Peroxisome proliferator-activated receptor γ (PPARγ) is expressed in a variety of cancer cells. The addition of ligand activates the receptor by inducing a conformational change in the receptor, which can be recapitulated by mutation. To investigate the role of activated PPARγ signaling in breast cancer, we compared the function of a constitutively active PPARγ (PγCA) mutant with the wild-type PPARγ in ErbB2-induced mammary tumorigenesis in vivo. Tumor cells transduced with either PPARγ or PγCA were implanted into immunocompetent FVB mice. Enhanced tumor growth was observed in PγCA-transduced cells, which was associated with increased angiogenesis and endothelial stem cells as evidenced by increased number of cells stained with von Willebrand factor, c-Kit, CD133, and CD31. Genome-wide expression profiling identified a group of genes within the angiogenesis pathway, including Angptl4, as targets of activated PPARγ; PγCA also induced Angptl4 protein secretion in ErbB2-transformed mammary epithelial cells. Angptl4 promoted vascular endothelial cell migration; conversely, immunodepletion of Angptl4 reduced PγCA-mediated cellular migration. Collectively, these studies suggest that activated PPARγ induces Angptl4 to promote tumor growth through enhanced angiogenesis in vivo. [Cancer Res 2009;69(24):9236–44]

The peroxisome proliferator-activated receptors (PPAR) are ligand-activated nuclear receptors that include PPARα, PPARγ, and PPARδ (1). PPARγ, originally cloned as a transcription factor involved in fat cell differentiation (2), is activated by endogenous ligands, such as fatty acids produced by sterol response element binding protein 1 and fatty acid synthase pathway (3) and synthetic ligands including the thiazolidinediones. Viral transduction of the murine liver with PPARγ induces hepatic steatosis (4). This differentiation function of PPARγ is antagonized in cultured cells and in vivo by endogenous levels of cyclin D1 (5). PPARγ regulates expression of secreted proteins, such as ACRP30 (6), which in turn may affect heterotypic signaling from nearby cells.

It is estimated that at least 2 million patients take antidiabetic drugs that function as PPARγ ligands. Understanding the role of this receptor, including its possible role in cancer, is therefore important. Independent lines of evidence support a role for PPARγ to either inhibit or promote tumorigenesis. In studies of N-nitrosomethylurea–induced mammary tumorigenesis in rats, PPARγ ligands prevented tumor initiation (7) and 7,12-dimethylbenz(a)anthracene–induced mammary tumorigenesis was inhibited by a synthetic ligand, troglitazone (8). PPARγ is expressed in breast, prostate, and colonic epithelia and addition of ligands to cultured cancer cell lines derived from these tissues inhibits cellular proliferation (913). Heterozygous mutations of PPARγ have been detected in >80% of patients with colon cancer (14) and chromosomal translocation between PAX8 and PPARγ in follicular thyroid cancer enhanced cellular proliferation (15).

In contrast with these findings, several studies suggest that PPARγ activation may enhance tumor growth. PPARγ ligands increased polyp numbers in the ApcMin model of familial adenomatosis (16, 17) and PPARγ inhibits β-catenin abundance only in the presence of wild-type APC (17), raising the possibility that the genetic environment of the host may determine the outcome of PPARγ activation. PPARγ ligands may function independently of the PPARγ receptor with growth inhibition shown in cells lacking PPARγ (1820). To determine the role of the PPARγ and avoid the potential spurious effect of receptor-independent functions, a constitutively active PPARγ (PγCA) was generated, which selectively activated a PPARγ response element without regulating other nuclear receptors. When targeted to mammary epithelium, PγCA collaborated in mammary oncogenesis with polyoma middle T antigen (21).

Human breast cancer is associated with ErbB2 amplification or overexpression in >30% of patients. Although PPARγ is reduced in ErbB2-induced tumor, the role of PPARγ in growth control of ErbB2-induced tumorigenesis is poorly understood. Herein, we show that PPARγ enhanced ErbB2-induced mammary tumorigenesis in vivo. PγCA promoted tumor angiogenesis through induction of the proangiogenic factor Angptl4. Genome-wide expression profiling identified Angptl4 and Wnt5a as targets of PPARγ. Angptl4 promoted migration of vascular endothelial cells and immunodepletion of Angptl4 diminished the effect of PγCA in promoting cellular migration. Thus, activated PPARγ enhances angiogenesis and tumor growth in vivo. As PPARγ-activating ligands are widely used to treat diabetes, a careful further analysis of breast cancer risk may be warranted.

Mice

All animal experiments were done following animal protocols that had been approved by the Institutional Animal Care and Use Committee at Thomas Jefferson University.

Cell culture

Human embryonic kidney 293T was maintained in DMEM containing penicillin and streptomycin (100 mg of each per 1 liter) and supplemented with 10% fetal bovine serum (5). MCF10A and MCF10A transformed with oncogene NeuT (MCF10A/NeuT) were cultured in DMEM/F-12 (50:50) supplemented with 5% horse serum, 5% fetal bovine serum, insulin (10 μg/mL), epidermal growth factor (20 ng/mL), hydrocortisone (0.5 μg/mL), and cholera toxin (100 ng/mL). Human umbilical vein endothelial cells (HUVEC) were purchased from Lifeline Cell Technology and maintained in VascuLife Basal Medium Supplemented with SMC LifeFactors. All cells were cultured in humidified atmosphere with 5% CO2 at 37°C.

Plasmids

pCMX-mPPARγ1 was a gift from Dr. C. Glass. The cDNA of mouse PPARγ was amplified by PCR and subcloned into p3xFLAG CMV10 vector (Sigma) and pACT vector (Promega) to express 3xFLAG-tagged (FLAG-PPARγ) and VP16 fusion protein (PγCA), respectively. The coding region of 3xFLAG-PPARγ1 cDNA or PγCA cDNAs (5) was inserted into the MSCV-IRES-GFP vector at the EcoRI site (blunted) upstream of the IRES driving expression of green fluorescent protein (GFP).

S.c. tumor implantation

The NAFA cells derived from oncogenic MMTV-ErbB2 transgenic mice stably expressing PPARγ or PγCA were implanted by s.c. injection in the flank of female 6- to 8-week-old FVB mice. Comparisons were made for 10 to 15 animals in each group between NAFA/GFP, NAFA/PPARγ, and NAFA/PγCA. The tumor growth rates were examined using serial caliper measurements. The tumor volume was calculated using the equation: (a2 × b) / 2, where a and b are the length and width of the tumor, respectively. Tumors were weighed at the time of sacrifice. At the completion of the experiments, tumors were excised and weighed and statistical significance of differences in tumor volume was log-transformed and analyzed using a linear mixed model. Separate slope and intercepts were computed for each group (GFP, PPARγ, and PγCA) and then compared across groups using a global test followed by pair-wise comparisons via linear contrasts. Data before day 9 were ignored due to zeroes at day 0 (inability to take logarithms) and an initial nonlinearity/change in some of the animals' growth patterns before day 9. Thus, the intercept at day 9 is interpretable as initiation of growth and the slope is interpretable as rate of growth. For the models, we report coefficients, confidence intervals (CI) of coefficients, and two-sided P values. Analyses were completed in SAS version 9.1.3.

Western blot

Conditioned cell culture medium was concentrated with Amicon Ultra 10K device (Millipore) and separated by 10% SDS-PAGE and the proteins were transferred to nitrocellulose membrane for Western blot as described previously (22). The following antibodies were used for Western blot: anti-Angptl4 (Invitrogen) and mouse M2 anti-FLAG antibody (Sigma).

Retrovirus preparation and infection

The MSCV-IRES-GFP retrovirus vector and the vector pSV-ψ-E-MLV that provides ecotropic packaging helper function and infection methods were described previously (23). Retroviruses were prepared by transient cotransfection of vector expressing PPARγ, PγCA, and empty vector together with the helper viral vector into 293T cells using calcium phosphate precipitation. The retroviral supernatants were harvested 48 h after transfection and filtered through a 0.45 μm filter. Mammary epithelial cells MCF10A, MCF10A/NeuT, or NAFA were incubated with fresh retroviral supernatants in the presence of 8 μg/mL polybrene for 24 h, cultured for a further 48 h, and subjected to fluorescence-activated cell sorting (FACStar Plus; BD Biosciences) to select for cells expressing GFP if infection efficiency was lower than expected (<80% GFP-positive cells). GFP-positive cells were used for subsequent analysis.

Nile Red staining of intracellular lipid

Staining was carried out on living cells. When cells had grown to the desired confluence, dye was added to the cell culture at a final concentration of 100 ng/mL and incubated for a minimum of 5 min. Fluorescent and phase-contrast imaging were collected using the ×20 objective of an Olympus IX-70 laser confocal scanning microscope. Nile Red exhibits red fluorescence.

Immunofluorescent staining

Tumors from PPARγ, PγCA, and GFP vector control group were collected and fixed. The antibodies were directed to von Willebrand factor (vWF; DAKO), c-Kit, CD133, and CD31 (Biocare Medical; ref. 24). Heat-induced epitope retrieval was done on formalin-fixed and paraffin-embedded sections by incubating the sections in citrate buffer with 5% Tween 20 (pH 6.0) at 100°C for 20 min and allowed to cool for 20 min before staining. The sections were then incubated with 3% hydrogen peroxide diluted in 0.02 mol/L PBS (pH 7.4) for 5 min at room temperature, rinsed in 1× TBS for 15 min, and incubated with normal goat serum (PBS containing 10% normal goat serum and 2% bovine serum albumin) for 30 min at room temperature to block nonspecific antibody binding. The slides were rinsed and incubated with primary antibody diluted to a concentration of 1:500 with PBS containing 2% goat serum overnight at 4°C. The slides were then rinsed as above and incubated with horseradish peroxidase–conjugated secondary antibody according to the manufacturer's protocol (Dako EnVision). The signal was visualized using a Cy3-labeled tyramide amplification system (Perkin-Elmer Life Sciences). Sections were mounted in aqueous medium containing 4′,6-diamidino-2-phenylindole as a nuclear counterstain (Vector Labs). A negative control was done to ensure the specificity of fluorescent immunostaining by replacing primary antibody with a normal rabbit IgG. Sections were examined with an Olympus BX 61 confocal microscope, and representative areas were photographed using ×10 and ×20 objectives. The microvessel density of the tumors was analyzed based on vWF staining as described previously (25). Vessels were quantified at ×200 using a linear encoded motorized XY stage. At least 20 fields were quantified in four different tumor sections. The number of capillary vessels per square millimeter of tumor tissue was quantified using ×10 eyepieces equipped with 10 × 1 mm by 10 × 1 mm graticule and was expressed as a percentage of area of the grid. For control, 30 fields were analyzed in four different tumor samples. At least 40 fields were counted in three tumor sections for each experimental group, and significance was analyzed using the Student's t test. A difference of P < 0.05 was considered to be statistically significant. All analyses were done with SPSS 11.5 software. Data are expressed as mean ± SE.

Microarray analysis

Microarray analysis was conducted as described (26). Total RNA was isolated from tumors using Trizol and used to probe Affymetrix 430 2.0 arrays. RNA quality was determined by gel electrophoresis. Probe synthesis and hybridization were done according to the manufacturer's manual. Three arrays were used for each condition. Analysis of the arrays was done using the statistical package R statistics package and the limma library of the Bioconductor software package. Arrays were normalized using Robust Multiarray Analysis and the genes were ranked using fold change and T test for differentially expressed genes. These genes were then subjected to hierarchical clustering with “complete” agglomeration and each cluster was further analyzed based on known function of the genes contained in the cluster.

Migration assay

Transwell migration assays were done as described before (25). Briefly, 4 × 104 GFP-positive cells were seeded on 8 μm pore-sized Transwell filter insert (Costar). After 16 h of incubation at 37°C and 5% CO2, cells adherent to the top surface of the filter were removed using a cotton applicator. Cells were fixed with 3.7% formaldehyde and stained with crystal violet, and the numbers of cells on the bottom were counted. Data were from three experiments done in triplicate (mean ± SE).

PγCA induces lipid formation in normal and oncogene-transformed mammary epithelial cells

To examine the effect of PγCA on human breast cancer cellular proliferation, immortal MCF10A cells were transduced with expression vectors encoding either PPARγ or PγCA. Cells were selected by GFP expression from the viral expression vector. MCF10A cells transduced with PPARγ were more polarized and less spread than the vector control cells (Fig. 1A). PγCA induced a similar less spread morphology. Multiple small oil droplets, which stained positively with Nile Red (Fig. 1A), were identified in both PPARγ and PγCA transduced cells. MCF10A cells transformed with an activating ErbB2 mutant, NeuT, adopt a spindle-shaped and more polarized morphology (Fig. 1B). Transduction with PPARγ reduced the polarized morphology of MCF10A/NeuT cells (Fig. 1B) accompanied by the formation of a small number of lipid droplets. PγCA also reduced the polarized fibroblastoid morphology of MCF10A/NeuT cells with a relatively greater abundance of Nile Red–positive oil droplets (Fig. 1B). The accumulation of oil droplets is consistent with the known ability of liganded PPARγ to induce lipid metabolism in fibroblasts and enhanced transactivation of the PγCA in the absence of ligand.

Figure 1.

PγCA induces lipid formation of mammary epithelial cells transformed by ErbB2 oncogene. (A) MCF10A or (B) MCF10A/NeuT cells were infected with retroviral vector expressing GFP, PPARγ, and PγCA. Forty-eight hours post-infection, cells were stained with Nile Red (100 ng/mL). Images were taken under a fluorescent microscope. GFP-positive cells were analyzed for accumulation of oil droplets within the cells as shown by Nile Red staining.

Figure 1.

PγCA induces lipid formation of mammary epithelial cells transformed by ErbB2 oncogene. (A) MCF10A or (B) MCF10A/NeuT cells were infected with retroviral vector expressing GFP, PPARγ, and PγCA. Forty-eight hours post-infection, cells were stained with Nile Red (100 ng/mL). Images were taken under a fluorescent microscope. GFP-positive cells were analyzed for accumulation of oil droplets within the cells as shown by Nile Red staining.

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PγCA promotes tumor growth in vivo

The immortal and transformed MCF10A cells, grown in culture with matrix-rich basement membrane constituents, formed spherical structures in which the outermost layer forms polarized epithelium with the basal surface associating with surrounding basement member proteins and the apical surface pointing inward as shown previously (27). PγCA expression disorganized the polarized structure of MCF10A and MCF10A/NeuT cells (data not shown). The effect of PγCA on ErbB2-induced tumor was next examined in vivo in immunocompetent animals (Fig. 2). ErbB2-expressing mammary tumor cells (NAFA) derived from MMTV-ErbB2 (FVB) transgenic mice were implanted into FVB animals. The size of PγCA-expressing tumors was greater than either wild-type PPARγ or GFP control group (Fig. 2A). Tumor volume was enhanced ∼2-fold by PγCA compared with wild-type PPARγ (Fig. 2B). Supplementary Figure S1A-C displays the individual animal growth profiles (dashed lines) as well as the group-specific linear growth models (solid lines). Supplementary Fig. 1D displays the fitted growth models for all three groups. Table 1 provides the parameter estimates from each of these models in addition to information on statistical comparisons. PPARγ-expressing tumors showed significantly lower initial growth rates than the GFP group (intercept difference = −0.66; P = 0.01; 95% CI = −1.18 to −0.14). PγCA-transduced tumors had significantly lower initial growth rates than GFP-expressing tumors (difference = −0.77; P = 0.003; 95% CI = −1.27 to −0.27). This finding is consistent with our observation that NAFA cells expressing PγCA had decreased proliferation rates compared with the control group (data not shown). The PPARγ-expressing tumor growth rates did not differ from GFP, whereas PγCA-expressing tumors had a significantly higher rate of growth than either GFP (slope = 0.07; P < 0.001; 95% CI = 0.04-0.10) or PPARγ (slope = 0.06; P< 0.001; 95% CI = 0.03-0.09). Collectively, these studies show PγCA promotes ErbB2-induced tumor growth in immunocompetent animals.

Figure 2.

PγCA promotes tumor growth in vivo. A, NAFA cells transduced with MSCV-IRES-GFP vector encoding either PPARγ, PγCA, or empty vector were implanted into FVB by injecting 2 × 106 cells s.c. Tumor growth was measured every 3 d by digital caliper and tumor volume was calculated. B, tumor volumes were log-transformed and analyzed using a linear mixed model. Separate slope and intercepts were computed for each group (GFP, PPARγ, and PγCA) and then compared across groups using a global test followed by pair-wise comparisons via linear contrasts. Analyses were completed in SAS version 9.1.3, and detailed analysis can be found in Table 1 and Supplementary Fig. S1.

Figure 2.

PγCA promotes tumor growth in vivo. A, NAFA cells transduced with MSCV-IRES-GFP vector encoding either PPARγ, PγCA, or empty vector were implanted into FVB by injecting 2 × 106 cells s.c. Tumor growth was measured every 3 d by digital caliper and tumor volume was calculated. B, tumor volumes were log-transformed and analyzed using a linear mixed model. Separate slope and intercepts were computed for each group (GFP, PPARγ, and PγCA) and then compared across groups using a global test followed by pair-wise comparisons via linear contrasts. Analyses were completed in SAS version 9.1.3, and detailed analysis can be found in Table 1 and Supplementary Fig. S1.

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Table 1.

Linear mixed model coefficients for growth models

GroupCoefficientSEtP*Difference vs GFP (95% CI)PDifference vs PPARγ (95% CI)P
GFP 5.22 0.19 28.02 <0.001 — (—) — — (—) — 
PPARγ 4.55 0.18 13.99 <0.001 -0.66 (-1.18 to -0.14) 0.01 — (—) — 
PγCA 4.45 0.16 12.67 <0.001 -0.77 (-1.27 to -0.27) 0.003 -0.11 (-1.48 to -0.01) 0.61 
GFP 0.08 0.01 6.68 <0.001 — (—) — — (—) — 
PPARγ 0.09 0.01 7.51 <0.001 0.01 (-0.02 to 0.06) 0.26 — (—) — 
PγCA 0.15 0.01 14.11 <0.001 0.07 (0.04-0.10) <0.001 0.06 (0.03-0.09) <0.001 
GroupCoefficientSEtP*Difference vs GFP (95% CI)PDifference vs PPARγ (95% CI)P
GFP 5.22 0.19 28.02 <0.001 — (—) — — (—) — 
PPARγ 4.55 0.18 13.99 <0.001 -0.66 (-1.18 to -0.14) 0.01 — (—) — 
PγCA 4.45 0.16 12.67 <0.001 -0.77 (-1.27 to -0.27) 0.003 -0.11 (-1.48 to -0.01) 0.61 
GFP 0.08 0.01 6.68 <0.001 — (—) — — (—) — 
PPARγ 0.09 0.01 7.51 <0.001 0.01 (-0.02 to 0.06) 0.26 — (—) — 
PγCA 0.15 0.01 14.11 <0.001 0.07 (0.04-0.10) <0.001 0.06 (0.03-0.09) <0.001 

*P value is based on testing significance of individual coefficient using t test.

P value is based on testing significance of comparing coefficients across two groups by t test.

PγCA promotes angiogenesis

Growth and formation of capillary blood vessels are essential steps required for sustained tumor growth and metastasis and contribute to cancer progression in vivo. To determine the mechanism(s) through which PγCA enhanced ErbB2-mediated tumor growth in vivo, we analyzed blood vessels located in both the periphery and the interior of tumors. vWF is broadly accepted as a marker for endothelial cells. Immunofluorescent staining using anti-vWF antibody showed increased blood vessel density in PγCA-expressing tumors compared with GFP control tumors. Tumors expressing wild-type PPARγ alone did not affect vWF expression (Fig. 3A). Immunohistochemistry staining with an antibody against platelet endothelial cell adhesion molecule-1 (also known as CD31; Fig. 3B) showed a similar result. Blood vessels in tumors derived from endothelial progenitor cells (EPC) are characterized by CD133 and c-Kit (28, 29). Increased expression levels of both CD133 and c-Kit were shown in the PγCA-expressing tumors but not in GFP control or PPARγ-expressing tumors (Fig. 3C). This finding suggests that circulating EPCs released from the bone marrow may contribute to the increased vasculogenesis in ErbB2-induced tumors expressing active PPARγ.

Figure 3.

PγCA induces mammary tumor angiogenesis. A, immunostaining was conducted using antibodies against vWF for microvessel formation. Microvessel density was quantitated showing increased microvessel formation in PγCA group (22 ± 1.5) compared with GFP control or PPARγ groups. B, immunostaining was conducted in tumor samples to detect the expression levels of platelet endothelial cell adhesion molecule-1 (CD31) and CD133 (C).

Figure 3.

PγCA induces mammary tumor angiogenesis. A, immunostaining was conducted using antibodies against vWF for microvessel formation. Microvessel density was quantitated showing increased microvessel formation in PγCA group (22 ± 1.5) compared with GFP control or PPARγ groups. B, immunostaining was conducted in tumor samples to detect the expression levels of platelet endothelial cell adhesion molecule-1 (CD31) and CD133 (C).

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Expression profiling of PPARγ target genes in mammary tumors

To investigate the molecular mechanism(s) by which PγCA regulated angiogenesis/vasculogenesis, a genome-wide expression array analysis was conducted. Total RNA was prepared from extirpated tumors grown in immunocompetent mice. Gene expression profiles were compared between PPARγ and PγCA tumors (Fig. 4). Gene targets differentially regulated by PγCA convey functions in cell communication, angiogenesis, cell cycle, signal transduction, and cell differentiation. Genes regulated by PγCA in the angiogenesis pathway include angiopoietin-like 4 (Angptl4), fibroblast growth factor 9 (Fgf9), fibroblast growth factor 1 (Fgf1), adrenomedullin (Adm), matrix metallopeptidase 12 (Mmp12), Bcl-2/adenovirus E1B 19-kDa interacting protein 3 (Bnip3), pleiotrophin (Ptn), chemokine (C-X-C motif) ligand 2 (Cxcl2), and vascular endothelial growth factor A (Vegfa; Fig. 4B). We validated several genes including Angptl4, Fgf1, and Ptn by quantitative reverse transcription-PCR on additional tumor samples (Fig. 4C). Angptl4 was cloned as a PPARγ target gene and initially termed as PPARγ angiopoietin related and fasting-induced adipose factor according to the nature of how this gene was discovered (30, 31). We have shown previously that Angptl4 was upregulated in primary culture of mammary epithelial cells transduced with PγCA (32). Angptl4 is a secreted protein that belongs to the family of angiopoietin-like proteins. We further showed that Angptl4 secretion was increased in PγCA transduced NAFA cells compared with PPARγ and vector control (Fig. 4D , top).

Figure 4.

Gene expression profiling of tumors. A, tumors from PPARγ and PγCA groups were collected when the tumor implantation experiment completed. Total RNA prepared from fresh tumors was used for gene profiling using Affymetrix gene expression array. B, heat map was generated based on the genes differentially regulated by PγCA and PPARγ. C, quantitative reverse transcription-PCR was done to quantitatively measure the mRNA abundance of Angptl4, Fgf1, and Ptn. D, 10 mL of conditioned medium were concentrated to 100 μL. Fifty microliters were used for Western blot to determine the abundance of the secreted form of Angptl4 (top). Ten microliters of medium were separated on 10% SDS-PAGE gel followed by staining the gel with Coomassie blue to show the equal loading (bottom).

Figure 4.

Gene expression profiling of tumors. A, tumors from PPARγ and PγCA groups were collected when the tumor implantation experiment completed. Total RNA prepared from fresh tumors was used for gene profiling using Affymetrix gene expression array. B, heat map was generated based on the genes differentially regulated by PγCA and PPARγ. C, quantitative reverse transcription-PCR was done to quantitatively measure the mRNA abundance of Angptl4, Fgf1, and Ptn. D, 10 mL of conditioned medium were concentrated to 100 μL. Fifty microliters were used for Western blot to determine the abundance of the secreted form of Angptl4 (top). Ten microliters of medium were separated on 10% SDS-PAGE gel followed by staining the gel with Coomassie blue to show the equal loading (bottom).

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PγCA promotes endothelial cell migration through induction of secreted factors

As PγCA induced the expression of genes governing angiogenesis, we hypothesized that secreted factors were involved. We examined the conditioned medium from PγCA-expressing cells in endothelial cell migration assays. Transwell migration assays were conducted. Conditioned medium was collected from MCF10A/NeuT cells expressing GFP, PPARγ, or PγCA and applied to either the top chamber or the bottom chamber (Fig. 5A). HUVECs were seeded in the Boyden chamber and the cells migrating across the membrane were stained with crystal blue and counted (Fig. 5B). When conditioned medium from cells expressing PγCA were added to the bottom chamber, the number of migrating cells compared with GFP and PPARγ groups was significantly increased (Fig. 5B,, left). When HUVECs were seeded in the conditioned medium in the top chamber, the number of cells migrating toward the serum-free medium (in the bottom chamber) was significantly reduced (Fig. 5B , right). These observations suggest that PγCA expression in breast cancer cells induces secreted factors that promote endothelial cell migration.

Figure 5.

PγCA induction of proangiogenic factor Angptl4 in tumor cells is required for HUVEC cell migration. Transmigration assay was done with HUVECs. Conditioned medium was collected from MCF10A/ErbB2 cells transduced PPARγ, PγCA, GFP control. Conditioned medium was applied to either bottom or top chamber of Transwell device. Transmigrating cells were visualized by staining with crystal violet (A) and quantified (B). Purified Angptl4 was included as positive control. C, conditioned medium was pretreated with antibody to deplete Angptl4. Conditioned medium was applied to the top chamber of Transwell device. Transmigrating cells were visualized by staining with crystal violet. D, schematic representation of active PPARγ promotion of tumor growth through enhanced angiogenesis by inducing Angptl4 production.

Figure 5.

PγCA induction of proangiogenic factor Angptl4 in tumor cells is required for HUVEC cell migration. Transmigration assay was done with HUVECs. Conditioned medium was collected from MCF10A/ErbB2 cells transduced PPARγ, PγCA, GFP control. Conditioned medium was applied to either bottom or top chamber of Transwell device. Transmigrating cells were visualized by staining with crystal violet (A) and quantified (B). Purified Angptl4 was included as positive control. C, conditioned medium was pretreated with antibody to deplete Angptl4. Conditioned medium was applied to the top chamber of Transwell device. Transmigrating cells were visualized by staining with crystal violet. D, schematic representation of active PPARγ promotion of tumor growth through enhanced angiogenesis by inducing Angptl4 production.

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Angptl4 mediates PγCA induction of angiogenesis

Angptl4 stimulates endothelial cell growth and tubule formation, which is required for neovascularization and adipose tissue expansion (33). Angptl4 functions to regulate angiogenesis/vasculogenesis (3335), which contributes to solid tumor growth (35). As PγCA induced Angptl4 expression and secretion, we determined the role of Angptl4 in PγCA-induced endothelial cell migration. To deplete Angptl4 proteins, the conditioned medium from MCF10A/NeuT/PγCA cell culture was incubated with an Angptl4 antibody conjugated to protein A agarose beads. Conditioned medium depleted of Angptl4 was assessed in HUVEC Transwell migration assays. As we expected, more cells cultured in Angptl4-depleted medium transmigrated to the bottom chamber compared with the IgG control (Fig. 5C), indicating Angptl4-containing medium is more favorable to endothelial cells. These studies suggest that PγCA induction of Angptl4 in breast cancer cells promotes endothelial cellular migration.

The current studies showed that the growth of ErbB2-induced mammary tumors implanted in immunocompetent animals is promoted by an activated PPARγ (PγCA) in contrast with a cytostatic effect of PγCA on cancer cell proliferation in tissue culture. The role of PPARγ in tumorigenesis remains poorly understood in regards to growth inhibition effects in cultured cells and in N-nitrosomethylurea–induced or 7,12-dimethylbenz(a)anthracene–induced mammary tumorigenesis. Genetic removal of one allele of the PPARγ gene predisposes mice to gastric cancer (36). The tumor implantation analysis showed that early growth initiation by PPARγ and PγCA was significantly lower than the initial growth rate of mammary tumors expressing GFP. These findings are consistent with prior studies in which PPARγ ligands enhanced 7,12-dimethylbenz(a)anthracene–induced rat mammary adenocarcinoma (8) and promoted colonic tumor growth in ApcMin mice fed a high-fat diet (17). Targeted expression of an activated PPARγ to the mammary gland also enhanced tumorigenesis induced by polyoma middle T antigen (21). It is not feasible to directly compare the effect of PPARγ ligands with a PPARγ mutant that mimics the ligand-activated state, as some PPARγ ligands may possess receptor-independent functions. However, collectively these studies suggest that activated PPARγ can enhance mammary tumor growth induced by either ErbB2 or polyoma middle T antigen. The molecular mechanisms by which PγCA enhanced mammary tumorigenesis were investigated using gene expression profiling and we identified the induction of a proangiogenic pathway. In a prior study with a similar PPARγ mutant, gene expression analysis identified induction of Wnt signaling (21). It was proposed that the tumor-promoting effects of active PγCA in the mammary epithelium was due to enhanced Wnt signaling (21).

PPARγ inhibits angiogenesis in vitro (37). PPARγ is expressed in endothelial cells and PPARγ ligands, 15-deoxy-Δ12,14-prostaglandin J2 and thiazolidinedione, inhibit tubular structure formation and proliferation of endothelial cell (HUVEC) in tissue culture (38). Consistent with our findings, a proangiogenic effect of PPARγ ligands was reported (39, 40). PPARγ ligands promote angiogenesis via induction of VEGF in cardiac myofibroblasts and human vascular smooth muscle cells but inhibit VEGF-mediated blood vessel formation in a chick chorioallantoic membrane model (41) suggesting cell type–specific effects. The current studies of breast cancer in vivo using genome-wide expression screening showed that PγCA induced the proangiogenic genes VEGFa, Fgf1, Fgf4, and Angptl4. Angptl4 belongs to an angiopoietin-like protein family and is secreted by adipocytes, hepatocytes, the placenta, as well as cancer cells. Although the role of Angptl4 in angiogenesis is controversial, Angptl4 promotes angiogenesis in obesity (3335) and contributes to solid tumor growth (35).

Our studies suggest that PγCA induces the number of EPCs within mammary tumors. Because PPARγ increases hematopoietic cell differentiation toward endothelial lineage (42), blood vessels in tumors may be derived from EPCs. Downregulation of PPARδ in EPCs reduced prostacyclin biosynthesis and decreased capillary formation in nude mice in vivo (43). Activation of PPARγ or PPARδ promotes angiogenic progenitor cell differentiation toward endothelial cell lineage and facilitates vasculogenesis by increasing the population of hematopoietic stem cells in bone marrow and EPCs in peripheral blood (42, 44). Patients with coronary artery disease who received pioglitazone had an increased number of EPCs compared with the placebo group and rosiglitazone increased the number and migratory activity of EPCs in type II diabetic patients (45, 46).

The value of PPARγ as a potential target for the prevention and treatment of cancer using differentiation-based therapies revealed interesting preliminary observations (47), in which PPARγ ligand was used to treat human liposarcoma (48). However, a subsequent phase II trial failed to show an effect of rosiglitazone in liposarcoma (49) or in refractory breast cancer (50). The spread of obesity and increased prevalence of a sedentary lifestyle has substantially increased the incidence of type II diabetes. PPARγ agonists are increasingly used to enhance insulin sensitivity and improve glucose tolerance and thereby reduce complications, including diabetic neuropathy, cardiovascular disease, and retinopathy with blindness. As the current studies show that a ligand-activated PPARγ mutant promotes ErbB2 oncogene-induced tumorigenesis, further epidemiologic studies are required in patients treated with PPARγ agonists for possible effects on tumor development.

No potential conflicts of interest were disclosed.

Grant support: Susan Komen Breast Cancer Foundation awards BCTR0504227 (C. Wang), R01CA70896, R01CA75503, R01CA86072 (R.G. Pestell), and R01CA80250. Work conducted at the Kimmel Cancer Center was supported by the NIH Cancer Center core grant P30CA56036 (R.G. Pestell). This project is funded in part by the Dr. Ralph and Marian C. Falk Medical Research Trust (R.G. Pestell) and Pennsylvania Department of Health (C. Wang and R.G. Pestell).

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.

1
Rosen
ED
,
Spiegelman
BM
. 
PPARγ: a nuclear regulator of metabolism, differentiation, and cell growth
.
J Biol Chem
2001
;
276
:
37731
4
.
2
Tontonoz
P
,
Hu
E
,
Spiegelman
BM
. 
Stimulation of adipogenesis in fibroblasts by PPARγ2, a lipid-activated transcription factor
.
Cell
1994
;
79
:
1147
56
.
3
Kim
JB
,
Wright
HM
,
Wright
M
,
Spiegelman
BM
. 
ADD1/SREBP1 activates PPARγ through the production of endogenous ligand
.
Proc Natl Acad Sci U S A
1998
;
95
:
4333
7
.
4
Yu
S
,
Matsusue
K
,
Kashireddy
P
, et al
. 
Adipocyte-specific gene expression and adipogenic steatosis in the mouse liver due to peroxisome proliferator-activated receptor 1 (PPAR1) overexpression
.
J Biol Chem
2003
;
278
:
498
505
.
5
Wang
C
,
Pattabiraman
N
,
Zhou
JN
, et al
. 
Cyclin D1 repression of peroxisome proliferator-activated receptor γ expression and transactivation
.
Mol Cell Biol
2003
;
23
:
6159
73
.
6
Brakenhielm
E
,
Veitonmaki
N
,
Cao
R
, et al
. 
Adiponectin-induced antiangiogenesis and antitumor activity involve caspase-mediated endothelial cell apoptosis
.
Proc Natl Acad Sci U S A
2004
;
101
:
2476
81
.
7
Suh
N
,
Wang
Y
,
Williams
CR
, et al
. 
A new ligand for the peroxisome proliferator-activated receptor-γ (PPAR-γ), GW7845, inhibits rat mammary carcinogenesis
.
Cancer Res
1999
;
59
:
5671
3
.
8
Pighetti
GM
,
Novosad
W
,
Nicholson
C
, et al
. 
Therapeutic treatment of DMBA-induced mammary tumors with PPAR ligands
.
Anticancer Res
2001
;
21
:
825
9
.
9
Brockman
JA
,
Gupta
RA
,
Dubois
RN
. 
Activation of PPARγ leads to inhibition of anchorage-independent growth of human colorectal cancer cells
.
Gastroenterology
1998
;
115
:
1049
55
.
10
Mueller
E
,
Sarraf
P
,
Tontonoz
P
, et al
. 
Terminal differentiation of human breast cancer through PPARγ
.
Mol Cell
1998
;
1
:
465
70
.
11
Elstner
E
,
Muller
C
,
Koshizuka
K
, et al
. 
Ligands for peroxisome proliferator-activated receptor γ and retinoic acid receptor inhibit growth and induce apoptosis of human breast cancer cells in vitro and in BNX mice
.
Proc Natl Acad Sci U S A
1998
;
95
:
8806
11
.
12
Ricote
M
,
Huang
J
,
Fajas
L
, et al
. 
Expression of the peroxisome proliferator-activated receptor γ (PPARγ) in human atherosclerosis and regulation in macrophages by colony stimulating factors and oxidized low density lipoprotein
.
Proc Natl Acad Sci U S A
1998
;
95
:
7614
9
.
13
Sarraf
P
,
Mueller
E
,
Jones
D
, et al
. 
Differentiation and reversal of malignant changes in colon cancer through PPARγ
.
Nat Med
1998
;
4
:
1046
52
.
14
Sarraf
P
,
Mueller
E
,
Smith
WM
, et al
. 
Loss-of-function mutations in PPARγ associated with human colon cancer
.
Mol Cell
1999
;
3
:
799
804
.
15
Kroll
TG
,
Sarraf
P
,
Pecciarini
L
, et al
. 
PAX8-1 fusion oncogene in human thyroid carcinoma [corrected]
.
Science
2000
;
289
:
1357
60
.
16
Lefebvre
AM
,
Chen
I
,
Desreumaux
P
, et al
. 
Activation of the peroxisome proliferator-activated receptor γ promotes the development of colon tumors in C57BL/6J-ApcMin/+ mice
.
Nat Med
1998
;
4
:
1053
7
.
17
Saez
E
,
Tontonoz
P
,
Nelson
MC
, et al
. 
Activators of the nuclear receptor PPARγ enhance colon polyp formation
.
Nat Med
1998
;
4
:
1058
61
.
18
Abe
A
,
Kiriyama
Y
,
Hirano
M
, et al
. 
Troglitazone suppresses cell growth of KU812 cells independently of PPARγ
.
Eur J Pharmacol
2002
;
436
:
7
13
.
19
Chawla
A
,
Barak
Y
,
Nagy
L
,
Liao
D
,
Tontonoz
P
,
Evans
RM
. 
PPAR-γ dependent and independent effects on macrophage-gene expression in lipid metabolism and inflammation
.
Nat Med
2001
;
7
:
48
52
.
20
Moore
KJ
,
Rosen
ED
,
Fitzgerald
ML
, et al
. 
The role of PPAR-γ in macrophage differentiation and cholesterol uptake
.
Nat Med
2001
;
7
:
41
7
.
21
Saez
E
,
Rosenfeld
J
,
Livolsi
A
, et al
. 
PPARγ signaling exacerbates mammary gland tumor development
.
Genes Dev
2004
;
18
:
528
40
.
22
Lee
RJ
,
Albanese
C
,
Stenger
RJ
, et al
. 
pp60(v-src) induction of cyclin D1 requires collaborative interactions between the extracellular signal-regulated kinase, p38, and Jun kinase pathways. A role for cAMP response element-binding protein and activating transcription factor-2 in pp60(v-src) signaling in breast cancer cells
.
J Biol Chem
1999
;
274
:
7341
50
.
23
Neumeister
P
,
Pixley
FJ
,
Xiong
Y
, et al
. 
Cyclin D1 governs adhesion and motility of macrophages
.
Mol Biol Cell
2003
;
14
:
2005
15
.
24
Chizea
J
,
Dailey
VK
,
Williams
E
,
Johnson
MD
,
Pestell
RG
,
Ojeifo
JO
. 
Endothelial progenitor cells significantly contribute to vasculatures in human and mouse breast tumors
.
Open Hematol J
2008
;
2
:
30
62
.
25
Ju
X
,
Katiyar
S
,
Wang
C
, et al
. 
Akt1 governs breast cancer progression in vivo
.
Proc Natl Acad Sci U S A
2007
;
104
:
7438
43
.
26
Sakamaki
T
,
Casimiro
MC
,
Ju
X
, et al
. 
Cyclin D1 determines mitochondrial function in vivo
.
Mol Cell Biol
2006
;
26
:
5449
69
.
27
Liu
M
,
Ju
X
,
Willmarth
NE
, et al
. 
Nuclear factor-κB enhances ErbB2-induced mammary tumorigenesis and neoangiogenesis in vivo
.
Am J Pathol
2009
;
174
:
1910
20
.
28
Gao
D
,
Nolan
DJ
,
Mellick
AS
,
Bambino
K
,
McDonnell
K
,
Mittal
V
. 
Endothelial progenitor cells control the angiogenic switch in mouse lung metastasis
.
Science
2008
;
319
:
195
8
.
29
Hilbe
W
,
Dirnhofer
S
,
Oberwasserlechner
F
, et al
. 
CD133 positive endothelial progenitor cells contribute to the tumour vasculature in non-small cell lung cancer
.
J Clin Pathol
2004
;
57
:
965
9
.
30
Kersten
S
,
Wahli
W
. 
Peroxisome proliferator activated receptor agonists
.
Exs
2000
;
89
:
141
51
.
31
Yoon
JC
,
Chickering
TW
,
Rosen
ED
, et al
. 
Peroxisome proliferator-activated receptor γ target gene encoding a novel angiopoietin-related protein associated with adipose differentiation
.
Mol Cell Biol
2000
;
20
:
5343
9
.
32
Zhou
J
,
Zhang
W
,
Liang
B
, et al
. 
PPARγ activation induces autophagy in breast cancer cells
.
Int J Biochem Cell Biol
2009
;
41
:
2334
42
.
33
Gealekman
O
,
Burkart
A
,
Chouinard
M
,
Nicoloro
SM
,
Straubhaar
J
,
Corvera
S
. 
Enhanced angiogenesis in obesity and in response to PPARγ activators through adipocyte VEGF and ANGPTL4 production
.
Am J Physiol Endocrinol Metab
2008
;
295
:
E1056
64
.
34
Hermann
LM
,
Pinkerton
M
,
Jennings
K
, et al
. 
Angiopoietin-like-4 is a potential angiogenic mediator in arthritis
.
Clin Immunol
2005
;
115
:
93
101
.
35
Le Jan
S
,
Amy
C
,
Cazes
A
, et al
. 
Angiopoietin-like 4 is a proangiogenic factor produced during ischemia and in conventional renal cell carcinoma
.
Am J Pathol
2003
;
162
:
1521
8
.
36
Lu
M
,
Kwan
T
,
Yu
C
, et al
. 
Peroxisome proliferator-activated receptor γ agonists promote TRAIL-induced apoptosis by reducing survivin levels via cyclin D3 repression and cell cycle arrest
.
J Biol Chem
2005
;
280
:
6742
51
.
37
Giaginis
C
,
Margeli
A
,
Theocharis
S
. 
Peroxisome proliferator-activated receptor-γ ligands as investigational modulators of angiogenesis
.
Expert Opin Investig Drugs
2007
;
16
:
1561
72
.
38
Xin
X
,
Yang
S
,
Kowalski
J
,
Gerritsen
ME
. 
Peroxisome proliferator-activated receptor γ ligands are potent inhibitors of angiogenesis in vitro and in vivo
.
J Biol Chem
1999
;
274
:
9116
21
.
39
Yamakawa
K
,
Hosoi
M
,
Koyama
H
, et al
. 
Peroxisome proliferator-activated receptor-γ agonists increase vascular endothelial growth factor expression in human vascular smooth muscle cells
.
Biochem Biophys Res Commun
2000
;
271
:
571
4
.
40
Chintalgattu
V
,
Harris
GS
,
Akula
SM
,
Katwa
LC
. 
PPAR-γ agonists induce the expression of VEGF and its receptors in cultured cardiac myofibroblasts
.
Cardiovasc Res
2007
;
74
:
140
50
.
41
Aljada
A
,
O'Connor
L
,
Fu
YY
,
Mousa
SA
. 
PPARγ ligands, rosiglitazone and pioglitazone, inhibit bFGF- and VEGF-mediated angiogenesis
.
Angiogenesis
2008
;
11
:
361
7
.
42
Wang
CH
,
Ciliberti
N
,
Li
SH
, et al
. 
Rosiglitazone facilitates angiogenic progenitor cell differentiation toward endothelial lineage: a new paradigm in glitazone pleiotropy
.
Circulation
2004
;
109
:
1392
400
.
43
He
T
,
Lu
T
,
d'Uscio
LV
,
Lam
CF
,
Lee
HC
,
Katusic
ZS
. 
Angiogenic function of prostacyclin biosynthesis in human endothelial progenitor cells
.
Circ Res
2008
;
103
:
80
8
.
44
Han
JK
,
Lee
HS
,
Yang
HM
, et al
. 
Peroxisome proliferator-activated receptor-δ agonist enhances vasculogenesis by regulating endothelial progenitor cells through genomic and nongenomic activations of the phosphatidylinositol 3-kinase/Akt pathway
.
Circulation
2008
;
118
:
1021
33
.
45
Pistrosch
F
,
Herbrig
K
,
Oelschlaegel
U
, et al
. 
PPARγ-agonist rosiglitazone increases number and migratory activity of cultured endothelial progenitor cells
.
Atherosclerosis
2005
;
183
:
163
7
.
46
Sorrentino
SA
,
Bahlmann
FH
,
Besler
C
, et al
. 
Oxidant stress impairs in vivo reendothelialization capacity of endothelial progenitor cells from patients with type 2 diabetes mellitus: restoration by the peroxisome proliferator-activated receptor-γ agonist rosiglitazone
.
Circulation
2007
;
116
:
163
73
.
47
Sporn
MB
,
Suh
N
,
Mangelsdorf
DJ
. 
Prospects for prevention and treatment of cancer with selective PPARγ modulators (SPARMs)
.
Trends Mol Med
2001
;
7
:
395
400
.
48
Demetri
GD
,
Fletcher
CD
,
Mueller
E
, et al
. 
Induction of solid tumor differentiation by the peroxisome proliferator-activated receptor-γ ligand troglitazone in patients with liposarcoma
.
Proc Natl Acad Sci U S A
1999
;
96
:
3951
6
.
49
Debrock
G
,
Vanhentenrijk
V
,
Sciot
R
,
Debiec-Rychter
M
,
Oyen
R
,
Van Oosterom
A
. 
A phase II trial with rosiglitazone in liposarcoma patients
.
Br J Cancer
2003
;
89
:
1409
12
.
50
Burstein
HJ
,
Demetri
GD
,
Mueller
E
,
Sarraf
P
,
Spiegelman
BM
,
Winer
EP
. 
Use of the peroxisome proliferator-activated receptor (PPAR) γ ligand troglitazone as treatment for refractory breast cancer: a phase II study
.
Breast Cancer Res Treat
2003
;
79
:
391
7
.

Supplementary data