Characterization of OCT3/4, Nestin, NANOG, CD44 and CD24 as stem cell markers in canine prostate cancer

https://doi.org/10.1016/j.biocel.2019.01.002 Get rights and content

Highlights

  • Nanog and Nestin are potential cancer stem cell markers in canine prostate cancer.

  • Prostate cancer presented OCT3/4 decreased expression.

  • CD44 high expression was related to poor patient outcome.

Abstract

The cancer cell population is heterogeneous, and cancer stem cells (CSCs) are important for tumor growth and maintenance. The CSC population is associated with different neoplastic characteristics, such as cell migration, resistance to apoptosis, radiation therapy and chemotherapy. To increase the knowledge of CSCs in canine prostate cancer (PC), we characterized CSC markers in canine PC tissues and tumorspheres. We performed immunohistochemistry of OCT3/4, Nestin, NANOG, CD44 and CD24 in 10 normal canine prostatic tissue samples, 10 prostatic hyperplastic (PH) tissue samples and 28 PC tissue samples. Then, we established two canine prostate cancer cell cultures and characterized the CSC profile of tumorspheres grown from these cultures. Normal and PH tissues were positive for Nestin, NANOG, CD44 and CD24 only in the basal cell layer. OCT3/4 was expressed in the luminal cells of normal and PH tissues. There was no significant difference in Nestin expression among the prostatic tissues. However, we found higher expression of NANOG and CD44 in canine PC tissues than that in normal and PH tissues. Tumorspheres from canine prostate cancer cells express OCT3/4, Nestin, NANOG and CD44, indicating that these markers may be potential cancer stem cell markers in canine PC. The results obtained can be useful to better characterize the stem cell population in canine prostatic cancer and to guide future studies in comparative oncology.

Introduction

Human prostate cancer (PC) is the most common cancer subtype in the western world and has a high mortality rate (Siegel et al., 2018). The greatest therapeutic challenge of human PC is the development of resistance to standard androgen-deprivation therapy (Cattrini et al., 2017). Studies performed on animal models of prostate cancer have increased the understanding of tumor biology, metastasis and therapeutic targets (Facina et al., 2018; McClurg et al., 2018), and dogs can provide an interesting spontaneous model for human PC (Fonseca-Alves, 2018).

Canine PC is a multifactorial disease with a heterogeneous cancer cell population, which may promote tumor proliferation, invasion and metastasis (Keller et al., 2013). Cancer stem cells (CSCs) maintain the capacity for cellular proliferation, survival and motility and thereby contribute to carcinogenesis in many tumors, including prostate cancer in men (Kleeberger et al., 2007). The presence of CSCs could explain the long life of cancer and its immortalization since CSCs are resistant to apoptosis and chemotherapeutic agents (Jaworska et al., 2015; Jeter et al., 2015; Klarmann et al., 2009). In human PC, neoplastic cells with stem cell properties can express different CSC markers, such as OCT3/4, Nestin, NANOG, CD44 and CD24 (Miyazawa et al., 2014). However, limited information is available on the existence of CSCs in canine PC. Since canine PC can be an important model for the human counterpart, the characterization of canine PC is an important step for comparative studies.

The gene expression of CD133, CD44, C-KIT, CD34, ITGA6, OCT4, DDX5 and MELK (Moulay et al., 2013) and the protein expression of Survivin and Sox9 (Bongiovanni et al., 2018) were previously investigated in canine PC. CD117 gene and protein expression were also previously demonstrated. However, a loss of CD117 was found in PC samples, indicating that CD117 has no role in prostate tumorigenesis (Fonseca-Alves et al., 2017). However, these authors did not perform functional studies to associate staining with stem cell properties. Usui et al. (2017) established an organoid model of canine PC and demonstrated high CD44 expression, indicating CD44 as a cancer stem cell marker in canine PC.

OCT3/4 (also known as Oct4 and Pou5F1) is a transcriptional regulator of genes involved in maintaining an undifferentiated pluripotent state and is characterized by two isoforms: OCT4A (isoform 1), which is localized in the nucleus, and OCT4B (isoform 2), which is mainly localized in the cytoplasm (Lee et al., 2006; Monsef et al., 2009). In the human prostate, OCT4 isoform 1 is associated with self-renewal and pluripotency of CSCs (Lee et al., 2006).

Nestin is an intermediate filament involved in motility, cellular stress, signal transduction and myogenesis regulation (Hyder et al., 2014). Nestin has been reported in prostate cancer in association with cell migration, metastasis (Kleeberger et al., 2007) and the self-renewal of prostate CSCs (Kimbro and Simons, 2006; Mabjeesh and Amir, 2007), and it is considered a prognostic marker in other cancers (Hope et al., 2016; Bernal and Arranz, 2018; Bien-Möller et al., 2018).

NANOG is a divergent homeobox domain protein transcription factor that functions in association with OCT4 to form an embryonic stem cell identity (Gong et al., 2015; Amini et al., 2014). NANOG also plays an important role in cell proliferation, tumorigenicity, clonogenic growth, invasiveness and therapeutic resistance (Jeter et al., 2015).

CD44 is a cell surface marker and transmembrane glycoprotein involved in various cellular activities by binding with hyaluronan or other extracellular molecules (Yu et al., 2012; Moura et al., 2015). CSC properties have been explored in canine mammary gland tumors, and the CD44+/CD24- phenotype is a feature of tumors with more aggressive behaviors (Magalhães et al., 2013; Figueroa et al., 2015; Rybicka and Król, 2016).

The gene expression of the stem cell markers CD44, CD133, CD34, c-KIT, OCT4, ITGA6, MELK and DDX5 has been evaluated in different canine prostatic cell lines (Liu et al., 2007). However, no studies have characterized the stem cell population of tumorspheres or canine prostatic tissues. To identify potential cancer stem cells in canine PC, we analyzed the expression of OCT3/4, NANOG, Nestin, CD44 and CD24 in normal, hyperplastic and neoplastic canine prostate samples, as well as tumorspheres derived from two canine prostate cancer cell lines.

Section snippets

Formalin-fixed paraffin-embedded tissue samples

This study was approved by the animal ethics committee of the School of Veterinary Medicine and Animal Science of the São Paulo State University (Protocol: 0004/2017). To evaluate the expression of stem cell markers in canine prostatic tissue, 48 formalin-fixed paraffin-embedded canine prostatic tissues were retrieved from the archives of the Veterinary Pathology Service, São Paulo State University and the School of Veterinary Science, The University of Queensland. Ten normal prostatic tissues,

Clinical and histopathologic analyses

Based on the clinical records, metastases were present in 14 out 28 PC cases. Six out of 14 (42.9%) of these patients had metastases at the time of diagnosis, and the most commonly affected sites were the lung (4/6), bone (3/6) and intestine (2/6). Eight animals underwent chemotherapy (8/14), and, in three of them (3/14), only a surgical procedure was performed. The remaining three animals (3/14) received only palliative treatments. The main clinical signs in the dogs with metastases were

Discussion

The present study identified a group of cells that express OCT3/4, CD44+/CD24, NANOG and Nestin in canine PC tissues, with the potential to be a cancer stem cell population. No previous literature has shown a strong link between the different CSC markers and stem cell properties in canine PC. Moulay et al. (Moulay et al., 2013) evaluated the gene expression of several stem cell markers in canine monolayer prostatic cell lines and, among them, identified high transcript levels of CD44. However,

Conclusion

Canine prostate cancer does express a population of cells with a stem cell phenotype, confirmed by in vitro tests on tumorsphere formations. This new information can be useful for future studies in comparative oncology, as well as studies on the role of CSCs in prognosis (survival time), tumor chemoresistance and metastatic potential in canine PC.

Acknowledgment

The authors would like to thank the São Paulo Research Foundation (FAPESP # 2015/25400-7 and 2017/25822-4) for financial support.

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