Circulating osteocyte‐related biomarkers (vitamin D, sclerostin, dickkopf-1), hepcidin, and oxidative stress markers in early breast cancer: Their impact in disease progression and outcome

Highlights

• Breast cancer is associated with altered circulating osteocyte‐related biomarkers, hepcidin, and oxidative stress parameters.

• Wnt inhibitors and hepcidin are inversely correlated to vitamin D in early BC.

• Vitamin D and SOD have a good predictive of outcome in early BC.

• Hepcidin and iron metabolism are crucial elements in BC development via oxidative stress.

• Circulating DKK1, hepcidin, and SOD differ between benign breast disease and controls.

Abstract

Breast cancer (BC) is a major concern to female health worldwide. We assessed the circulating osteocyte‐related biomarkers, hepcidin, and oxidative stress status among early-stage BC patients in aspects of clinical severity and impact on the outcome. The study incorporated 73 patients categorized into 57 early-stage BC and 16 benign breast diseases and 30 healthy controls. Serum 25-hydroxyvitamin D [25(OH)D], sclerostin (SOST), dickkopf-1(DKK1), and hepcidin were measured using ELISA, while, serum oxidative stress markers were assessed by spectrophotometry. Our results show that patients with BC showed significant increase in the mean levels of DKK1, SOST, hepcidin, and LPER and significant decrease in the mean levels of 25(OH)D, SOD, GPx, and Hb when compared with controls and benign breast diseases. Significantly higher DKK1, hepcidin, and SOD levels among benign breast diseases were found in comparison to control group. There were significantly lower levels of 25(OH)D, SOD, and Hb and significantly higher levels of SOST, DKK1, hepcidin, No, and LPER with advanced grade. Lower levels of 25(OH)D, SOD and higher levels of SOST, hepcidin were observed with increasing the malignant stage. Reduced levels of 25(OH)D, and SOD were significantly associated with poor prognosis and were strong predictors among BC. There were significant negative correlations between 25(OH)D with LPER, SOST, and hepicidin. We conclude that low 25(OH)D, high SOST, DKK1, and hepcidin, and dysregulated oxidative stress could be helpful in early detection and assessment of BC. 25(OH)D, and SOD were the most relevant to tumor progression and prognosis which indicate a significant role in the BC pathogenesis and could be promising targets in management. Our research paves the way to disrupt vicious circle between these biomarkers to obtain the best care of BC.

Introduction

Breast cancer (BC) remains the most widespread malignancy among women all over the world, with more than 268,600 new cases diagnosed in the US in 2017 [1,2]. It is the second most commonly leading cause of cancer deaths in females. Despite advancements in diagnostic and therapeutic approaches, breast cancer incidence is still rising at an amazing rate. Therefore, the scientific search persists for the finding of biomarkers which can be useful with optimum sensitivity and specificity in diagnosing or predicting the tumor behavior and treatment response, or even as therapeutic targets [3]. In Egypt, it is accounting for 29.1 % of cancer deaths and affects 37.7 % of all females [4].

Vitamin D is known to be a vital micronutrient hormone with a significant function in human health. Throughout the last decades, the general public and the medical community have been increasingly concerned over the research of the effects of vitamin D deficiency as a risk factor in many autoimmune, metabolic, and neoplastic pathologies [5]. Accumulating evidence indicates that vitamin D can control the entire tumorigenesis cycle, from initiation to invasion and the interactions between the cells and microenvironments. These processes involve regulating cell behaviors such as differentiation, proliferation, autophagy, apoptosis, and epithelial -mesenchymal transition (EMT), and moderation interactions between cells and microenvironments inducing angiogenesis, production of inflammatory cytokines, and modulation of the immune system [6].

The canonical Wnt–β-catenin signaling pathway is a pathway that plays a role in accelerating cell transcription. The canonical signaling is triggered by the attachment of Wnt ligands to the double receptor complex including frizzled (FZD) protein and either low-density lipoprotein receptor-related protein 5 or 6 (LRP5/6). This results in the disruption of the multiprotein β-catenin degradation complex, thereby relieving β-catenin from its proteosomal digestion. This allows the accumulation of β-catenin in the cytoplasm and its subsequent translocation into the nucleus to serve as a transcriptional coactivator for transcription factors belonging to the T-cell factor / lymphoid enhancing factor (TCF / LEF) family to control target gene transcription [7]. Canonical wnt pathway activity is absolutely necessary for the growth of mammary glands and works during pre-and post-natal development at various critical time periods. Many epigenetic defects in the genes of the Wnt pathway have also been found in human breast carcinogenesis [8,9].

Dickkopf-related protein 1 (DKK1) and sclerostin (SOST) were among the various Wnt inhibitors explored. During embryogenesis, their expression begins in the limb bud and persists postnatally in the developed skeleton inside osteoblasts and osteocytes [10]. Sclerostin is a crucial bone remodeling regulator, prevents bone formation through Wnt signaling inhibition. It blocks canonical Wnt signaling by binding to LRP5/6. Sclerostin not only influences the bone formation and mineralization but also affects serum levels of hormones that control mineral accumulation, like calcitriol and fibroblast growth factor 23 (FGF23) [11]. While DKK1 lessens Wnt signaling by connecting to LRP5/6 and the Kremen-1 cell surface co-receptor, thus fostering receptor complex internalization [10]. Sclerostin and DKK1 expression aren't limited to bone [12]. Various studies have shown that SOST and DKK1 were involved in the control of different pathological and physiological processes like hippocampal neurogenesis, osteoclastogenesis, proliferation of tumor cells, survival, and bone metastasis [10,11,13,14]. However, the function of DKK1 in tumor biology is contentious [15]. Regarding early-stage BC, the precise roles of SOST and DKK1 in disease progression and clinical prognosis are still unclear.

Hepcidin, is a 25 amino acid hormone, synthesized by hepatocyte and secreted into the blood stream [16]. Hepcidin is encoded on the human chromosome 19 by the hepcidin antimicrobial peptide (HAMP) gene. This protein, originally known as an antimicrobial peptide, is now recognized as a type 2 acute-phase reactant due to its control by interleukin 6 (IL-6). Hepcidin attaches to the ferroportin protein (FPN), the main transmembrane iron export-protein on the iron-regulated cells. The binding causes internalization and subsequent degradation of the hepcidin-ferroportin complex resulting in blocked cellular iron efflux to the plasma and reduction of iron blood concentration. In various cancers, the alterations of iron homeostasis by hepcidin-ferroportin have been actively analyzed [17]. In BC, bone morphogenetic protein 7 (BMP 7) is overexpressed in breast tissue and linked with increased expression of hepcidin and cancer metastasis [18].

Oxidative stress (OS) develops when the reactive oxygen species (ROS) levels exceed the antioxidant defense systems because of increased ROS output and/or deterioration of the cell's antioxidant ability. This OS plays a crucial role in carcinogenesis [19]. OS can cause oxidative damages in lipids, proteins, and DNA, such as deletion, chromosomal rearrangements, mutations of vital tumor suppressor genes or proto-oncogenes and persuades stimulation of Akt/PI3K/mTOR signaling pathway, which are all hallmarks of cancer development and progression (including BC) [20]. The present study aimed to assess the circulatory osteocyte‐related biomarkers, hepcidin and oxidative stress markers among early-stage breast cancer with the possible comparison of these biochemical parameters with the detected clinicopathological findings and prognosis in these patients which provide a well understanding of the pathogenesis of BC and may be beneficial if targeted in BC management hoping to direct clinical decision-making.