PG545 enhances anti-cancer activity of chemotherapy in ovarian models and increases surrogate biomarkers such as VEGF in preclinical and clinical plasma samples

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Abstract

Background

Despite the utility of antiangiogenic drugs in ovarian cancer, efficacy remains limited due to resistance linked to alternate angiogenic pathways and metastasis. Therefore, we investigated PG545, an anti-angiogenic and anti-metastatic agent which is currently in Phase I clinical trials, using preclinical models of ovarian cancer.

Methods

PG545’s anti-cancer activity was investigated in vitro and in vivo as a single agent, and in combination with paclitaxel, cisplatin or carboplatin using various ovarian cancer cell lines and tumour models.

Results

PG545, alone, or in combination with chemotherapeutics, inhibited proliferation of ovarian cancer cells, demonstrating synergy with paclitaxel in A2780 cells. PG545 inhibited growth factor-mediated cell migration and reduced HB-EGF-induced phosphorylation of ERK, AKT and EGFR in vitro and significantly reduced tumour burden which was enhanced when combined with paclitaxel in an A2780 model or carboplatin in a SKOV-3 model. Moreover, in the immunocompetent ID8 model, PG545 also significantly reduced ascites in vivo. In the A2780 maintenance model, PG545 initiated with, and following paclitaxel and cisplatin treatment, significantly improved overall survival. PG545 increased plasma VEGF levels (and other targets) in preclinical models and in a small cohort of advanced cancer patients which might represent a potential biomarker of response.

Conclusion

Our results support clinical testing of PG545, particularly in combination with paclitaxel, as a novel therapeutic strategy for ovarian cancer.

Introduction

Significant progress in understanding the molecular biology of epithelial ovarian cancer (OVCA), has not yet translated into improvements in disease outcomes. Most patients respond initially to therapy, however the majority relapse, making OVCA essentially incurable [1]. As a result, efforts are ongoing to identify novel agents that target the tumour microenvironment to impact angiogenesis, invasion and metastasis. While antiangiogenic therapy is emerging as a viable option for OVCA, other growth factors either remain functionally active or increase as a mode of resistance to anti-VEGF therapies [1]. The resistance mechanisms associated with the use of anti-VEGF therapies have been reported to include FGF-2 [2] and HB-EGF [3]. Therefore, a more effective treatment strategy would be to target key angiogenesis pathways simultaneously while reducing metastatic spread via inhibition of heparanase, an enzyme whose expression correlates with poor survival in metastatic gynaecological adenocarcinomas [4] and may contribute to the proliferation and metastasis of ovarian cancer [5]. Heparan sulfate proteoglycans (HSPG) play an important role in modulating heparan sulfate-binding growth factor (GF) signalling and heparanase activity [6], [7], in the extracellular matrix [8], and are implicated in angiogenesis and metastasis [9], [10], [11]. Thus, the development of therapeutics that inhibit these growth factors plus heparanase activity may have an advantage over existing antiangiogenic agents [12].

PG545 is a sulfated tetrasaccharide optimised for potency through the addition of a lipophilic moiety to attain potent in vivo activity, long plasma half-life and low anticoagulant potential [13], [14]. It inhibits angiogenesis via inhibition of VEGF and FGF-2 while preventing metastasis through blockade of heparanase [13], [15]. PG545 inhibits in vivo angiogenesis, attenuates tumour growth and/or metastasis in various cancer models including lung, hepatocellular, prostate, colon, melanoma, pancreatic and head and neck cancers [16], [17]. In a skin carcinogenesis model, PG545 inhibited the heparanase-dependent formation of tumour lesions providing further validation for the targeting of this enzyme in the development of cancer therapeutics [18]. It also reduced heparanase expression in a model of metastatic breast cancer [17]. However, PG545 activity in OVCA has not yet been studied. This is particularly relevant as previous investigations supporting the efficacy of anti-angiogenic agents in OVCA [19], [20], [21] would suggest an additional treatment benefit could be achieved with dual targeting of the angiogenic (VEGF) and metastatic (heparanase) pathways. Therefore, the aim of this study was to investigate the activity of PG545 in OVCA by, firstly, studying its impact on ovarian tumour cell growth, cellular migration and invasion, secondly, further define the molecular downstream signalling effects of PG545, and, thirdly, explore the impact of PG545 as a single agent and in combination with cytotoxic therapy in OVCA preclinical models. Finally, a preliminary assessment of putative biomarkers for PG545 activity was performed by analysing GFs and heparanase in the plasma samples from mice and a small cohort of advanced cancer patients treated with PG545 from a previous Phase I trial (ClinicalTrials.gov Identifier: NCT01252095). The safety and tolerability of intravenously-infused PG545 is currently being assessed in patients with advanced solid tumours (ClinicalTrials.gov Identifier: NCT02042781).

Section snippets

Cell culture

SKOV3, OV202, A2780, and ID8 cells were cultured as previously described [22], [23]. A2780 cells were obtained from Fox Chase Cancer Center and ID8 cells from Dr. Katherine Roby [24].

Growth factors & reagents

HB-EGF, HGF, FGF-2, VEGF, SDF-1, FGF-2, human recombinant VEGF165, stromal cell-derived factor-1 were purchased from R&D Systems (Minneapolis, MN). PG545 was synthesised by Progen Pharmaceuticals (Brisbane, QLD, Australia). All drugs (PG545, cisplatin (Calbiochem, Millipore, Billerica, MA), carboplatin (50 mg/5 ml,

PG545 inhibits OVCA cell viability in vitro as single agent and in combination with chemotherapeutic agents

The potential of PG545 to synergise with the chemotherapeutic agents cisplatin and paclitaxel against OVCA cells was investigated using cell viability assays. Firstly, we showed that PG545 exhibited concentration-dependent growth inhibition (Supplementary Fig. 1A and D) in A2780 and SKOV3 cell lines. In combination with cisplatin or paclitaxel, PG545 at 10 μM increased the cytotoxic effect of cisplatin or paclitaxel over a range of concentrations in A2780 and SKOV3 cell lines (Supplementary Fig.

Discussion

Multiple clinical trials have demonstrated efficacy of the antiangiogenic drug bevacizumab, in the treatment of advanced OVCA [19], [20], [21]. However, despite initial responses, once bevacizumab treatment was stopped, most patients recurred which resulted in bad prognosis. Genomic and molecular complexity of epithelial OVCA and its ability to metastasise via multiple routes (intraperitoneal, hematogenous, lymphatic) might contribute to some of the resistance against single target anti

Role of funding source

The work conducted by the laboratory head Dr. Viji Shridhar is supported in part by the grants from the National Institutes of Health CA123249, and Minnesota Cancer Alliance (MOCA) to (VS) and Gynecologic Oncology Fellowship fund to (BW) from the Department of Obstetrics and Gynecology at Mayo Clinic.

Conflict of interest statement

Edward Hammond, Keith Dredge and Darryn Bampton are employed by, or act as consultants for Progen Pharmaceuticals, which developed PG545. None of the other authors have any conflicts of interest.

Acknowledgements

The authors wish to acknowledge the technical assistance of Jacie Maguire, Takayo Ota, Jenifer Baranski, Erin Trachet (Charles River Discovery Service, Morrisville, NC), Kay Meshaw (Charles River Discovery and Imaging Services, Ann Arbor, MI) on the A2780 s.c. model, Ralf Brandt (vivoPharm, Bundoora,, VIC, Australia) for the SKOV3 model, Maree Smith, Debra Siebert and Linda Wright (TetraQ, Brisbane, Australia) for the bioanalysis of the PG545 samples.

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    1

    These authors contributed equally to this work.

    2

    Current address: Department of Obstetrics, Gynecology and Women’s Health, University of Minnesota, USA.

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