacrificed on day 3 and the implanted chamber was removed. A ring without filters was placed on the same site and then photographed. The newly formed blood vessels in the air sac fascia were morphologically distinguishable from the pre-existing background vessels by their zigzagging characters.

Co-immunoprecipitation assay to detect binding between p16 and HIF-1α proteins

MDA-231 cells were seeded onto a 10-cm cell culture dish at a density of 5 × 10⁵ cells/dish and incubated for 24 hr in complete media. The cells were either untreated or transduced with AdRSVp16 or AdRSVlacZ at moi of 200 for 24 hr, respectively. The medium was changed and the cells were then transfected with 15-μg HIF-1α (pCEP4 expression vector expressing human HIF-1α; see (13)), or empty vector pCEP4 (Invitrogen, San Diego, CA) by Fugen6 according to the manufacturer’s instruction. At 48 hr after transfection, the cells were harvested and lyzed at 4°C for 30 min with lysis buffer containing 1% Brij 97 (Sigma), 0.25-M Sucrose, 10-mM Tris-Cl pH 8.0, 0.05-mM CaCl₂, 0.02% azide, 0.1-mM phenylmethyl-sulfonyl fluoride, 0.2-mM sodium vanadate, and a protease and phosphatase inhibitor cocktail (Sigma). The lysates (500 μg in 100 μL) were precleared with 50 μL of a combination of protein A-Sepharose and protein G-Sepharose beads (protein A+G Sepharose beads) (Amersham Bioscience) on ice for 60 min. After removing the insoluble material by 10,000 rpm for 10 min at 4°C, the supernatant was used as the cleared lysate. At the same time, 50-μL protein A+G Sepharose beads were precleared with lysis buffer and incubated with 1-μg anti-HIF-1α antibody (Cat# NB100-105, Novus, Littleton, CO) for 1 hr at 4°C. These antibody–preabsorbed protein A+G Sepharose beads (50 μL) were incubated with the cleared lysate (500 μg in 100 μL) overnight at 4°C. The precipitates were washed thrice with lysis buffer, dissolved at 1:1 ratio (v:v) in 2x Laemnli sample buffer (10% sodium dodecylsulfate (SDS), 20% glycerol, 1M Tris-HCL pH 6.8, and 0.01% Bromphebol Blue), heated at 90°C for 10 min, electrophoresized on a NuPage 4–12% Bis-Tris Gel (Invitrogen), and electrically transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore). The membrane was immunoblotted with rabbit anti-human p16 antibody (Santa Cruz Biotech, Santa Cruz, CA) at 1:1000 dilution, and then followed by horseradish peroxide-conjugated anti-rabbit IgG at 1:2000 dilution, and processed to detection with chemiluminescent reagents (ECL kit, Amersham) according to the manufacturer’s protocol. The same immunoblot was stripped and reprobed with antibody against HIF-1α (see below).

Western blot analysis

Cells were extracted and processed for gel electrophoresis as described previously (29). Cell extract lysates (100 μg) were loaded on polyacrylamide gels and subjected to SDS gel electrophoresis, then transferred to a nitrocellulose membrane (Bio-Rad Laboratories, Hercules, CA). The membrane was treated with blocking solution (15% non-fat milk, and 0.02% sodium azide in phosphate-buffered saline) overnight at 4°C. The membrane was incubated for 1 hr at room temperature with the primary antibody, mouse anti-human HIF-1α antibody (1:1000 dilution, BD Biosciences). The membrane was then incubated for 1 hr at room temperature with goat anti-mouse secondary antibody coupled to peroxidase, followed by detection with chemiluminescent reagents (ECL kit, Amersham).

Immunofluorescent staining of p16 and HIF-1α and confocal microscopy

MDA-231 cells were seeded in four-chamber slide flask (BD Biosciences, Bedford, MA) at 5 × 10⁴ cells/per chamber. Cells were either untreated or transduced with AdRSVp16 plus Ad-HIF-1α (moi = 100 for each virus). After 72 hr of viral transduction, the cells were fixed with 3% paraformadehyde for 15 min at room temperature. The cells were then permeabilized with 0.1% brij 98 for 2 min at room temperature, followed by blocking with 20% goat serum in 1x PBS for 60 min. The cells were incubated with mouse anti-HIF-1α (1:1,000 dilution, BD Biosciences) and rabbit anti-p16 (1:100 dilution, Santa Cruz Biotechnology, Santa Cruz, CA) for 1 hr at room temperature, respectively. After washing, the cells were incubated with goat anti-mouse IgG FITC (1:100 dilution, Sigma), and goat anti-rabbit IgG’s Rhodamine-B conjugate (1:100 dilution, Biosource, Camarillo, CA) for 1 hr at room temperature, respectively. After washing with PBS, the slides were mounted using Fluor Saver Reagent (Calbiochem, La Jolla, CA) and observed under a fluorescent microscope (BioRad 1024 Confocal Microscope, software Lasersharp 2000).

Induction of ecotopic expression of p16 protein in breast cancer cells

Consistent with the other report (9), we observe an apparent p16 expression in normal human breast tissue and little, if any, p16 expression in breast cancer tissues; in contrast, normal breast tissue had sparse VEGF expression, whereas the cancerous breast tissue had an overwhelming high expression of VEGF (not shown). To facilitate the induction of p16 expression, a replication-defective recombinant adenovirus-expressing human wild-type p16 under the control of a RSV promoter, AdRSVp16, was used to transduce breast cancer cells. To demonstrate that AdRSVp16 is able to transfer and express p16 protein in breast cancer cells, human breast cancer MDA-MB-231 cells were transduced with AdRSVp16 at moi of 200. Three days later, the cells were processed for immunohistochemical staining for p16 protein using primary antibody against p16. As shown in Figure 1, cells transduced by AdRSVp16 expressed a positive staining (dark brown color) for p16 protein (Figure 1(b)), while control untreated cells did not have the p16 staining (Figure 1(a)). We also established a stably transfected breast cancer line expressing p16 under Dox induction, MDA-MB-231/Tet-on p16. The expression of p16 protein in this line can be turned on and off by incubating cells with (Figure 1(d)) or without Dox (Figure 1(c)), the inducer for Tet-on promoter that controls p16 expression.

p16 downregulates VEGF secretion

To determine whether p16 modulates VEGF gene expression at the protein level, MDA-MB-231 cells were either left untreated or transduced by control virus or AdRSVp16, and 72 hrs later the cell culture media were collected to analyze the secreted form of VEGF protein by ELISA assay. The ELISA results showed that AdRSVp16-transduced MDA-MB-231 cells had significantly less VEGF protein secreted into the media (about 66% reduction compared to the untreated control cells at the same amount of cells) (Figure 2(a)).

p16 appears to specifically downregulate HIF-1α-mediated VEGF expression at the transcriptional level

To examine whether p16 regulates VEGF gene expression at the transcriptional level, we transiently cotransfected a 2.4-kb VEGF promoter/luciferase reporter chimeric construct, pVEGF/Luc (13) with either control vector (pSV40βgal), or p16 expression vector pAvsp16 (25), into MDA-MB-231 cells. As shown in Figure 2(b), there was a moderate luciferase activity in the absence of p16 expression (lane 1, control group, Figure 2(b)), reflecting a native (basal) moderately active transactivation activity in MDA-MB-231 cells for VEGF transcription. In contrast, p16-expressing cells had a significantly reduced luciferase activity (67.2% inhibition as compared to the control group), implying that p16 downregulates VEGF expression at the transcriptional level (lane 2, Figure 2(b)). In addition, to confirm that HIF-1α stimulates VEGF gene transcription, we also cotransfected a VEGF/Luc construct with a HIF-1α expression vector (13). We found that ectopic HIF-1α expression alone stimulated VEGF gene transcription by more than threefold (lane 3, Figure 2(b)), and this HIF-1α-mediated transactivation of VEGF promoter can be neutralized by p16 when both ectopic HIF-1α and p16 expression were introduced (lane 4, Figure 2(b)). To exclude the possibility that downregulation of VEGF gene transcription by p16 is due to an indirect effect through a p16-mediated cell-cycle arrest, we also cotransfected a pVEGF/Luc into MDA-MB-231 cells with a vector-expressing p21Waf1, another CDK inhibitor gene that also causes cell-cycle arrest (30). p21Waf1 did not show any inhibitory effect on the VEGF gene transcription (data not shown), implying that this p16-mediated downregulation of VEGF expression is specific (possibly via regulation of HIF-1α) and independent of p16’s cell-cycle arrest effect. To avoid the possible artificial effect due to the overexpression system mediated by adenoviral transduction, we also performed the similar experiment in the stably transfected breast cancer line, MDA-MB-231/Tet-on p16. Likewise, in the MDA-MB-231/Tet-on p16 cell system, the p16-expressing cells (+Dox) had a significantly reduced luciferase activity compared to its p16-nonexpressing counterpart (−Dox) (Figure 2(c)). Together, these results indicate that p16 expression can downregulate VEGF gene expression at the transcriptional level.

p16 inhibits angiogenesis in vivo

To determine whether p16 inhibits tumor cell-induced angiogenesis, the effect of p16 on neovascularization of tumor-surrounding cells were examined by dorsal air sac assay (31). MDA-MB-231 cells were transduced with AdRSVp16. After 48 hr, the cells were harvested and injected into a chamber that was wrapped with a semi-permeable membrane allowing for the diffusion of growth factor, such as VEGF, but not cells. The chamber was implanted into a dorsal air sac in nude mice, and the newly formed blood vessels on the undersurface of the chamber were examined 3 days later. As shown in Figure 3, PBS-treated mice (as a negative control) did not have any obvious neovascularization (Figure 3(a)). However, the mice injected with MDA-MB-231 cells developed tumor cell-induced neovascularization as evidenced by the newly formed “zigzagging-shape” small vessels in the air sac fascia (exampled by arrow, Figure 3(b)). A similar high neovessel formation was observed in MDA-231 cells transduced with control virus (exampled by arrow, Figure 3(c)). In contrast, mice with AdRSVp16-transduced MDA-MB-231 cells developed much less newly formed blood vessels (Figure 3(d) and (f)), compared with mice injected with MDA-MB-231 cells alone (Figure 3(b)), or mice injected with control viral-transduced MDA-MB-231 cells (Figure 3(c) and (e)); both of the latter two groups induced a more extensive capillary network. When mice were injected with MDA-MB-231 cells along with neutralized anti-VEGF antibody, this tumor-induced angiogenesis nearly disappeared (Figure 3(g)). By using matrigel plug assay in another experiment to analyze the in vivo angiogenic ability of tumor cells in the mice, the angiogenic ability of MDA-MB-231 cells can be measured and quantitated as microvessel density. In comparison with the untreated control, the control virus-treated group had a 5% inhibition of angiogenesis and AdRSVp16-treated group had a 72% inhibition (not shown). Taken together, these results demonstrate that breast cancer cells can induce neovascularization around the tumor by secreting VEGF to the surrounding environment, and p16 can inhibit this tumor cell-induced neovascularization.

p16 appears to form a complex with HIF-1α in MDA-MB-231 cells

To explore how p16 regulates VEGF gene expression, we examined the possibility that p16 may regulate VEGF expression by modulating its transcriptional factor HIF-1α. To study whether p16 forms a complex with HIF-1α (as one potential mechanism to alter HIF-1α’s ability to either locate its normal cellular location or its transactivation ability to VEGF promoter), MDA-MB-231 cells were transduced with AdRSVp16 and transfected with HIF-1α expression vector (13) for 72 hr. The nuclear extracts were immunoprecipitated with either anti-HIF-1α (lane 1–3, Figure 4) or with preimmune IgG as a negative control (lane 4, Figure 4). The immunoprecipitates (lane 1–4) were then analyzed by Western blotting using anti-p16. As a positive control for the p16 band, cell extracts over-expressing p16 protein (we used AdRSVp16-transduced PC-3 cells, see (25)) were also used directly for Western blotting by anti-p16. Indeed, we saw a complex of HIF-1α and p16 (lane 3, Figure 4), demonstrating that p16 binds to HIF-1α in cells expressing both p16 and HIF-1α.

Colocalization

To further confirm the interaction between p16 and HIF-1α, we performed colocalization in MDA-MB-231 cells that were transduced by both AdRSVp16 and Ad-HIF-1α. For every cell transduced by both AdRSVp16 (red, Figure 5(a)) and Ad-HIF-1α (green, Figure 5(b)), there is a yellow color (the merged color of red and green, Figure 5(c)). Some cells may be transduced only by AdRSVp16 but not by Ad-HIF-1α (as demonstrated by an arrow, Figure 5(a)), correspondingly, no yellow color was shown in that particular cell. Thus, there is a clear colocalization for both protein p16 and HIF-1α in cells expressing both (yellow color, Figure 5(c) and (f)), strongly implying that p16 may interact with HIF-1α protein in the cells. Together with result from Figure 4, these results demonstrate that p16 binds HIF-1α inside the cells, which may provide one mechanism for how p16 modulates VEGF gene expression: via interaction of HIF-1α, the transcriptional activator for VEGF, may somehow affect HIF-1α’s ability to transactivation of its targeted gene VEGF.

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