Elsevier

Research in Veterinary Science

Volume 135, March 2021, Pages 297-303
Research in Veterinary Science

Nucleolin as a potential biomarker for canine malignant neoplasia

https://doi.org/10.1016/j.rvsc.2020.10.004Get rights and content

Highlights

  • Nucleolin is markedly expressed in PBMCs cells of dogs with neoplasia.

  • Nucleolin mRNA expression in canine PBMCs is higher than controls.

  • Higher mass bands of nucleolin were detected in PBMCs of canine neoplastic.

  • T-cell lymphoma has nucleolin expression higher in all cells analyzed.

  • Nucleolin amplicons obtained by RT-PCR was confirmed by Sanger sequencing.

Abstract

Human nucleolin (NCL) is a multifunctional protein that is involved in diverse pathological processes. Recent evidences have shown that NCL is markedly overexpressed on the surface of most human cancer cells when compared to normal cells, being overexpressed in several malignant cells. Based on the exposed, the purpose of this pilot study is to investigate the expression pattern of NCL in canine malignant neoplasia and control groups. NCL expression at both messenger RNA and protein levels in the subcellular fractions were respectively detected by RT-PCR and western blotting, allowing to infer the NCL positivity rate in canine neoplasia. The identity of NCL amplicons obtained by RT-PCR was confirmed by Sanger sequencing and found to correspond to Canis lupus familiaris. Using flow cytometry, the blood cells expressing NCL from canine neoplasms were also identified using several cell surface markers and their levels quantified. These results showed that NCL expressed in lymphocytes, monocytes and neutrophils in dogs with malignant neoplasia is higher (> 50%) when compared with the control group. We found an increased expression of surface and cytoplasmic NCL in canine malignant neoplasia group, while nuclear NCL is predominantly found in the control group. Overall, this study discloses and identifies for the first time the presence of NCL in canine blood.

Introduction

Cancer is a major cause of disease and a leading cause of death in aging humans and older pet animals (Snyder and Johnson, 2014). Currently, it is estimated that each year, around 6 out of 70 million dogs in USA will develop cancer (Center for Cancer Research, 2019). For pet dogs, both lifespan and cancer incidence vary between breeds and countries (Kent et al., 2018). As expected, risk increased with age, and 50% of the dogs over 10 years of age died of cancer (Merlo et al., 2008). Despite all studies seem to agree with susceptibility of certain breeds to develop cancer, there is no consensus about a specific ranking (Adams et al., 2010; Bonnett et al., 1997). For instance, breeds at highest risk of developing pyometra and mammary tumors include Leonbergers, Irish wolfhounds, Bernese mountain dogs, great Danes, Rotweillers, among others (Jitpean et al., 2012). Although universal updated data is scarce, a recent study in northern Italy with 10,095 dogs for a 90-month period showed that cancer incidence rate was 804 per 100,000 dog-years being cutaneous mastocytoma and hemangiopericytoma, mammary gland complex carcinoma, and simple carcinoma the most common cancer types (Baioni et al., 2017). Higher cancer rates were observed in purebred dogs, particularly in Yorkshire terrier and Boxer breed (Baioni et al., 2017). Another study compiled the cancer incidence for other countries such as the UK, USA (State of California) and Canada (Ontario province) with incidence rates of 747.9, 381 and 850 dog-years, respectively (Merlo et al., 2008). For these reasons, minimally invasive biomarkers that can be measured in blood and used in early diagnosis of cancer have become an important subject in human and veterinary medicine (Kycko and Reichert, 2014). However, although human and canine cancer present several similarities at the level of their microenvironment and histopathology, clinically useful cancer biomarkers identified so far in humans have not been transposed to veterinary oncology. To date, only two circulating microRNAs have demonstrated potential biomarkers for a broad spectrum of canine cancers, according to a study recently published (Heishima et al., 2017). Mammary tumors are the most frequent neoplasia in dogs, being also the most studied (Kaszak et al., 2018). The most reliable biomarkers for mammary tumors are antigen Ki-67 (Carvalho et al., 2016), human epidermal growth factor receptor 2 (HER-2) (Ressel et al., 2013) and cyclooxygenase 2 (COX-2) (Carvalho et al., 2017; Guimarães et al., 2014), which can be detected in both serum and tissue samples. Other multiple biomarkers combined have been shown to be most sensitive and specific, such as C-reactive protein, thymidine kinase 1, and haptoglobin, which have been most extensively studied and commercialized in diagnostic tests for canine lymphoma (Bryan, 2016; Lee et al., 2019). These tests are relatively sensitive to the presence of high-grade lymphoma in the body, but they did not distinguish aggressive lymphomas or immunophenotype of lymphoma. Further studies are necessary to determine if early intervention guided by biomarker elevation will improve the quantity or quality of life for dogs with lymphoma (Bryan, 2016; Lee et al., 2019). Circulating heat shock protein 70 (Hsp70) was recently measured in liquid biopsies of canine tumor patients as a potential biomarker (Salvermoser et al., 2019).

Nevertheless, the efforts to develop new biomarkers especially by biotech/pharmaceutical industries for canine neoplasia have been limited due to difficulties in investing in clinical trials, rigor in disease staging, follow-up and lack of biological markers, among others (Klingemann, 2018). Altogether, these reasons emphasize the urgent need to identify novel biomarkers for improving the diagnostic and prognostic of these diseases.

NCL is a multifunctional phosphoprotein found in nucleus, nucleoplasm, cytoplasm and cell membrane of eukaryotic cells (Ugrinova et al., 2018). The phosphorylation and glycosylation conditions of different domains of NCL affect the distribution of the protein. The protein is constituted by four RNA-binding domains (RBDs), which are involved in the interaction with nucleic acids (Jia et al., 2017; Ugrinova et al., 2018). According to its cellular location, NCL has been in described to be involved in different cellular processes. In the nucleus and nucleoplasm, NCL is involved in ribosome biogenesis, chromatin organization and stability, DNA and RNA metabolism and microRNA processing (Jia et al., 2017). In the cytoplasm, NCL is correlated with mRNA stability and regulation of translation (Abdelmohsen et al., 2011; Chen et al., 2012). Cell surface NCL serves as a ligand of extracellular molecules implicated in cell differentiation, adhesion, and leukocyte trafficking, inflammation, angiogenesis and tumorigenesis (Koutsioumpa and Papadimitriou, 2014). Interestingly, human NCL is known to shuttle between nucleus, cytoplasm and cell surface and it is markedly overexpressed on the surface of most cancer cells when compared to normal cells (Mongelard and Bouvet, 2007). By taking advantage of such fact, NCL is an attractive target for diagnosis purposes. Recently, NCL staining was used to separate circulating prostate cancer cells from white blood cells (WBCs) in human patients with metastatic disease (Chalfin et al., 2017). Other studies identified NCL as a candidate biomarker for the diagnosis of hepatocellular carcinoma (Song et al., 2009). However, and to our best knowledge, there are no studies involving NCL detection in the blood of dogs with neoplasia.

Herein, we report the presence of circulating NCL in the blood of dogs with malignant neoplasia. Initially, a complete physical examination and biochemical profile were performed by veterinarians, envisaging to establish the animal groups in this study. Two groups were established in this study: dogs diagnosed with malignant neoplasia and control group. Western blot, RT-PCR, flow cytometry and fluorescence-activated cell sorting (FACS) analysis and sequencing of peripheral blood mononuclear cells (PBMC) were performed. The expression of NCL was higher (> 50%) in PBMC of dogs with neoplasia than in the control group. These results pave the way for using NCL as a potential low invasive biomarker of canine malignant neoplasia that can be easily detected in blood samples and measured with sensitive, specific and cost-effective assays.

Section snippets

Materials and methods

The PBMC cells were separated from whole blood and plasma by Ficoll-Isopaque centrifugation. NCL was detected in isolated canine PBMC cells using three distinct methods, namely: Western-blot, which is based on the detection of NCL protein using an antibody; RT-PCR by detecting NCL mRNA expression; and flow cytometry, in which the percentage of cells expressing surface NCL was evaluated.

Results

All dogs included in this study were evaluated by complete physical, hemogram and biochemical analysis. None of the animals had received any immunosuppressive treatment, including surgery, chemotherapy, or radiotherapy, at the time blood samples were collected. Animals were included in two groups: control group and dogs diagnosed with malignant neoplasia. The neoplasia classification was performed according to histological type and topography as shown in Table S1. During the course of the

Discussion

Based on previous studies in humans, it is known that NCL plays an essential role in cell survival, growth and proliferation and is localized primarily in the nucleus of normal cells, while on cancer cells it is present in the cytoplasm and at the cell surface (Ugrinova et al., 2018). In our recent work, we showed that cell surface human NCL is a potential target of nucleic acid secondary structures and could be successfully used in cancer therapeutics and diagnosis (Santos et al., 2019). In

Conclusions

The preliminary results of our pilot study showed experimental evidence of the potential exploitation of NCL as a cancer biomarker of canine malignant neoplasia. The higher molecular mass (>110 kDa) bands of NCL were detected by western blot in PBMC cells of canine neoplastic diseases, while NCL lower molecular mass bands (< 75 kDa) were found in control group. The RT-PCR analysis also detected significant differences in the expression levels of NCL mRNA. Furthermore, the profile of NCL mRNA

Declaration of Competing Interest

The authors declare no conflict of interest.

Acknowledgements

The authors acknowledge Jean-Louis Mergny for the valuable discussion and contribution to the proof-reading of the manuscript. This work was supported by FCT project “Projeto de Investigação Exploratória” ref. IF/00959/2015 entitled “NCL targeting by G-quadruplex aptamers for cervical cancer therapy” financed by Fundo Social Europeu e Programa Operacional Potencial Humano, MIT Portugal FCT project BIODEVICE ref. MIT-EXPL/BIO/0008/2017 and UTAustin Portugal Program Exploratory project DREAM ref.

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