PEDF: a potential therapeutic agent for osteosarcoma

by

Peter F. M. Choong, M.D.

Professor

and

Crispin R. Dass, Ph.D.

Senior Research Officer

Orthopaedics-Research

St. Vincent’s Hospital Melbourne,

Fitzroy, Victoria, Australia 

Introduction

Osteosarcoma (OS) is the most common malignancy of bone and is the second highest cause of cancer-related deaths in the paediatric age group. 465 new cases of OS occurred in Australia between 1995-99, an increase of 15% in comparison to a similar period a decade earlier (Australian Institute of Health and Welfare, Cancer in Australia, 2001). The burden to patients and the community is high with disabling surgery, prolonged rehabilitation and treatment that is expensive, protracted, intensive and requiring inpatient monitoring. Importantly, an average of 17 life-years per patient is lost due to sarcomas, compared to 6.5 for bowel, lung and breast cancer (Canstat Victoria, 2001 data). This contributes to a disproportionate cost to the individual and community and targets this type of cancer as an age-related phenomenon which is now a major public health issue. 

Treatment

While surgery remains the mainstay of OS treatment, chemotherapy has delivered significant improvements in survival. Despite this, fatality from recurrent disease still approaches 1/3 of those afflicted. Intensification of chemotherapy has failed to demonstrate significant survival improvement in patients with aggressive relapse highlighting the need for novel therapies that increase the effectiveness while reducing the toxicity of treatment (Ek and Choong 2006). The importance of translating basic science to clinical practice is becoming a priority for government (Victorian Cancer Agency, 2006), and funding bodies (NHMRC). Identifying targeted therapies that aim to enhance the effect or reduce the toxicity of conventional chemotherapy when treating primary or metastatic OS is a primary goal of this project.  

Cartilage is a barrier to OS growth

OS arises most commonly around the growing ends of long bones (metaphysis) adjacent to the growth plate cartilage (GPC) suggesting a disruption of physiologic control in the region earmarked for normal proliferation. Despite the destructive capacity of OS, cartilage acts as a strong barrier against invasion and is only penetrated as a late local event. We were the first to link the impediment to advance of OS through the GPC with regulator(s) of bone maturation and the state of cartilage lacking an adequate supply of blood vessels (Quan et al. 2003). In analysing the molecular components of the GPC, we identified for the first time the presence of a protein called pigment epithelium-derived factor (PEDF), the strongest physiologic inhibitor of angiogenesis known, in the part of the GPC lacking blood vessels (Quan et al. 2005). PEDF has been shown to be more than twice as potent as angiostatin, and more than seven times as potent as endostatin, two endogenous molecules that are potently anti-angiogenic in their own right (Dawson et al. 1999). We demonstrated that OS regularly stopped advancing when it met the layer of the GPC at which PEDF protein was abundant (Fig. 1). In contrast, OS grew in that part of bone where a protein called vascular endothelial growth factor (VEGF), a potent pro-angiogenic factor, was present at high levels (Quan et al. 2005). The interface between PEDF and VEGF was the point of cessation of tumor advancement suggesting a balance between opposing pro-and anti-angiogenic forces and this is consistent with the fundamental role of angiogenesis in tumor biology that is also applicable to OS.   

Figure 1:OS growth stops at the GPC and correlates with PEDF expression. A: MRI showing GPC impeding advance of OS in the tibia (shin-bone). B: Histological section of mouse bone depicting growth inhibition of OS at the GPC (starting at the dotted line). C: PEDF protein is abundantly present at the GPC where blood vessels are lacking (brown staining indicates presence of PEDF). D: VEGF protein is abundantly present below the GPC where blood vessels are present (brown staining indicates presence of VEGF). 

Our data on PEDF and osteosarcoma 

We have compiled a considerable amount of published data that demonstrates the effectiveness of PEDF in inhibiting migration, invasion and proliferation of OS cells in vitro (Fig. 2) and these actions can be replicated in vivo.    

Figure 2: Forcibly expressing PEDF in OS cells reduces ability of cells to proliferate. L, Proliferation of rat OS (UMR106-01) parental, PEDF-overexpressingand empty vector-transfected cells at 3 days post-seeding. R, Proliferation of human OS (SaOS-2) parental, PEDF-overexpressing and empty vector-transfected cells at 3 days post-seeding. Data shown are mean + s.d., n = 4, * p < 0.05. 

Recently, we reported that by forcibly expressing (overexpressing) PEDF in our OS model, tumor growth and metastasis were profoundly inhibited (Fig. 3). A similar effect was also noted when we exposed developing tumor cells to recombinant PEDF protein (rPEDF). To improve the effectiveness of our strategy, we isolated shorter derivatives of the PEDF protein which contained the regions responsible for anti-angiogenic and anti-proliferative/apoptotic (programmed cell death) activity. By exposing cells in vitro and tumor in vivo, significant antitumoral activity was seen with the less expensive peptides. 

Figure 3: Forcibly expressing PEDF in OS cells reduces tumor growth in mice. Growth of human OS (SaOS-2) parental, PEDF-overexpressing and empty vector-transfected tumor cells in mice 5 weeks after implantation of cells.  Representative mice were chosen for presentation, n = 5. 

To see if apoptosis is associated with PEDF treatment, we treated SaOS-2 cells with rPEDF and noted a dose-related increase in apoptosis (Fig. 4). A specific inhibitor of PEDF, an antibody, inhibited the apoptotic effect of rPEDF noted in earlier experiments, confirming that the pro-apoptotic effect was indeed due to PEDF activity. Interrogating the Fas/Fas ligand cell death pathway would be a focus of this project as apoptosis by the induction of Fas ligand by PEDF has been alluded to before (Doll et al. 2003). In addition, we will also look at other cell death pathways such as tumor necrosis factor (TNF)/TNF receptor and TNF-related apoptosis-inducing ligand (TRAIL) and receptors.

Figure 4: Treatment of OS cells with PEDF protein causes cell death. Human OS (SaOS-2) cells were treated with increasing doses of PEDF and the effect on cell death (apoptosis) was examined. * p < 0.01, n = 4   

There are a number of important interactions between PEDF, VEGF and the latter’s receptor. Reports infer a regulatory action of PEDF on VEGF as well as an inverse relationship between the two (Ohno-Matsui et al. 2001). More recent reports suggest a role for PEDF in regulation of VEGF gene expression (Ohno-Matsui et al. 2003). We have demonstrated the ability of rPEDF to inhibit angiogenesis by disrupting the expression of VEGF in OS cells (Fig. 5). We would like to further tease out the mechanistic interplay between PEDF and VEGF.  

Figure 5. Treatment of OS cells with PEDF protein reduces cellular VEGF protein level. Rat (UMR106-01) and human (SaOS-2) OS cells were treated with increasing doses of PEDF and the effect on the level of VEGF protein within cells was examined, n = 4. 

To date, experimental administration of PEDF to tumors in vivo has been either via direct injection of rPEDF into the tumor or gene transfer via plasmid DNA or viruses in animal models of disease (Ek et al. 2006). Viral vectors suffer from the risk of de novo cancer initiation via recombination within the patient’s DNA. Plasmid vectors are safer, but are often hindered by low efficiency of gene expression. The use of recombinant protein is attractive in that it avoids the above difficulties and may be produced in bulk via established industrial methods, is easier to handle, and thus, facilitates compliance by treating teams and patients. Recombinant proteins, however, are often unstable and inherently susceptible to biological degradation, while high molecular weight proteins are often associated with low tissue penetration, poor availability at the target site, and uptake into target cells. Production of larger proteins is frequently hampered by high costs and difficulties with handling and storage as has been the case with endostatin, a 20 kDa polypeptide. As PEDF is a 50 kDA protein, it is potentially more vulnerable to the constraints facing endostatin. A more rational and advantageous approach would be to use active smaller peptide derivatives of PEDF that maintain bioactivity. We generated four 25-mer synthetic peptides (SVO 1-4) derived from parent PEDF corresponding to functional regions, which suppressed OS cell proliferation, inhibited cell invasion, increased cell adhesion to type-1 collagen and reduced VEGF protein (Ek et al. 2007). SVO2 and SVO3 demonstrated consistently the highest activity in vitro, and in vivo exhibited a greater than 30% reduction in tumor volume by day 32 and a marked reduction in pulmonary metastases (Fig. 6). These results demonstrate successful translation of our initial basic work to a treatment strategy for OS. The implementation of systemic delivery of peptide derivatives will be a major initiative of this project.

Figure 6: Treatment with short peptides based on PEDF in OS cells reduces tumor growth in mice. Human (SaOS-2) OS cells were mixed with the short peptides (SVO2 and SVO3) and the effect on tumor size (A) and number of lung metastasis (B) was examined, n = 5, * p < 0.05, ** p , 0.01. 

Significance of project 

Our research project thus combines basic science investigations with a prominent translational element. We aim to build on the preceding data by testing rPEDF and its cheaper derivatives on established primary and metastatic OS in our model, individually and in combination with doxorubicin. This series of experiments will analyse the important role of rPEDF and its ability to reduce the toxicity of doxorubicin when used in combination. The molecular postulates that we believe to underpin the effectiveness of PEDF and which we shall explore include the regulation of pro-angiogenic factors and the induction of apoptosis by PEDF. The main focus in our lab is to progress our PEDF research findings towards clinical evaluation and hopefully provide the first instance of successful molecular therapy for OS using endogenous biologicals.

References

Cai J, Jiang WG, Grant MB, Boulton M. Pigment epithelium-derived factor inhibits angiogenesis via regulated intracellular proteolysis of vascular endothelial growth factor receptor 1. J. Biol. Chem. 281:3604-13, 2006 

Dawson DW, Volpert OV, Gillis P, Crawford SE, Xu H, Benedict W, Bouck NP. Pigment epithelium-derived factor: a potent inhibitor of angiogenesis. Science 285:245-8, 1999

Doll JA, Stellmach VM, Bouck NP, Bergh AR, Lee C, Abramson LP, Cornwell ML, Pins MR, Borensztajn J, Crawford SE. Pigment epithelium-derived factor regulates the vasculature and mass of the prostate and pancreas. Nat. Med. 9:774-80, 2003

Ek ETH, Choong, PFM. The role of high-dose therapy and autologous stem cell transplantation for pediatric bone and soft tissue sarcomas. Expert Rev. Anticancer Ther. 6:225-37, 2006

Ek ETH, Dass CR, Choong, PFM. PEDF: a potential molecular therapeutic target with multiple anti-cancer activities. Trends Mol. Med. 12:497-502, 2006

Ek ETH, Dass CR, Contreras KG, Choong PFM. PEDF-derived synthetic peptides exhibit antitumor activity in an orthotopic model of human osteosarcoma. J. Orthop. Res. 25:1671-80, 2007 

Garcia, M., Garcia M, Fernandez-Garcia NI, Rivas V, Carretero M, Escamez MJ, Gonzalez-Martin A, Medrano EE, Volpert O, Jorcano JL, Jimenez B, Larcher F, Del Rio M. Inhibition of xenografted human melanoma growth and prevention of metastasis development by dual antiangiogenic/antitumor activities of pigment epithelium-derived factor. Cancer Res. 64:5632-42, 2004 

Ohno-Matsui K, Morita I, Tombran-Tink J, Mrazek D, Onodera M, Uetama T, Hayano M, Murota SI, Mochizuki M. Novel mechanism for age-related macular degeneration: an equilibrium shift between the angiogenesis factors VEGF and PEDF. J. Cell. Physiol. 189:323-33, 2001

Ohno-Matsui, K., Ohno-Matsui K, Yoshida T, Uetama T, Mochizuki M, Morita I. Vascular endothelial growth factor upregulates pigment epithelium-derived factor expression via VEGFR-1 in human retinal pigment epithelial cells. Biochem. Biophys. Res. Commun. 303:962-7, 2003 

Quan GM, Ojaimi J, Nadesapillai AP, Zhou H, Choong PF. (2003). Resistance of epiphyseal cartilage to invasion by osteosarcoma is likely to be due to expression of antiangiogenic factors. Pathobiology 70:361-7, 2003

Quan GM, Ojaimi J, Li Y, Kartsogiannis V, Zhou H, Choong PF. (2005). Localization of pigment epithelium-derived factor in growing mouse bone. Calcif. Tissue Int. 76:146-53, 2005 

Tombran-Tink J, Barnstable CJ. Osteoblasts and osteoclasts express PEDF, VEGF-A isoforms, and VEGF receptors: possible mediators of angiogenesis and matrix remodeling in the bone. Biochem. Biophys. Res. Commun. 316:573-9, 2004

V5N4 ESUN Copyright © 2008 Liddy Shriver Sarcoma Initiative.