Co-Variation of Serum Osteoprotegerin and Pigment-Epithelial Derived Factor as Biomarker of Pancreatic Cancer

Research Article

Austin J Gastroenterol. 2022; 9(1): 1118.

Co-Variation of Serum Osteoprotegerin and Pigment-Epithelial Derived Factor as Biomarker of Pancreatic Cancer

Edderkaoui M1*, Chheda C1, Lim A1, Pandol SJ1 and Murali R1,2*

¹Departments of Medicine, Biomedical Sciences, Samuel Oschin Comprehensive Cancer Center, Cedars-Sinai Medical Center, USA

²Research Division of Immunology, Cedars-Sinai Medical Center, USA

*Corresponding author: Mouad Edderkaoui, Departments of Medicine, Biomedical Sciences, Cedars-Sinai Medical Center, 8700 Beverly Blvd, Los Angeles CA 90048, USA

Ramachandran Murali, Departments of Medicine, Biomedical Sciences, Research Division of Immunology, Cedars-Sinai Medical Center, USA

Received: December 15, 2021; Accepted: January 12, 2022; Published: January 19, 2022

Abstract

Pancreatic cancer is one the most lethal cancers. Currently, there are reliable predictive markers to assess cancer development. Widely used CA19- 9 molecular marker has been less effective in the diagnosis of early stages of cancer.

Objective: To study if the soluble Osteoprotegerin (OPG) and pigmentepithelial derived factor (PEDF) levels in serum will be an indicator of cancer progression.

Methods: Soluble OPG and PEDF were measured from human pancreatic cancer patients by ELISA.

Results: We show that while OPG has been less predictive features, PEDF is more sensitive than CA19-9 in cancer detection. More importantly, PEDF and CA19-9 as combined markers showed higher sensitivity in stratifying early stages of pancreatic cancer.

Conclusion: Results from the pilot studies suggest that PEDF is useful biomarker for pancreatic cancer.

Keywords: Pancreatic cancer; Pigment-epithelial derived factor; Osteoprotegerin

Introduction

Pancreatic cancer has extremely poor prognosis and is the fourth leading cause of cancer-related death in the US [1]. Pancreatic ductal adenocarcinoma (PDAC) comprises more than 85% of all pancreatic cancer and the five-year survival rate for pancreatic cancer patients is estimated at a mere 6-7.7%, and complete cures are rarely if ever achieved [2].

A major challenge in the clinics is the lack of effective methods for early detection of the disease. Currently, CA19-9 (carbohydrate antigen 19-9, also called cancer antigen 19-9 or Sialyl-LewisA antigen) is the recommended marker for pancreatic cancer diagnosis in symptomatic patients and for monitoring therapy. However, it has been shown to be less reliable in predicting the progression of pancreatic cancer. Other modalities are imaging with computed tomography, magnetic resonance imaging, endoscopic ultrasound and positron emission tomography. However, these imaging tools are expensive and not suitable for early diagnosis in a wider population. There is an urgent need to identify biomarkers that can augment detection of pancreatic cancer and guide therapeutic options.

Malignant pancreatic cancer progression depends on tumor cells’ ability to disseminate via blood and lymph vessels and the perineural space; this process is facilitated via disrupted structural elements of basement membranes and extracellular matrix (ECM) by heparinase, enzymes produced by the cancer cells [3]. The main components of basement membranes and ECM are glycoproteins decorated with glycosaminoglycan (GAG) that are largely consist of the heparan sulfate (HS) type (HSPG) [4]. HSPGs containing HS are negative-charged and bind to several factors that carry positively-charged proteins, termed as “Heparin binding proteins (HSBD)” [5,6]. These proteins binding to HSPG can regulate many diverse functions from blood coagulations, infection, immunity and cancer [7] Among HSBD, of particular interest related to pancreatic cancer are the secreted proteins that contains heparin binding motif, XBBBXXBX and XBBXBX, where B is Arg/Lys are Osteoprotegerin (OPG) [8,9] and pigment epithelial-derived factor (PEDF) [10,11]. These blood proteins can competitively bind to extracellular matrix collagen and HSPG through heparin-binding motifs and regulate fibrosis, inflammation, and metastasis. For example, IL-6 which plays a critical role in PDAC progression and metastasis has been shown to bind GAG through haparin-binding-domain [12]. These observations suggest that dysregulated soluble Heparin- Binding Domain containing Proteins (sHBDP) will influence the development of pancreatic cancer.

Osteoprotegerin (OPG) is a pleotropic factor that regulates dendritic cell maturation, osteoblast and vascular functions. It is a major decoy receptor to RANKL and TRAIL, a proapoptotic cytokines through N-terminal domain. The c-terminal domain contains putative GAG binding signature. The precise role of the C-terminus of sOPG is not known. In pancreatic cancer, OPG is overexpressed in pancreatic cancer patients’ serum [8,13]. Since OPG is also secreted by immune cells such as dendric cells, we wanted to know whether the source of secreted/soluble OPG (sOPG) is pancreatic specific. For this purpose, we investigated the expression of OPG in panel of pancreatic cancer cells. We have shown that K-ras activation is positively correlated with the secretion of sOPG. By cancer cells, and not by normal cells. More importantly, it was shown that overexpression of sOPG promote pancreatic cancer growth by blocking TRAIL-induced apoptosis [9]. These observations suggested that one of the major sources for the observed excess secretion of OPG is by pancreatic cancer cells [8].

Pigment-epithelial derived factor (PEDF) is a pleotropic heparinbinding soluble protein, and it has been implicated in development of cancer by acting as linker in ECM; PEDF could link collagens with GAGs by simultaneously binding to both collagens and GAGs and could be important mediator of angiogenesis [14]. The expression of PEDF in prostate and lung cancers show that lower expression in cancer cells compared to normal cells indicated poor prognosis. In pancreatic cancer, as in other cancers, PEDF plays an important role in cancer development; unlike sOPG which is overexpressed, the loss of PEDF is correlated with invasive pancreatic cancer presumably modifying pancreatic cancer stroma to become highly vascularized (i.e. fibrotic and angiogenic). Due its role in cancer development, we investigated soluble factor sOPG and sPEDF in serum of pancreatic cancer patients. Here, we show that monitoring the expression of PEDF in solid tumor can be a useful biomarker. Based on these observations we hypothesized that the co-variation of heparinbinding proteins, in particular, the sOPG and the sPEDF expression level in serum will be a novel biomarker to stratify PDAC progression.

Materials and Methods

Quantitative real-time PCR (RT-PCR)

Total RNA was extracted using Trizol (ThermoFisher, Canoga Park, CA, USA), and reverse transcription reaction was carried out using High-Capacity Reverse Transcription Kit (Thermo Fisher, Canoga Park, CA, USA). Real-time quantitative PCR (RT-qPCR) was used for quantifying mRNA levels using the iTaq Universal SYBR Green Supermix (Bio-Rad, Hercules, CA, USA) and BioRad cfx96 platform according to the manufacturer’s protocol. Gene expression levels were normalized to that of GAPDH. Primers were purchased from Integrated DNA technologies (IDT), Coralville, IA, USA. The sequences of primers used for RT-PCR were as follow: h-PEDF-F; T A T G A C C T G T A C C G G G T G C G A T, h-PEDF-R; C C A C A C T G A G A G G A G A C A G G A G C [6], h-OPG-F; A A G A C C G T G T G C G C C C C T T G, h-OPG-R; A C G C G G T T G T G G G T G C G A T T [15], h-GAPDH-F; C C A G G T G G T C T C C T C T G A C T T C A A C A, h-GAPDH-R; A G G G T C T C T C T C T T C C T C T T G T G C T C.

Collection of human blood samples

Blood samples were collected from patients during their routine blood collections. The normal control group had no history of acute or chronic pancreatitis, diabetes, pancreatic surgery, and no family history of cancer. Patients with pancreatic cystic lesions, benign tumors, or marked pancreatic atrophy or fat degeneration on MRI images were excluded. This prospective study was approved by the local institutional review board. Written informed consents were obtained from all participants. Blood samples were provided by the Pancreatic Biomarker Bank, or “Panc-Bank,” at the Cedars-Sinai Medical Center (CSMC) with IRB protocol number: 41571.

PEDF and OPG ELISA measurement

PEDF and OPG levels were measured in the patient’s serum using the PEDF ELISA kit (Cat #: MBS760087, MyBiosource, San Diego, CA) and the OPG ELISA kit (Cat #: MBS2508007, MyBiosource, San Diego, CA), respectively following the protocol instructions.

Results

PEDF is increased in PDAC tissues compared to normal pancreatic tissues

First, we measured the mRNA level pf PEDF and OPG in mouse and human pancreatic normal and cancer tissues. We found a significant increase in PEDF levels in PDAC samples compared to normal pancreatic tissues in mice. The increase in PEDF mRNA levels was 10-fold in mice (Figure 1A) and 2-fold in human tissues (Figure 1C). Differently from PEDF, OPG levels showed a different ratio in mice and human tissues. In mice we found a significant increase in OPG in PDAC tissues compared to normal tissues (Figure 1B); while in humans, OPG was lower in PDAC tissues compared to normal tissues (Figure 1D).