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[Cancer Research 66, 5599-5607, June 1, 2006]
© 2006 American Association for Cancer Research

Molecular Biology, Pathobiology, and Genetics

SSeCKS Metastasis-Suppressing Activity in MatLyLu Prostate Cancer Cells Correlates with Vascular Endothelial Growth Factor Inhibition

Bing Su1, Qiao Zheng1, Mary M. Vaughan2, Yahao Bu1 and Irwin H. Gelman1

Departments of 1 Cancer Genetics, and 2 Pharmacology and Therapeutics, Roswell Park Cancer Institute, Buffalo, New York

Requests for reprints: Irwin H. Gelman, Department of Cancer Genetics, Roswell Park Cancer Institute, Buffalo, NY 14263. Phone: 716-845-7681; Fax: 716-845-2342; E-mail: Irwin.gelman{at}roswellpark.org.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
SSeCKS, a Src-suppressed protein kinase C substrate with metastasis suppressor activity, is the rodent orthologue of human gravin/AKAP12, a scaffolding protein for protein kinase A and protein kinase C. We show here that the tetracycline-regulated reexpression of SSeCKS in MatLyLu (MLL) prostate cancer cells suppressed formation of macroscopic lung metastases in both spontaneous and experimental models of in vivo metastasis while having minimal inhibitory effects on the growth of primary-site s.c. tumors. SSeCKS decreased angiogenesis in vitro and in vivo by suppressing vascular endothelial growth factor (VEGF) expression in MLL tumor cells as well as in stromal cells. The forced reexpression of VEGF165 and VEGF121 isoforms was sufficient to reverse aspects of SSeCKS metastasis-suppressor activity in both the experimental and spontaneous models. SSeCKS reexpression in MLL cells resulted in the down-regulation of proangiogenic genes, such as osteopontin, tenascin C, KGF, angiopoietin, HIF-1{alpha}, and PDGFRß, and the up-regulation of antiangiogenic genes, such as vasostatin and collagen 18a1, a precursor of endostatin. These results suggest that SSeCKS suppresses formation of metastatic lesions by inhibiting VEGF expression and by inducing soluble antiangiogenic factors. (Cancer Res 2006; 66(11): 5599-607)


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The Src-suppressed C-kinase substrate (SSeCKS), originally identified as a gene down-regulated in response to Src and Ras activation (1), is the rodent orthologue of human gravin, an autoantigen in some cases of myasthenia gravis (2). Besides binding protein kinase C (PKC; ref. 3), gravin and SSeCKS are also known as AKAP12 because of their ability to scaffold protein kinase A through a COOH-terminal RII subunit binding motif (4). gravin is a single-copy gene mapping to 6q24-25.2, a hotspot for deletion in advanced prostate, breast, and ovarian cancer (5, 6) and a region known to harbor tumor- or metastasis-suppressing functions (7–11). The expression of SSeCKS/gravin/AKAP12 is down-regulated by several oncogenes and strongly suppressed in various cancers, including prostate, ovary, and breast (6, 12–14).

SSeCKS reexpression suppresses Src-induced oncogenesis by reducing anchorage-independent growth in soft agar and Matrigel invasiveness and by restoring normal cell morphology and cytoskeletal architecture (12). The tetracycline-regulated reexpression of SSeCKS in the rat prostate cancer cell line MatLyLu (MLL) reduced anchorage-independent growth in cell culture, and in nude mice, SSeCKS reexpression severely decreased the formation of lung metastases without significantly affecting the growth of primary s.c. tumors (6). These findings led to the suggestion that SSeCKS functions as a potential metastasis suppressor, although its mechanism of action remains unclear (15).

Cancer cell metastasis involves a complex cascade of events including escape from the primary tumor site, intravasation into the circulatory system, trapping in the microcirculation, extravasation into peripheral tissue compartments, proliferation, and finally neovascularogenesis. Failure of any single step will lead to suppression of peripheral metastasis formation. Much attention has been given to the critical role played by tumor angiogenesis in promoting primary and metastatic tumor growth (16). Specifically, tumor neovascularization is required to provide nutrients and diffusible oxygen to rapidly growing tumor masses. Therefore, the therapeutic targeting of the migration or proliferation of endothelial cells that produce neovascularization is an attractive means to inhibit primary tumor and metastatic progression (17).

Based on a recent report showing that SSeCKS regulates the formation of the blood-brain barrier by inhibiting brain angiogenesis through the reduction of vascular endothelial growth factor (VEGF) expression (18), we hypothesized that SSeCKS might suppress metastasis through antiangiogenic mechanisms. Here, we show that SSeCKS decreases angiogenesis in vitro and in vivo by suppressing VEGF expression in MLL tumor cells as well as in stromal cells. The forced reexpression of VEGF isoforms is sufficient to reverse the metastasis-suppressor activity of SSeCKS in spontaneous and experimental models of metastasis in vivo. These data strongly suggest that SSeCKS suppresses neovasculature formation induced by tumor cells at peripheral sites, thereby inhibiting metastasis progression.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture. Rat MLL (19) prostate cancer cells were grown in DMEM supplemented with 10% bovine serum. MLL and NIH 3T3 cell lines expressing tetracycline-regulated (tet-OFF) SSeCKS (MLL/SSeCKS and S24, respectively) were previously described (6, 20). Primary human microvessel endothelial cells (passage 4) were purchased from Cambrex BioScience (East Rutherford, NJ) and grown in Clonetics EGM-2-MV medium (Cambrex). The rat smooth muscle cell line A7r5 (ATCC, CRL-1444) was grown in DMEM with 4 mmol/L L-glutamine adjusted to contain 1.5 g/L sodium bicarbonate and 4.5 g/L glucose, 10% fetal bovine serum (FBS). The procurement and growth of the human prostate cancer cell lines was previously described (6).

In vivo metastasis assay. For spontaneous metastasis, 5-week-old female nude mice (Taconic Farms, Germantown, NY) were injected s.c. into the left and right flank regions with 5 x 104 MLL/SSeCKS cells via a 27-gauge needle. The viability of the cells was >90% as determined by trypan blue exclusion. Mice were fed water containing 100 µg/mL tetracycline (tet) plus 5% sucrose until the primary tumors were palpable (2-4 mm), at which point the mice were divided into two groups (six mice per group). One group received regular water and another received water with tet as control. Tumor volume (cubic millimeters) was measured using a caliper, applying the formula [volume = 0.52 x (width)2 x (length)] for approximating the volume of a spheroid. Tumor burden per mouse was calculated by accumulating the tumor volume thrice weekly. Three weeks later, the animals were sacrificed and lungs were examined for visible metastatic foci after H&E staining.

For experimental metastasis, 104 cells were injected into tail veins (i.v.) of nude mice (six mice per group). After sacrifice, the removed lungs were stained with India ink and fixed in Fekete's solution (100 mL of 70% alcohol, 10 mL formalin, and 5 mL glacial acetic acid); metastases were scored as surface lesions excluding ink. All animal experiments were done under the supervision of the Institutional Animal Care and Use Committee of Roswell Park Cancer Institute.

Clonogenic assays were done by plating equal numbers of single-cell lung suspensions (produced by collagenase treatment) on media selective for the MLL/SSeCKS growth (puromycin), followed by counting colony number after roughly 10-day growth.

Tumor doubling time was calculated by producing a linear growth curve during the logarithmic growth phase (48-120 hours). Doubling time was calculated as log 2/slope and the significance of mean doubling times was calculated by t test. P < 0.05 was considered statistically significant.

Tissue staining. Primary tumor tissues were doubly stained for CD31 (endothelial cells) and apoptosis-related terminal deoxyribonucleotidyl transferase–mediated dUTP nick end labeling (TUNEL) activity. Apoptotic cells were detected using the ApopTag in situ detection kit (Chemicon International, Temecula, CA). The tissues were fixed with a zinc fixative (BD PharMingen 550523; BD PharMingen, San Jose, CA) according to the protocol of the manufacturer. Sections were cut at 5 µm, placed on charged slides, and dried at 60°C for 1 hour. Sections were then deparaffinized, rehydrated, and washed in PBS. Endogenous peroxidase activity was quenched with 3% H2O2 in PBS for 10 minutes. The 3'-hydroxy DNA strand breaks were enzymatically labeled with digoxygenin-dUTP with terminal deoxynucleotidyl transferase for 70 minutes at 37°C (negative control, PBS), and the reaction was terminated with a stop wash buffer. Sections were incubated with peroxidase-antidigoxygenin for 30 minutes at room temperature, washed, and 3,3'-diaminobenzidine substrate was used as the chromogen (brown product). Slides were rinsed in distilled water and then stained for CD31 on the DAKO autostainer as follows: slides were washed once in PBS/T (PBS + 0.05% Tween 20) followed by a 30-minute incubation with casein (0.03% in PBS/T). The slides were then incubated for 1 hour with 10 µg/mL anti-CD31 (BD PharMingen) or an isotype-matched control (10 µg/mL rat immunoglobulin G), washed with PBS/T, then incubated for 30 minutes with biotinylated antirat immunoglobulin. After washing with PBS/T, the slide was incubated for 30 minutes with alkaline phosphatase–conjugated streptavidin complex (DAKO, Carpinteria, CA), washed with PBS/T, then stained for 5 minutes with Fast Red (DAKO) resulting in a pink/magenta product. Slides were counterstained with Mayer's hematoxylin (Lillie's modification) and coverslipped with aqueous mountant. Lung tissues were stained with etoposide (VP16; Santa Cruz Biotechnology, Santa Cruz, CA; dilution, 1:40), antihuman VEGF (Santa Cruz Biotechnology; dilution, 1:800), or appropriate preimmune sera (immunoglobulin) as negative controls.

Tube formation assay. Conditioned medium was obtained by incubating MLL/vector or MLL/SSeCKS cells (70% confluence in 6 cm dishes) for 24 hours in medium containing 1% FBS followed by filtration through a 0.45-µm low protein-binding filter (Pall/Gelman, Ann Arbor, MI). Cells were further incubated for 24 hours and media were collected and filtered. Human microvessel endothelial cells (104) seeded in triplicate on a layer of previously polymerized Matrigel (12.2 mg/mL) in 96 wells were incubated overnight with different concentrations of conditioned media and then photographed under phase-contrast microscopy.

Transient transfection. Plasmid DNA (2 µg) of either pBABE/hygro/SSeCKS or empty vector was transiently transfected into A7r5 cells (106/6-cm dish) using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA). After 36 hours, the cells were harvested and lysed.

Stable reexpression of VEGF isoforms. A human VEGF165 cDNA (kindly provided by Prof. M. Shibuya, Institute of Medical Science, University of Tokyo, Tokyo, Japan) was excised and inserted as a HindIII/XbaI fragment into pcDNA3.1(+) (Invitrogen). A retrovirus expression vector encoding the human VEGF121 isoform (pG1EN-hVEGF121) was kindly provided by Dr. D. Fraker (Department of Surgery, University of Pennsylvania, Philadelphia, PA). Stable MLL/SSeCKS/VEGF expressor cell lines were produced by either transfecting with 2 µg of pcDNA/VEGF165 plasmid DNA in Lipofectamine 2000 (Invitrogen) or by infecting with pG1EN-hVEGF121 virus packaged in 293GPG cells (21) and then selecting in 1 mg/mL G418 (Mediatech, Inc., Herndon, VA). VEGF165– and VEGF121– expressing clones were verified by immunoblotting using antihuman VEGF monoclonal antibody (mAb; BD PharMingen, G153-694; working dilution, 1:300). MLL cells exclusively express the rodent VEGF121 isoform (22).

Western blot analysis. Proteins were immunoblotted as previously described using polyclonal anti-SSeCKS antibody (ref. 23; working dilution, 1:1,000), monoclonal anti-actin (Sigma clone AC40; working dilution, 1:1,000; Sigma, St. Louis, MO), and anti-VEGF (Santa Cruz Biotechnology; working dilution, 1:400) antibody.

Differential gene expression profiling. Total cellular RNA was isolated using TRIzol reagent (Life Technologies, Carlsbad, CA) from MLL/SSeCKS cells grown in the presence or absence of tet for 2 days, and then cDNA probes were synthesized with [32P]dCTP by reverse transcription-PCR (RT-PCR). Equal total counts of the tet+ or tet– probes were hybridized to GEArray Q Series Mouse Angiogenesis Gene Arrays membrane (SuperArray Bioscience Corp., Frederick, MD) overnight at 60°C with continuous agitation. The membranes were washed twice with wash solution 1 (2x SSC, 1% SDS) and twice with wash solution 2 (0.1x SSC, 0.5% SDS) at 60°C with agitation for 15 minutes each. The membranes were phosphorimaged (Storm-860, GE Healthcare, Piscataway, NJ) and the data analyzed with GEArray Analyzer software. All hybridization signals were normalized to those of the housekeeping genes, ß-actin, GADPH, cyclophilin A, and ribosomal protein L13a.

Semiquantitative RT-PCR. One microgram of total cellular RNA from MLL/SSeCKS cells grown in the presence or absence of tet for 2 days was converted to cDNA as previously described (1). One microliter of the resulting cDNA was used as template for each PCR reaction. PCR conditions were optimized for each primer pair (Table 1 ) using MJ Research PTC-200 DNA thermal cycler (Watertown, MA) to identify cycle numbers at the high end of mid-log phase amplification. ß-Actin primers (24 cycles) were used as an internal control for cDNA normalization. The PCR products were analyzed on 2% agarose gels and the products were digitally imaged and quantified on a Chemi-Genius2 Bio-Imager (Syngene, Frederick, MD) using GeneTools software.


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  		Table 1. Primers used for semiquantitative RT-PCR

 
VEGF ELISA. Human or rodent VEGF was quantified using species-specific ELISA kits (R&D Systems, Minneapolis, MN) according to the instructions of the manufacturer. Triplicate six-well plates were seeded with 105 cells per well containing 2 mL/well of DMEM plus 1% FBS, incubated overnight, and then 200 (human) or 50 (rodent) µL of conditioned medium per well were assayed for VEGF in a 96-well format using a Bio-Rad model 680 spectrophotometer at 450 nm.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Overexpression of SSeCKS inhibits metastasis in vivo. We previously developed an experimental metastasis model in which MLL rat prostate cancer cells with tetracycline-regulated (tet-OFF) SSeCKS reexpression (MLL/SSeCKS) were injected s.c. in nude mouse flanks, and then tumor and lung metastasis formations were monitored in groups of mice receiving drinking water containing 100 µg/mL tetracycline (tet) plus 5% sucrose (no SSeCKS reexpression) or regular water (SSeCKS reexpression; ref. 6). SSeCKS reexpression severely inhibited the generation of macroscopic lung metastases while having no significant effect on the growth of the primary lesions at the s.c. site. Furthermore, SSeCKS reexpression neither inhibited MLL cell migration in vitro nor extravasation to the lung as determined by clonogenic assay. Thus, our data strongly suggested that the tumor cells were metastasizing to the lung after SSeCKS reexpression but then failing to produce macroscopic lesions at peripheral sites. It should be stressed that in our MLL/SSeCKS system, SSeCKS is reexpressed to physiologic levels in the absence of tet (i.e., similar to levels in untransformed rat prostatic epithelial cells; ref. 6).

To determine whether SSeCKS could suppress experimental metastases, nude mice were injected i.v. into tail veins with 104 MLL/SSeCKS cells and then half received either tet- or regular water for 10 days. Figure 1A shows that all of the mice receiving tet-water developed multiple macroscopic lung metastases. In contrast, all the mice receiving regular water showed no macroscopic lung lesions. Mice injected i.v. with parental MLL cells and then fed with either tet- or regular water showed statistically similar numbers of lung lesions as the MLL/SSeCKS mice receiving tet-water (data not shown), indicating that the metastasis suppression was due to SSeCKS reexpression rather than effects of tet treatment.


Figure 1
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  		Figure 1. Reexpression of SSeCKS inhibits metastasis in vivo. Experimental metastasis model. A, macroscopic appearance of lung metastatic nodules 10 days after tail vein injection of 104 MLL/SSeCKS cells in mice fed tet-water (–SSeCKS; top) or regular drinking water (+SSeCKS; bottom). B, spontaneous metastasis model. Top, columns, average number of metastatic lung foci per mouse 21 days after s.c. injection of 105 MLL/SSeCKS cells (six mice per group); bars, SD. Bottom, average sizes (in micrometers) of lung lesions from the experiment in the top; data reflect the sum of two random tissue slices stained with H&E for each of four mice per group. P = 0.0007. C, representative H&E-stained lung sections 21 days after s.c. injection of 105 MLL/SSeCKS cells. Bar, 198 µm (10x); 50 µm (40x). D, single MLL/SSeCKS tumor cells (arrow) could be identified by VP16 staining in the lungs of mice receiving regular drinking water 21 days after s.c. injection. For comparison, a small lung lesion, typical of those arising after s.c. injection of MLL/SSeCKS cells in the absence of tet, is stained for VP16. The matched immunoglobulin (Ig) controls are shown at right. Bar, 100 µm.

 
We then quantified the effect of SSeCKS reexpression on the number and size of lung metastases produced in a spontaneous metastasis model. Three weeks after s.c. injection of 5 x 104 MLL/SSeCKS cells, lungs from sacrificed nude mice were analyzed for microscopic lesions (microscopic analysis of H&E-stained tissue slices). Figure 1B (top) shows that SSeCKS reexpression inhibited formation of microscopic lung lesions by at least 4-fold. Whereas most microscopic lungs lesions of the tet-water mice were >100 µm with roughly a third of the lesions >200 µm, the majority of the lesions from the SSeCKS reexpressors were <30 µm (Fig. 1B, bottom). Moreover, the SSeCKS-reexpressor lungs contained roughly one fifth the number of microscopic lesions when compared with the lungs of the tet-water mice. Figure 1C shows similar results from lungs from the experimental metastasis model described in Fig. 1A: whereas the tet-water mice (–SSeCKS) contained large lung lesions, the SSeCKS-reexpressor mice contained significantly smaller and fewer lesions. The 4- to 5-fold decrease in microscopic lesions in SSeCKS reexpressors, especially after i.v. tumor cell injection, suggests that either many of these cells fail to survive in the lung (e.g., apoptosis) or remain as single, dormant cells. Indeed, many single, SSeCKS-reexpressing tumor cells were as identified by staining for the VP16 component of the tet transactivator, tTA (Fig. 1D), suggesting that many survive in dormancy. We also showed that SSeCKS reexpression had no effect on the colonization of MLL cells to the lung based on clonogenic assays done 3 days after tail vein injection of MLL/SSeCKS cells in mice receiving normal or tet-water (data not shown). Although SSeCKS reexpression did not increase the apoptotic index of primary s.c. tumors (Fig. 2C ), we cannot rule out the possibility that SSeCKS increases the apoptotic index of tumor cells at peripheral sites. Taken together, these data strongly suggest that SSeCKS suppresses the growth of lesions once tumor cells metastasize to peripheral sites.


Figure 2
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  		Figure 2. SSeCKS decreases tumor angiogenesis. A, left, endothelial cell tube formation (24 hours) by human microvessel endothelial cells plated onto Matrigel in the presence of 30% conditioned media from MLL/SSeCKS cells grown in the presence or absence of tet. Right, mean number of tubes per visual field (five fields per group counted) from three independent experiments. Bars, SE. *, P < 0.001. B, double CD31 (magenta) and TUNEL (brown; arrows) staining of primary s.c. tumors (21 days postimplantation) of mice receiving tet-water (+tet) or regular drinking water (–tet). C, average values of microvessel density (top left), vessel length in micrometers (bottom left), apoptotic index (top right), and vessel diameter in micrometers (bottom right) were derived from the experiment in (B). Columns, mean of six random visual fields of three lungs per group; bars, SE. *, P < 0.001. Bar, 100 µm. D, average tumor volumes of primary s.c. tumors of mice receiving tet-water or regular drinking water. E, expression of SSeCKS protein was examined by immunoblotting of primary s.c. tumors (21 days postimplantation) recovered from mice receiving tet-water or regular drinking water. Actin immunoblotting was used as a loading control.

 
SSeCKS decreases tumor angiogenesis. Given the role of SSeCKS in controlling VEGF-dependent brain angiogenesis and in the formation of the blood-brain barrier (18), and given that tumor dormancy has been explained by a lack of vascularization at micrometastasis sites (24), we addressed whether SSeCKS metastasis-suppressor activity was due to antiangiogenesis. Figure 2A shows that conditioned media from MLL/SSeCKS grown in the absence of tet decreased the ability of human microvascular endothelial cells plated onto Matrigel-coated wells to form capillary-like tube structures in comparison with conditioned media from MLL/SSeCKS grown in tet ("MLL/Control") or parental MLL cells grown in either the presence or absence of tet (not shown).

SSeCKS also exhibited antiangiogenic activity in vivo. Twenty-one days after s.c. inoculation of 5 x 104 MLL/SSeCKS cells, tumors were removed, fixed, and then stained for endothelial cells (CD31) or for apoptosis (TUNEL; Fig. 2B). Although SSeCKS reexpression did not affect the apoptotic index in the primary tumors, it did decrease microvessel density as well as average vessel length and diameter (Fig. 2C). Although SSeCKS decreased the vascularization of primary-site tumors, it had only marginal effects on primary tumor growth (Fig. 2D) as we previously showed (6). As a control, Fig. 2E shows sustained and consistent SSeCKS reexpression among primary tumors of mice receiving regular water ("+SSeCKS") compared with those receiving tet-water ("–SSeCKS"). Collectively, these data indicate that SSeCKS inhibits vascular differentiation in vitro and tumor vascularization in vivo.

SSeCKS inhibits expression of VEGF. VEGF is a key effector of angiogenesis: formation of new blood vessels requires VEGF to induce the recruitment, survival, and proliferation of endothelial cells to sites of neovascularization (25). Even the 50% loss of VEGF in engineered mouse heterozygotes results in early embryonic lethality at E11/E12 due to impaired angiogenesis and the formation of blood pools (26). There is now a large body of data showing that the ability of many tumor cells to up-regulate VEGF expression is critical to the formation of peripheral-site metastases (27). Moreover, VEGF-mediated recruitment of endothelial cells to metastatic sites requires activated Src kinase (28), an oncogenic signal mediator known to severely inhibit SSeCKS expression (1). Therefore, we analyzed how changes in SSeCKS expression affected levels of intracellular VEGF mRNA and protein as well as secreted VEGF. VEGF transcript levels were decreased roughly 3-fold following the induction of SSeCKS expression in MLL/SSeCKS cells grown in the absence of tet (Fig. 3A ). Growth of MLL/SSeCKS in increasing concentrations of tet (0-0.7 µg/mL) resulted in a concentration-dependent loss of ectopic SSeCKS concomitant with a 2.5-fold increase in intracellular VEGF (Fig. 3B, top and middle) and a >4-fold increase in secreted VEGF (Fig. 3B, bottom). Moreover, the change in intracellular VEGF levels was almost double when cells were grown in 0.5% serum (data not shown). The effect of SSeCKS on VEGF secretion is probably more relevant to the rate of tumor neovascularization than the intracellular VEGF concentration because angiogenesis requires the binding of secreted VEGF to endothelial cell receptors (29). These data indicate that SSeCKS suppresses VEGF expression at both mRNA and protein levels in MLL cells.


Figure 3
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  		Figure 3. Suppression of VEGF expression by SSeCKS. A, VEGF mRNA expression was determined by semiquantitative RT-PCR of RNA from MLL/SSeCKS grown in the presence or absence of tet. Relative transcript levels were determined using GeneTools software (Syngene) on images captured on a Chemi-Genius2 (Syngene). B, top, MLL/SSeCKS cells were exposed to the different concentrations of tet for 24 hours and then cell lysates were probed by immunoblotting for SSeCKS, VEGF, and actin. Middle, the relative level of intracellular VEGF in the experiment in the top. Typical results of three independent experiments. Bottom, secreted VEGF (assayed by quantitative ELISA to rodent VEGF) from the media of the MLL/SSeCKS cells grown in the top, normalized to cell number as described in Materials and Methods. C, rat smooth muscle cells (A7r5) transiently transfected with vector alone or an SSeCKS expression plasmid, or NIH 3T3 fibroblasts with tet-regulated SSeCKS (S24) grown in the presence or absence of tet, were probed for VEGF or actin by immunoblotting. Typical results of three independent experiments. D, MEF lysates stably expressing an SSeCKS shRNA (or pSHAG vector alone as a control) were probed for SSeCKS, VEGF, or actin by immunoblotting.

 
Tumor angiogenesis is thought to involve a complex interplay between multiple secreted mediators such as VEGF and platelet-derived growth factor (PDGF)-BB as well as their respective receptors, ultimately leading to a tumor-induced recruitment of stromal and endothelial cells to sites of neovascularization (30, 31). For example, the complete suppression of human rhabdomyosarcoma growth and angiogenesis in a xenograft model was dependent on the inhibition of VEGF expression by the tumor and the host (32). Nonetheless, stromally derived VEGF from host fibroblasts and smooth muscle cells could play a significant role in tumor angiogenesis involving Ras-transformed VEGF-deficient adult dermal fibroblasts (33). Thus, as most models indicate that stroma-derived VEGF plays a significant role in the signaling crosstalk in tumor angiogenesis (31), we addressed whether SSeCKS could control VEGF expression in smooth muscle and fibroblast cell lines. Indeed, the transient overexpression of SSeCKS in A7r5 rat smooth muscle cells and in NIH 3T3 fibroblasts decreased intracellular VEGF protein levels 3- to 4-fold (Fig. 3C, top and bottom). Moreover, the short hairpin RNA (shRNA)–mediated suppression of SSeCKS levels in NIH 3T3 fibroblasts resulted in a 2.5-fold increase in intracellular VEGF protein levels (Fig. 3D). These data clearly show a reciprocal relationship between SSeCKS and VEGF levels, strongly suggesting that SSeCKS controls angiogenesis at metastatic sites through the attenuation of VEGF expression.

The reexpression of VEGF rescues metastatic growth suppressed by SSeCKS. To address whether VEGF attenuation was sufficient for SSeCKS-mediated metastasis suppression, the MLL/SSeCKS cells were transduced with cDNAs expressing either the human VEGF165 or VEGF121 isoform, or empty vector, as a negative control. Both VEGF isoforms can stimulate angiogenesis in in vitro and in vivo systems (25). Indeed, the increased tumorigenicity induced by the ectopic overexpression of VEGF165 (34) and VEGF121 (35, 36) correlated with increased tumor angiogenesis. Figure 4A (top) shows that VEGF165 induced 3.5-fold higher numbers of lung metastases compared with the MLL/SSeCKS/vector cells 13 days after tail vein injection in mice receiving only regular water. Interestingly, the VEGF165 lung metastases were typical of the small (<20 µm) lesions induced by the parental MLL/SSeCKS cells (Fig. 4A, middle). However, VEGF121 expression did not increase the total number of experimental lung metastases formed but did induce an increase in the relative size of the lesions (roughly 75% >50 µm; Fig. 4A, middle). In sharp contrast, both VEGF165 and VEGF121 reexpression induced significant increases in the number of macroscopic lung metastases after s.c. injection (Fig. 4A, bottom). It should be noted that the difference in metastasis formation by the VEGF reexpressors is likely not significant because although their respective primary tumors grew at the same rate (Fig. 4C), the onset of tumor take in the VEGF165 mice was slightly earlier than that of the VEGF121 mice. Thus, several of the VEGF165 mice had to be sacrificed at day 16 rather than day 19 after tumor injection.


Figure 4
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  		Figure 4. The forced reexpression of VEGF rescued metastatic growth suppressed by SSeCKS. A, left, average numbers of lung metastasis in individual mice carrying tumors of control and SSeCKS cells at 13 days after i.v. injection. Middle, the different sizes of metastatic foci in the lung. Bottom, average numbers of metastatic foci in the lung of individual mice carrying tumors of control and SSeCKS cells after s.c. injection. Bars, SD. *, P < 0.01. B, immunohistochemical analysis of vascularization of primary tumors stained with anti-CD31. Blood vessel density and diameter were quantified by morphometric analysis. Bars, SE. *, P < 0.01. C, relative change in the primary tumor volumes of MLL/SSeCKS and the VEGF isoform-rescued lines. D, lung metastases stained with antihuman VEGF mAb from mice injected s.c. with VEGF165- or VEGF121-reexpressing MLL/SSeCKS cells that received regular drinking water (MLL/SSeCKS/VEGF165, 16 days postinjection; MLL/SSeCKS/VEGF121, 19 days postinjection). Insets, top, H&E stain; bottom, isotype control mAb (Ig). Bar, 100 µm. E, expression of exogenous human VEGF isoforms (hVEGF) examined by immunoblotting from cell lines (top) or primary tumors (bottom) using anti-hVEGF mAb. Recombinant human VEGF165 (Clontech) is shown as a control.

 
The primary tumors formed from the VEGF165 and VEGF121 cell lines were compared with the parental MLL/SSeCKS cells for the ability to induce tumor angiogenesis as defined by CD31 staining. Figure 4B indicates that both VEGF isoforms significantly increased the microvessel density when compared with the parental MLL/SSeCKS tumors in mice receiving regular drinking water (–tet). Although both VEGF isoforms also increased the average vessel length (Fig. 4B, bottom right), the vessels induced by VEGF165 were uniformly longer than those of the parental MLL/SSeCKS tumors in mice receiving tet-water. However, VEGF121 induced wider vessels similar to those of the parental MLL/SSeCKS tumors in mice receiving tet-water, consistent with previous reports (37) and our data (Fig. 4A) that MLL cells predominantly express VEGF121. The wide vessel formation exhibited by the MLL/SSeCKS/VEGF121-induced tumors is consistent with recent associations of VEGF121 with the wide vasculature that typifies tumor angiogenesis rather than the ordered vasculature associated with VEGF165 (38). As a control, we confirmed that the lesions induced by either VEGF165 or VEGF121 MLL/SSeCKS cells retained VEGF expression in the lung metastases (Fig. 4D) and in the primary-site s.c. tumors (Fig. 4E, bottom) using a human VEGF-specific mAb. Collectively, these data indicate that VEGF reexpression is sufficient to reverse the ability of SSeCKS to suppress metastasis formation and tumor angiogenesis.

Expression profile of angiogenesis-control genes regulated by SSeCKS reexpression. In addition to VEGF, tumor angiogenesis can be promoted by many host factors capable of regulating the recruitment of endothelial and stromal cells to tumor sites (39). To query which angiogenesis-regulating genes are modulated by reexpression of SSeCKS in MLL cells, a focused GEArray Q Series Mouse Angiogenesis Gene Array was hybridized with equal amounts of 32P-labeled cDNA from MLL/SSeCKS cells grown in the presence or absence of tet. SSeCKS-induced changes in signal hybridization were determined after normalization to four housekeeping genes: ß-actin, GADPH, cyclophilin A, and ribosomal protein L13a. The blots were scanned (Fig. 5A ) and the signals quantified digitally, and then the blots were reprobed with newly prepared 32P-labeled cDNA. Figure 5C shows that SSeCKS reexpression results in the down-regulation of several proangiogenic genes (osteopontin, tenascin C, KFG/FGF7, and Adamts1) as well as the up-regulation of vasostatin and collagen type 18a1 (a precursor for endostatin). In addition to confirming many of these expression changes by semiquantitative RT-PCR (Fig. 5B), several other genes known to be up-regulated in tumor angiogenesis [hypoxia-inducible factor 1{alpha} (HIF-1{alpha}), PDGFRß, and angiopoietin] were shown by RT-PCR to be down-regulated 3.1- to 5.7-fold by SSeCKS reexpression. These data are consistent with the concept that SSeCKS functions as a master controller of angiogenesis-regulating genes.


Figure 5
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  		Figure 5. Expression profile of angiogenesis-related genes associated with SSeCKS reexpression in MLL cells. A, total cellular RNA was isolated from MLL/SSeCKS cells grown for 48 hours in the presence or absence of tet, converted into 32P-labeled cDNA probes, and the probes used to hybridize to GEArray Q Series Mouse Angiogenesis Gene Arrays (SuperArray Bioscience). The digitized image is typical of two independent hybridizations with independently derived cDNA probes. B, validation of cDNA expression results by semiquantitative RT-PCR using primers sets described in Table 1. C, quantitative (fold) changes in angiogenesis-related genes. Positive values describe genes of which the expression is induced by SSeCKS reexpression whereas the negative values describe genes down-regulated by SSeCKS. The SuperArray data represent two independent hybridizations whereas the RT-PCR data are the average of three independent reactions.

 
Inverse correlation between SSeCKS/gravin/AKAP12 and VEGF expression in human prostate cancer cell lines. Given the ability of SSeCKS to suppress VEGF expression and our previous data indicating that SSeCKS/gravin/AKAP12 is down-regulated in rat and human prostate cancer cell lines and tumor tissues (6), we addressed whether there was an inverse relationship between levels of SSeCKS/gravin/AKAP12 and levels of intracellular or secreted VEGF in a panel of human prostate cancer lines with varying tumorigenic and metastatic potentials. Figure 6 shows that the prostate cancer cell lines with the lowest levels of SSeCKS/gravin/AKAP12—DU145, LNCaP, and C4-2—have the highest levels of secreted VEGF compared with the SV40-immortalized prostate epithelial line, P69SV40T. PC-3, which showed a 5-fold decrease in the relative SSeCKS levels and had much higher levels of secreted VEGF than untransformed control cells yet less than half the levels of the other prostate cancer cells, as was previously reported (40). These data indicate a loose inverse correlation between SSeCKS/gravin/AKAP12 levels and the ability of prostate cancer cells to secrete VEGF.


Figure 6
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  		Figure 6. Inverse relationship between SSeCKS/Gravin/AKAP12 protein levels and VEGF secretion levels in human prostate cancer cells. A, expression of human SSeCKS protein (Gravin/AKAP12) in prostate cancer cells by Western blot analysis. B, relative SSeCKS protein level from (A) normalized to actin protein levels. Columns, mean of three independent experiments; bars, SE. C, level of secreted human VEGF (normalized to cell number) was measured by ELISA in conditioned media as described in Materials and Methods. Columns, mean of triplicate measurements; bars, SE.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The current study employs a MLL prostate cancer model to show that SSeCKS can suppress the formation of distal metastases through the down-regulation of VEGF and possibly other proangiogenic factors. Unlike tumor suppressors that inhibit migration, such as RASSF1A (41), LGI1 (42), and zyxin (43), SSeCKS reexpression did not prevent migration of metastatic cells from primary s.c. tumors to the lung. SSeCKS reexpressor tumor cells injected i.v. formed greater numbers of micrometastases that were at least 3- to 4-fold smaller than the lesions formed by the parental MLL cells, underlining that the SSeCKS-induced block to metastasis relates to a suppression of growth at distal sites. Although SSeCKS reexpression resulted in 3- to 5-fold fewer macroscopic and microscopic lung lesions than the parental MLL cells in both the experimental and spontaneous metastasis models, there was a significant increase in the number of single, presumably dormant, tumor cells found in lung sections after SSeCKS reexpression. This suggests that SSeCKS may suppress metastatic growth at two levels (i.e., by inhibiting proliferation of individual metastatic cells at distal sites and by preventing progression of distal lesions).

Our findings typify the many examples in the literature in which histologic examination of experimental models that yield no macroscopic metastases nonetheless uncovers multiple micrometastases. Indeed, Zetter (44) reviews a number of studies in which typically avascular lesions survive for long periods without significant expansion. Although these lesions are functionally "dormant," they continue to express proliferation markers at levels found in rapidly expanding tumors.

Our data strongly suggest that SSeCKS inhibits metastasis formation by suppressing expression of VEGF, isoforms of which are critical to the formation of tumor-associated neovasculature (reviewed in refs. 45, 46). Indeed, VEGF can induce metastasis formation as a paracrine factor that facilitates endothelial and stromal cell activation and recruitment; as an autocrine factor that stimulates hematopoietic cell differentiation and survival; as an inducer of vascular permeability; and as a recruiter of bone marrow-derived premetastatic niches (47, 48). We show that SSeCKS can inhibit VEGF transcript, intracellular and secreted protein levels in the MLL/SSeCKS cells and intracellular VEGF protein levels in A7r5 and NIH 3T3 cells, used as examples of rodent smooth muscle cells and fibroblasts, respectively. Moreover, the forced decrease of SSeCKS in 3T3 cells using a specific shRNA results in increased VEGF protein levels, strengthening the concept of a reciprocal relationship between SSeCKS and VEGF. We are currently investigating whether SSeCKS-mediated inhibition of c-jun NH2-terminal kinase activity and activator protein expression is sufficient to suppression VEGF expression, as was shown previously in astrocytes (18).

The SSeCKS-mediated decrease in VEGF correlated with decreased vascularization and decreased vascular maturation in primary-site tumors. This correlated with only a small delay in tumor onset at the primary site but no diminution in the rate of primary tumor growth. In contrast, the loss of VEGF-mediated angiogenic activity resulted in a severe decrease in metastatic potential, as determined by the formation of macroscopic and microscopic lung lesions. Thus, in our model, the primary tumors are less dependent on angiogenic signals than metastases, a finding consistent with other animal metastasis models (49). One explanation for this difference might be that angiogenesis at the primary tumor site is abetted by the initial inflammatory response resulting from the physical injection of tumor cells whereas single metastatic cells in the lung are required to induce a de novo angiogenic cascade.

The supposition that SSeCKS-mediated down-regulation of VEGF is responsible, at least in part, for the suppression of metastasis is strengthened by our finding that VEGF reexpression can rescue the metastatic potential of MLL cells already reexpressing SSeCKS. Indeed, the forced expression of VEGF has been shown in other tumor systems to induce metastatic potential (50–54). Interestingly, there is a difference in the ability of two major VEGF isoforms, VEGF165 and VEGF121, to qualitatively and quantitatively affect metastasis formation by MLL/SSeCKS cells: whereas VEGF165 rescues the frequency of lung metastasis formation in both spontaneous and experimental models, VEGF121 induces high numbers of lung metastases in the spontaneous model yet only induces larger lesions in the experimental model. One possible explanation is that VEGF121 induces shorter yet more dilated vessels whereas VEGF165 induces longer, narrower vessels. The effect of VEGF121 has been reported to correlate with the increased intratumoral pressure that results from the dysregulated vasculature found in many tumors (55).

The deficiency of SSeCKS-reexpressing MLL cells to grow as distal metastases, even after i.v. injection, is reminiscent of recent studies indicating that Src activity is required for VEGF-mediated angiogenesis at metastatic sites (49, 56). Given that SSeCKS expression is severely down-regulated by activated Src (1), that SSeCKS reexpression suppresses v-Src-induced oncogenic transformation without inhibiting the ability of Src to phosphorylate cellular substrates (12), and that activated Src is required for endothelial cells to be recruited to sites of tumor angiogenesis by VEGF (49), our current data suggest that SSeCKS plays a direct role in attenuating metastatic progression via control of a VEGF/Src angiogenesis axis. The data further imply that metastatic progression through activated Src pathways requires the down-regulation of SSeCKS.

Interestingly, major tumor suppressor factors, such as p53, von Hippel-Lindau, and phosphatase and tensin homologue, inhibit expression of HIF-1{alpha} (57, 58), a transcription factor required for VEGF expression, yet none of these has been specifically associated with metastasis suppression. This may be due to the ability of SSeCKS to attenuate multiple proangiogenic factors and to induce antiangiogenic factors as shown by our microarray results.


   Acknowledgments
 
Grant support: NIH grant CA94108 and Department of Defense grant PC040256 (I.H. Gelman).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank M. Shibuya and D. Fraker for the VEGF plasmids, Y.Z. Liu for help with constructing SSeCKS shRNA, and Jun Yang (Department of Health Behavior, Cancer Prevention and Population Science, Roswell Park Cancer Institute, Buffalo, NY) for help with statistical analyses.

Received 11/16/05. Revised 4/ 7/06. Accepted 4/ 7/06.


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