TRAIL and TRAIL Receptors as Prognostic Markers in Breast Cancer Patients

Background: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a key player in the extrinsic pathway of apoptosis; it selectively damages cancer cells through binding to its surface receptors, however, cancers can escape this pathway through expression of dysfunctional decoy receptors. Purpose: The present study directed mainly to elucidate the serum TRAIL levels in breast cancer patients and to explore the variation in gene expression of TRAIL death and decoy receptors in breast cancer tissues, and to explore their role as prognostic markers in breast cancer as well as to detect their correlation with Patients’ Clinical Characteristics. Subjects and Methods: TRAIL levels were assayed in the sera of 124 breast cancer patients and 150 healthy females. Moreover, the expression of TRAIL death and decoy receptors was determined in both malignant and adjacent normal breast tissues collected from patients. ER, PR and Her-2 expression in breast cancer tissue were performed using immunohistochemical method. Apoptotic index (AI) was analyzed using H&E stain under light microscopy. Results: Serum levels of TRAIL in breast cancer patients were significantly lower than controls (P < 0.001), additionally, the expression of DR4, DR5 and DcR1 were significantly filing of TRAIL receptors expression may serve as prognostic markers in breast cancer patients.


Introduction
One of the most essential hallmarks of cancer is apoptosis-resistance [1]. Cancer cells escape apoptosis either through the overexpression of anti-apoptotic proteins or the inhibition of the pro-apoptotic signals. With regard to breast cancer (BC), previous studies reported resistance of BC cells to receptor-mediated apoptosis, even with the presence of receptors in cancer cells [2].
Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is expressed mainly by immune system cells; it triggers the extrinsic pathway of apoptosis through interacting with its surface receptors expressed on the target cells. TRAIL binds to two pro-apoptotic receptors known as death receptor 4 (DR4/ TRAIL-R1), death receptor 5 (DR5/TRAIL-R2), and two decoy receptors DcR1 (TRAIL-R3), DcR2 (TRAIL-R4) [3]. As their names imply, only two of these receptors (TRAIL-R1 and TRAIL-R2) are functional death receptors as they possess the functional cytoplasmic domain and are capable of transmitting the apoptotic signals into cells. Conversely, TRAIL-R3 and TRAIL-R4 are decoy receptors that lack the functional death domains (DD), and their overexpression may result in blocking of TRAIL-mediated apoptosis [4].
Interestingly, TRAIL preferentially induce apoptosis and kill cancer cells without affecting normal cells, thus rendering it a highly promising approach in cancer treatment [5]. However, cancer cells express innate or acquired resistance to TRAIL-induced apoptosis through the expression of the dysfunctional decoy receptors (DCR1 and DcR2); these receptors compete with the functional death receptors (DR4 and DR5) for the binding sites on TRAIL. Binding to decoy receptors disrupts TRAIL-induced apoptosis through either ability to dilute out TRAIL ligands (like DcR1) or supply anti-apoptotic signals (like DcR2) to cells [6].
On the other side, TRAIL-induced apoptosis is crucial for tumor surveillance through induction of immune-mediated clearance of metastatic cells [7], loss of TRAIL receptor-expression is associated with worse prognosis and tumor recurrence in patients suffering from different tumors [8]. Additionally, increased expression of decoy receptors correlated with both tumor stage and metastasis in patients suffering from colorectal cancer [9]. In spite of the increasing interest for TRAIL-receptors as therapeutic approach in management of cancer, studies on the genetic expression, distribution and relevance of TRAIL-receptors as prognostic markers in breast cancer are still lacking.
To the best of our knowledge, few previous studies concern with the variation

Sample Collection
After providing an inforned consent a blood sample was withdrawn from all enrolled participants. Subjects' sera were kept at −80˚C until the time of use. All enrolled patients underwent modified radical mastectomy. During surgery, two tissue samples were taken, one from the resected tumor and the other from the adjacent apparently normal tissue. A part of each tissue was embedded in paraffin for histopathological examinations, while the rest was immediately transferred to −80˚C for further analysis. Patients were clinically followed up for a mean time of 60 months.

Apoptotic Index (AI)
From the paraffin embedded tissue, two 4 µm thickness sections were stained with hematoxylin and eosin (H&E) for calculating the apoptotic index (AI). The H&E stained sections were examined using oil immersion lenses (×100). From each section, 10 fields devoid of artifacts were selected. 1000 cells were evaluated for presence of apoptotic cells and apoptotic bodies. The AI was expressed as a percentage of the total number of non-apoptotic cells counted.  Table 1.

Serum TRAIL Quantification Assay
Quantitative determination of serum levels of TRAIL in the sera of enrolled patients as well as control subjects was done using an Enzyme-linked Immunosorbent Assay (ELISA) Kit (Uscn Life Science Inc., China) according to the manufacturer's instructions.

Statistical Analyses
Data were analyzed using SPSS software package version 20.0 (IBM Corporation, Chicago, Illinois, USA). The distributions of quantitative variables were tested for normality using Kolmogorov-Smirnov test. Mann-Whitney test was used to compare between two studied groups, for Kruskal Wallis test was used to compare between more than two groups. Wilcoxon signed ranks test was used to compare expression levels between cancer and adjacent normal tissues, Spearman correlation test was used to study the correlation with the clinicopathological parameters. Cox proportional hazards regression model analysis was done to investigate the prognostic value of studied parameters. At all statistical analyses, p value was considered significant at ≤0.05.

Patients' Clinical Characteristics
The clinicopathological characteristics of the enrolled patients are presented in Table 2. The median age of enrolled patients didn't differ significantly from that

Decreased Serum Levels of TRAIL in Sera of Breast Cancer Patients Compared to Healthy Subjects
The serum TRAIL levels in breast cancer patients were found to be significantly lower than those in the healthy control (P < 0.001) (Figure 1(a)).

Genetic Expression of TRAIL Death/Decoy Receptors and Apoptotic Index in Breast Cancer Tissues
The apoptotic index in breast cancer tissues was significantly higher than that in the corresponding normal tissues (P < 0.001) (Figure 1(b)). Moreover, the expression of DR4, DR5 and DcR1 showed a significant up-regulation in cancerous tissues as compared to normal tissues (p < 0.001, p < 0.001, p = 0.039, respectively), however, the genetic expression of DcR2 didn't show any significant difference between cancerous tissues and normal tissues (p > 0.05) (Figure 1(c)).

TRAIL, DR4, DR5, DcR1 and DcR2 Are Correlated to Apoptotic Index
Apoptotic index showed significant positive correlations with both serum TRAIL levels and expression of DR4; however, it had a significant negative correlation with expression of DcR1. On the other hand, the expression of DR5 and DcR2 didn't show any significant correlation with apoptotic index (Table 3).

TRAIL, DR4, DR5, DcR1 and DcR2 Are Correlated to Clinicopathological Characteristics
Serum TRAIL levels showed a significant negative correlation with tumor size, grade, and clinical stage, but the correlation with receptor status, lymph node invasion, and vascular invasion was found to be insignificant. On the other hand, the expression of DcR1 showed a significant positive correlation with tumor size, however, TRAIL DR4, DR5 and DcR2 didn't show any significant correlation with any of the clinicopathological parameters (Table 3). On the other side, Apoptotic index correlated negatively with the grade, while positively correlated with the positive expression of ER and PR (Table 3).

Association between TRAIL, DR4, DR5, DcR1, DcR2 and Apoptotic Index and Clinicopathological Characteristics
Stratification analyses revealed significant association between serum TRAIL levels with lower histological grade and clinical stage, and between the expression of DcR1 with the tumor stage. Moreover, both serum TRAIL and DcR1 expression showed a significant association with mortality among BC patients (Table 4). American Journal of Molecular Biology

Decreased Serum TRAIL and Overexpression of DcR1 Are Prognostic Factors for Poor Overall Survival among BC Patients
Univariate and multivariate analyses were performed on BC cases to identify factors that correlate with prognosis using Cox proportional hazards regression model analysis. The results showed that decreased serum TRAIL (p ≤ 0.001), increased expression of DcR1 (p ≤ 0.001), were associated with worse overall survival. Moreover, the multivariate analysis indicated that they can be independent prognostic factors for overall survival among BC patients (Table 5).

Discussion
TRAIL is a key member of TNF family that induces apoptosis upon binding to its surface receptors; because of the unique selectivity of TRAIL in damaging cancer cells without any harmful effects to the normal cells, TRAIL became a promising therapeutic approach in cancer management; moreover, it has been already entered in some clinical trials for treatment of many solid tumors. However, some tumors unfortunately develop resistance to TRAIL-mediated apoptosis through expression of decoy receptors instead of death receptors [12]. Therefore, the expression of TRAIL death receptors and the downstream signaling molecules of the TRAIL pathway represent prerequisite for TRAIL-based therapies. Furthermore, the expression ratio of death and decoy receptors may be decisive for the sensitivity of tumor cells to TRAIL [13]. Therefore, we investigated the serum levels of TRAIL in BC patients in addition to analysis of the expression pattern of death as well as decoy receptors in BC tissues. The present study revealed a significant decrease in serum TRAIL level in breast cancer patients; these findings are in accordance with those of Toiyama D who demonstrated that serum TRAIL levels in RCC patients were markedly lower than the healthy controls [14]. Additionally, few previous studies reported a decrease in serum TRAIL levels in many cancers including metastatic melanoma [15] and chronic myeloid leukemia [16] as compared to its levels in healthy individuals.
Decreased TRAIL increase the ability of cancerous cells to evade immune surveillance and apoptosis, this in turn prompts cancer development and progression [14]. In RCC, serum TRAIL levels increased significantly after the complete resection of the primary tumor. This may be attributed to the involvement of cancer cells in the inhibition of TRAIL production circulation via two possible effects. First, cells may secrete some soluble factors that can inhibit immune cells production of TRAIL, such as lymphocytes. The second possible mechanism may be through sequestration of TRAIL-producing immune, resulting in decreased serum TRAIL levels [14]. This hypothesis can be supported by the finding of Liu et al. who reported decreased expression of TRAIL in breast cancer tissues comparing to the adjacent normal tissues [17]. Moreover, removal of the renal tumor might result in the return of circulating TRAIL levels to baseline values [14].
The significant upregulation of TRAIL DR4 and DR5 may be attributed to the fact that the increased expression of TRAIL receptors may enhance the apoptotic response induced by TRAIL and lead to an increase in the apoptotic rate among tumor cells [18] [19]. These results were supported by the positive correlation between the expressions of TRAIL DR4 and the apoptotic index in the breast cancer tissues. Previous studies indicated possible selectivity of DR4 vs. DR5 apoptotic signaling in tumor cells from different origins. Tumors such as pancreatic carcinoma [20], chronic lymphocytic leukemia or mantle cell lymphoma prefer DR4 for apoptosis induction [21] [22]; other types, including colorectal or other epithelial cancer cells appear to prefer DR5 as an apoptosis inducer [23]  DR5 are required for rhTRAIL to induce the maximal killing effect of breast cancer cells, moreover, the loss of cell surface expression of DR4 or DR5 lead to cellular resistance to their corresponding antibody and correlates with a decreased sensitivity of cells to TRAIL [25].
On the other hand, the significant overexpression of DcR1 overexpression in malignant tissues contributes to ability of cancerous cells to evade the TRAILmediated apoptosis. TRAIL-R3 and TRAIL-R4 decoy receptors lack the functional intercellular death domain, this in turn prevent their association with procaspases 8 and 10 to form DISC which is mandatory for activating downstream signaling pathway [26]. A previous study documented a significant upregulation of decoy-R1 (DcR1) but a downregulation in decoy-R2 (DcR2) in head and neck cancers [27]. Moreover, the expression of DcR2 was reported to be significantly increased in high grade meningiomas [28], DcR2 binds to TRAIL-R2 via the pre-ligand-binding assembly domain (PLAD) [29], thus the overexpression of DcR2 neutralizes the apoptosis-inducing capacity of TRAIL-DR5 and confers TRAIL resistance in higher tumor grades. Accordingly, DcR2 overexpression has been reported to inhibit TRAIL-induced apoptosis in many tumors [30] [31].
The negative correlation between serum TRAIL level and with tumour size, grade, and clinical stage, are in accordance with Toiyama D who reported inverse correlation between the serum TRAIL level the progression and aggressiveness of RCC. These findings suggest that decreased TRAIL may allow cancer cells to escape immune surveillance and apoptosis [14]. Verim A also indicated a statistically significant association between TRAIL, and clinical parameters including stage, lymph node, metastasis, perineural invasion and degree of tumor differentiation in Lx SCC patients [32].
Decreased serum TRAIL levels and overexpression of DcR1 were associated with decreased overall survival among breast cancer patients, these findings are in accordance partly with Toiyama D who reported that decreased serum levels of TRAIL correlated to survival among RCC [14]. Furthermore, Liu et al. reported increased survival rates in breast cancer patients with positive expression of TRAIL in their tissues over patients lacking tissue-TRAIL expression [17] On the contrary; Koschny reported the lack of correlation between TRAIL receptors with the progression-free or overall survival among meningioma patients [28].
In conclusion, serum TRAIL levels and TRAIL receptor expression pattern might be useful prognostic markers to follow up disease progression by virtue of their connection to clinical staging and pathologic grading in BC patients, moreover, profiling of TRAIL receptor expression be helpful in determining ideal patients' targets for a TRAIL-mediated gene therapy.

Ethical Approval
All procedures performed in studies involving human participants were in accordance with the ethical standards of local ethics committee, Medical Research Institute, Alexandria University, and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.

Data Availability
The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.